InteractiveFly: GeneBrief

branchless : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

branchless is Drosophila's only known fibroblast growth factor (FGF), and as its name implies, is involved in branching morphogenesis. Branching morphogenesis is an essential part of development in organs such as the trachea and lungs. Before taking a closer look at the gene branchless, a brief overview of branching morphogenesis and the role of fibroblast growth factors in general: FGFs constitute a large family of peptide growth factors, with nine different mammalian FGF genes and four genes encoding FGF receptors (Johnson, 1993). FGFs function as mitogens, trophic factors and differentiation factors. FGFs are involved in several processes of branching morphogenesis: in lung morphogenesis, in branching of seminiferous tubules and as stimulators of angiogenesis.

The Drosophila FGF receptor Breathless (BTL), expressed in the developing tracheal system, promotes branching at several stages of tracheal development, and plays a permissive role in the formation of certain branches (Reichman-Fried, 1994 and 1995). A second FGF receptor, Fibroblast growth factor receptor 1, is necessary for heart formation and mesodermal cell migration (Beiman, 1996 and Gisselbrecht, 1996).

The Drosophila tracheal (respiratory) system is a tubular epithelial network that delivers oxygen to internal tissues. Trachea form from segmentally repeated clusters of tracheal precursor cells, which give rise to the tracheal system by cell migration and elongation. Each cluster invaginates from the ectoderm and forms an epithelial sac of about 80 cells. The six main (primary) branches of each sac begin to form as one or two lead cells from any of five or six positions migrate out in stereotyped directions, similar from segment to segment. A small number of cells follow the lead cells and organize into tubes as they migrate. Several hours later, secondary branches sprout from the primary branches. Subsequently secondary branches ramify into dozens of terminal branches, which are long cytoplasmic extension that form a lumen and transport oxygen directly to the fly's tissues. Each level of tracheal branching is controlled by a particular set of genes, which have provided molecular markers for the different branch types. For example, breathless is necessary for primary branching, pointed is required to form secondary branches (Samakovlis, 1996) and Serum response factor (Guillemin, 1996) regulates terminal branch formation (Sutherland, 1996).

In the absence of branchless, tracheal cells fail to migrate and branch. bnl is expressed not in the trachea but in ectodermal cells that overly the migrating and branching trachea. The striking feature of bnl expression is its spatial complexity, one of the most complex patterns known in the embryo. For example, at stage 11, just before tracheal branching begins, bnl expression appears in five small clusters of epidermal cells arrayed around the tracheal sac, at positions where the five primary tracheal branches will soon bud. As the primary branches grow by cell migration over the next 2 hours (stages 12 and 13), expression in the clusters decreases. This appears to occur in a specific spatial pattern: the bnl-expressing cells closest to or contacting the growing tracheal branches lose expression first, with the tracheal cells continuing to migrate toward the remaining bnl-expressing cells. Two more cell clusters begin expressing bnl as expression in the other clusters turns off, presaging the subsequent outgrowth of additional specific branches (Sutherland, 1996).

What regulates the complex expression pattern of bnl? The answer is not yet known, but most likely a conbination of segmentation and dorsal-ventral patterning genes act on the promoter sequence of bnl. The even more striking dynamic nature of bnl expression, with new patches developing bnl expression even as expression in older patches declines, suggests that there is a complex temporal scheme of bnl expression added on top of the spatial one, and that there is feedback between branching and bnl expression, resulting in the diminished expression overlying positions where branching has taken place (Sutherland, 1996).

In Drosophila, Branchless acts as a guidance molecule controlling tracheal cell migration, but does not act as a mitogen, a documented role of FGFs in vertebrates. As expected for a chemoattractant, the concentration of BNL is critical for migration: migration of tracheal branches is sensitive to the level of bnl expression. BNL acts not only on initial tracheal branching, but acts during formation of secondary and tertiary branching. At each stage in branching, different genes, each dependent on bnl function, are expressed in temporal sequence (Sutherland, 1996). What regulates the temporal progression of bnl dependent gene expression? As descrubed below, one of the critical regulatiors of bnl levels is the local oxygen supply.

Oxygen regulation of airway branching in Drosophila Is mediated by Branchless FGF

Sprouting of the major tracheal branches is stereotyped and controlled by hard-wired developmental cues. Ramification of the fine terminal branches is variable and regulated by oxygen, and this process is controlled by a local signal or signals produced by oxygen-starved cells. Evidence is provided that the critical signal is Branchless (Bnl) FGF, the same growth factor that patterns the major branches during embryogenesis. During larval life, oxygen deprivation stimulates expression of Bnl, and the secreted growth factor functions as a chemoattractant that guides new terminal branches to the expressing cells. Thus, a single growth factor is reiteratively used to pattern each level of airway branching, and the change in branch patterning results from a switch from developmental to physiological control of its expression (Jarecki, 1999).

There were 68% more cytoplasmic extensions and mature branches in first instar larvae grown for 20 hr under 5% O2 compared to siblings grown under normal atmospheric oxygen (21% O2). Conversely, larvae grown under high oxygen tension (60% O2) had fewer branches than normoxic controls. Similar effects are observed at each larval stage and at different positions in the animal. The morphology of the branches is also affected by oxygen. Under high oxygen tension the terminal branches are shorter and straight with few side branches, whereas under the low oxygen condition they are long and tortuous with many side branches. Thus, oxygen is an important regulator of terminal branching, influencing the initial budding and the final number and morphology of the branches (Jarecki, 1999).

To determine if the effects of oxygen on terminal branching are globally regulated or mediated by local signals produced by oxygen-starved tissues, small regions of the larvae were deprived of their normal oxygen supply. This was accomplished by generating clones of blistered- tracheal cells, which are unable to form terminal branches, leaving the surrounding region without its normal tracheation. The blistered+ tracheal cells in neighboring segments grow into the detracheated region, sprouting 40% more terminal branches than normal, whereas blistered+ tracheal cells not bordering the clone are unaffected. To determine if it was the absence of terminal branches or just the absence of an oxygen supply that induced neighboring branches to grow, clones of synaptobrevin- tracheal cells that extend terminal branches but are unable to form a lumen and deliver oxygen to the target were tested. Even in the presence of the nonfunctional synaptobrevin- terminal branches, neighboring wild-type tracheal cells grow into the oxygen-starved region. This implies that it is lack of oxygen delivery to the region, not some other function of missing terminal branches, that causes neighboring tracheal cells to respond. It is concluded that terminal branching is regulated by local oxygen need, and that oxygen-starved cells produce a signal or signals that can attract tracheal branches from as far as one segment away (Jarecki, 1999).

When bnl expression is examined by in situ hybridization at larval stages of development, it is found that the gene turns back on by the first larval instar and continues to be expressed throughout larval life. However, in contrast to its highly restricted expression pattern in the embryo, the gene is broadly expressed in the larva, including all tissues that become heavily tracheated with terminal branches. In particular, the three most highly tracheated tissues—gut, muscles, and central nervous system (CNS)—all show generalized expression of bnl during the first and second larval instars when terminal branches are sprouting. Thus, bnl is expressed in the appropriate tissues at the right time to regulate terminal branching in the larva. Several tissues that are not tracheated, including the epidermis and salivary gland, do not express bnl, strengthening the correlation between bnl expression and terminal branching. The correlation is not absolute though, as several other tissues with few or no branches, including the imaginal discs, heart, and fat body, do express significant levels of BNL mRNA. However, in no case does a terminally tracheated tissue not express bnl (Jarecki, 1999).

The arborization pattern of terminal branches is remarkably complex. Each terminal tracheal cell sprouts dozens of branches that spread out and contact nearly every cell in the target. At first glance, the complex and variable nature of the branch pattern suggests that the process is highly random, with the constraint only that branches fill the available space. The results presented here support a model in which the final pattern, although variable, is not at all random. Instead, branching is precisely controlled during development to meet the oxygen needs of the target cells. A model is proposed in which each cell experiencing an oxygen debt senses the impending crisis and responds by upregulating expression of Bnl. Bnl FGF diffuses to nearby tracheal cells and stimulates new tracheal branches to form and grow toward each signaling source. This supplies oxygen to the hypoxic cells and shuts off the signal. The process is dynamic and repeats itself many times due to the constantly changing balance in cell oxygen need and supply. Over the course of development, most cells would become hypoxic, serve as an FGF signaling center, and receive an appropriate tracheal supply. Thus, the ultimate pattern of tracheal branches would reflect the complex history of the oxygen needs and Bnl expression pattern of the tissues (Jarecki, 1999).

Drosophila glypican Dally-like acts in FGF-receiving cells to modulate FGF signaling during tracheal morphogenesis

Studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. This study shows that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, the role of Dlp was analyzed in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, these results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila (Yan, 2007).

There are three main important findings in this work. First, Dlp was identified as an essential molecule required for tracheal development. Dlp is required for Btl-mediated tracheal branching during embryogenesis while both Dlp and Dally are involved in the formation of air sac tracheoblasts in the wing disc. Second, the data show that other HSPGs cannot replace Dlp for Btl signaling during embryogenesis and that both Dlp and Dally are not essential for Htl-mediated mesodermal cell migration. These data demonstrate that different FGFs may require different HSPGs to execute their effective signaling activities during development. Third and most importantly, strong evidence is provided that Dlp controls Btl signaling only in FGF-receiving cells in both embryonic and larval tracheal systems. This mechanism of HSPG activity in FGF signaling is very different from its roles in regulating the signaling activities of morphogens including Wnt, Hh and Dpp. Together, these new findings further define novel mechanisms and the specificities of HSPGs in FGF signaling during development (Yan, 2007).

Extensive biochemical and cell culture studies suggest that HSPGs are the part of the FGF/FGFR signaling complex. However, the mechanisms of HSPGs in FGF signaling during development are less known. Embryos mutant for two HSPG biosynthesis enzymes, sgl and sfl, exhibit defects in both Btl- and Htl-mediated FGF signaling. An important issue remaining to be solved is which HSPG core proteins are involved in these signaling events. The data in this work provide strong evidence that Dlp is the key molecule required for Btl signaling during embryonic tracheal development, while both Dlp and Dally are involved in the Btl mediated air sac tracheoblasts formation in the wing disc. The results provide several novel insights into the specificity of individual HSPG in FGF signaling. First, Dlp is involved in Btl signaling, but not in Htl signaling. These findings indicate that different FGF/FGFR complexes may require different HSPGs for their signaling activities. Second, Dlp is highly active and specific for Btl signaling; overexpression of the other three Drosophila HSPGs fail to rescue tracheal defects in dlp embryos. The specific activity of Dlp in Btl signaling could be due to the Dlp protein core or the HS GAG chains attached to the Dlp core protein. In this regard, it is especially surprising that Dally, which has 22% identity with Dlp and also bears a GPI anchor, cannot rescue tracheal phenotypes associated with dlp embryos. As Dlp is involved in several other signaling pathways such as Hh, it is unlikely that Dlp core protein interacts with the ligands directly. In this regard, it is worthwhile to note that ectopic expression of Dally also fails to rescue Hh signaling in dlp embryos. It is proposed that Dlp may have unique HS GAG chains that might provide high and specific activity for ligands such as Bnl and Hh (Yan, 2007).

The biosynthesis of HS GAG chains is determined by the HSPG protein core in which the GAG attachment sites and other protein parts such as the N-terminal cystenine-rich domain control both quantity and quality of the attached GAG chains. Detailed structure and functional studies of Dlp will further help to define specific requirements of the core protein or GAG attachment sites in FGF signaling. Furthermore, the unique GAG chains may be modified by specific enzymes. In this regard, it is particularly important to note that 6-O sulfation of HS is critical for Btl signaling, as Drosophila heparan sulfate 6-O-sulfotransferase is specifically expressed in embryonic tracheal system and is required for Btl signaling during embryogenesis. Recent study has shown that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts. In this regard, it is suggested that Dlp may be the optimal substrate for sulfation enzymes during embryogenesis. Therefore, the activity of Dlp in FGF signaling during embryogenesis cannot be replaced by other HSPGs including Dally, Syndecan and Perlecan (Yan, 2007).

Although Dlp is essential for Btl signaling during embryogenesis, both Dally and Dlp are involved in Btl signaling in air sac tracheoblast cells. Similarly, previous studies have shown that both Dally and Dlp are involved in regulating Wg, Hh and Dpp distribution in the wing disc. The different functions of the same HSPG in embryos and discs may reflect temporal and developmental stage dependent regulation of HSPG functions (Yan, 2007).

While it is well established that HSPGs can regulate FGF signaling by facilitating FGF/FGFR interaction, it is unknown whether HSPGs can also control FGF distribution, thereby modulating FGF signaling. This is a particularly important issue as in many developmental contexts FGF ligand is produced in one type of cell and acts on other cells to initiate its biological activity. One important finding of this work is that HSPGs control tracheal morphogenesis by regulating FGF signaling only in FGF-receiving cells, but not by regulating the secretion or distribution of FGF ligand in its producing cells and surrounding cells. Several important results support these conclusion: (1) dlp mutant embryos can suppress the phenotype of overexpressing Bnl in the tracheal cells. (2) Ectopic expression of Dlp in tracheal cells, rather than FGF expression cells, can effectively restore tracheal defects associated with dlp embryos. (3) Embryos rescued by prd-Gal4/UAS-dlp in dlp backbround is very similar to btl mutant embryos rescued by prd-Gal4/UAS-btl-GFP. (4) HSPGs are required for FGF signaling in its receiving cells in the air sac, but are dispensable in the columnar epithelial layer which includes FGF producing cells and other surrounding cells. Detailed analyses thus demonstrate the specific and distinct requirement of HSPGs in FGF signaling during tracheal development. Moreover, embryonic and larval data together suggest this is likely a general mechanism for HSPG function in FGF signaling in Drosophila (Yan, 2007).

Two major models are proposed for the role of HSPGs in FGF signaling. In one model, low affinity HS/GAG chains on the cell surface limit the diffusion of FGF ligand, thereby increasing its local concentration and the probability that it will interact with high-affinity FGFRs. In the second model, HSPGs facilitate the dimerization or oligomerization of FGF ligands thereby inducing receptor clustering and signal transduction. The experimental data cannot exclude either of these mechanisms. However, the results are in favour of the second case, since it is shown that HSPGs are not required in FGF concentration gradient in FGF producing cells, but are essential in FGF-receiving cells. Finally, a recent study showed that dynamin-mediated vesicle internalization is a crucial step to regulate FGF signaling in Drosophila tracheal system. Mutants in awd (abnormal wing disc) or shi (shibire), which encodes for a nucleoside diphosphate kinase and Drosophila dynamin, respectively, have increased levels of Btl in tracheal cell surface, increased FGF signaling activity and ectopic tracheal branching. In this regard, HSPGs may control FGF signaling by stabilizing the FGF/FGFR complex from degradation or internalization in FGF receiving cells. Further experiments using HSPG and awd/shi double mutant are needed to test this possibility (Yan, 2007).

Over the past several years, extensive studies in Drosophila and other model systems have established the essential roles of HSPGs in developmental signaling pathways including Wg, Hh and Dpp. In Drosophila embryo and wing imaginal disc, HSPGs are involved in the transport of morphogens including Wg, Hh and Dpp by a restricted diffusion mechanism. Narrow stripes of clones mutant for HSPGs can impede the movement of morphogens to further cells. However, in all of these cases, the first mutant cells adjacent to the morphogen source can still transduce signals arguing that HSPGs are not essential for morphogen signaling activity, but rather control the distributions or local concentrations of morphogens. The novel results from this work point out a major difference for a role of HSPGs in FGF signaling from their roles in morphogen signaling, as removal of HSPGs (dally-dlp or sfl) from FGF receiving cells can effectively block FGF signaling. Although the graded FGF activity may play an essential role in tracheal morphogenesis, the data from this work argue that the main function of HSPGs in FGF signaling is not to regulate the distribution of FGF ligand. Consistent with the different roles of HSPGs in FGF and morphogen signaling, it was found that Dlp acts cell-autonomously in FGF signaling while it functions non-autonomously in Hh signaling in embryos. These results suggest that Bnl transportation may be different from morphogen movement in the epithelial cells of the wing pouch. Indeed, morphogen molecules diffuse through the same layer of cells, columnar epithelial cells, while FGF is transported between different layers of tissues, from columnar epithelia to tracheoblasts. Moreover, leading air sac cells are always in close proximity with underlying columnar epithelia. They also extend multiple filopodia toward ligand gradient and presumably actively pursue the FGF ligands while wing disc morphogens including Wg, Hh and Dpp need to transport many cell diameters from their sources to reach their receiving cells. Studies in vertebrate also suggest that a graded distribution of FGF8 protein can be generated by the decay of fgf8 mRNA and this RNA gradient is translated into a protein gradient. In this case, no active transport mechanism is required to form a FGF gradient. In mammalian limb and lung development different FGFs are often expressed in different layers of cells, such as epithelium and mesenchyme, and signal through each other. It is interesting to determine whether HSPGs function similarly in these systems as in Drosophila (Yan, 2007).

Specificity of Drosophila cytonemes for distinct signaling pathways

Cytonemes are types of filopodia in the Drosophila wing imaginal disc that are proposed to serve as conduits in which morphogen signaling proteins move between producing and target cells. The specificity was investigated of cytonemes that are made by target cells. Cells in wing discs made cytonemes that responded specifically to Decapentaplegic (Dpp) and cells in eye discs made cytonemes that responded specifically to Spitz (the Drosophila epidermal growth factor protein). Tracheal cells had at least two types: one made in response to Branchless (a Drosophila fibroblast growth factor protein, Bnl), to which they segregate the Bnl receptor, and another to which they segregate the Dpp receptor. It is concluded that cells can make several types of cytonemes, each of which responds specifically to a signaling pathway by means of the selective presence of a particular signaling protein receptor that has been localized to that cytoneme (Roy, 2011).

Cells in developing tissues are influenced by multiple signals that they process and integrate to control cell fate, proliferation, and patterning. An example is in the Drosophila wing imaginal disc, where cells depend on several signaling systems that are intrinsic to the disc. Dpp, Wingless (Wg), Hedgehog (Hh), and epidermal growth factor (EGF) are produced and released by different sets of disc cells, and receipt of these signaling proteins programs their neighbors to develop and grow. The mechanisms by which morphogen signaling proteins influence target cells must ensure both specificity and accuracy, and one possibility is that these proteins transfer at points of direct contact. Imaginal discs are flattened sacs that have a monolayer of columnar cells on one side and squamous peripodial cells on the other. Many cells in wing discs make filopodial extensions that lie along the surfaces of the monolayers, oriented toward morphogen-producing cells. These extensions have been termed cytonemes to denote their appearance as cytoplasmic threads and to distinguish them as specialized structures that polarize toward morphogen-producing regions (Roy, 2011).

In wing discs dissected from third instar larvae, cytonemes can be seen as filaments extending from randomly generated somatic clones engineered to express a fluorescent protein such as soluble, cytoplasmic green fluorescent protein (GFP) or a membrane-bound form such as mCD8:GFP (the extracellular and transmembrane domains of the mouse lymphocyte protein CD8 fused to GFP). To image disc cytonemes, unfixed discs were placed peripodial side down on a coverslip, covered with a 1-mm-square glass, and mounted over a depression slide with the disc hanging from the coverslip. Because fluorescence levels in cytonemes were low relative to background, recorded images were processed to increase intensity and were subjected to de-convolution. Expression of CD8:GFP in wing disc clones revealed cytonemes emanating from both the apical and basal surfaces of columnar cells, as well as from peripodial cells (whose apical and basal surfaces could not be distinguished). Most cytonemes were perpendicular to the anterior/posterior (A/P) axis of the disc and oriented toward the cells that produce Dpp at the A/P compartment border; others were oriented toward the cells that produce Wingless at the dorsal/ventral (D/V) compartment border. Disc-associated myoblasts also had filopodia (Roy, 2011).

In the eye disc, cells in the columnar layer organize into ommatidial clusters as a wave of differentiation [the morphogenetic furrow (MF)] passes from posterior to anterior. A second axis, centered at the equator, is orthogonal to the MF and defines a line of mirror-image symmetry where dorsal and ventral ommatidia are juxtaposed. The columnar cells divide during the third instar period but stop or divide only once after the MF passes. CD8:GFP expression was induced in somatic clones and the columnar cells were examined. Whereas clones of six to eight cells were present on both sides of the MF, only cells anterior to the MF had visible cytonemes. Cytonemes emanating from these clones oriented either toward the axis defined by the MF or toward the axis defined by the equator. Single clones with cytonemes oriented both toward the MF and toward the equator were not observed, and there was no apparent correlation between clone position and cytoneme orientation or cytoneme length. Cells in the peripodial layer of the eye disc also had cytonemes (Roy, 2011).

The EGF pathway is a key signaling system for eye development, and cells in the MF express the EGF protein Spitz (Spi). Because one of the two types of anterior cell cytonemes extended toward the MF and to explore the distribution of membrane-bound receptor proteins, clones were induced that expressed an epidermal growth factor receptor:GFP (EGFR:GFP) fusion protein. Anterior cells expressing EGFR:GFP had cytonemes that oriented toward the MF, and most of these cytonemes had fluorescent puncta; no cytonemes that were marked by EGFR:GFP oriented toward the equator. Other than their 'furrow-only' orientation, the cytonemes marked by EGFR:GFP were similar to those marked by CD8:GFP. In contrast, co-expression of CD8:GFP with (nonfluorescent) EGFR marked both furrow-directed and equator-directed cytonemes. Thus, expression of EGFR:GFP does not eliminate the equator-directed cytonemes, suggesting that the specific localization of EGFR:GFP to furrow-directed cytonemes is not a consequence of ectopic (over)expression of this fusion protein (Roy, 2011).

Evidence that the furrow-directed cytonemes depend on Spi/EGF signaling was obtained by expressing a dominant negative form of EGFR. Although EGFR is required for cell proliferation in the disc, small clones expressing EGFR^DN were recovered that co-expressed EGFR^DN and CD8:GFP; in these clones, only cytonemes that appeared to be randomly oriented were present, indicating that the long, furrow-directed cytonemes may require EGFR signal transduction in the cytoneme-producing cells (Roy, 2011).

Wing disc-associated tracheal cells also make cytonemes. The transverse connective (TC) is a tracheal tube that nestles against the basal surface of the wing disc columnar epithelium and that sprouts a new branch [the air sac primordium (ASP)] during the third instar period in response to Branchless (Bnl) expressed by the wing disc. Tracheal tubes are composed of a monolayer of polarized cells whose apical surfaces line a lumen. Expression of CD8:GFP throughout the trachea (btl-Gal4 UAS-CD8:GFP) made it possible to detect GFP fluorescence in several types of cytonemes emanating from the basal surfaces of the TC and ASP. Cytonemes at the tip of the ASP (length range, 12 to 50 μm; average length of 23 μm) contained the Breathless (Btl); the Drosophila fibrobast growth factor receptor (FGFR) and appeared to contact disc cells that express Bnl. Short cytonemes (length range, 2 to 15 μm; average length of 8.5 μm) extended from the TC cells in the vicinity of the ASP (Roy, 2011).

Tests were carried out to se whether Dpp, Spi, Bnl, and Hh affected wing disc, eye disc, and tracheal cytonemes differentially. Ubiquitous expression of Spi, Bnl, or Hh (induced by heat shock) did not alter the A/P-oriented apical cytonemes in the wing disc, and, in the eye disc, the long cytonemes of the columnar layer were unaltered after ubiquitous expression of Dpp, Bnl, or Hh. In contrast, long oriented cytonemes were absent in wing discs after ubiquitous expression of Dpp, and only short cytonemes that appeared to be randomly oriented were observed. Similarly, 0.5 to 3 hours after cSpi, a constitutively active form of EGF, was expressed ectopically by heat shock induction, clones expressing CD8:GFP in the eye disc had many short cytonemes that lacked apparent directional bias; in contrast to controls, no long cytonemes oriented toward the MF were observed. Cytonemes with normal orientation and length (including MF-directed cytonemes) were present in eye discs that were examined later, 8 hours after a pulse of cSpi expression. To monitor EGFR-containing cytonemes for sensitivity and responsiveness to Spi, cSpi was expressed by heat shock induction, and cells in clones expressing EGFR:GFP were examined. After a pulse of cSpi expression, the extensions oriented outward without apparent directional bias, and the EGFR:GFP puncta were present in all cytonemes (Roy, 2011).

To examine responses of the ASP tip cytonemes, Hh, Spi, Dpp, and Bnl were overexpressed by heat shock and GFP-marked cytonemes at the ASP tip were examined. No differences in number of cytonemes were detected until about 3 hours after heat shock. Four to 5 hours after heat shock, expression of Bnl increased the number of tip cytonemes by ~2.6 times, and although most of the cytonemes were <30 μm, the cytonemes >30 μm also increased (~3.2 times). Most of the long cytonemes in these preparations were oriented in directions other than toward the cells that normally express Bnl. The number of long cytonemes >30 μm did not change after overexpression of Hh, Spi, and Dpp (0.6 to 0.8 times); the number of short cytonemes increased after Dpp overexpression (~1.7 times) but not after overexpression of Hh or Spi (Roy, 2011).

Thus, the responses of apical wing disc cytonemes to overexpressed Dpp, of eye disc cytonemes to ubiquitous Spi, and of ASP tip cytonemes to exogenous Bnl (Drosophila FGF) are similar. These results suggest that the cytonemes detected in the wing discs and eye discs may have orientations and lengths that are dependent specifically on the respective sources of Dpp and Spi, whereas the ASP may extend cytonemes in response to more than one signaling protein. These results are, however, complicated by the heat shock mode of induction because both the cells that expressed GFP (and extended marked cytonemes) as well as the surrounding cells expressed the signaling proteins. To overcome this problem, a method was developed to induce two types of somatic clones in the same tissue, one that expressed GFP and another that expressed Dpp (Roy, 2011).

The GAL4 system was used to label cytonemes with CD8:GFP. Clones of GAL4-expressing cells were generated with heat shock-induced flippase (FLP recombinase). The second type of clone expressed a Dpp:Cherry fusion and was generated with a variant Cre-progesterone receptor recombinase that could be activated with a regime of heat shock and RU486. By adjusting the timing and strength of induction, wing discs were produced with small, independent, and relatively infrequent clones. In discs with clones that expressed ectopic Dpp as well as clones that expressed CD8:GFP, apical cytonemes tagged with GFP were detected that oriented toward nearby Dpp:Cherry-expressing cells and not toward either the A/P or D/V signaling centers. Such 'abnormally directed' cytonemes were never observed in control discs. The abnormally oriented cytonemes suggest that apical cytonemes in the wing blade respond directly to sources of Dpp and that their orientation reflects extant sources of signaling protein (Roy, 2011).

To characterize the relationship between tracheal ASP tip cytonemes and FGF signaling from the wing disc, the distribution of Btl (FGFR) was examined in ASP cells and in ASP cytonemes. In preparations from larvae with tracheal expression of both CD8:GFP and Btl:Cherry (btl-GAL4 UAS-CD8:GFP;UAS-Btl:Cherry), cytonemes were marked by CD8:GFP, some of which had fluorescent Btl:Cherry puncta. Each ASP had only a few long (>30 μm) cytonemes, most of which contained Btl:Cherry puncta. Few of the more numerous short cytonemes (<30 μm) contained Btl:Cherry puncta. To characterize Btl:Cherry after overexpression of Bnl, focus was placed on preparations obtained 1 to 2 hours post-induction (genotype btl-GAL4 UAS-CD8:GFP/HS-Bnl;UAS-Btl:Cherry/Gal80^ts), because during this time interval the ASP morphology was close to normal but cytonemes had changed. ASPs were ignored after longer postinduction intervals because of major malformations to ASP morphology after 3 to 4 hours. Long cytonemes with Btl:Cherry puncta were present 1 hour after a pulse of Bnl expression; but 2 hours after the pulse, most ASPs had no long cytonemes, and the number of short puncta-containing cytonemes increased at the tip and along the shaft of the ASPs. After control heat shock or heat shock-induced expression of Dpp, the distribution of Btl:Cherry puncta in the ASP tip cytonemes was similar to normal controls: Long cytonemes had Btl:Cherry puncta, but most short cytonemes did not (Roy, 2011).

Because the number of small cytonemes at the ASP tip may have increased after ectopic Dpp expression, whether the thickveins (tkv) gene, which encodes a subunit of the Dpp receptor, is expressed in the ASP was investigated. Expression of the tkv reporter, tkv-lacZ (P{lacW}tkv¹⁶⁷¹³), was detected in the ASP. When Tkv:GFP and Btl:Cherry were expressed together, Tkv:GFP and Btl:Cherry segregated to separate tip cytonemes at the ASP tip. Whereas Tkv-containing cytonemes were short (<30 μm), most of the Btl-containing cytonemes were longer (three of four of the Btl:Cherry-containing cytonemes were longer than 30 μm), and they lay in focal planes closer to the disc. These properties were consistent in all preparations examined in which both green Tkv and red Btl cytonemes were intact. Imaging these marked ASPs revealed that overexpressed Tkv:GFP and Btl:Cherry were present not only in the plasma membranes (as expected) but also in separate puncta in the cell bodies. This shows that Tkv and Btl receptors also segregated to separate locations in the ASP cell bodies (Roy, 2011).

These findings suggest that the ASP has long cytonemes that are specific to Bnl and specifically harbor Btl-containing puncta and that the ASP also has cytonemes that are specific to Dpp and specifically harbor Tkv. Similarly in the eye disc, the presence of EGFR:GFP in furrow-oriented cytonemes and not in equator-oriented cytonemes suggests that cytonemes in the eye disc also selectively localize receptors. And as was previously shown, apical cytonemes in the wing disc selectively localize Tkv. The apparent ligand specificities and contrasting makeup of these cytonemes suggest a diversity of functionally distinct subtypes: Cells appear to make cytonemes that respond specifically to the Dpp, EGF, or Bnl signaling proteins. The basal filopodia implicated in Delta-Notch signaling in the wing disc may represent yet another type (Roy, 2011).

The mechanism that endows cytonemes with specificity for a particular signaling protein cannot be based solely on tissue-specific expression of a receptor. Spi, Dpp, and Hh are active in eye discs, but only changes in Spi signaling affected the furrow-directed cytonemes. And in the wing disc, both the Hh and EGF signal transduction pathways are active in cells at the A/P compartment border, but the apical cytonemes only responded to overexpressed Dpp. The findings that tracheal cells in the ASP respond to both Dpp and Bnl and that the Tkv and Btl receptors are present in different cytonemes that the ASP cells extend suggest that specificity may be a consequence of the constitution of the cytoneme, not on which receptors the cells make. The mechanism that localizes receptors to different cytonemes is not known, but because the marked receptors that were expressed also segregated to different intracellular puncta, the processes that concentrate these receptors in separate locations may not be exclusive to cytonemes. There is a precedent for segregation of proteins to different cellular extensions, neurons segregate proteins to dendrites or axons, so extending projections with specific and distinct attributes may be a general property of cells (Roy, 2011).

A single-cell survey of Drosophila blood

Drosophila blood cells, called hemocytes, are classified into plasmatocytes, crystal cells, and lamellocytes based on the expression of a few marker genes and cell morphologies, which are inadequate to classify the complete hemocyte repertoire. This study used single-cell RNA sequencing (scRNA-seq) to map hemocytes across different inflammatory conditions in larvae. Plasmatocytes were resolved into different states based on the expression of genes involved in cell cycle, antimicrobial response, and metabolism, together with the identification of intermediate states. Further, rare subsets within crystal cells and lamellocytes were discovered that express fibroblast growth factor (FGF) ligand branchless and receptor breathless, respectively. These FGF components were identified as required for mediating effective immune responses against parasitoid wasp eggs, highlighting a novel role for FGF signaling in inter-hemocyte crosstalk. This scRNA-seq analysis reveals the diversity of hemocytes and provides a rich resource of gene expression profiles for a systems-level understanding of their functions (Tattikota, 2020).

Previous studies have identified three major Drosophila blood cell types essential for combating infections in this species. This study used scRNA-seq of larval fly blood to gain deeper insights into the different cell types and their transition states in circulation during normal and inflammatory conditions. Comprehensive scRNA-seq data provide information on subpopulations of plasmatocytes and their immune-activated states. Importantly, scRNA-seq could precisely distinguish mature crystal cells and lamellocytes from their respective intermediate states, which are less well understood and for which marker genes were not previously available. Thus, new marker genes identified in this study should facilitate further study of these states. Moreover, it was possible to identify the gene signature of self-renewing plasmatocytes and suggest their role as extra-lymph gland oligopotent precursors. In addition to the identification of various states of mature cell types, this study also suggests novel roles for a number of genes and pathways in blood cell biology. In particular, a putative new Mtk-like anti-microbial peptide (AMP) was identified, and a role was proposed for the FGF signaling pathway in mediating key events leading to the melanization of wasp eggs. Finally, a user-friendly searchable online data mining resource was developed that allows users to query, visualize, and compare genes within the diverse hemocyte populations across conditions (Tattikota, 2020).

Blood cell types are dynamic in nature and several transient intermediate states exist in a continuum during the course of their maturation in several species. This scRNA-seq analysis provides a framework to distinguish cell types from their various states including oligopotent, transient intermediate and activated states (Tattikota, 2020).

Oligopotent state: ScRNA-seq analysis identified PM2 as the oligopotent state of plasmatocytes based on the enrichment of several cell cycle genes including polo and stg. This signature suggests that PM2 corresponds to self-renewing plasmatocytes located in the circulatory and sessile compartments of the Drosophila hematopoietic system where plasmatocytes are the only dividing cells identified. Further, previous studies suggested that lamellocytes derived from embryonic-lineage hemocytes are readily detectable in circulation prior to their release from the lymph gland, and that terminally differentiated crystal cells can also derive from preexisting plasmatocytes in the sessile hub. Hence, it is proposed that PM2 corresponds to the oligopotent state that not only drives expansion of plasmatocytes, but importantly can also give rise to crystal cells and lamellocytes. Monocle3 analysis indicates that cell cycle genes decrease over pseudotime and there is ample evidence in support of the notion that cell cycle arrest may be required for terminal differentiation of various cell types in flies and vertebrates. Our in vivo data also indicates that cell cycle arrest can lead to the generation of terminally differentiated lamellocytes. Interestingly, recent evidence in hemocytes suggests that perturbing cell cycle by knocking down jumu, which is upstream of polo, can also lead to the generation of lamellocytes by activating Toll. In contrast, forced expression of certain oncogenes such as activated Ras and Hopscotch/JAK in hemocytes can also lead to overproduction of plasmatocytes and lamellocytes. It is, however, speculated that the proliferation and differentiation of hemocytes in these contexts may be linked to cell cycle. Thus, it is important to address this paradoxical role of cell cycle in the maintenance of oligopotency and transdifferentiation of plasmatocytes. Studies using lineage tracing methods such as G-TRACE or CRISPR-based in vivo cellular barcoding techniques may help further characterize the contribution of proliferating oligopotent plasmatocytes to blood cell lineages (Tattikota, 2020).

Immune-activated states: PM5 from the scRNA-seq data is enriched in several genes that encode glutathione S-transferase family of metabolic enzymes, which are known to catalyze the conjugation of reduced glutathione (GSH) to xenobiotics for their ultimate degradation. It has been demonstrated that a subset of hemocytes accumulate high GSH levels in Drosophila, in support of these data. Further, the two AMP clusters PM6-7 (PM^AMP) were identified as part of the immune-activated states of plasmatocytes. A recent study has demonstrated that AMPs are highly specific and act in synergy against various pathogens. The scRNA-seq analysis reveals the remarkable difference in the expression of a set of AMPs in the two clusters. Future studies with PM^AMP-specific perturbation of various AMPs identified within plasmatocytes should clarify their contribution in killing specific pathogens. Moreover, the role of Mtkl against pathogens needs further characterization. Pseudotime analysis showed that PM^AMP ends in the same lineage as lamellocytes suggesting a common mode of activation for these cell types and states. Interestingly, induction in hemocytes of Toll, which is upstream of Drs, can lead to the production of lamellocytes, suggesting that LM^int cells may act as the common branch point between immune-activated states and lamellocytes (Tattikota, 2020).

Transient intermediate states: In addition to the oligopotent and immune-activated states, plasmatocytes showed several subpopulations, which most likely are transient intermediate states. Although it remains to be seen whether they exist throughout the larval development, it is possible that these transient states exist along the continuum of cell maturation process. On the other hand, the transcriptomic composition of CC1 and LM1 clusters suggested the presence of intermediates for crystal cells and lamellocytes, respectively. Further analysis by Monocle3, which placed these clusters prior to their terminally differentiated cell types, confirmed the hypothesis that CC1 and LM1 correspond to CC^int and LM^int states, respectively. In the context of the CC lineage within the lymph gland, ultrastructural studies have revealed the presence of immature crystal cells, called procrystal cells, alongside mature crystal cells. This study furthered this observation by demonstrating in vivo that crystal cells exist in a continuum (PPO1^low to PPO1^high), validating the Monocle3 and scRNA-seq data. Moreover, clear gene signatures between the CC^int and LM^int states and their mature counterparts revealed that these intermediates most likely emerge from preexisting Hml⁺ plasmatocytes. With regards to the LM lineage, several groups have speculated that intermediates, called podocytes, or also lamelloblasts, may exist based on cell morphology and size. The scRNA-seq and Monocle3-based data clearly demarcate mature lamellocytes from LM^int at the transcriptomic level. In addition, sub-clustering analysis revealed that LM^int possessed a PM signature demonstrating that these intermediates are presumably derived from PM2 (Tattikota, 2020).

A novel role for the FGF signaling pathway in hemocyte crosstalk: In addition to the known hemocyte - tissue crosstalk, Drosophila hemocytes must act in a coordinated fashion to combat harmful pathogens and foreign entities such as wasp eggs. However, the signaling pathways that mediate the interactions among hemocytes and wound sites or wasp eggs have been unclear. The scRNA-seq uncovered a novel role for the FGF signaling pathway in controlling hemocyte differentiation and subsequent effects on the melanization of wasp eggs. The FGF ligand bnl and its receptor btl were among the genes identified in rare subsets of crystal cells and lamellocytes, respectively, highlighting the power of scRNA-seq in capturing and detecting these small populations of cells. Based on the in vivo data, it is proposed that Bnl⁺crystal cells interact with Btl⁺ lamellocytes to coordinate lamellocyte differentiation and possible migration towards parasitoid wasp eggs. Furthermore, because lamellocytes are also enriched in additional core components of the FGF signaling pathway, future studies involving a comprehensive analysis of this pathway will advance understanding of blood cell communication, differentiation, and migration in the context of immune response (Tattikota, 2020).

In summary, these scRNA-seq data provides a resource for a comprehensive systems-level understanding of Drosophila hemocytes across various inflammatory conditions (Tattikota, 2020).

REGULATION

Transcriptional regulation

DPP controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo

Decapentaplegic controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo. The requirement for Dpp is revealed by two manipulations: (1) the overexpression of Dpp using a heat-shock promoter and (2) use of mutations in the Dpp receptors thickveins and punt. The failure of tracheal cells to receive the DPP signal from adjacent dorsal and ventral cells results in the absence of dorsal and ventral migrations. Ectopic Dpp signaling can reprogram cells in the center of the placode to adopt a dorsoventral migration behavior. The effects observed in response to ectopic Dpp signaling are also observed upon the tracheal-specific expression of a constitutive active Dpp type I receptor (TKV[Q253D]). The alterations in migration behavior are similar for constitutively active receptor and for Dpp ectopic expression, indicating that the Dpp signal is received and transmitted in tracheal cells to control their migration behavior. Whereas, lack of Dpp signaling results in a failure of tracheal cells to migrate along the dorsoventral axis without significantly affecting anterior migrations, ubiquitous Dpp signaling suppresses anterior migrations without interfering with dorsoventral migration (Vincent, 1997).

Dpp signaling determines localized gene expression patterns in the developing tracheal placode, and is also required for the dorsal expression of the Branchless (Bnl) guidance molecule, the ligand of the Breathless (Btl) receptor. spalt (sal) is strongly expressed in dorsal trunk cells in stage 14 embryos and is necessary for the directed anterior migration of these cells. sal is expressed in the dorsal trunk in punt and tkv mutant embryos, indicating that Dpp does not regulate sal expression. However, embryos in which the Dpp signaling pathway has been activated in all tracheal cells at the placode stage fail to accumulate Sal. This lack of Sal expression correlates with the absence of the dorsal trunk upon ectopic Dpp signaling. In contrast to sal the gene knirps is activated in the developing tracheal system in all the branches (dorsal branch, ganglionic branch, and lateral trunk) that are thought to be under the control of Dpp. kni expression is lost in tkv mutants; (kni expression only persists in the visceral branches of tkv mutants). kni expression is turned on in all tracheal cells after constitutive Dpp signaling. The requirement for Dpp for the correct expression of the ligand branchless was revealed using tkv and punt mutants. The dorsal-most patches of bnl expression which prefigure the formation of the dorsal branches, are severely reduced in punt mutant embryos and absent in tkv mutants. Thus, Dpp plays a dual role during tracheal cell migration. It is required to control the dorsal expression of the Bnl ligand. In addition, the Dpp signal recruits groups of dorsal and ventral tracheal cells and programs them to migrate in dorsal and ventral directions (Vincent, 1997).

Sex-specific deployment of FGF signaling in Drosophila recruits mesodermal cells into the male genital imaginal disc

A central issue in developmental biology is how the deployment of generic signaling proteins produces diverse specific outcomes. Drosophila FGF is used, only in males, to recruit mesodermal cells expressing the FGF receptor to become part of the genital imaginal disc. Male-specific deployment of FGF signaling is controlled by the sex determination regulatory gene doublesex. The recruited mesodermal cells become epithelial and differentiate into parts of the internal genitalia. These results provide exceptions to two basic tenets of imaginal disc biology -- that imaginal disc cells are derived from the embryonic ectoderm and that they belong to either an anterior or posterior compartment. The recruited mesodermal cells migrate into the disc late in development and are neither anterior nor posterior (Ahmad, 2002).

The extensive sexual dimorphisms of the genitalia and analia suggest that the genital disc is relatively enriched in genes expressed downstream of dsx. To identify such genes, a random collection of enhancer traps was screened for sex-specific expression patterns in late third instar genital discs. Enhancer trap insertions in the bnl and btl genes were both isolated as enhancer traps expressed in male but not female genital discs. The sex specificity and the spatial patterns of expression of these enhancer traps accurately reflect the expression of the bnl and btl genes in the genital disc. Of the three primordia that comprise the genital disc, bnl and btl are both expressed in only one: the A9-derived developing 'male' primordium. bnl and btl are also expressed in adjacent domains: bnl is expressed at the base of two bilateral bowl-like infoldings of the disc epithelium, while btl is expressed in a group of loosely packed cells that fills these bowls and extends over the anterior and ventral surfaces of the disc (Ahmad, 2002).

The juxtaposition of btl- and bnl-expressing cells suggested that their proximity to one another might be the result of FGF-mediated cell-cell signaling. The locations of btl-expressing cells in male genital discs were determined at different stages of larval development. At early third instar (70-75 hr after egg laying), while a few btl-expressing cells are associated with the external surface of the disc, none are detected inside the disc. In mid-third instar (89-99 hr AEL), the btl-expressing cells are lying on the external surface of the disc, as well as adjacent to, and filling shallow invaginations in the disc epithelium. And by late third instar (110-120 hr AEL), these invaginations have become much deeper and are completely filled by btl-expressing cells. Thus, these btl-expressing cells are not originally a part of the disc epithelium but are recruited into invaginations in the epithelium during the third instar. Unlike the disc epithelium, the btl-expressing cells in the third instar disc do not express escargot (esg), a classical marker for ectoderm-derived imaginal cells, indicating that the btl-expressing cells have a different origin than do the other cells of the disc. The btl-expressing cells are, in fact, mesodermal in origin and derived from the adepithelial cells associated with the genital disc (Ahmad, 2002).

A priori, there are two possible explanations for the male-specific expression of FGF. One possibility is that bnl is an A9-specific gene, being expressed only in males where the A9-derived primordium grows significantly. The other possibility is that bnl is a target of the sex determination hierarchy, being either repressed by the female-specific Dsx protein (Dsx^F) in females and/or activated by the male-specific Dsx protein (Dsx^M) in males. To distinguish between these possibilities, feminized (Tra protein-expressing) clones of cells were generated in the A9-derived primordium of wild-type male genital discs and the effects of these clones on bnl expression were examined. Whenever feminized clones overlapped domains of bnl expression, the expression of bnl was repressed, indicating that it is cell-autonomous regulation by the sex determination hierarchy that is responsible for the male-specific expression of bnl in the genital disc (Ahmad, 2002).

When a feminized clone completely eliminated bnl expression from one side of a male disc, the lobe lacking bnl expression looked flattened. This was a consequence of btl-expressing cells not migrating into this lobe in the absence of Bnl protein, showing that bnl expression is not simply sufficient, but also necessary for the recruitment of btl-expressing cells. This observation suggests that btl, unlike bnl, is not a target of the sex determination hierarchy, and that the male-specific presence of btl-expressing cells in the genital disc is solely a consequence of Bnl recruiting the btl-expressing cells (Ahmad, 2002).

To examine how dsx regulates bnl expression, bnl expression was examined in wild-type genital discs and discs lacking dsx function. bnl is expressed in the A9-derived primordium of a wild-type male disc, where Dsx^M is present, but is not expressed in the A8-derived primordium of a wild-type female disc, where Dsx^F is expressed. However, in a disc in which neither Dsx protein is expressed, both the A8 and A9 primordia proliferate and bnl expression is seen in both primordia. That the A8 primordium grows in both wild-type and dsx mutant females but bnl is expressed in the A8 primordium only when the Dsx^F protein is absent, implies that bnl expression is repressed in the female genital disc by the presence of Dsx^F protein (Ahmad, 2002).

The ectopic expression of bnl in the A8-derived 'female' primordia of discs lacking dsx function offers an explanation for a puzzling observation: while wild-type males have only two paragonia (mesodermally derived components of the male disc), dsx mutant flies often have as many as four paragonia-like structures. The finding that the ectopic expression of bnl in flies mutant for dsx results in btl-expressing cells from the ventral surface of the disc being recruited into two ectopic invaginating pockets in the A8-derived female primordium of the disc, in addition to the original bowls in the A9-derived primordium, suggests that these ectopic pockets of btl-expressing cells give rise to the supernumerary paragonia when taken together with the observation that the extra paragonia in dsx mutants arise from the female primordium (Ahmad, 2002).

It is concluded that the sex-specific deployment bnl in the genital disc depends on the sex of the individual bnl-expressing cells. Given that bnl is regulated cell autonomously by Dsx^F, an obvious question is whether the Dsx^F protein directly represses bnl. In this regard, it is noted that 0.7 kb and 1.6 kb upstream of the putative bnl transcriptional start site, there are clusters of 5 and 4 sites respectively with at most a 1 bp mismatch to the 13 bp consensus Dsx binding site sequence. This is reminiscent of the 3 Dsx binding sites in a 76 bp stretch of an enhancer for the Yolk protein (Yp) genes, the only known direct targets of dsx (Ahmad, 2002).

The Drosophila sex determination hierarchy acts at multiple levels to control sexual differentiation. Some terminal differentiation genes like the Yp genes are direct transcriptional targets of the Dsx proteins and are continuously subject to their regulation. In other cases, the direct targets of dsx appear to be genes involved in initiating the differentiation of sex-specific tissues; genes expressed subsequently in these sex-specific tissues are governed by a tissue differentiation program, rather than being directly controlled by the sex hierarchy. It seems likely that the targets through which dsx initiates formation of such sex-specific tissues will be the genes where information from several developmental hierarchies are integrated to direct the differentiation of tissues (Ahmad, 2002).

These results suggest that bnl is one of the genes used by the sex determination hierarchy to direct the construction of sex-specific tissues. Bnl recruits btl-expressing cells into the male genital disc, and the recruited cells eventually form the paragonia and vas deferens (another mesodermally derived gonadal organ), tissues that are present only in males. Moreover, three genes expressed in the paragonia, the male-specific transcripts (msts) 316, 355a, and 355b, have been shown to be regulated in a tissue-specific rather than sex-specific manner: while transcription of these three male-specific RNAs begins in the late pupal period, their expression is governed by the sex hierarchy acting earlier, during the third larval instar -- the period when the expression of bnl recruits the paragonia-forming btl-expressing cells into the male genital disc. Thus, the sex-specific expression of the msts is achieved by dsx acting through bnl to generate the sex-specific tissue, the paragonia, in which the msts are subsequently expressed (Ahmad, 2002).

bnl also appears to be a gene where information from other regulatory hierarchies and the sex determination hierarchy are integrated in the male genital disc. The genetic hierarchies that control pattern formation and confer positional identity in the thoracic imaginal discs have previously been shown to function analogously in the genital disc. The fact that the bnl expression domain is limited to two specific subsets of the ectoderm-derived disc epithelia in males implies that bnl is also regulated by these pattern formation hierarchies. One area of future exploration will be examining how this coordinated regulation of bnl by dsx and the genes involved in pattern formation is brought about (Ahmad, 2002).

An intriguing aspect of these findings is the gradual transition of the btl-expressing cells, upon recruitment into the male genital disc, from twi-expressing mesodermal cells to epithelial cells with septate junctions. It is not clear if this transformation is also a consequence of FGF signaling, or if it is brought about by a different process. However, three separate observations suggest a role for bnl and btl in this mesoderm-epithelial transition: (1) FGF signaling mediates this process in mice -- during kidney development, FGF2 and leukemia inhibiting factor (LIF) secreted from the epithelial ureteric bud induce the conversion of the undifferentiated mesoderm-derived metanephric mesenchyme to the epithelial tubular structures of the nephron; (2) the converse process can also be mediated by FGF signaling -- FGFR1 regulates the morphogenetic movement and cell fate specification events during gastrulation in mice; it orchestrates the epithelial to mesenchymal transition during morphogenesis at the primitive streak and specifies the mesodermal cell fate of these mesenchymal cells, and (3) stumps, a gene acting downstream of the FGFR-encoding btl, has its expression elevated in the btl-expressing cells undergoing the transition into epithelial cells in the genital disc (Ahmad, 2002 and references therein).

Finally, it is noted that there are striking parallels between the roles of the FGF in sexual differentiation in the fly and FGF9 in sexual differentiation in mice. FGF9 is required for testicular embryogenesis in mice, and in its absence, XY mice undergo male-to-female sex reversal. FGF9 is expressed in the early embryonic gonads of male mice, not in the gonads of female mice, and not in the mesonephros of either sex, while bnl is expressed in the male genital disc, not in the female genital disc, and not in the btl-expressing mesodermal cells that are recruited into the male disc. The mesonephric cells migrate into only the male gonads, and the btl-expressing cells are recruited only into the male genital disc. Exogenous FGF9 induces mesonephric cell migration into female gonads, while ectopic expression of bnl is sufficient to recruit the btl-expressing cells into the female primordium of a dsx disc. The btl-expressing cells are mesodermal in origin, eventually undergo a transition into epithelial cells, and give rise to the vascular paragonia and vas deferens. The mesonephros, too, is derived from the mesoderm, and mesonephric cell migration into the testis contributes to the vascular endothelial, myoepithelial, and peritubular myoid cell populations. Given that there is considerable variation in the earlier aspects of sex determination across species, these findings suggest a possible conserved role for FGF signaling in later aspects of sexual differentiation (Ahmad, 2002 and references therein).

Genome-wide identification of in vivo Drosophila Engrailed-binding DNA fragments and related target genes

Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. This approach was validated by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Strong evidence is presented showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo (Solano, 2003).

the expression of 14 genes was studied that are localized close to the genomic DNA fragments isolated in the library and tested previously for their Engrailed-specific binding ability. The results are shown for four genes (frizzled2, hibris, branchless, frazzled) that are representative of the different pathways where engrailed seems to be involved. frizzled 2 expression is activated in the presence of (VP16-En) and repressed in the presence of En. This suggests that engrailed might act as a repressor on fz2 expression. hibris is expressed along the wing margin and in the presumptive region of wing vein L3 and L4 in wild type. This expression is slightly activated in the presence of (VP16-En), but strongly repressed when En is overexpressed, suggesting that hbs expression is regulated by engrailed in vivo. branchless is essentially expressed in a dorsal/posterior territory surrounding the wing pouch in wild type. In the presence of (VP16-En), several additional patches of bnl expression are detected within the wing pouch, whereas no activation of bnl is observed after wild type En overexpression. As expected, because MS1096 drives Gal4 expression only in the wing pouch, endogenous bnl expression outside the wing pouch is not affected, showing the specificity of the experiment. Finally, frazzled is slightly expressed in wild-type wing disc. This expression is activated when (VP16-En) is overexpressed, and repressed upon En overexpression (Solano, 2003).

Functional subdivision of trunk visceral mesoderm parasegments in Drosophila is required for gut and trachea development

In Drosophila, trunk visceral mesoderm, a derivative of dorsal mesoderm, gives rise to circular visceral muscles. It has been demonstrated that the trunk visceral mesoderm parasegment is subdivided into at least two domains by connectin expression, which is regulated by Hedgehog and Wingless emanating from the ectoderm. These findings have been extended by examining a greater number of visceral mesodermal genes, including hedgehog and branchless. Each visceral mesodermal parasegment appears to be divided in the A/P axis into five or six regions, based on differences in expression patterns of these genes. Ectodermal Hedgehog and Wingless differentially regulate the expression of these metameric targets in trunk visceral mesoderm. hedgehog expression in trunk visceral mesoderm is responsible for maintaining its own expression and con expression. hedgehog expressed in visceral mesoderm parasegment 3 may also be required for normal decapentaplegic expression in this region and normal gastric caecum development. branchless expressed in each trunk visceral mesodermal parasegment serves as a guide for the initial budding of tracheal visceral branches. The metameric pattern of trunk visceral mesoderm, organized in response to ectodermal instructive signals, is thus maintained at a later time via autoregulation, is required for midgut morphogenesis and exerts a feedback effect on trachea and ectodermal derivatives (Hosono, 2003).

Metameric RNA expression of bnl, which encodes a ligand for Breathless FGF receptor, is first observed as 12 patches at mid stage 11. bnl RNA expression becomes homogeneous and then diminished during stage 12. tin is a homeobox gene that is required for dorsal mesodermal development. At early stage 10, tin is expressed throughout the dorsal mesoderm from which VM is derived. Metameric Tin expression becomes evident by early stage 11. Tin expression decreases during stage 12. Expression of bap, another homeobox gene required for VM development, can be monitored by bap 4.5#230; (bap-lacZ). Staining for Tin and bap-lacZ or bnl RNA indicates that tin, bap and bnl are co-expressed in VM-PS3-12 during stage 11; in VM-PS2, only bnl is expressed. Stage 11-12 VM also stains for Tin and VM-hh-lacZ. Tin and VM-hh-lacZ expression partially overlaps. VM-hh expression in the anterior terminal region of VM-PSs indicates that each tin/bnl/bap trio expression domain straddles the VM-PS boundary (Hosono, 2003).

