Delta
Maternal 5.4 and 4.5 kb Delta transcripts are uniformly distributed throughout the embryo during the first nine nuclear divisions of the syncytial blastoderm (Kopczynski, 1989). Zygotic transcripts begin to accumulate at the start of cellularization (early stage 5), and are localized in lateral bands bordering the prospective mesoderm. After gastrulation Delta is found in ventral neurogenic region and in a pair-rule-like banded structure in the ectoderm, distributed in a ventral to dorsal gradient (Haenlin, 1990).
By stage 6, there is accumulation in the anterior gnathal segments [Images] and procephalic neurogenic ectoderm From stage 8 through 11, Delta transcripts accumulate in endodermal derivatives (anterior and posterior midgut). By stage twelve, Delta transcripts are reduced throughout the ventral epidermis. Delta accumulates at high levels in the mesoderm by stage 10 (Kopczynski, 1989).
Big brain and Delta proteins colocalize. In the prospective mesoderm just before gastrulation, Bib
protein disappears from the plasma membrane and is present in punctate cytoplasmic structures basal to the
nucleus. The Delta protein is expressed in a similar manner. To address whether Bib and Dl colocalize,
embryos were simultaneously labeled with Bib and Dl antibodies.
The Bib and Dl proteins did in fact colocalize in the plasma membrane and in the punctate cytoplasmic structures of prospective mesoderm cells, although the intensity
of the two signals was not always similar. By immunoelectron microscopy, it was
found that bib is associated with the plasma membrane and concentrated in apical
adherens junctions as well as in small cytoplasmic vesicles (Doherty, 1997).
For information about the role of Delta (reviewed by Hartenstein, 1992) in the development of specific tissues see:
Notch signalling is an evolutionarily conserved cell interaction mechanism; its role in controlling cell fate choices has been studied extensively. Recent studies in both vertebrates and invertebrates have revealed additional functions of Notch in proliferation and apoptotic events. Evidence suggests an essential role of the Notch signalling pathway during morphogenetic cell movements required for the formation of the foregut-associated proventriculus organ in the Drosophila embryo. The activation of the Notch receptor occurs in two rows of boundary cells in the proventriculus primordium. The boundary cells delimit a population of foregut epithelial cells that invaginate into the endodermal midgut layer during proventriculus morphogenesis. Notch receptor activation requires the expression of its ligand Delta in the invaginating cells and apical Notch receptor localization in the boundary cells. The movement of the proventricular cells is dependent on the short stop gene that encodes the Drosophila plectin homolog of vertebrates and is a cytoskeletal linker protein of the spectraplakin superfamily. short stop is transcriptionally activated in response to the Notch signalling pathway in boundary cells, and it has been shown that the localization of the Notch receptor and Notch signalling activity depend on short stop activity. These results provide a novel link between the Notch signalling pathway and cytoskeletal reorganization controlling cell movement during the development of foregut-associated organs (Fuss, 2004).
The proventriculus is a multiply folded, cardia-shaped organ that functions as a valve to regulate food passage from the foregut into the midgut of Drosophila larvae. It is derived from the stomodeum, which gives rise to the foregut tube and to parts of the anterior midgut in the early embryo. Cell shape changes are initiated at stage 12 when cell proliferation has been completed within the proventriculus primordium. Anti-Forkhead (Fkh)/anti-Defective proventriculus (Dve) double immunostainings which specifically visualize ectodermal and endodermal cells, respectively, reveal that the first step of proventriculus morphogenesis involves the formation of a ball-like evagination at the ectoderm/endoderm boundary of the posterior foregut tube. The formation of this evagination is initiated by a local constriction of apical membranes at the ectoderm/endoderm boundary leading to an accumulation of membrane-associated markers such as Arm towards the luminal (apical) side. It is notable that the ectodermal part of the ball-like evagination localizes in a mesoderm-free region, whereas the surrounding cells of the developing foregut and the midgut are covered by visceral mesoderm. At stage 14, a constriction forms at the boundary of the ectodermal and the endodermal cells. This results in the formation of the 'keyhole' structure. From stage 14 onward, cells from the anterior portion of the ectodermal keyhole part (in the mesoderm-free area) begin to move inward into the endodermal part of the keyhole and a heart-like structure is formed. The ectodermal keyhole cells continue to move inward until late stage 17 and give rise to the recurrent layer of the proventriculus; it links the outer endodermal layer (derived from the endodermal keyhole cells) and the inner layer of the proventriculus which is a continuation of the esophagus. The cells at the tip of the invaginating ectodermal keyhole cells thatderive from the most anterior region of the keyhole, are not covered by visceral mesoderm. It has been observed before that these cells assume a stretched appearance with long cytoplasmic extensions (Fuss, 2004 and references therein).
Immunohistochemical analysis demonstrates that the ligands of the Notch
receptor, Delta and Serrate are expressed in the ectodermal keyhole cells that invaginate into the endodermal cell layer during proventriculus development. Their expression becomes downregulated in the anterior and posterior boundary cells in which the Notch receptor is elevated and in which the Notch signalling pathway is activated, as demonstrated by the Notch-dependent Gbe-Su(H)m8-lacZ reporter construct. Whereas there is no proventricular phenotype in Ser mutants, the invagination movement of the ectodermal keyhole cells is defective in mutants of other components of the Notch signalling pathway, such as Notch, Delta, fng or Su(H). This strongly suggests that the boundary cells play a crucial role for cell movement during proventriculus development. It is not known whether
the cell movements are driven by the anterior boundary cells, dragging the
esophageal cells behind or whether the major force for the inward movement is contributed by the ectodermal foregut cells changing their shapes from a cuboidal to a more stretched appearance. The latter is known to occur during mid and late stages of embryogenesis when the foregut and the hindgut elongate dramatically increasing their size by two- to threefold. It has been shown for dorsal closure that multiple forces contribute to cell sheet morphogenesis. A similar scenario may apply for proventriculus morphogenesis. Genetic mosaic studies have revealed that the activity of the Notch receptor occurs in cells that are adjacent to the ligand-expressing cells. Therefore, the downregulation of Delta in the boundary cells may be a prerequisite for Notch signalling and cell movement, which would be consistent with the observation that a Notch-like proventriculus phenotype is induced when Delta expression is maintained in the anterior and posterior boundary cells (Fuss, 2004).
The spatial and temporal coordination of patterning and morphogenesis is often achieved by paracrine morphogen signals or by the direct coupling of cells via gap junctions. How paracrine signals and gap junction communication cooperate to control the coordinated behavior of cells and tissues is mostly unknown. This study found that Hedgehog signaling is required for the expression of wingless and of Delta/Notch target genes in a single row of boundary cells in the foregut-associated proventriculus organ of the Drosophila embryo. These cells coordinate the movement and folding of proventricular cells to generate a multilayered organ. hedgehog and wingless regulate gap junction communication by transcriptionally activating the innexin2 gene, which encodes a member of the innexin family of gap junction proteins. In innexin2 mutants, gap junction-mediated cell-to-cell communication is strongly reduced and the proventricular cell layers fail to fold and invaginate, similarly as in hedgehog or wingless mutants. It was further found that innexin2 is required in a feedback loop for the transcriptional activation of the hedgehog and wingless morphogens and of Delta in the proventriculus primordium. It is proposed that the transcriptional cross regulation of paracrine and gap junction-mediated signaling is essential for organogenesis in Drosophila (Lechner, 2007).
In both vertebrates and invertebrates, the posterior foregut constitutes a center of organogenesis from which gut-associated organs such as the lung in vertebrates or the proventriculus in Drosophila develop. Proventriculus development involves the folding and invagination of epithelial cell layers to generate a multiply-folded organ. Two cell populations, the anterior and the posterior boundary cells, were shown previously to control cell movement and the folding of the proventriculus organ. In the posterior boundary cells, which organize the endoderm rim of the proventriculus, the JAK/STAT signaling cascade cooperates with Notch signaling to control the expression of the gene short stop encoding a cytoskeletal crosslinker protein of the spectraplakin superfamily. Thereby the Notch signaling pathway is connected to cytoskeletal organization in the posterior boundary cells, which have to provide a stiffness function to enable the invagination of the ectodermal foregut cells. The findings in this paper provide evidence that hedgehog is essential for the Notch signaling-dependent allocation of the anterior boundary cells. In amorphic hedgehog mutants, evagination and the formation of the constriction at the ectoderm/endoderm boundary are not affected, however, the inward movement of the anterior boundary cells is not initiated at the keyhole stage. The lack of cell movement of the ectodermal proventricular cells is consistent with the finding that hedgehog specifically controls Notch target gene activity in the anterior boundary cells. Genetic experiments further identify wingless as a target gene of hedgehog in the anterior boundary cells. wingless, in turn, controls the transcription of the innexin2 gene, which is expressed in the invaginating proventricular cells. When wingless is re-supplied in the genetic background of hedgehog mutants, innexin2 expression is rescued, providing further evidence that innexin2 is a target gene of wingless in the proventriculus primordium. Innexin2 encodes a member of the innexin family of gap junction proteins and is essential for the development of epithelial tissues. In the proventriculus, innexin2 mRNA is initially expressed in the early evagination stage in a broad domain covering both the ectodermal and endodermal precursor cells of the proventriculus primordium. When the ectodermal cells start to invaginate into the proventricular endoderm, innexin2 expression is upregulated in the ectodermal cell layer. Invagination of the ectodermal cells fails in hedgehog, wingless and kropf mutant proventriculi and dye tracer injection experiments demonstrate that hedgehog and kropf mutants show a strong reduction of gap junction communication. These data suggest that the direct coupling of cells via Innexin2-containing gap junctions, which are induced in response to hedgehog and wingless activities, is important for the coordinated movement of the ectodermal cell layer. It is known from extensive studies in mammals that the coupling of cells and tissues via gap junctions enables the diffusion of second messengers, such as Ca2+, inositol-trisphosphate (IP3) or cyclic nucleotides to allow the rapid coordination of cellular behavior during morphogenetic processes such as cell migration and growth control. Cell movement and folding involves a modulation of cell adhesion and of cytoskeletal architecture of the proventricular cells. A functional interaction of innexin2 with the cell adhesion regulator DE-cadherin, which is a core component of adherens junctions has been shown recently by co-immunoprecipitation, yeast two-hybrid studies, and genetic analysis. In mutants of DE-cadherin, Innexin2 is mislocalized and vice versa suggesting that the regulation of cell adhesion and gap junction-mediated communication may be linked. Similar evidence for a coordinated regulation of connexin activity and N-cadherin has been obtained in mammals during migration of neural crest cells (Lechner, 2007).
In kropf mutants or innexin2 knockdown animals, hedgehog, wingless and Delta transcription is strongly reduced as shown by in situ hybridization and by quantitative RT PCR experiments using mRNAs isolated from staged embryos. Furthermore, hedgehog, wingless and Delta are ectopically expressed and their mRNA is upregulated in embryos in which innexin2 is overexpressed. In summary, these experiments provide strong support that the gap junction protein Innexin2 plays an essential role enabling or promoting transcriptional activation of hedgehog, wingless and Delta. These data point towards an essential requirement of gap junction communication for the transcriptional activation of morphogen-encoding genes activating evolutionary conserved signaling cascades essential for patterning in animals. It is of note that gap junctions are established at very early stages of embryonic development, correlating with a maternal and zygotic expression of innexin2 and other innexin family members. kropf mutant animals, which are devoid of maternal and zygotic innexin2 expression are early embryonic lethal and develop no epithelia, consistent with a fundamental role of gap junctions in development, on top of which pattern formation of tissues and organs may occur. It has been shown previously that gap junctions are essential for C. elegans, Drosophila, and vertebrate embryogenesis from early stages onwards (Lechner, 2007 and references therein).
In the nematode C. elegans, a transient network formed by the innexin gap junction protein NSY-5 was recently shown to coordinate left-right asymmetry in the developing nervous system. Previous findings in chick and Xenopus laevis embryos have suggested an essential role of connexin43-mediated gap junction for the determination of the left-right asymmetry of the embryos. Treatment of cultured chick embryos with lindane, which results in a decreased gap junctional communication, frequently unbiased normal left-right asymmetry of Sonic hedgehog and Nodal gene expression, causing the normally left-sided program to be recapitulated. An important role of connexin43 (Cx43)-dependent gap junction communication for sonic hedgehog expression was also observed in limb patterning of the chick wing. Additionally, modulation of gap junctions in Xenopus embryos by pharmacological agents specifically induced heterotaxia involving mirror-image reversals of the heart, gut, and gall bladder. These data in combination with the current findings indicate that the transcriptional regulation of hedgehog and other morphogen-encoding genes by gap junction proteins may be evolutionary conserved between deuterostomes (vertebrates) and protostomes (Drosophila), although the Drosophila innexin gap junction genes share very little sequence homology with the connexin genes. The molecular mechanism underlying innexin2-mediated transcriptional regulation of hedgehog, wingless and Delta is not clear. It has been proposed that the nuclear localization of the carboxy-tail of connexin43 may exert effects on gene expression and growth in cardiomyocytes and HeLa cells. This would infer a cleavage of connexin43 to release the C-terminus, however, in vivo evidence for this event is still lacking. Sequence analysis reveals a nuclear receptor recognition motif within the C-terminus of Innexin2. It has been demonstrated that this recognition motif mediates the interaction of coactivators with nuclear receptors. However, there is no immunohistochemical evidence for a nuclear localization of Innexin2 or the Innexin2 C-terminus in Drosophila embryonic cells indicating that a direct involvement of Innexin2 in regulating transcription of target genes may not occur. The direct association of a transcription factor with gap junctions has been recently proposed for the mouse homolog of ZO-1-associated nucleic acid-binding protein (ZONAB). This transcription factor binds to ZO-1, which is associated with oligodendrocyte, astrocyte and retina gap junctions. It is possible that innexin2-dependent transcriptional regulation may involve a similar type of mechanism: a still unknown transcriptional regulator associated with the C-terminus of innexin2-containing gap junctions could be released upon modulation of gap junction composition thereby modulating the transcription of innexin2-dependent target genes (Lechner, 2007).