In summary, VM-PSs in thorax and abdomen, respectively, are subdivided into five or six regions with respect to differential expression of VM-metameric genes at stages 11-12. Detailed analysis of VM-hh, bnl, tin and bap expression in addition to con indicates that trunk visceral mesodermal genes are classified into three distinct groups -- tin/bnl/bap, VM-hh and con -- and each VM-PS is subdivided into five or six regions, which become apparent during mid stage 11 to stage 12 (Hosono, 2003).

VM is presently considered to develop in two steps under the control of ectodermal Hh and Wg signals. First, by stage 10 (when four mesodermal primordia have become specified), VM competent or bap expression regions are promoted by hh but repressed by wg, via a direct targetor, slp. The second surge of hh and wg activity at stages 10-11 is responsible for subdividing VM-PSs into two regions: con positive and negative. These results indicate that the expression of four other VM-metameric genes, hh, tin, bnl and bap, is also regulated by the second surge of hh and wg activity at stages 10-11 (Hosono, 2003).

In view of morphological changes in a VM competent region and consideration of these findings on VM gene regulation, the following model for VM-PS cell specification is proposed. At stage 10 to early stage 11, anterior terminal cells of VM-PSs are presumed to be situated near an ectodermal AP border, where they are capable of continuously receiving Wg and Hh signals, and Wg confers competence on these cells to express tin/bnl/bap. Wg and Hh are responsible for inducing VM-hh, and Hh, for con expression. In the anterior-most cells, con expression is reduced, which would be expected in view of repression by high Wg signal. The different thresholds of hh for con and VM-hh expression may explain why the con area expands more posteriorly compared with that of VM-hh. Posterior terminal VM cells, when formed, are situated far from Wg expressed on the ectodermal PS border. But as they migrate posteriorly and close to the posteriorly neighboring AP border by early stage 11, they become capable of receiving Wg and acquire competence to express tin/bnl/bap. Thus, the tin/bnl/bap domain would appear regulated by spatially and temporally distinct Wg signals. The two-step induction of tin/bnl/bap expression is supported by experiments using the wg^ts mutant, where, either posterior or anterior expression within one patch can be differentially turned off. Indeed, a stepwise activation of tin/bnl expression is seen in VM-PSs around stage 11. tin and bnl metameric expression became apparent almost simultaneously at mid-stage 11, and preliminary experiments have shown that neither tin nor bnl misexpression can induce the ectopic expression of any other metameric genes examined here. Thus, tin and bnl expression might be initiated in a mutually independent manner (Hosono, 2003).

Reiterative bnl expression in VM is likely to be a determinant of the particular mode of visceral branches (VB) migration of the trachea, an ectodermal organ. The tip of VB first comes in touch with the vicinity of the posterior end of the tin/bnl/bap expressing alpha region, where all the five metameric genes examined are expressed. Bnl misexpression with VM-specific-GAL4 drivers induces VB misrouting and bifurcation, but neither hh misexpression nor transient loss of Hh activity during stage 11 has any effect on VB budding. BNL misexpression brings about no significant change in expression of tin, while restriction of the tin/bnl/bap expression domain using either dTCF-DeltaN or wg^ts causes a shift in the first VB/VM contact point. Furthermore, under a wg mutant condition, no change is detected in VM-hh or in con expression. Thus, only the bnl expression appears to be closely correlated with VB budding, strongly suggesting that BNL serves as a chemoattractant for initial VB migration (Hosono, 2003).

Tramtrack regulates different morphogenetic events during Drosophila tracheal development

Tramtrack (Ttk) is a widely expressed transcription factor, the function of which has been analysed in different adult and embryonic tissues in Drosophila. So far, the described roles of Ttk have been mainly related to cell fate specification, cell proliferation and cell cycle regulation. Using the tracheal system of Drosophila as a morphogenetic model, a detailed analysis of Ttk function was undertaken. Ttk is autonomously and non-autonomously required during embryonic tracheal formation. Remarkably, besides a role in the specification of different tracheal cell identities, it was found that Ttk is directly involved and required for different cellular responses and morphogenetic events. In particular, Ttk appears to be a new positive regulator of tracheal cell intercalation. Analysis of this process in ttk mutants has unveiled cell shape changes as a key requirement for intercalation and has identified Ttk as a novel regulator of its progression. Moreover, Ttk was defined as the first identified regulator of intracellular lumen formation and; it is autonomously involved in the control of tracheal tube size by regulating septate junction activity and cuticle formation. In summary, the involvement of Ttk in different steps of tube morphogenesis identifies it as a key player in tracheal development (Araújo, 2007).

As with the transcription factors Trh and Vvl, which are involved in orchestrating early events of tracheal development, Ttk plays a role in orchestrating several late tracheal events. Ttk69 has been found to act mostly as a repressor. This study identified Ttk targets that appear to be negatively regulated (such as mummy (mmy), encodes a UDP-N-acetylglucosamine pyrophosphorylase enzyme required for the synthesis of the building blocks of chitin, and escargot (esg) whereas others appear to be positively regulated (such as polychaetoid (pyd) and branchless (bnl). In this latter case, Ttk might be converted into a positive regulator, as already described during photoreceptor development (Araújo, 2007).

This study identified multiple tracheal requirements for Ttk. Interestingly, most of them depend on Ttk regulating events downstream of cell fate specification, at the level of cellular responses. Additionally, a few other requirements depend on cell fate specification, as has been described for most other functions of Ttk in other developmental situations. For instance, Ttk regulates fusion cell specification by acting as a target and mediator of Notch, as occurs during sensory organ development and oogenesis. Such regulation of Ttk by N might be post-transcriptional, as occurs during sensory organ development. Remarkably, it was found that, although Ttk is sufficient to repress esg expression in fusion cells, it might not be the only esg- and fusion fate-repressor, because absence of Ttk does not increase the number of Esg-positive cells, as does downregulating N. Other N targets might be redundant with Ttk, and such redundancy could reinforce N-mediated repression of fusion fate in positions in which inductive signals (such as Bnl, Dpp and Wg) are very high, particularly near the branch tips (Araújo, 2007).

Cell rearrangements during development are common to most animals and ensure proper morphogenesis. During tracheal development, many branches grow and extend by cell intercalation. Several cellular and genetic aspects of tracheal intercalation have been well described. However, targets of Sal (which inhibits intercalation) are currently unknown (Araújo, 2007).

This study identified Ttk as a new and positive regulator of intercalation. Ttk is involved in cell junction modulation by transcriptionally regulating pyd, the only junctional protein shown, so far, to affect intercalation. In fact, modulation of AJs has been proposed to play a role during intercalation. However, Pyd cannot be the only Ttk effector of intercalation, because the pyd mutant phenotype is much weaker than that of ttk mutants. Accordingly, it was found that, in ttk mutants, cells in branches that usually intercalate remain paired and cuboidal, and appear unable to change shape and elongate. Although other explanations could account for the impaired intercalation detected in ttk mutants, it is proposed that inefficient cell shape changes represent the main cause, and might prevent the proper accomplishment of several events, such as the sliding of cells, formation of a first autocellular contact and zipping up, thereby blocking intercalation. Hence, it is proposed that cell shape changes, particularly cell elongation, are an obligate requisite for different steps of intercalation. Other targets of Ttk might presumably be regulators or components of the cytoskeleton involved in cell shape changes. It is relevant to point out here that Ttk has also been proposed to regulate morphogenetic changes required for dorsal appendage elongation (Araújo, 2007).

How does Ttk relate to the known genetic circuit (Sal-dependent) involved in intercalation? Being a transcription factor, Ttk initially appeared as an excellent candidate to participate in this genetic network by regulating sal and/or kni expression. However, both these genes to be normally expressed in ttk mutants, and several differences were detected in the intercalation phenotype of ttk loss versus sal upregulation. For instance, although both situations block intercalation, cells expressing sal, unlike those lacking ttk, are still able to undergo a certain change in shape, from cuboidal to elongated. Therefore, the results fit a model in which Ttk acts in a different and parallel pathway to Sal during intercalation. Consistent with this model, it was found that Ttk is not sufficient to promote intercalation on its own, because its overexpression cannot overcome the inhibition of intercalation imposed by Sal in the DT. Finally, genetic interactions also favour this model, because it was found that: (1) ttk overexpression did not rescue lack of intercalation produced by sal overexpression (even though it rescued the intercalation defects of ttk mutants), and (2) absence of sal (by means of the constitutive activation of the Dpp pathway) does not overcome the intercalation defects of ttk mutants. Therefore, it is proposed that Ttk promotes intercalation by endorsing changes in cell shape, but absence of Sal is still required to allow other aspects of intercalation to occur (Araújo, 2007).

Tube size regulation is essential for functionality. It was found that Ttk is involved in such regulation. Tube expansion and extension relies on a luminal chitin filament that assembles transiently in the tracheal tubes. The metabolic pathway that leads to chitin synthesis involves several enzymes, among which are Mmy and krotzkopf verkehrt (Kkv, a Chitin synthase). In addition, other proteins are known to participate in the proper assembly and/or modification of the chitin filament, such as Knk, Rtv, Verm and Serp. SJs are also required to regulate tube size and it was proposed that they exert this activity, at least partly, via the control of the apical secretion of chitin modifiers. The current results revealed that ttk acts as a key gene in tube size control, playing at least two roles: it regulates chitin filament synthesis and septate junction (SJ) activity (Araújo, 2007).

SJ regulation by Ttk appears functional rather than structural: mild defects were detected in the accumulation of only some SJ markers and there was a loss of the transepithelial diffusion barrier, whereas accumulation of other markers and SJ localisation remained apparently unaffected. It is speculated that Ttk transcriptionally controls one or several SJ components that contribute to maintain the paracellular barrier and to control a specialised apical secretory pathway. As a result, chitin binding proteins such as Verm or Serp are not properly secreted (Araújo, 2007).

It was also found that mmy is transcriptionally regulated by Ttk. mmy tracheal expression positively depends on a mid-embryonic peak of the insect hormone 20-hydroxyecdysone. Therefore, it is proposed that Ttk and ecdysone exert opposing effects on chitin synthesis. Excess of mmy mRNA results in the abnormal deposition of the chitin filament, as occurs in ttk mutants. Defects in chitin deposition might lead to the irregular organisation of taenidia and the faint larval cuticle observed in ttk mutants. Strikingly, Ttk is also required for normal chorion production, which represents another specialised secreted layer (Araújo, 2007).

ttk mutants are defective in the formation of terminal and fusion branches. These defects are due, in part, to non-autonomous, secondary and/or pleiotropic effects of ttk. For instance, ttk mutants exhibit a dorsal closure defect, which prevents the approach and fusion of contralateral dorsal branches. Additionally, terminal and fusion branches depend on correct cell type specification, which did not reliably occur in ttk mutants. For instance, DSRF (Blistered) was missing in some presumptive terminal cells of ttk mutants, impairing terminal branch formation. These tracheal cell identity specification defects might be related to non-autonomous requirements of ttk. For instance, DSRF is not properly expressed in ttk mutants because of an abnormal expression of its regulator, Bnl (Araújo, 2007).

It is important to note that, in spite of these non-autonomous and cell fate specification defects, two pieces of evidence indicate that ttk also plays a specific and autonomous role in the formation of terminal and fusion tubes. First, markers for fusion and terminal cell specification were expressed in many tracheal cells of ttk mutants, but yet most of these cells did not form terminal or fusion branches. Second, only the tracheal expression of ttk in ttk mutants (but not the constitutive activation of the btl pathway, which regulates the terminal and fusion identity) was able to restore the formation of terminal branches (Araújo, 2007).

A common feature of terminal and fusion branches is that they both display intracellular lumina that lack detectable junctions. The cellular events that precede the formation of fusion and terminal branches differ, but the mechanisms by which their intracellular lumina form has been proposed to be comparable. It was found that, in ttk mutants, terminal and fusion cells engage in the correct cellular changes before intracellular lumen formation. However, neither of these two cell types finalised the cellular events leading to tube formation. It has been proposed that the lumen of terminal and fusion branches forms by the coalescence of intracellular vesicles that use a 'finger' tip provided by the neighbouring stalk cell as a nucleation point. Interestingly, it was found that vesicles containing luminal material are less abundant in ttk mutants. These observations suggest a new role for Ttk in the formation of intracellular lumina in distinct cell types. Intracellular lumen formation also occurs in other branched tubular structures, such as in vertebrate endothelial cells and in the excretory cell of Caenorhabditis elegans, presumably by the coalescence of vesicles. Importantly, a crucial role for vesicle formation and their fusion during intracellular tube formation has been demonstrated (Araújo, 2007).

ttk is the first gene described to be involved in intracellular lumen formation during tracheal development. Possible targets of Ttk might be genes related to the apical surface and the underlying cytoskeleton, because several of these genes are involved in C. elegans excretory canal formation. Additionally, genes involved in intracellular vesicle trafficking might also be good candidates. In this respect, several abnormalities have been detected in ttk mutants that might reflect defects in vesicle trafficking (Araújo, 2007).

Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting

Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. This study shows that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-alpha homolog Similar (Sima) and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. It is proposed that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting (Dekanty, 2008).

To address whether Sima and Fatiga participate in the regulation of tracheal terminal sprouting, phenotypic alterations of the tracheae of third-instar larvae exposed to hypoxia were examined in detail. For quantitative purposes, focus was placed on the dorsal branch of the third segment, whose terminal cell has a characteristic branching pattern, typically comprising of a main cellular process from which straight cellular extensions of about 1 μm diameter (hereafter, 'Thick Terminal Branches' [TTBs]) project and thinner extensions ramify thereafter. The average number of TTBs at the third dorsal branch of wild-type larvae maintained in normoxia was 5.65. In hyperoxia (60% O₂), a slight but significant reduction of TTBs (4.82) was observed, whereas in hypoxia (5% O₂), the average TTB number increased to 8.76. Of note, this increase in the number of TTBs was paralleled by a similar increase in the number of thinner terminal projections. Third-instar larvae displayed an average of 17.0 ± 2.7 thin projections at the third dorsal branch in normoxia and an average of 30.7 ± 3.4 thin projections at 5% O₂, implying that, upon hypoxic exposure, the number of thin terminal projections increases to an extent similar to that of the TTBs. These results confirm the tight correlation between tracheal terminal branching and oxygen levels that has been known since Wigglesworths' pioneering studies in the 1950s, and they establish the number of TTBs as a good read out to analyze the extent of terminal branching. Next, it was tested whether fatiga mutant larvae, which are known to accumulate high levels of Sima protein in normoxia, have alterations in the number of ramifications. The fga¹/fga⁹ allelic combination that can develop to the third-larval instar was used, and it was observed that, under normoxic conditions, these larvae displayed an average of 9.49 TTBs and 43.6 ± 5.3 thin terminal projections at the third dorsal branch, an overall number of ramifications even higher than that of wild-type larvae exposed to hypoxia. To test whether increased levels of Sima can account for the excess of TTBs in fga mutant larvae, fga sima double homozygous individuals, which are viable to adulthood, were uxamined. In fga sima double mutant larvae, the number of TTBs (5.14) and thin terminal projections (18.2 ± 3.3) reverted to wild-type levels, which, in turn, was very similar to those of sima homozygous mutants. These findings suggest that the extraterminal sprouting phenotype observed in fga mutants is due to increased levels of Sima protein (Dekanty, 2008).

To gather additional evidence of the participation of the Fatiga (Fga)-Sima system in tracheal hypoxia-dependent plasticity, focus was placed on a different oxygen-dependent modification that is typical of hypoxic larvae: upon exposure to 5% O₂, most tracheal branches become tortuous and, in particular, ganglionic branches (the branches that reach the central nervous system) adopt a ringlet appearance. The proportion of larvae exhibiting at least one ringlet-shaped ganglionic branch (RSGB) was quantitated and it was found that, in normoxic larvae, all ganglionic branches were straight, whereas upon exposure to hypoxia, 36.4% of the larvae displayed at least one RSGB. Among fga¹/fga⁹ larvae, the proportion of individuals exhibiting at least one RSGB dramatically increased, with 69.2% of them being RSGB positive. Strikingly, in fga sima double mutants, the incidence of RSGBs was reduced again to almost wild-type levels, suggesting that RSGBs in fga mutant larvae are also provoked by increased levels of Sima. These results paralleled those regarding the regulation of terminal sprouting, and they support the notion that oxygen-dependent tracheal plasticity of Drosophila larvae is controlled by the oxygen-sensing prolyl-4-hydroxylase Fga, through the regulation of Sima protein abundance (Dekanty, 2008).

This study has analyzed the role of the Drosophila HIF-α homolog Sima and the oxygen-sensing prolyl-4-hydroxylase Fga in tracheal terminal branching. It is assumed that during embryonic stages, tracheal development depends on hard-wired developmental cues, and, later, in larval stages, tracheal terminal branching is driven by local hypoxia in the target tissues. The observations carried out in this study indicate that the tracheal system of sima mutant third-instar larvae is indistinguishable from that of wild-type individuals, including the pattern of terminal branches. Thus, the results imply that if terminal branching during normal development is mediated by tissue hypoxia, the mechanism involved in such a local response should be Sima independent. This is a remarkable difference between Drosophila tracheogenesis and the development of the mammalian vascular system, in which HIF proteins are critically required for both vasculogenesis and developmental angiogenesis (Dekanty, 2008).

Sima does play a cardinal role in hypoxia-dependent tracheal terminal branch sprouting, as well as in the formation of terminal branches that compensate for poor oxygenation in exceptional situations in which a neighboring branch is missing. Sima-dependent extra sprouting is negatively regulated by the oxygen-sensing prolyl-4-hydroxylase Fga, since fga mutants displayed an extra sprouting phenotype that was even stronger than that observed in wild-type individuals exposed to hypoxia. This extra sprouting phenotype is the first demonstration that loss of function of a HIF-prolyl hydroxylase can provoke an angiogenic-like phenotype. Thus, it seems reasonable to expect that conditional knockdown of mammalian PHDs in an appropriate cell type will promote angiogenesis (Dekanty, 2008).

The long-standing paradigm for mammalian angiogenesis is that low oxygen levels trigger HIF accumulation in target tissues, which, in turn, mediates VEGF induction that, upon binding to VEGF receptors on endothelial cells, attracts the outgrowth of newly formed blood capillaries. Nevertheless, this apparently passive role of endothelial cells has recently been challenged. It has been demonstrated that in endothelial cell-specific HIF-α knockout mice the angiogenic response is impaired, highlighting a central role of the oxygen-sensing machinery in endothelial cells (Dekanty, 2008).

This study has shown that the specialized Drosophila tracheal cells that respond to hypoxia by projecting angiogenic-like subcellular processes (i.e., the terminal branches) are apparently more sensitive to hypoxia than any other cell type in the larva. The sensory threshold to induce Sima-driven gene activation in these cells is shifted to near-normoxic oxygen tension. An alternative interpretation of these data is that tracheal terminal cells are similarly sensitive but more hypoxic than other cells, thereby inducing hypoxia-dependent transcription with higher sensitivity. In either case, the results suggest that Sima-dependent transcription within the tracheal terminal cells is part of the mechanism of oxygen sensing and tracheal extra sprouting (Dekanty, 2008).

To test this hypothesis directly, EGFP-labeled sima homozygous mutant terminal cells were generated, and it was found that the ability of these cells to ramify upon a hypoxic stimulus is largely impaired. Furthermore, whether overexpression of Sima in the tracheae can provoke the angiogenic-like response was investigated, and it was found that, indeed, expression of Sima restricted to the tracheal system is sufficient to induce extra sprouting. In contrast, overexpression of Sima -- or of a nondegradable variant of Sima -- in flip-out random clones outside the tracheae failed to provoke a similar phenotype, suggesting that accumulation of Sima in these cells is not sufficient for extra sprouting. Interestingly, in these Sima flip-out clones, a cell-autonomous response was observed, in which long subcellular processes projected from the cells that overexpressed Sima. Thus, although it is clear that bnl is induced in hypoxia and attracts the extension of terminal branches, the data support the notion that Sima is necessary, but not sufficient, for bnl induction in hypoxia (Dekanty, 2008).

Which Sima target genes might be responsible for tracheal extra sprouting was investigated in fga mutants or upon exposure of wild-type larvae to hypoxia. Northern blot analyses indicated that bnl and btl are both upregulated in mildly hypoxic larvae or fga mutants. However, bnl homozygous EGFP-labeled terminal cells of larvae exposed to hypoxia retained their branching capacity, suggesting that extra sprouting in hypoxia is not mediated by an autocrine effect of Bnl, upon Sima-dependent induction in tracheal cells. In contrast, btl is directly induced by Sima in tracheal cells, and, consistent with this, overexpression of Btl in tracheal cells was sufficient to mimic the phenotypes of larvae exposed to hypoxia. Thus, the data suggest that Sima-dependent transcriptional induction of btl in tracheal terminal cells is a critical step of the angiogenic-like response of the tracheal system in hypoxic larvae (Dekanty, 2008).

In summary, it is proposed that tracheal cells respond to hypoxia in an autonomous manner, by promoting the accumulation of Sima, which induces expression of the receptor Btl, thereby increasing sensitivity of these cells to the ligand Bnl. Concomitantly, Bnl is induced in hypoxic target tissues through a mechanism that also involves the participation of Sima, and serves to cue the outgrowth of terminal branches toward O₂-starved areas (Dekanty, 2008).

During angiogenesis, vertebrate VEGF receptors are upregulated in endothelial cells of blood vessels that invade hypoxic tissues, and, particularly, Flt-1 induction is HIF dependent. Endothelial-specific overexpression of VEGF receptors might reveal to what extent this induction is a cardinal step in the angiogenic response to hypoxia (Dekanty, 2008).

Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting

Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. This study show that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-α homolog Sima and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. It is proposed that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting (Centanin, 2008).

This study has analyzed the role of the Drosophila HIF-α homolog Sima and the oxygen-sensing prolyl-4-hydroxylase Fga in tracheal terminal branching. It is assumed that during embryonic stages, tracheal development depends on hard-wired developmental cues, and, later, in larval stages, tracheal terminal branching is driven by local hypoxia in the target tissues. The observations carried out in this study indicate that the tracheal system of sima mutant third-instar larvae is indistinguishable from that of wild-type individuals, including the pattern of terminal branches. Thus, the results imply that if terminal branching during normal development was mediated by tissue hypoxia, the mechanism involved in such a local response should be Sima independent. This is a remarkable difference between Drosophila tracheogenesis and the development of the mammalian vascular system, in which HIF proteins are critically required for both vasculogenesis and developmental angiogenesis (Centanin, 2008).

It was also shown that Sima does play a cardinal role in hypoxia-dependent tracheal terminal branch sprouting, as well as in the formation of terminal branches that compensate for poor oxygenation in exceptional situations in which a neighboring branch is missing. Sima-dependent extra sprouting is negatively regulated by the oxygen-sensing prolyl-4-hydroxylase Fga, since fga mutants displayed an extra sprouting phenotype that was even stronger than that observed in wild-type individuals exposed to hypoxia. This extra sprouting phenotype is the first demonstration that loss of function of a HIF-prolyl hydroxylase can provoke an angiogenic-like phenotype. Thus, it seems reasonable to expect that conditional knockdown of mammalian PHDs in an appropriate cell type will promote angiogenesis (Centanin, 2008).

The long-standing paradigm for mammalian angiogenesis is that low oxygen levels trigger HIF accumulation in target tissues, which, in turn, mediates VEGF induction that, upon binding to VEGF receptors on endothelial cells, attracts the outgrowth of newly formed blood capillaries. Nevertheless, this apparently passive role of endothelial cells has recently been challenged. It has been demonstrated that in endothelial cell-specific HIF-α knockout mice the angiogenic response is impaired, highlighting a central role of the oxygen-sensing machinery in endothelial cells (Centanin, 2008).

This study has shown that the specialized Drosophila tracheal cells that respond to hypoxia by projecting angiogenic-like subcellular processes -- i.e., the terminal branches -- are apparently more sensitive to hypoxia than any other cell type in the larva. The sensory threshold to induce Sima-driven gene activation in these cells is shifted to near-normoxic oxygen tension. An alternative interpretation of the data is that tracheal terminal cells are similarly sensitive but more hypoxic than other cells, thereby inducing hypoxia-dependent transcription with higher sensitivity. In either case, the results suggest that Sima-dependent transcription within the tracheal terminal cells is part of the mechanism of oxygen sensing and tracheal extra sprouting (Centanin, 2008).

To test this hypothesis directly, EGFP-labeled sima homozygous mutant terminal cells were generated, and it was found that the ability of these cells to ramify upon a hypoxic stimulus is largely impaired. Furthermore, whether overexpression of Sima in the tracheae can provoke the angiogenic-like response was examined, and it was found that, indeed, expression of Sima restricted to the tracheal system is sufficient to induce extra sprouting. In contrast, overexpression of Sima -- or of a nondegradable variant of Sima -- in flip-out random clones outside the tracheae failed to provoke a similar phenotype, suggesting that accumulation of Sima in these cells is not sufficient for extra sprouting. Interestingly, in these Sima flip-out clones, a cell-autonomous response was observed, in which long subcellular processes projected from the cells that overexpressed Sima. Thus, although it is clear that bnl is induced in hypoxia and attracts the extension of terminal branches, the data support the notion that Sima is necessary, but not sufficient, for bnl induction in hypoxia (Centanin, 2008).

This study investigated which Sima target genes might be responsible for tracheal extra sprouting in fga mutants or upon exposure of wild-type larvae to hypoxia. Northern blot analyses indicated that bnl and btl are both upregulated in mildly hypoxic larvae or fga mutants. However, bnl homozygous EGFP-labeled terminal cells of larvae exposed to hypoxia retained their branching capacity, suggesting that extra sprouting in hypoxia is not mediated by an autocrine effect of Bnl, upon Sima-dependent induction in tracheal cells. In contrast, btl is directly induced by Sima in tracheal cells, and, consistent with this, overexpression of Btl in tracheal cells is sufficient to mimic the phenotypes of larvae exposed to hypoxia. Thus the data suggest that Sima-dependent transcriptional induction of btl in tracheal terminal cells is a critical step of the angiogenic-like response of the tracheal system in hypoxic larvae (Centanin, 2008).

In summary, it is proposed that tracheal cells respond to hypoxia in an autonomous manner, by promoting the accumulation of Sima, which induces expression of the receptor Btl, thereby increasing sensitivity of these cells to the ligand Bnl. Concomitantly, Bnl is induced in hypoxic target tissues through a mechanism that also involves the participation of Sima, and serves to cue the outgrowth of terminal branches toward O₂-starved areas (Centanin, 2008).

During angiogenesis, vertebrate VEGF receptors are upregulated in endothelial cells of blood vessels that invade hypoxic tissues, and, particularly, Flt-1 induction is HIF dependent. Endothelial-specific overexpression of VEGF receptors might reveal to what extent this induction is a cardinal step in the angiogenic response to hypoxia (Centanin, 2008).

The female-specific doublesex isoform regulates pleiotropic transcription factors to pattern genital development in Drosophila.

Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in Drosophila is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, genome-wide transcriptional profiling was performed of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, 23 genes that have sex-differential transcript levels at all three time points were identified, of which 13 encode transcription factors, a significant enrichment. This study focused on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. In female genital discs, Dsx activates lz and represses Dr and AP-2. It was further shown that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes observed upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. This time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors (Chatterjee, 2011).

A common theme in the evolution of development is that a limited 'toolkit' of regulatory factors is deployed for different purposes during morphogenesis. It is therefore not surprising that the key regulators of genital morphogenesis that this study identified are pleiotropic factors with roles in other developmental processes (Chatterjee, 2011).

Two genes that are expressed sex-differentially in the genital disc, branchless (bnl) and dachshund (dac), provide the best picture of how dsx controls genital morphogenesis. Bnl, which is the fly fibroblast growth factor (FGF), is expressed in two bowl-like sets of cells in the A9 primordium in male discs; there is no expression in female discs because DsxF cell-autonomously represses bnl. Bnl recruits mesodermal cells expressing the FGF receptor Breathless (Btl) to fill the bowls; these Btl-expressing cells develop into the vas deferens and accessory glands (Chatterjee, 2011 and references therein).

Dac, a transcription factor, is expressed in male discs in lateral domains of the A9 primordium and in female discs in a medial domain of the A8 primordium. These lateral and medial domains correspond to regions exposed to high levels of the morphogens Decapentaplegic (Dpp) and Wingless (Wg), respectively. Dsx determines whether these signals activate or repress dac. Male dac mutants have small claspers with fewer bristles and lack the single, long mechanosensory bristle. Female dac mutants have fused spermathecal ducts (Chatterjee, 2011 and references therein).

As with bnl and dac, it remains to be determined whether these downstream genes are direct Dsx targets. Each contains at least one match within an intron to the consensus Dsx binding sequence ACAATGT. Future work will determine whether these matches are indeed contained within Dsx-regulated genital disc enhancers. Moreover, efforts are underway to define Dsx binding locations genome-wide through experiments rather than bioinformatics (B. Baker and D. Luo, personal communication to Chatterjee, 2011); combined with the current expression data, these binding data could speed the discovery of a large number of sex-regulated genital disc enhancers (Chatterjee, 2011).

An important future direction will be to determine how spatial and temporal cues are integrated with dsx to regulate downstream genes. Because lz is expressed in the anterior medial region of the female disc, it is hypothesized that, like dac, it is activated by Wg and repressed by Dpp. Such combinatorial regulation could explain the spatially restricted competence of cells in the male disc to activate lz in response to DsxF. Although Dr, AP-2 and lz are expressed at L3, P6 and P20, many other genes are differentially expressed at only one or two of these time points. How these timing differences are regulated is an important unanswered question, especially for genes such as ac, which shifts from highly female biased at P6 to highly male biased at P20. The finding that Dsx binding sites are most enriched in genes with sex-biased expression at L3 suggests that indirect regulation through a cascade of interactions might contribute to expression timing differences (Chatterjee, 2011).

It has already been shown that DsxF indirectly represses bnl by repressing Dr. To date, Dr has been shown to repress, but not activate, transcription. Therefore, activation of bnl by Dr might itself be indirect, via repression of a repressor. The regulation of bnl by Dr is sufficient to explain the sex-specific expression of bnl. However, upstream of bnl are two sequence clusters that match the consensus binding motif of Dsx. Thus, bnl might be repressed both directly and indirectly by Dsx, in a coherent feed-forward loop (FFL). FFLs attenuate noisy input signals. An FFL emanating from Dsx could provide a mechanism of robustly preventing bnl activation in female discs, despite potential fluctuations in DsxF levels (Chatterjee, 2011).

Understanding how Dr controls the morphogenesis of external structures is also important. The posterior lobe will be of particular interest because it is the most rapidly evolving morphological feature between D. melanogaster and its sibling species. Mutations in Poxn and sal also impair posterior lobe development. Understanding how these two regulators work with Dr to specify and pattern the developing posterior lobe could substantially advance efforts to understand its morphological divergence. Likewise, understanding how lz governs spermathecal development could advance evolutionary studies, as this organ also shows rapid evolution (Chatterjee, 2011).

The extent to which the regulators that were identified play deeply conserved roles in genital development remains to be determined. Although sex-determination mechanisms evolve rapidly, some features are shared by divergent animal lineages. The observation that FGF signaling is crucial to male differentiation in mammals, or that mutations in a human sal homolog cause anogenital defects, could reflect ancient roles in genital development or convergent draws from the toolkit (Chatterjee, 2011).

Whether AP-2, Dr and lz play conserved roles in vertebrate sexual development is similarly uncertain. In mice, an AP-2 homolog is expressed in the urogenital epithelium (albeit in both sexes) and at least one AP-2 homolog shows sexually dimorphic expression (albeit in the brain). The mouse Dr homolog Msx1 is expressed in the genital ridge and Msx2 functions in female reproductive tract development. In chick embryos, Msx1 and Msx2 are expressed male specifically in the Müllerian ducts. The mouse lz homolog Aml1 (Runx1) is expressed in the Müllerian ducts and genital tubercle. As more data accumulate on the genetic mechanisms controlling genital development in other taxa, the question of how deeply these mechanisms are conserved might be resolved (Chatterjee, 2011).

The bHLH transcription factor, hairy, refines the terminal cell fate in the Drosophila embryonic trachea

The pair-rule gene, hairy, encodes a basic helix-loop-helix transcription factor and is required for patterning of the early Drosophila embryo and for morphogenesis of the embryonic salivary gland. Although hairy was shown to be expressed in the tracheal primordia and in surrounding mesoderm, whether hairy plays a role in tracheal development is not known. This study reports that hairy is required for refining the terminal cell fate in the embryonic trachea and that hairy's tracheal function is distinct from its earlier role in embryonic patterning. In hairy mutant embryos where the repressive activity of hairy is lost due to lack of its co-repressor binding site, extra terminal cells are specified in the dorsal branches. hairy was shown to function in the muscle to refine the terminal cell fate to a single cell at the tip of the dorsal branch by limiting the expression domain of branchless (bnl), encoding the FGF ligand, in surrounding muscle cells. Abnormal activation of the Bnl signaling pathway in hairy mutant tracheal cells is exemplified by increased number of dorsal branch cells expressing Bnl receptor, Breathless (Btl) and Pointed, a downstream target of the Bnl/Btl signaling pathway. hairy genetically interacts with bnl in TC fate restriction, and overexpression of bnl in a subset of the muscle surrounding tracheal cells phenocopies the hairy mutant phenotype. These studies demonstrate a novel role for Hairy in restriction of the terminal cell fate by limiting the domain of bnl expression in surrounding muscle cells such that only a single dorsal branch cell becomes specified as a terminal cell. These studies provide the first evidence for Hairy in regulation of the FGF signaling pathway during branching morphogenesis (Zhan, 2010).

To date hairy function in epithelial morphogenesis is limited to a previous study on hairy's role in the regulation of apical membrane growth during embryonic salivary gland development. The current study demonstrate a novel function for hairy in refinement of the terminal cell fate to a single cell at the tip of the dorsal branch through restriction of bnl expression domains in muscle cells surrounding tracheal cells. Due to the strong requirement for hairy in early embryonic patterning, it was necessary to distinguish hairy function in tracheal development from its earlier patterning role. Thus, this analysis of hairy function in the trachea was focused on mutations that did not perturb segmentation of the embryo. h⁴⁷ mutant embryos had no patterning defect, and yet, extra TCs were specified. h^ACT, which not only lacks the WRPW motif but also contains the transcriptional activation domain of VP16 induces ectopic expression of target genes when expressed in the late blastoderm stage, the time when endogenous Hairy is expressed and is active. Expression of h^ACT in mid-embryogenesis, prior to specification of the terminal cell fate led to ectopic expression of bnl in muscle cells and specification of extra TCs. Since h^ACT was induced after patterning of the early embryo was complete, there were no segmentation defects in h^ACT-expressing embryos and yet, extra TCs were specified. Similar to h^ACT, in h⁶⁷⁴ mutant embryos with no segmentation defect, bnl expression domain was expanded and extra TCs were specified. Since, h⁶⁷⁴, like h^ACT, lacks the C-terminal co-repressor binding WRPW tetrapeptide it is possible that the Hairy mutant protein of h⁶⁷⁴ embryos also acts as an activator and induces expression of downstream target genes. Thus, the h⁴⁷, h⁶⁷⁴and h^ACT mutant embryos which are segmented properly and yet show specification of extra TCs in the tracheal dorsal branches provide evidence that hairy's role in early patterning and in tracheal development are indeed distinct (Zhan, 2010).

In addition to a role for hairy in refinement of the terminal cell fate through regulation of bnl expression in muscle cells, evidence is provided that hairy and bnl act antagonistically to regulate terminal branch lumen length. These studies provide the first evidence for a role for bnl in tracheal lumen size control. Although hairy mutant tracheal cells invaginated completely, they did so in an uncoordinated manner compared to wild-type. Thus, hairy function is required at multiple stages of tracheal development (Zhan, 2010).

The data support a model where Hairy in the somatic muscle, normally refines the spatial expression of bnl in the muscle cells that are in close proximity to the migrating tracheal branches, such that only a single cell at the tip of each dorsal branch becomes specified as the terminal cell. Upon loss of Hairy's repressive activity, bnl expression expands in the muscle cells and abnormally activates the bnl/btl signaling pathway, such that extra TCs become specified. These data do not suggest whether bnl is a direct or indirect transcriptional target of Hairy; future studies will distinguish between these two possibilities. It is also possible that Hairy regulates bnl/btl signaling and TC specification via mechanisms other than control of bnl expression. For example, it was recently shown that in the developing tracheal air sac of Drosophila larvae, matrix metalloprotease Mmp2 spatially restricts FGF signaling. Thus, hairy may modulate the extent of bnl/btl signaling in tracheal cells in a post-translational manner as well (Zhan, 2010).

Sequoia establishes tip-cell number in Drosophila trachea by regulating FGF levels

Competition and determination of leading and trailing cells during collective cell migration is a widespread phenomenon in development, wound healing and tumour invasion. This issue was analyzed during in vivo ganglionic branch cell migration in the Drosophila tracheal system. Sequoia (Seq) was identified as a negative transcriptional regulator of Branchless (Bnl), a Drosophila FGF homologue, and it was observed that modulation of Bnl levels determines how many cells will lead this migrating cluster, regardless of Notch lateral inhibition. These results show that becoming a tip cell does not prevent others in the branch taking the same position, suggesting that leader choice does not depend only on sensing relative amounts of FGF receptor activity (Araujo, 2011).

Although high levels of FGF can induce the terminal cell fate in all tracheal cells, small variations in FGF levels can also establish how many cells in the migrating cluster behave as leading cells irrespective of Notch inhibition. This argues for different mechanism being responsible for the branching morphogenesis of ganglionic branches (GBs) and dorsal branches (DBs). In the case of the DBs, it has been proposed that tip cell choice requires Notch-driven selection of a leader cell. This study reports that GB tip-cell selection is not dependent on a Notch lateral inhibitory mechanism. DB migration is in many ways very different from GB migration. For most of their migration, DBs maintain two leading cells, the one that will become the terminal cell and the one that will take the fusion cell fate. Notch lateral inhibition plays a crucial role in singling out the fusion cell. Thus, Notch-mediated effects in DB migration might be associated with this fate choice rather than with tip-cell selection. Fusion cells are not present at the tip of GBs, and therefore their migration is not affected autonomously by Notch. In addition, Notch signalling has a non-autonomous effect in tracheal development by negatively regulating bnl expression, which might mask its real autonomous effects in DB migration. In this scenario, it is proposed that the Notch-independent mechanism, observed for GB migration, is likely to be the norm for most tracheal clusters and other migratory cell groups where fate choices are not an issue (Araujo, 2011).

In conclusion, FGF signals received by tracheal cells, associated with FGF receptor activation, can induce more than one tip cell, irrespective of the migratory behaviour of the neighbouring tracheal cells. These results show that one cell becoming the tip cell does not inhibit others in the migrating cluster taking up the same position. This suggests that the distinction between leading and trailing cells could depend not only on a competition mechanism sensing the relative amounts of FGFR activity, but also on a level of FRGR activity above a critical threshold, induced by a variation in levels of Bnl (Araujo, 2011).

Targets of Activity

branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and pattern of branching

pointed and Serum response factor (also known as pruned, blistered and DSRF) are targeted by BNL. To define the role of bnl in later branching events, expression of secondary (pointed) and terminal (Srf) branch genes in bnl loss-of-function mutants was assayed. pnt and Srf fail to be expressed in the tracheal system of bnl mutants. In contrast, in embryos that ectopically express bnl, both markers are activated throughout the tracheal system; expressing cells later give rise to secondary and terminal branches. These results support the hypothesis that bnl expression near the ends of the primary branches not only guides primary branch outgrowth, but also activates the program of secondary and terminal branching in cells at these positions (Sutherland, 1996).

adrift is expressed in the leading cells of growing tracheal branches, near clusters of branchless FGF-expressing cells and in a pattern very similar to that of several known branchless-induced genes including pointed, DSRF/pruned and sprouty. This suggested that adrift expression might also be induced by the bnl signaling pathway. Expression of an adrift lacZ reporter was examined in embryos mutant for four components of the branchless pathway: bnl, breathless, pnt and pruned. Initial expression of the adrift reporter in stage 11 tracheal cells is normal in all four mutants, but subsequent expression in the leading cells of the branches is absent in bnl, btl and pnt mutants. Expression in pruned mutants is unaffected. In a complementary experiment in which bnl was misexpressed under the control of the hsp70 promoter, expression of the adrift reporter expands to include additional cells in each branch. Thus, the Branchless FGF pathway induces adrift expression in the leading cells of tracheal branches, and this induction requires the bnl FGF, the btl FGF receptor and the pointed ETS domain transcription factor (Englund, 1999).

Expression of the blistered/DSRF gene is controlled by different morphogens during Drosophila trachea and wing development

blistered is expressed in the precursors of the terminal tracheal cells and in the future intervein territories of the third instar wing imaginal disc. Dissection of the blistered regulatory region reveals that a single enhancer element, which is under the control of the fibroblast growth factor (FGF)-receptor signaling pathway, is sufficient to induce blistered expression in the terminal tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is active in the central intervein sector and is induced by the Hedgehog signaling pathway. The other element is under the control of Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector. Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and the localized expression of a gene required for the final differentiation of this territory (Nussbaumer, 2000).

A 500 bp enhancer element (TCE) has been isolated whose activity reproduces the blistered expression pattern in terminal tracheal cells, with respect to its temporal, spatial and regulatory cues. The TCE is the first enhancer identified in Drosophila that responds to FGF signaling. It has been reported that the FGF signaling cascade activates the MAP kinase pathway and that the Ets-domain containing protein Pnt is a target for the ERK-MAP kinase. Therefore, the expression of the TCE is likely to be controlled by FGF signaling which, through the ERK-MAP kinase pathway, activates a Pnt-DNA-bound complex in conjunction with other factors. Further dissection of the enhancer and the identification of the individual DNA sites and the relevant transcription factors should help to elucidate how FGF triggers specific nuclear responses. Interestingly, during early mesoderm induction in Xenopus laevis, an Ets-SRF complex has been implicated in transducing FGF signaling. Therefore, blistered might not only be a target gene whose transcription is activated in response to FGF signaling, but it might also encode a protein that assembles into a complex to integrate the FGF signal. However, the TCE does not require Blistered itself for signal induction since it is still active in a blistered loss-of-function mutant. Nevertheless, other putative target genes induced via FGF signaling in the terminal tracheal cells could require the activation of an Ets-Blistered-DNA complex (Nussbaumer, 2000).

Protein Interactions

branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and pattern of branching

Breathless is the FGF receptor that serves to transduce branchless signals to the interior of the cell. breathless-branchless double mutants exhibit a tracheal phenotype similar to either one's loss-of-function mutation, as expected if the two function in the same signaling pathway. Reduction in the level of breathless can exacerbate the bnl-signaling defects, suggesting that bnl is insufficient in the haploid (single gene copy) state. A constitutively activated form of the BTL receptor can partially ameliorate the effect of the absence of bnl. Under such conditions there is a modest restoration of branching, reflecting the that the normal spatial distribution of activated receptor is not restored. BNL can activate the BTL receptor in vivo. In wild type extracts, a low level of phosphorylated BTL is detected, presumably due to its activation by endogenous BNL. In transgenic embryos that express bnl throughout the body, the level of phosphorylated BTL is increased about 8 fold (Sutherland, 1996).

Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development

The Drosophila sugarless and sulfateless genes encode enzymes required for the biosynthesis of heparan sulfate glycosaminoglycans. Biochemical studies have shown that heparan sulfate glycosaminoglycans are involved in signaling by fibroblast growth factor receptors, but evidence for such a requirement in an intact organism has not been available. sugarless and sulfateless mutant embryos are shown to have phenotypes similar to those lacking the functions of two Drosophila fibroblast growth factor receptors, Heartless and Breathless. sfl and sgl mutants are shown to phenocopy the mesoderm migration defect associated with loss of heartless function and sfl and sgl are required for Btl-dependent tracheal cell migration. Moreover, both Heartless- and Breathless-dependent MAPK activation are significantly reduced in embryos which fail to synthesize heparan sulfate glycosaminoglycans. Consistent with an involvement of Sulfateless and Sugarless in fibroblast growth factor receptor signaling, a constitutively activated form of Heartless partially rescues sugarless and sulfateless mutants, and dosage-sensitive interactions occur between heartless and the heparan sulfate glycosaminoglycan biosynthetic enzyme genes. Overexpression of Branchless, the Breathless ligand, can partially overcome the requirement of Sugarless and Sulfateless for Breathless activity. These results provide the first genetic evidence that heparan sulfate glycosaminoglycans are essential for fibroblast growth factor receptor signaling in a well defined developmental context, and support a model in which heparan sulfate glycosaminoglycans facilitate fibroblast growth factor ligand and/or ligand-receptor oligomerization (Lin, 1999).

There are two well characterized HSPGs in Drosophila: Dally, a Glypican-like cell surface molecule that has been implicated in both Decapentaplegic and Wg signaling, and a transmembrane proteoglycan related to the vertebrate Syndecan family. It has been suggested that syndecans particpate in signaling by vertebrate FGFRs, although other HSPGs may also be involved in this process. It is also possible that different HSPGs could be specific for particular FGF ligand-receptor combinations in individual tissues or at distinct developmental stages. Genetic analysis in Drosophila should provide a useful approach for addressing these important questions (Lin, 1999 and references).

Specific and flexible roles of heparan sulfate modifications in Drosophila FGF signaling

Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, this study characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. It was found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts (Kamimura, 2006; full text of article).

It was asked whether FGF signaling is impaired in mutant animals using an antibody that specifically recognizes the diphosphorylated form of MAP kinase (dpMAPK). In wild-type embryos, dpMAPK is detected in the tracheal placodes at stage 10, reflecting activation of Egfr. This dpMAPK signal was not diminished in the Hs2st; Hs6st embryos, showing that Egfr signaling is not affected by the double mutations. At stage 12, wild-type embryos show a strong dpMAPK signal in the migrating tip cells of each primary branch due to activation of FGF signaling. In contrast, the btl-dependent MAPK activation in the tip cells is disrupted in the Hs2st; Hs6st embryos. In situ RNA hybridization experiments revealed that bnl expression is not altered in the double mutant embryos, confirming that the branching defects observed in the double mutants are caused by disruption of FGF reception but not FGF expression. These results showed that HS with neither 2-O nor 6-O sulfate groups lost the ability to mediate Btl signaling (Kimimura, 2006).

DEVELOPMENTAL BIOLOGY

Embryonic

Early branchless expression (Stage 5) occurs around the dorsal aspect of the cephalic furrow [Images] and the posterior transverse furrow. By stage 8 ventral expression occurs around the cephalic furrow, while dorsal furrow expression diminishes.

branchless is expressed in epidermal clusters outside the developing tracheal system at essentially every position where a major tracheal branch will bud and grow, suggesting that BNL is an attractive factor that induces and guides the outgrowth of the major branches. The expression pattern is complex and dynamic. At stage 11, just before tracheal branching begins, bnl expression appears in five small clusters of epidermal cells arrayed around the tracheal sac, at positions where the five primary tracheal branches will soon bud. As the primary branches grow by cell migration over the next 2 hours (stages 12 and 13), expression in the clusters decreases. This appears to occur in a specific spatial pattern: the bnl-expressing cells closest to or contacting the growing tracheal branches lose expression first, with the tracheal cells continuing to migrate toward the remaining bnl-expressing cells. Two more cell clusters begin expressing bnl as expression in the other clusters turns off, presaging the subseuent outgrowth of specific branches. For example, the ganglionic branch, which initially grows toward one cluster, continues toward a cluster that expresses bnl later, finally reaching the central nervous system. A branch of bnl-expressing cells in the head marks the path of the pharyngeal branch.

branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and pattern of branching

Localized ectopic expression of bnl induces branching toward the ectopic source, while general overexpression disrupts the normal pattern of branching, with fine branches growing out in random directions. Expression is observed in a metameric pattern in the central nervous system (Sutherland, 1996).

branchless activates late programs of tracheal branching. Secondary branches sprout from the ends of primary branches and from a few internal positions. Ectodermal bnl is expressed near positions where secondary branch markers begin to be expressed, suggesting that bnl might also play a role in selecting where secondary branches sprout (Sutherland, 1996).

In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis

In the developing tracheal system of Drosophila, six major branches arise by guided cell migration from a sac-like structure. The chemoattractant Branchless/FGF (Bnl) appears to guide cell migration and is essential for the formation of all tracheal branches, while Decapentaplegic signaling is strictly required for the formation of a subset of branches, the dorsal and ventral branches. Using in vivo confocal video microscopy, it has been found that the two signaling systems affect different cellular functions required for branching morphogenesis. Bnl/FGF signaling affects the formation of dynamic filopodia, possibly controlling cytoskeletal activity and motility as such, and Dpp controls cellular functions allowing branch morphogenesis and outgrowth (Ribeiro, 2002).

To investigate possible dynamic cell shape changes accompanying tracheal cell movement in vivo and link them to the different signaling systems, three-dimensional reconstructions were used of confocal images of living embryos expressing different GFP-tagged proteins in the developing tracheal system. Expression of GFP-actin, driven in tracheal cells by the btl-Gal4 driver line, revealed fine cellular protrusions from cells at the tip of growing branches after initiation of germ band retraction when migration starts. Such cell extensions are most prominently observed in the developing dorsal and ganglionic branches as well as in the dorsal trunk anterior and posterior. During the early stages of branch outgrowth, these cellular extensions were generally short and relatively few in number (Ribeiro, 2002).

In order to visualize possible cell shape changes during later migratory phases, a GFP protein fused to the myristilation site of the Src protein was expressed under the indirect control of the btl enhancer. This GFP fusion protein labels cellular membranes and thus traces the outline of tracheal cells. Three-dimensional reconstruction of dorsal branches using a stack of optical sections through a living embryo expressing this construct revealed that the two leading cells form numerous membranous extensions in all directions; extensions from more proximal cells of the dorsal branch or from cells of the dorsal trunk were only seen very rarely. To ascertain that these membranous extensions contain actin, embryos of the same developmental stage expressing the GFP-actin construct were also examined. Clearly, a similar network of cell extensions was also discernable with actin-coupled GFP. The diameter of these extensions was in the range of 0.3 to 0.4 µm (Ribeiro, 2002).

To investigate the dynamics of the formation of these cellular extensions, a time-lapse confocal analysis was performed of actin cytoskeletal activity in tracheal cells during the migration process, with special emphasis on dorsal and ganglionic branches. In both cases, actin-containing extensions were seen most prominently in the cells at the tip of the branches. Each of the two leading cells in the dorsal branches formed numerous dynamic cellular outgrowths. In the ganglionic branches, cell extensions were most prominently seen in the single leading cell. The formation of cell extensions was extremely dynamic and their topology changed dramatically with time. Some extensions were found to be short lived; others were more stable and rather long (up to 20 µm). It is concluded from these data that tracheal cell migration is accompanied by the formation of thin, dynamic actin-containing cell extensions, referred to here as filopodia (Ribeiro, 2002).

Until now, the only known chemoattractant for tracheal cells is the FGF-like protein encoded by the branchless (bnl) gene. Since bnl is expressed in nontracheal cells adjacent to the tip of migrating branches, it is likely that tracheal cells form extensions as a result of the activation of the Bnl/FGF signaling cascade. To test this prediction, the cytoskeletal activity of tracheal cells was examined in mutant embryos in which Bnl/FGF signaling was disrupted. The failure of tracheal cells to migrate in btl mutants is accompanied by a failure to form filopodial extensions. Lack of filopodia is also observed in embryos mutant for bnl and dof. These experiments demonstrate that Bnl/FGF signaling is required for filopodia formation. Ectopic expression of bnl in all tracheal cells leads to the formation of ectopic filopodia. These experiments provide clear evidence that tracheal cells react to the Bnl chemoattractant with the formation of dynamic actin-containing filopodial extensions. Further experiments show that Bnl/FGF signaling induces cytoskeletal dynamics in the absence of transcriptional induction of any known gene, and it is likely that the signaling input directly influences cytoplasmic events in the absence of changes in nuclear transcription (Ribeiro, 2002).

The formation of tracheal branches via directed cell migration requires input from other signaling systems in addition to Bnl/FGF. Activation of the Dpp signal transduction cascade is essential in dorsal and ventral tracheal cells prior to migration for the subsequent formation of dorsal and ventral (ganglionic and lateral trunk anterior and posterior) branches. In the absence of the Dpp receptors Thick veins (Tkv) or Punt (Put), dorsal branches completely fail to develop and ventral branches are strongly affected. Dpp induces the expression of the genes kni and knrl in the ventral and dorsal cells of the placode; in the absence of these two nuclear proteins, dorsal branches are absent and ventral branches are strongly abnormal (Ribeiro, 2002).

Knowing that Bnl/FGF acts as a chemoattractant for tracheal cells, and having shown above that Bnl/FGF signaling induces filopodial activity, one must wonder why cells need input from the Dpp signaling cascade for a directed movement to the Bnl/FGF source. Is the Dpp response a prerequisite for the subsequent induction of filopodia by Bnl/FGF? Or do dorsal branch cells respond to Bnl/FGF with the formation of filopodia even in the absence of Dpp signaling input, yet fail to migrate properly? In order to find out how these different signaling systems interact in vivo, the cytoskeletal activity of tracheal cells was examined in the absence of Dpp signaling, with particular emphasis on dorsal branches. However, both tkv and put mutants lack dorsal expression of bnl; therefore, they not only lack the Dpp signaling input but also the Bnl/FGF signaling input. In line with the absence of dorsal bnl expression, cellular extensions were not observed in dorsal tracheal cells in put mutants when analyzed in vivo using the GFP-actin fusion protein (Ribeiro, 2002).

In the absence of Dpp signaling, tracheal cells close to the dorsal bnl-expressing ectodermal cells are able to form actin-containing filopodial extensions and initiate dorsal migration. However, the lack of Dpp signaling, which results in the lack of expression of the kni/knrl target genes, leads to failure to form a dorsal branch, and the short, bud-like dorsal outgrowths eventually reintegrate into the main dorsal trunk. Consistent with this interpretation, cells forming the initial dorsal outgrowth in Dad-expressing embryos in rare cases generated a dorsal trunk-sized lumen. These dorsally directed stumps of dorsal trunk were also visible in third instar larvae. Such dorsal trunk-like buds are also seen in mutants that lack Dpp-induced kni/knrl in the tracheal system, indicating that dorsal migration also takes place in these mutants. These buds are never observed in put mutants, presumably due to the lack of dorsal expression of the chemoattractant Bnl/FGF (Ribeiro, 2002).