This paper investigated the developmental-genetic steps that shape the entero-endocrine system of Drosophila melanogaster from the embryo to the adult. The process starts in the endoderm of the early embryo where precursors of endocrine cells and enterocytes of the larval midgut, as well as progenitors of the adult midgut, are specified by a Notch signaling-dependent mechanism. In a second step that occurs during the late larval period, enterocytes and endocrine cells of a transient pupal midgut are selected from within the clusters of adult midgut progenitors (AMPd). As in the embryo, activation of the Notch pathway triggers enterocyte differentiation and inhibits cells from further proliferation or choosing the endocrine fate. The third step of entero-endocrine cell development takes place at a mid-pupal stage. Before this time point, the epithelial layer destined to become the adult midgut is devoid of endocrine cells. However, precursors of the intestinal midgut stem cells (pISCs) are already present. After an initial phase of symmetric divisions which causes an increase in their own population size, pISCs start to spin off cells that become postmitotic and express the endocrine fate marker, Prospero. Activation of Notch in pISCs forces these cells into an enterocyte fate. Loss of Notch function causes an increase in the proliferatory activity of pISCs, as well as a higher ratio of Prospero-positive cells (Takashima, 2011).
The function of the intestinal tract of all animals is regulated by two closely interrelated systems, the autonomic nervous system and the endocrine system. The endocrine system is formed by specialized endocrine glands, like the islets of Langerhans in vertebrates' pancreas, or the corpora cardiaca in insects, as well as scattered entero-endocrine cells that form part of the intestinal epithelium. These cells, which typically outnumber cells of all other endocrine organs combined, represent the diffuse endocrine system (DES). Entero-endocrine cells produce different peptide hormones with specific regional distributions and functions. Well known examples from vertebrates are secretin or CKK (produced in the duodenum; stimulates pancreatic bicarbonate secretion), or gastrin (produced in the stomach; increases acid secretion from parietal cells). The peptide hormones produced by entero-endocrine cells also occur as neuro-transmitters in neurons and are therefore frequently referred to as 'brain-gut peptides'. For example, the peptides of the insect tachykinin family are found both in midgut entero-endocrine cells, as well as in neurons of the central nervous system (Winther, 2003). Tachykinin released from neurons locally affects gut muscle contractility; systemic release into the hemolymph acts on many effector organs, including the excretory Malpighian tubules, the heart, and the somatic musculature (Takashima, 2011).
The Drosophila midgut consists of three major cell types, enterocytes, entero-endocrine cells, and progenitors/stem cells. These cells are generated three times during development, when generating a larval midgut, transient pupal midgut, and adult midgut. The mechanisms that specify the cell types, and the morphogenetic process by which these cells actually separate from each other, appear to be highly conserved during each of these phases. Thus, in a first step, a pool of undifferentiated progenitor cells increases its population by cell division. During this phase (early embryonic endoderm; early larval AMPs, late larval AMPs) all cells express esg. In a second step, enterocytes (the outer endoderm layer in embryo; peripheral cells in larva; and prospective adult enterocytes in early pupa) become postmitotic, lose esg expression, and physically split from another group of cells which maintain esg expression and continue to divide (inner layer of embryonic endoderm; inner AMPs in larva; pISCs in pupa). In a third step, the esg-positive cells give off endocrine cells which become Pros-positive and eventually lose esg, as well as progenitor/stem cells that continuously remain esg-positive (Takashima, 2011).
Notch/Delta signaling mediates the decision of midgut cell fates produced by the ISCs in the adult. After each ISC division, Delta is maintained in one of the two daughter cells as a renewed ISC and activates Notch pathway in the other, neighboring daughter cell. This second daughter, called enteroblast, is inhibited by Notch activity from further division. In addition, the level of Notch activity determines whether the enteroblast differentiates as enterocyte (high Notch activity) or entero-endocrine cell (low Notch activity). Thus, a key element of the mechanism underlying midgut fate determination is the level and localization of Delta. In the adult midgut, upstream acting signaling events emanating from the visceral musculature that surrounds the gut are responsible in maintaining (various levels of) Delta expression in the ISCs. It is proposed that the same mechanism could start acting during the pupal stage. During early metamorphosis, the larval visceral musculature de-differentiates; myofibrils and extracellular matrix is lost, even though the muscle cells themselves survive. Around 40 hours APF these cells re-differentiate into adult visceral muscle. This is about the time when pISC change their proliferation pattern from symmetric self renewal to a mode that produces endocrine cells. It is possible that the re-occurrence of visceral muscle structure may differentially activate Delta protein in pISC to change the fate of their daughter cells (Takashima, 2011).
It is likely that the level and spatial distribution of the Delta signal is also instrumental in determining the right number and patterning of enterocytes vs endocrine cells vs progenitors in the larva and embryo. In the larva, complex signaling events between the emerging peripheral cells and the central cells of the AMP islands could focus expression of Delta towards the latter. Again, it is possible that the larval visceral musculature also plays a role in adjusting Delta expression. Delta in turn activates the Notch cascade in the periphery of the AMP islands, resulting in a certain number of transient pupal enterocytes and endocrine cells to develop within each island (Takashima, 2011).
In the embryo, Delta is required to trigger high Notch activity in endoderm cells that separate as outer layer and differentiate into enterocytes. Delta is expressed widely in the endoderm during the stage when inner and outer layers split, and it is not clear how differences in Delta level correlate with different locations of cells within the endoderm. It should be noted that the situation is complicated by the fact that several different types of enterocytes develop which will populate the different segments of the midgut along the antero-posterior axis; for example, one these cell types, the so called interstitial cells which come to lie within the middle of the gut tube, split from the remainder of the enterocytes even earlier than the endocrine cells and the AMPs. More work, based on additional fate-specific markers, is required to reconstruct in detail the mechanics of the Notch-Delta signaling step that specifies fate in the embryonic endoderm (Takashima, 2011).
In the vertebrate gut, entero-endocrine cells are formed within the same endodermal primordium that gives rise to the enterocytes. As in Drosophila, all endodermal cells of the early vertebrate embryo proliferate. Subsequently, as the endodermal epithelium gets folded into villi and crypts, proliferation gets restricted to the crypts. In the mature gut, crypts contain small numbers of slowly cycling stem cells (ISCs), surrounded by a progeny that divides fast (amplifying progenitors) and at the same time adopts different fates. As cells differentiate, they move upward to the apices of the villi, from which they are eventually sloughed off (Takashima, 2011).
Recent studies in vertebrates have started to elucidate the mechanism by which different intestinal cell fates, including enterocytes, entero-endocrine cells (and other secretory cell types), and proliferatory stem cells are related. Labeled clones have been shown to contain both enterocytes and entero-endocrine cells. This suggests that at the level of the progenitor or stem cells in which the clones were induced, a decision between endocrine and enterocyte fate had not yet been made. In line with this conclusion, other studies showed that cell-cell interactions among intestinal cell precursors involving the Notch signaling pathway specify intestinal cell fate. Endocrine precursors (including still proliferatory as well as postmitotic cells) express bHLH transcription factors ('pro-endocrine factors'), in particular Math-1 and neurogenin 3, as well as the Notch ligand, Delta. Loss of Delta or Notch function increases the number of endocrine cells, often at the expense of enterocytes; loss of 'proendocrine factors' has the opposite phenotype. For example, in mouse, Math-1 is expressed in the zone of transient amplifying progenitors and then becomes restricted to postmitotic exocrine and endocrine cells. Loss of Math-1 results in the absence of both cell populations. Another proneural gene, neurogenin 3, may act downstream of Math-1 in a more restricted progenitor populations that include only endocrine cell types. Thus, loss of neurogenin 3 in mouse results in the absence or strong reduction of several endocrine cell populations, in particular glucagon, somatostatin, and gastrin expressing cells. The Notch ligand Delta (DeltaD in zebrafish; Delta1 in mouse) is expressed in enteroendocrine and secretory cells. Disruption of Delta function (by depleting the gene mib in zebrafish) converts gut enterocytes into secretory (exocrine/endocrine) cells. These and other data indicate that the cell fate determining mechanism is highly conserved in vertebrates and Drosophila (Takashima, 2011 and references therein).
A general conclusion that can be drawn from this study, as well as similar studies in vertebrate intestinal development, is that the selection of endocrine cells from epithelial enterocytes follows a similar mechanism, and is controlled by the same gene cassette, as the selection of neural precursors from the neurectoderm. The 'developmental-genetic scenario' that encountered during early neurogenesis (bHLH gene expression-proneural cluster-Notch signaling lateral inhibition-high Notch activity-epithelial cells low Notch activity-delaminating neurons/neuroblasts) appears very similarly in gut development. Here, the epithelial cells depending on high Notch activity are the enterocytes; the cells requiring bHLH genes, and low Notch activity, are the endocrine precursors, which also (at least partially) delaminate from the epithelium. It has long been realized that neurons and endocrine cells share the expression of numerous molecular and ultrastructural features. However, given the developmental findings, the extent of 'genetic overlap' between endocrine and neural cells may be even greater than previously expected. The similarities between neurons and endocrine cells probably reflect a common phylogenetic origin of these cell types. The communication among cells that is mediated by secreted, diffusible signals is phylogenetically older than neural transmission. Animals without nervous system (e.g., sponges) and even protists produce many secreted molecules which are often molecularly homologous to hormones or neural transmitters found in multicellular animals. One may speculate that, during an early step of evolution that occurred in primitive multicellular animals, specialized epithelial cells within the epidermis and the gut reacted to certain stimuli by secreting metabolites that diffused throughout the body and evoked adaptive responses in other tissues. A direct line of evolution led from these cells to the entero-endocrine cells is still found today in all animals. A second line of evolution transformed subsets of the hypothetical 'endocrine-neural forebears' into neurons, cells which elaborate processes and form specialized synaptic contacts. According to this view it would be expected that a conserved genetic mechanism is employed to separate endocrine cells and neurons from the respective epithelium into which they are embedded (Takashima, 2011).
Interactions between niche cells and stem cells are vital for proper control over stem cell self-renewal and differentiation. However, there are few tissues where the initial establishment of a niche has been studied. The Drosophila testis houses two stem cell populations, which each lie adjacent to somatic niche cells. Although these niche cells sustain spermatogenesis throughout life, it is not understood how their fate is established. This study shows that Notch signaling is necessary to specify niche cell fate in the developing gonad. Surprisingly, these results indicate that adjacent endoderm is the source of the Notch-activating ligand Delta. Niche cell specification occurs earlier than anticipated, well before the expression of extant markers for niche cell fate. This work further suggests that endoderm plays a dual role in germline development. The endoderm assists both in delivering germ cells to the somatic gonadal mesoderm, and in specifying the niche where these cells will subsequently develop as stem cells. Because in mammals primordial germ cells also track through endoderm on their way to the genital ridge, this work raises the possibility that conserved mechanisms are employed to regulate germline niche formation (Okegbe, 2011).
The data reveal that Notch signaling is necessary to specify hub cell fate. A similar conclusion has recently been reached by Kitadate (2010). It is interesting to note that in three well-characterized stem cell-niche systems in Drosophila, including the transient niche for adult midgut progenitors, the female gonad and now the developing male gonad, Notch signaling is directly responsible for niche cell specification. Moreover, Notch has been found to play a role in the maintenance of various mammalian stem cell populations, including neural stem cells, HSCs and hair follicle stem cells. However, owing to difficulty in performing lineage-specific knockouts in these systems, it remains unclear which cells require Notch activity. As the various cases in Drosophila all require direct Notch activation for niche cell specification, perhaps this reveals a conserved role for Notch signaling in other, more complex stem cell systems (Okegbe, 2011).
Notch signaling specifies niche cells in both the male and female Drosophila gonad; however, it is important to note that there are still some differences. For the ovary, only Delta is required to activate the Notch receptor for proper niche cell specification. For the testis, both ligands contribute to the process, although, here too, it appears that Delta is the dominant ligand employed. Interestingly, depleting Delta or (genetically) separating the endoderm from SGPs (somatic gonadal precursors) both led to a 70% reduction in hub cell number, while depleting Serrate yielded a 30% reduction. Perhaps Delta-Notch signaling from the endoderm accounts for two-thirds of hub cell specification, while Serrate-Notch signaling accounts for only one-third of this process. Although the source of Serrate could not be identifed in this study, Kitadate (2010) has shown that Serrate mRNA is expressed from SGPs after gonad coalescence. Perhaps, this late expression accounts for the modest role Serrate plays in hub specification. That study did not explore in detail a potential role for Delta in hub specification, and the current data suggests that that role is carried out at earlier stages, and from outside the gonad proper (Okegbe, 2011).
In the ovary, cells within the developing gonad appear to present the Notch-activating ligand, although it is unclear whether germ cells or somatic cells are the source of Delta. The current data suggests that cells from a distinct germ layer, the endoderm, present Delta to SGPs in the male gonad. These differences may indicate distinct evolutionary control over gonadal niche development between the sexes (Okegbe, 2011).
Although the gonad first forms during mid-embryogenesis, hub cells only become identifiable just prior to hatching of the larvae, some 6 hours later. At that time, hub cells begin to tightly pack at the anterior of the gonad, upregulate several cell adhesion and cytoskeletal molecules (Fascilin 3, Filamin, DN-Cadherin, DE-Cadherin) as well as induce Upd expression and other markers of hub fate. Surprisingly, the current data reveal that most hub cells are specified well before these overt signs of hub cell differentiation, as judged by Notch reporter activation and Notch rescue. Although it was previously thought that SGPs were equivalent at the time of gonad coalescence it is now clear that due to Notch activity, the SGPs are parsed into a group of either hub cells or cyst cells before gonad coalescence occurs (Okegbe, 2011).
Thus, it is believed that a series of steps must occur before the hub can function as a niche. First, the PMG (posterior midgut) presents Delta, leading to Notch activation in some SGPs as they are carried over these endodermal cells during germ band retraction. Activation might be dependent on, for example, length of time in contact with passing PMG cells. At the present time, it is unclear whether all SGPs are activated for Notch (Kitadate, 2010), or only some of them (this study). After gonad coalescence, activated SGPs must then migrate anteriorly. Although it is known that integrin-mediated adhesion is required to maintain the hub at the anterior (Tanentzapf, 2007), no cues have been identified that could guide the migration of the Notch-activated SGPs. Next, as the cells reach the anterior of the gonad they must execute a mesenchymal-to-epithelial transition, as evidenced by the upregulation of cell-adhesion molecules and preferential associations between hub cells. This step occurs independently of the integrin-mediated anchoring at the anterior. Finally, the hub cells must induce Upd expression and recruit neighboring cells to adopt stem cell fate. The apparent delay between the activation of the Notch pathway and the initiation of the hub cell gene expression program might suggest that initiating that hub program first requires that the cells coalesce into an epithelium. Such a mechanism would prevent precocious or erroneous stem cell specification within the gonad (Okegbe, 2011).