These results demonstrate that while Bnl/FGF signaling is necessary and sufficient for the induction of filopodial activity in tracheal cells and for cell migration in the strictest sense (cells do start to migrate dorsally when Dpp signaling is inhibited by Dad), Bnl/FGF is apparently not sufficient to allow productive dorsal branch outgrowth. For dorsal branches to grow out and form, Dpp signaling input is strictly required, in addition to filopodial activity induced by Bnl/FGF. Thus, Dpp signaling does not appear to collaborate with Bnl/FGF in filopodia production and motility, but instead to target cellular functions distinct from those targeted by Bnl/FGF signaling. Thus, despite the essential and crucial role of Bnl/FGF, chemoattraction is not sufficient for successful tracheal branching and, despite the requirement of Dpp for dorsal branch formation, migration per se is not affected (Ribeiro, 2002).

Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea

Oxygen delivery in many animals is enabled by the formation of unicellular capillary tubes that penetrate target tissues to facilitate gas exchange. The tortuous outgrowth of tracheal unicellular branches towards their target tissues is controlled by complex local interactions with target cells. Slit, a phylogenetically conserved axonal guidance signal, is expressed in several tracheal targets and is required both for attraction and repulsion of tracheal branches. Robo and Robo2 are expressed in different branches, and are both necessary for the correct orientation of branch outgrowth. At the CNS midline, Slit functions as a repellent for tracheal branches and this function is mediated primarily by Robo. Robo2 is necessary for the tracheal response to the attractive Slit signal and its function is antagonized by Robo. It is proposed that the attractive and repulsive tracheal responses to Slit are mediated by different combinations of Robo and Robo2 receptors on the cell surface (Englund, 2002).

The tracheal system develops from 20 clusters of ectodermal cells, each containing about 80 cells. After invagination and without further cell division, each epithelial cluster sequentially extends primary, secondary, fusion and terminal branches to generate the tubular network that facilitates larval respiration. The regular outgrowth pattern of the primary branches is determined by the localized expression of signaling factors in the surrounding tissues. Among these signals, Branchless (Bnl), a member of the Fibroblast Growth Factor family, first directs the outgrowth of multicellular branches to its site of expression, and it then induces the activation of a set of terminal branching genes in the leading cells of the primary branches. Single terminal cells then form a unicellular branch, migrate over substantial distances and finally stretch and bind to distinct parts of the target tissue to facilitate respiration. A single terminal cell of each ganglionic branch (GB), for example, targets each hemisegment of the embryonic ventral nerve cord (VNC). A cluster of bnl-expressing cells just outside the CNS attracts the GB toward the CNS. The GB cells migrate ventrally along the intersegmental nerve (ISN), but just before reaching the entry point into the CNS, they break their contact with ISN and turn posteriorly to associate with the segmental nerve (SN). This substrate switch is promoted by the expression of adrift (aft), a bnl-induced gene required in the trachea for efficient entry into the CNS. Inside the CNS, the GB1 cell extends over a distance of about 50 µm, from the entry point into the CNS via four different neural and glial substrata to its target on the dorsal side of the neuropil. During the first 20 µm of its journey inside the CNS, the GB1 cell moves its cell body and nucleus along the exit glia, the SN and ventral longitudinal glia towards the midline. The rest of the path is covered by a long cytoplasmic projection that turns dorsally at the midline and reaches the dorsal part of the neuropil by the end of embryogenesis. The signals that guide GB1 migration inside the CNS are not known but the substrata that the GB contacts along its path could potentially provide important guidance cues (Englund, 2002).

Stripe provides cues synergizing with Branchless to direct tracheal cell migration

The Drosophila tracheal system is an interconnected tubular respiratory network, which is formed by directed stereotypic migration and fusion of branches. Cell migration and specification are determined by combinatorial signaling of several morphogens secreted from the ectoderm. A group of ectodermal cells, marked by Stripe (Sr) expression coordinates tracheal cell migration in the dorsoventral axis. Sr, an EGR family transcription factor, is known to regulate muscle migration. Sr ectodermal cells also provide signals that are utilized for tracheal migration. These cues are separated in the time course of embryonic development. Initially, tendon-precursor cells are in close proximity to the tracheal cells, and later, when tracheal migration is complete, the muscles displace the trachea and attach to the tendon cells. sr-mutant embryos exhibit defects in migration of all tracheal branches. Although the FGF ligand Branchless (Bnl) is expressed in a subset of tendon-precursor cells independently of Sr, Bnl functions cooperatively with proteins induced by Sr in attraction of tracheal branches (Dorfman, 2002).

Sr mutant and misexpression experiments imply that Sr induces an essential, yet noninstructive, signal for tracheal migration. To assess whether Sr is able to modulate or enhance the chemoattractive ability of Bnl, Sr or Bnl were expressed alone or simultaneously, in the salivary glands of larvae, which normally lack any trachea. Tracheal branches are attracted to the salivary glands upon ectopic expression of Bnl. Misexpression of Sr in the salivary glands attracts muscles. However, misexpression does not attract tracheal branches. In contrast, misexpression of both Sr and Bnl leads to dramatic tracheal sprouting in the salivary glands, much more pronounced than following Bnl misexpression alone. This experiment demonstrates that Sr can enhance the chemoattractive activity of Bnl. One possible way by which Sr cells may facilitate Bnl activity is through expression of an extracellular component by the tendon-precursor that is able to trap secreted Bnl, thus increasing the local concentration of this potent chemoattractant. Similarly, the activity of Bnl has been shown to depend on heparan sulfate proteoglycans. This mode of action is also employed by tendon cells via the activity of Kakapo to concentrate Vein in tendon/muscle junctions. An alternative way for Sr to provide synergizing cues to the tracheal cells is through induction of cell-cell contact or cell-matrix interaction between the tendon and the tracheal cells. However, the putative Sr-target genes that may mediate the tendon-tracheal interaction remain to be identified (Dorfman, 2002).

In conclusion, it has been shown that ectodermal cells expressing Sr provide sequential signals for migration of tracheal and muscle cells. While the signal for muscles is instructive, the cue for tracheal migration synergizes with the restricted and dynamic Bnl signal. Sr and Bnl functioned cooperatively to attract trachea to the salivary glands upon ectopic expression. Based on this result, it is tempting to speculate that, in a similar manner, the tendon cells normally mark the correct path to the tracheal cells. This may be achieved by expression of cell surface molecules that restrict the diffusion of Bnl and thus necessitate tight association between the trachea and ectoderm for proper migration (Dorfman, 2002).

Drosophila tracheal system formation involves FGF-dependent cell extensions contacting bridge-cells

Development of the ectodermally derived Drosophila tracheal system is based on branch outgrowth and fusion that interconnect metamerically arranged tracheal subunits into a highly stereotyped three-dimensional tubular structure. Recent studies have revealed that this process involves a specialized cell type of mesodermal origin, termed the bridge-cell. Single bridge-cells are located between adjacent tracheal subunits and serve as guiding posts for the outgrowing dorsal trunk branches. Bridge-cell-approaching tracheal cells form filopodia-like cell extensions, which attach to the bridge-cell surface and are essential for the tracheal subunit interconnection. The results of both dominant-negative and gain-of-function experiments suggest that the formation of cell extensions require Cdc42-mediated Drosophila fibroblast growth factor activity (Wolf, 2002).

Drosophila FGF signalling is used reiteratively during the different developmental steps of tracheal organogenesis. It triggers primary branch outgrowth, controls secondary branch sprouting and mediates terminal branching in response to the signals produced by oxygen-starved cells. Evidence is provided that FGF also acts as a growth factor that stimulates the development of tracheal cell extensions necessary for tracheal branch fusion. This conclusion is based on the observations that tracheal cell extensions are missing in FGF-signalling mutants while the formation of ectopic extensions is induced by ectopic FGF/Bnl. Gain-of-function experiments suggest that the FGF/Bnl-dependent cell extension formation is mediated via the Rho-like GTPase Cdc42 (Wolf, 2002).

What is the function of the tracheal cellular extensions? During early tracheal development, FGF/Bnl is instructive for tracheal branch outgrowth. However, gain-of-function experiments indicate that the dorsal trunk forms independently of FGF/Bnl-guidance, suggesting that FGF/Bnl provides a permissive rather than an instructive signal for the dorsal trunk formation. The mesodermal bridge-cell guides the dorsal trunk branches. The results establish that the FGF/Bnl-induced tracheal cell extensions are necessary for bridge-cell-mediated dorsal trunk formation. Several observations support this conclusion. (1) Bridge-cells are in direct contact with the leading edges of the outgrowing dorsal trunk branches that form cell extensions. (2) While the extensions grow out in an anterior or a posterior direction, they are in direct association with the bridge-cells. (3) The cell extensions interconnect adjacent tracheal metameres ~2.5 h before the dorsal trunk branches fuse. (4) Ectopic expression of dominant-negative Cdc42, which represses the formation of cell extensions, frequently induces the lack of dorsal trunk branch interconnections. (5) Cdc42-activated ectopic extensions partially rescue fusion of dorsal trunk rudiments in embryos that lack FGF/Bnl (Wolf, 2002).

The ability of cell extensions to mediate branch fusion via bridge-cells is restricted to dorsal trunk formation. This specific function is likely to be essential since dorsal trunk branch fusion is a multicellular process while all other tracheal interconnections are single cell fusions. Furthermore, dorsal trunk fusion precedes the other fusion processes although all branches bud out at the same time. In addition, different surrounding cell matrices may require various mechanisms for branch outgrowth. In fact, previous work has shown that dorsal branch cells follow a path along a pattern of grooves left between the muscle precursor cells of adjacent metameres, whereas the dorsal trunk branches remain in association with a contiguous population of mesodermal cells (Wolf, 2002).

Interestingly, FGF signalling has also been implicated in the outgrowth of cytonemes, which are thought to function in the distribution of morphogens during Drosophila imaginal disc development. However, cytonemes are remarkably long and microtubule-free cell extensions with a diameter of 0.2 microm. Thus, they differ from the cell extensions described here in size and cytoskeletal composition, i.e., tracheal cell extensions are more than double in diameter and contain microtubules in addition to filamentous actin (Wolf, 2002).

It is speculated that the bridge-cell recognition, the sliding of the tracheal cell extensions along the bridge-cell surface and finally the fusion process are likely to involve extracellular matrix and/or cell adhesion molecules that are associated with the tubular cell extensions and the bridge-cell surface. The functional characterization of such proteins will provide further insights into the guidance mechanisms of cell extensions along specialized cells (Wolf, 2002).

Spatial restriction of FGF signaling by a matrix metalloprotease controls branching morphogenesis

FGF signaling is a central regulator of branching morphogenesis processes, such as angiogenesis or the development of branched organs including lung, kidney, and mammary gland. The formation of the air sac during the development of the Drosophila tracheal system is a powerful genetic model to investigate how FGF signaling patterns such emerging structures. This article describes the characterization of the Drosophila matrix metalloprotease Mmp2 as an extracellular inhibitor of FGF morphogenetic function. Mmp2 expression in the developing air sac is controlled by the Drosophila FGF homolog Branchless and then participates in a negative feedback and lateral inhibition mechanism that defines the precise pattern of FGF signaling. The signaling function for MMPs described here may not be limited to branching morphogenesis processes (Wang, 2010).

To explore their potential role in morphogenesis, the expression patterns of Drosophila mmp1 and mmp2 were studied using GFP reporter strains. Consistent with previously published in situ hybridization data (Page-McCaw, 2003), it was found that both Drosophila mmp genes are active in the larval air sac primordium (ASP) as this structure forms and migrates across the wing imaginal disc, invading larval tissues. In the course of air sac outgrowth, mmp1 is evenly expressed throughout the tubular structure, whereasmmp2 levels become progressively more prominent in the distal end of the air sac and the tip cells (Wang, 2010).

To investigate whether the striking expression pattern of the Drosophila mmp genes in the ASP might point to a role in air sac formation, Mmp1 or Mmp2 function in the tracheal system of Drosophila third instar larvae was disrupted. This was achieved by the expression of specific RNAi constructs for either gene (UAS-mmp1RNAi and UAS-mmp2RNAi, respectively) under the spatial control of the trachea-specific btlGal4 driver and the temporal control of a temperature-sensitive Gal80ts suppressor. The efficacy of the RNAi-mediated knockdown was validated by PCR and antibody staining. Whereas knockdown of mmp1 had only a subtle effect, the phenotype caused by loss of mmp2 was dramatic: air sac extension was impaired, resulting in a severely deformed structure. The characteristic elongated and pointed shape of the air sac was lost. Instead of a single well-defined tip, mmp2- deficient air sacs displayed multiple tips and sometimes had a multilobed appearance. Proliferation appeared unaffected upon knockdown of mmp2 in the ASP as shown by anti-phospho H3 staining, indicating that the mmp2 loss-of-function (LOF) phenotype is not caused by an insufficient supply of tracheoblasts for air sac development. Expression of Drosophila Timp, a specific MMP inhibitor, under btlGal4/Gal80ts control resulted in a multitip phenotype indistinguishable from the one elicited by mmp2RNAi. Coexpression of Mmp2, but not Mmp1, largely reverted the ASP defect caused by TIMP. Moreover, the function of Mmp2 is continuously required to maintain the ordered outgrowth of the ASP (Wang, 2010).

In order for outgrowth to proceed normally, the ASP has to be patterned into stalk and tip cells. Under wild-type conditions, characteristic actin-rich filopodia emanate from the migrating ASP tip and extend toward the source of Bnl/FGF signaling. The multitipped, migration-deficient ASP caused by mmp2 LOF, however, are characterized by the widespread appearance of such filopodia. It is concluded that the patterning into stalk and tip cells might be disturbed under mmp2 LOF conditions, resulting in an expansion of tip territory. To confirm this interpretation, the expression of the tip cell marker esg was monitored in mmp2 LOF air sacs using a GFP reporter under the control of an esgGal4 driver or a straight esg-LacZ reporter. Expression of mmp2RNAi either in the tip cell domain (using the esgGal4 driver) or throughout the tracheal system (using btlGal4) causes a significant expansion of esg expression (Wang, 2010).

It is concluded that Mmp2 function is required to spatially constrain the tip cell region. It is plausible that the failure to migrate and the multitip phenotype are direct consequences of an expansion of the tip cell domain at the expense of the stalk cells (Wang, 2010).

The esg transcription factor controls tip-cell-specific functions in the developing tracheal system of the Drosophila embryo. It is reasonable to assume that esg would similarly confer tip cell specification in the larval ASP. If that were the case, one might expect that the expanded domain of esg expression that was have seen under conditions of reduced mmp2 expression might be causal for the multitip phenotype. To test this possibility, esg was overexpressed throughout the third instar ASP under the control of btlGal4/tubGal80ts, thereby expanding the expression of esg beyond the prospective tip domain. Interestingly, this manipulation caused a multitip phenotype resembling that of mmp2 LOF conditions. It is concluded that the expansion of the esg expression domain is sufficient to mediate the mmp2 LOF phenotype. This finding supports the idea that the mmp2 mutant phenotype is a consequence of a patterning defect caused by an expansion of tip cell fate (Wang, 2010).

Tip cell fate is specified by FGF signaling. Therefore, the expansion of the tip cell domain under mmp2 LOF conditions might be caused by a spread of FGF signaling activity. Such FGF signaling activity in the tip region can be visualized by staining with a phospho-specific (dpERK) antibody, which recognizes the doubly phosphorylated, active form of Drosophila ERK. Consistent with previous reports, dpERK staining was found to be restricted to the tip area of the wild-type ASP. However, upon suppression of Mmp2 activity, either by expression of mmp2RNAi or of timp, dpERK staining was expanded broadly throughout the air sac. This result suggests that the mmp2 LOF phenotype is caused by an expansion of FGF signaling. Consequently, tracheoblasts that would otherwise become part of the stalk are misspecified and adopt ectopic tip cell fates. Consistent with this interpretation, deliberate activation of FGF signaling by overexpression of FGF receptor throughout the air sac can phenocopy the Mmp2 LOF phenotype. These data suggest that Mmp2 can prevent FGF signaling in prospective stalk cells and thereby restrict FGF activity to the tip cell domain (Wang, 2010).

To directly confirm that Mmp2 can suppress FGF signaling, experiments were conducted in Drosophila S2 cells. Transient coexpression of Bnl and Btl potently activates ERK phosphorylation, as monitored by immunoblotting with the dpERK antibody. This ERK response can be abrogated by coexpression with Mmp2. A catalytically inactive mutant, Mmp2E258A, however, has no effect. In agreement with the in vivo data, this result suggests that Mmp2 can interfere with Bnl/Btl signaling. To investigate whether this effect is specific for the FGF pathway or whether other receptor tyrosine kinases might also be affected, a similar experiment was conducted in which ERK was activated by expression of Drosophila EGF receptor and a soluble form of its ligand, Keren. Expression of EGF-R and sKrn causes ERK phosphorylation to a similar degree as Bnl/Btl expression. Significantly, however, this activation is insensitive to the presence of Mmp2. It is concluded that the regulatory function of Mmp2 on air sac development is selective for the FGF pathway. Mmp2 signaling might therefore control the balance between EGF-regulated cell proliferation and FGF-mediated patterning and cell migration (Wang, 2010).

Many examples of branching morphogenesis require a lateral inhibition process that serves to spatially restrict a tip domain in the outgrowing organ. Lateral inhibition is mediated by an inhibitory signal that is released by distal cells once they have adopted tip cell identity, to stop their neighbors from doing the same. In this manner, spreading of tip fate into the adjacent stalk cell area is prevented, assuring the correct patterning and structure of the forming organ. The data presented so far are compatible with the idea that Mmp2 is part of a tip-cell-specific lateral inhibition mechanism. Consistent with this model, this study found that FGF signaling itself can induce Mmp2 expression in the ASP. This conclusion was further confirmed by real-time RT-PCR and western blotting (Wang, 2010).

The lateral inhibition model predicts that Mmp2 expression is required in the tip cells themselves, to restrict expansion of tip cell territory. To test this notion, a clonal analysis strategy was adopted. Using MARCM technology, random clones of GFP-marked cells were generated that were homozygous for themmp2 LOF allele mmp2G535R or the mmp1 LOF allele mmp1Q273*. In parallel, mmp2RNAi or Timp was clonally expressed using the flp-out Gal4 driver system in developing larvae. All strategies resulted in the generation of GFP-labeled clones that lacked the capacity to express Mmp2 activity. The location of these clones within the mRFP-labeled air sac was recorded. Both strategies showed that the mmp2-deficient cells rarely contributed to tip territory, whereas control clones that lack Mmp1 function or express GFP only were randomly distributed across the whole area of the air sac, including the tip. This is interpretated to mean that mmp2-deficient cells, even if they were the first to receive an FGF signal, would not be able to maintain tip fate, as they could not inhibit FGF signaling in their wild-type neighbors. Those neighbor cells expressing Mmp2 normally would then exert a lateral inhibition effect preventing mmp2-deficient clonal cells from receiving FGF signaling. In other words, cells of the ASP compete with each other to contribute to the tip. Cells that lack Mmp2 activity are at a disadvantage and will likely lose the ability to respond to FGF signals, and assume a subsidiary stalk cell role (Wang, 2010).

Finally, an experiment wss designed to directly visualize the paracrine effect of Mmp2 on FGF signaling in the ASP. To this end, a small number of clones expressing Mmp2 were induced in the ASP. The strain used here also carried the hs-bnl transgene. Thereby, Bnl expression can be ubiquitously activated by exposing wandering third instar larvae to a mild heat treatment. The resulting elevated levels of FGF throughout the ASP made it easier to observe inhibitory functions of Mmp2. Strikingly, areas were found of diminished ERK phosphorylation adjacent to Mmp2-expressing clones, as monitored by dpERK staining. Control clones that expressed only GFP never caused such an effect (Wang, 2010).

Several observations in this experiment are noteworthy. First, the inhibitory effect of Mmp2 expression on ERK signaling is strictly nonautonomous. Only cells adjacent to the Mmp2- expressing clones showed decreased ERK activity. The clonal cells themselves are impervious to the inhibitory activity of Mmp2. This finding explains the persistent FGF activity in the ASP tip cells even after Mmp2 expression is induced, and supports the concept that tip cells act as classical organizers that secrete signals to which neighboring cells respond but to which they themselves are insensitive (Wang, 2010).

Second, the paracrine inhibitory effect that Mmp2-expressing cells exert on FGF signaling in their neighbors is not gradual. The affected cells adjacent to the Mmp2-expressing clones have either normal or dramatically decreased ERK signaling activity, but none show intermediary levels. This suggests that Mmp2 activity influences a yes/no decision. Such a mechanism would be consistent with the proposed function of the FGF-Mmp2 signaling circuit to distinguish between two distinct cell fate choices: tip or stalk (Wang, 2010).

Third, not all cells touching Mmp2-expressing clones show decreased ERK activity. The basis for this anisotropic effect of Mmp2 is not clear, but it might be related to the previous point: cells can adopt either an ERK on (tip cell) or an ERK off (stalk cell) state, a decision that is influenced by the interplay between FGF, FGF receptor, and Mmp2. Stochastic variations in signaling might tip the balance one way or the other, especially in the experimental setting employed here, in which high ubiquitous levels of FGF are present (Wang, 2010).

Matrix metalloproteases have long been implicated in invasion and branching morphogenesis. Whereas many studies focus on MMP-dependent extracellular matrix (ECM) remodeling in this context, a different role for Mmp2 was documented in this study: controlling the spatial pattern of FGF signaling. It should be noted that the signaling function of Mmp2 documented here by in vivo and cell-culture evidence does not rule out a mechanical contribution of MMP to air sac outgrowth and invasion. Interestingly, Guha (2009) has very recently reported that Mmp2 clears ECM components around the outgrowing ASP, which may facilitate the movement of the structure (Wang, 2010).

The function of Mmp2 as a modulator of FGF signaling and as part of a lateral inhibition mechanism can be explained by the following model (Figure 4C): tracheal cells that receive the FGF signal first will activate ERK to induce gene expression programs that direct budding and air sac formation. Among the activated transcription units is the mmp2 gene, which is required for the release of an inhibitory signal that nonautonomously prevents further FGF responses in adjacent cells. This Mmp2-mediated lateral inhibition mechanism would thereby restrict the spreading tip cell fate through the prospective air sac. The nature of the inhibitory signal that is delivered by the Mmp2-expressing tip cells is still unknown (Wang, 2010).

It is likely that the mechanisms described here for the Drosophila air sac are also employed by other species and developmental processes. For example, it has been shown that cells with high levels of FGF activity have a competitive advantage in populating the tips or 'terminal end buds' of invading ducts during murine mammary development (Lu, 2008), a finding that is indicative of a lateral inhibition process. Interestingly, it is well established that both MMPs and FGF signaling make critical contributions to mammary development. It is therefore tempting to speculate that the regulatory interplay between MMPs and FGFs operates broadly in invasive growth and branching morphogenesis (Wang, 2010).

The Drosophila homologue of SRF acts as a boosting mechanism to sustain FGF-induced terminal branching in the tracheal system

Recent data have demonstrated a crucial role for the transcription factor SRF (serum response factor) downstream of VEGF and FGF signalling during branching morphogenesis. This is the case for sprouting angiogenesis in vertebrates, axonal branching in mammals and terminal branching of the Drosophila tracheal system. However, the specific functions of SRF in these processes remain unclear. This study establish the relative contributions of the Drosophila homologues of FGF [Branchless (BNL)] and SRF [Blistered (BS)] in terminal tracheal branching. Conversely to an extended view, it was shown that BNL triggers terminal branching initiation in a DSRF-independent mechanism and that DSRF transcription induced by BNL signalling is required to maintain terminal branch elongation. Moreover, increased and continuous FGF signalling can trigger tracheal cells to develop full-length terminal branches in the absence of DSRF transcription. These results indicate that DSRF acts as an amplifying step to sustain the progression of terminal branch elongation even in the wild-type conditions of FGF signalling (Gervais, 2011).

The results contribute to clarification of the roles of BNL and DSRF in terminal branch formation. First, DSRF transcription is dispensable for terminal branch initiation but is a crucial requirement for the progression of this process in wild-type embryos. Second, a constitutively activated form of DSRF still requires BNL signalling to achieve terminal branch formation, thereby indicating that an additional outcome from BNL signalling is required for terminal branch development. Third, high levels of BNL signalling give rise to terminal branches independently of DSRF transcription. All these observations indicate that branches with an intracellular lumen can initiate their development in the absence of DSRF activity. As these are the specific features of terminal cells, it can be concluded that DSRF is not a general determinant of terminal cell fate (Gervais, 2011).

On the basis of these results, the following model is proposed for terminal branch development. On the one hand, BNL signalling triggers the initial phases of cell elongation and intracellular lumen formation. This step is independent of DSRF transcription, probably because BNL levels at this stage are high enough to promote terminal branch initiation. On the other hand, BNL signalling activates DSRF, which in turn allows the progression of cell elongation and intracellular lumen formation. Indeed, as mechanical tension has been proposed as a means to active DSRF expression, it could well be that the same elements involved in the triggering of cell elongation by BNL signalling might also mediate activation of DSRF expression in the terminal cell. Irrespective of the mechanism promoting its expression, DSRF activity can be considered to be a boosting mechanism that, together with other outputs from BNL signalling, ensures that the cellular modifications required for elongation and intracellular lumen formation are kept active in the wild-type conditions of BNL signalling. Consequently, this process appears to spatially restrict induction of terminal branching to places of high BNL signalling, which are often found at the tip of the branches. Finally, although high levels of BNL signalling do not reproduce physiological wild-type conditions, the observation that such high levels bypass the requirement of DSRF transcription for the growth of terminal branches can be of relevance in stress conditions, such as in hypoxia. Likewise, a similar mechanism could be of significance for the induction of angiogenesis in disease (Gervais, 2011).

Specification of leading and trailing cell features during collective migration in the Drosophila trachea

The role of tip and rear cells in collective migration is still a matter of debate and their differences at the cytoskeletal level are poorly understood. This study analysed these issues in the Drosophila trachea, an organ that develops from the collective migration of clusters of cells that respond to Branchless (Bnl), a FGF homologue expressed in surrounding tissues. Individual cells in the migratory cluster were tracked and their features were characterized; two prototypical types of cytoskeletal organization were unveiled that account for tip and rear cells respectively. Indeed, once the former are specified, they remain as such throughout migration. Furthermore, it was shown that FGF signalling in a single tip cell can trigger the migration of the cells in the branch. Finally, specific Rac activation was found at the tip cells, and how FGF-independent cell features such as adhesion and motility act on coupling the behaviour of trailing and tip cells was analyzed. Thus, the combined effect of FGF promoting leading cell behaviour and the modulation of cell properties in a cluster can account for the wide range of migratory events driven by FGF (Lebreton, 2013).

Among the tracheal branches from each placode, two grow towards the ventral side of the embryo, one in the anterior and the other in the posterior region of the segment, the lateral trunk anterior (LTa) and the lateral trunk posterior (LTp) respectively. By a combination of migration, intercalation and elongation, the tip cell of the LTp migrates towards the central nervous system (CNS), and the resulting ganglionic branch (GB) connects the CNS to the main tracheal tube. Another cell from the LTp migrates towards the LTa of the adjacent posterior metamere and makes a fusion branch that connects the two LT branches. This study focused on this branch (LTp/GB) because its complex morphology and pattern of migration make it particularly appropriate for analysing the morphology and behaviour of the tip and trailing cell during tracheal collective migration (Lebreton, 2013).

The FGF signalling pathway is involved in many morphogenetic events requiring collective migration of cell clusters. However, it is not entirely clear whether in these events FGF signalling is directly involved in triggering cell migration, or alternatively if it is required for other processes such as cell determination which only affect cell migration indirectly. Moreover, while FGF might be required it is not clear either whether all the cells or just a subset of those need to directly receive the signal to sustain the migration of the entire cluster. One well-studied case is the role of FGF in the development of the zebra fish lateral line. In that case, FGF appears to be produced by the leading cells which signal to the trailing cells, the cells where FGF signalling is active. Restriction of FGF signalling is thereafter required for the asymmetric expression of the receptors for the chemokines that guide migration (Lebreton, 2013).

A very different scenario applies in the case of Drosophila tracheal migration. On the one hand, FGF is expressed in groups of cells outside the migrating cluster. On the other hand the results in the LTp/GB indicate that FGF signalling is required and sufficient in the leading cells, and not in the trailing cells, for the migration of the whole cell cluster. Therefore, in spite of its widespread involvement, the mechanisms triggered by FGF signalling in collective migration appear to be quite different (Lebreton, 2013).

Rho inactivation produced breaks and detachment in the LTp/GB cluster while its constitutive activation led these cells to hold together impairing migration. Likewise, upon Cdc42 inactivation LTp/GB cells were associated by thin extensions associated in some cases with breaks, while upon its constitutive activation, the LTp/GB transient pyramidal organisation did not evolve, or evolved much more slowly, towards branch elongation. However, the phenotypes from each RhoGTPase mutants don't look alike and the detailed analysis suggests that Rho impinges primarily on cell adhesion while Cdc42 does so on cell motility (Lebreton, 2013).

These results are consistent with previous findings that show a role for Rho in regulating adherens junctions stability and for Cdc42 as the main mediator of filopodia formation. It is noted, however, that Cdc42 was found to exert in the LTp/GB an opposite effect to the one identified in other systems, as Cdc42DN mutants showed more protrusions and were more actin-enriched basally than wild-type cells and Cdc42ACT mutants showed a reduced the motility of LTp/GB (Lebreton, 2013).

There is an increasing amount of data pointing to the different effect of RhoGTPases in vitro versus in vivo models and also among various cell types. A unidirectional assignment between a specific cellular process in vivo and a single RhoGTPase is probably an oversimplification and this was not the aim of the current study. Rather the study relied on mutant forms of the RhoGTPases to modulate cell features, either individually or collectively, to assess their role in the overall behaviour of the cell cluster. In doing so, the results point to a critical role for a balance between cell adhesion and cell motility for the collective migration of a cell cluster (Lebreton, 2013).

The results support the following model for the specification, features and behaviour of leading cells in the migration of the LTp/GB branch. Upon signalling from the FGF pathway, tip cells reorganise their cytoskeleton features (actin enrichment at the basal membrane, small apical surface and an apicobasal polarity along the proximo-distal axis), thereby enabling them to acquire leading behaviour. Indeed, FGF can induce migratory capacity to the whole cluster by signalling only the tip cells, where a dynamic transition between states of Rac activity is needed to acquire a leading role. How the behaviour of tip cells leads collective migration thereafter depends on the features of the cells in the cluster, which are determined by various regulators (among these, the RhoGTPases) which act, at least in part, in an FGF-independent manner. Ultimately, the balance between individual cell properties such as cell adhesion, motility and apicobasal polarity will (1) determine the net movement of the overall cell bodies or alternatively changes in cell shape in terms of elongation, (2) control the migratory speed and (3) define whether cells will migrate individually or in clusters and whether clusters will bifurcate in different paths. The combined effect of the changes promoting leading cell behaviour and modulation of cell features is likely to be a widely exploited mechanism in collective migration. In particular, the actual balance between these cell features may dictate the specifics of each migratory process and, consequently, the final shape of the tissues and organs they contribute to generate (Lebreton, 2013).

FGF is an essential mitogen and chemoattractant for the air sacs of the Drosophila tracheal system

The Drosophila adult has a complex tracheal system that forms during the pupal period. The derivation of part of this sytem, the air sacs of the dorsal thorax, has been studied. During the third larval instar, air sac precursor cells bud from a tracheal branch in response to FGF, and then they proliferate and migrate to the adepithelial layer of the wing imaginal disc. In addition, FGF induces these air sac precursors to extend cytoneme-like filopodia to FGF-expressing cells. These findings provide evidence that FGF is a mitogen in Drosophila; they correlate growth factor signaling with filopodial contact between signaling and responding cells, and suggest that FGF can act on differentiated tracheal cells to induce a novel behavior and role (Sato, 2002).

Metamorphosis presents special challenges to the tracheal system. The process that transforms the Drosophila larva into an adult fly consumes larval tissues and creates new organs using imaginal cells that were prepared during larval development. A new tracheal system that will satisfy the aerobic requirements of the specialized tissues of the adult must be built. But, in addition, the organs of the pupa must be kept oxygenated while the adult develops. Transformation of the tracheal system begins during the third larval instar, when imaginal tracheoblasts start to divide. These proliferating tracheoblasts spread over the larval tracheal system, using it as a scaffold to form an extensive branching network before the larval cells histolyze. Some tracheoblasts elaborate coiled structures that are unique to the pupa. Others grow to form the air sacs of the adult. Air sacs are large reservoirs that are juxtaposed with major muscle systems and with the brain. These structures have been thought to form as dilations of the main tracheal trunks, which are the direct descendants of the main tracheal trunks of the embryo and larva. The work described in this study shows that in the dorsal thorax these air sacs originate independently from a distinct population of cells (Sato, 2002).

The majority of the adult thorax, including most of the dorsal thoracic epidermis, the wing, and flight muscle, is produced by the wing imaginal disc. This organ arises as a tubular invagination of the epidermis, and when it grows and flattens during the larval periods, it develops four distinct cell types. It has squamous peripodial cells on one surface, columnar epithelial cells on the other surface, a distinct group of adepithelial cells that nestle against the most proximal columnar epithelial cells, and stalk cells that connect the disc to the epidermis. A large tracheal branch attaches in some manner to the columnar epithelial surface. It orients along the dorsal/ventral axis of the disc, but it does not ramify to generate multiple contacts with the disc cells. Therefore, no framework exists to serve as a template for the tracheolar network that provides oxygen to the thoracic cells of the adult. The mechanisms responsible for the branch formation and path finding that produce the extensive and complex tracheolar network in the adult thorax remain to be identified (Sato, 2002).

The work described in this study represents an effort to understand the role of FGF in wing disc development. These investigations led to the identification of a new cell type in the wing disc that migrates and proliferates in response to FGF. These cells contact FGF-expressing cells across at least one cell layer by extending long cytoneme-like filopodia. These cells are destined to form the air sacs that associate with the flight muscles in the adult thorax, but they are distinct from the cells that form the larval trachea or from the group of imaginal precursors that are programmed to generate tracheae in the pupa and adult (Sato, 2002).

To investigate the role of FGF in Drosophila wing disc development, patterns of expression of bnl and the two FGF receptor genes, breathless (btl) and heartless (htl), were examined in third instar discs. bnl/FGF expression is restricted to a small group of cells in the columnar epithelium. An exact count of their number was problematic: since the apparent level of expression in many cells is very low, their number is roughly estimated to be between 15 and 60 in early third instar discs and between 80 and 150 in late third instar discs. These cells straddle the anterior/posterior compartment border and are dorsal to the region of the prospective wing blade. The cells that express bnl most strongly are ventral to the progenitor of the aPA machrochaete, as indicated by double staining for Achaete protein. They are in a region that contributes to the notal wing processes: the cuticle located between the adult scutum and wing hinge (Sato, 2002).

Expression of btl and Htl is restricted to cells in the adepithelial layer of the wing disc and is absent from the cells of the columnar epithelium. The adepithelial cells give rise to the adult musculature; they also express twist, which controls htl and is a signature of all mesodermal cells and muscle precursors. It was confirmed that the wing disc adepithelial cells express both Htl and Twist. In addition, a small group of adepithelial cells was identified that expresses a btl enhancer trap line but expressed neither Htl nor Twist. Stumps, a putative adapter protein required for both Btl and Htl signaling, is expressed in both btl- and Htl-expressing adepithelial cells (Sato, 2002).

The btl-expressing cells in the adepithelial layer had not been identified previously, and their presence was unexpected, since one of the principal domains of btl expression is the trachea. Tracheal cells are almost invariably associated with tubules with cuticle-lined lumen, and the adepithelial cells have no such distinct structures. Nevertheless, the adepithelial btl-positive cells appear to maintain continuity with the cells of the main tracheal branch. Evidence is presented that the btl-expressing adepithelial cells are the precursors of the adult tracheal air sacs (Sato, 2002).

To better understand the origin and fate of the btl-expressing adepithelial cells, they were tracked during larval and pupal development. In early third instar wing discs, no btl expression was detected in adepithelial cells; only tracheal branch cells are btl positive. However, as third instar discs mature, btl-positive cells are detected budding from the tracheal branch that adheres to the wing disc. This bud forms at a stereotypical position just dorsal to the wing hinge progenitors and adjacent to the group of 15–60 bnl-expressing cells in the columnar epithelium. During development of the third instar, the number of btl-expressing cells increase, and the bud expands posteriorly toward the region of greatest bnl expression. In late third instar discs, the btl-expressing cells form a coherent group surrounded by Htl-expressing cells. These btl-expressing cells do not express Htl. Possible explanations for the complementarity of the patterns of btl and Htl expression are that btl cells displace Htl-expressing cells or that the expression of these genes is mutually exclusive. However, in the early third instar discs, Htl expression is already absent from the region that will be occupied by btl-expressing cells in older discs; therefore, these are not likely to be sufficient explanations (Sato, 2002).

To characterize these cells further, their relationship to the larval tracheal system was examined. The larval tracheal branch that adheres to the wing disc is called the first transverse connective. It has a small offshoot called the spiracular branch, where imaginal tracheoblasts, precursors of the adult tracheae, are located. These imaginal tracheoblasts do not express btl but do express escargot (esg) and trachealess (trh). Since esg inhibits endoreplication of imaginal cells and the imaginal tracheoblasts are assumed to be diploid, expression of esg in these cells was not unexpected. The presence of Trh, a transcription factor that directly activates btl transcription, may presage btl expression at a subsequent stage. The btl-expressing adepthelial cells also express Trh. They do not express esg early in the third instar; therefore, it is unlikely that they derive from the imaginal tracheoblasts. However, in mid third instar and thereafter, esg expression is evident in the most posterior cells of the group (Sato, 2002).

The capacity of tracheal branch cells to proliferate was unexpected because the cells lining the main tracheal branches had been considered to be both terminally differentiated and polyploid. Neither state is expected to be compatible with a mitotic cell cycle program. The morphology and fluorescence of DAPI-stained discs was examined to better understand the nature of the disc-associated tracheal cells. Most of the cells that line the lumen of the main tracheal branch, the first transverse connective, as well as the imaginal tracheoblasts, have nuclei that are similar in diameter (5–6 µm), are relatively small, and have a similar level of fluorescence. Some cells with large nuclei and >3× the fluorescence intensity of the smaller nuclei were observed populating other branches that connect to the first transverse connective. Assuming that the imaginal tracheoblasts are diploid, these observations suggest that the cells that respond to Bnl-FGF have a similar ploidy and that the tracheal branches that associate with the wing disc include both diploid and polyploid cells (Sato, 2002).

To determine the fate of the btl-positive adepithelial cells, their behavior and movements were examined throughout pupal development. They are located next to the posterior part of the prospective wing hinge in the late third instar disc. Analysis of fixed wing discs prepared at various times during the first 12 hr after puparium formation (APF) revealed that they remain in this location as a tight, rounded cluster of cells next to the prospective wing hinge (Sato, 2002).

To continue following the fate of these cells, five individual pupae that expressed btl-Gal4 UAS-GFP were observed during the pupal period, and photographs were taken at regular intervals. The pupal case is transparent to the GFP fluorescence; therefore, no surgical manipulations were necessary. The btl-positive adepithelial cells were identified by their proximity to the wing hinge at 12 hr APF, consistent with the observations of dissected, fixed discs. These btl-positive cells migrate dorsally between 12 and 23 hr APF then anteriorly and posteriorly to form three branches. At 32 hr APF, they cease their migrations and began to elaborate into air sacs. It is concluded that the btl-positive adepithelial cells are the precursors of the adult air sacs and that the air sacs of the adult thorax are derived from cells that are distinct from the imaginal tracheoblasts (Sato, 2002).

Although the air sac tracheoblasts do not form a tubular structure at 12 hr APF, these pupae do have numerous tracheae that project to the developing muscles. These tracheae are clearly distinct from the air sac tracheoblasts. They derive from the second dorsal branch and are present only during the pupal period. Air sacs are prominent and extensive in older pupae and in adults and are associated with numerous bundles of tracheae that extend from the air sacs and extensively interdigitate with flight muscle cells. At present, the structure of the air sacs is not understood enough to know how these tracheae either connect with or contribute to the function of the air sacs (Sato, 2002).

Thus, bnl/FGF expression is detected in a small group of columnar epithelial cells during the third instar and pupal periods. Although their fate in the adult has not been established, these cells are small in number and therefore cannot produce more than a small part of the adult cuticle. Nevertheless, their effect on the adult is profound. Through the action of Bnl-FGF, they induce a group of tracheal cells to initiate a program of proliferation and migration and to join with the cells in the disc adepithelial layer. Despite this intimate association with these mesodermal progenitors, the FGF-responsive cells retain their tracheal identity and go on to form the prominent adult tracheal air sacs that extend throughout much of the dorsal thorax (Sato, 2002).

The tracheal system of the Drosophila embryo has ten interconnected metameric units on either side of the animal; one unit derives from the second, or mesothoracic, segment. This mesothoracic component consists of portions of the dorsal and lateral trunks, a transverse connective that links these trunks to each other, a dorsal branch that connects the left and right sides, and numerous branches that radiate out to various tissues. During larval development, this general structure is retained, and, although the tracheal cells do not divide, many new branches form, and the diameter of the more proximal tracheae increase. The wing imaginal disc attaches to the transverse connective. Imaginal tracheoblast precursors of the adult tracheae populate a small spiracular branch at a location just dorsal to the disc attachment (Sato, 2002).

In constructing the adult tracheal system, the imaginal tracheoblasts use parts of the larval framework as templates and, in effect, remodel the dorsal and lateral trunks, the transverse connective, the dorsal branch, and the main pupal branches to the wing and leg. In contrast, the large and extensive air sacs do not correspond to earlier branches in any obvious way and have no apparent antecedent. The airs sacs of the dorsal thorax form de novo from a small group of wing imaginal disc cells. This study chronicles the transformation of these air sac tracheoblasts from a tight cluster of adepithelial disc cells to sculpted air sacs. These observations were made by expressing GFP under btl control and by following the GFP-containing cells through the pupal period. It was possible to directly account for three branches of the dorsal thoracic air sacs (e.g. the medioscutal, lateroscutal, and scutellar sacs) as products of the wing disc air sac tracheoblasts. All of the air sacs in the notum appeared to contain GFP in these animals, but the resolution of the analysis was not sufficient to make this a definitive conclusion nor did it allow for a conclusion that all of the air sacs derive from disc tracheoblasts. Nevertheless, this study did establish that the disc tracheoblasts generate air sacs and, by some process that is as yet unknown, form a tracheal lumen and tracheal network. It will be interesting to identify the intrinsic and extrinsic systems that direct the genesis of the air sacs, since they apparently develop in the absence of a preexisting framework (Sato, 2002).

Evidence is presented for air sac tracheoblasts in the wing imaginal disc. Data suggest that there may be similar strategies to make air sacs in other regions of the fly. This statement is based on the presence of nontracheal btl-positive cells and bnl-expressing cells in other imaginal discs. In leg discs, bnl expression is found in the stalk region, and the pattern of expression becomes more extensive and complex in the disc epithelium of pupal discs. In third instar discs, btl-positive cells are localized to an offshoot of a tracheal branch that attaches near the stalk and adjacent to the bnl-expressing cells. After puparium formation, the btl-positive cells migrate along the basal surface of the disc columnar epithelium to a position that roughly correlates with the region where air sacs will later form. In eye-antenna discs, bnl is expressed in cells surrounding the ocelli progenitors. Although btl- expressing cells were not found in larval eye-antenna discs, it was observed that in early pupae btl-positive cells assume a position underlying the presumptive ocelli cells. Air sacs in the adult head underlie most of the medial head cuticle and encircle the region where the ocelli form. These observations led to the suggestion that the process that induces the air sac tracheoblasts in the wing disc may be common to other discs as well (Sato, 2002).

Bnl-FGF is the key determinant of tracheal branching as the preadult tracheal system matures. In the embryo, it guides the migration of tracheal cells to form primary branches, induces secondary branches as the primary branches approach the cells expressing bnl, and regulates the process that generates terminal branches. To produce these different outcomes, the FGF signaling pathway acts through different, but related, mechanisms, but its role is, in effect, a single one -- to mold the tracheal cells and to influence where and how they extend. The roles that it plays during the early stages of air sac morphogenesis are different. As a signal in the wing imaginal disc, Bnl-FGF functions as a chemoattractant, inducing these cells to migrate from outside the imaginal disc to a location within the adepithelial layer. And it acts as a mitogen, inducing the tracheoblasts to proliferate. These properties are not manifested by Bnl-FGF during earlier stages of tracheal development. Thus, the morphogenic process that generates the air sacs is distinguished both by its independence from the tracheal framework of the embryo and larva and by the roles that FGF plays (Sato, 2002).

Perhaps the most surprising behavior this work identified is the response of the larval tracheal cells to Bnl-FGF. These cells form a tripartite tube that consists of an external basal lamina, a squamous epithelium, and a complex, multilayered luminal cuticle. They had been considered to be terminally differentiated and incapable of proliferation, having ceased cell division in the early embryo. It was found, instead, that many of the wing disc-associated tracheal cells can respond to Bnl-FGF by migrating out of the tracheal branch to embark on a program of proliferation and morphogenesis. Remarkably, this latent capacity for conversion to proliferative tracheoblast is shared by many (and perhaps all) of the tracheal cells that populate the disc-associated branch. These cells require only the action of Bnl-FGF to initiate the process. Although the possibility of a separate and distributed population of tracheal stem cells cannot be excluded, it is thought that the widespread capacity of tracheal cells to adopt a program of proliferation and migration makes this possibility unlikely. Instead, it is proposed that Bnl-FGF acts as an instructional determinant, reprogramming the cells in the tracheal branch to become air sac tracheoblasts. It is thought that the activity of Drosophila FGF to induce cells to dedifferentiate has no precedent (Sato, 2002).

Actin-based filopodia have been observed in many cells that send or receive signals. For purposes of illustration, four are mentioned here. (1) Neuronal growth cones are populated with many active filopodia that appear to probe their environment for guidance cues. (2) Long and highly dynamic filopodia extend from primary mesenchyme cells in the interior of early sea urchin embryos, apparently to contact and explore the overlying ectoderm. They are thought to relay information from the peripheral ectoderm that patterns internal skeletal elements. (3) Dendritic cells, professional antigen-presenting cells of the vertebrate immune system, are defined in part by their Medussa-like morphology. Their unusually large number of finger-like projections may maximize the likelihood that antigen presentation finds a suitable target cell. (4) Drosophila wing imaginal disc cells have thin filopodial extensions, 'cytonemes', that appear to connect cells with the organizer region at the anterior/posterior compartment border. Although the filopodia produced by these various cell types are similar in gross morphology, it is not known whether they all have a role in trafficking signals, have a common mechanism that receives and transduces signals, or are regulated in a similar manner (Sato, 2002).

The filopodia that the wing disc tracheoblasts produce have a number of properties in common with cytonemes. Both are actin-based, have a comparable size and appearance, and are similarly sensitive to standard conditions of fixation. Functional analysis indicates that the tracheoblast filopodia are dependent on Bnl-FGF and upon the ability of the tracheoblasts to carry out FGF signal transduction. There is no direct evidence that signaling cannot occur if they are absent, but the correlation between these structures and active signaling is strong. Moreover, their presence offers a possible mechanism to move the FGF signal from its source in the columnar epithelial cell layer across the adepithelial cell layer to the tracheal target cells. It is not known what alerts the target cells to the presence of a source of FGF. Two possibilities are that nonspecific cytoneme-like filopodia explore the extracellular environment for potential sources or that less efficient signaling can occur in the absence of direct contacts. It is assumed that, if this second possible mechanism is operative, once filopodia are induced and make appropriate contacts, signaling will be accelerated and more productive. The question of how the filopodia are induced and oriented is certainly important, but the very presence of these cellular extensions that make contact across these distances offers a mechanism to facilitate the ordered movement of signals and the concerted and directed migration of cells (Sato, 2002).

A signaling network for patterning of neuronal connectivity in the Drosophila brain

The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. This question was investigated in the developing adult brain of Drosophila. Extension and retraction is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern (Srahna, 2006).

Based on the observation that blocking Fz2 results in decreased numbers of dorsal cluster neuron (DCN) axons in the medulla, it was reasoned that Fz2 could be a receptor for a putative stabilization signal. Since Fz2 and Fz are partially redundant receptors for the canonical Wnt signaling pathway, expression of the canonical Wnt ligand Wingless (Wg) was investigated in the brain during pupation. However, no Wg expression was detected in the pupal optic lobes, suggesting that Wg is unlikely to be involved in regulating DCN axon extension. Therefore, the expression of Wnt5, which has been shown to be involved in axon repulsion and fasciculation in the embryonic CNS, was investigated. Anti-Wnt5 staining revealed widely distributed Wnt5 expression domains beginning at PF and lasting throughout pupal development and into adult life. Wnt5 is strongly expressed in the distal medulla and is also present on axonal bundles crossing the second optic chiasm.The number of DCN axons crossing to the medulla was examined in wnt5 mutant flies. The number of DCN axons crossing the optic chiasm is reduced from 11.7 to 7.9 in the absence of wnt5, suggesting that it may play a role in stabilizing DCN axons (Srahna, 2006).

Next, the requirement of the Wnt signaling adaptor protein Dsh was tested. In animals heterozygous for dsh⁶, a null allele of dsh, the average number of DCN axons crossing between the lobula and the medulla is reduced from 11.7 to 7.6 with 78.5% showing less than eight axons crossing. Signaling through Dsh is mediated by one of two domains. Signaling via the DIX (Disheveled and Axin) domain is thought to result in the activation of Armadillo/β-Catenin. DEP (Disheveled, Egl-10, Pleckstrin) domain-dependent signaling results in activation of the JNK signaling pathway by regulation of Rho family GTPase proteins during, for example, convergent extension movements in vertebrates. To uncover which of these two pathways is required for DCN axon extension the dsh¹ mutant, deficient only in the activity of the DEP domain, was tested. Indeed, in brains from dsh¹ heterozygous animals the number of extending axons was reduced from 11.7 to 7.4. In flies homozygous for the dsh¹ allele the average number of axons crossing was further reduced to 4.7, with all the samples having less than six axons crossing. In contrast, the DCN-specific expression of Axin, a physiological inhibitor of the Wnt canonical pathway, did not affect the extension of DCN axons. Similarly, expression of a constitutively active form of the fly β-Catenin Armadillo also had no apparent effect on DCN extension. Finally, whether Wnt5 and Dsh interact synergistically was tested. To this end, wnt5, dsh¹ trans-heterozygous animals were generated. These flies show the same phenotype as flies homozygous for dsh¹, suggesting that Wnt5 signals through the Dsh DEP domain (Srahna, 2006).

To determine if dsh is expressed at times and places suggested by its genetic requirement in DCN axon outgrowth, the distribution of Dsh protein during brain development was examined. Dsh protein is ubiquitously expressed during brain development. High expression of Dsh is detected in the distal ends of DCN axons at about 15% PF shortly before they extend across the optic chiasm toward the medulla. In general, higher levels of Dsh were observed in the neuropil than in cell bodies (Srahna, 2006).

In summary, these data indicate that the stabilization of DCN axons is dependent on the Dsh protein acting non-canonically via its DEP domain. Importantly, the axons that do cross in dsh mutant brains do so along the correct paths. This suggests that, like JNK signaling, Wnt signaling regulates extension, but not guidance, of the DCN axons (Srahna, 2006).

Wnt signaling to Dsh requires the Fz receptors. To examine if the effect of Wnt5 on DCN axon extension is also mediated by Fz receptors, the number of DCN axons crossing the optic chiasm in was counted fz, fz2, and fz3 mutants. There was no significant change in the number of axons crossing in the brain of fz3 homozygous animals. In contrast, in brains heterozygous for fz and fz2, the number of the axons crossing was reduced from 11.7 to 6.6 (fz) and 6.9 (fz2), with 71% and 85.7%, respectively, showing less than eight axons crossing. These data suggest that DCN axons respond to Wnt5 using the Fz and Fz2 receptors, but not Fz3. To determine whether the Fz receptors act cell-autonomously in individual DCNs, single-cell clones doubly mutant for fz and fz2 were generated and the number of DCN axons crossing the optic chiasm was counted. In contrast to wild-type cells, where 37% of all DCN axons cross, none of the fz, fz2 mutant axons reach the medulla. To test whether wnt5, fz, and fz2 genetically interact in DCNs, flies trans-heterozygous for wnt5 and both receptors were examined. Flies heterozygous for both wnt5 and fz mutations show a strong synergistic loss of DCN axons (11.7 to 3.7) and in fact have a phenotype very similar to that of flies homozygous for dsh¹. Flies doubly heterozygous for wnt5 and fz2 also show a significant decrease in DCN axons (5.7), compared with either wnt5 (~8) or fz2 (8.5) mutants. These data indicate that the genetic interaction between wnt5 and fz is stronger than the interaction between wnt5 and fz2 (Srahna, 2006).

Examination of the expression domains of Fz and Fz2 in the developing brain supports the possibility that they play roles in stabilizing DCN axons. Both Fz and Fz2 are widely expressed in the developing adult brain neuropil. In addition, Fz is expressed at higher levels in DCN cell bodies (Srahna, 2006).

The observation that the wnt5 null phenotype can be enhanced by reduction of Fz, Fz2, or Dsh suggests that another Wnt may be partially compensating for the loss of Wnt5. To test this possibility, flies heterozygous for either wnt2 or wnt4 were examined. wnt2 heterozygotes display a reduction of DCN axon crossing from 11.7 to 7.3, whereas no phenotype was observed for wnt4. Thus, wnt2 and wnt5 may act together to stabilize the subset of DCN axons that do not retract during development. In summary, these results support the model that Wnt signaling via the Fz receptors transmits a non-canonical signal through Dsh resulting in the stabilization of a subset of DCN axons (Srahna, 2006).

Data is provided that supports the hypothesis that the regulation of JNK by Rac1 modulates DCN axon extension. As such attempts were made to determine how Wnt signaling might interact with Rac1 and JNK. The opposite phenotypes of dsh and Rac1 loss-of-function suggest that they might act antagonistically. To determine if Rac1 is acting upstream of, downstream of, or in parallel to Dsh in DCN axon extension, dominant-negative Rac1 was expressed in dsh¹ mutant flies. If Rac1 acts upstream of Dsh, the dsh¹ phenotype (i.e., decreased numbers of axons crossing the optic chiasm) is expected. If Rac1 acts downstream of Dsh, the Rac1 mutant phenotype (i.e., increased number of axons crossing) would be expected If they act in parallel, an intermediate, relatively normal phenotype is expected. Increased numbers of axon crossing were observed, suggesting that Rac1 acts downstream of Dsh during DCN axon extension and that Dsh may repress Rac1 (Srahna, 2006).

Next, whether Dsh control of DCN axon extension is mediated by the JNK signaling pathway acting downstream of Wnt signaling was tested, as the similarity of their phenotypes suggests. If this were the case, activating JNK signaling should suppress the reduction in Dsh levels. Conversely, reducing both should show a synergistic effect. Therefore the JNKK hep was expressed in dsh¹ heterozygous flies and it was found that the hep gain-of-function is epistatic to dsh loss-of-function. Furthermore, reducing JNK activity by one copy of BSK-DN in dsh¹ mutant animals results in a synergistic reduction of extension to an average of 0.8 axons with 60% showing no axons crossing and no samples with more than three axons. In summary, the results of genetic analyses suggest that Wnt signaling via Dsh enhances JNK activity through the suppression of Rac1 (Srahna, 2006).

Dsh appears to promote JNK signaling and to be expressed in DCN axons prior to their extension toward the medulla early in pupal development. Since JNK signaling is required for this initial extension, it may be that Dsh also plays a role in the early extension of DCN axons. To test this possibility, DCN axon extension was examined at 30% pupal development in dsh¹ mutant brains. In wild-type pupae, essentially all (~40) DCN axons extend toward the medulla. In contrast, in dsh¹ mutant pupae, a strong reduction in the number of DCN axons crossing the optic chiasm between the lobula and the medulla was observed (Srahna, 2006).

Although the genetic data indicate that Dsh- and Rac-mediated signaling have sensitive and antagonistic effects on the JNK pathway, they do not establish whether the Dsh-Rac interaction modulates JNK's intrinsic activity. To test this, the amount of phosphorylated JNK relative to total JNK levels in fly brains was evaluated by Western blot analysis using phospho-JNK (P-JNK) and pan-JNK specific antibodies. Then it was determined if Dsh is indeed required for increased levels of JNK phosphorylation. Dsh¹ mutant brains showed a 25% reduction in P-JNK consistent with a stimulatory role for Dsh on JNK signaling. The reduction caused by loss of Dsh function is reversed, when the amount of Rac is reduced by half, consistent with a negative effect of Rac on JNK signaling downstream of Dsh. These data support the conclusion that Dsh and Rac interact to regulate JNK signaling by modulating the phosphorylated active pool of JNK (Srahna, 2006).

Taken together, these data suggest that during brain development DCN axons extend under the influence of JNK signaling. A non-canonical Wnt signal acting via Fz and Dsh ensures that JNK signaling remains active by attenuating Rac activity. In contrast, activation of the FGFR activates Rac1 and suppresses JNK signaling. These data support a model whereby the balance of the Wnt and FGF signals is responsible for determining the number of DCN axons that stably cross the optic chiasm. To test this model, FGFR levels were reduced, using the dominant-negative btl transgene, in dsh¹ heterozygous flies. It was found that simultaneous reduction of FGF and Wnt signaling restored the number of axons crossing the optic chiasm to almost wild-type levels (10.2, with 33% of the samples indistinguishable from wild-type, suggesting that the two signals in parallel, act to control the patterning of DCN axon connectivity (Srahna, 2006).

These data suggest the following model of DCN axon extension and retraction. DCN axons extend due to active JNK signal. These axons encounter Wnt5 and probably Wnt2 as well, resulting in activation of Disheveled. Disheveled, via its DEP domain, has a negative effect on the activity of the Rac GTPase, thus keeping JNK signaling active. After DCN axons cross the second optic chiasm they encounter a spatially regulated FGF/Branchless signal that activates the FGFR/Breathless pathway. Breathless in turn activates Rac, which inhibits JNK signaling in a subset of axons. These axons then retract back toward the lobula. The wide expression of the different components of these pathways and the modulation of JNK phosphorylation by Dsh and Rac in whole-head extracts strongly suggests that this model may apply to many neuronal types (Srahna, 2006).

Branchless and Hedgehog operate in a positive feedback loop to regulate the initiation of neuroblast division in the Drosophila larval brain

The Drosophila central nervous system is produced by two rounds of neurogenesis: one during embryogenesis to form the larval brain and one during larval stages to form the adult central nervous system. Neurogenesis caused by the activation of neural stem division in the larval brain is essential for the proper patterning and functionality of the adult central nervous system. Initiation of neuroblast proliferation requires signaling by the Fibroblast Growth Factor homolog Branchless and by the Hedgehog growth factor. The Branchless and Hedgehog pathways form a positive feedback loop to regulate the onset of neuroblast division. This feedback loop is initiated during embryogenesis. Genetic and molecular studies demonstrate that the absolute level of Branchless and Hedgehog signaling is critical to fully activate stem cell division. Furthermore, over-expression and mutant studies establish that signaling by Branchless is the crucial output of the feedback loop that stimulates neuroblast division and that Branchless signaling is necessary for initiating the division of all mitotically regulated neuroblasts in the brain lobes. These studies establish the molecular mechanism through which Branchless and Hedgehog signaling interface to regulate the activation of neural stem cell division (Barretta, 2008).

These studies have demonstrated that Hh and Bnl act in a positive feedback loop in the larval brain to control the onset of neuroblast proliferation. The feedback loop acts at the transcriptional level, such that Hh signaling activity is essential to control the level of bnl expression and vice versa. Double mutant analyses showed that an absolute level of signaling by both Bnl and Hh are required to maintain normal neuroblast activation, rather than other possible models that would suggest a certain balance of signaling activity (for example more Bnl than Hh) is sufficient regardless of the exact magnitude of signaling activity. The discovery that Bnl signaling is the critical output of the feedback loop suggests that the main function of Hh signaling is to maintain the proper level of Bnl production and signaling. Furthermore, the observation that only the mushroom body and ventral lateral neuroblasts continue to divide in bnl null mutants regardless of the level of Hh signaling indicates that all the regulated neuroblasts, both optic lobe and central brain sets, require the input of the Bnl pathway to enter S phase. Thus the Hh-Bnl feedback loop appears to control cell cycle progression in all the mitotically arrested neuroblasts that begin cell division in first instar (Barretta, 2008).

Other developmental events that require Hedgehog and FGF signaling have used those pathways in different manners to achieve their goals. For example, in the mouse ventral telencephalon, Hedgehog and FGF/MAPK signaling operate as two independent pathways. FGF signaling is independent of Sonic Hedgehog (SHH) and does not affect expression of either SHH itself or its target gene and effector GLI1. Other systems have shown a linear dependence of FGF expression on SHH signaling and vice versa. During budding morphogenesis in the mouse lung Hedgehog signaling inhibits expression of FGF10 but up-regulates FGF7. In the Xenopus eye, expression of Banded Hedgehog increases expression of FGF8. In the zebrafish forebrain inhibition of Hh signaling decreases expression of FGF3, FGF8 and FGF19. Hedgehog also regulates FGF expression in the zebrafish mid/hindbrain. However, in the zebrafish forebrain HH expression requires FGF signaling. Inhibition of both FGF3 and FGF8 expression resulted in a downregulation of SHH. Alternatively, the HH and FGF pathways can integrate at the level of intracellular components. FGF has been shown to induce expression of GLI2, a transcription factor and HH signaling effector in ventroposterior development in zebrafish (Barretta, 2008).

Of course the classic example of FGF and SHH interplay is the development of the chick limb bud. In this system, several FGFs set up a signaling center at the tip of the bud that turns on expression of SHH in the posterior limb mesenchyme. In turn, SHH signaling is required for maintenance of FGF4, FGF9 and FGF17 expression in the bud tip. This function of SHH occurs through the expression of Gremlin, an inhibitor of Bone Morphogenetic Protein signaling. Gremlin inhibition of Bone Morphogenetic Protein signaling prevents down-regulation of the FGFs. Thus a positive feedback loop exists between SHH and FGFs, mediated by Gremlin (Barretta, 2008).

The model of the Hh-Bnl feedback loop proposed in this study is most similar to the classic SHH-FGF feedback loop described in the vertebrate limb bud. In is not yet known whether the regulation of bnl expression by Hh signaling is direct or if it is mediated by another signaling pathway such as the Gremlin/Bone Morphogenetic Protein connection that operates in the limb bud. However, like the distinct domains of FGF and SHH in the limb bud, bnl and hh expression also occur in distinct regions of the brain lobe. The fact that the Hh-Bnl feedback loop is activated during embryogenesis, but that the first regulated neuroblasts do not enter S phase until 8-10 h after larval hatching also suggests that additional events must take place downstream of Bnl signaling to permit mitotically arrested stem cells to transit through G1 to S phase. One such possibility is exposure to the steroid hormone ecdysone, which is necessary during first larval instar for the initiation of neuroblast division a few hours later. Both SHH and FGF2 have been shown to be necessary for the division of different subsets of neural stem cells in many different vertebrate and mammalian models and in multiple contexts. This is the first time that the interactions between these two pathways necessary to stimulate the reactivation of stem cell division in quiescent neural stem cells have been elucidated. The next challenge will be to determine whether different molecular mechanisms tying these two signaling pathways are used for different developmental decisions such as progeny cell fate, initiation of cell division and maintenance of stem cell identity (Barretta, 2008).

EFFECTS OF MUTATION

branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and pattern of branching

branchless was identified as a P transposon-induced mutation that reduces or eliminates tracheal branching, just like breathless mutation. At stage 16 in wild-type embryos, primary branches have budded and grown out from the tracheal sacs; secondary and some terminal branches have formed, and branch fusion has taken place to form the dorsal and lateral tracheal trunks. In branchless mutants, none of these events occur normally: almost every tracheal metamere appears as an unbranched, elongated sac of tracheal cells. Tracheal cells invaginate and form tracheal sacs normally in branchless mutants, but branches fail to grow out. As with breathless mutants, specific defects appear in the central nervous system (Sutherland, 1996).

Study of the posterior spiracles of Drosophila as a model to understand the genetic and cellular mechanisms controlling morphogenesis

The development of the posterior spiracles of Drosophila may serve as a model to link patterning genes and morphogenesis. A genetic cascade of transcription factors downstream of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic mechanisms. The inner cells that give rise to the spiracular chamber invaginate by elongating into 'bottle-shaped' cells. The surrounding cells give rise to a protruding stigmatophore by changing their relative positions in a process similar to convergent extension. In the larvae the spiracular chamber forms a very refractile filter, the filzkorper. The opening of the spiracular chamber, the stigma, is surrounded by four sensory organs; the spiracular hairs. Clones labeling the spiracular hairs show that each one is formed by four cells related by lineage, two neural and two support cells, the typical structure of a type I external sensory organ. When the larva is buried in the semi-liquid medium on which it feeds, the stigmatophore periscopes out of the medium allowing the larva to continue breathing. The genetic cascades regulating spiracular chamber, stigmatophore, and trachea morphogenesis are different but coordinated to form a functional tracheal system. In the posterior spiracle, this coordination involves the control of the initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly. As a result, the opening of the tracheal system shifts back from the spiracular branch of the trachea into the posterior spiracle cells (Hu, 1999).

The connection of the posterior spiracle to the trachea is a regulated event. In mutants for the Drosophila FGF and FGF-receptor homologs branchless and breathless the tracheal pits do invaginate, but since they do not migrate toward one another, they do not form a continuous network. In contrast, in btl mutants, the posterior spiracle connects normally to the A8 spiracular branch of the trachea. In mutants for Abd-B the stigma of A8 does not slide posteriorly, but stays in the same position as in anterior abdominal segments, where the spiracular branch attaches to the outside epidermis. The contribution of the ems gene to coordination of morphogenetic movements has been examined. The spiracle-trachea connection occurs in cut and sal mutants but not in ems mutants. In ems mutants, invagination of the spiracle cells adjacent to the trachea does not occur, but more posterior cells of the spiracle invaginate normally. The elongation does not occur simultaneously in all cells, but starts in the more anterior ones and, in general, the invaginating cells keep contact with the external surface of the embryo. This results in the cells that have invaginated earlier being deeper in the spiracular chamber and more elongated. The defective invagination in ems mutants results in a spiracle without a lumen and with the tracheal opening located outside it. The results show that cell elongation and formation of a lumen are two independently controlled processes. The spiracles provide a good model for the study of cellular and molecular mechanisms controlling cell shape and cell rearrangements, two mechanisms which are used during the morphogenesis of a variety of organisms (Hu, 1999).

Rac promotes epithelial cell rearrangement during tracheal tubulogenesis in Drosophila

Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested it to be a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. The tracheal system of Drosophila was used to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. A reduced Rac activity leads to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibits dynamic epithelial cell rearrangement. In contrast, hyperactivation of Rac inhibits assembly of newly synthesized E-Cadherin into cell junctions and causes loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules (Chihara, 2003).

p21-activated kinase (Pak) is known as a mediator of the activity of Rac GTPase. Tracheal defects similar to those of Rac1, 2 mutants are found in pak mutants. Furthermore, Rac1, 2 and Pak mutations synergistically enhance tracheal defects. Such results suggest that Rac and Pak are required for directed movement of tracheal branches (Chihara, 2003).

The loss of Rac activity also causes a defect in cell differentiation. Tips of dorsal branch 1-9 are normally capped with terminal cells that extend terminal branch in the ventral direction. In Rac 1, 2 mutant embryos, the loss of terminal branches was observed with high penetrance. Consistently, serum response factor (SRF), a marker protein for the terminal cell, also disappears, suggesting that terminal cell differentiation does not occur (Chihara, 2003).

Since directed cell migration and terminal cell differentiation are processes requiring FGF signaling, it was asked whether Rac is involved in FGF signaling (a strong genetic interaction). Although tracheal patterning is only mildly affected by half dose reductions of bnl (ligand), btl (receptor) and dof (intracellular effector), the phenotype is strongly enhanced by introducing one copy of Rac1, 2 mutant chromosome from mothers. A similar genetic interaction was found between pak and bnl. These genetic interactions suggest that Rac and Pak are required for the migration of tracheal branches in response to FGF signaling (Chihara, 2003).

To determine the epistatic relationship between Rac and FGF signaling, the effect of constitutive activation of Rac was tested in btl mutants. In the btl mutant, tracheal branching does not proceed beyond the invagination at stage 11, and MAP kinase activation is absent (Chihara, 2003).

Expression of Rac1V12 partially restores the movement of tracheal cells, and activates MAP kinase, as revealed by staining with the antibody against the diphosphorylated form of MAP kinase (dp-MAPK). These results suggest that Rac activation is an essential downstream event of tracheal cell motility induced by FGF signaling (Chihara, 2003).

Extracellular signals that promote tracheal branching are good candidates for regulators of Rac in tracheal cells. In this regard, the strong genetic interaction between Rac and FGF signaling components observed suggests an intriguing possibility that FGF signaling activates Rac within tracheal cells to promote both cell motility and cell rearrangement. In support of this idea, it was found that activated Rac 1 partially rescues tracheal cell motility and MAP kinase activation in btl mutants. Involvement of Rac in FGF-dependent events may not be limited to cell motility. Expression of SRF, the product of one of the target genes activated by FGF signaling in the tracheal system, is lost in the mutant trachea with reduced Rac activity because of Rac 1, 2 mutation or Rac 1N17. This result suggests that Rac also regulates transcription (Chihara, 2003).

Several lines of evidence suggest that FGF signaling is activated locally at the tip of branches, and activation of FGF signaling in all tracheal cells prevents branching, suggesting that localized activation of FGF signaling is essential for branching. Therefore the proposed function of Rac in transducing FGF signaling must be localized at the tip of branches. How does the proposed function of Rac in transducing FGF signaling relate to the Rac function in regulating cell rearrangement? Since the effect of Rac 1N17 is most clearly observed in cells destined to become tracheal stalk cells, the location of tracheal cells requiring two of the Rac functions appears to be different. One idea is that FGF signaling activated at the tracheal tip is transmitted to tracheal stalk cells by a secondary signal that activates Rac to promote cell rearrangement. It will be important to identify the upstream signal regulating Rac in stalk cells (Chihara, 2003).

Grainy head controls apical membrane growth and tube elongation in response to Branchless/FGF signalling

Epithelial organogenesis involves concerted movements and growth of distinct subcellular compartments. Apical membrane enlargement is critical for lumenal elongation of the Drosophila airways, and is independently controlled by the transcription factor Grainy head. Apical membrane overgrowth in grainy head mutants generates branches that are too long and tortuous without affecting epithelial integrity, whereas Grainy head overexpression limits lumenal growth. The chemoattractant Branchless/FGF induces tube outgrowth -- it upregulates Grainy head activity post-translationally, thereby controlling apical membrane expansion to attain its key role in branching. A two-step model for FGF in branching is favored: first, induction of cell movement and apical membrane growth, and second, activation of Grainy head to limit lumen elongation, ensuring that branches reach and attain their characteristic lengths (Hemphälä, 2003).

Bnl is the key morphogen co-ordinating branching that acts via the receptor tyrosine kinase Breathless (Btl) and the adaptor protein Dof/Stumps. This pathway leads to phosphorylation and activation of MAPK, which in turn may alter the activity of regulatory proteins to control cell behavior. During primary branching, actin-rich basal extensions are sent by the tracheal cells towards the sources of Bnl, a process that is likely to involve cytoskeletal modulation by the Rho family GTPases. Bnl signalling is also required for the expression of cell-fate determining genes in specific subsets of tracheal cells in each primary branch. Analysis of these genes has identified key components of the patterning and guidance of the unicellular secondary and terminal branches. However, the role of Bnl in the movement of the cell bodies and the growth of the branch lumen remains unknown (Hemphälä, 2003).

One possible mechanism for regulation of Grh activity is through Bnl signalling, which is instrumental in the formation and extension of all tracheal branches. Initially, it was established that apical cell surface growth is an intrinsic component of Bnl-induced tube extension, by combining alleles of grh and bnl. This revealed that a subset of the branch outgrowth defects seen in embryos that carry only one copy of the bnl gene are partially rescued by a reduction in grh function (grh^s2140/grh^s2140; bnl^P1/+). Thus, in embryos heterozygous for bnl, 40% of the ganglionic branches fail to reach the CNS, whereas the simultaneous removal of grh restores this phenotype so that 78% of the branches now enter the CNS. These data therefore show that Grh-mediated modulation of the apical cell surface has an active inhibitory role on Bnl-induced branch extension (Hemphälä, 2003).

In order to analyse whether tracheal Grh activity could be targeted by Bnl/Btl signal transduction, GBE-lacZ expression was analyzed in embryos with altered levels of Bnl and Btl activity. When Bnl is ectopically expressed in all tracheal cells, GBE-lacZ expression becomes significantly upregulated, although the levels of Grh protein are not altered. This suggests that Bnl controls Grh activity post-translationally, and surprisingly, upregulates the expression of this artificial Grh target. Nevertheless, the effects of Btl appear specific since with more limited Bnl expression using the Term-Gal4 driver, GBE-lacZ expression becomes enhanced specifically in the cells that respond to Bnl by ectopically expressing the terminal marker DSRF. Similar enhancement of GBE-lacZ expression is evident upon tracheal expression of an activated form of the Btl receptor itself (UASBtl-Tor). In all instances the augmented GBE-lacZ expression is dependent on Grh, since embryos that express ectopic Bnl or the activated form of Btl, but lack Grh activity, do not express GBE-lacZ. Furthermore, ectopic activation of Dpp, another signalling pathway that promotes the growth of dorsal and ganglionic branches during tracheal development, has no effect on GBE-lacZ, indicating that the effects on GBE-lacZ are specific for Bnl/Btl (Hemphälä, 2003).

Whether Bnl signalling is a prerequisite for the transcriptional activity of Grh was tested by analysing the levels of GBE-lacZ expression in mutants for bnl, btl or pointed (pnt). Tracheal GBE-lacZ expression is both reduced and uniform in bnl and btl mutant embryos, but is unchanged in pnt embryos that lack the activity of a downstream transcriptional effector of the ETS family. Since Grh is a substrate for activated MAPK (ERK2) in vitro, its activity could be modulated directly during branching by Bnl-induced phosphorylation. This would account for the fact that GBE-lacZ expression is affected by mutations in bnl and btl, but not by mutations in the nuclear effector pnt (Hemphälä, 2003).

It is concluded that Bnl signalling converts Grh to a more potent activator of its GBE-lacZ target. Since Grh becomes phosphorylated by MAPK in vitro, and MAPK is a downstream effector of Btl signal transduction, the alteration in Grh activity may be brought about by MAPK-mediated phosphorylation of the Grh protein (Hemphälä, 2003).

Currently, two ways of explaining the biological consequence of the regulation of Grh have been suggested. In the first model, the regulation of Grh by Bnl increases its activity, and thereby delimits lumen growth. This invokes a hierarchical two step function for Bnl in which it first promotes branching and tube elongation and it then activates Grh to halt excess apical surface growth and establish a functional lumen. In this model active restriction of morphogenetic processes is required to achieve stereotyped tube dimensions and is an intrinsic part of the program that induces branching morphogenesis. In the second model, regulation by Bnl has differential consequences on Grh, activating some functions (like the one necessary for GBE-lacZ expression) and inactivating others, necessary for inhibiting apical membrane growth. In this model, high levels of Btl signalling would temporarily inactivate Grh, in order to allow for apical membrane expansion during the process of branch extension. Both models are consistent with the genetic interactions, which indicate an antagonistic relationship between grh and bnl, and add the control of apical membrane growth to the repertoire of cellular activities regulated by FGF signalling during morphogenesis (Hemphälä, 2003).

Of the two models, the former, where Btl coordinates branching through a sequence of activities, is currently favored since this model is consistent with the activation of the GBE-lacZ reporter. It can also be well integrated with the apical overgrowth phenotype of grh mutants, which becomes apparent first in the branches that have reached their final length and only after the completion of branch elongation at stage 16. If Grh were acting to restrict membrane growth continuously, the grh mutant phenotype would be expected to appear at earlier stages. A two step model could also explain the inhibiting effect on tube elongation that is seen upon expression of activated forms of Btl receptors in all tracheal cells of wild-type embryos (Hemphälä, 2003).

Drosophila Perlecan modulates FGF and Hedgehog signals to activate neural stem cell division

Mutations in the Drosophila terribly reduced optic lobes (trol) gene cause cell cycle arrest of neuroblasts in the larval brain. trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF (Branchless) and Hedgehog (Hh) signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system (Park, 2003).

Trol appears to display functions similar to mammalian Perlecans, which are known to bind FGF-2 and to be required for FGF signaling. Dominant enhancement of the neuroblast proliferation phenotype of two different trol alleles has been observed with mutations in bnl and the Bnl receptor breathless (btl), but not with mutations in the orphan heartless (htl) receptor. The neuroblast proliferation phenotype of trol⁸ mutant brains was rescued in culture to control levels by addition of human FGF-2. Addition of the MAPK inhibitor PD98059 at 10 hs post-hatching decreased the number of S-phase neuroblasts. Biochemical analysis has shown that FGF-2 can be coimmunoprecipitated with Trol and that the binding of FGF-2 to Trol can be competed by added heparin. This suggests that, like mPerlecan, Trol binds FGF-2 through heparan sulfate residues. These results demonstrate that Trol-mediated FGF signaling is required for initiation of neuroblast proliferation sometime in first larval instar. This similarity to the function of mPerlecans in mammalian FGF signaling and the implications of up-regulation of mPerlecan in tumors strongly imply that trol encodes a functional Drosophila Perlecan homolog (Park, 2003).

Social interactions among epithelial cells during tracheal branching morphogenesis

Many organs are composed of tubular networks that arise by branching morphogenesis in which cells bud from an epithelium and organize into a tube. Fibroblast growth factors (FGFs) and other signalling molecules have been shown to guide branch budding and outgrowth, but it is not known how epithelial cells coordinate their movements and morphogenesis. Genetic mosaic analysis has been used in Drosophila to show that there are two functionally distinct classes of cells in budding tracheal branches: cells at the tip that respond directly to Branchless FGF and lead branch outgrowth, and trailing cells that receive a secondary signal to follow the lead cells and form a tube. These roles are not pre-specified; rather, there is competition between cells such that those with the highest FGF receptor activity take the lead positions, whereas those with less FGF receptor activity assume subsidiary positions and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibition that prevents extra cells from assuming the lead. There may also be cooperation between budding cells, because in a mosaic epithelium, cells that cannot respond to the chemoattractant, or respond only poorly, allow other cells in the epithelium to move ahead of them (Ghabrial, 2006).

The Drosophila tracheal system develops from epithelial sacs of about 80 cells from which primary, secondary and terminal branches sprout without cell division or cell death. Primary branch sprouting is induced by Branchless (Bnl) FGF, a chemoattractant secreted by clusters of cells surrounding each sac, which activates Breathless (Btl) FGF receptor (FGFR), a receptor tyrosine kinase expressed on tracheal cells. Primary branches contain 3-20 cells that organize into a tube as they migrate out from the sac. Bnl also induces the expression of secondary branching genes, such as the transcription factor pointed (pnt), and specifies terminal cells at the ends of outgrowing branches. Terminal cells ramify in the larva in response to Bnl to form fine terminal branches. Other cells at the ends of primary branches become fusion cells that connect with neighbouring branches to form a continuous tracheal network. Terminal and fusion cell fate decisions are also influenced by the Notch, Dpp and Wingless signalling pathways. Dorsal branches, the primary branches that were focused on here in this study, typically consist of five or six cells: two cells near the branch tip, one (DB1) that becomes a terminal cell and another (DB2) that becomes a fusion cell, and three or four cells (DB3-DB6) that form the branch stalk (Ghabrial, 2006).

In a genetic mosaic screen, six mutants (724, 788, 1118, 1187, 1476 and 1684) were identified with a subtle phenotype: mosaic branches (+ / + , +/ - , -/ - cells) were grossly normal, yet homozygous mutant clones (- / - cells) rarely if ever included terminal cells. These were neither general nor terminal-cell-specific lethal mutations because homozygous mutant cells were readily recovered in all other tracheal positions, and there was no decrease in the overall number of cells in mosaic dorsal branches or the number of terminal cells. It was difficult to imagine how mutations could block clone generation in specific cells. It seemed more likely that the mutations caused cells otherwise destined to become terminal cells to switch fates with other tracheal cells (Ghabrial, 2006).

The six mutations compose a single lethal complementation group that mapped to the left arm of chromosome 3 and failed to complement breathless^LG18. DNA sequencing identified a single nucleotide change in each mutant resulting in a nonsense or missense mutation in btl. Five mutations (724, 788, 1118, 1476 and 1684) appear to be null btl mutations, whereas the sixth mutation (1187) causes partial loss of function. Thus, the 'no mutant terminal cells' gene is btl (Ghabrial, 2006).

The distribution of cells homozygous was quantified for btl null mutations (724 and 788), or homozygous for a wild-type btl allele as a control, in mosaic dorsal branches. Control clones were evenly distributed throughout the branch at the expected frequencies; for example, the ratio of stalk-cell to terminal-cell clones was about 3:1. By contrast, btl^-/- cells showed a nearly complete bias against the DB1 position: the ratio of stalk-cell to terminal-cell clones was 51:1. The three exceptional mutant terminal cells may be cases in which the clone was induced after btl began to be expressed, allowing wild-type btl gene products to perdure in mutant cells. Hundreds of mosaic branches with one or more btl^-/- cells present in positions DB2-DB6 were recovered without affecting cell or branch morphology. Indeed, branches composed largely or exclusively of btl^-/- cells, except for a wild-type terminal cell, were morphologically indistinguishable from wild-type branches. Thus, although all tracheal cells normally express btl, and the receptor is activated by Bnl in most or all cells of budding branches, the receptor appears to be required in just a single leading cell (DB1). All other cells can migrate normally and form tubes in the absence of btl. It is concluded that there are two functionally distinct classes of cells in budding primary branches: lead cells, which require Btl FGFR and directly respond to Bnl FGF, and trailing cells, which do not require Btl but follow the lead cell and form the stalk (Ghabrial, 2006).

What does it take to become the leader? The lead cell (DB1) is specified to become a terminal cell by Bnl-Btl signalling. If terminal cell specification is required, then null mutations in the downstream gene pnt, which abolish this function, should have the same effect as btl mutations. Cell clones homozygous for pnt^Delta88 or two new pnt alleles isolated in the screen (198 and 1318) failed to develop as terminal cells, as expected. However, unlike btl mosaic branches, pnt mosaic branches often lacked a terminal cell. When a terminal cell was missing, there was usually a pnt^-/- cell in the stalk position nearest the tip, presumably the DB1 cell that failed to differentiate into a terminal cell. This suggests that pnt^-/- cells are able to assume the lead position but fail to differentiate as terminal cells, and that the bias against btl mutant terminal cells is due to the earlier, pnt-independent, function of Btl in primary branch budding and outgrowth. If cells lacking Btl cannot migrate in response to Bnl during budding, they should not be able to move to the lead position necessary to be selected as a terminal cell. Consistent with this, genetic mosaic analysis of stumps (dof/heartbroken), which encodes a Btl adaptor required for cell migration, showed a dearth of terminal cell clones similar to btl (Ghabrial, 2006).

Two results demonstrate that the ability to sense Bnl and migrate in response to it is not enough to become the leader: cells compete for the lead position. The first involves btl^BN (E796K mutation in the kinase domain), a weak btl allele isolated in a separate screen. Unlike btl^-/- animals, which die in first larval instar and lack virtually all branches, btl^BN homozygotes survived until L3 larval stage or beyond and had a normally patterned tracheal system with a full complement of terminal cells. The only defects detected were a reduced number and altered morphology of terminal branches, presumably due to the dosage-sensitive function of btl in terminal branch outgrowth. The late and subtle phenotype demonstrates that Btl^BN protein retains sufficient activity for early migration and terminal cell specification events. However, in genetic mosaic animals, in which btl^BN/BN cells must compete with btl^BN/+ and btl^+/+ tracheal cells, btl^BN/BN cells rarely acquired the lead position (DB1) and developed as terminal cells. Indeed, homozygosity for btl^BN conferred nearly as complete a bias against becoming a terminal cell as total loss of btl. Thus, Btl activity above the threshold necessary for migration and terminal cell specification is not sufficient to acquire the lead position and become a terminal cell: a cell must have more Btl activity than other cells in the branch (Ghabrial, 2006).

Similar conclusions derive from a second experiment in which marked wild-type (btl^+/+) cells were analysed in heterozygous (btl^+/-) animals. Whereas btl^+/+ clones in control (btl^+/+) animals were distributed evenly throughout the branch, btl^+/+ clones in btl^788/+ heterozygotes preferentially localized to the tip. Cells that did not occupy the lead (DB1) position took positions close to the tip. Similar results were obtained for btl^+/+ clones in animals heterozygous for btl¹¹⁸⁷, a partial-loss-of-function allele. Clones mutant for sprouty, an FGF feedback inhibitor, also preferentially populated the tip. Together, the data show that there is competition for the lead position: cells with highest btl activity assume positions at or near the tip of the branch, whereas those with less or no activity segregate towards its base (Ghabrial, 2006).

Because small differences in btl dosage or activity affect a cell's ability to compete for the lead, whether lateral inhibitory mechanisms that amplify small differences in signalling might be operative was investigated. Data suggest that the Notch pathway, a lateral signalling pathway implicated in cell specification events including cell fate determination at tracheal branch tips, also affects cell arrangement. N^ts embryos shifted to the restrictive temperature during budding formed branches in which most DB cells behaved like lead cells, resulting in large clusters of cells congregated at the lead position, whereas expression of constitutively active N^ACT throughout the tracheal epithelium had the opposite effect, arresting outgrowth and stalling cells near the base of the branch. It is proposed that Notch-mediated lateral inhibition among tracheal cells prevents extra cells from assuming the lead position (Ghabrial, 2006).

These results provide evidence for social stratification and dynamic social interactions between epithelial cells during branching morphogenesis. First, the results show that budding cells are functionally specialized. A cell at the branch tip requires btl and leads outgrowth towards the Bnl signalling center. Trailing cells do not require btl but nevertheless follow the lead cell towards the Bnl source. Because tracheal cells do not migrate or form tubes in btl^-/- animals, trailing cells must receive a secondary signal generated by the lead cell that induces them to migrate and also activates their tubulogenesis program. This could be a secreted molecule or physical stimulus such as pulling or stretching the trailing cells (Ghabrial, 2006).

Second, these roles are not pre-specified. Rather, there is competition between cells such that those with high Btl FGFR activity become lead cells whereas those with less or no btl FGFR activity become trailing cells and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibitory signalling between tracheal cells, and it may also be influenced by positive feedback mechanisms such as increased activation and expression of Btl as cells approach the Bnl source. Third, there may be cooperation between cells, because in a genetically mosaic epithelium, tracheal cells with less Btl activity allow those with more activity to move ahead of them (Ghabrial, 2006).

There may be similar social interactions between budding cells in other branching organs. Studies of other branching processes have identified genes selectively expressed in tip cells of budding branches, and in some cases these cells display morphological specializations indicating that they might actively lead outgrowth. However, because most budding branches contain hundreds or thousands of cells, it is difficult to track and manipulate individual cells to investigate social behaviours like those described here. Recent analyses of chimaeric Ret⁺/Ret^- mouse renal ureteric buds in culture and btl mosaic air sacs reveal that cells lacking these receptor tyrosine kinases are excluded from branch tips, indicating that RTK-dependent interactions similar to those described here might be operative in more complex branching events (Ghabrial, 2006).

FGF ligands in Drosophila have distinct activities required to support cell migration and differentiation

Fibroblast growth factor (FGF) signaling controls a vast array of biological processes including cell differentiation and migration, wound healing and malignancy. In vertebrates, FGF signaling is complex, with over 100 predicted FGF ligand-receptor combinations. Drosophila presents a simpler model system in which to study FGF signaling, with only three ligands and two FGF receptors (FGFRs) identified. This study analyzed the specificity of FGFR [Heartless (Htl) and Breathless (Btl)] activation by each of the FGF ligands [Pyramus (Pyr), Thisbe (Ths) and Branchless (Bnl)] in Drosophila. It was confirmed that both Pyr and Ths can activate Htl, and that only Bnl can activate Btl. To examine the role of each ligand in supporting activation of the Htl FGFR, genetic approaches were utilized that focus on the earliest stages of embryonic development. When pyr and ths are equivalently expressed using the Gal4 system, these ligands support qualitatively different FGFR signaling responses. Both Pyr and Ths function in a non-autonomous fashion to support mesoderm spreading during gastrulation, but Pyr exhibits a longer functional range. pyr and ths single mutants exhibit defects in mesoderm spreading during gastrulation, yet only pyr mutants exhibit severe defects in dorsal mesoderm specification. This study demonstrated that the Drosophila FGFs have different activities and that cell migration and differentiation have different ligand requirements. Furthermore, these FGF ligands are not regulated solely by differential expression, but the sequences of these linked genes have evolved to serve different functions. It is contended that inherent properties of FGF ligands make them suitable to support specific FGF-dependent processes, and that FGF ligands are not always interchangeable (Kadam, 2009).

These experiments demonstrate that the Drosophila FGFs Pyr, Ths and Bnl have different functions and that the activation of FGF receptors by specific ligands affects particular biological processes. Examination of an allelic series of pyr and ths mutants suggests that pyr and ths are not redundant in function: both influence mesoderm spreading, whereas pyr is the dominant player controlling Eve⁺ cell specification within the dorsal mesoderm. It has been demonstrated that ectopic expression of ths by twist-Gal4 and 69B-Gal4 in the Df(2R)BSC25 mutant background can support Htl FGFR activation. However, this study assayed whether the expression supported in distinct domains would support Htl activation. By a series of 'rescue' experiments, through ectopic expression of one ligand in the Df(2R)BSC25 mutant background, evidence was obtained that localized expression of the ligands is important for proper mesoderm spreading. It was found, surprisingly, that the ligands exhibit differences in their functional range of action. In addition, using this same approach, it was found that either Pyr or Ths can support Eve⁺ cell specification within the dorsal mesoderm, but that Bnl cannot. Collectively, these data suggest that the Pyr and Ths FGFs function as ligands for the Htl FGFR and that specificity of FGF-FGFR interactions exists in Drosophila (Kadam, 2009).

The results demonstrate that both Pyr and Ths FGF ligands can activate the Htl FGFR, whereas only the Bnl FGF ligand can activate the Btl FGFR. Specificity of FGFR activation was observed: pyr or ths, but not bnl, expression is able to activate Htl to affect expression of Eve, and bnl, but neither pyr nor ths, is able to support tracheal specification. No evidence was obtained that other cross-interactions occur (i.e. Pyr-Btl, Ths-Btl or Bnl-Htl), which demonstrates that Gal4-mediated ectopic expression does not simply 'swamp the system'. This experimental approach also 'levels the playing field', since expression of each ligand is driven at the same time and place and presumably at similar levels. It is concluded that only three FGF-FGFR combinations function in Drosophila (i.e. Pyr-Htl, Ths-Htl and Bnl-Btl), which supports the idea that FGFRs exhibit ligand-binding preferences. Previous studies have investigated FGF signaling specificity by analyzing the ability of other receptor tyrosine kinases to support cell migration or by activating particular intracellular signaling pathways to examine which are required to effect FGFR-dependent cell migration versus cell differentiation. This work analyzed the specificity of FGF ligand-receptor interactions and how they contribute to particular developmental processes (Kadam, 2009).

When ligand expression is supported by twist-Gal4, Htl FGFRs presumably become saturated because dpERK is ectopically activated in all cells and spreading is negatively affected. One explanation for why this might affect mesoderm cell spreading is that these FGF-saturated mesoderm cells may no longer be competent to respond to endogenous ligands that provide directional cues. Recently, it has been shown that movement of the mesoderm cells during gastrulation is in fact directional (McMahon, 2008). Pyr and Ths ligands are differentially expressed during gastrulation and this might provide the necessary positional information required to direct migration of the mesoderm. It is proposed that Pyr and Ths have different activities that fulfil aspects of FGFR activation required to support cell migration. Ectopic expression of Pyr within the ectoderm negatively affects mesoderm spreading, which suggests that the refined expression domain of pyr within cells of the dorsal ectoderm is normally required to guide the mesoderm cells toward dorsal regions. However, even though ectopic expression of ths in the ectoderm has no effect on mesoderm spreading, ths mutants also exhibit defects in mesoderm spreading, demonstrating that both genes are required, perhaps to control different aspects of the migration. The 'rescue' experiments using the zenVRE.Kr-Gal4 driver support the view that Pyr has a longer functional range than Ths. These differences in range of function might correlate with different diffusion capabilities, but an alternative explanation is that the ligands activate the receptor with different affinities. Additional experiments will be necessary to distinguish their exact functions and to uncover the molecular basis for the differential functions of Pyr and Ths; it is suggested that in vivo imaging and quantitative analysis (McMahon, 2008) of single-mutant phenotypes will provide insights (Kadam, 2009).

With regard to the FGF-dependent cell differentiation, the 'rescue' experiments suggest that ectopic expression of either Pyr or Ths is sufficient to support Eve⁺ cell specification. The reason why loss of ths has less of an effect on Eve⁺ cell specification is most likely because pyr is prominently expressed in the vicinity of the future Eve⁺ cells; normally, Pyr supports this function, but Ths can support this activity if presented at sufficient levels within the correct domain. Furthermore, it is proposed that FGF signaling might not play an instructive role in supporting eve expression. Other signaling pathways already provide positional information required for the specification of Eve⁺ cells; FGF signaling pathway activation might simply serve a permissive role, and in this context either ligand would suffice (Kadam, 2009).

Mitotic cell rounding accelerates epithelial invagination

Mitotic cells assume a spherical shape by increasing their surface tension and osmotic pressure by extensively reorganizing their interphase actin cytoskeleton into a cortical meshwork and their microtubules into the mitotic spindle. Mitotic entry is known to interfere with tissue morphogenetic events that require cell-shape changes controlled by the interphase cytoskeleton, such as apical constriction. However, this study shows that mitosis plays an active role in the epithelial invagination of the Drosophila tracheal placode. Invagination begins with a slow phase under the control of epidermal growth factor receptor (EGFR) signalling; in this process, the central apically constricted cells, which are surrounded by intercalating cells, form a shallow pit. This slow phase is followed by a fast phase, in which the pit is rapidly depressed, accompanied by mitotic entry, which leads to the internalization of all the cells in the placode. It was found that mitotic cell rounding, but not cell division, of the central cells in the placode is required to accelerate invagination, in conjunction with EGFR-induced myosin II contractility in the surrounding cells. It is proposed that mitotic cell rounding causes the epithelium to buckle under pressure and acts as a switch for morphogenetic transition at the appropriate time (Kondo, 2013).

The invagination of epithelial placodes converts flat sheets into the three-dimensional structures that form complex organs, and it is a key morphogenetic process in animal development. A major mechanism of invagination is apical constriction, which is driven by actomyosin contraction. However, not all constricted cells invaginate, and some cell internalization occurs without apical constriction, suggesting that additional mechanisms of inward cell movement contribute to invagination (Kondo, 2013).

To obtain three-dimensional information about cell behaviour during invagination, live imaging was performed of the Drosophila tracheal placode. Ten pairs of tracheal placodes, each of which is composed of about 40 cells, are formed in the ectoderm at mid-embryogenesis, and each placode initiates invagination simultaneously. Using an adherens junction marker, DE-cadherin-green fluorescent protein (E-cad-GFP), it was found that the adherens junctions of the central placode cells slowly created a depression by apical constriction, which became the tracheal pit. After 30 to 60 min of slow movement (slow phase), the tracheal pit was suddenly enlarged, and the tracheal cells were rapidly internalized (fast phase) and eventually formed L-shaped tube structures (Kondo, 2013).

After the fast transition, all the tracheal cells and surrounding epidermal cells entered mitosis 16, the final round of embryonic mitosis. It was noticed that the fast invagination was always associated with the mitotic entry of central cells that were frequently the first to enter mitosis 16. Intriguingly, mitotic rounding of the central constricted cells occurred simultaneously with the rapid depression of their apices, followed by chromosome condensation 10 min later. In this study, this atypical mitotic rounding associated with apical depression in an internalized cell is called 'rounding', to distinguish it from canonical surface mitosis (surface cell rounding) (Kondo, 2013).

To determine whether cell rounding is required for invagination, zygotic mutants were examined of the cell-cycle gene Cyclin A (CycA), which fail to enter mitosis 16, and double parked^a3 (dup^a3), which show a prolonged S phase 16 and delayed entry into mitosis 16. Tracheal invagination was initiated normally in the CycA and dup^a3 mutants, but proceeded more slowly than in controls, indicating that entry into mitosis 16 is required for proper timing of the fast phase (Kondo, 2013).

Although delayed, the accelerated invagination in the CycA or dup^a3 mutants eventually occurred, allowing the formation of tube structures and suggesting that additional mechanisms are involved. After invagination, fibroblast growth factor (FGF) signalling is activated in the tracheal cells to induce branching morphogenesis through chemotaxis. To examine the contribution of FGF signalling to invagination, mutants of the FGF ligand branchless (bnl) or the FGF receptor breathless (btl) were analyzed. These mutants invaginated normally, indicating that chemoattraction to FGF is dispensable for invagination (Kondo, 2013).

Next, to assess FGF's role in the mitosis-defective condition, double mutants were analyzed for CycA and bnl or CycA and btl, and it was found that they showed slower invagination than CycA single mutants. Furthermore, the invagination in these double mutants was incomplete, in that the cells failed to form L-shaped tubular structures. Therefore, FGF signalling is critical for invagination when mitosis is blocked, serving a back-up role. Tracheal-specific CycA expression rescued the defects in invagination speed and tube structure in the CycA btl mutants. In addition, mitosis of cells outside the pit was occasionally observed that occurred before the mitosis of the central apically constricted cells and was not correlated with the fast invagination phase. Thus, mitosis of the surrounding epidermal cells is dispensable for tracheal invagination. Taken together, it is concluded that mitotic entry of central cells is a major mechanism for accelerating tracheal invagination (Kondo, 2013).

To distinguish the role of cell rounding from that of cell division in the fast phase, the microtubule inhibitor colchicine was used to arrest the cell cycle after cell rounding. Colchicine treatment after mitosis 15 induced M-phase arrest at mitosis 16, but the fast invagination movement accompanied by cell rounding was not affected. This result indicates that cell rounding, but not cell division, is responsible for the acceleration phase of the tracheal invagination (Kondo, 2013).

Mitosis of cells in the columnar epithelium normally occurs at the apical surface after surface rounding. It was next asked how the apical surface of the central cells becomes depressed during internalized cell rounding. One possible model explains internalized cell rounding as cell-autonomously controlled by the association of the cells with the basement membrane or underlying mesodermal cells. However, genetic removal of basement-membrane adhesion by the maternal and zygotic mutation of βPS-integrin (also known as mys) did not compromise the speed of invagination, and snail-twist double-mutant embryos, which lack mesodermal cells, still showed tracheal invagination with internalized cell rounding. These results suggest that anchoring to the basal side is probably not required (Kondo, 2013).

A second model proposes that the apical depression of the rounding cells is driven by local planar interactions among the tracheal cells. Before and during tracheal invagination, myosin II is enriched at the cell boundaries tangential to the centre of the placode and regulates cell intercalation. It was noted that the myosin II level in the central cells was lower than in the surrounding, intercalating cells. Nevertheless, the apices of the central cells were constricted during the slow phase, strongly suggesting that the surrounding cells exerted centripetal pressure on the central cells through myosin II cables. Myosin II cables fail to form in EGFR signalling mutants (such as rho, the rhomboid endopeptidase required for EGF ligand maturation, and Egfr), and apical constriction is impaired in these mutants. The first few cells undergoing mitosis 16 in the tracheal placode of rho or Egfr mutants showed surface cell rounding with expanded apices, indicating that EGFR signalling is required to couple the mitotic cell rounding with fast apical depression. It is speculated that the columnar shape of the central cells resists centripetal movements, resulting in the accumulation of inward pressure during the slow phase. The existence of such resistance was supported by the results of a physical perturbation experiment using a pulsed ultraviolet lase. The cell rounding associated with mitotic entry would release the stored inward pressure by means of cytoskeletal remodelling that causes rapid depression of apical surface together with the active shortening of cell height, leading to rapid buckling of the apical surface and the fast phase of invagination (Kondo, 2013).

Even with the loss of both EGFR and FGF signalling, the tracheal placodes form moderately invaginated structures, compared to the flat tracheal placode observed in the rho-bnl-CycA triple mutant at the same stage, indicating that cells needed to undergo mitosis 16 to induce invagination, independent of EGFR and FGF signalling. In rho bnl double mutants, although the cells undergoing the earliest mitoses showed surface cell rounding, some of the subsequent mitotic events were coupled to apical depression and internalized cell rounding. Unlike the earlier mitotic events on the surface, the internalized rounding cells in the rho bnl embryos showed constricted apices and were surrounded by apically rounded cells before mitosis. Internalized rounding with a constricted apical surface were shared properties of cells in mitoses leading to invagination, in both control and rho bnl embryos. It is suggested that the first few cells undergoing surface cell rounding compress the adjacent interphase cells and restrict their apical area, so that they are forced to move internally after rounding, causing the epithelial layer to buckle and invaginate (Kondo, 2013).

Although invagination was largely blocked in the rho-bnl-CycA triple mutants, any double mutant combination permitted invagination to some degree, indicating that three qualitatively distinct mechanisms, mitotic cell rounding, myosin II contractility (EGFR) and active cell motility (FGFR), can independently trigger invagination. In the normal context of wild-type development the combination of cell rounding and EGFR signalling may optimize the timing and speed of invagination, and then invaginated tracheal sacs subsequently respond to FGF emanating from several target tissues guiding branching morphogenesis (Kondo, 2013).

These observations demonstrates a new role for mitosis in tissue morphogenesis to generate mechanical force through cell rounding, independent of cell division. This is distinct from previously described invagination mechanisms involving cell-autonomous constriction by the apical activation of actomyosin contractility, which is incompatible with mitosis. Mitosis 16 outside the tracheal placode occurs in clusters on the ectoderm surface, but does not lead to invagination, suggesting that the tracheal placode is sensitized to invaginate upon mitosis, independent of EGFR and FGFR signalling. Future research to uncover the properties of the tracheal placode that enables it to respond to clustered mitosis will explain not only this new mode of morphogenesis, but also the homeostasis mechanisms of epithelial architecture (Kondo, 2013).

Progenitor outgrowth from the niche in Drosophila trachea is guided by FGF from decaying branches

Although there has been progress identifying adult stem and progenitor cells and the signals that control their proliferation and differentiation, little is known about the substrates and signals that guide them out of their niche. By examining Drosophila tracheal outgrowth during metamorphosis, this study showed that progenitors follow a stereotyped path out of the niche, tracking along a subset of tracheal branches destined for destruction. The embryonic tracheal inducer branchless FGF (fibroblast growth factor) is expressed dynamically just ahead of progenitor outgrowth in decaying branches. Knockdown of branchless abrogates progenitor outgrowth, whereas misexpression redirects it. Thus, reactivation of an embryonic tracheal inducer in decaying branches directs outgrowth of progenitors that replace them. This explains how the structure of a newly generated tissue is coordinated with that of the old (Chen, 2014).

Many adult stem cells reside in specific anatomical locations, or niches, and are activated during tissue homeostasis and after injury. Although considerable effort has been made to identify factors that control stem cell proliferation and differentiation, how stem or progenitor cells move out of the niche and how they form new tissue are not well understood. Tissue formation in mature animals faces challenges not present in the embryo. The new cells migrate longer distances and navigate around and integrate into a complex milieu of differentiated tissues. This work investigated the substratum and signals that guide Drosophila tracheal imaginal progenitor cells into the posterior during metamorphosis to form the pupal abdominal tracheae (PAT) that replace the posterior half of the larval tracheal system (tracheal metameres Tr6 to Tr10), which decays at this time (Chen, 2014).

The PAT extend from the transverse connective (TC) branches in Tr4 and Tr5. Each PAT consists of a multicellular stalk with many secondary branches, each of which has dozens of terminal cells that form numerous fine terminal branches (tracheoles). There are two known tracheal progenitor populations at metamorphosis: dedifferentiated larval tracheal cells and spiracular branch (SB) imaginal tracheal cells set aside during embryonic tracheal development. Lineage tracing showed that PAT derive from imaginal progenitors (Chen, 2014).

To determine how progenitors in Tr4 and Tr5 reach the posterior, a btl-RFP-moe transgene (RFP, red fluorescent protein) was used to label activated progenitor cells, and ppk4>GFP (GFP, green fluorescent protein) was used to label larval tracheal branches. Before metamorphosis, there are 7 to 10 quiescent progenitor cells in each SB niche. In early third larval instar (L3), progenitors proliferate but remain in the niche. Later in L3, progenitors leave the niche, moving onto the larval TC branches toward the dorsal trunk (DT), while progenitors within the niche continue to proliferate. Progenitors in other metameres also proliferate but do not move out of the niche. Migrating progenitors in Tr4 and Tr5 crawl along the basal surface of larval tracheal cells, with cytoplasmic projections emanating from cells at the leading edges of the progenitor cluster. Progenitors maintain epithelial polarity and a lumen continuous with the SB and TC branches, forming a saclike structure. By wandering L3, progenitors reach the DT, where they pause (~12 hours) until the onset of puparium formation (Chen, 2014).

Around 1 hour after puparium formation (APF), progenitors move onto the DT and turn posteriorly. Posterior migration continues for 9 hours, extending half the animal's length (~0.8 mm) past Tr9. Live imaging showed that progenitors move at ~1.7 μm/min, crawling along and wrapping around the DT as they migrate (Chen, 2014).

Differentiation begins as progenitors migrate. At the beginning of puparium formation (0 hours APF), a subset of progenitors that have exited the niche begins to express the terminal cell master regulator Pruned (Blistered) SRF (serum response factor), initiating cell specialization). As progenitors migrate along the DT, budlike structures composed of Pruned-expressing cells are detected at the tips of progenitor clusters, whereas Pruned-negative cells form the stalks of new trachea. By 6 hours APF, Pruned-expressing progenitors in the tips adopt an elongated and differentiated morphology, flattening along the DT as they extend further posteriorly. Around 13 hours APF, the PAT mature and fill with gas as posterior tracheal branches collapse (Chen, 2014).

What guides tracheal progenitors on their stereotyped path along specific branches of the larval tracheal system? Expression of breathless (btl) FGFR (fibroblast growth factor receptor) is induced in PAT progenitors, as shown by the btl-RFP-moe reporter. Whether the Btl pathway, which directs tracheal branch outgrowth in embryos and larvae and induces adult air-sac primordium formation, is involved was tested. Expression of dominant-negative Btl FGFR in the progenitors and their descendants blocked migration and diminished or eliminated PAT formation. To determine the source of the only known Btl ligand, Branchless (Bnl) FGF, a bnl reporter, bnl-Gal4 enhancer trap line NP2211 driving UAS-GFP was used. Unlike previously described examples of tracheal outgrowth, bnl was not expressed in surrounding tissue. Instead, it was expressed within the tracheal system, specifically by larval tracheal cells along which progenitors migrate. The expression pattern is dynamic and precise, almost perfectly matching the positions and timing of progenitor migration. In L3 animals, when progenitors are observed along the TC branches, bnl>GFP was expressed in TC larval cells in Tr4 and Tr5, but not in other metameres. Shortly after puparium formation, when PAT progenitors turn to migrate toward the posterior, DT larval cells in the segment just posterior to PAT progenitors express bnl>GFP. As progenitors continue along the DT, DT larval cells activate bnl>GFP expression one segment at a time from anterior to posterior, matching progenitor movement (Chen, 2014).

This dynamic bnl expression along the migration path is required for progenitor outgrowth. Knockdown of bnl expression by RNA interference (RNAi) in larval tracheal cells blocked migration and resulted in diminished or absent PAT. Mosaic expression of bnl RNAi in small patches along the path also arrested migration, so long as the patch encompassed the full DT circumference. Thus, Bnl is required all along the migration path, and the signal does not cross even short gaps (Chen, 2014).

Ectopic bnl expression in GFP-labeled clones of larval tracheal cells induced by dfr-FLP redirected progenitor migration. Depending on the location of the clones, ectopic bnl caused incorrect exit from the niche, premature entry onto the DT, or wrong turns on the DT. Dual clones induced bifurcation with groups of progenitors moving toward each ectopic bnl source. Clones in Tr3 and posterior metameres caused progenitors in these regions to leave the niche, even though they do not normally do so. When there was a large clone, progenitors migrated throughout the clone, implying that progenitors do not require a gradient and will spread to cover an entire region of cells expressing bnl at equivalent levels. When bnl-expressing clones failed to induce migration, the clones appeared to be too far from the progenitors or there was competition from another clone close by. Ectopic bnl expression within the progenitor cluster arrested migration (Chen, 2014).