Although these data reveal Notch-activated SGPs at all positions within the gonad and that some of these become hub cells, it is unclear how hub cell number is tightly regulated. Potentially, SGP migration over endodermal cells could induce Notch activation among SGPs throughout the forming gonad, potentiating these cells to become hub cells. However, solely relying on that mechanism could lead to the specification of too many hub cells. It appears, though, that specification is regulated by EGFR pathway activation (Kitadate, 2010). EGFR protein is observed on most SGPs throughout the embryonic gonad, beginning at gonad coalescence (stage 13). The EGFR ligand Spitz is expressed from all germ cells during gonad coalescence and activates EGFR among posterior SGPs. This activity antagonizes Notch and that appears to regulate final hub cell number. How EGFR activation is restricted or enhanced only among posterior SGPs is at present unclear (Okegbe, 2011).
Given that this study found that hub cell specification occurs prior to gonad coalescence, it is also possible that Notch and EGFR act in a temporal sequence. In this case, early Notch-activated SGPs, perhaps even those in the posterior will adopt hub cell fate. But, as EGFR becomes activated, further induction of the Notch pathway in the posterior is antagonized, prohibiting the specification of too many hub cells. Such a temporal inhibition might be important, as Serrate is expressed on the SGPs (Kitadate, 2010) and both Delta and Serrate are robustly expressed on tracheal cells, the activity of which might otherwise lead to excess hub cell induction. Perhaps during later stages of gonadogenesis (stages 14-16) a small number of anterior SGPs become Notch activated due to the activity of Serrate-Notch signaling from other SGPs, supplementing the hub cells previously specified by Delta-Notch signaling (Okegbe, 2011).
Given that niche cells in the Drosophila ovary become activated via Delta-Notch signaling by neighboring somatic cells, it was initially expected that Notch would be activated in a subset of SGPs by ligand presented from other SGPs. However, this study could not detect Delta nor Serrate expression among SGPs. Furthermore, although nearby tracheal cells expressed both ligands robustly, that expression appears later than Notch rescue suggests would be necessary, and genetic ablation of tracheal cells did not influence hub cell number (Okegbe, 2011).
Instead, this study found that a crucial signal for niche cell specification is presented from the endoderm, as Delta is expressed robustly on posterior midgut cells, at a time consistent with the requirement for Notch function. Furthermore, these endodermal cells are close enough to SGPs for productive Delta-Notch signaling to occur. Although visceral mesodermal cells are also close to the PMG and the SGPs, this tissue does not affect hub specification, since this study found that brachyenteron mutants exhibited normal hub cell number. By contrast, in mutants that do not internalize the gut (fog), and thus would not present Delta to SGPs, a drastic reduction was seen in hub cell number (Okegbe, 2011).
Additionally, it is noted that absolute hub cell number varies among animals and according to genetic background. This is attributed to normal biological variation, just as germline stem cell number varies. Potentially, this variation could be caused by the robustness with which the Notch pathway is activated in SGPs, as they are carried over the midgut cells. It will be interesting to test this hypothesis by genetically manipulating the number of midgut cells or the time of contact between endoderm and SGPs. Additionally, the antagonistic effects of EGFR signaling might account for some of the observed variation. In fact, gonads heterozygous for Star, a component of the EGFR pathway, exhibit increased hub cell number (Okegbe, 2011).
Finally, it is interesting to consider why the endoderm would be crucial for the proper specification of the GSC niche. In Drosophila, as in many animals, there is a special relationship between the gut and the germ cells. Primordial germ cells in mammals and in Drosophila must migrate through the endoderm to reach the gonadal mesoderm. In fact, in Drosophila, the gut exercises elaborate control over germ cell migration. As the germ cells begin their transepithelial migration and exit from the midgut pocket, tight connections between midgut cells are dissolved, allowing for easy germ cell passage. Germ cells then migrate on the basal surface of endodermal cells and midgut expression of wunens (which encodes lipid phosphate phosphatases) repels germ cells, driving them into the mesoderm. Thus, the endoderm not only delivers germ cells to the somatic mesoderm, but the same endoderm specifies niche cells from among the somatic mesoderm wherein germ cells can subsequently develop into stem cells. In mammals, although the exact make-up of the spermatogonial stem cell niche has not been determined, it must (in part) derive from cells of the genital ridge. It will be interesting to determine whether proximity to the gut endoderm is important for the specification of this niche (Okegbe, 2011).
Delta and Notch are required for partitioning of vein and intervein cell fates within the provein during Drosophila metamorphosis. Partitioning of these fates is dependent on Delta-mediated signaling from 22 to 30 hours after puparium formation at 25 degrees C. Within the provein, Delta is expressed more highly in central provein cells (presumptive vein cells) and Notch is expressed
more highly in lateral provein cells (presumptive intervein cells). Accumulation of Notch in presumptive intervein cells is dependent on
Delta signaling activity in presumptive vein cells; constitutive Notch receptor activity represses Delta accumulation in presumptive
vein cells. When Delta protein expression is elevated ectopically in presumptive intervein cells, complementary Delta and Notch
expression patterns in provein cells are reversed, and vein loss occurs because central provein cells are unable to stably adopt the vein
cell fate. These findings imply that Delta-Notch signaling exerts feedback regulation on Delta and Notch expression during metamorphic
wing vein development, and that the resultant asymmetries in Delta and Notch expression underlie the proper specification of vein and
intervein cell fates within the provein (Huppert, 1997).
Notch activation at the midline plays an essential role both
in promoting the growth of the eye primordia and in regulating eye patterning. Specialized cells are established along the dorsal-ventral midline
of the developing eye by Notch-mediated signaling between dorsal and ventral cells. D-V signaling in the eye shares many similarites with D-V signaling in the wing. In both cases an initial asymmetry is set up by Wingless expression. Both Eye and wing cells then go through a distinct intermediate step: in the wing, Wingless represses the expression of Apterous, a positive regulator of fringe (fng) expression; in the eye, Wingless promotes the expression of mirror (mrr), which encodes a negative regulator of fringe (unpublished observations of McNeill, Chasen, Papayannopoulos, Irvine, and Simon, cited by Papayannopoulos, 1998). Both wing and eye cells share a Fng-Ser-Dl-Notch signaling cassette to effect signaling between dorsal and ventral cells and establish Notch activation along the D-V midline. Local activation of Notch leads to production of diffusible, long-range signals that direct growth and patterning, which in the wing include Wingless, but in the eye remain unknown. At least one downstream target of D-V midline signaling, four jointed (fj), is also conserved. four jointed is also expressed in the wing and its expression there is indirectly influenced by Notch (Papayannopoulos, 1998 and references).
During early eye development, fringe is expressed by ventral cells. This expression appears to be complementary to that of the dorsally expressed gene mrr. During early to mid-third instar, additional expression of fng appears in the posterior of the eye disc. This line of posterior fng expression is just in front of the morphogenetic furrow and moves across the eye ahead of the furrow. In the wing disc, Dl and Ser induce each other's expression, and become up-regulated along the D-V border where they can productively signal. Dl and Ser are also preferentially expressed along the D-V midline during eye development. Ser expression, like fng expression, is complementary to that of mrr, whereas Dl expression partially overlaps that of mrr. The spatial relations among fng, Ser, and Dl expression in the eye are thus similar to those in the wing, although in the wing, their expressions are inverted with respect to the D-V axis (Papayannopoulos, 1998).
The four-jointed gene is expressed in a gradient during early eye development, with a peak of expression along the D-V midline. Together with Ser and Dl, Fj serves as a molecular marker of midline fate. Ubiquitous expression of Fng during early eye development, generated by placing fng under the control of an eyeless enhancer, eliminates detectable expression of Ser and Dl along the midline. Conversely, misexpression of Fng in clones of cells, can result in ectopic expression of Ser and fj that is centered along novel borders of Fng expression in the dorsal eye. Ectopic Ser and fj expression can also be detected along the borders of fng mutant clones in the ventral eye. These observations show that Fng expression borders play an essential and instructive role in establishing a distinct group of cells along the D-V midline of the developing eye. Animals with reduced fng activity have small eyes. Moreover, ubiquitous fng expression also results in a dramatic loss of tissue. Tissue loss is detectable in the developing imaginal disc, before the morphogenetic furrow moves across the eye. Moreover, eye loss is observed when fng is ectopically expressed during early development, but not when fng is ectopically expressed behind the furrow. These observations indictate that a Fng expression border is required for eye growth, specifically during early eye development (Papayannopoulos, 1998).
Fng differentially modulates the action of Notch ligands in the eye just as it does in the wing.
Clones of cells ectopically expressing Dl can induce Ser expression in ventral, Fng-expressing cells, but not in dorsal cells. Fng alone can induce Ser expression in dorsal cells, but only near the D-V midline. When Fng and Dl are co-misexpressed, Ser expression can be induced in dorsal cells even when the clones are far from the D-V midline. Clones of cells ectopically expressing Ser are able to induce increased expression of Dl in dorsal cells but not in ventral, Fng expressing cells. However, if Ser is ectopicallly expressed in fng mutant animals, it can induce Dl expression in ventral cells (Papayannopoulos, 1998).
Notch function is also necessary for normal D-V midline cell fate. The ability of Ser and Dl to induce one another's expression indicates that the expression of either one is a marker for Notch activation in the eye. Analysis of loss-of-function mutants of Notch and its ligands, as well as ectopic expression studies, indicate that Notch activation also regulates eye growth. Several observations indicate that the D-V midline is the focus of Notch activation required for growth. Moreover, the midline corresponds to a fng expression border, which is essential for growth and modulates Notch signaling during early eye development. Because local activation of Notch has long-range effects on growth and four-jointed expression, it is inferred that Notch induces the expression of a diffusible growth factor at the midline. Notch activation influences ommatidial chirality. fng mutant clone borders within the ventral eye can be associated with reversals of ommatidial chirality, whereas mutant clones that cross the D-V midline disrupt the normal equator. The equatorial bias in the influence of ectopic Notch activation implies that the equator is the normal source of a Notch-dependent, chirality-determining signal (Papayannopoulos, 1998).
In Drosophila, a wave of differentiation progresses across the retinal field in response to signals from posterior cells. Hedgehog (Hh), Decapentaplegic (Dpp) and Notch (N) signaling all contribute. Clones of cells mutated for receptors and nuclear effectors of one, two or all three pathways were studied to define systematically the necessary and sufficient roles of each signal. Hh signaling alone is sufficient for progressive differentiation, acting through both the transcriptional activator Ci155 and the Ci75 repressor. In the absence of Ci, Dpp and Notch signaling together provide normal differentiation. Dpp alone suffices for some differentiation, but Notch is not sufficient alone and acts only to enhance the effect of Dpp. Notch acts in part through downregulation of Hairy; Hh signaling downregulates Hairy independently of Notch. One feature of this signaling network is to limit Dpp signaling spatially to a range coincident with Hh (Fu, 2003).
Comparison between Mad Su(H) ci cells and Su(H) ci cells
shows that Dpp signaling is sufficient to initiate eye differentiation in its
normal location in the absence of Hh or N signals, but such differentiation is delayed. The normal timing of differentiation is restored by combined Dpp and N signals (in ci clones). This is the basis for the ectopic
differentiation on co-expression of Dpp and Dl ahead of the furrow (Fu, 2003).
Dl, Hh and Dpp are generally thought to signal over very different
distances. How can signals of such different range substitute for one another
to permit normal eye development? Dpp is
transcribed in response to Hh signaling and is produced where Ci155 levels are highest. Dl is regulated by Hh indirectly through Ato and
Ato-dependent Egfr activity in differentiating cells. Hh is
expressed most posteriorly of the three, in differentiating photoreceptors (Fu, 2003).
Eye differentiation uses Hh to progress through cells unable to respond to
Dpp (tkv, Mad) or N (Su(H)). The range of Hh diffusion
depends in part on the shape of the morphogenetic furrow cells. The Dpp
that drives differentiation through ci-mutant cells unable to respond
to Hh must diffuse from outside the ci clones because Dpp synthesis
is Hh dependent. Large ci clones develop normally so Dpp diffusion
cannot be limiting (dpp-mutant clones offer no information about the
range of Dpp because they express and differentiate in response to Hh).
Instead, the rate of progression in response to Dpp is controlled by Dl. Dl
signals over (at most) one or two cell diameters at the morphogenetic furrow (Fu, 2003).
The previous view of eye patterning was influenced by the morphogen
function of Hh and Dpp in other discs. It was thought that domains of Ato and Hairy expression reflected increasing concentrations of Hh and Dpp. The data shows that, in the eye, the combination of signals is important. Differentiation is triggered where Dl and/or Hh synergize with Dpp, regardless of where the source of Dpp is. The additional requirements limit Dpp to initiating differentiation at the same locations that Hh does (Fu, 2003).
> Epsin is part of a protein complex that performs endocytosis in eukaryotes. Drosophila epsin, Liquid facets (Lqf), was identified because it is essential for patterning the eye and other imaginal disc derivatives. Previous work has provided only indirect evidence that Lqf is required for endocytosis in Drosophila. Epsins are modular and have an N-terminal ENTH (epsin N-terminal homology) domain that binds PIP2 at the cell membrane and four different classes of protein-protein interaction motifs. The current model for epsin function in higher eukaryotes is that epsin bridges the cell membrane, a transmembrane protein to be internalized, and the core endocytic complex. This study shows directly that Drosophila epsin (Lqf) is required for endocytosis. Specifically, Lqf is essential for internalization of the Delta (Dl) transmembrane ligand in the developing eye. Using this endocytic defect in lqf mutants, a transgene rescue assay has been developed and a structure/function analysis of Lqf has been performed. When Lqf is divided into two pieces, an ENTH domain and an ENTH-less protein, each part retains significant ability to function in Dl internalization and eye patterning. These results challenge the model for epsin function that requires an intact protein (Overstreet, 2003).
To test for endocytosis defects in lqf- mutants, the localization of the transmembrane receptor Dl was monitored in developing eyes. Dl normally undergoes endocytosis in the eye, and as the internalized protein is not degraded rapidly, internalized Dl can be detected in vesicles (Overstreet, 2003).
The Drosophila eye, composed of 800 identical 22-cell ommatidia, or facets, develops in the larval and pupal stages in a monolayer epithelium called the eye imaginal disc. Eye development occurs as a wave, where the morphogenetic furrow forms at the posterior of the disc, and moves anteriorly into the monolayer of undifferentiated cells. Rows of ommatidia assemble stepwise posterior to the furrow one or two cells at a time, starting with the eight photoreceptors (R1-R8) (Overstreet, 2003).