The results show that the embryonic tracheal inducer Bnl FGF guides tracheal progenitors out of the niche and into the posterior during tracheal metamorphosis. The source of Bnl is the larval tracheal branches destined for destruction, which serve both as the source of the chemoattractant and as the substratum for progenitor migration. Several days earlier in embryos, these larval tracheal branches were themselves induced by Bnl provided by neighboring tissues. But after embryonic development, most tracheal cells, including those in the decaying larval branches, down-regulate btl FGFR expression and thus do not respond to (or sequester) the Bnl signal they later express. One of the most notable aspects of this larval Bnl is its exquisitely specific pattern in decaying larval branches, which presages progenitor outgrowth. It is unclear how Bnl expression is controlled, though it does not appear to require signals from migrating progenitors because the bnl reporter expression front progressed normally when progenitor outgrowth was stalled by a tracheal break. Perhaps expression of Bnl involves gradients in the tracheal system or spatial patterning cues established during embryonic development in conjunction with temporal signals mediated by molting hormones (Chen, 2014).

Because the signal guiding progenitor migration is provided by tracheae destined for destruction, progenitors become positioned along the larval branches they replace. Perhaps during tissue repair and homeostasis, recruitment of adult stem or progenitor cells from the niche is similarly guided by signals from decaying tissue, thereby ensuring that new tissue is directed to the appropriate sites (Chen, 2014).

EVOLUTIONARY HOMOLOGS

Glypican 4 modulates FGF signalling and regulates dorsoventral forebrain patterning in Xenopus embryos

Heparan sulphate proteoglycans such as glypicans are essential modulators of intercellular communication during embryogenesis. In Xenopus laevis embryos, the temporal and spatial distribution of Glypican 4 (Gpc4) transcripts during gastrulation and neurulation suggests functions in early development of the central nervous system. The role of Xenopus Gpc4 has been functionally analyzed by using antisense morpholino oligonucleotides; Gpc4 is shown to be part of the signalling network that patterns the forebrain. Depletion of GPC4 protein results in a pleiotropic phenotype affecting both primary axis formation and early patterning of the anterior central nervous system. Molecular analysis shows that posterior axis elongation during gastrulation is affected in GPC4-depleted embryos, whereas head and neural induction are apparently normal. During neurulation, loss of GPC4 disrupts expression of dorsal forebrain genes, such as Emx2, whereas genes marking the ventral forebrain and posterior central nervous system continue to be expressed. This loss of GPC4 activity also causes apoptosis of forebrain progenitors during neural tube closure. Biochemical studies establish that GPC4 binds FGF2 and modulates FGF signal transduction. Inhibition of FGF signal transduction, by adding the chemical SU5402 to embryos from neural plate stages onward, phenocopies the loss of gene expression and apoptosis in the forebrain. It is proposed that GPC4 regulates dorsoventral forebrain patterning by positive modulation of FGF signalling (Galli, 2003).

HSPG synthesis by zebrafish Ext2 and Extl3 is required for Fgf10 signalling during limb development

Heparan sulphate proteoglycans (HSPGs) are known to be crucial for signalling by the secreted Wnt, Hedgehog, Bmp and Fgf proteins during invertebrate development. However, relatively little is known about their effect on developmental signalling in vertebrates. This study reports the analysis of daedalus, a novel zebrafish pectoral fin mutant. Positional cloning identified fgf10 as the gene disrupted in daedalus. fgf10 mutants strongly resemble zebrafish ext2 and extl3 mutants, which encode glycosyltransferases required for heparan sulphate biosynthesis. This suggests that HSPGs are crucial for Fgf10 signalling during limb development. Consistent with this proposal, a strong genetic interaction is observed between fgf10 and extl3 mutants. Furthermore, application of Fgf10 protein can rescue target gene activation in fgf10, but not in ext2 or extl3 mutants. By contrast, application of Fgf4 protein can activate target genes in both ext2 and extl3 mutants, indicating that ext2 and extl3 are differentially required for Fgf10, but not Fgf4, signalling during limb development. This reveals an unexpected specificity of HSPGs in regulating distinct vertebrate Fgfs (Norton, 2005).

The secreted serine protease xHtrA1 stimulates long-range FGF signaling in the early Xenopus embryo

The secreted serine protease xHtrA1, expressed in early Xenopus embryos and transcriptionally activated by FGF signals, promotes posterior development in mRNA-injected embryos. xHtrA1 mRNA leads to the induction of secondary tail-like structures, expansion of mesoderm, and formation of ectopic neurons in an FGF-dependent manner. An antisense morpholino oligonucleotide or a neutralizing antibody against xHtrA1 has the opposite effects. xHtrA1 activates FGF/ERK signaling and the transcription of FGF genes. Xenopus Biglycan, Syndecan-4, and Glypican-4 are proteolytic targets of xHtrA1 and heparan sulfate and dermatan sulfate trigger posteriorization, mesoderm induction, and neuronal differentiation via the FGF signaling pathway. The results are consistent with a mechanism by which xHtrA1, through cleaving proteoglycans, releases cell-surface-bound FGF ligands and stimulates long-range FGF signaling (Hou, 2007).

HtrA1 belongs to the HtrA (High temperature requirement-A) family of serine proteases that is well conserved from bacteria to humans. HtrA1 was originally isolated as a gene downregulated in SV40-transformed human fibroblasts. Overexpression of HtrA1 in cancer cells suppresses growth and proliferation in vivo, suggesting that HtrA1 is a candidate tumor suppressor. More recently, a single nucleotide polymorphism in the HtrA1 promoter has been presented as a major risk factor for age-related macular degeneration. HtrA1 binds to and inactivates members of the TGFβ family and modulates insulin-like growth factor (IGF) signals, but its biological function is not yet known (Hou, 2007).

The Xenopus homolog of HtrA1 (xHtrA1) was identified in a direct screen for secreted proteins. xHtrA1 is a modulator of FGF signaling that participates in axial development, mesoderm formation, and neuronal differentiation. xHtrA1 is activated by FGF signals and induces ectopic FGF4 and FGF8 transcription. Biglycan, Syndecan-4, and Glypican-4 are proteolytic targets of xHtrA1; pure heparan sulfate and dermatan sulfate phenocopy xHtrA1 and FGF activities in Xenopus embryos. The results suggest that xHtrA1 acts as a positive regulator of FGF signals and, through proteolytic cleavage of proteoglycans, allows long-range FGF signaling in the extracellular space (Hou, 2007).

Phosphatidylinositol-3 kinase acts in parallel to the ERK MAP kinase in the FGF pathway during Xenopus mesoderm induction

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that can phosphorylate phosphaditylinositides leading to the cell type-specific regulation of intracellular protein kinases. PI3Ks are involved in a wide variety of cellular events including mitogenic signaling, regulation of growth and survival, vesicular trafficking, and control of the cytoskeleton. Some of these enzymes also act downstream of receptor tyrosine kinases or G-protein-coupled receptors. Using two strategies to inhibit PI3K signaling in embryos, the role of PI3Ks during early Xenopus development has been analyzed. A class 1A PI3K catalytic activity is required for the definition of trunk mesoderm during the blastula stages, but is less important for endoderm and prechordal plate mesoderm induction or for organizer formation. It is required in the FGF signaling pathway downstream of Ras and in parallel to the extracellular signal-regulated kinase (ERK) MAP kinases. In addition, ERKs and PI3Ks can synergise to convert ectoderm into mesoderm. These data provide the first evidence that class 1 PI3Ks are required for a specific set of patterning events in vertebrate embryos. Furthermore, they bring new insight into the FGF signaling cascade in Xenopus (Carballada, 2001).

While PI3K has been shown to be required for signal transduction in response to RTK ligands such as PDGF, insulin or EGF, its role in FGF signaling is much less clear cut. On the one hand, several molecules able to bind PI3K subunits, such as dof, act downstream of FGF or are found associated with the FGF receptor. Also, treatment of cultured cell lines with basic FGF can lead to a modest increase in PI3K activity. On the other hand, inhibition of PI3K signaling seldom has a demonstrated direct effect on the response to FGF and in the few cases where this appears to be the case, the role of PI3K is limited to the reorganization of the cytoskeleton or the regulation of exocytosis. In no case has the direct activation by FGF of a target gene been shown to be PI3K dependent. In contrast to the controversial role of PI3K in FGF signaling, activation of the MAP kinase pathway plays a crucial role in FGF signaling. On the basis of the overexpression of activated MAP kinase, it has been suggested that the Ras-dependent activation of this kinase is sufficient to account for the FGF-mediated induction of mesoderm induction and for the direct activation of Xbra (Carballada, 2001).

Using two different strategies to interfere with PI3K signaling, this study provides the first demonstration that PI3K signaling is crucial for the direct activation by FGF of Xbra. PI3K signaling is not involved in the activation of ERK by FGF but rather acts in parallel to the MAP kinase pathway. In contrast to what has been previously proposed, these results thus indicate that, during mesoderm induction, the FGF signaling pathway splits upstream of ERK into at least two cooperating branches. Several questions remain to be addressed. (1) The weak mesoderm induction obtained when both the ERK and PI3K pathways are activated suggests the existence of additional parallel effector pathways downstream of Ras. Several effector pathways, including Ral, Rac/Rho and phospholipase D, have been shown to act downstream of Ras and probably in parallel to ERK and PI3K. It will be important to test the role of these pathways in Xenopus mesoderm formation. It will also be important to position PI3K with respect to laloo, a recently described src-family tyrosine kinase acting in the FGF pathway. (2) PI3K is required for FGF signaling, whether this PI3K activity is modulated by FGF signaling has not been addressed. This could be the case, since in other systems FGF can stimulate, albeit weakly, PI3K activity. In addition, p85 is associated to the FGF receptor in Xenopus embryos during gastrulation. (3) The components acting downstream of PI3K in mesoderm induction must be identified. Several downstream effectors of PI3K have been characterized in cultured cells including GSK3, PKB/Akt, p70 S6k and the GTPases Rac and Rho. The results presented here do not support a role for GSK3 downstream of PI3K in early embryos, since expression of Siamois, a direct target of the beta-catenin/GSK3 pathway, is not affected by treatment with LY294002. It will be interesting to test a potential role for PKB/Akt and Rac/Rho in mesoderm induction downstream of PI3K. The availability of constitutively active or dominant negative forms of proteins acting in the Ras and PI3K pathways in other systems, coupled with the convenience of the Xenopus system, will help shed light on these issues (Carballada, 2001).

Negative feedback regulation of FGF signaling levels by Pyst1/MKP3 in chick embryos

The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. High-level expression of Chicken Pyst1/Mkp3 in neural plate correlates with active MAPK. FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). by applying a specific MAP kinase kinase inhibitor (PD184352) it has been shown that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. It is concluded that Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. These results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates (Eblaghie, 2003).

Lipoprotein receptors and a Disabled family cytoplasmic adaptor protein regulate EGL-17/FGF export in C. elegans

Growth factors and morphogens need to be secreted to act on distant cells during development and in response to injury. Evidence is presented that efficient export of a fibroblast growth factor (FGF), EGL-17, from the C. elegans developing vulva requires the lipoprotein receptor-related proteins Ce-LRP-1 and Ce-LRP-2 and a cytoplasmic adaptor protein, Ce-DAB-1 (Disabled). Lipoprotein receptors are transmembrane proteins best known for their roles in endocytosis. Ce-LRP-1 and Ce-LRP-2 possess a conserved intraluminal domain that can bind to EGL-17, as well as a cytosolic FXNPXY motif that can bind to Ce-DAB-1. Ce-DAB-1 contains signals that confer subcellular localization to Golgi-proximal vesicles. These results suggest a model in which Ce-DAB-1 coordinates selection of receptors and cargo, including EGL-17, for transport through the secretory pathway (Kamikura, 2003).

Dab family adaptor proteins interact functionally with lipoprotein receptors in both nematodes and mammals, even though the biological processes they mediate vary greatly. Ce-DAB-1 regulates secretion, Dab2 regulates endocytosis in the kidney, and Dab1 relays extracellular signals during brain development, each via lipoprotein receptors. Although the role of Ce-DAB-1 in signaling is unclear, the high degree of functional conservation across species suggests that vertebrate Dab family members or other PTB-containing proteins may participate in regulated traffic of lipoprotein receptors and associated cargoes to the cell surface. Indeed, it is possible that an early embryonic requirement for Dab2 might be a consequence of altered protein traffic in polarized epithelial cells of the embryo (Kamikura, 2003).

Sensitized genetic backgrounds reveal a role for C. elegans FGF EGL-17 as a repellent for migrating CAN neurons

Although many molecules are necessary for neuronal cell migrations in C. elegans, no guidance cues are known to be essential for any of these cells to migrate along the anteroposterior (AP) axis. The fibroblast growth factor (FGF) EGL-17, an attractant for the migrating sex myoblasts (SMs), repels the CANs, a pair of neurons that migrate posteriorly from the head to the center of the embryo. Although mutations in genes encoding EGL-17/FGF and a specific isoform of its receptor EGL-15/FGFR had little effect on CAN migration -- they enhanced the CAN migration defects caused by mutations in other genes. Two cells at the anterior end of the embryo express EGL-17/FGF, raising the possibility that EGL-17/FGF functions as a repellent for migrating CANs. Consistent with this hypothesis, ectopic expression of EGL-17/FGF shifted the final CAN cell positions away from these novel sites of expression. Cell-specific rescue experiments demonstrated that EGL-15/FGFR acts in the CANs to promote their migration. The tyrosine phosphatase receptor CLR-1 regulates CAN migration by inhibiting EGL-15/FGFR signaling, and the FGFR adaptor protein SEM-5/GRB2 may mediate EGL-15/FGFR signaling in CAN migration. Thus, EGL-17/FGF signaling through an EGL-15/FGFR isoform and possibly SEM-5/GRB2 mediates both attraction of the SMs and repulsion of the CANs. This study also raises the possibility that several guidance cues regulate cell migrations along the C. elegans AP axis, and their role in these migrations may only be revealed in sensitized genetic backgrounds (Fleming, 2005).

In C. elegans, the gene vab-8 is both necessary and sufficient for posteriorly directed migrations of cells and growth cones. Most posterior migrations require vab-8, and ectopic expression of vab-8 can reroute anteriorly projected axons towards the posterior. The vab-8 locus encodes at least two novel intracellular proteins that act in the cells to promote their migration. How these proteins regulate these migrations, however, remains unknown (Fleming, 2005).

Unlike VAB-8, the C. elegans transmembrane protein MIG-13 plays a nonautonomous role in guiding cell migrations along the AP axis. mig-13 loss-of-function alleles display more specific defects than vab-8 mutants, disrupting the anterior directed migrations of only the BDU neurons and descendants of the right Q neuroblast. Ectopic expression of mig-13 from a heat shock promoter, however, induces an anterior shift in the final positions of neurons that migrate in either direction along the AP axis, indicating that MIG-13 plays a broader role than was suggested by the effects of mig-13 mutants. As with VAB-8, the role of MIG-13 in these migrations remains unclear (Fleming, 2005).

In C. elegans, the fibroblast growth factor (FGF) homolog EGL-17 functions as an attractant for the precise positioning of the anteriorly directed migrations of the sex myoblasts (SMs). In early larval stages, the SMs migrate from the posterior midbody to positions flanking the center of the gonad. During SM migration, EGL-17 is expressed in the primary vulval precursor cells (VPCs) and the dorsal uterine (DU) cells of the somatic gonad, which define the final destination of the SMs. EGL-17 signals through the FGF receptor (FGFR) EGL-15 to attract SMs (Fleming, 2005).

In an effort to understand AP guidance in C. elegans, focus was placed on the posterior migrations of the CANs, a pair of bilaterally symmetric neurons that are born in the head and migrate to the middle of the embryo. Although a previous screen for CAN migration mutants identified a number of genes, none of them encoded guidance cues. One explanation for this outcome is that multiple cues contribute to CAN migration, and therefore removing one might result in only subtle CAN migration defects. To test this hypothesis, sensitized genetic backgrounds, specifically a vab-8 mutation was used to re-evaluate the potential role of secreted molecules in CAN migration. The use of these sensitized backgrounds revealed a role for FGF in CAN migration (Fleming, 2005).

FGF negatively regulates muscle membrane extension in Caenorhabditis elegans

Striated muscles from Drosophila and several vertebrates extend plasma membrane to facilitate the formation of the neuromuscular junction (NMJ) during development. However, the regulation of these membrane extensions is poorly understood. In C. elegans, the body wall muscles (BWMs) also have plasma membrane extensions called muscle arms that are guided to the motor axons where they form the postsynaptic element of the NMJ. To investigate the regulation of muscle membrane extension, 871 genes were screened by RNAi for ectopic muscle membrane extensions (EMEs) in C. elegans. An FGF pathway, including let-756(FGF), egl-15(FGF receptor), sem-5(GRB2) and other genes were found to negatively regulate plasma membrane extension from muscles. Although compromised FGF pathway activity results in EMEs, hyperactivity of the pathway disrupts larval muscle arm extension, a phenotype called muscle arm extension defective or MAD. Expression of egl-15 and sem-5 in the BWMs are each necessary and sufficient to prevent EMEs. Furthermore, let-756 expression from any one of several tissues can rescue the EMEs of let-756 mutants, suggesting that LET-756 does not guide muscle membrane extensions. The screen also revealed that loss-of-function in laminin and integrin components results in both MADs and EMEs, the latter of which are suppressed by hyperactive FGF signaling. These data are consistent with a model in which integrins and laminins are needed for directed muscle arm extension to the nerve cords, while FGF signaling provides a general mechanism to regulate muscle membrane extension (Dixon, 2006).

FGF signaling regulates Wnt ligand expression to control vulval cell lineage polarity in C. elegans

The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. This study shows that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/beta-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, it was shown that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans (Minor, 2013).

A Twist-like bHLH gene is a downstream factor of an endogenous FGF and determines mesenchymal fate in the ascidian embryos

Ascidian larvae develop mesenchyme cells in their trunk. A fibroblast growth factor (FGF9/16/20) is essential and sufficient for induction of the mesenchyme in Ciona savignyi. Two basic helix-loop-helix (bHLH) genes named Twist-like1 and Twist-like2 have been identified as downstream factors of this FGF. These two genes are phylogenetically closely related to each other, and are expressed specifically in the mesenchymal cells after the 110-cell stage. Gene-knockdown experiments using a specific morpholino oligonucleotide demonstrate that Twist-like1 plays an essential role in determination of the mesenchyme and that Twist-like2 is a downstream factor of Twist-like1. In addition, both overexpression and misexpression of Twist-like1 converts non-mesenchymal cells to mesenchymal cells. The upstream regulatory mechanisms of Twist-like1 are different between B-line mesenchymal cells and the A-line mesenchymal cells called 'trunk lateral cells'. FGF9/16/20 is required for the expression of Twist-like1 in B-line mesenchymal precursor cells, whereas FGF, FoxD and another novel bHLH factor called NoTrlc (for No Trunk lateral cells, because it is essential for the differentiation of TLCs, which give rise to blood cells and body-wall muscles after metamorphosis) are required for Twist-like1 to be expressed in the A-line mesenchymal precursor cells. Therefore, two different but partially overlapping mechanisms are required for the expression of Twist-like1 in the mesenchymal precursors, that triggers the differentiation of the mesenchyme in Ciona embryos (Imai, 2003).

Ephrin signaling establishes asymmetric cell fates in an endomesoderm lineage of the Ciona embryo

Mesodermal tissues arise from diverse cell lineages and molecular strategies in the Ciona embryo. For example, the notochord and mesenchyme are induced by FGF/MAPK signaling, whereas the tail muscles are specified autonomously by the localized determinant, Macho-1. A unique mesoderm lineage, the trunk lateral cells, develop from a single pair of endomesoderm cells, the A6.3 blastomeres, which form part of the anterior endoderm, hematopoietic mesoderm and muscle derivatives. MAPK signaling is active in the endoderm descendants of A6.3, but is absent from the mesoderm lineage. Inhibition of MAPK signaling results in expanded expression of mesoderm marker genes and loss of endoderm markers, whereas ectopic MAPK activation produces the opposite phenotype: the transformation of mesoderm into endoderm. Evidence is presented that a specific Ephrin signaling molecule, Ci-ephrin-Ad, is required to establish asymmetric MAPK signaling in the endomesoderm. Reducing Ci-ephrin-Ad activity via morpholino injection results in ectopic MAPK signaling and conversion of the mesoderm lineage into endoderm. Conversely, misexpression of Ci-ephrin-Ad in the endoderm induces ectopic activation of mesodermal marker genes. These results extend recent observations regarding the role of Ephrin signaling in the establishment of asymmetric cell fates in the Ciona notochord and neural tube (Shi, 2008).

This study presents evidence that competition between Eprhin and FGF signaling is important for the asymmetric specification of endoderm and mesoderm lineages from a common endomesoderm progenitor cell, the A6.3 blastomere. A similar mechanism was recently invoked to account for the asymmetric specification of the notochord and nerve cord from common A6.2 and A6.4 progenitors (Picco, 2007). In both cases, a localized Ephrin-Ad signal produced by the primitive ectoderm (the animal blastomeres) competes with FGF9 signals from the primitive gut. Those cells in extended contact with the ectoderm lack MAPK activation, whereas those cells in contact with the endoderm experience MAPK activation and follow a different fate. It is conceivable that this interplay of Ephrin and FGF signaling is used in other systems to produce asymmetric cell fates (Shi, 2008).

Ephrins have been implicated in a variety of cellular processes, including axonal guidance, repulsive cell-cell interactions, and adhesion. Different Ephrin family members can activate or inhibit RTK signaling in different cellular contexts. The present study, along with the recent analysis of Ci-Bra regulation (Picco, 2007), suggest that Ci-ephrin-Ad functions as a localized inhibitor of FGF signaling to produce asymmetric cell fates in Ciona. The presumptive endoderm/endomesoderm produces a localized source of FGF9/16/20, which induces the specification of diverse mesoderm lineages, including the notochord and mesenchyme. Evidence is presented that Ephrin also controls the subdivision of the A6.3 endomesoderm (Shi, 2008).

A model is presented for the specification of the A7.6 blastomere. Previous studies have shown that Nodal is essential for the expression of several A7.6 marker genes, including Hand-like (also known as NoTrlc), FGF8 and Delta-like. Nodal is expressed in the A6.3 blastomere of 32-cell embryos, as well as in the other progenitors of the endoderm. FGF/MAPK signaling is also active in the A6.3 at this stage, as judged by anti-dpERK staining. Ephrin-Ad produced by b6.5 (and other animal blastomeres) inhibits MAPK in A7.6, thereby permitting Nodal to activate the A7.6 group genes. Nodal signaling in A7.6 might be reinforced by Nodal expression in the b6.5 lineage. Thus, the inhibition of FGF signaling by Eprhin-Ad, along with augmented levels of Nodal signal, might be responsible for the activation of A7.6 group genes. However, evidence is presented that Nodal in b6.5 is not essential for A7.6 group gene expression. Instead, it would appear that the combination of endogenous Nodal in the A6.3 progenitor, along with the localized inhibition of MAPK in A7.6 by Eprhin-Ad, is the decisive determinant of A7.6 specification (Shi, 2008).

Inhibition of MAPK signaling via drug treatment or ectopic expression of Ephrin-Ad leads to misexpression of A7.6 marker genes in the anterior endoderm, where Nodal is normally inactive owing to FGF/MAPK signaling. Posterior endoderm cells also contain Nodal but fail to express A7.6 marker genes upon inhibition of MAPK. This might reflect the restricted distribution of additional activators required for A7.6 gene expression. For example, Hand-like is activated by the combination of Nodal signaling and the FoxA transcription factor. FoxA expression is restricted to the anterior endoderm, dorsal mesoderm and future CNS floorplate, but is absent from the posterior endoderm. This is consistent with the result of ectopic Hand-like and Delta-like activation in the A-line neural lineage by expression of the FoxD::Nodal transgene (Shi, 2008).

A7.6 expresses a number of localized determinants, including two crucial signaling molecules, FGF8 and Delta-like. A7.6 is located in a strategically important position within the vegetal hemisphere. It contacts components of all three germ layers: the endoderm, ectoderm and mesenchyme. The Delta-like ligand expressed in A7.6 induces the secondary notochord lineage via Notch signaling, and also induces the lateralmost neural fate. Similarly, FGF8 expression is required for maintaining the primary notochord fate. Because these signaling pathways require either direct cell-cell contact (Notch) or act over one or two cell diameters (FGF), it is crucial to activate the expression of Delta-like and FGF8 to A7.6, but not in its sibling A7.5 endoderm blastomere. The activities of three pathways, Ephrin, MAPK and Nodal signaling, are employed to achieve this precise asymmetric cell-fate specification event (Shi, 2008).

Recent phylogenetic analysis suggests that tunicates (e.g., Ciona) are the closest living relatives of the vertebrates. As a result, it is possible that vertebrates employ a mechanism for the specification and subdivision of the endomesoderm that is similar to the one used in Ciona. The A6.3 endomesoderm cell is established by the action of a localized maternal determinant, β-Catenin, which activates the expression of multiple signaling molecules including Nodal and FGF9. Nodal is required to activate A7.6-specific genes such as Hand-like, FGF8 and Delta-like. The failure of Nodal to activate A7.6 group genes in the endoderm is due to MAPK signaling. FGF signaling either directly or indirectly inhibits Nodal. As a result, Nodal signaling is blocked in A6.3, but is activated in A7.6 owing to the localized inhibition of FGF signaling by Eprhin-Ad (Shi, 2008).

Most or all metazoan embryos possess a transient endomesoderm that generates specific mesodermal derivatives. In vertebrates, the presumptive endomesoderm gives rise to blood, heart and muscle. Formation of the vertebrate endomesoderm depends on TGF-β signaling molecules such as Xnrs in Xenopus and Squint and Cyclops (Nodal-related 1 and 2, respectively; ZFIN) in zebrafish. The subsequent subdivision of the endomesoderm is not clearly understood, but might depend on FGF signaling. It remains to be seen if competitive interactions between Nodal (or some other TGF-β signaling molecule) and FGF lead to the subdivision of endomesoderm in vertebrate embryos (Shi, 2008).

The cellular and molecular etiology of the cleft secondary palate in Fgf10 mutant mice

Mammalian palatogenesis depends on interactions between the stomodium-derived epithelium and the cranial neural crest-derived ectomesenchyme. Fibroblast growth factor 10 (FGF10) is a mesenchymal signaling factor that guides the morphogenesis of multiple organs through tissue-tissue interactions. This is consistent with widespread agenesis and dysgenesis of organs observed in Fgf10^−/− mice. A wide-open cleft secondary palate is present in Fgf10 homozygous null mutant mice. Fgf10 transcripts are detected in the palatal mesenchyme from E11.5 to E13.5 during normal palatogenesis and are enriched in the anterior and middle portions of the palatal shelves. In Fgf10^−/− embryos, histological analyses revealed aberrant adhesion of the palatal shelves with the tongue in the anterior and fusion with the mandible in the middle and posterior beginning at E13.5, which could prevent normal elevation of the palatal shelves leading to a cleft palate. TUNEL and BrdU assays demonstrate significant levels of apoptosis in the medial edge epithelium (MEE) but unaltered cell proliferation in mutant palatal shelves. At the molecular level, Fgf10 is shown to be epistatic to Jagged2 and Tgfβ3 in the developing palate. Notably, the expression of Jagged2 is downregulated throughout the palate epithelium in Fgf10 mutants while Tgfβ3 is misexpressed in the palatal epithelium at the oral side. These results demonstrate that mesenchymally expressed Fgf10 is necessary for the survival of MEE cells and for the normal expression of Jagged2 and Tgfβ3 in the palatal epithelium during mammalian palatogenesis (Alappat, 2004).

FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst

Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the formation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. Cell lineage from the eight-cell stage was followed by live cell tracing, and no clear linkage was found between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, a model is proposed in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner (Yamanaka, 2010).

Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers

Members of the POU and SOX transcription factor families exemplify the partnerships established between various transcriptional regulators during early embryonic development. Although functional cooperativity between key regulator proteins is pivotal for milestone decisions in mammalian development, little is known about the underlying molecular mechanisms. In this study, focus was placed on two transcription factors, Oct4 and Sox2, since their combination on DNA is considered to direct the establishment of the first three lineages in the mammalian embryo. Using experimental high-resolution structure determination, followed by model building and experimental validation, it was found that Oct4 and Sox2 were able to dimerize onto DNA in distinct conformational arrangements. The DNA enhancer region of their target genes is responsible for the correct spatial alignment of glue-like interaction domains on their surface. Interestingly, these surfaces frequently have redundant functions and are instrumental in recruiting various interacting protein partners (Reményi, 2003).

The interaction of Oct1 and Oct4 with Sox2 was investigated on two different DNA enhancers to test whether a previously discovered regulation mechanism of DNA-mediated swapping of the arrangement of homodimers may also be applicable for unrelated transcription factor assemblies. The crystal structure of the ternary Oct1/Sox2/FGF4 enhancer element complex was solved and then homology modeling tools were used to construct an Oct4/Sox2/FGF4 as well as an Oct4/Sox2/UTF1 structural model. These models reveal that the FGF4 and the Undifferentiated Transcription Factor 1 (UTF1) enhancers mediate the assembly of distinct POU/HMG complexes, leading to different quaternary arrangements by swapping protein-protein interaction surfaces of Sox2. Moreover, it has been demonstrated that Sox2 uses one of its two protein interacting surfaces to assemble a ternary complex with another unrelated transcription factor on a late-embryonic-stage-specific enhancer (Pax6/Sox2 on the DC5 element). These findings outline a simple mechanism for promiscuous yet highly specific assembly of transcription factors, in which the sequence of DNA enhancers governs a combinatorial use of redundant protein-protein interaction surfaces (Reményi, 2003).

Pax3 regulation of FGF signaling affects the progression of embryonic progenitor cells into the myogenic program

Pax3/7-dependent stem cells play an essential role in skeletal muscle development. Fgfr4 lies genetically downstream from Pax3 and is a direct target. In chromatin immunoprecipitation (ChIP)-on-chip experiments, Pax3 binds to a sequence 3' of the Fgfr4 gene that directs Pax3-dependent expression at sites of myogenesis in transgenic mouse embryos. The activity of this regulatory element is also partially dependent on E-boxes, targets of the myogenic regulatory factors, which are expressed as progenitor cells enter the myogenic program. Other FGF signaling components, notably Sprouty1, are also regulated by Pax3. In vivo manipulation of Sprouty expression reveals that FGF signaling affects the balance between Pax-positive progenitor cells and committed myoblasts. These results provide new insight into the Pax-initiated regulatory network that modulates stem cell maintenance versus tissue differentiation (Lagha, 2008).

CRTC1 nuclear translocation following learning modulates memory strength via exchange of chromatin remodeling complexes on the Fgf1 gene

Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. This study finds that the transcription cofactor CRTC1 (see Drosophila Crtc) is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB (see Drosophila CrebB), and histone acetyltransferase CBP (see Drosophila Nejire) induces transient transcription. Strong training later substitutes KAT5 (see Drosophila Tip60) for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets (Uchida, 2017).

Integration of multiple signal transducing pathways on Fgf response elements of the Xenopus caudal homologue Xcad3

Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. The nature of Fgf response element (FRE) has been investigated in a posterior neural gene, Xcad3 (Xenopus caudal homolog), which plays a crucial role of posterior neural development. Evidence suggests that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s) (Haremaki, 2003).

The reporter constructs containing the FREs exhibit high dose dependence on Fgf similar to that shown for endogenous Xcad3, when examined in the embryonic cell culture assay. Sequence and mutagenesis analyses reveal that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs (EBMs and TLBMs respectively) that lie in juxtaposition. The EBM is known to serve as the binding site for Ets family transcription factors that are nuclear effectors of the Fgf/Ras/Mapk pathway. Indeed, functional and physical analyses show that Ets proteins are involved in the Fgf response of Xcad3 as transcriptional activators, and that Xcad3 is directly targeted by the Fgf signaling pathway. This conclusion is consistent with the previous observation that Fgf can induce Xcad3 expression in the animal cap assay within 2 hours of its addition and even in the presence of the protein synthesis inhibitor cycloheximide, which indicates that Xcad3 is an immediate early target of Fgf signaling (Haremaki, 2003 and references therein).

TLBMs could serve as the binding sites for Tcf/Lef family transcription factors that are nuclear effectors of the Wnt/ß-catenin pathway. It was anticipated that XTcf3 would functioned as a co-activator of Ets proteins, since Wnt signaling has been suggested as being involved in activation of posterior neural genes. Surprisingly, however, functional analysis reveals that XTcf3 acts as a repressor of Xcad3. The data suggest that the endogenous pool of ß-catenin in ectoderm cells is considerably smaller compared with that of XTcf3 co-repressors such as XCtBP and Groucho. This in turn implies that Wnt signaling could activate Xcad3 expression in embryonic cells, when they are provided with a larger pool of ß-catenin. Marginal zone cells of the early gastrula embryo, where Xcad3 is initially expressed, are among such candidate cells, since a relatively large amount of ß-catenin is translocated into the nucleus in these cells. Recently, a mutant function of Tcf3 as a repressor has revealed in the zebrafish headless mutant that carries a mutation in Tcf3. In this mutant, expression of midbrain-hindbrain boundary genes such as En2 and Pax2 is de-repressed in more anterior neural region, leading to severe head defects. It would be interesting to know whether similar anterior expansion is seen in Cdx gene expression in this mutant (Haremaki, 2003 and references therein).

Sox2 is de-repressed by Bmp antagonists in the neurogenic region of ectoderm during neural induction. Sox2, which shares a cognate DNA bindings motif with Tcf/Lef family members, is required as a co-activator for the Fgf response of Xcad3. Sox2 is likely to compete with XTcf3 for TLBMs in the composite FREs to cooperate with Ets proteins that bind to adjacent EBMs. Physical analysis supports this idea. Both Sox and Ets family transcription factors interact with specific partner factors to direct signals to target genes, but direct partnership between them has not been reported. Collectively, these results indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on the FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox proteins and negatively acting Tcf protein (Haremaki, 2003).

FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis

Branching morphogenesis of mouse submandibular glands is regulated by multiple growth factors. Ex vivo branching of intact submandibular glands decreases when either FGFR2 expression is downregulated or soluble recombinant FGFR2b competes out the endogenous growth factors. However, a combination of neutralizing antibodies to FGF1, FGF7 and FGF10 is required to inhibit branching in the intact gland, suggesting that multiple FGF isoforms are required for branching. Exogenous FGFs added to submandibular epithelial rudiments cultured without mesenchyme induce distinct morphologies. FGF7 induces epithelial budding, whereas FGF10 induces duct elongation, and both are inhibited by FGFR or ERK1/2 signaling inhibitors. However, a PI3-kinase inhibitor also decreases FGF7-mediated epithelial budding, suggesting that multiple signaling pathways exist. FGF receptors were immunolocalized and changes in FGFR, FGF and MMP gene expression were analyzed to identify the mechanisms of FGF-mediated morphogenesis. FGFR1b and FGFR2b are present throughout the epithelium, although FGFR1b is more highly expressed around the periphery of the buds and the duct tips. FGF7 signaling increases FGFR1b and FGF1 expression, and MMP2 activity, when compared with FGF10, resulting in increased cell proliferation and expansion of the epithelial bud, whereas FGF10 stimulates localized proliferation at the tip of the duct. FGF7- and FGF10-mediated morphogenesis is inhibited by an MMP inhibitor and a neutralizing antibody to FGF1, suggesting that both FGF1 and MMPs are essential downstream mediators of epithelial morphogenesis. Taken together, these data suggests that FGFR2b signaling involves a regulatory network of FGFR1b/FGF1/MMP2 expression that mediates budding and duct elongation during branching morphogenesis (Steinberg, 2005).

Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage

Lineage formation in the lung mesenchyme is poorly understood. Using a transgenic mouse line expressing LacZ under the control of Fgf10 regulatory sequences, the pool of Fgf10-positive cells in the distal lung mesenchyme has been shown to contain progenitors of the parabronchial smooth muscle cells. Fgf10 gene expression is slightly repressed in this transgenic line. This allowed creation of a hypomorphic Fgf10 phenotype by expressing the LacZ transgene in a heterozygous Fgf10 background. Hypomorphic Fgf10 mutant lungs display a decrease in ß-galactosidase-positive cells around the bronchial epithelium associated with an accumulation of ß-galactosidase-expressing cells in the distal mesenchyme. This correlates with a marked reduction of alpha smooth muscle actin (SMA) expression, thereby demonstrating that FGF10 is mostly required for the entry of mesenchymal cells into the parabronchial smooth muscle cell lineage. The failure of exogenous FGF10 to phosphorylate its known downstream targets ERK and AKT in lung mesenchymal cultures strongly suggests that FGF10 acts indirectly on the progenitor population via an epithelial intermediate. This study provides support for a role of epithelial BMP4 in mediating the formation of parabronchial smooth muscle cells (Mailleux, 2005).

The results indicate a decrease in Bmp4 expression in Fgf10^LacZ/– embryos. This reduction in Bmp4 expression seems to primarily occur in the epithelium. These results are consistent with previous reports showing that FGF10 upregulates epithelial Bmp4 transcription. Overexpression of Bmp4 in the distal lung epithelium using the surfactant protein C promoter leads to ectopic expression of alpha-SMA in the distal mesenchyme. While addition of recombinant SHH induces alpha-SMA expression on isolated lung mesenchymal explants, overexpression of Shh in the distal lung epithelium in vivo does not modify alpha-SMA expression. This may be explained by the lack of upregulation of Bmp4 in the epithelium or the mesenchyme upon overexpression of Shh in vivo, by contrast to the induction of Bmp4 expression by SHH in vitro (Mailleux, 2005).

Consistent with a major role for Bmp4 in SMC differentiation, recombinant BMP4 induces alpha-SMA expression in lung mesenchyme explants in vitro after 48 hours of culture. These results strongly suggest that BMP4 induces SMC formation by acting directly on the mesenchyme. It is therefore proposed that FGF10 expressed by the distal mesenchyme may contribute to parabronchial SMC formation via the upregulation of BMP4 synthesis by the epithelium. The failure to induce alpha-SMA expression in all cells can be explained by the presence of other cell types in the mesenchymal explants, e.g. the endothelial cells. In addition, these explants also contain a layer of mesothelium, producing FGF9, which has been shown to prevent the differentiation of the smooth muscle cells (Mailleux, 2005).

Posteriorization by FGF, Wnt, and Retinoic Acid is required for neural crest induction

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. The roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos, was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, is transformed into neural crest by posteriorizing signals. This transformation is mimicked by bFGF, Wnt-8, or retinoic acid treatment and is also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells is also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor has been shown to directly affect the ectoderm. A two-step model is proposed for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. A unifying model is discussed where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction (Villanueva, 2002).

It is suggested that at the early-gastrula stage, a gradient of BMP activity is established in the ectoderm, which specifies the neural plate, the neural plate border, and the epidermis at progressively higher concentrations of BMP. The neural plate border, induced at a precise location within the mediolateral axis of the ectoderm, has an anterior character. Later, between early and midgastrula stage, signals presumably originating from the ventrolateral mesoderm transform a region of the anterior neural plate border into prospective neural crest cells. A role for this mesoderm in neural crest induction has been shown. The spread of these molecules from the mesoderm into the ectoderm consequently locates them only in large animal caps, explaining why the neural crest was not induced when small animal caps were used. These signals could correspond to Wnt8 and eFGF, since it is known that they are expressed in the ventrolateral mesoderm, and could correspond to lateralizing signals. However, the neural crest is not specified at this stage; this does not occur until the end of gastrulation. Thus, additional signals are required for the final induction of the neural crest. Finally, as gastrulation proceeds, the ventrolateral mesoderm becomes localized to the posterior region of the embryo, where it continues to produce Wnt8, eFGF, and possibly retinoic acid, as well as another, as yet unknown, posteriorizing agent(s) that generates an anterior-posterior gradient of these morphogenes. This gradient would be required for the final specification of the neural crest in the most posterior region of the neural plate border. Thus, the lateral-posterior regions of the neural plate border receive the lateralizing/posteriorizing signals for an extended period of time, finally specifying them as neural crest. In contrast, the anterior neural plate border does not receive such signals or these are inhibited by other agents produced by the anterior regions of the embryo, such as cerberus or dkk1, two known Wnts inhibitors, and, as a consequence, this border region does not develop as neural crest cells. It is tempting to speculate that the anterior-posterior differences within the neural crest could be controlled by a similar mechanism (Villanueva, 2002).

Requirement for endoderm and FGF3 in ventral head skeleton formation

The vertebrate head skeleton is derived in part from neural crest cells, which physically interact with head ectoderm, mesoderm and endoderm to shape the pharyngeal arches. The cellular and molecular nature of these interactions is poorly understood, and the function of endoderm in this process has been explored. By genetic ablation and reintroduction of endoderm in zebrafish, it has been shown that endoderm is required for the development of chondrogenic neural crest cells, including their identity, survival and differentiation into arch cartilages. Using a genetic interference approach, Fgf3 has been identified as a critical component of endodermal function that allows the development of posterior arch cartilages. Together, these results reveal that the endoderm provides differential cues along the anteroposterior axis to control ventral head skeleton development and demonstrate that this function is mediated in part by Fgf3 (David, 2002).

In vertebrates, endoderm and mesoderm are specified by Nodal-related signals, members of the transforming growth factor ß superfamily. Nodal activities are mediated by type I TGFß receptors ALK4 and ALK7 in mammals and most likely by their relative, Taram-A (tar) in zebrafish. Activation of the Nodal pathway by expression of a constitutively active version of tar (tar*) leads to a respecification of early zebrafish blastomeres to an endodermal fate, consistent with the model that high levels of Nodal signaling are sufficient to direct cells to become endoderm. Downstream of nodal signaling, endoderm formation further involves the homeobox transcription factors Mixer/Bonnie and clyde (bon) and the recently identified Sox-related factor Casanova (cas). Zebrafish embryos in which Nodal signals are inactive develop neither endoderm nor mesoderm while, in contrast, bon or cas mutants form mesoderm but little or no endoderm. In particular, cas mutants lack expression of all endodermal markers and derivatives. Interestingly, cas embryos are not rescued by activation of Nodal-related ligands or of the Tar cascade, consistent with its proposed function downstream. However, they provide a permissive environment for endoderm development since tar*-activated wild-type blastomeres can autonomously restore endoderm formation when grafted into cas embryos (David, 2002 and references therein).

cas and bon mutants were used to test whether or not pharyngeal endoderm is required for formation of the pharyngeal skeleton. Both mutants lack most of the ventral cartilage of the head skeleton and these gene functions are required after cephalic neural crest migration, when crest normally contacts the endoderm. Neural crest has been fate mapped in cas and it has been show that, in the absence of endoderm, cephalic neural crest cells remain as a cluster on the surface of the yolk sac and down regulate the expression of pre-chondrogenic markers. cas neural crest cells still have the ability to develop into cartilage when transplanted into wild-type embryos, showing that the cas gene is not required autonomously in neural crest cells but rather in their environment. Furthermore, endoderm can rescue head cartilage formation when reintroduced by grafting wild-type tar*-injected cells into cas embryos. Finally, FGF has been implicated in signaling from endoderm to neural crest by demonstrating that endodermal expression of FGF3 is specifically required for the formation of the posterior, branchial arches. Altogether, these results demonstrate a requirement for pharyngeal endoderm and FGF signaling in the control of head neural crest fates and cartilage induction, and identify FGF3 as the first endodermal signal with an AP restricted function in the pharyngeal region (David, 2002).

FGF/MAPK signaling is required in the gastrula epiblast for avian neural crest induction

Neural crest induction involves the combinatorial inputs of the FGF, BMP and Wnt signaling pathways. Recently, a two-step model has emerged where BMP attenuation and Wnt activation induces the neural crest during gastrulation, whereas activation of both pathways maintains the population during neurulation. FGF is proposed to act indirectly during the inductive phase by activating Wnt ligand expression in the mesoderm. This study used the chick model to investigate the role of FGF signaling in the amniote neural crest and uncovered a novel requirement for FGF/MAPK signaling. Contrary to current models, it was demonstrated that FGF is required within the prospective neural crest epiblast during gastrulation and is unlikely to operate through mesodermal tissues. Additionally, it was shown that FGF/MAPK activity in the prospective neural plate prevents the ectopic expression of lateral ectoderm markers, independently of its role in neural specification. The temporal participation of BMP/Smad signaling was investigated, and a later involvement in neural plate border development is suggested, likely due to widespread FGF/MAPK activity in the gastrula epiblast. These results identify an early requirement for FGF/MAPK signaling in amniote neural crest induction and suggest an intriguing role for FGF-mediated Smad inhibition in ectodermal development (Stuhlmiller, 2012).

Dynamic control of head mesoderm patterning

The embryonic head mesoderm gives rise to cranial muscle and contributes to the skull and heart. Prior to differentiation, the tissue is regionalised by the means of molecular markers. This pattern is shown to be established in three discrete phases, all depending on extrinsic cues. Assaying for direct and first-wave indirect responses, it was found that the process, analyzed in the chicken, is controlled by dynamic combinatorial as well as antagonistic action of retinoic acid (RA), Bmp and Fgf signalling. In phase 1, the initial anteroposterior (a-p) subdivision of the head mesoderm is laid down in response to falling RA levels and activation of Fgf signalling. In phase 2, Bmp and Fgf signalling reinforce the a-p boundary and refine anterior marker gene expression. In phase 3, spreading Fgf signalling drives the a-p expansion of bHLH transcription factor MyoR (musculin) and Tbx1 expression along the pharynx, with RA limiting the expansion of MyoR. This establishes the mature head mesoderm pattern with markers distinguishing between the prospective extra-ocular and jaw skeletal muscles, the branchiomeric muscles and the cells for the outflow tract of the heart (Bothe, 2011).

Expression of Fgf and Bmp responsive molecules indicated that the anterior head mesoderm receives Fgf and Bmp signals for the first time during phase 2 when Alx4 and MyoR are upregulated. Suppression of Bmp signalling prevented, and elevated Bmp signalling advanced, Alx4 activation. Thus, Bmp is necessary and sufficient to control Alx4. MyoR, however, was repressed by suppression of either Bmp or Fgf signalling. Elevation of Bmp or Fgf signalling promoted MyoR, albeit only at the stage at which the gene is normally expressed; premature MyoR expression could only be achieved by combinatorial application of Bmp and Fgf. Thus, combined Fgf and Bmp activity is required to activate MyoR (Bothe, 2011).

Expression analysis showed that the onset of MyoR is rather sudden. The bead implantation experiments indicated that in the anterior head mesoderm, Fgf enhanced the expression of Bmp responsive genes and Bmp upregulated genes indicative of active Fgf signalling. This suggests that Bmp and Fgf reinforce each other, possibly creating the appropriate setting to activate MyoR. Studies on mouse mutants placed Pitx2 upstream of MyoR. Thus, it is conceivable that, in addition to Bmp and Fgf, the earlier activation of Pitx2 is a further prerequisite for the activation of MyoR (Bothe, 2011).

In the posterior head mesoderm, Bmp strongly suppressed Tbx1. Fgf signalling, however, was unaffected, suggesting that Bmp controls the anterior border of Tbx1 expression, possibly directly targeting Tbx1. Tbx1, by contrast, has recently been suggested to suppress Bmp signalling by preventing Smad1-Smad4 interaction. This suggests that Tbx1 indirectly controls the extension of Bmp dependent markers (Bothe, 2011).

When Bmp and Fgf signalling commences in the anterior head mesoderm, Fgf signalling levels increase significantly in the posterior domain, owing to the positive Fgf-Tbx1 feedback loop. After applying Fgf to the anterior head mesoderm, i.e. elevating the Fgf level beyond that which is normally found there, it was noticed that Pitx2 and Alx4 expression declined. Thus, although Fgf is necessary for the activation of MyoR, high Fgf levels prevent the molecular set-up of the anterior head mesoderm. This infers that, whereas Bmp controls the anterior border of the posterior head mesoderm marker, Fgf controls the posterior border of the two anterior markers Pitx2 and Alx4 (Bothe, 2011).

In phase 3, extension of MyoR and Tbx1 expression is concomitant with the spread of high-level Fgf signalling along the floor of the pharynx. Fgf application was found to accelerate the MyoR-Tbx1 spread, and suppression of Fgf signalling prevented it. This suggests that Fgf signalling is key to establishing the final head mesoderm pattern. Notably, MyoR remained sensitive to RA. In the embryo, however, the site of RA production continuously recedes posteriorly during phases 2 and 3, suggesting that the posterior extension of MyoR expression occurs at a rate set by RA (Bothe, 2011).

The anteriorly spreading Fgf signals will eventually reach the Pitx2-Alx4 domain. Both genes were negatively regulated by high Fgf levels in phases 1 and 2; yet, in phase 3 the genes remain expressed. Likewise, Tbx1 spreads anteriorly although this territory is controlled by Bmp. Notably, Fgf levels vary along the anteroposterior extent of the pharynx; at HH13, for example, Fgf signalling appears lower in the anterior compared with the posterior pharyngeal arches. Thus, it is possible that in the anterior head mesoderm, Fgf levels might remain low enough to allow Pitx2 and Alx4 expression, but rise sufficiently to override the Bmp effect on Tbx1. Conversely, the Fgf levels in the posterior head mesoderm might by so high that MyoR expression can spread, whereas Pitx2 and Alx4 remain repressed. It cannot be excluded that additional signals restrict Pitx2 and Alx4 expression. Yet, the spread of MyoR outside of the Pitx2 territory indicates that in phase 3 MyoR expression has become independent from its former upstream regulator (Bothe, 2011).

RA, Bmp and Fgf signalling play multiple roles during development. RA, in many settings, promotes cell differentiation; in the head, RA first suppresses cardiac markers to set the posterior limit of the heart field, but then specifies the sinoatrial region of the heart. Moreover, RA has the capacity to provide cells with a more posterior positional identity. Bmp is a crucial regulator of cardiac development and has been suggested to recruit head mesodermal cells into the cardiac lineage. Fgf promotes the secondary heart field and keeps cells proliferative and undifferentiated. Therefore whether the observed changes in head mesodermal marker expression occurred because of cell recruitment into cardiac lineage, premature differentiation or posteriorisation was tested. RA or Fgf treatment was found not to change cell fate or differentiation status. Bmp induced cardiac marker gene expression only when applied during phase 0. When applied in phase 1, i.e. just before Bmp signalling is normally activated in the head mesoderm, Bmp did not induce cardiac markers unless the dosage was increased. This suggests that, possibly, cardiac induction can occur from exposure to higher Bmp levels and/or longer exposure times. Taken together, this study suggests that RA, Bmp and Fgf specifically control head mesoderm patterning with the cells remaining undifferentiated and competent to enter any of the possible mesodermal lineages (Bothe, 2011).

Male-to-female sex reversal in mice lacking Fibroblast growth factor 9

Fgfs direct embryogenesis of several organs, including the lung, limb, and anterior pituitary. Male-to-female sex reversal occurs in mice lacking Fibroblast growth factor 9 (Fgf9), demonstrating a novel role for FGF signaling in testicular embryogenesis. Fgf9-/- mice also exhibit lung hypoplasia and die at birth. Reproductive system phenotypes range from testicular hypoplasia to complete sex reversal, with most Fgf9-/- XY reproductive systems appearing grossly female at birth. Fgf9 appears to act downstream of Sry to stimulate mesenchymal proliferation, mesonephric cell migration, and Sertoli cell differentiation in the embryonic testis. While Sry is found only in some mammals, Fgfs are highly conserved. Thus, Fgfs may function in sex determination and reproductive system development in many species (Colvin, 2001a).

Male and female mouse gonads at embryonic day 11.0 (E11.0) are morphologically identical in different gonads medial to each mesonephros. By E13.5, the testis is twice the size of the ovary and exhibits morphologically complex testicular cords. Three male-specific events are known to direct early testiculogenesis: cell proliferation, cell migration, and testicular cord formation. An increase in proliferation at the coelomic lining of the gonad (the coelomic epithelium) occurs between E11.3 and E12.1. This proliferation gives rise to Sertoli cells (a supporting cell lineage) early on and to interstitial cells throughout this period. Cells contributing to the interstitium, including vascular endothelial cells and peritubular myoid cells, migrate into the testis from the mesonephros and are required for testicular cord formation. Testicular cord development begins at about E12.0 with clustering of Sertoli and germ cells, followed by rearrangement so that Sertoli cells surround the germ cells. Testicular cords isolate male germ cells from interstitial cells, and prevent male germ cells from entering meiosis. Ovarian germ cells, which are not enclosed by supporting cells, progress by E13.5 to the first meiotic division (Colvin, 2001a and references therein).

The testis regulates further male reproductive development. Until E13.5, both sexes have Mullerian and Wolffian ducts in each mesonephros. Sertoli cells produce Mullerian inhibiting substance (MIS). MIS causes regression of the Mullerian ducts, which, in the absence of MIS, form the oviducts, uterus, and upper vagina. Interstitial Leydig cells produce testosterone, which induces formation of Wolffian duct derivatives, including the epididymis, vas deferens, and seminal vesicles. In females, the absence of testicular MIS and testosterone results in development of Mullerian structures and regression of the Wolffian ducts. Targeted deletion of Mis or its receptor results in development of Mullerian structures in XY mice (Colvin, 2001a and references therein).

Testicular expression of Sry, a transcription factor gene on the Y chromosome, is essential for increased proliferation in, and mesonephric cell migration into, the mouse testis. Sry is expressed in mouse testis between E10.5 and E12.5 and is necessary and sufficient to induce male development. Deletion of Sry generates XY ovaries and mice with a female phenotype, and addition of an Sry transgene generates XX males. A potential downstream target of Sry is Sox9, an autosomal transcription factor expressed in Sertoli cells. Mutations in SRY and SOX9 have been identified in human XY females with gonadal dysgenesis (Colvin, 2001a and references therein).

Fgf9 appears to act downstream of Sry, but the signaling relationship between Sox9 and Fgf9 is unclear. Sry is essential for each mode of mesenchymal expansion in the early testis: proliferation and mesonephric cell migration. Thus, reduced mesenchyme in Fgf9-/- XY gonads suggests that Sry and Fgf9 act along the same developmental pathway. Testicular Fgf9 expression begins shortly after the onset of Sry expression at E10.5, consistent with Fgf9 acting downstream of Sry. Some Fgf9-/- XY gonads exhibit aberrant Sox9 expression, but Fgf9 is not required to induce Sox9 expression in the testis or to maintain Sox9 expression through E18.5. Analysis of Fgf9 expression in Sox9-deficient gonads would determine if Sox9 is required to induce testicular Fgf9 expression. Unfortunately, Sox9 heterozygous mice die at birth precluding the generation of homozygous mutant embryos, and embryos derived by introducing Sox9 homozygous mutant ES cells into tetraploid blastocysts, die by E11.5. Correlation between testicular cord formation and Sox9 expression in Fgf9-/- XY gonads suggests that Fgf9 may regulate Sox9 expression indirectly by facilitating testicular development (Colvin, 2001a).