In wild-type, Dl is detected exclusively as intracellular dots within developing ommatidial clusters throughout the eye disc. In larval eye discs homozygous for lqfFDD9, a weak, viable mutant allele, Dl is detected mainly at the membranes of cells just posterior to the furrow. In clones of cells homozygous for lqfARI, a strong, lethal mutant allele, similar membrane localization of Dl is observed. It is concluded that lqf+ is required for Dl internalization (Overstreet, 2003).
All epsins have an amino-terminal ENTH domain that binds PIP2 at the cell membrane and three or four types of protein-protein interaction motifs, whose copy numbers vary among different epsins. The ubiquitin interaction motifs (UIMs) bind ubiquitin (Ub) noncovalently. There are also clathrin binding motifs (CBMs), DPW motifs that bind the core endocytic adaptor complex, AP-2, and NPF motifs that bind Eps15, another accessory factor (Overstreet, 2003).
A step toward understanding the role of Lqf in endocytosis is the identification of the modules of Lqf protein that are required. In yeast, there are straightforward assays for the function of the two epsins (Ent1 and Ent2). Structure/function analyses have demonstrated that the ENTH domain of Ent1 is necessary and sufficient to rescue the lethality of ent1Δent2Δ double mutants. Moreover, the ENTH domain and to a lesser extent the UIMs have been shown to be required for endocytosis. Because there are mechanistic differences between endocytosis in yeast and higher eukaryotes, the yeast epsins might function somewhat differently from vertebrate epsins and Drosophila Lqf. The major difference between these systems is that the AP-2 core adaptor complex in yeast has no known function in endocytosis, and, accordingly, the yeast epsins lack DPW motifs. As in yeast, structure/function analyses of epsins in vertebrate cell culture have pointed to the importance of the ENTH domain. These assays, however, rely on dominant-negative effects of mutant epsin proteins on endocytosis, and their interpretation is difficult (Overstreet, 2003).
Either the ENTH domain alone, or an ENTH-less Lqf protein, rescues the patterning and Dl endocytosis defects in lqfFDD9 homozygous eyes. Since experimental results in yeast and in vertebrates have emphasized the importance of the ENTH domain, the most remarkable result is that an ENTH-less Lqf protein can function. The simplest interpretation of the rescue results is that LqfΔENTH can function independently of the ENTH domain (Overstreet, 2003).
Transgenes that express Rat epsin1 or human epsin 2b in Drosophila with pRO, each as full-length proteins or without the ENTH domain, rescue the eye defects in lqfFDD9 homozygotes. Thus, there is unlikely to be a significant functional difference between the Drosophila and vertebrate epsins in the region C-terminal to the ENTH domain. In addition, the ENTH domains of Lqf and yeast epsin are functionally similar. It was shown previously that expression of the ENTH domain of Ent1, but not the complementary portion of the protein, restores viability to ent1Δent2Δ yeast. Similarly, expression of full-length Lqf or LqfENTH rescues ent1Δent2Δ lethality but LqfΔENTH expression does not (Overstreet, 2003).
Thus Drosophila epsin, Lqf, is essential for endocytosis of Dl in the developing eye. Moreover, the ENTH domain alone and an ENTH-less Lqf protein each retain significant function. The prevailing model in vertebrates is that epsin functions like a bridge, where the ENTH domain links the membrane to clathrin, a cell surface protein to be internalized, and to AP-2. Since this model requires an intact epsin protein, the results presented here suggest that the prevailing model cannot be the whole story. Moreover, the observation that either the ENTH domain or the remainder of the protein, which are functionally distinct, can be deleted without destroying Lqf function completely suggests that each fragment of Lqf may be partially redundant with another Drosophila endocytic protein. Candidates for the other endycotgic protein include the other ENTH domain protein in Drosophila, Epsin-2 and the Drosophila homolog of AP180, which, like the ENTH-less Lqf protein, binds clathrin and AP-2 (Overstreet, 2003).
Cell proliferation in animals must be precisely controlled, but the signaling
mechanisms that regulate the cell cycle are not well characterized. A regulated
terminal mitosis, called the second mitotic wave (SMW), occurs during Drosophila
eye development, providing a model for the genetic analysis of proliferation
control. This study reports a cell cycle checkpoint at the G1-S transition that initiates
the SMW; Notch signaling is required for cells to
overcome this checkpoint. Notch triggers the onset of proliferation by multiple
pathways, including the activation of dE2F1, a member of the E2F transcription
factor family. Delta to Notch signaling derepresses the inhibition of dE2F1 by
RBF, and Delta expression depends on the secreted proteins Hedgehog and Dpp.
Notch is also required for the expression of Cyclin A in the SMW (Baonza, 2005).
This work identifies a new cell cycle checkpoint in the second mitotic wave
and describes how intercellular signaling overcomes this checkpoint. Delta
signaling to Notch triggers a progression from G1 arrest in the morphogenetic
furrow into the S phase of the terminal mitosis. Two
effectors of this Notch requirement have been identified, dE2F1 transcriptional activity and
cyclin A expression. Although the data imply that at least one other
target also exists, this is unidentified. The data preclude this additional factor from being
Cyclin E. Previous work has identified a later SMW checkpoint, at the G2-M
transition. Together, Notch and
the EGFR therefore coordinately provide spatial and temporal control of the cell
cycle in the SMW (Baonza, 2005).
These results led to a
proposal of the following course of events. Notch is activated by the uniform band
of Delta in all cells as they emerge from the morphogenetic furrow. Cells that
are uncommitted thereby enter S phase, whereas cells that are part of the
precluster are blocked from responding and remain in G1. It has been
shown that the G1 arrest of precluster
cells is dependent on EGFR activation, although the details of the mechanism
remain unclear. One of the consequences of EGFR activation in precluster cells
is the upregulation of Delta expression. Cells between the preclusters would
therefore end up initially receiving low-level uniform Delta, later reinforced
by the upregulated Delta in the adjacent preclusters. Together, these provide a
robust and modulated activation of Notch in cells that will enter the SMW (Baonza, 2005).
It is
emphasized that this work uncovers a normal developmental function for Notch
signaling only in the control of a specific terminal mitotic cycle. The fact
that clones of Notch and Delta mutant cells can be generated
implies that they are not required for the earlier, unpatterned proliferation
ahead of the morphogenetic furrow. Similarly, the ability to make clones in
other imaginal discs indicates that there is no requirement for Delta/Notch
signaling in most cell proliferation in Drosophila. Rather, this signal
requirement, and the subsequent EGFR-dependent entry into mitosis, is
superimposed upon normal controls in this regulated terminal mitosis. Moreover,
the ability of Notch signaling to initiate S phase is restricted to a short
period. Notch has other functions later in eye development, and it has been
shown that later ectopic signaling does not lead to additional proliferation.
Nevertheless, Delta-expressing clones in other tissues also hyperproliferate,
suggesting that ectopic Notch activity has a wider ability to trigger
inappropriate proliferation (Baonza, 2005).
The EGFR and Notch signal systems play distinct
roles in regulating the SMW. After completing its preliminary role in
maintaining cells in G1 arrest, EGFR signaling ensures that cells only undergo
mitosis if they are adjacent to developing clusters, thereby matching the number
of cells born with the number that will be required to complete ommatidial
differentiation. In
contrast, Notch initiates the whole process by regulating whether cells emerging
from the morphogenetic furrow enter the SMW or remain arrested in G1 and start
to differentiate (Baonza, 2005).