Fgf9 affects early steps in testiculogenesis, including Sertoli cell development, gonadal cell proliferation, and mesonephric cell migration. Pre-Sertoli cells originate from multipotential cells in the coelomic epithelium and proliferate at the coelomic epithelium between E11.3-E11.5. Impaired Fgf9-/- Sertoli cell development suggests that Fgf9 could directly induce Sertoli cell specification, proliferation, and/or maintenance of differentiation. Loss of signaling from Sertoli cells could then secondarily impair mesenchymal proliferation and mesonephric cell migration. Full Sertoli cell differentiation probably requires testicular cord formation, and maintenance of Sertoli differentiation may require contact with peritubular myoid cells and the basal lamina. Thus, Fgf9 could also facilitate Sertoli cell differentiation by promoting mesenchymal expansion and testicular cord formation (Colvin, 2001a).

Proliferation at the coelomic epithelium gives rise to Sertoli and interstitial cells during an initial burst of proliferation (E11.3-E11.5), and to interstitial cells after this time. Proliferation below the coelomic epithelium in E12.5 Fgf9-/- XY gonads is reduced relative to controls, indicating that Fgf9 is essential for normal proliferation at this stage. Decreased numbers of Sertoli and interstitial cells are observed in Fgf9-/- gonads by E12.5. This, and the onset of testicular Fgf9 expression between E10.5-E11.5, suggests that Fgf9 may mediate the initial stage of proliferation as well (Colvin, 2001a).

Mesonephric cell migration into the testis at E11.3-E16.5 contributes to interstitial cell populations, including vascular endothelial, myoepithelial, and peritubular myoid cells. Exogenous FGF9 induces mesonephric cell migration into E11.5 XX gonads, suggesting that FGF9 in the early testis could act as a chemotactic factor for mesonephric cells. When mesonephric migration into XX gonads is artificially induced, XX gonads exhibit testicular cord formation and increased Sox9 expression. Conversely, blocking mesonephric cell migration in culture impairs testicular cord formation, indicating that impaired mesonephric cell migration could contribute to Fgf9-/- sex reversal. Analysis of mesonephric cell migration into Fgf9-/- XY gonads will test this hypothesis. Mesonephric cells that migrate into the testis are proliferating, suggesting that one molecular signal could induce both migration and proliferation. In the embryonic lung, FGF10 stimulates both migration and proliferation of epithelial cells (Colvin, 2001a).

Fgf9 induces proliferation and nuclear localization of FGFR2 in Sertoli precursors during male sex determination

Loss of Fgf9 has been shown to result in a block of testis development and a male to female sex-reversed phenotype; however, the function of Fgf9 in sex determination was unknown. Fgf9 is now shown to be necessary for two steps of testis development just downstream of the male sex-determining gene, Sry: (1) for the proliferation of a population of cells that give rise to Sertoli progenitors; and (2) for the nuclear localization of an FGF receptor (FGFR2) in Sertoli cell precursors. The nuclear localization of FGFR2 coincides with the initiation of Sry expression and the nuclear localization of SOX9 during the early differentiation of Sertoli cells and the determination of male fate (Schmahl, 2004).

It has been known for some time that many cell-surface growth factor receptors can accumulate within the nucleus. However, the biological relevance of this event is not known. It has been speculated that nuclear growth factor receptors may act as weak transcription factors, topoisomerases and/or nuclear kinases. Nuclear FGF receptors have been observed in spliceosomes. This is particularly interesting, since several components of the sex-determination pathway (including SRY, SOX9 and the +KTS isoform of WT) have been shown to associate with splicing factors, and have been demonstrated to have splicing activity. Another function of nuclear FGF receptors may be to phosphorylate nuclear substrates; forced nuclear translocation of FGF receptors leads to an increase in the phosphorylation of nuclear proteins, and some activities of the nuclear receptor are abolished by deactivation of the kinase domain. The discovery of the sex-specific subcellular localization of FGFR2 in the nuclei of Sertoli precursors provides a well-characterized biological context in which to study the function of nuclear growth factor receptors. In this context, the transition of FGFR2 from the cell membrane to the nucleus suggests that the nuclear localization of cell-surface receptors is linked to the initiation of cell differentiation. It is not yet clear how proliferation of Sertoli cell precursors in the coelomic epithelium and subsequent commitment to the Sertoli fate are interwoven; however, these findings suggest that FGF signaling may be involved in bridging these two processes essential to testis development (Schmahl, 2004).

In mouse embryogenesis, Sry is transiently activated in a center-to-pole wavelike manner along the anteroposterior (AP) axis of developing XY gonads. However, the mechanism and significance of the center-to-pole expansion of testis initiation pathways downstream of Sry expression remain unclear. This study demonstrates that FGF9 can act as a diffusible conductor for a poleward expansion of tubulogenic programs at early phases of testis differentiation. In XY genital ridge cultures of anterior, middle and posterior segments at 11.0-11.25 days post-coitum, male-specific activation of Sry and its target gene, Sox9, was still observed in both anterior and posterior pole segments despite their isolation from the central domain. However, high-level Sox9 expression was not maintained, resulting in the failure of testis cord organization in most pole segments. A reconstruction experiment using ROSA:lacZ middle segments showed rescue of the tubulogenic defect in the poles without any appreciable contribution of lacZ-positive gonadal parenchyma cells. A partition culture assay also showed a possible contribution of soluble/diffusible factors secreted from the gonadal center domain to proper tubulogenesis in the poles. Among various signaling factors, Fgf9 expression was significantly lower in both anterior and posterior pole segments than in the central domain. The supportive role of the central domain could be substituted by exogenous FGF9 supply, whereas reduction of Wnt4 activity did not rescue the tubulogenesis defect in the pole segments. These observations imply that center-to-pole FGF9 diffusion directs a poleward expansion of testiculogenic programs along the AP axis of developing XY gonads (2010).

The role of Fgf10 signaling in branching morphogenesis and gene expression of the rat prostate gland: lobe-specific suppression by neonatal estrogens

Brief exposure of rats to high-dose estrogen during the neonatal period interrupts prostate development in a lobe-specific manner and predisposes the gland to dysplasia with aging, a phenomenon referred to as developmental estrogenization. These effects are initiated through altered steroid receptor expression; however, the immediate downstream targets remain unclear. Developmental expression of Shh-ptc-gli has been shown to be downregulated in the dorsolateral prostate following estrogenization, and this is responsible, in part, for branching deficits observed in that prostatic region specifically. In the present study, the roles were examined of Fgf10 signaling during rat prostate development and as a mediator of the developmental estrogenized phenotype. Fgf10 and Fgf R2iiib localize to the distal signaling center of elongating and branching ducts in separate prostate lobes where they regulate the expression of multiple morphoregulatory genes including Shh, ptc, Bmp7, Bmp4, Hoxb13, and Nkx3.1. Ventral and lateral lobe organ cultures and mesenchyme-free ductal cultures demonstrate a direct role for Fgf10/FgfR2iiib in ductal elongation, branching, epithelial proliferation, and differentiation. Based on these findings, a model is proposed depicting the localized expression and feedback loops between several morphoregulatory factors in the developing prostate that contribute to tightly regulated branching morphogenesis. Similar to Shh-ptc-gli, neonatal estrogen exposure downregulates Fgf10, Fgf R2iiib, and Bmp7 expression in the dorsolateral prostate while ventral lobe expression of these genes is unaffected. Lateral prostate organ culture experiments demonstrate that growth and branching inhibition as well as Fgf10/FgfR2iiib suppression are mediated directly at the prostatic level. Furthermore, exogenous Fgf 10 fully rescues the growth and branching deficits due to estrogen exposure. Together, these studies demonstrate that alterations in Fgf10 signaling are a proximate cause of Shh-ptc-gli and Bmp7 downregulation that together result in branching inhibition of the dorsolateral prostate following neonatal estrogen exposure (Huang, 2005).

A crucial role for Fgfr2-IIIb signalling in epidermal development and hair follicle patterning

To understand the role Fgf signalling in skin and hair follicle development, the phenotype was analyzed of mice deficient for Fgfr2-IIIb and its main ligand Fgf10. These studies show that the severe epidermal hypoplasia found in mice null for Fgfr2-IIIb is caused by a lack of the basal cell proliferation that normally results in a stratified epidermis. Although at term the epidermis of Fgfr2-IIIb null mice is only two to three cells thick, it expresses the classical markers of epidermal differentiation and establishes a functional barrier. Mice deficient for Fgf10 display a similar but less severe epidermal hypoplasia. By contrast, Fgfr2-IIIb^–/– (but not Fgf10^–/–) mice produce significantly fewer hair follicles, and their follicles were developmentally retarded. Following transplantation onto nude mice, grafts of Fgfr2-IIIb^–/– skin show impaired hair formation, with a decrease in hair density and the production of abnormal pelage hairs. Expression of Lef1, Shh and Bmp4 in the developing hair follicles of Fgfr2-IIIb^–/– mice is similar to wild type. These results suggest that Fgf signalling positively regulates the number of keratinocytes needed to form a normal stratified epidermis and to initiate hair placode formation. In addition, Fgf signals are required for the growth and patterning of pelage hairs (Petiot, 2003).

FGF signaling is required for initiation of feather placode development

Morphogenesis of hairs and feathers is initiated by an as yet unknown dermal signal that induces placode formation in the overlying ectoderm. To determine whether FGF signals are required for this process soluble versions of FGFR1 or FGFR2 were overexpressed in the skin of chicken embryos. This produced a complete failure of feather formation prior to any morphological or molecular signs of placode development. Fgf10 is expressed in the dermis of nascent feather primordia, and anti-FGF10 antibodies block feather placode development in skin explants. In addition FGF10 can induce expression of positive and negative regulators of feather development and can induce its own expression under conditions of low BMP signaling. Together these results demonstrate that FGF signaling is required for the initiation of feather placode development and implicate FGF10 as an early dermal signal involved in this process (Mandler, 2004).

WNT5A/JNK and FGF/MAPK pathways regulate the cellular events shaping the vertebrate limb bud

The vertebrate limb is a classical model for understanding patterning of three-dimensional structures during embryonic development. Although decades of research have elucidated the tissue and molecular interactions within the limb bud required for patterning and morphogenesis of the limb, the cellular and molecular events that shape the limb bud itself have remained largely unknown. This study shows that the mesenchymal cells of the early limb bud are not disorganized within the ectoderm as previously thought but are instead highly organized and polarized. Using time-lapse video microscopy, it was demonstrated that cells move and divide according to this orientation. The combination of oriented cell divisions and movements drives the proximal-distal elongation of the limb bud necessary to set the stage for subsequent morphogenesis. These cellular events are regulated by the combined activities of the WNT and FGF pathways. WNT5A/JNK is necessary for the proper orientation of cell movements and cell division. In contrast, the FGF/MAPK signaling pathway, emanating from the apical ectodermal ridge, does not regulate cell orientation in the limb bud but instead establishes a gradient of cell velocity enabling continuous rearrangement of the cells at the distal tip of the limb. Together, these data shed light on the cellular basis of vertebrate limb bud morphogenesis and uncover new layers to the sequential signaling pathways acting during vertebrate limb development (Gros, 2010).

Vertebrate limb bud formation is initiated by localized epithelial-to-mesenchymal transition

Vertebrate limbs first emerge as small buds at specific locations along the trunk. Although a fair amount is known about the molecular regulation of limb initiation and outgrowth, the cellular events underlying these processes have remained less clear. This study shows that the mesenchymal limb progenitors arise through localized epithelial-to-mesenchymal transition (EMT) of the coelomic epithelium specifically within the presumptive limb fields. This EMT is regulated at least in part by Tbx5 and Fgf10, two genes known to control limb initiation. This work shows that limb buds initiate earlier than previously thought, as a result of localized EMT rather than differential proliferation rates (Gros, 2014).

Glypican-3 modulates BMP- and FGF-mediated effects during renal branching morphogenesis

The kidney of the Gpc3-/- mouse, a novel model of human renal dysplasia, is characterized by selective degeneration of medullary collecting ducts preceded by enhanced cell proliferation and overgrowth during branching morphogenesis. Cellular and molecular mechanisms underlying this renal dysplasia have been identified. Glypican-3 (GPC3) deficiency is associated with abnormal and contrasting rates of proliferation and apoptosis in cortical (CCD) and medullary collecting duct (MCD) cells. In CCD, cell proliferation is increased threefold. In MCD, apoptosis was increased 16-fold. Expression of Gpc3 mRNA in ureteric bud and collecting duct cells suggests that GPC3 can exert direct effects in these cells. Indeed, GPC3 deficiency abrogates the inhibitory activity of BMP2 on branch formation in embryonic kidney explants, converts BMP7-dependent inhibition to stimulation, and enhances the stimulatory effects of KGF (FGF-7). Similar comparative differences are found in collecting duct cell lines derived from GPC3-deficient and wild type mice and induced to form tubular progenitors in vitro, suggesting that GPC3 directly controls collecting duct cell responses. It is proposed that GPC3 modulates the actions of stimulatory and inhibitory growth factors during branching morphogenesis (Grisaru, 2001).

The molecular basis for observations in ureteric bud and collecting duct cells remains to be determined. The demonstration that bFGF forms a molecular complex with cell surface heparan sulfate and the FGF cell surface receptor suggests that GPC3 may physically interact with receptors that bind BMP2, BMP7, and KGF. The opposite response of collecting duct cells to GPC3 deficiency, that is, inhibition of BMP2 activity and enhancement of KGF activity, suggests that the consequences of these interactions may differ. A second possibility is that GPC3 may function via independent signaling pathways that physically interact at the postreceptor level with BMP and KGF signaling intermediates. Alternatively, the GPC3 and growth factor-signaling pathways may interact indirectly by regulating competing or complementary gene products. Increasing evidence regarding the nature of inhibitory and stimulatory BMP-dependent signaling pathways in collecting duct cells provides a basis to determine the nature of GPC3 interactions with BMP2 and BMP7 (Grisaru, 2001).

Conditional ablation of Pten in osteoprogenitors stimulates FGF signaling

PTEN is a direct antagonist of phosphatidylinositol 3 kinase. Pten is a well recognized tumor suppressor and is one of the most commonly mutated genes in human malignancies. More recent studies of development and stem cell behavior have shown that PTEN regulates the growth and differentiation of progenitor cells. Significantly, PTEN is found in osteoprogenitor cells that give rise to bone-forming osteoblasts; however, the role of PTEN in bone development is incompletely understood. To define how PTEN functions in osteoprogenitors during bone development, Pten was conditionally deleted in mice using the cre-deleter strain Dermo1cre, which targets undifferentiated mesenchyme destined to form bone. Deletion of Pten in osteoprogenitor cells led to increased numbers of osteoblasts and expanded bone matrix. Significantly, osteoblast development and synthesis of osteoid in the nascent bone collar was uncoupled from the usual tight linkage to chondrocyte differentiation in the epiphyseal growth plate. The expansion of osteoblasts and osteoprogenitors was found to be due to augmented FGF signaling as evidenced by (1) increased expression of FGF18, a potent osteoblast mitogen, and (2) decreased expression of SPRY2, a repressor of FGF signaling. The differentiation of osteoblasts was autonomous from the growth plate chondrocytes and was correlated with an increase in the protein levels of GLI2, a transcription factor that is a major mediator of hedgehog signaling. Evidence is provided that increased GLI2 activity is also a consequence of increased FGF signaling through downstream events requiring mitogen-activated protein kinases. To test whether FGF signaling is required for the effects of Pten deletion, one allele of fibroblast growth factor receptor 2 (FGFR2) was deleted. Significantly, deletion of FGFR2 caused a partial rescue of the Pten-null phenotype. This study identifies activated FGF signaling as the major mediator of Pten deletion in osteoprogenitors (Guntur, 2011).

Tbx1 has a dual role in the morphogenesis of the cardiac outflow tract

Dysmorphogenesis of the cardiac outflow tract (OFT) causes many congenital heart defects, including those associated with DiGeorge syndrome. Genetic manipulation in the mouse and mutational analysis in patients have shown that Tbx1, a T-box transcription factor, has a key role in the pathogenesis of this syndrome. Tbx1 function during OFT development have been dissected using genetically modified mice and tissue-specific deletion, and have defined a dual role for this protein in OFT morphogenesis. Tbx1 regulates cell contribution to the OFT by supporting cell proliferation in the secondary heart field, a source of cells fated to the OFT. This process might be regulated in part by Fgf10, which is a direct target of Tbx1 in vitro. Tbx1 expression is required in cells expressing Nkx2.5 for the formation of the aorto-pulmonary septum, which divides the aorta from the main pulmonary artery. These results explain why aortic arch patterning defects and OFT defects can occur independently in individuals with DiGeorge syndrome. Furthermore, the data link the function of the secondary heart field to congenital heart disease (Xu, 2004).

Foxp1 coordinates cardiomyocyte proliferation through both cell-autonomous and nonautonomous mechanisms

Cardiomyocyte proliferation is high in early development and decreases progressively with gestation, resulting in the lack of a robust cardiomyocyte proliferative response in the adult heart after injury. Little is understood about how both cell-autonomous and nonautonomous signals are integrated to regulate the balance of cardiomyocyte proliferation during development. This study shows that a single transcription factor, Foxp1, can control the balance of cardiomyocyte proliferation during development by targeting different pathways in the endocardium and myocardium. Endocardial loss of Foxp1 results in decreased Fgf3/Fgf16/Fgf17/Fgf20 expression in the heart, leading to reduced cardiomyocyte proliferation. This loss of myocardial proliferation can be rescued by exogenous Fgf20, and is mediated, in part, by Foxp1 repression of Sox17. In contrast, myocardial-specific loss of Foxp1 results in increased cardiomyocyte proliferation and decreased differentiation, leading to increased myocardial mass and neonatal demise. Nkx2.5 is a direct target of Foxp1 repression, and Nkx2.5 expression is increased in Foxp1-deficient myocardium. Moreover, transgenic overexpression of Nkx2.5 leads to increased cardiomyocyte proliferation and increased ventricular mass, similar to the myocardial-specific loss of Foxp1. These data show that Foxp1 coordinates the balance of cardiomyocyte proliferation and differentiation through cell lineage-specific regulation of Fgf ligand and Nkx2.5 expression (Zhang, 2010).

Onset of neuronal differentiation is regulated by paraxial mesoderm and requires attenuation of FGF signaling

While many neuronal differentiation genes have been identified, little is known about what determines when and where neurons will form and how this process is coordinated with the differentiation of neighboring tissues. In most vertebrates the onset of neuronal differentiation takes place in the spinal cord in a head to tail sequence. The changing signaling properties of the adjacent paraxial mesoderm control the progression of neurogenesis in the chick spinal cord. An inverse relationship is found between the expression of caudal neural genes in the prospective spinal cord, which is maintained by underlying presomitic mesoderm and FGF signaling, and neuronal differentiation, which is repressed by such signals and accelerated by somitic mesoderm. Key to this interaction is the ability of somitic mesoderm to repress Fgf8 transcription in the prospective spinal cord. These findings further indicate that attenuation of FGF signaling in the prospective spinal cord is a prerequisite for the onset of neuronal differentiation and may also help to resolve mesodermal and neural cell fates. However, inhibition of FGF signaling alone does not promote the formation of neurons, which requires still further somite signaling. A model is proposed in which signaling from somitic tissue promotes the differentiation of the spinal cord and serves to co-ordinate neural and mesodermal development (Diez del Corral, 2002).

Two lines of evidence are presented that strongly suggest that signals from the differentiating somitic mesoderm regulate the onset of neuronal differentiation in the developing spinal cord: (1) somitic signals accelerate the appearance of neurons in caudal neural plate (CNP) explants, as indicated by the swift onset of Delta 1 in single cells, NeuroM expression and the appearance of cells with neurofilament-positive fine processes; and (2) these signals are also required in vivo for the normal onset of neuronal differentiation, as revealed by the strikingly few NeuroM-positive cells in neural tube forming in the absence of the differentiating somitic mesoderm. Removal of somites flanking the later neural tube also depletes the number of NeuroM-expressing cells, while Sox2 and Delta 1 expression remain unaltered, indicating that there is a continuing requirement for somite signals for neurogenesis progression. However, it is likely that somites become dispensable for the production of neurons at later stages, because their removal at stages 12-16 does not alter the number of motor neurons. This suggests that the influence of somite-derived factors is confined to the first born neurons (future reticular and spinal interneurons). Since explants of the neural tube readily form neurons in vitro, this requirement for somite signals in vivo suggests that these signals normally act to oppose other signals present in the neural tube that repress neuronal differentiation (Diez del Corral, 2002).

Evidence is also presented that somitic signals repress the caudal neural gene cash4, consistent with its down regulation in vivo prior to neuronal differentiation. Sax1 expression was not consistently altered by such signals. This apparent difference in regulation may reflect the later onset of Sax1 expression or its regulation by other signals in the embryo. Indeed, removal of the presomitic mesoderm in vivo leads to the loss of cash4 and Sax1, suggesting that during normal development repression of caudal neural genes is mediated by a combination of 1) signal loss, as the presomitic mesoderm differentiates, and 2) exposure to signals from the newly formed somites. In this context it is striking that removal of the presomitic mesoderm also leads to the precocious appearance of NeuroM-positive cells in the preneural tube in a small number of cases. This suggests that the presomitic mesoderm not only maintains caudal neural genes, but also represses neuronal differentiation. This in vivo situation contrasts with CNP explants cultured alone in vitro (for 24 hours), which maintain cash4 and Sax1 and do not contain neurons. This difference may be explained if mesoderm cells in CNP explants (which in vivo would have been displaced during gastrulation) provide signals that maintain caudal neural genes and repress NeuroM. In addition other tissues/signals may be present in the embryo, which normally oppose signals from the presomitic mesoderm and which act swiftly following removal of this tissue in vivo. These opposing signals could be provided by the notochord/floor plate at the ventral midline and/or by abutting NeuroM-positive spinal cord (which has been exposed to somites). Together, these findings indicate that the onset of neuronal differentiation in the spinal cord is regulated by a balance between signals provided by the presomitic mesoderm and the somites and that both these tissues may act in the embryo to counter opposing signals (Diez del Corral, 2002).

Specific regulation of cyclins D1 and D2 by FGF and Shh signaling coordinates cell cycle progression, patterning, and differentiation during early steps of spinal cord development

In the vertebrate embryo, spinal cord elongation requires FGF signaling that promotes the continuous development of the posterior nervous system by maintaining a stem zone of proliferating neural progenitors. Those escaping the caudal neural stem zone initiate ventral patterning in the neural groove before starting neuronal differentiation in the neural tube. The integration of D-type cyclins, known to govern cell cycle progression under the control of extracellular signals, in the program of spinal cord maturation was investigated. In chicken embryo, it was found that cyclin D2 is preferentially expressed in the posterior neural plate, whereas cyclin D1 appears in the neural groove. Loss- and gain-of-function experiments demonstrate that FGF signaling maintains cyclin D2 in the immature caudal neural epithelium, while Shh activates cyclin D1 in the neural groove. Moreover, forced maintenance of cyclin D1 or D2 in the neural tube favors proliferation at the expense of neuronal differentiation. These results contribute to the understanding of how the cell cycle control can be linked to the patterning programs to influence the balance between proliferation and neuronal differentiation in discrete progenitors domains (Labjois, 2004)

Initiation of neural induction by FGF signaling before gastrulation

During neural induction, the 'organizer' of the vertebrate embryo instructs neighboring ectodermal cells to become nervous system rather than epidermis. This process is generally thought to occur around the mid-gastrula stage of embryogenesis. The isolation of ERNI, an early response gene to signals from the organizer (Hensen's node), is reported in this study. Using ERNI as a marker, evidence is provided that neural induction begins before gastrulation -- much earlier in development than previously thought. The organizer and some of its precursor cells produce a fibroblast growth factor signal, which can initiate, and is required for, neural induction (Streit, 2000).

The prevailing model for neural induction suggests that cells differentiate into neural fates by default, but are normally inhibited by bone morphogenetic proteins (BMPs). The organizer, by emitting BMP antagonists, allows cells in its vicinity to execute their default neural program. However, other work suggests a more complex mechanism. In the chick embryo, naive epiblast cells do not respond to BMP antagonists unless previously exposed to organizer signals for five hours. A differential screen has been designed for genes that are induced in the epiblast by a grafted organizer within this time period. Among the complementary DNAs isolated is the gene ERNI (for early response to neural induction); it is not homologous to any known sequence and contains a putative coiled-coil domain and tyrosine phosphorylation site. When transfected into COS cells, ERNI protein is found throughout the cytoplasm in most cells, but is restricted to the nucleus in about 10% of cells, which are invariably smaller and fibroblast-like. The predicted structure and subcellular localization suggest that ERNI could be part of a protein complex that travels from the cytoplasm to the nucleus (Streit, 2000).

Induction is defined as "an interaction between two tissues, as a result of which the responding cells change their fate." To ensure that the responses to the grafted organizer are due to induction, rather than recruitment of cells from the neural plate, the screen was designed using the extra-embryonic region, which does not normally contribute to the nervous system. Therefore any gene identified in this screen that is relevant to neural induction should be expressed at some stage in the prospective neural plate of the normal embryo. Indeed, cERNI begins to be expressed at pre-primitive streak stages, throughout the region that will contribute to the nervous system; by streak stages, its distribution coincides with the known limits of the prospective neural plate. Shortly thereafter, expression clears from the center of the neural plate and becomes confined to its border; transcripts disappear by early somite stages. A quail Hensen's node induces cERNI expression in chick extra-embryonic epiblast in as little as 1-2 h. By 5h cERNI induction is most intense, and by 8h it begins to clear from the center, forming a ring resembling its normal expression at the border of the neural plate. These findings make cERNI the earliest known marker for a response to organizer signals, even earlier than Sox3 (whose early expression it resembles and which is induced by the node in 3 h (Streit, 2000).

Which signaling molecules from the organizer are responsible for inducing cERNI? One approach to identifying candidate factors is to assess the ability of embryonic tissues to induce cERNI, to map the distribution of inducing factors. Head process, notochord, prechordal mesendoderm (stage 5; 14/14) and presomitic mesoderm all induce cERNI, whereas other tissues tested have either reduced or no ability to generate ectopic cERNI expression. This distribution of inducing ability is reminiscent of sites of fibroblast growth factor (FGF) activity. Indeed, FGF8-coated beads induce cERNI expression as strongly and as quickly as does the node, within 1-2 h, without inducing the mesodermal markers brachyury or Tbx6L. In contrast, ectopic expression of cERNI is never observed after misexpression of the BMP antagonists chordin, noggin or cerberus, or of BMP4 or hepatocypte growth factor/scatter factor (HGF/SF). FGF8 also strongly induces the expression of Sox3, but not the later neural marker Sox-2. Together, these findings implicate FGFs as possible early signals in the neural induction cascade. Of the members of this family, FGF8 is the best candidate endogenous inducer because at primitive streak stages it is expressed in the anterior part of the streak including the node, and is downregulated as the node begins to lose neural inducing ability (Streit, 2000).

Is FGF expression in Hensen's node required to induce cERNI and  Sox3? Two different loss-of-function approaches have been used: the FGF-receptor inhibitor SU5402, which specifically interferes with the FGF signaling pathway, and cells secreting the extracellular portion of the FGF receptor. SU5402 greatly reduced the frequency of induction by a grafted node of Sox3 and of cERNI. Moreover, cells secreting chimaeric FGF receptor markedly reduced induction by the node of Sox3 and of cERNI. A marked reduction in the level of expression of both cERNI and Sox3 occurs in the normal neural domain of the host embryo in the presence of FGF inhibitors (Streit, 2000).

These findings suggest that neural induction is initiated before the beginning of gastrulation by FGF emanating from a population of organizer precursors at the posterior margin of the embryo (perhaps reinforced by the spreading hypoblast). The coming together of this cell population with a second precursor population in the epiblast generates a fully functional organizer that provides the remaining signals in a cascade, including BMP antagonists. These results provide an explanation for the hitherto unexplained finding that in Xenopus, BMP antagonists do not induce neural tissue in the presence of dominant-negative FGF receptors and for controversial reports of FGF as a direct neural inducer. FGF signals are clearly not sufficient to generate a complete nervous system. But are they sufficient to sensitize the epiblast to BMP antagonists and to generate expression of later neural markers? The finding that msx1 is upregulated by FGF8 raises the possibility that this is part of a mechanism that leads to self-maintaining activation of BMP signaling, which would be a required first step if the BMPs are later to be inhibited. In contrast, neither FGF nor 5 h of signals from the node followed by BMP antagonists is sufficient to generate induction of Sox2 or later neural markers. It is proposed that neural induction is a multi-step process of considerable complexity. FGF mimics the first 5 h of signals from the organizer, but further steps remain to be discovered (Streit, 2000).

Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless

Fgf-10-deficient mice (Fgf-10(-/-)) were generated to determine the role(s) of Fgf-10 in vertebrate development. Limb bud initiation was abolished in Fgf-10(-/-) mice. Strikingly, Fgf-10-/- fetuses continue to develop until birth, despite the complete absence of both forelimbs and hindlimbs. Fgf-10 is necessary for apical ectodermal ridge (AER) formation and acts epistatically upstream of Fgf-8, the earliest known AER marker in mice. Fgf-10-/- mice exhibit perinatal lethality associated with complete absence of lungs. Although tracheal development is normal, main-stem bronchial formation, as well as all subsequent pulmonary branching morphogenesis, is completely disrupted. Lack of lungs and main-stem bronchi in Fgf-10-/- mice is very reminiscent of the Drosophila mutant bnl. Both genetic and biochemical evidence indicates that Bnl is a ligand for Breathless (Btl), an Fgf receptor. Drosophila btl mutants exhibited a phenotype that was very similar to that of bnl. Mammalian Fgfr2b is highly expressed in the epithelium throughout embryonic lung development. Transgenic mice expressing a dominant-negative form of FGFR2b splice variant under control of the SP-C promotor exhibit perinatal lethality and failed to develop lungs, indicating that FGFR2b is a receptor necessary for pulmonary branching morphogenesis. Ligands of this receptor include FGF-1, FGF-7, and FGF-10, and all three have been shown to promote expansion and/or budding of endodermal cells in lung explant studies. Fgfr2b transgenic mice exhibit trachea formation and bifurcation of main-stem bronchi, unlike the Fgf-10 knockout mice, which only develop a trachea without further branching. This difference was most likely caused by spatial and temporal differences between Fgf-10 expression and SP-C promotor activity. The SP-C promotor drives transgene expression in distal lung epithelium starting at E10. In contrast, Fgf-10 is already expressed in the distal mesenchymal cells of developing respiratory tract buds at E9.5. Presumably, by E10-E10.5, the formation of the primordial bronchi has already occurred. The impaired pulmonary development observed in Fgfr2b transgenic mice, coupled with similarities in pulmonary phenotypes of Fgf-10 knockout mice and Drosophila bnl and btl mutants, suggests striking functional similarities in the signaling pathways of mammalian Fgf-10 and Drosophila bnl (Min, 1998).

FGF signaling and the anterior neural induction in Xenopus

FGF is capable of inducing Xenopus gastrula ectoderm cells in culture to express position-specific neural markers along the anteroposterior axis in a dose-dependent manner. However, conflicting results have been obtained concerning involvement of FGF signaling in the anterior neural induction in vivo using the same dominant-negative construct of Xenopus FGF receptor type-1 (delta XFGFR-1 or XFD) as was used in the in vitro study. This issue has been explored by employing in addition a similar construct of another receptor, XFGFR-4a, since expression of XFGFR-4a is seen to peak between gastrula and neurula stages, when the neural induction and patterning take place, whereas expression of XFGFR-1 does not have a distinct peak during that period. Further, these two FGFRs are distantly related within the Xenopus FGFR family. When mRNA of a dominant-negative version of XFGFR-4a (delta XFGFR-4a) is injected into eight animal pole blastomeres at the 32-cell stage, anterior defects including loss of normal structure in telencephalon and eye regions become prominent as examined morphologically or by in situ hybridization. Overexpression of delta XFGFR-1 appears far less effective than that of delta XFGFR-4a. Requirement of FGF signaling in ectoderm for anterior neural development was further confirmed in culture: when ectoderm cells that overexpress delta XFGFR-4a are cocultured with intact organizer cells from either early or late gastrula embryos, expression is inhibited of anterior and posterior neural markers, respectively. delta XFGFR-4 strongly suppresses autonomous neuralization of the anterior-type observed in ectoderm cells that have been subjected to prolonged dissociation. This suppression is even stronger than that exerted by delta XFGFR-1. It is thus indicated that FGF signaling in ectoderm, mainly through XFGFR-4, is required for the anterior neural induction by organizer (Hongo, 1999).

The present results seem, at first glance, inconsistent with the neural default model, recently proposed for the molecular mechanism of neural induction. This model features central roles for BMP signaling within ectoderm and for BMP antagonists, such as noggin and chordin, secreted by the organizer. BMP signaling alone induces ectoderm to form epidermis and suppresses ectoderm neuralization, whereas noggin and chordin work by locally antagonizing the BMP signaling; they act by directly binding BMP4 to prevent it from activating its receptor; this allows dorsal ectoderm to follow its default neural fate. It is argued that neural fate, specifically anterior neural fate, is the default fate of gastrula ectoderm in the sense that the neural induction, at least its initial step, requires only the absence of epidermal-inducing signals. However, the present observations indicate that the presence of FGF signaling in ectoderm is also required for neural induction. One basis for the neural default model is the finding that ectodermal cells, subjected to prolonged dissociated culture during gastrula stages, form histologically recognizable neural tissue after reaggregation. It has also been found that ectodermal explants from gastrula embryos are neuralized by expression of a dominant-negative version of a BMP receptor. In both cases, ectoderm adopts an anterior neural fate in the absence of neural inducing signals from the organizer. Thus it has been postulated that deprivation of endogenous neural-inhibiting signaling (such as BMP signaling) in ectoderm cells as a result of prolonged dissociation or overexpression of a dominant-negative receptor, is enough to cause their neuralization. However, the present studies show that autonomous neuralization in dissociated ectoderm cells requires FGF signaling. Interestingly, Xenopus gastrula ectoderm cells have been shown to express several members of the FGF family in addition to BMPs, though the level of their expression is considerably lower than that in the organizer region. It should be noted that these FGF family members have the common property of binding to components of extracellular matrix, such as heparin; these are characteristics that would make them not readily released from the cell surface, as compared to BMPs even in prolonged dissociated culture. More interesting is the fact that gastrula ectoderm cells contain novel types of ligands for the FGF receptor: FRL1 and FRL2. These ligands have an N-terminal signal sequence and can be anchored to the cell membrane by their C-termini. It is possible that some ligands of the two FGF receptors listed above support constitutive FGF signaling in ectoderm cells, contributing their neuralization without signals from the organizer. The neural default model and the present data can be reconciled by simply postulating that the default state of ectoderm is endowed with constitutive FGF signaling. According to this idea, both BMP and FGF signaling are working constitutively in intact ectoderm in either autocrine or paracrine manner (Hongo, 1999 and references therein).

Smad10 is required for formation of the frog nervous system

Before the nervous system establishes its complex array of cell types and connections, multipotent cells are instructed to adopt a neural fate and an anterior-posterior pattern is established. Smad10, a Medea related member of the Smad family of intracellular transducers of TGFß signaling, is required for formation of the nervous system. In addition, two types of molecules proposed as key to neural induction and patterning, bone morphogenetic protein (BMP) antagonists and fibroblast growth factor (FGF), require Smad10 for these activities. These data suggest that Smad10 may be a central mediator of the development of the frog nervous system (LeSueur, 2002).

Two separate classes of organizer-derived secreted molecules -- FGFs and BMP antagonists -- are thought to be key to neural induction and anterior-posterior patterning. In Xenopus, FGFs induce posterior neural tissue, and FGFs are required for neural induction in the chick and spinal cord formation in the frog. Since Smad10 is necessary for formation of neural structures, including the spinal cord, it follows that FGF might require Smad10 for its neuralizing properties. To test this notion, embryos were injected with control morpholino-modified oligonucleotide (morpholinos or MO), Smad10 MO, DNS10 mRNA, or ß-galactosidase mRNA and explanted animal caps. During gastrulation, the caps were cultured in the presence of FGF under conditions that induce formation of neural tissue and then the caps were analyzed for expression of neural markers. Smad10 does not block all FGF activities, but rather is required specifically for neural induction by FGF (LeSueur, 2002).

If Smad10 transduces an RTK signal, what ligand might activate the cascade? One plausible candidate is FGF. FGFs signal via an RTK pathway that involves phosphorylation and activation of Erks. This FGF pathway has similar biological functions to Smad10; both induce posterior neural fates. Furthermore, FGF requires Smad10 for this activity. These data, coupled with the in vitro phosphorylation results and the inability of the Smad10-PXAPx3 mutant to induce spinal cord formation, are consistent with the idea that FGF initiates an RTK pathway that leads to activation of Erk, subsequent phosphorylation of Smad10, and induction of posterior neural fates. Additional biochemical experiments will be required to test this hypothesis (LeSueur, 2002).

The data suggest that a RTK pathway may regulate the function of Smad10 and may do so in a direct biochemical sense. Smad10 contains Erk consensus phosphorylation sites, Erk2 directly phosphorylates Smad10 in vitro, and the PX(S/T)P consensus phosphorylation sites on Smad10 are required for this Erk-dependent phosphorylation. Of note, when the Erk consensus sites are mutated to alanine, Smad10 remains functional and generates anterior neural fates; however, the mutant no longer produces posterior neural fates. This suggests that the nonphosphorylated and phosphorylated forms of Smad10 might interact with different subsets of transcription factors to generate distinct cell fates. Erks often phosphorylate and activate transcription factors that regulate gene expression. Smad10 may be another example of such a transcription factor. Taken together, these data suggest that an RTK signal, rather than a TGFß signal, might control Smad10's biological function in anterior versus posterior neural development (LeSueur, 2002).

Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors

In chordates, formation of neural tissue from ectodermal cells requires an induction. The molecular nature of the inducer remains controversial in vertebrates. Using the early neural marker Otx as an entry point, the neural induction pathway in the simple embryos of Ciona intestinalis was dissected. The regulatory element driving Otx expression in the prospective neural tissue was isolated; this element directly responds to FGF signaling and FGF9/16/20 acts as an endogenous neural inducer. Binding site analysis and gene loss of function established that FGF9/16/20 induces neural tissue in the ectoderm via a synergy between two maternal response factors. Ets1/2 mediates general FGF responsiveness, while the restricted activity of GATAa targets the neural program to the ectoderm. Thus, this study identifies an endogenous FGF neural inducer and its early downstream gene cascade. It also reveals a role for GATA factors in FGF signaling (Bertrand, 2003).

Otx expression starts in the animal a6.5 pair of blastomeres as they become restricted to anterior neural fate, at the onset of the neural induction process. At this stage, Otx is also activated in the animal b6.5 pair of blastomeres (precursors of the posterior dorsal neural tube and of the dorsal midline which constitutes a neurogenic region and in some vegetal B-line blastomeres (precursors of the posterior mesendoderm). Interestingly, Otx activation in b6.5, as in a6.5, requires an induction from vegetal blastomeres (Bertrand, 2003 and references therein).

The region in Otx located between -1541 and -1417 is required for expression in the a6.5 lineage, and is referred to as the a-element. Consistent with the simultaneous induction of Otx in a6.5 and b6.5 by vegetal cells, deletion of the a-element also reduces the activity in the b6.5 lineage. Finally, regions located between positions -1417 to -1133, and -706 to -271 are required for expression in A-line, and B/b-lines respectively (Bertrand, 2003).

Otx activation in the a6.5 neural precursors requires an interaction with the anterior vegetal blastomeres (A-line). Thus, the inducing FGF should be expressed in A-line blastomeres, before the onset of Otx expression at the 32-cell stage. The Ciona intestinalis genome contains 6 members of the FGF family. By in situ hybridization, only detect one FGF, FGF9/16/20, could be detected that was expressed at the right time and place to be the inducer. Its expression starts at the 16 cell-stage in the A-line and some B-line cells. Expression is stronger in the A-line than in the B-line, and this difference is further enhanced at the early 32-cell stage. This expression pattern is similar to that of the Ciona savignyi ortholog and is consistent with a role for FGF9/16/20 as endogenous neural inducer (Bertrand, 2003).

By both gene loss of function and binding sites analysis it has been determined that cooperation between the maternal transcription factors, Ets1/2 and GATAa, mediates the initial transcriptional response to FGF. Ets transcription factors have already been shown to act in the FGF pathway in vertebrates, and the members of the Ets1/2 subfamily can be directly phosphorylated and activated by Erk. A role for GATAa in this process was more unexpected, since GATA factors have so far not been implicated in the FGF pathway. However, the fact that multimerized GATA binding sites mediate FGF responsiveness indicate that, in this system, GATA does not act solely to modify or enhance Ets activity but functions as an FGF-activated transcription factor. Consistent with the proposal of a direct involvement of GATA factors in the FGF pathway in vivo, it has recently been shown, in vitro, that vertebrate GATA4 can be directly phosphorylated and activated by Erk (Bertrand, 2003).

Could members of the Ets1/2 and GATA families also play a role in neural induction in vertebrates? Ets2 messenger is present maternally in Xenopus eggs and has recently been shown to be required for the induction of Brachyury by FGF in mesodermal cells. It will be interesting to test whether it also acts in the neural induction pathway. Vertebrate GATA factors are thought to antagonize rather than promote neural tissue formation; GATA1/2/3 family members are expressed during gastrulation in the nonneural ectoderm in zebrafish, Xenopus, and chick and GATA1 has an antineuralizing activity when overexpressed in Xenopus. However, GATA2 has no antineuralizing activity, showing that this is not a general property of GATA factors. GATA2 and GATA5 are present in Xenopus eggs but the early role of these maternal GATA factors has not been studied, leaving open the possibility of an involvement in neural induction. Finally, it is proposed that, in ascidians, the use of different response factors accounts for the activation of different target genes in neuroectoderm and mesoderm. It will be interesting to test whether the same logic is used in vertebrates or whether the increase in gene number has led to the recruitment of different FGF inducers or receptors in these two lineages (Bertrand, 2003 and references therein).

Convergent inductive signals specify midbrain, hindbrain, and spinal cord identity in gastrula stage chick embryos

In the chick embryo, neural cells acquire midbrain, hindbrain, and spinal cord character over a ~6 hr period during gastrulation. The convergent actions of four signals appear to specify caudal neural character. Fibroblast growth factors (FGFs) and a paraxial mesoderm-caudalizing (PMC) activity are involved, but neither signal is sufficient to induce any single region. FGFs act indirectly by inducing mesoderm that expresses PMC and retinoid activity and also directly on prospective neural cells, in combination with PMC activity and a rostralizing signal, to induce midbrain character. Hindbrain character emerges from cells that possess the potential to acquire midbrain character upon exposure to higher levels of PMC activity. Induction of spinal cord character appears to involve PMC and retinoid activities (Muhr, 1999).

Are FGF and PMC activities in combination able to generate cells of midbrain and hindbrain character? On its own, stage 4 anterior primitive streak (itself a source of FGFs and PMC activity), as well as stage 4 caudal paraxial mesoderm in the presence of FGF2, induce Otx2+/En1/2+ cells and Krox20+ cells in stage 3 R explants. Both stage 4 streak and paraxial mesoderm killed by freeze/thawing, in combination with FGF2, are sufficient to induce Otx2+/En1/2+ and Krox20+ cells, suggesting that PMC activity is expressed by cells in the anterior streak by stage 4. These results suggest that PMC activity and FGF, in combination, act directly on epiblast cells to induce midbrain and hindbrain character in stage 3 R explants. They also support the idea that the differential response of stage 3 C and 3 R explants to stage 4 caudal paraxial mesoderm reflects the prior exposure of C cells to FGFs. The effects of SU5402, an inhibitor of FGF receptor signaling, were examined on the induction of midbrain and hindbrain character in caudal epiblast cells. SU5402 blocks the FGF2-mediated induction of midbrain and hindbrain character in stage 3 R epiblast explants grown with stage 4 paraxial mesoderm but does not block the induction of Isl1+/HB9+ motor neurons in stage 4 C epiblast explants exposed to Shh-N. Therefore, it was asked whether SU5402 inhibits the induction of cells of midbrain and hindbrain character in stage 3 C explants grown together with stage 4 caudal paraxial mesoderm. SU5402 completely blocks the generation of Otx2+/En1/2+ and Krox20+ cells in these conjugates, and no expression of Hoxb8 is detected. These results provide evidence that FGF signaling in caudal epiblast cells is required to induce cells of midbrain and hindbrain character. They also suggest that FGF signaling is still ongoing in stage 3 C explants grown in vitro. However, in stage 4 C explants exposed to stage 4 caudal paraxial mesoderm in the presence of SU5402, Krox20+ cells are generated, indicating that a brief period of FGF signaling is sufficient for the generation of hindbrain character. In contrast, the generation of Otx2/En1/2+ cells is still inhibited, suggesting a requirement for a more prolonged period of FGF signaling in the generation of cells of midbrain character. Stage 7 paraxial mesoderm still induces Hoxb8+ expression in stage 3 C and 3 R explants in the presence of SU5402, suggesting that the induction of spinal cord identity by paraxial mesoderm is independent of FGF signaling (Muhr, 1999).

These findings suggest that at stages 2 and 3, prospective caudal neural plate cells are specified as cells of anterior prosencephalic-like character and begin to be exposed to FGFs derived from the primitive streak. By stage 3, caudal epiblast cells that migrate through the primitive streak may be exposed to high levels of FGF and differentiate into caudal paraxial mesodermal cells, which express PMC activity. The specification of caudal neural cells appears to be initiated at ~stage 3+ when caudal epiblast cells are first exposed to PMC activity that derives from the nascent paraxial mesoderm. At this stage, prospective caudal neural plate cells acquire predominantly midbrain character, yet 2 hr later, they have also acquired the potential to generate cells of hindbrain character. The differentiation of the epiblast into cells with these two regional characters coincides with the temporal increase in the level of PMC activity and with the gradual restriction of a rostralizing (midbrain-promoting) activity to the anterior tip of the primitive streak. Changes in the level of exposure of caudal epiblast cells to these two activities between stages 3 and 4 may, therefore, contribute to the initial differentiation of cells of midbrain and hindbrain character. After another ~2-3 hr, the caudal paraxial mesoderm starts to express high levels of RALDH2 and thus acquires retinoid synthetic capacity. he combined actions of PMC activity and retinoids provided by the paraxial mesoderm appear to induce spinal cord character in caudal epiblast cells, at the expense of midbrain and hindbrain character (Muhr, 1999).

Ras-mediated FGF signaling is required for the formation of posterior but not anterior neural tissue in Xenopus laevis

Fibroblast growth factor (FGF) has been proposed to be involved in the specification and patterning of the developing vertebrate nervous system. There is conflicting evidence, however, concerning the requirement for FGF signaling in these processes. To provide insight into the signaling mechanisms that are important for neural induction and anterior-posterior neural patterning, the dominant negative Ras mutant, N17Ras, was employed, in addition to a truncated FGF receptor (XFD). Both N17Ras and XFD, when expressed in Xenopus laevis animal cap ectoderm, inhibit the ability of FGF to generate neural pattern. They also block induction of posterior neural tissue by XBF2 and XMeis3. However, neither XFD nor N17Ras inhibits noggin, neurogenin, or XBF2 induction of anterior neural markers. MAP kinase activation has been proposed to be necessary for neural induction, yet N17Ras inhibits the phosphorylation of MAP kinase that usually follows explantation of explants. In whole embryos, Ras-mediated FGF signaling is critical for the formation of posterior neural tissues but is dispensable for neural induction (Ribisi, 2000).

Posterior mesoderm tissue induces midbrain and hindbrain fates from prospective forebrain, an activity that is mimicked in explant culture by bFGF. Treatment of early gastrula age animal cap ectoderm with bFGF protein induces the expression of the spinal cord marker hoxB9. Late gastrula-age (stage 11) animal cap ectoderm treated with bFGF expresses the midbrain and hindbrain marker genes en2 and krox20, in addition to hoxB9, and the forebrain marker otx2 is not induced. The combination of somite tissue with animal caps of gastrula age (stage 10.5) induces the expression of hindbrain-specific genes and low levels of spinal cord-specific genes in the animal cap tissue and this induction is partially sensitive to XFD. Keller explants faithfully recapitulate the A-P distribution of neural markers observed in the whole embryo: blocking FGF signaling using XFD eliminates posterior neural development in Keller explants. The claim that FGF signaling is required for the formation of posterior neural tissue is supported by the results of explant assays. The induction of posterior neural markers requires FGF and Ras signaling. Animal caps do not express posterior neural markers in response to either XBF2 or XMeis3 when either FGF or Ras signaling is blocked. In addition, when MAPK activation is directly inhibited by MAP kinase phosphatase, the ability of XMeis3 to induce the expression of posterior neural markers is greatly curtailed (Ribisi, 2000 and references therein).

Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups

Initiation of Hox genes requires interactions between numerous factors and signaling pathways in order to establish their precise domain boundaries in the developing nervous system. There are distinct differences in the expression and regulation of members of the Hox gene family within a complex, suggesting that multiple competing mechanisms are used to initiate Hox gene expression domains in early embryogenesis. In this study, by analyzing the response of HoxB genes to both RA and FGF signaling in neural tissue during early chick embryogenesis (HH stages 7-15), two distinct groups of Hox genes have been defined based on their reciprocal sensitivity to RA or FGF during this developmental period. The sharp reciprocal transition from RA to FGF responsiveness in moving from the 3' (Hoxb1 to Hoxb5) to the 5' (Hoxb6-Hoxb9) Hox genes is surprising. In mouse the 3' Hox genes do not respond uniformly to RA treatment, since there is a progressive temporal shift in their competence or ability to respond to RA during gastrulation, such that successively more 5' genes respond in later time windows. Hence, it had been suggested that the most posterior 5' Hox genes might also be progressively sensitive to RA in later stages at the end of or after gastrulation. The expression domain of 5' members from the HoxB complex (Hoxb6-Hoxb9) can be expanded anteriorly in the chick neural tube up to the level of the otic vesicle following FGF treatment and these same genes are refractory to RA treatment at these stages (Bel-Vialar, 2002).

The chick caudal-related genes, cdxA and cdxB, are also responsive to FGF signaling in neural tissue and their anterior expansion is also limited to the level of the otic vesicle. Using a dominant negative form of a Xenopus Cdx gene (XcadEnR) it has been found that the effect of FGF treatment on 5' HoxB genes is mediated in part through the activation and function of CDX activity. Conversely, the 3' HoxB genes (Hoxb1 and Hoxb3-Hoxb5) are sensitive to RA but not FGF treatments at these stages. In ovo electroporation of a dominant negative retinoid receptor construct (dnRAR) shows that retinoid signaling is required to initiate expression. Elevating CDX activity by ectopic expression of an activated form of a Xenopus Cdx gene (XcadVP16) in the hindbrain ectopically activates and anteriorly expands Hoxb4 expression. In a similar manner, when ectopic expression of XcadVP16 is combined with FGF treatment, it was found that Hoxb9 expression expands anteriorly into the hindbrain region. These findings suggest a model whereby, over the window of early development examined, all HoxB genes are actually competent to interpret an FGF signal via a CDX-dependent pathway. However, mechanisms that axially restrict the Cdx domains of expression, serve to prevent 3' genes from responding to FGF signaling in the hindbrain. FGF may have a dual role in both modulating the accessibility of the HoxB complex along the axis and in activating the expression of Cdx genes. The position of the shift in RA or FGF responsiveness of Hox genes may be time dependent. Hence, the specific Hox genes in each of these complementary groups may vary in later stages of development or other tissues. These results highlight the key role of Cdx genes in integrating the input of multiple signaling pathways, such as FGFs and RA, in controlling initiation of Hox expression during development and the importance of understanding regulatory events/mechanisms that modulate Cdx expression (Bel-Vialar, 2002).

An expanded domain of fgf3 expression in the hindbrain of zebrafish valentino mutants results in mis-patterning of the otic vesicle

The valentino (val) mutation in zebrafish perturbs hindbrain patterning and, as a secondary consequence, also alters development of the inner ear. The relationship between these defects and expression of fgf3 and fgf8 in the hindbrain were examined. The otic vesicle in val/val mutants is smaller than normal, yet produces nearly twice the normal number of hair cells, and some hair cells are produced ectopically between the anterior and posterior maculae. Anterior markers pax5 and nkx5.1 are expressed in expanded domains that include the entire otic epithelium juxtaposed to the hindbrain, and the posterior marker zp23 is not expressed. In the mutant hindbrain, expression of fgf8 is normal, whereas the domain of fgf3 expression expands to include rhombomere 4 through rhombomere X (an aberrant segment that forms in lieu of rhombomeres 5 and 6). Depletion of fgf3 by injection of antisense morpholino (fgf3-MO) suppresses the ear patterning defects in val/val embryos: excess and ectopic hair cells are eliminated, expression of anterior otic markers is reduced or ablated, and zp23 is expressed throughout the medial wall of the otic vesicle. By contrast, disruption of fgf8 does not suppress the val/val phenotype but instead interacts additively, indicating that these genes affect distinct developmental pathways. Thus, the inner ear defects observed in val/val mutants appear to result from ectopic expression of fgf3 in the hindbrain. These data also indicate that val normally represses fgf3 expression in r5 and r6, an interpretation further supported by the effects of misexpressing val in wild-type embryos. This is in sharp contrast to the mouse, in which fgf3 is normally expressed in r5 and r6 because of positive regulation by kreisler, the mouse ortholog of val. Implications for co-evolution of the hindbrain and inner ear are discussed (Kwak, 2002).

Unique and combinatorial functions of Fgf3 and Fgf8 during zebrafish forebrain development

Complex spatiotemporal expression patterns of fgf3 and fgf8 within the developing zebrafish forebrain suggest their involvement in its regionalization and early development. These factors have unique and combinatorial roles during development of more posterior brain regions, and similar findings have been made for the developing forebrain. Fgf8 and Fgf3 regulate different aspects of telencephalic development, and Fgf3 alone is required for the expression of several telencephalic markers. Within the diencephalon, Fgf3 and Fgf8 act synergistically to pattern the ventral thalamus, and are implicated in the regulation of optic stalk formation, whereas loss of Fgf3 alone results in defects in zona limitans intrathalamica development. Forebrain commissure formation is abnormal in the absence of either Fgf3 or Fgf8; however, most severe defects are observed in the absence of both. Defects are observed in patterning of both the midline territory, within which the commissures normally form, and neuronal populations, whose axons comprise the commissures. Analysis of embryos treated with an FGFR inhibitor suggests that continuous FGF signalling is required from gastrulation stages for normal forebrain patterning, and identifies additional requirements for FGFR activity (Walshe, 2003).

Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension

Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however, molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore, somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. retinoic acid (RA) has been identified as this somitic signal; retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation, including ventral neural patterning, while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm, where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. These data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension (Diez del Corral, 2003).

FGF can maintain an undifferentiated cell state, and retinoids can drive differentiation in many different contexts; for example, mouse ES cells form neural precursors in vitro under the influence of FGF signaling, while exposure to RA promotes neuronal differentiation. In the mouse embryo, excess RA, due to mutation of the RA-metabolizing enzyme CYP26, has been shown to repress Fgf8 expression in the tail bud, and RA downregulates Fgf8 in both neural and mesodermal tissues in vitro. Although Fgf8 expression perdures in caudal regions of Vitamin A-deficient (VAD) quails, it is still eventually lost from the presomitic mesoderm and neuroepithelium. This suggests either that somites provide another signal that can repress Fgf8 or that Fgf8 transcripts normally decay and that RA acts to accelerate this process. In the presomitic mesoderm, Fgf8 can be induced by FGF8 and so RA could effect Fgf8 reduction by interfering with the FGF signaling pathway. Conversely, FGF8 controls RA synthesis by inhibiting onset of Raldh2 in the paraxial mesoderm. Furthermore, exposure to FGF also blocks neuronal differentiation in explants of neural tube that do not express Raldh2, suggesting that FGF can also oppose RA activity in the neuroepithelium. It is proposed that during normal extension of the axis, a slight decline in Fgf8 transcripts (facilitated by regression of the primitive streak that expresses FGFs able to induce Fgf8) is sufficient for Raldh2 onset. As presomitic mesoderm begins to synthesize RA, retinoid signaling then accelerates Fgf8 downregulation in both the paraxial mesoderm and adjacent preneural tube. This mutual opposition of FGF and RA pathways thus helps to ensure the coordinated differentiation of mesodermal and neural tissues (Diez del Corral, 2003).

The level of FGF signaling in the presomitic mesoderm controls where a somite boundary will form, and the ability of RA to attenuate Fgf8 in the paraxial mesoderm identifies a role for the retinoid pathway in this process. According to the current model, somite size is determined by two components: the period of oscillation of transiently expressed mRNAs associated with Notch signaling such as Hairy1 (the segmentation clock) and the speed of FGF decline in the presomitic mesoderm (the maturation wavefront). Changes in FGF signaling do not alter the period of oscillation: resulting segmentation defects are due to alteration in the speed at which FGF levels fall below a threshold. Since RA downregulates Fgf8 in the presomitic mesoderm, it must set the rate of maturation wavefront progression and thereby influence somite size. Further, since FGF and RA pathways are mutually inhibitory, this could create a sharp transition in cell signaling in the presomitic mesoderm, and one possibility is that this change precisely defines the future somite border. Consistent with this, in VAD embryos where Fgf8 expression is prolonged and wavefront progression is slowed, initial somite size is smaller (Diez del Corral, 2003).

Finally, opposition of FGF and RA pathways may be a conserved mechanism for controlling differentiation and maintaining progenitor pools in the developing embryo. A striking analogy can be drawn with the proximo-distal extension of the limb in which distal FGF signaling provided by the apical ridge restricts RA synthesis and RARß receptor expression to the proximal limb. FGF signaling also opposes RA in the forming hindbrain, preserving rhombomere1 as a site of FGF activity that undergoes extensive proliferation to generate the cerebellum. The data suggest that mutual inhibition and opposing activities of FGF and RA pathways act to maintain a critical balance between preservation of the progenitor pool/stem zone and the progressive differentiation of neural and mesodermal tissues during the extension of the embryonic axis (Diez del Corral, 2003).

Cdx-Hox code controls competence for responding to Fgfs and retinoic acid in zebrafish neural tissue

Fibroblast growth factor (Fgf) and retinoic acid (RA) signals control the formation and anteroposterior patterning of posterior hindbrain. They are also involved in development processes in other regions of the embryo. Therefore, responsiveness to Fgf and RA signals must be controlled in a context-dependent manner. Inhibiting the caudal-related genes cdx1a and cdx4 in zebrafish embryos caused ectopic expression of genes that are normally expressed in the posterior hindbrain and anterior spinal cord, and ectopic formation of the hindbrain motor and commissure neurons in the posteriormost neural tissue. Combinational marker analyses suggest mirror-image duplication in the Cdx1a/4-defective embryos, and cell transplantation analysis further revealed that Cdx1a and Cdx4 repress a posterior hindbrain-specific gene expression cell-autonomously in the posterior neural tissue. Expression of fgfs and retinaldehyde dehydrogenase 2 suggested that in the Cdx1a/4-defective embryos, the Fgf and RA signaling activities overlap in the posterior body and display opposing gradients, compared with those in the hindbrain region. Fgf and RA signals were required for ectopic expression. Expression of the posterior hox genes hoxb7a, hoxa9a or hoxb9a, which function downstream of Cdx1a/4, or activator fusion genes of hoxa9a or hoxb9a (VP16-hoxa9a, VP16-hoxb9a) suppressed this loss-of-function phenotype. These data suggest that Cdx suppresses the posterior hindbrain fate through regulation of the posterior hox genes; the posterior Hox proteins function as transcriptional activators and indirectly repress the ectopic expression of the posterior hindbrain genes in the posterior neural tissue. These results indicate that the Cdx-Hox code modifies tissue competence to respond to Fgfs and RA in neural tissue (Shimizu, 2006).

FGF-dependent midline-derived progenitor cells in hypothalamic infundibular development

The infundibulum links the nervous and endocrine systems, serving as a crucial integrating centre for body homeostasis. This study shows that the chick infundibulum derives from two subsets of anterior ventral midline cells. One set remains at the ventral midline and forms the posterior-ventral infundibulum. A second set migrates laterally, forming a collar around the midline. Collar cells are composed of Fgf3⁺ SOX3⁺ proliferating progenitors, the induction of which is SHH dependent, but the maintenance of which requires FGF signalling. Collar cells proliferate late into embryogenesis, can generate neurospheres that passage extensively, and differentiate to distinct fates, including hypothalamic neuronal fates and Fgf10⁺ anterior-dorsal infundibular cells. Together, this study shows that a subset of anterior floor plate-like cells gives rise to Fgf3⁺ SOX3⁺ progenitor cells, demonstrates a dual origin of infundibular cells and reveals a crucial role for FGF signalling in governing extended infundibular growth (Pearson, 2011).

Involvement of Hedgehog and FGF signalling in the lamprey telencephalon: evolution of regionalization and dorsoventral patterning of the vertebrate forebrain

Dorsoventral (DV) specification is a crucial step for the development of the vertebrate telencephalon. Clarifying the origin of this mechanism will lead to a better understanding of vertebrate central nervous system (CNS) evolution. Based on the lamprey, a sister group of the gnathostomes (jawed vertebrates), three lamprey Hedgehog (Hh) homologues were identified, that are thought to play central signalling roles in telencephalon patterning. However, unlike in gnathostomes, none of these genes, nor Lhx6/7/8, a marker for the migrating interneuron subtype, was expressed in the ventral telencephalon, consistent with the reported absence of the medial ganglionic eminence (MGE) in this animal. Homologues of Gsh2, Isl1/2 and Sp8, which are involved in the patterning of the lateral ganglionic eminence (LGE) of gnathostomes, were expressed in the lamprey subpallium, as in gnathostomes. Hh signalling is necessary for induction of the subpallium identity in the gnathostome telencephalon. When Hh signalling was inhibited, the ventral identity was disrupted in the lamprey, suggesting that prechordal mesoderm-derived Hh signalling might be involved in the DV patterning of the telencephalon. By blocking fibroblast growth factor (FGF) signalling, the ventral telencephalon was suppressed in the lamprey, as in gnathostomes. It is concluded that Hh- and FGF-dependent DV patterning, together with the resultant LGE identity, are likely to have been established in a common ancestor before the divergence of cyclostomes and gnathostomes. Later, gnathostomes would have acquired a novel Hh expression domain corresponding to the MGE, leading to the obtainment of cortical interneurons (Sugahara, 2011).

Independently specified Atoh1 domains define novel developmental compartments in rhombomere 1

The rhombic lip gives rise to neuronal populations that contribute to cerebellar, proprioceptive and interoceptive networks. Cell production depends on the expression of the basic helix-loop-helix (bHLH) transcription factor Atoh1. In rhombomere 1, Atoh1-positive cells give rise to both cerebellar neurons and extra-cerebellar nuclei in ventral hindbrain. The origin of this cellular diversity has previously been attributed to temporal signals rather than spatial patterning. This study shows that in both chick and mouse the cerebellar Atoh1 precursor pool is partitioned into initially cryptic spatial domains that reflect the activity of two different organisers: an isthmic Atoh1 domain, which gives rise to isthmic nuclei, and the rhombic lip, which generates deep cerebellar nuclei and granule cells. A combination of in vitro explant culture, genetic fate mapping and gene overexpression and knockdown was used to explore the role of isthmic signalling in patterning these domains. An FGF-dependent isthmic Atoh1 domain was shown to be the origin of distinct populations of Lhx9-positive neurons in the extra-cerebellar isthmic nuclei. In the cerebellum, ectopic FGF induces proliferation while blockade reduces the length of the cerebellar rhombic lip. FGF signalling is not required for the specification of cerebellar cell types from the rhombic lip and its upregulation inhibits their production. This suggests that although the isthmus regulates the size of the cerebellar anlage, the downregulation of isthmic FGF signals is required for induction of rhombic lip-derived cerebellar neurons (Green, 2014).

A bi-modal function of Wnt signalling directs an FGF activity gradient to spatially regulate neuronal differentiation in the midbrain

FGFs and Wnts are important morphogens during midbrain development, but their importance and potential interactions during neurogenesis are poorly understood. This study employed a combination of genetic and pharmacological manipulations in zebrafish to show that during neurogenesis FGF activity occurs as a gradient along the anterior-posterior axis of the dorsal midbrain and directs spatially dynamic expression of the Hairy gene her5. As FGF activity diminishes during development, Her5 is lost and differentiation of neuronal progenitors occurs in an anterior-posterior manner. Mathematical models were generated to explain how Wnt and FGFs direct the spatial differentiation of neurons in the midbrain through Wnt regulation of FGF signalling. These models suggested that a negative-feedback loop controlled by Wnt is crucial for regulating FGF activity. Sprouty genes were tested as mediators of this regulatory loop using conditional mouse knockouts and pharmacological manipulations in zebrafish. These reveal that Sprouty genes direct the positioning of early midbrain neurons and are Wnt responsive in the midbrain. A model is proposed in which Wnt regulates FGF activity at the isthmus by driving both FGF and Sprouty gene expression. This controls a dynamic, posteriorly retracting expression of her5 that directs neuronal differentiation in a precise spatiotemporal manner in the midbrain (Dyer, 2014).

Nodal and FGF coordinate ascidian neural tube morphogenesis

This study investigated the formation of the neural tube in Ciona intestinalis. Previous studies have implicated Nodal and FGF signals in the specification of lateral and ventral neural progenitors. This study showed that these signals also control the detailed cellular behaviors underlying morphogenesis of the neural tube. Live imaging experiments show that FGF (see Drosophila Branchless) controls the intercalary movements of ventral neural progenitors, while Nodal is essential for the characteristic stacking behavior of lateral cells. Ectopic activation of FGF signaling is sufficient to induce intercalary behaviors in cells that have not received Nodal. In the absence of FGF and Nodal, neural progenitors exhibit a default behavior of sequential cell divisions, and fail to undergo the intercalary and stacking behaviors essential for normal morphogenesis. Thus, cell specification events occurring prior to completion of gastrulation coordinate morphogenetic movements underlying the organization of the neural tube (Navarrete, 2016).

Tracing of her5 progeny in zebrafish transgenics reveals the dynamics of midbrain-hindbrain neurogenesis and maintenance

The midbrain-hindbrain domain (MH) of the vertebrate embryonic neural tube develops in response to the isthmic organizer (IsO), located at the midbrain-hindbrain boundary (MHB). MH derivatives are largely missing in mutants affected in IsO activity; however, the potentialities and fate of MH precursors in these conditions have not been directly determined. To follow the dynamics of MH maintenance in vivo, artificial chromosome transgenesis was used in zebrafish to construct lines where egfp transcription is driven by the complete set of regulatory elements of her5, the first known gene expressed in the MH area. In these lines, egfp transcription faithfully recapitulates her5 expression from its induction phase onwards. Using the stability of GFP protein as lineage tracer, her5, first demonstrated at gastrulation, is a selective marker of MH precursor fate. By comparing GFP protein and her5 transcription, the spatiotemporal dynamics of her5 expression that conditions neurogenesis progression towards the MHB over time was further revealed. The molecular identity of GFP-positive cells was traced in the acerebellar (ace) and no-isthmus (noi) mutant backgrounds to analyze directly fgf8 and pax2.1 mutant gene activities for their ultimate effect on cell fate. Most MH precursors are maintained in both mutants but express abnormal identities, in a manner that strikingly differs between the ace and noi contexts. These observations directly support a role for Fgf8 in protecting anterior tectal and metencephalic precursors from acquiring anterior identities, while Pax2.1 controls the choice of MH identity as a whole. Together, these results suggest a model where an ordered MH pro-domain is identified at gastrulation, and where cell identity choices within this domain are subsequently differentially controlled by Fgf8 and Pax2.1 functions (Tallafuß, 2003).

vhnf1 and Fgf signals synergize to specify rhombomere identity in the zebrafish hindbrain

Vertebrate hindbrain segmentation is a highly conserved process but the mechanism of rhombomere determination is not well understood. Recent work in the zebrafish has shown a requirement for fibroblast growth factor (Fgf) signaling and for the transcription factor variant hepatocyte nuclear factor 1 (vhnf1) in specification of rhombomeres 5 and 6 (r5+r6). vhnf1 functions in two ways to subdivide the zebrafish caudal hindbrain domain (r4-r7) into individual rhombomeres: (1) vhnf1 promotes r5+r6 identity through an obligate synergy with Fgf signals to activate valentino and krox20 expression; (2) vhnf1 functions independently of Fgf signals to repress hoxb1a expression. Although vhnf1 is expressed in a broad posterior domain during gastrulation, it promotes the specification of individual rhombomeres. This is achieved in part because vhnf1 gives cellular competence to respond to Fgf signals in a caudal hindbrain-specific manner (Wiellette, 2003).

Morphogenetic movements underlying eye field formation require interactions between the FGF and ephrinB1 signaling pathways

Normal growth and morphogenesis of the cranial vault reflect a balance between cell proliferation in the sutures and osteogenesis at the margins of the cranial bones. In the clinical condition craniosynostosis, the sutures fuse prematurely as a result of precocious osteogenic differentiation and craniofacial malformation results. Mutations in several fibroblast growth factor receptor (FGFR) genes have now been identified as being responsible for the major craniosynostotic syndromes. A grafting technique was used to manipulate the levels of endogenous FGF-2 ligand in embryonic chick cranial vaults and thereby perturb morphogenesis. Implantation of beads loaded with FGF-2 does not affect normal cranial development at physiological concentrations, although they elicit a morphogenetic response in the limb. Implantation of beads loaded with a neutralizing antibody to FGF-2 generates a concentration-dependent response. When a single bead was implanted, the grafts grew to a massive size as a result of increased cell division in the tissue. With greater inactivation of FGF-2 protein (two to three beads implanted), all further bone differentiation and cell proliferation is blocked. These data further support the emerging idea that the intensity of FGF-mediated signaling determines the developmental fate of the skeletogenic cells in the cranial vault. High and low levels correlate with differentiation and proliferation, respectively. A balance between the two ensures normal cranial vault morphogenesis. This is consistent with the observation that several FGFR mutations causing craniosynostosis result in constitutive activation of the receptor (Moore, 2002).

Fgf signaling is required for zebrafish tooth development

Fibroblast growth factor (FGF) signaling during the development of the zebrafish pharyngeal dentition were investigated with the goal of uncovering novel roles for FGFs in tooth development as well as phylogenetic and topographic diversity in the tooth developmental pathway. The tooth-related expression of several zebrafish genes is similar to that of their mouse orthologs, including both epithelial and mesenchymal markers. Additionally, significant differences in gene expression between zebrafish and mouse teeth are indicated by the apparent lack of fgf8 and pax9 expression in zebrafish tooth germs. FGF receptor inhibition with SU5402 at 32 h blocks dental epithelial morphogenesis and tooth mineralization. While the pharyngeal epithelium remains intact as judged by normal pitx2 expression, not only is the mesenchymal expression of lhx6 and lhx7 eliminated as expected from mouse studies, but the epithelial expression of dlx2a, dlx2b, fgf3, and fgf4 is as well. This latter result provides novel evidence that the dental epithelium is a target of FGF signaling. However, the failure of SU5402 to block localized expression of pitx2 suggests that the earliest steps of tooth initiation are FGF-independent. Investigations of specific FGF ligands with morpholino antisense oligonucleotides revealed only a mild tooth shape phenotype following fgf4 knockdown, while fgf8 inhibition revealed only a subtle down-regulation of dental dlx2b expression with no apparent effect on tooth morphology. These results suggest redundant FGF signals target the dental epithelium and together are required for dental morphogenesis. Further work will be required to elucidate the nature of these signals, particularly with respect to their origins and whether they act through the mesenchyme (Jackman, 2004).

Intrinsic, Hox-dependent cues determine the fate of skeletal muscle precursors

It is generally held that vertebrate muscle precursors depend totally on environmental cues for their development. Instead, it is shown that somites are predisposed toward a particular myogenic program. This predisposition depends on the somite's axial identity: when flank somites are transformed into limb-level somites, either by shifting somitic boundaries with FGF8 or by overexpressing posterior Hox genes, they readily activate the program typical for migratory limb muscle precursors. The intrinsic control over myogenic programs can only be overridden by FGF4 signals provided by the apical ectodermal ridge of a developing limb (L. Alvares, 2003).

Since the competence to start a particular program of hypaxial myogenesis is linked to the position of the somites along the anteroposterior body axis, it was reasoned that this competence may be a consequence of the somite's axial identity. Indeed, when the ability was exploited of FGF8 to move the somitic boundaries, thereby changing the axial identity of somites in the flank into the identity of hindlimb-level somites, these somites gained the ability to produce Lbx1-expressing migratory muscle precursors (MMPs). This suggests that the positional values intrinsic to the somites determine their competence to generate either migratory or nonmigratory hypaxial muscle precursors (L. Alvares, 2003).

It is established that the axial identity of somites is controlled by the overlapping expression of Hox/HOM genes. In particular, a code of Hox gene expression is conserved for crucial anatomical landmarks such as the neck-thorax transition. Significantly, conserved Hox gene expression boundaries also demarcate the transition of limb-flank somites. Moreover, Hox gene expression patterns are maintained when somites are heterotopically grafted, as is the ability to express the MMP marker Lbx1. It was therefore reasoned that Hox genes may control the somitic competence to initiate a particular myogenic program. To test this possibility, expression constructs were engineered for HoxD9 and HoxA10, both normally present in hindlimb-level somites but not in the flank. Upon electroporation of either of the constructs into flank somites, these somites readily expressed Lbx1, while control constructs were unable to evoke expression of the Lbx1 gene. Thus, Lbx1 expression-directly or indirectly-is under the control of Hox genes (L. Alvares, 2003).

Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension

Differentiation onset in the vertebrate body axis is controlled by a conserved switch from fibroblast growth factor (FGF) to retinoid signalling, which is also apparent in the extending limb and aberrant in many cancer cell lines. FGF protects tail-end stem zone cells from precocious differentiation by inhibiting retinoid synthesis, whereas later-produced retinoic acid (RA) attenuates FGF signalling and drives differentiation. The timing of RA production is therefore crucial for the preservation of stem zone cells and the continued extension of the body axis. Canonical Wnt signalling mediates the transition from FGF to retinoid signalling in the newly generated chick body axis. FGF promotes Wnt8c expression, which persists in the neuroepithelium as FGF signalling declines. Wnt signals then act here to repress neuronal differentiation. Furthermore, although FGF inhibition of neuronal differentiation involves repression of the RA-responsive gene, retinoic acid receptor β (RARβ), Wnt signals are weaker repressors of neuron production and do not interfere with RA signal transduction. Strikingly, as FGF signals decline in the extending axis, Wnt signals now elicit RA synthesis in neighbouring presomitic mesoderm. This study identifies a directional signalling relay that leads from FGF to retinoid signalling and demonstrates that Wnt signals serve, as cells leave the stem zone, to permit and promote RA activity, providing a mechanism to control the timing of the FGF-RA differentiation switch (Olivera-Martinez, 2007).

Chordin, FGF signaling, and mesodermal factors cooperate in zebrafish neural induction

The ectoderm gives rise to both neural tissue and epidermis. In vertebrates, specification of the neural plate requires repression of bone morphogenetic protein (BMP) signaling in the dorsal ectoderm. The extracellular BMP antagonist Chordin and other signals from the dorsal mesoderm play important roles in this process. Zebrafish mutant combinations that disrupt Chordin and mesoderm formation were used to reveal additional signals that contribute to the establishment of the neural domain. Fibroblast growth factor (FGF) signaling accounts for the additional activity in neural specification. Impeding FGF signaling results in a shift of ectodermal markers from neural to epidermal. However, following inhibition of FGF signaling, expression of anterior neural markers recovers in a Nodal-dependent fashion. Simultaneously blocking, Chordin, mesoderm formation, and FGF signaling together eliminates neural marker expression during gastrula stages. FGF signaling is required for chordin expression but it also acts via other mechanisms to repress BMP transcription during late blastula stages. Activation of FGF signaling is also able to repress BMP transcription in the absence of protein synthesis. These results support a model in which specification of anterior neural tissue requires early FGF-mediated repression of BMP transcript levels and later activities of Chordin and mesodermal factors (Londin, 2005).

A balance of FGF, BMP and WNT signalling positions the future placode territory in the head

The sensory nervous system in the vertebrate head arises from two different cell populations: neural crest and placodal cells. By contrast, in the trunk it originates from neural crest only. How do placode precursors become restricted exclusively to the head and how do multipotent ectodermal cells make the decision to become placodes or neural crest? At neural plate stages, future placode cells are confined to a narrow band in the head ectoderm, the pre-placodal region (PPR). The head mesoderm is identified as the source of PPR inducing signals, reinforced by factors from the neural plate. Several independent signals are needed: attenuation of BMP and WNT is required for PPR formation. Together with activation of the FGF pathway, BMP and WNT antagonists can induce the PPR in naive ectoderm. WNT signalling plays a crucial role in restricting placode formation to the head. Finally, the decision of multipotent cells to become placode or neural crest precursors is demonstrated to be mediated by WNT proteins: activation of the WNT pathway promotes the generation of neural crest at the expense of placodes. This mechanism explains how the placode territory becomes confined to the head, and how neural crest and placode fates diversify (Litsiou, 2005).

This study finds that FGF signalling cooperates with WNT and BMP antagonists to impart generic placode character to uncommitted ectoderm. In the chick, activation of the FGF pathway in naive ectoderm leads to rapid expression of pre-neural markers such as Sox3 and Erni, both of which are later co-expressed at the border of the neural plate. However, activation of the FGF pathway is not sufficient to specify cells (neural crest and placode precursors) that arise from this border. The observation that continued FGF signalling is not required for pre-placodal Six4 expression, but can directly induce Eya2, suggests that FGFs may play a dual role. Early FGF signalling may confer 'border character' to ectodermal cells to make them responsive to PPR and crest inducing signals. The finding that ectopic PPR induction occurs only in the presence of active FGF signalling supports this notion. Later, FGFs from the head mesoderm, probably FGF4, initiate the expression of Eya2 in the placode territory as a crucial step to activate downstream target genes (Litsiou, 2005).

Simultaneously, the head mesoderm provides both BMP and WNT antagonists, most likely DAN and Cerberus, to counteract the inhibitory effect of both factors on the generation of placode precursors. The results show that attenuation of either the BMP or WNT pathway leads to an expansion of the PPR into the adjacent ectoderm. However, while the expansion in response to BMP inhibition is limited to the head ectoderm, WNT antagonism also results in the expression of PPR specific genes in the trunk. This is in agreement with recent findings in Xenopus reporting that simultaneous overexpression of BMP and WNT antagonist expands Six1 expression posteriorly along the induced secondary axis. In the chick, Wnt8c is expressed in trunk mesoderm and the mesoderm lateral to the heart primordium, whereas Wnt6 is found in trunk ectoderm. It is proposed that WNT activity from surrounding tissues is essential to restrict the placode territory to the head ectoderm next to the neural plate and thus ensure that sensory placodes are confined to the head. To allow placode formation, WNT antagonists in cooperation with FGF and anti-BMPs from the head mesoderm protect placode precursors from this inhibitory influence (Litsiou, 2005).

Fgf10 regulates transition period of cortical stem cell differentiation to radial glia controlling generation of neurons and basal progenitors

Radial glia (RG), the progenitors of cortical neurons and basal progenitors (BPs), differentiate from neuroepithelial cells (NCs) with stem cell properties. The morphogen Fgf10 is transiently expressed by NCs coincident with the transition period of NC differentiation into RG. Targeted deletion of Fgf10 delays RG differentiation, whereas overexpression has opposing effects. Delayed RG differentiation in Fgf10 mutants occurs selectively in rostral cortex, paralleled by an extended period of symmetric NC divisions increasing progenitor number, coupled with delayed and initially diminished production of neurons and BPs. RG eventually differentiate in excess number and overproduce neurons and BPs rostrally resulting in tangential expansion of frontal areas and increased laminar thickness. Thus, transient Fgf10 expression regulates timely differentiation of RG, and through this function, determines both length of the early progenitor expansion phase and onset of neurogenesis and ultimately the number of progenitors and neurons fated to specific cortical areas (Sahara, 2009).

Morphogenetic movements underlying eye field formation require interactions between the FGF and ephrinB1 signaling pathways

The definitive retinal progenitors of the eye field are specified by transcription factors that both promote a retinal fate and control cell movements that are critical for eye field formation. However, the molecular signaling pathways that regulate these movements are largely undefined. Both the FGF and ephrin pathways impact eye field formation. Activating the FGF pathway before gastrulation represses cellular movements in the presumptive anterior neural plate and prevents cells from expressing a retinal fate, independent of mesoderm induction or anterior-posterior patterning. Inhibiting the FGF pathway promotes cell dispersal and significantly increases eye field contribution. EphrinB1 reverse signaling is required to promote cellular movements into the eye field, and can rescue the FGF receptor-induced repression of retinal fate. These results indicate that FGF modulation of ephrin signaling regulates the positioning of retinal progenitor cells within the definitive eye field (Moore, 2004).

Retinal development consists of a series of steps that progressively restrict the available cell fates. First, a subset of embryonic cells are prevented from expressing a retinal fate by inherited maternal factors, whereas others become biased toward retinal fates due to their position within the neural inductive field of the animal hemisphere. As the CNS is regionalized, part of the anterior neural plate is specified as the eye field. Potential retinal progenitors need to be positioned within the eye field to receive the local environmental signals that will direct their ultimate fates. Only after these steps are accomplished do the steps of eye organogenesis, cellular lamination, and phenotype specification occur. Although there has been great progress in understanding how retinal cell type specification occurs, the molecular mechanisms that control which embryonic cells become specified as the definitive retinal progenitors in the eye field remain largely undefined (Moore, 2004).

An accepted hypothesis of how the eye field forms is that signals from surrounding anterior structures regionalize the anterior neural plate. The presumptive eye field then expresses several transcription factors that initiate the retina developmental program, e.g., rx1, pax6, and six3. However, cellular movements during gastrulation and neurulation, directed in part by eye field transcription factors, also are critical, and the signaling factors involved in these early steps of eye field formation have not been identified (Moore, 2004).

Several FGF family members have been implicated in affecting cell movements during gastrulation, and the anterior expression patterns of some FGFs and their receptors are consistent with a role in the morphogenetic movements of eye field cells. Therefore, whether FGF signaling prior to gastrulation plays a role in determining which embryonic cells form the eye field was investigated. Using a constitutively active FGF receptor, enhanced FGF signaling was demonstrated to prevent the normal retinal progenitors from populating the presumptive eye field, suggesting that low levels of FGF signaling are normally required for cells to adopt a retinal fate. This was confirmed by demonstrating that reduced FGF signaling, accomplished by expression of a dominant-negative receptor, enhances the number of cells that become retinal progenitors. It is further reported that ephrinB1 signaling during gastrulation is required for retinal progenitors to move into the eye field, and that this movement can be modified by activating the FGF pathway. These results demonstrate that FGF modulation of ephrin signaling is important for establishing the bona fide retinal progenitors in the anterior neural plate (Moore, 2004).

FGF signaling is required for brain left-right asymmetry and brain midline formation

Early disruption of FGF signaling alters left-right (LR) asymmetry throughout the embryo. This study has uncovered a role for FGF signaling that specifically disrupts brain asymmetry, independent of normal lateral plate mesoderm (LPM) asymmetry. When FGF signaling is inhibited during mid-somitogenesis, asymmetrically expressed LPM markers southpaw and lefty2 are not affected. However, asymmetrically expressed brain markers lefty1 and cyclops become bilateral. FGF signaling controls expression of six3b and six7, two transcription factors required for repression of asymmetric lefty1 in the brain. Analysis of Z0-1, atypical PKC (aPKC) and beta-catenin protein distribution revealed a midline structure in the forebrain that is dependent on a balance of FGF signaling. Ectopic activation of FGF signaling leads to overexpression of six3b, loss of organized midline adherins junctions and bilateral loss of lefty1 expression. Reducing FGF signaling leads to a reduction in six3b and six7 expression, an increase in cell boundary formation in the brain midline, and bilateral expression of lefty1. Together, these results suggest a novel role for FGF signaling in the brain to control LR asymmetry (Neugebauer, 2014).

Fgf/MAPK signaling is a crucial positional cue in somite boundary formation

The temporal and spatial regulation of somitogenesis requires a molecular oscillator, the segmentation clock. Through Notch signaling, the oscillation in cells is coordinated and translated into a cyclic wave of expression of hairy-related and other genes. The wave sweeps caudorostrally through the presomitic mesoderm (PSM) and finally arrests at the future segmentation point in the anterior PSM. By experimental manipulation and analyses in zebrafish somitogenesis mutants, a novel component involved in this process has been found. The level of Fgf/MAPK activation (highest in the posterior PSM) serves as a positional cue within the PSM that regulates progression of the cyclic wave and thereby governs the positions of somite boundary formation (Sawada, 2001).

Modulating Fgf signaling resulted in alterations in somite size. Detailed analyses of gene expressions in manipulated wild-type and mutant embryos reveal a novel function of Fgf/MAPK signaling in the PSM: the maintenance of cells in an immature state that allows the her1 wave to sweep through the PSM. Suppression of Fgf signaling posteriorizes the domain shift of her1 expression, as well as the expression of other segmentation genes such as mesp, a bHLH transcription factor crucial for segmentation initiation, and paraxial protocadherin. This leads to a posterior shift in segment border formation and larger somites. These results are complementary to those obtained with transplantation of Fgf beads, strengthening the idea that an Fgf signal determines the position of segment border formation by negatively regulating the maturation of the PSM. Since Fgf signal is known to have profound effects on many developmental processes such as cell growth and maintenance of progenitor cells, it is possible that manipulation of an Fgf signal locally changes the cell number in the PSM by regulating cell proliferation and/or cell migration within the mesoderm (axial, paraxial and lateral plate mesoderm). This could cause alterations in somite size. However, no such effect was observed in manipulated PSM, indicating that an Fgf signal in the PSM simply regulates the maturation status of cells without affecting cell proliferation or migration (Sawada, 2001).

The data are largely consistent with the 'clock-and-wavefront' model in which a cyclic wave operates in conjunction with a maturation wavefront that gradually moves posteriorly, resulting in arrest of the cyclic wave and initiation of segment furrow formation. Fgf/MAPK signaling negatively regulates the wavefront activity and restricts it to the anterior PSM that is devoid of MAPK activation. In zebrafish, the essential components of a conserved somite-making mechanism, the segmentation clock and wavefront are Notch- and fused somites-dependent, respectively. Zebrafish after eight/deltaD mutation desychronizes the oscillation wave, while, in the absence of Fused somites, the anterior PSM fails to acquire the wavefront activity. How could Fgf/MAPK signal interact with these components? In fact, it has been reported that the Ras/MAPK pathway interacts with the Notch pathway in C. elegans vulval development and malignant transformation of cultured cells. However, no interaction between Fgf/MAPK and Notch or Fss pathways could be demonstrated in this study: modulating Fgf signaling exerts identical effects on wild-type and after eight/DeltaD or fused somites mutants in terms of gene expression. Furthermore, the patterns of ERK activation and fgf8 expression in the PSM is not affected by after eight/DeltaD or fused somites mutations. Thus, it is concluded that the activation and action of Fgf/MAPK signaling in the PSM are not mediated by Notch or Fss pathway (Sawada, 2001).

The fact that four to five somites are normally formed after SU5402 treatment indicates that the positioning of furrow formation is already specified or Fgf insensitive at least at the position -IV to -V in the PSM. The result also indicates that ERK activation in segmented somites is not involved in segment border formation. Interestingly, the Fgf-sensitive region corresponds approximately to the heat-shock sensitive zone in zebrafish; that is, the initial defects in the segmental pattern of somite boundaries are observed five somites caudal to the forming somite at the time of heat shock. These data suggest that position -IV to -V represents a position at which the level of Fgf/MAPK activation drops below a threshold, rendering the cells competent to maturation signals. In support of this, transplanted Fgf8 beads exert their effects only when they are located in the Fgf-negative anterior PSM. Importantly, the relative position of MAPK activation domain to the newly formed somite is kept constant in the PSM as the embryos extend. These observations are consistent with the idea that the level of Fgf/MAPK activation serves as a positional cue within the PSM (Sawada, 2001).

FGF signaling acts upstream of the NOTCH and WNT signaling pathways to control segmentation clock oscillations in mouse somitogenesis

Fibroblast growth factor (FGF) signaling plays a crucial role in vertebrate segmentation. The FGF pathway establishes a posterior-to-anterior signaling gradient in the presomitic mesoderm (PSM), which controls cell maturation and is involved in the positioning of segmental boundaries. In addition, FGF signaling was shown to be rhythmically activated in the PSM in response to the segmentation clock. This study shows that conditional deletion of the FGF receptor gene Fgfr1 abolishes FGF signaling in the mouse PSM, resulting in an arrest of the dynamic cyclic gene expression and ultimately leading to an arrest of segmentation. Pharmacological treatments disrupting FGF signaling in the PSM result in an immediate arrest of periodic WNT activation, whereas Notch-dependent oscillations stop only during the next oscillatory cycle. Together, these experiments provide genetic evidence for the role of FGF signaling in segmentation, and identify a signaling hierarchy controlling clock oscillations downstream of FGF signaling in the mouse (Wahl, 2007).

The initiation and propagation of Hes7 oscillation are cooperatively regulated by Fgf and notch signaling in the somite segmentation clock

Periodic formation of somites is controlled by the segmentation clock, where the oscillator Hes7 regulates cyclic expression of the Notch modulator Lunatic fringe. This study shows that Hes7 also regulates cyclic expression of the Fgf signaling inhibitor Dusp4/MKP2 (MAP kinase phosphatase 2) and links Notch and Fgf oscillations in phase. Strikingly, inactivation of Notch signaling abolishes the propagation but allows the initiation of Hes7 oscillation. By contrast, transient inactivation of Fgf signaling abolishes the initiation, whereas sustained inactivation abolishes both the initiation and propagation of Hes7 oscillation. It is thus proposed that Hes7 oscillation is initiated by Fgf signaling and propagated/maintained anteriorly by Notch signaling (Niwa, 2007).

Emergence of traveling waves in the zebrafish segmentation clock

The spatial and temporal periodicity of somite formation is controlled by the segmentation clock, in which numerous cells cyclically express hairy-related transcriptional repressors with a posterior-to-anterior phase delay, creating 'traveling waves' of her1 expression. In zebrafish, the first traveling wave buds off from the synchronous oscillation zone in the blastoderm margin. This study shows that the emergence of a traveling wave coincides with the anterior expansion of Fgf signaling; transplanted Fgf8b-soaked beads induce ectopic traveling waves. It is thus proposed that as development proceeds, the activity of Fgf signaling gradually expands anteriorly, starting from the margin, so that cells initiate her1 oscillation with a posterior-to-anterior phase delay. Furthermore, it is suggested that Fgf has an essential role in establishing the period gradient that is required for the her1 spatial oscillation pattern at the emergence of the traveling wave (Ishimatsu, 2010).

Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro

The CNS is thought to develop from self-renewing stem cells that generate neurons, astrocytes, and oligodendrocytes. Other data, however, have suggested that astrocytes and oligodendrocytes are generated from separate progenitor populations. To reconcile these observations, progenitors have been prospectively isolated that do or do not express Olig2, an oligodendrocyte bHLH determination factor. Both Olig2 non-expressing and Olig2 expressing progenitors can behave as tripotential CNS stem cells (CNS-SCs) in vitro. Growth in FGF-2 causes induction of Olig2 in the former population, permitting oligodendrocyte differentiation; extinction of Olig2 in the latter cells permits astrocyte differentiation. The induction of Olig2 by FGF-2 is mediated, in part, via endogenous Sonic Hedgehog. These data indicate that clonogenic competence to generate neurons, astrocytes, and oligodendrocytes reflects a deregulation of dorsoventral patterning during expansion in vitro, raising the question of whether such trifatent cells actually exist in vivo (Gabay, 2003).

Mice expressing green fluorescent protein (GFP) in the Olig2 expression domain were used to prospectively isolate Olig2-expressing and nonexpressing progenitor cells by fluorescence-activated cell sorting (FACS). Both Olig2+ and Olig2- progenitors were found to be able to form neurospheres and can behave in vitro as tripotential CNS-SCs. In the case of initially Olig2- cells, the acquisition of oligodendrocyte capacity is caused by an unexpected effect of FGF-2, a mitogen commonly used to expand CNS-SCs, to induce expression of Olig2. This induction is mediated, at least in part, by endogenous Sonic Hedgehog (Shh) and reflects a ventralization of positional identity. Conversely, the ability of initially Olig2+ cells to generate astrocytes in vitro reflects the extinction of Olig2 expression. These data indicate that competence to clonogenically generate neurons, astrocytes, and oligodendrocytes, an identifying hallmark of CNS-SCs, reflects a deregulation of dorsoventral patterning caused by expansion in FGF-2 (Gabay, 2003).

FGF-dependent generation of oligodendrocytes by a hedgehog-independent pathway

During development, spinal cord oligodendrocyte precursors (OPCs) originate from the ventral, but not dorsal, neuroepithelium. Sonic hedgehog (SHH) has crucial effects on oligodendrocyte production in the ventral region of the spinal cord; however, less is known regarding SHH signalling and oligodendrocyte generation from neural stem cells (NSCs). NSCs isolated from the dorsal spinal cord can generate oligodendrocytes following FGF2 treatment, a MAP kinase dependent phenomenon that is associated with induction of the obligate oligogenic gene Olig2. Cyclopamine, a potent inhibitor of hedgehog signalling, does not block the formation of oligodendrocytes from FGF2-treated neurosphere cultures. Furthermore, neurospheres generated from SHH null mice also produced oligodendrocytes, even in the presence of cyclopamine. These findings are compatible with the idea of a hedgehog independent pathway for oligodendrocyte generation from neural stem cells (Chandran, 2003).

Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects

Human embryonic stem cells (hESCs) offer a platform to bridge what has been learned from animal studies to human biology. Using oligodendrocyte differentiation as a model system, this study shows that sonic hedgehog (SHH)-dependent sequential activation of the transcription factors OLIG2, NKX2.2 and SOX10 is required for sequential specification of ventral spinal OLIG2-expressing progenitors, pre-oligodendrocyte precursor cells (pre-OPCs) and OPCs from hESC-derived neuroepithelia, indicating that a conserved transcriptional network underlies OPC specification in human as in other vertebrates. However, the transition from pre-OPCs to OPCs is protracted. FGF2, which promotes mouse OPC generation, inhibits the transition of pre-OPCs to OPCs by repressing SHH-dependent co-expression of OLIG2 and NKX2.2. Thus, despite the conservation of a similar transcriptional network across vertebrates, human stem/progenitor cells may respond differently to those of other vertebrates to certain extrinsic factors (Hu, 2009).

Inaccessibility to human embryo experimentation calls for an alternative, in vitro model to study human cells or tissues directly. In recent years, directed neural differentiation from human embryonic stem cells (hESCs) has allowed a re-examination of the fundamental principles of early neural development learned from vertebrate studies. The present study revealed that human oligodendrocyte development involves a transcriptional network of nearly identical sequence to that observed in vertebrate models. Following expression of OLIG2 and genesis of motoneurons, the human OLIG2 progenitors become pre-OPCs by co-expressing NKX2.2, and finally differentiate into OPCs by activation of SOX10 and PDGFRα. Blocking OLIG2 expression inhibits OPC production, confirming the requirement of OLIG2 for human OPC specification. The vast majority of human OLIG2 progenitors are generated in a SHH-dependent manner, because cultures without exogenous SHH, or those in which endogenous SHH signaling has been blocked with cyclopamine, have few OLIG2 progenitors and OPCs. It was also found that SHH is not only crucial for efficiently inducing pre-OPCs, but it is also required for the transition from pre-OPCs to OPCs, for which endogenous SHH signaling is sufficient. Thus, the SHH-dependent signaling network underlying vertebrate OPC development is conserved in humans (Hu, 2009).

This study also reveals unique aspects of human OPC generation. In human OPC differentiation cultures, NKX2.2 is the first OPC-related transcription factor that is co-expressed with OLIG2 at the fifth week, preceding the expression of PDGFRα. This expression pattern resembles that in the chick and in the mouse hindbrain, but differs from that in the mouse spinal cord, where NKX2.2 expression is induced after PDGFRα+ OPCs are formed. This might be partly related to the hindbrain/cervical spinal identity of the human progenitors patterned by RA, although species differences cannot be excluded. A protracted transition period was found from pre-OPCs at the fifth week to human OPCs at the fourteenth week. In chick and mouse brainstem, the OLIG2+ NKX2.2+ progenitors quickly express PDGFRα and become migrating OPCs. OLIG2 progenitors differentiated from mESCs also co-express PDGFRα and NG2 shortly following motoneuron differentiation. It is possible that the serum-free culture conditions are not optimal for acquisition of the OPC identity at an earlier time. The addition of FGF2, EGF, SHH or noggin, however, did not accelerate the transition of pre-OPCs to OPCs. Similarly, removal of PDGF, NT3 and/or IGF1 did not alter the time course of OPC generation either. Alternatively, this protracted transition might be intrinsically controlled. The long OPC-specification process (3 months) appears to match the earliest appearance of OPCs in human embryos at the end of the first trimester. One potential explanation for the late appearance of OPCs is a need for expanding neurogenic progenitors, as large numbers of neurons are needed for the evolutionarily enlarged human brain and spinal cord (Hu, 2009).

FGF2 is a mitogen for rodent and human neural stem/progenitor cells and enhances the generation of OPCs in rodents. In the present study, it is interesting that following the induction of OLIG2, FGF2 nearly completely blocked motoneuron differentiation and preferentially increased the proportion of OLIG2 progenitors. The inhibition of motoneuron differentiation is not attributable to the mitogenic effect of FGF2 preventing the OLIG2 progenitors from exiting the cell cycle. Nor does FGF2 preferentially promote the survival of the OLIG2 progenitors. It is likely that FGF2 enhances the transition from the neurogenic OLIG2 progenitors to pre-OPCs. Although fine dissection of the mechanism is not possible with the available system, this finding provides, as it stands, a simple way of switching neurogenesis to gliogenesis from a pool of progenitors (Hu, 2009).

Continued use of FGF2 inhibited the generation of OPCs from hESCs. This is reminiscent of observations in the past decade that human neural stem/progenitor cells, following expansion with FGF2, or FGF2 and EGF, rarely produce oligodendrocytes in vitro. Even when the human neural progenitor cultures are enriched for OPCs, the OPCs quickly disappear after culturing in the presence of FGF2. The present finding that co-expression of OLIG2 and NKX2.2 is suppressed by FGF2 in the pre-OPCs explains the phenomenon. The inhibitory effect appears specific to FGF2, as EGF did not affect the co-expression of OLIG2 and NKX2.2 and subsequent generation of OPCs. FGF2 induces OPC generation from mouse neural stem/progenitor cells by activating endogenous SHH signaling or by as yet unknown SHH-independent pathways. In the human cell differentiation system, FGF2 inhibits endogenous SHH expression and significantly increases the level of GLI2 and GLI3, which are downstream repressors of SHH signaling, thus disrupting co-expression of OLIG2 and NKX2.2 in pre-OPCs. This results in the loss of pre-OPCs and the conversion to progenitors expressing OLIG2 (at a low level) or NKX2.2, which generates astrocytes or neurons. Nevertheless, the possibility was not excluded that FGF does this through other oligodendroglial transcription factors, such as SOX10 and ASCL1, or by selectively promoting the survival/expansion of neuronal progenitors in long-term cultures (Hu, 2009).

The present study has developed an effective strategy for reproducibly directing hESCs to an enriched population of OPCs with an unequivocal oligodendrocyte identity and myelination potential. In the previous reports of OPC differentiation from hESCs, SHH was not applied and the expression of OLIG2 in neural progenitors was not examined by single-cell-resolution immunocytochemistry, but instead by PCR on bulk cultures. Based on these findings, it is believed that the OPCs described in those reports are differentiated from OLIG2 progenitors spontaneously induced by endogenous or alternatively activated SHH signaling. The near pure population of 'OPCs' generated from hESCs without application of SHH cannot be explained by the current model, nor has the result been replicated by in other reports. The identity of the OPCs in that report has not been unequivocally verified either (Hu, 2009).

The SHH-dependent transcriptional network underlying human OPC specification and the time course of OPC generation, consistent with that in human embryo development, revealed in the present study suggest that the hESC differentiation system is a useful tool for understanding the biology of human cells. The divergent responses to common growth factors such as FGF2 between human and other vertebrate cells might be related to the in vitro system, but may also reflect the nature of normal human development. Similar to the present finding, the maintenance of human and mouse ESCs depends on the same transcriptional network. However, the common extrinsic factor, BMP, maintains the self-renewal of mESCs but induces cell differentiation of hESCs. This seemingly 'trivial' deviation has slowed down the translation of findings from mESCs to the establishment of hESCs. Similarly, over the past decade, many laboratories have stumbled in trying to replicate the finding in rodents, so as to differentiate human neural stem/progenitors to OPCs. Thus, the confirmation of conserved principles and the revelation of 'nuances' using the hESC differentiation system might bear significant consequences (Hu, 2009).

FGF22 and its close relatives are presynaptic organizing molecules in the mammalian brain

Target-derived cues promote local differentiation of axons into nerve terminals at sites of synaptic contact. Using clustering of synaptic vesicles in cultured neurons as an assay, putative target-derived presynaptic organizing molecules were purified from mouse brain and FGF22 was identified as a major active species. FGF7 and FGF10, the closest relatives of FGF22, share this activity; other FGFs have distinct effects. FGF22 is expressed by cerebellar granule cells during the period when they receive synapses. Its receptor, FGFR2, is expressed by pontine and vestibular neurons when their axons (mossy fibers) are making synapses on granule cells. Neutralization of FGF7, -10, and -22 inhibits presynaptic differentiation of mossy fibers at sites of contact with granule cells in vivo. Inactivation of FGFR2 has similar effects. These results indicate that FGF22 and its relatives are presynaptic organizing molecules in the mammalian brain and suggest new functions for this family of signaling molecules (Umemori, 2004).

WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo

A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm (IM) and the lateral plate mesoderm (LPM) in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo (Kawakami, 2001).

Axial tissues medial to the LPM (such as the IM and somites), have been shown to produce factors that initiate limb formation, operating on cells of the LPM to maintain and restrict expression of the Fgf-10 gene. FGF proteins such as FGF-2, FGF-4, and FGF-8, expressed in the IM and the somites adjacent to the limb forming areas, are capable of inducing Fgf-10, and thus they have been postulated as the endogenous inducers of limb initiation. Once Fgf-10 expression has been consolidated and restricted to the LPM of the presumptive limb areas, FGF-10 operates on the overlying surface ectoderm to induce expression of another Fgf gene, Fgf-8. This induction is concomitant with the appearance of the AER, and expression of Fgf-8 in the AER is required for the maintenance of Fgf-10 in the nascent limb mesenchyme and the localization of Shh to the posterior margin of the limb bud. Thus, a regulatory loop between FGFs is established so that FGF-8 (and probably other FGFs) produced by the IM and/or the somites adjacent to the limb forming areas, signal to the LPM to maintain and restrict expression of Fgf-10, which, in turn, induces Fgf-8 in the overlying nascent limb ectoderm. The regulatory loop is completed by FGF-8 (and other FGFs produced in the AER) signaling back to the limb mesenchyme to maintain limb bud outgrowth (Kawakami, 2001 and references therein).

Even though in the last few years this model has constituted an excellent framework for the analysis of limb initiation and AER induction, several interesting problems still remain to be solved; from among these, three were chosen for further study: (1) conflicting reports have been published on the role of the IM in limb initiation, so that the exact contribution of the IM and the somites remains unclear; (2) the relatively long times of induction of Fgf-10 (in the LPM) by FGF-8 and of Fgf-8 (in the ectoderm) by FGF-10 clearly suggest the existence of molecular mediators of the FGF-8/FGF-10 regulatory loop; (3) although FGF-10 appears to mediate initiation of both forelimbs and hindlimbs, it is unclear whether the same upstream mechanism of regulation of Fgf-10 (i.e., induction by FGF-8) operates in both cases. Thus, it has been proposed that FGF-8 may initiate both the forelimb (coming from the IM) and the hindlimb (coming from the primitive streak and other caudal embryonic structures (Kawakami, 2001 and references therein).

The results presented here provide novel insights into all these problems. (1) A limb-inducing gene, Wnt-2b, has been identified that is expressed in the somites, the IM, and the LPM of the forelimb level. This opens the door to further molecular studies aimed at clarifying the exact role of these three tissues (and the genes expressed in them) in forelimb induction. (2) It has been demonstrated that both Wnt-2b and Wnt-8c mediate the FGF-8/FGF-10 regulatory loop that controls limb initiation. Both Wnt-2b (expressed in the forelimb area) and Wnt-8c (expressed in the hindlimb area) act through beta-catenin to control Fgf-10 in the LPM of the prospective limb territories. A beta-catenin dependent activity is a common (and necessary) requirement for both forelimb and hindlimb induction, since antagonism of beta-catenin by Axin severely interferes with early limb development. (3) Another Wnt gene, Wnt-3a, mediates the induction of Fgf-8 in the limb ectoderm by FGF-10. Thus, three Wnt genes that signal through beta-catenin act as key molecular mediators of the FGF regulatory loop that controls both limb initiation and AER induction. Of the many signaling processes of regionalization that operate in the vertebrate embryo, these results also illustrate how a unique signaling mechanism (WNT/beta-catenin), which is essential for limb induction, is triggered by two different WNT ligands at two different locations in the embryo (Kawakami, 2001).

These results allow for the proposal of an expanded model of limb initiation and AER induction in the chick embryo. WNT and FGF signaling pathways interact in a way that ensures the adequate transference of inductive signals between the different tissues involved in these crucial morphogenetic processes. Prior to limb initiation, Fgf-10 is expressed in a wide region that includes the segmental plate (SP), IM and LPM, without any specific restriction to the presumptive limb areas. At limb bud initiation, Fgf-10 expression becomes confined to the LPM of the presumptive limb bud by signals emanating from the axial structures medial to the LPM. Two members of the WNT family, WNT-2B and WNT-8C, contribute to restrict and/or maintain Fgf-10 expression at the appropriate (fore and hindlimb) levels of the LPM. Both WNT-2B and WNT-8C inductive activities are mediated by beta-catenin, whose activity is absolutely required for the maintenance of Fgf-10 expression in the presumptive limb regions. Finally, once limb initiation is underway, and after Fgf-10 expression has been restricted to the LPM that corresponds to the presumptive limb areas, FGF-10 signals to the overlying ectoderm to induce expression of Wnt-3a, which eventually will become restricted to the AER. WNT-3A then signals through beta-catenin to activate Fgf-8 expression. To complete the loop, FGF-8 signals back to the mesenchyme of the nascent limb bud, where it contributes to maintain expression of Fgf-10 and to initiate and/or maintain Shh expression (Kawakami, 2001).

Transcriptional cascades responsible for initiating the formation of vertebrate embryonic structures such as limbs are not well established. Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations along the anteroposterior axis of the embryo is not known. The T-box transcription factor TBX5 is important for normal heart and limb formation, but its role in early limb development is not well defined. Mouse embryos lacking Tbx5 do not form forelimb buds, although the patterning of the lateral plate mesoderm into the limb field is intact. Tbx5 is not essential for an early establishment of forelimb versus hindlimb identity. In the absence of Tbx5, the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression. Tbx5 directly activates the Fgf10 gene via a conserved binding site, providing a simple and direct mechanism for limb bud initiation. Lef1/Tcf1-dependent Wnt signaling is not essential for initiation of Tbx5 or Fgf10 transcription, but is required in concert with Tbx5 for maintenance of normal levels of Fgf10 expression. It is conclude that Tbx5 is not essential for the early establishment of the limb field in the lateral plate mesoderm but is a primary and direct initiator of forelimb bud formation. These data suggest common pathways for the differentiation and growth of embryonic structures downstream of T-box genes (Agarwal, 2003).

Fgf signaling controls the number of phalanges and tip formation in developing digits

Tetrapods have two pairs of limbs, each typically with five digits, each of which has a defined number of phalanges derived from an archetypal formula. Much progress has been made in understanding vertebrate limb initiation and the patterning processes that determine digit number in developing limb buds, but little is known about how phalange number is controlled. An additional phalange can be induced in a chick toe if sonic hedgehog protein is applied in between developing digit primordia. This study shows that formation of an additional phalange is associated with prolonged Fgf8 expression in the overlying apical ridge and that an Fgf receptor inhibitor blocks phalange formation. The additional phalange is produced by elongation and segmentation of the penultimate phalange, suggesting that the digit tip forms when Fgf signaling ceases by a special mechanism, possibly involving Wnt signaling. Consistent with this, Fgfs inhibit tip formation whereas attenuation of Fgf signaling induces tip formation prematurely. It is proposed that duration of Fgf signaling from the ridge, responsible for elongation of digit primordia, coupled with a characteristic periodicity of joint formation, generates the appropriate number of phalanges in each digit. It is also proposed that the process that generates the digit tips is independent of that which generates more proximal phalanges. This has implications for understanding human limb congenital malformations and evolution of digit diversity (Sanz-Ezquerro, 2003).

The fact that terminal phalanges are formed by a mechanism that is completely different from that which generates proximal phalanges has not been widely appreciated, despite many observations suggesting that this is so. This study provides experimental evidence reinforcing the idea that there is a special program for making a digit tip. The fact that members of the Wnt signaling pathway, such as Wnt5a, their receptors Frizzleds (e.g., Fz4), and secreted inhibitors (e.g., FrzB) are expressed at digit tips suggests that Wnt signaling is part of this program, and the results suggest that Wnt14 may be involved. Moreover, transgenic mice expressing an inhibitor of Wnt signaling (Dickkopf) in the skin lack nails, consistent with Wnt-Wnt antagonist expression in digit tips being related to nail induction. Interestingly, regeneration of limbs in higher vertebrates is confined to digit tips. Therefore, understanding the molecular basis of tip formation could lead to development of new strategies to enhance the regenerative ability of limbs. Finally, similar mechanisms could also operate in the development of other appendages where a distal Fgf source directs outgrowth, such as genital tubercule, facial primordia, and tail. It is interesting to note that the tail tip also seems to express a special set of genes, suggesting an independent program for its formation (Sanz-Ezquerro, 2003).

BMP signals control limb bud interdigital programmed cell death by regulating FGF signaling

In vertebrate limbs that lack webbing, the embryonic interdigit region is removed by programmed cell death (PCD). Established models suggest that bone morphogenetic proteins (BMPs) directly trigger such PCD, although no direct genetic evidence exists for this. Alternatively, BMPs might indirectly affect PCD by regulating fibroblast growth factors (FGFs), which act as cell survival factors. The mouse BMP receptor gene Bmpr1a was inactivated specifically in the limb bud apical ectodermal ridge (AER), a source of FGF activity. Early inactivation completely prevents AER formation. However, inactivation after limb bud initiation causes an upregulation of two AER-FGFs, Fgf4 and Fgf8, and a loss of interdigital PCD leading to webbed limbs. To determine whether excess FGF signaling inhibits interdigit PCD in these Bmpr1a mutant limbs, double and triple AER-specific inactivations of Bmpr1a, Fgf4 and Fgf8 were performed. Webbing persists in AER-specific inactivations of Bmpr1a and Fgf8 owing to elevated Fgf4 expression. Inactivation of Bmpr1a, Fgf8 and one copy of Fgf4 eliminates webbing. It is concluded that during normal embryogenesis, BMP signaling to the AER indirectly regulates interdigit PCD by regulating AER-FGFs, which act as survival factors for the interdigit mesenchyme (Pajni-Underwood, 2007).

Fgf-dependent Etv4/5 activity is required for posterior restriction of Sonic Hedgehog and promoting outgrowth of the vertebrate limb

Crosstalk between the fibroblast growth factor (FGF) and Sonic Hedgehog (Shh) pathways is critical for proper patterning and growth of the developing limb bud. This study shows that FGF-dependent activation of the ETS transcription factors Etv4 and Etv5 contributes to proximal-distal limb outgrowth. Surprisingly, blockage of Etv activity in early distal mesenchyme also resulted in ectopic, anterior expansion of Shh, leading to a polydactylous phenotype. These data indicate an unexpected function for an FGF/Etv pathway in anterior-posterior patterning. FGF activity in the limb is not only responsible for maintaining posterior-specific Shh expression, but it also acts via Etvs to prevent inappropriate anterior expansion of Shh. This study identifies another level of genetic interaction between the orthogonal axes during limb development (Mao, 2009).

These data support a role for an FGF/Etv pathway in repressing Shh expression in the anterior mesenchyme, thereby contributing to the posterior restriction of a Shh-producing organizing center. Based on the temporal pattern of ectopic Shh expression seen in the limb bud after EtvEnR misexpression, it is speculated that endogenous Etv4/5 acts to repress Shh in two distinct domains, one extending from the ZPA across the distal limb bud, and a second, later domain in the anterior of the limb bud. The later timing of ectopic anterior Shh expression is consistent with the fact that in other mutants exhibiting ectopic anterior Shh activity, the anterior domain is observed at a later time in limb development than the normal domain at the posterior margin. Both of these functions of Etv4/5, in the distal and anterior limb, must occur before ~E10.5. Thus, there is an early time window, prior to Shh activation, when all distal mesenchyme is competent to activate Shh but broad activation is blocked by Fgf/Etv4/5 action. This broad competence is then lost shortly thereafter. Whether the loss of competence reflects a change in the intrinsic properties of mesenchyme, or a redundant mechanism for restricting Shh expression to the posterior margin, is unclear (Mao, 2009).

FGF-regulated Etv genes are essential for repressing Shh expression in mouse limb buds

Anterior-posterior (A-P) patterning of the vertebrate limb is controlled by sonic hedgehog (SHH) signaling, and the precise restriction of Shh expression to the posterior limb bud is essential for its polarizing effect. Fibroblast growth factor (FGF) signaling, a key control of proximal-distal (P-D) limb outgrowth, is known to promote Shh expression in the posterior limb bud. This study shows that conditional knockout of the FGF-activated transcription factor genes Etv4 and Etv5 in mouse leads to ectopic Shh expression in the anterior limb bud and a preaxial polydactyly (PPD) skeletal phenotype. These unexpected results suggest that ETV4 and ETV5 act downstream of FGF signaling to inhibit Shh expression in the anterior limb bud. This finding elucidates a novel aspect of the mechanism coordinating limb development along the A-P and P-D axes (Zhang, 2009).

A BMP-Shh negative-feedback loop restricts Shh expression during limb development

Normal patterning of tissues and organs requires the tight restriction of signaling molecules to well-defined organizing centers. In the limb bud, one of the main signaling centers is the zone of polarizing activity (ZPA) that controls growth and patterning through the production of sonic hedgehog (SHH). The appropriate temporal and spatial expression of Shh is crucial for normal limb bud patterning, because modifications, even if subtle, have important phenotypic consequences. However, although there is a lot of information about the factors that activate and maintain Shh expression, much less is known about the mechanisms that restrict its expression to the ZPA. This study shows that BMP activity negatively regulates Shh transcription and that a BMP-Shh negative-feedback loop serves to confine Shh expression. BMP-dependent downregulation of Shh is achieved by interfering with the FGF and Wnt signaling activities that maintain Shh expression. FGF induction of Shh requires protein synthesis and is mediated by the ERK1/2 MAPK transduction pathway. BMP gene expression in the posterior limb bud mesoderm is positively regulated by FGF signaling and finely regulated by an auto-regulatory loop. These study emphasizes the intricacy of the crosstalk between the major signaling pathways in the posterior limb bud (Bastida, 2009).

Gli2 functions in FGF signaling during antero-posterior patterning

Patterning along the anteroposterior (A/P) axis involves the interplay of secreted and transcription factors that specify cell fates in the mesoderm and neuroectoderm. While FGF and homeodomain proteins have been shown to play different roles in posterior specification, the network coordinating their effects remains elusive. The function of Gli zinc-finger proteins in mesodermal A/P patterning has been examined. Gli2 is sufficient to induce ventroposterior development, functioning in the FGF-brachyury regulatory loop. Gli2 directly induces brachyury, a gene required and sufficient for mesodermal development, and Gli2 is in turn induced by FGF signaling. Moreover, the homeobox gene Xhox3, a critical determinant of posterior development, is also directly regulated by Gli2. Gli3, but not Gli1, has an activity similar to that of Gli2 and is expressed in ventroposterior mesoderm after Gli2. These findings uncover a novel function of Gli proteins, previously only known to mediate hedgehog signals, in the maintenance and patterning of the embryonic mesoderm. More generally, these results suggest a molecular basis for an integration of FGF and hedgehog inputs in Gli-expressing cells that respond to these signals (Brewster, 2000).

Previous work has shown that Gli2 can be induced by SHH signaling in frog embryos, and that it can mediate some of the effects of SHH. FGF also induces Gli2, although it remains unclear which factors directly initiate its expression in mesoderm, as this is difficult to separate from the general induction of mesoderm by FGF or TGFbeta family signals. While Gli2/3 function in mesoderm may have nothing to do with HH signaling, HH genes have been reported to be expressed at low levels throughout the gastrula marginal zone, raising the possibility that Gli2 and Gli3 activity in mesoderm could be responsive to HH signals by analogy with some of its later roles in neural development. For example, a low tonic HH signal throughout the marginal zone could attenuate the formation of putative Gli3 repressors, a process regulated by the SHH signaling pathway, thus allowing Gli2 and Gli3 activator forms to function in ventroposterior development. The fact that misexpression of HHs at early stages has no obvious consequence on mesodermal development could be consistent with this possibility if repressor forms were not required in mesoderm. In mice, loss of SHH, Gli2 or Gli3 function does not appear to affect the early embryonic mesoderm, possibly indicating that Gli proteins could have partially divergent roles in different organisms (Brewster, 2000).

The role of Gli2 in FGF signaling, the ability of FGF and SHH to induce its expression and its partial mediation of SHH functions suggest a mechanism for a possible integration of FGF and HH signaling in tissues in which these signals act on the same Gli-expressing cells. SHH can act through Gli1, and Gli3 has an antagonistic relationship with SHH/Gli1. In contrast, SHH can also act through Gli2 in some contexts, but in others, Gli2 can instead antagonize the actions of SHH and Gli1. A context-dependent function of Gli2 could therefore underlie the sometimes synergistic and sometimes antagonistic effects of FGFs and HHs. Similarly, antagonism between HH and FGF signaling could result from their use of Gli1 and Gli3, respectively. This model may be particularly relevant for Gli-expressing precursor cells. For example, SHH is a known mitogen for cerebellar granule precursors and FGF can partially inhibit this effect. Because Wnt signaling has been recently suggested to affect Gli2 and Gli3 expression in chick somites, a challenge of ongoing studies is to elucidate how different signaling inputs regulate Gli function in vertebrate development and disease (Brewster, 2000).

Early embryonic expression of FGF4/6/9 gene and its role in the induction of mesenchyme and notochord in Ciona savignyi embryos

In early Ciona savignyi embryos, nuclear localization of ß-catenin is the first step of endodermal cell specification, and triggers the activation of various target genes. A cDNA for Cs-FGF4/6/9, a gene activated downstream of ß-catenin signaling, was isolated and shown to encode an FGF protein with features of both FGF4/6 and FGF9/20. The early embryonic expression of Cs-FGF4/6/9 is transient and the transcript is seen in endodermal cells at the 16- and 32-cell stages, in notochord and muscle cells at the 64-cell stage, and in nerve cord and muscle cells at the 110-cell stage; the gene is then expressed again in cells of the nervous system after neurulation. When the gene function is suppressed with a specific antisense morpholino oligo, the differentiation of mesenchyme cells is completely blocked, and the fate of presumptive mesenchyme cells appears to change into that of muscle cells. The inhibition of mesenchyme differentiation is abrogated by coinjection of the morpholino oligo and synthetic Cs-FGF4/6/9 mRNA. Downregulation of ß-catenin nuclear localization results in the absence of mesenchyme cell differentiation due to failure of the formation of signal-producing endodermal cells. Injection of synthetic Cs-FGF4/6/9 mRNA in ß-catenin-downregulated embryos evokes mesenchyme cell differentiation. These results strongly suggest that Cs-FGF4/6/9 produced by endodermal cells acts an inductive signal for the differentiation of mesenchyme cells. In contrast, the role of Cs-FGF4/6/9 in the induction of notochord cells is partial; the initial process of the induction is inhibited by Cs-FGF4/6/9 morpholino oligo, but notochord-specific genes are expressed later to form a partial notochord. Thus, Ciona FGF4/6/9 is a target of ß-catenin signaling, is expressed transiently in endodermal cells of early embryos, and functions as an inductive signal for the differentiation of mesenchyme cells (Imai, 2002a).

An essential role of a FoxD gene in notochord induction in Ciona embryos

A key issue for understanding the early development of the chordate body plan is how the endoderm induces notochord formation. In the ascidian Ciona, nuclear accumulation of ß-catenin is the first step in the process of endoderm specification. Nuclear accumulation of ß-catenin directly activates the gene (Cs-FoxD) for a winged helix/forkhead transcription factor and this gene is expressed transiently at the 16- and 32-cell stages in endodermal cells. The function of Cs-FoxD, however, is not associated with differentiation of the endoderm itself but is essential for notochord differentiation or induction. In addition, it is likely that the inductive signal that appears to act downstream of Cs-FoxD does not act over a long range. It has been suggested that FGF or Notch signal transduction pathway mediates ascidian notochord induction. Previous work suggests that Cs-FGF4/6/9 is partially involved in the notochord induction. The present experimental results suggest that the expression and function of Cs-FGF4/6/9 and Cs-FoxD are not interdependent, and that the Notch pathway is involved in B-line notochord induction (B-line cells represent a secondary notochord lineage) downstream of Cs-FoxD (Imai, 2002b).

Suppression of macho-1-directed muscle fate by FGF and BMP is required for formation of posterior endoderm in ascidian embryos

Specification of germ layers is a crucial event in early embryogenesis. In embryos of the ascidian, Halocynthia roretzi, endoderm cells originate from two distinct lineages in the vegetal hemisphere. Cell dissociation experiments suggest that cell interactions are required for posterior endoderm formation, which has hitherto been thought to be solely regulated by localized egg cytoplasmic factors. Without cell interaction, every descendant of posterior-vegetal blastomeres, including endoderm precursors, assumes muscle fate. Cell interactions are required for suppression of muscle fate and thereby promote endoderm differentiation in the posterior endoderm precursors. The cell interactions take place at the 16- to 32-cell stage. Inhibition of cell signaling by FGF receptor and MEK inhibitor also support the requirement of cell interactions. Consistently, FGF was a potent signaling molecule, whose signaling is transduced by MEK-MAPK. By contrast, such cell interactions are not required for formation of the anterior endoderm. Another redundant signaling molecule, most likely BMP, is be involved in the posterior endoderm formation. Suppression of the function of macho-1, an odd-paired-related muscle determinant in ascidian eggs, by antisense oligonucleotide is enough to allow autonomous endoderm specification. Therefore, the cell interactions induce endoderm formation by suppressing the function of macho-1, which is to promote muscle fate. These findings suggest the presence of novel mechanisms that suppress functions of inappropriately distributed maternal determinants via cell interactions after embryogenesis starts. Such cell interactions would restrict the regions where maternal determinants work, and play a key role in marking precise boundaries between precursor cells of different tissue types (Kondoh, 2003).

Combinatorial Fgf and Bmp signalling patterns the gastrula ectoderm into prospective neural and epidermal domains

Studies in fish and amphibia have shown that graded Bmp signalling activity regulates dorsal-to-ventral (DV) patterning of the gastrula embryo. In the ectoderm, it is thought that high levels of Bmp activity promote epidermal development ventrally, whereas secreted Bmp antagonists emanating from the organiser induce neural tissue dorsally. However, in zebrafish embryos, the domain of cells destined to contribute to the spinal cord extends all the way to the ventral side of the gastrula, a long way from the organiser. In vegetal (trunk and tail) regions of the zebrafish gastrula, neural specification is initiated at all DV positions of the ectoderm in a manner that is unaffected by levels of Bmp activity and independent of organiser-derived signals. Instead, Fgf activity is required to induce vegetal prospective neural markers and can do so without suppressing Bmp activity. Bmp signalling is shown to occur within the vegetal prospective neural domain and Bmp activity promotes the adoption of caudal fate by this tissue (Kudoh, 2004).

To explore the epistatic relationships between the Fgf and Bmp pathways, the consequences of locally activating or suppressing Fgf signalling were examined. fgf3-expressing ectodermal cells transplanted into animal pole regions of host embryos induce sox3 non-autonomously in surrounding host cells. This induction still occurs if the donor cells are from embryos co-expressing a truncated Fgf receptor, suggesting that the Fgf signal from the donor cells acts directly on the host. When both donor and host cells are overexpressing bmp2b, fgf3-expressing cells still induce sox3 and suppress expression of the epidermal marker gene, foxi1, suggesting that exogenous Bmp activity does not block induction of prospective neural marker genes by Fgf (Kudoh, 2004).

Next, Fgf signalling was locally suppressed by transplanting truncated Fgf receptor (XFD) expressing donor cells to various positions in the prospective neural ectoderm of host embryos. sox3 expression was suppressed in XFD-expressing cells transplanted to dorsal, lateral or ventral vegetal ectoderm, and foxi1 was ectopically induced in transplants targeted to ventral-vegetal ectoderm. These results suggest that ventral vegetal ectoderm needs to receive Fgf to express sox3, otherwise it expresses the prospective epidermal marker, foxi1 (Kudoh, 2004).

To directly assess if Fgf signals are essential for vegetal ectoderm to form neural tissue, the eventual fate was traced of XFD-expressing donor cells transplanted to wild-type hosts. In these experiments, labelled wild-type cells were co-transplanted with XFD-expressing cells to the same locations in the vegetal ectoderm of unlabelled host embryos at the end of blastula stage. When the donor cells were transplanted to the dorsal side, wild-type cells primarily contributed to the hindbrain, whereas the XFD-expressing cells localised more anteriorly, mainly in the midbrain. However, when transplanted to ventral vegetal ectoderm, wild-type donor cells contributed to spinal cord and muscle whereas XFD-expressing cells were excluded from the CNS and found in tissues such as the epidermis and fin. These results suggest that Fgf signalling is required for vegetal ectoderm to contribute to caudal neural tissue. They also suggest that the consequences of suppression of Fgf signalling in cells in dorsal and ventral domains of the vegetal ectoderm are different: dorsally, cells with compromised Fgf signalling frequently move into anterior neural tissue; ventrally, cells move into the prospective epidermis and are excluded from neural tissue. These results are consistent with analyses of embryos in which XFD is expressed ubiquitously and which show loss of posterior neural structures and anteriorisation of remaining CNS tissue on the dorsal side of the embryo (Kudoh, 2004).

Churchill, a zinc finger transcriptional activator, regulates the transition between gastrulation and neurulation

Gastrulation generates mesoderm and endoderm from embryonic epiblast; soon after, the neural plate is established within the epiblast-both events require FGF signaling. A zinc finger transcriptional activator, Churchill (ChCh), is described that acts as a switch between different roles of FGF. FGF induces ChCh slowly; this activates Smad-interacting-protein-1 (Sip1), which blocks further induction of the mesoderm markers brachyury and Tbx6L by FGF. ChCh is first expressed as cells stop migrating through the primitive streak, and it regulates cell ingression. A simple mechanism is proposed by which FGF sensitizes cells to BMP signals. These results reveal that neural induction requires cessation of mesoderm formation at the midline in addition to the decision between epidermis and neural plate (Sheng, 2003).

An anterior limit of FGF/Erk signal activity marks the earliest future somite boundary in zebrafish

Vertebrate segments called somites are generated by periodic segmentation of the anterior extremity of the presomitic mesoderm (PSM). During somite segmentation in zebrafish, mesp-b determines a future somite boundary at position B-2 within the PSM. Heat-shock experiments, however, suggest that an earlier future somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. This study characterized fibroblast growth factor (FGF) signal activity within the PSM, and demonstrated that an anterior limit of downstream Erk activity corresponds to the future B-5 somite boundary. Moreover, the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. These results provide the first molecular evidence of the future somite boundary at B-5, and it is proposed that clock-dependent cyclic inhibition of the FGF/Erk signal is a key mechanism in the generation of perfect repetitive structures in zebrafish development (Akiyama, 2014).

An Shp2/SFK/Ras/Erk signaling pathway controls trophoblast stem cell survival

Little is known about how growth factors control tissue stem cell survival and proliferation. Mice with a null mutation of Shp2 (Ptpn11), a key component of receptor tyrosine kinase signaling, were analyzed. Null embryos die peri-implantation, much earlier than mice that express an Shp2 truncation. Shp2 null blastocysts initially develop normally, but they subsequently exhibit inner cell mass death, diminished numbers of trophoblast giant cells, and failure to yield trophoblast stem (TS) cell lines. Molecular markers reveal that the trophoblast lineage, which requires fibroblast growth factor-4 (FGF4), is specified but fails to expand normally. Moreover, deletion of Shp2 in TS cells causes rapid apoptosis. Shp2 is required for FGF4-evoked activation of the Src/Ras/Erk pathway that culminates in phosphorylation and destabilization of the proapoptotic protein Bim. Bim depletion substantially blocks apoptosis and significantly restores Shp2 null TS cell proliferation, thereby establishing a key mechanism by which FGF4 controls stem cell survival (Yang, 2006).

Distinct GATA6- and laminin-dependent mechanisms function downstream of FGF to regulate endodermal and ectodermal embryonic stem cell fates

The establishment of alternative cell fates during embryoid body differentiation has been investigated, when embryonic stem (ES) cells diverge into two epithelia simulating the pre-gastrulation endoderm and ectoderm. Endoderm differentiation and endoderm-specific gene expression, such as expression of laminin 1 subunits, is controlled by GATA6 induced by FGF. Subsequently, differentiation of the non-polar primitive ectoderm into columnar epithelium of the epiblast is induced by laminin 1. Using GATA6 transformed Lamc1-null endoderm-like cells, it was demonstrated that laminin 1 exhibited by the basement membrane induces epiblast differentiation and cavitation by cell-to-matrix/matrix-to-cell interactions that are similar to the in vivo crosstalk in the early embryo. Pharmacological and dominant-negative inhibitors reveal that the cell shape change of epiblast differentiation requires ROCK, the Rho kinase. Pluripotent ES cells display laminin receptors; hence, these stem cells may serve as target for columnar ectoderm differentiation. Laminin is not bound by endoderm derivatives; therefore, the sub-endodermal basement membrane is anchored selectively to the ectoderm, conveying polarity to its assembly and to the differentiation induced by it. Unique to these interactions is stem cell flow through two cell layers connected by laminin 1 and stem cell involvement in the differentiation of two epithelia from the same stem cell pool: one into endoderm controlled by FGF and GATA6; and the other into epiblast regulated by laminin 1 and Rho kinase (Li, 2004).

The inner cell mass (ICM) of preimplantation and early postimplantation mammalian embryos contain cells ancestral to the entire individual, that undergo extensive morphological change prior to gastrulation. In the blastocyst and early egg cylinder the ICM consists of an aggregate of non-polar stem cells, which before gastrulation undergo epithelialization and cavitation, creating a pseudostratified columnar epithelium that surrounds a central cavity similar to the proamniotic canal of the early embryo. The pseudostratified columnar epithelium or epiblast attaches to the sub-endodermal basement membrane (BM). This polarized epithelium allows intermingling of clonal derivatives and is thought to be necessary for gastrulation. Much is known about the role of endoderm to ectoderm signalling in anteroposterior patterning of the early embryo. The establishment of major elements of the amniote body plan during gastrulation has been also studied in detail. However, the mechanism that precedes these changes and transforms the non-polar primitive ectoderm into the columnar polar epiblast is little understood (Li, 2004).

Embryonic stem cell derived embryoid bodies (EBs) are similar to the egg cylinder embryo, but, in contrast to it, they can be grown in large quantities, providing a useful model for early embryogenesis. The mechanism of EB differentiation has been set out as a model for pregastrulation development and tube formation by cavitation. EBs have an external endoderm that is similar to the primitive or visceral endoderm of the embryo and is separated from the inner columnar ectoderm by a basement membrane (BM). Using a genetically undefined spontaneous mutation, which fails to form the columnar ectoderm layer, it was proposed that cavitation is regulated by two signals: one emanating from the outer endoderm layer was thought to be responsible for the apoptotic signal/s of cavitation; the second, originating in the BM, was considered necessary for the maintenance and survival of the columnar ectoderm (Li, 2004 and references therein).

The work carried out in this study started as a study of the role of FGF signalling in EB differentiation and led to questions regarding BM assembly that were investigated using ES cells that express truncated Fgfr2 cDNA as a dominant-negative mutation. ES cells expressing dnFgfr fail to develop the two characteristic cell layers of the EB. They display a homogenous aggregate of non-polar cells and form no endoderm or ectoderm-like elements, but survive for weeks during cultivation. EBs formed by dnFgfr ES cells fail to synthesize laminin and collagen IV isotypes, which supply the protein network of the BM. Co-cultivating wild-type and dnFgfr ES cells rescued EB differentiation, suggesting that an FGF-controlled extracellular substance, subsequently identified as laminin 1, is required for epiblast differentiation. Exogenously added laminin 1 partially rescues the EB phenotype and induces epithelial transformation, demonstrating that laminin 1 produced by the endoderm is necessary and sufficient to induce epiblast polarization (Li, 2004).

Laminin 1 has been shown to be required for EB differentiation. Targeted disruption of ß1-integrin, which inhibits laminin alpha1 synthesis, interferes with epiblast differentiation. Disruption of Lamc1 encoding laminin gamma1, one of the three polypeptides of the laminin 1 heterotrimer, leads to a similar phenotype. Significantly, defective epiblast differentiation caused by loss of either gene was rescued by exogenously added laminin 1, which in turn could be inhibited by the E3 fragment of laminin alpha1 containing the heparin and sulfatide binding site of the LG4 globular domain of the laminin alpha1-chain. Recognising the potential importance of these findings for understanding epithelial differentiation and early development, it would help their analysis if the succession and main intermediates of EB differentiation were defined (Li, 2004).

In the present study, attempts were made to obtain a comprehensive view of the developmental interactions that precede gastrulation. To achieve this, several specific questions had to be answered. Is FGF signalling required for the differentiation of both epithelia and the pattern of their arrangement in the EB, or for only an initial step that is necessary for later events? Defective FGF signalling could be partially restored by exogenous laminin 1. The next question is can the same effect be obtained by laminin 1 presented by the BM in a physiological cell-matrix interaction? It was also important to determine whether laminin affects the stem cell directly, or whether it activates precursors after they reached a specific stage of FGF dependent differentiation. To answer these questions, mutant and wild-type ES cell lines were used , and their behaviour was studied as an effect of chemical inhibitors and co-cultivation experiments between mutant and wild-type cells (Li, 2004).

As an experimental system to elucidate interactions between the endoderm and primitive ectoderm GATA4- or GATA6-transformed endoderm-like cells co-cultivated with mutant ES cell lines were used. This system demonstrated that GATA4 and GATA6 transform ES cells into functional extra-embryonic endoderm that deposits a BM, which in turn mediates epiblast polarization. GATA transformed cells synthesize and later secrete laminin 1 and collagen IV into the culture supernatant, which could be used to rescue epiblast differentiation. Genetic evidence of laminin gamma1 null ES cells has demonstrated the specificity of mutant rescue. This experimental system thus recreated the physiological BM-mediated interaction and allowed the separation of endoderm and epiblast differentiation according to their respective FGF/GATA6 and laminin/Rho kinase-dependent mechanisms (Li, 2004).

Endoderm differentiation depends on FGF signalling, as demonstrated by the targeted disruption of Fgf4. Fgf4 is expressed in the ICM and contributes to the maintenance of the endoderm, where the multiple FGF receptors that read its signals are localized. Expression of GATA4 and GATA6, where GATA4 is regulated by GATA6, is controlled by FGF signalling. Nevertheless, the immediate downstream elements of FGF signalling are insufficiently understood in EB differentiation. In vitro evidence suggests that most FGF dependent signals go through Frs2a, a docking protein, which communicates with the Grb2 adaptor. Interestingly although null mutants of Fgf4 die with defective endoderm development shortly after implantation, Frs2a null embryos survive until advanced gastrulation, indicating that FGF signalling may exhibit unique characteristics in the early embryo. Analysis of signal transduction in dnFgfr ES cells revealed that PI3K-Akt/PKB rather than MAPK-ERK signalling is affected by defective FGF activity. In agreement, this study found that constitutively active Akt/PKB enhances endoderm development and the synthesis of laminin and collagen IV isotypes, indicating that the PI3K-Akt/PKB pathway predominates in FGF-dependent endoderm differentiation (Li, 2004).

GATA6 is an intermediary of FGF signalling. GATA6, which is transcribed already in the ICM, behaves as a master gene for endoderm differentiation. GATA6 activates the synthesis of all three polypeptide chains of laminin 1, which together with collagen IV, nidogen and perlecan assemble into the sub-endodermal BM. GATA factors induce endoderm differentiation and BM assembly even in dnFgfr ES cells, indicating that once activated, these transcription factors induce endoderm differentiation independently from FGF signalling. Because endoderm differentiation requires GATA6 and because cysts of GATA6 transformed cells contain only endoderm-like elements, it is concluded that GATA factors are required and sufficient to induce endoderm development and deposition of the subendodermal BM (Li, 2004).

Additional elements of this pathway are the transcription factors COUP-TFs I and II, which are upregulated by GATA4/6 during endoderm development and induce Lamc1 and Lamb1 expression. It follows that minimal elements of this interaction are, sequentially, Fgf4, multiple Fgfr, PI3K and AKT/PKB, GATA6 and GATA4, COUP-TFs I and II, as well as the genes encoding the three polypeptide chains of laminin 1 (Li, 2004).

Evidence demonstrates that E-cadherin is also required for early EB differentiation. E-cadherin-null ES cells fail to aggregate, do not form a normal ectoderm and do not undergo EB differentiation. Therefore, E-cadherin-dependent ES cell aggregation may be a prerequisite for the restriction of FGF signalling to the outer cells of the developing EB. E-cadherin is connected to the ß-catenin-GSK3-wnt pathway. Patterning events involving cadherin-Wnt/ß-catenin interactions have been shown to be controlled by FGF signalling (Li, 2004).

There is strong evidence for the epithelialization of ES cells by exogenous laminin 1. Laminin 1 can induce epiblast differentiation as part of the BM that mediates the physiological interaction of the endoderm with the epiblast. While laminin 1 binds to ES cells and their ectodermal derivatives, it does not associate with the primitive endoderm. Thus, the cell-binding domains of the laminin alpha1 chain determine the location of the subendodermal BM by interacting with their receptors displayed by the stem cells localized below the endoderm layer. This therefore defines the direction of laminin-mediated signalling, thereby determining the topographical relationship of endoderm and ectoderm (Li, 2004).

Besides inducing epiblast polarization, the BM affects the simple two-cell layer pattern of the EB and egg cylinder embryo. Since cell-to-matrix interactions take place through direct contact, epithelialization of residual stem cells is precluded, and a single epiblast monolayer develops from cells immediately adjacent to the BM. It has been proposed that the residual stem cells are removed by programmed cell death induced by factors derived from the endoderm, to form a central cavity. Investigation of the role of BMP signalling in cavitation indicates that BMP2 synthesizes in the endoderm, and BMP4 in the primitive ectoderm can both contribute to cavitation, although BMP4 is expressed only for a short period. The data indicate that cavitation and columnar ectoderm differentiation do not require the endoderm, provided that exogenous laminin 1 is presented. It is therefore possible that the developing ectoderm itself secretes the necessary apoptotic factors, such as BMP4, although inhibition of ROCK activity uncouples cavitation from full epithelialization of the primitive ectoderm and argues that cavitation may be either not different from necrosis, or it might be due to mechanical separation of the columnar ectoderm from the residual stem cells. This issue requires further study (Li, 2004).

Dominant-negative ROCK abolishes epiblast polarization without affecting endoderm differentiation, suggesting that it may be regulated separately in the two cell lineages. This assumption was supported by observing that ROCK expression and epiblast polarization does not require the endoderm for the laminin-induced differentiation of dnFgfr ES cells. Although ROCK is required for the epithelialization of the primitive ectoderm, it is not sufficient to induce this process, as suggested by the observation that dominant-active ROCK does not rescue dnFgfr differentiation. Although in the epiblast ROCK activity may be induced by laminin, in the endoderm it appears to be under FGF control and the resistance of endodermal differentiation to ROCK inhibition is consistent with the possibility that RAC1 or Cdc42, which are co-expressed in the endoderm, may have a role in endodermal differentiation (Li, 2004).

Separation of endoderm and epiblast differentiation has been repeatedly observed in this study. FGF signalling is shown to be required for endoderm differentiation but not for epiblast polarization, which is independently induced by laminin 1 of the sub-endodermal BM. The two lineages are also distinguished by laminin binding. ES cells and their ectodermal derivatives bind laminin, while the primitive and visceral endoderm do not, which defines the direction of laminin-induced differentiation. It follows that the extra-embryonic and embryonic epithelium of the EB and egg cylinder embryo develop by distinct mechanisms, which are connected by the inductive activity of the laminin component of their common BM. Future research will have to clarify whether other epithelial transitions are also controlled by laminin-dependent mechanisms (Li, 2004).

FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signaling

Members of the fibroblast growth factor (FGF) family induce lens epithelial cells to undergo cell division and differentiate into fibers; a low dose of FGF can stimulate cell proliferation (but not fiber differentiation), whereas higher doses of FGF are required to induce fiber differentiation. To determine if these cellular events are regulated by the same signaling pathways, the role of MAP kinase signaling in FGF-induced lens cell proliferation and differentiation was examined. FGF induces a dose-dependent activation of ERK1/2 as early as 15 minutes in culture, with a high (differentiating) dose of FGF stimulating a greater level of ERK phosphorylation than a lower (proliferating) dose. Subsequent blocking experiments using UO126 (a specific inhibitor of ERK activation) showed that activation of ERK is required for FGF-induced lens cell proliferation and fiber differentiation. Interestingly, inhibition of ERK signaling can block the morphological changes associated with FGF-induced lens fiber differentiation; however, it cannot block the synthesis of some of the molecular differentiation markers, namely, ß-crystallin. These findings are consistent with the in vivo distribution of the phosphorylated (active) forms of ERK1/2 in the lens. Taken together, these data indicate that different levels of ERK signaling may be important for the regulation of lens cell proliferation and early morphological events associated with fiber differentiation; however, multiple signaling pathways are likely to be required for the process of lens fiber differentiation and maturation (Lovicu, 2001).

The hedgehog pathway is a modulator of retina regeneration

The embryonic chick has the ability to regenerate its retina after it has been completely removed. A detailed characterization of retina regeneration in the embryonic chick has been carried out at the cellular level. Retina regeneration can occur in two distinct manners. The first is via transdifferentiation, which is induced by members of the Fibroblast growth factor (Fgf) family. The second type of retinal regeneration occurs from the anterior margin of the eye, near the ciliary body (CB) and ciliary marginal zone (CMZ). Regeneration from the CB/CMZ is the result of proliferating stem/progenitor cells. This type of regeneration is also stimulated by Fgf2. It can also be activated by Sonic hedgehog (Shh) overexpression when no ectopic Fgf2 is present. Shh-stimulated activation of CB/CMZ regeneration is inhibited by the Fgf receptor (Fgfr) antagonist, PD173074. This indicates that Shh-induced regeneration acts through the Fgf signaling pathway. In addition, the hedgehog (Hh) pathway plays a role in maintenance of the retina pigmented epithelium (RPE); ectopic Shh expression inhibits transdifferentiation and Hh inhibition increases the transdifferentiation domain. Ectopic Shh expression in the regenerating retina also results in a decrease in the number of ganglion cells present and an increase in apoptosis mostly in the presumptive ganglion cell layer (GCL). However, Hh inhibition increases the number of ganglion cells but does not have an effect on cell death. Taken together, these results suggest that the hedgehog pathway is an important modulator of retina regeneration (Spence, 2004).

Fgf and Hh signalling act on a symmetrical pre-pattern to specify anterior and posterior identity in the zebrafish otic placode and vesicle

Specification of the otic anteroposterior axis is one of the earliest patterning events during inner ear development. In zebrafish, Hedgehog signalling is necessary and sufficient to specify posterior otic identity between the 10 somite (otic placode) and 20 somite (early otic vesicle) stages. This study shows that Fgf signalling is both necessary and sufficient for anterior otic specification during a similar period, a function that is completely separable from its earlier role in otic placode induction. In lia-/- (fgf3-/-) mutants, anterior otic character is reduced, but not lost altogether. Blocking all Fgf signalling at 10-20 somites, however, using the pan-Fgf inhibitor SU5402, results in the loss of anterior otic structures and a mirror image duplication of posterior regions. Conversely, overexpression of fgf3 during a similar period, using a heat-shock inducible transgenic line, results in the loss of posterior otic structures and a duplication of anterior domains. These phenotypes are opposite to those observed when Hedgehog signalling is altered. Loss of both Fgf and Hedgehog function between 10 and 20 somites results in symmetrical otic vesicles with neither anterior nor posterior identity, which, nevertheless, retain defined poles at the anterior and posterior ends of the ear. These data suggest that Fgf and Hedgehog act on a symmetrical otic pre-pattern to specify anterior and posterior otic identity, respectively. Each signalling pathway has instructive activity: neither acts simply to repress activity of the other, and, together, they appear to be key players in the specification of anteroposterior asymmetries in the zebrafish ear (Hammond, 2011).

Fgf3 and Fgf10 are required for mouse otic placode induction

The inner ear, which contains the sensory organs specialized for audition and balance, develops from an ectodermal placode adjacent to the developing hindbrain. Tissue grafting and recombination experiments suggest that placodal development is directed by signals arising from the underlying mesoderm and adjacent neurectoderm. In mice, Fgf3 is expressed in the neurectoderm prior to and concomitant with placode induction and otic vesicle formation, but its absence affects only the later stages of otic vesicle morphogenesis. Mouse Fgf10 is expressed in the mesenchyme underlying the prospective otic placode. Embryos lacking both Fgf3 and Fgf10 fail to form otic vesicles and have aberrant patterns of otic marker gene expression, suggesting that FGF signals are required for otic placode induction and that these signals emanate from both the hindbrain and mesenchyme. These signals are likely to act directly on the ectoderm, since double mutant embryos show normal patterns of gene expression in the hindbrain. Cell proliferation and survival are not markedly affected in double mutant embryos, suggesting that the major role of FGF signals in otic induction is to establish normal patterns of gene expression in the prospective placode. Finally, examination of embryos carrying three out of the four mutant Fgf alleles revealed intermediate phenotypes, suggesting a quantitative requirement for FGF signalling in otic vesicle formation (Wright, 2003).

Requirements for FGF3 and FGF10 during inner ear formation

In the mouse, insertion of a neomycin resistance gene into the Fgf3 gene via homologous recombination results in severe developmental defects during differentiation of the otic vesicle. the precise roles of FGF3 and other FGF family members during formation of the murine inner ear has been addressed using both loss- and gain-of-function experiments. A new mutant allele lacking the entire FGF3-coding region was generated but surprisingly no evidence was found for severe defects either during inner ear development or in the mature sensory organ, suggesting the functional involvement of other FGF family members during its formation. Ectopic expression of FGF10 in the developing hindbrain of transgenic mice leads to the formation of ectopic vesicles, expressing some otic marker genes and thus indicating a role for FGF10 during otic vesicle formation. Expression analysis of FGF10 during mouse embryogenesis reveals a highly dynamic pattern of expression in the developing hindbrain, partially overlapping with FGF3 expression and coinciding with formation of the inner ear. However, FGF10 mutant mice have been reported to display only mild defects during inner ear differentiation. Thus double mutant mice were created for FGF3 and FGF10, that form severely reduced otic vesicles, suggesting redundant roles of these FGFs, acting in combination as neural signals for otic vesicle formation (Y. Alvarez, 2003).

A direct role for Fgf but not Wnt in otic placode induction

Induction of the otic placode, which gives rise to all tissues comprising the inner ear, is a fundamental aspect of vertebrate development. A number of studies indicate that fibroblast growth factor (Fgf), especially Fgf3, is necessary and sufficient for otic induction. However, an alternative model proposes that Fgf must cooperate with Wnt8 to induce otic differentiation. Using a genetic approach in zebrafish, the roles of Fgf3, Fgf8 and Wnt8 were tested. Localized misexpression of either Fgf3 or Fgf8 is sufficient to induce ectopic otic placodes and vesicles, even in embryos lacking Wnt8. Wnt8 is expressed in the hindbrain around the time of otic induction, but loss of Wnt8 merely delays expression of preotic markers and otic vesicles form eventually. The delay in otic induction correlates closely with delayed expression of fgf3 and fgf8 in the hindbrain. Localized misexpression of Wnt8 is insufficient to induce ectopic otic tissue. By contrast, global misexpression of Wnt8 causes development of supernumerary placodes/vesicles, but this reflects posteriorization of the neural plate and consequent expansion of the hindbrain expression domains of Fgf3 and Fgf8. Embryos that misexpress Wnt8 globally but are depleted for Fgf3 and Fgf8 produce no otic tissue. Finally, cells in the preotic ectoderm express Fgf (but not Wnt) reporter genes. Thus, preotic cells respond directly to Fgf but not Wnt8. It is proposed that Wnt8 serves to regulate timely expression of Fgf3 and Fgf8 in the hindbrain, and that Fgf from the hindbrain then acts directly on preplacodal cells to induce otic differentiation (Phillips, 2004).

The development of semicircular canals in the inner ear: role of FGFs in sensory cristae

In the vertebrate inner ear, the ability to detect angular head movements lies in the three semicircular canals and their sensory tissues, the cristae. The molecular mechanisms underlying the formation of the three canals are largely unknown. Malformations of this vestibular apparatus found in zebrafish and mice usually involve both canals and cristae. Although there are examples of mutants with only defective canals, few mutants have normal canals without some prior sensory tissue specification, suggesting that the cristae might induce the formation of their non-sensory components, the semicircular canals. The vertical canal pouch in chicken that gives rise to the anterior and posterior canals was fate-mapped, using a fluorescent, lipophilic dye (DiI), and a canal genesis zone was identified adjacent to each prospective crista that corresponds to the Bone morphogenetic protein 2 (Bmp2)-positive domain in the canal pouch. Using retroviruses or beads to increase Fibroblast Growth Factors (FGFs) for gain-of-function and beads soaked with the FGF inhibitor SU5402 for loss-of-function experiments, it was shown that FGFs in the crista promote canal development by upregulating Bmp2. It is postulated that FGFs in the cristae induce a canal genesis zone by inducing/upregulating Bmp2 expression. Ectopic FGF treatments convert some of the cells in the canal pouch from the prospective common crus to a canal-like fate. Thus, the first molecular evidence is provided whereby sensory organs direct the development of the associated non-sensory components, the semicircular canals, in vertebrate inner ears (Chang, 2004).

Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions

The mammalian inner ear comprises the cochleovestibular labyrinth, derived from the ectodermal otic placode, and the encasing bony labyrinth of the temporal bone. Epithelial-mesenchymal interactions are thought to control inner ear development, but the modes and the molecules involved are largely unresolved. During the precartilage and cartilage stages Fgf9 is expressed in specific nonsensory domains of the otic epithelium and its receptors, Fgfr1(IIIc) and Fgfr2(IIIc), widely in the surrounding mesenchyme. To address the role of Fgf9 signaling, the inner ears of mice homozygous for Fgf9 null alleles were analyzed. Fgf9 inactivation leads to a hypoplastic vestibular component of the otic capsule and to the absence of the epithelial semicircular ducts. Reduced proliferation of the prechondrogenic mesenchyme was found to underlie capsular hypoplasticity. Semicircular duct development is blocked at the initial stages, since fusion plates do not form. The results show that the mesenchyme directs fusion plate formation and they give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. In addition to the vestibule, in the cochlea, Fgf9 mutation caused defects in the interactions between the Reissner's membrane and the mesenchymal cells, leading to a malformed scala vestibuli. Together, these data show that Fgf9 signaling is required for inner ear morphogenesis (Pirvola, 2004).

Fgf3 is required for dorsal patterning and morphogenesis of the inner ear epithelium

The inner ear, which contains sensory organs specialized for hearing and balance, develops from an ectodermal placode that invaginates lateral to hindbrain rhombomeres (r) 5-6 to form the otic vesicle. Under the influence of signals from intra- and extraotic sources, the vesicle is molecularly patterned and undergoes morphogenesis and cell-type differentiation to acquire its distinct functional compartments. This study shows that mouse Fgf3, which is expressed in the hindbrain from otic induction through endolymphatic duct outgrowth, and in the prospective neurosensory domain of the otic epithelium as morphogenesis initiates, is required for both auditory and vestibular function. New morphologic data is provided on otic dysmorphogenesis in Fgf3 mutants, which show a range of malformations similar to those of Mafb (Kreisler), Hoxa1 and Gbx2 mutants, the most common phenotype being failure of endolymphatic duct and common crus formation, accompanied by epithelial dilatation and reduced cochlear coiling. The malformations have close parallels with those seen in hearing-impaired patients. The morphologic data, together with an analysis of changes in the molecular patterning of Fgf3 mutant otic vesicles, and comparisons with other mutations affecting otic morphogenesis, allow placement of Fgf3 between hindbrain-expressed Hoxa1 and Mafb, and otic vesicle-expressed Gbx2, in the genetic cascade initiated by WNT signaling that leads to dorsal otic patterning and endolymphatic duct formation. Finally, this study shows that Fgf3 prevents ventral expansion of r5-6 neurectodermal Wnt3a, serving to focus inductive WNT signals on the dorsal otic vesicle and highlighting a new example of cross-talk between the two signaling systems (Hatch, 2007).

FGF signaling regulates cytoskeletal remodeling during epithelial morphogenesis

Changes in the cytoskeletal architecture underpin the dynamic changes in tissue shape that occur during development. It is clear that such changes must be coordinated so that individual cell behaviors are synchronized; however, the mechanisms by which morphogenesis is instructed and coordinated are unknown. After its induction in non-neural ectoderm, the inner ear undergoes morphogenesis, being transformed from a flat ectodermal disk on the surface of the embryo to a hollowed sphere embedded in the head. Evidence that this shape change relies on extrinsic signals subsequent to genetic specification. By using specific inhibitors, it was found that local fibroblast growth factor (FGF) signaling triggers a phosphorylation cascade that activates basal myosin II through the activation of phopholipase Cgamma. Myosin II exhibits a noncanonical activity that results in the local depletion of actin filaments. Significantly, the resulting apical actin enrichment drives morphogenesis of the inner ear. Thus, FGF signaling directly exerts profound cytoskeletal effects on otic cells, coordinating the morphogenesis of the inner ear. The iteration of this morphogenetic signaling system suggests that it is a more generally applicable mechanism in other epithelial tissues undergoing shape change (Sai, 2008).

Basal actin depolymerization in otic ectoderm suggested a mechanism by which FGF signals, emanating from basally apposed mesoderm and neural ectoderm, remodeled the cytoskeleton. Somatic stage 10 otic regions were cultured with SU5402, a specific inhibitor of FGF signaling, for 4 hr. Such treatment does not affect genetic specification of the otic ectoderm. In otic region explants treated with carrier only (DMSO), actin filaments were apically biased and morphogenesis occurred as normal. In contrast, SU5402-treated otic regions showed loss of apical actin enrichment and a failure to invaginate. Only basal application of FGF beads could direct morphogenesis in stage 10 otic ectoderm isolates. Additionally, immunolocalization of FGFR1, the major FGF receptor in the otic placode at these stages, showed a basal bias, suggesting FGF is transduced basally. Thus mediators of FGF signaling were investigated. FGF signaling occurs through two distinct pathways: a Ras-dependent MAP kinase pathway and via the phosphorylation of phospholipase C gamma (PLCγ). Double phosphorylated Erk1/2 was detected in stage 10 otic ectoderm nuclei, but treatment of the stage 10 otic region with the MEK1/2 inhibitor U0126 only slightly altered F-actin localization and did not significantly inhibit invagination. Phosphorylated (and therefore active) PLCγ was detected at the basal side of otic placode cells. The basal localization of pPLCγ suggested activation by basal FGF signals. This was confirmed by treatment of stage 10 otic regions with SU5402. Phospho-PLCγ immunoreactivity was diminished in such treated otic regions (Sai, 2008).

Activated PLCγ affects the membrane translocation of typical protein kinase C (PKC). PKCα was detected basally in the stage 10 otic placode, reminiscent of the distributions of FGFR1 and phosphorylated PLCγ. A specific PLCγ inhibitor, U73122, caused a diminution of juxtamembrane PKCα immunoreactivity. In contrast, the inactive isomer U73343, used as a negative control, did not affect PKC localization. Whether PLCγ, acting through PKCα, leads to the asymmetric distribution of F-actin in otic placode cells was investigated. Actin polarization and otic invagination was lost in stage 10 otic regions treated with U73122. These data strongly suggest that localized PLCγ activation by FGF causes depletion of actin fibers on the basal side of the otic placode (Sai, 2008).

The involvement of actin and myosin in the closure of the mammalian neural plate and during Drosophila mesodermal invagination has been well documented, and in these cases F-actin and myosin II have been shown to colocalize and are thought to drive apical constriction. This is true during later inner ear morphogenesis, but during the earliest morphogenetic steps described in this study, F-actin and active myosin II are on opposite sides of the otic cell. Indeed, such a reciprocal localization can be detected in the early neural tube, even though during later morphogenesis colocalization is observed. Similarly, during the earliest steps of Drosophila gastrulation, myosin II is initially basal and its localization appears to be concomitant with basal F-actin loss and apical actin enrichment in the invaginating mesoderm. The signaling mechanisms that underlie these steps are still unclear, but recent data suggest the involvement of FGF receptor signal transduction in the closure of the neural tube, independent of any effect on cell fate specification. Taken together, these observations raise the intriguing possibility that the coordination of tissue morphogenesis by receptor signaling, through the activation of myosin II, may describe a general mechanism that underpins the cytoskeletal changes associated with epithelial morphogenesis (Sai, 2008).

Hey2 regulation by FGF provides a Notch-independent mechanism for maintaining pillar cell fate in the organ of Corti

The organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and, unlike other Hes/Hey factors, Hey2 expression is Notch independent. Hey2 is activated by FGF and blocks hair cell differentiation, whereas mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. It is speculated that co-option of FGF signaling to render Hey2 Notch independent also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear (Doetzlhofer, 2009).

The postnatal organ of Corti can be divided into four regions based on the expression of different combinations of Hes and Hey genes. Hes1 and HeyL define the neural region of the organ of Corti, being expressed in Kölliker's organ and inner phalangeal cells, whereas the abneural region is defined by the expression of Hes1 and Hey1 in Hensen's cells. Hes5, in combination with Hey1 and HeyL, defines the Deiters' cells that lie beneath outer hair cells, whereas Hey2 defines the pillar cell region. This combinatorial expression may have functional consequences, as Hes and Hey genes can form heterodimers that are often more stable than homodimers of each family member. The data also suggest a basis for the relatively mild cochlear phenotypes seen in single or double mutants of Hes1 and Hes5, since both Hes1 and Hes5 are expressed in supporting cells with an accompanying Hey gene family member (HeyL and Hey1, respectively), which might act redundantly with Hes1 or Hes5. Similarly, no hair cell phenotypes were observed in Hey1 or HeyL mutant mice and only very minor changes in hair cell density in Hey2 mutants. Future studies will address whether, at embryonic stages, signals initiating hair cell differentiation are responsible for the upregulation of Hes1, Hes5, and HeyL and/or for the restriction of Hey1 and Hey2 to specific cell types (Doetzlhofer, 2009).

The data reveal the existence of regulatory hierarchies between different Hes and Hey gene family members. In the absence of Hey2, the domain of Hes5 expression expanded laterally into the pillar cell region, suggesting that Hey2 can repress Hes5 expression. Such crossregulation may help to establish asymmetry in the organ of Corti, whereby inner hair cells are separated from outer hair cells by a hair cell-free region of Hey2-expressing pillar cells (Doetzlhofer, 2009).

It is interesting to note that, in contrast to the more recently derived cochlea, the mammalian vestibular system lacks pillar-like supporting cells, does not express Hey2, and contains no supporting cells that are resistant to DAPT (γ-secretase inhibitor IX). Based on the observation that extant basal monotreme mammals, such as the duck-billed platypus and echidna, have three to four rows of pillar cells separating inner from outer hair cells, it is speculated that co-option of Hey2 and its regulation by FGF signaling rather than the Notch pathway resulted in a lack of lateral inhibition between the multiple rows of pillar cells and their hair cell counterparts. In this evolutionary context, it would be interesting to determine whether Hey2 is expressed in the expanded pillar cell domain of monotremes and whether it plays a similar Notch-independent role in pattern formation in the monotreme inner ear (Doetzlhofer, 2009).

Progressive restriction of otic fate: the role of FGF and Wnt in resolving inner ear potential

The development of the vertebrate inner ear is an emergent process. Its progression from a relatively simple disk of thickened epithelium within head ectoderm into a complex organ capable of sensing sound and balance is controlled by sequential molecular and cellular interactions. Fibroblast growth factor (FGF) and Wnt signals emanating from mesoderm and neural ectoderm have been shown to direct inner ear fate. However, the role of these multiple signals during inner ear induction is unclear. This study demonstrates that the action of the FGFs and Wnts is sequential, and that their roles support a model of hierarchical fate decisions that progressively restrict the developmental potential of the ectoderm until otic commitment. Signalling by Fgf3 and Fgf19 is required to initiate a proliferative progenitor region that is a precursor to both the inner ear and the neurogenic epibranchial placodes. Significantly, it was found that only after FGF action is attenuated can the subsequent action of Wnt signalling allow otic differentiation to proceed. In addition, gain and loss of function of Wnt-signalling components show a role for this signalling in repressing epibranchial fate. This interplay of signalling factors ensures the correct and ordered differentiation of both inner ear and epibranchial systems (Freter, 2009).

Fgf3 signaling from the ventral diencephalon is required for early specification and subsequent survival of the zebrafish adenohypophysis

The pituitary gland consists of two major parts: the neurohypophysis, which is of neural origin, and the adenohypophysis, which is of non-neural ectodermal origin. Development of the adenohypophysis is governed by signaling proteins from the infundibulum, a ventral structure of the diencephalon that gives rise to the neurohypophysis. In mouse, the fibroblast growth factors Fgf8, Fgf10 and Fgf18 are thought to affect multiple processes of pituitary development: (1) morphogenesis and patterning of the adenohypophyseal anlage, and (2) survival, proliferation and differential specification of adenohypophyseal progenitor cells. The role of Fgf3 during pituitary development has been investigated in the zebrafish by analyzing lia/fgf3 null mutants. Fgf3 signaling from the ventral diencephalon has been shown to be required in a non-cell autonomous fashion to induce the expression of lim3, pit1 and other pituitary-specific genes in the underlying adenohypophyseal progenitor cells. Despite the absence of such early specification steps, fgf3 mutants continue to form a distinct pituitary anlage of normal size and shape, until adenohypophyseal cells die by apoptosis. It is further shown that Sonic Hedgehog (Shh) cannot rescue pituitary development, although it is able to induce adenohypophyseal cells in ectopic placodal regions of fgf3 mutants, indicating that Fgf3 does not act via Shh, and that Shh can act independently of Fgf3. In sum, these data suggest that Fgf3 signaling primarily promotes the transcriptional activation of genes regulating early specification steps of adenohypophyseal progenitor cells. This early specification seems to be essential for the subsequent survival of pituitary cells, but not for pituitary morphogenesis or pituitary cell proliferation (Herzog, 2004).

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