The secreted protein Hedgehog has a primary
role in the forward movement of the morphogenetic furrow.
Hedgehog also has an important function in
initiating and coordinating the onset of the SMW, specifically the initiation of
S phase. Hh and Dpp together lead to the expression of Delta
in the furrow. Furthermore, Hh is essential
for the expression of cyclin D and cyclin E in the morphogenetic
furrow, whereas Cyclin E
is the main cyclin that regulates S phase onset. These data imply that Hedgehog
signaling activates several independent branches of the pathway that lead to
the onset of S phase in the SMW. Incidentally, the observation that Cyclin E
accumulates in Notch mutant clones, which lack dE2F1 activity, indicates
that, at least in this context, Cyclin E is not sufficient to inhibit RBF and
thereby activate dE2F1 activity (Baonza, 2005).
Cyclin A is best characterized as
a mitotic cyclin, and its destruction is a key step in the completion of
mitosis. An additional function in the onset of S phase in Drosophila
remains enigmatic. Mammalian Cyclin A and its associated kinase Cdk2 can drive
G1 cell extracts into S phase, and anti-Cyclin A antibodies can block S phase in injected cells.
But, in Drosophila, S phase can proceed normally in the absence of Cyclin A and Cyclin A
does not bind Cdk2. Nevertheless, when overexpressed, Cyclin A can overcome the lack of
Cyclin E and allow cells to enter S phase. Furthermore, overexpression of the
Cyclin A inhibitor Roughex blocks entry into S phase in embryos, and
roughex mutants show precocious S phase entry in the SMW.
Ectopic BrdU incorporation is observed in the eye disc when Cyclin A is misexpressed.
The data indicating that Cyclin A is one of the targets of Notch
signaling further support the idea that Cyclin A is part of the machinery that
controls the onset of S phase in the SMW (Baonza, 2005).
Notch signaling in mammals, as in
flies, is pleiotropic and context dependent. This is highlighted in human
cancer, where Notch is oncogenic in a number of cases, particularly in
hematopoietic neoplasms, but in other contexts has tumor suppressor functions.
Moreover, although the current work highlights a proliferative function, it has been shown that
Notch inhibits proliferation in the wing disc. Notwithstanding this caveat, it
is striking that Notch activity can be hyperproliferative in humans and in
Drosophila, and little is known about this proliferative response. It has
recently been shown in the developing Drosophila central nervous system
that Notch activity can maintain cells in a proliferative state by antagonizing
the p21/p27 homolog Dacapo, thereby maintaining Cyclin E expression. Similarly,
the dacapo gene is downregulated in response to Notch in the
mitotic-to-endocycle transition in Drosophila follicle cells. This work
describes a different mechanism: Notch signaling overcomes a G1-S checkpoint via
the activation of universally conserved cell cycle components, RBF1, dE2F1, and
possibly Cyclin A. Although tempting to speculate that these data may provide
some insight into oncogenic mechanisms, it will be important to ascertain
whether the particular relationships between Notch and the core machinery that
triggers S phase is indeed conserved. In fact, the data imply that Notch
probably also influences the mitotic cycle at other points. If the only role of
Notch were to advance cells into S phase, they would simply arrest at the next
checkpoint, G2-M. The fact that Notch activity leads to overgrowth therefore
implies that Notch can also, directly or indirectly, drive cells through the
subsequent G2-M checkpoint (Baonza, 2005).
The developing Drosophila ommatidium is characterized by two distinct waves of pattern formation. In the first wave, a precluster of five cells is formed by a complex cellular interaction mechanism. In the second wave, cells are systematically recruited to the cluster and directed to their fates by developmental cues presented by differentiating precluster cells. These developmental cues are mediated through the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways and their combined activities are crucial in specifying cell type. The transcription factor Lozenge (Lz) is expressed exclusively in second wave cells. In this study Lz was ectopically supplied to precluster cells, and the various RTK/N codes that specify each of three second wave cell fates were concomitantly supplied. This protocol reproduced molecular markers of each of the second wave cell types in first wave precluster cells. Three inferences were drawn; (1) it was confirmed that Lz provides key intrinsic information to second wave cells, and this can now be combined with the RTK/N signaling to provide a cell fate specification code that entails both extrinsic and intrinsic information. (2) the reproduction of each second wave cell type in the precluster confirms the accuracy of the RTK/N signaling code, and (3) RTK/N signaling and Lz need only be presented to the cells for a short period of time in order to specify their fate (Mavromatakis, 2013).
This paper explored three inter-related themes bearing
on the nature of the signals that specify the cell types in the
Drosophila ommatidium. The ability of a transcription
factor to predispose the cellular responses to developmental signals was examined,
the accuracy of the signaling code that represents these
developmental signals was validated, and it was inferred that both the intrinsic and
extrinsic aspects are only required for a brief period of time (Mavromatakis, 2013).
Lz had long been assumed to be a key factor that distinguishes how
second wave cells differ from the precluster cells in their response
to developmental signals. This paper rigorously tested this
concept and reproduced features typical of the three second wave
cell types in the R3/4 precluster cells by supplying ectopic Lz along
with the appropriate RTK/N cell fate code. It is thus inferred that the
presence of Lz in R3/4 precluster cells is sufficient to endow them
with the second wave cell fate response repertoire. A number of issues related to these observations and their interpretations are discussed (Mavromatakis, 2013).
Normal R3/4 precursors undergo an N-Dl interaction that results in
the R4 precursor experiencing much higher levels of N activity than
the R3 precursor. When Lz was supplied to R3/4
precursors (sev.lz), the cell in the R4 position frequently transformed
into an R7, consistent with the requirement of high N for R7
specification. Less frequently, both R3/4 precursors adopted the R7
fate, and sometimes it was the cell in the R3 position alone that
generated an ectopic R7. These results suggest that in the sev.lz flies
the R3/R4 N-Dl interaction does not occur correctly. When the mδ0.5.lacZ reporter line was used as a reporter of N activity (which in wild-type larvae is robustly upregulated in
R4 precursors) an erratic pattern was observed, sometimes showing
the wild-type pattern, sometimes showing both R3/4 cells with high
levels of lacZ expression, and sometimes showing R3 alone with
high levels. Hence, by expressing Lz in the R3/4
precursors the cells were not only endowed with second wave response
abilities but also they were prevented from executing their N-Dl
interactions properly. Indeed, it was only when N was artificially
activated to a high level with activated Notch (sev.lz; sev.N**) that
the R7 fate was potently induced in both R3/4 precursors (Mavromatakis, 2013).
Native R7s critically require sev gene function; in its absence, they
differentiate as cone cells. However, some ectopic R7s were able to
differentiate when Lz was provided to the R3/4 precursors, even in
the absence of sev (sev0; sev.lz), suggesting that normal R7
specification was not fully reiterated here. Examination of these eye
discs suggested that some R3/4 precursors differentiated as R7s
whereas others became cone cells. Thus, these cells appear to be on
the cusp of the R7/cone cell fate choice, and some
cells expressing markers for both cell types were observed. In the cells that became
R7s, the presence was inferred of sufficient RTK activity, which was
likely to have been supplied by endogenous DER signaling active
in the precluster cells. Only when N activity was raised in these cells
(sev0; sev.lz; sev.N*) did their full sev dependence for the R7 fate
emerge, when all R3/4 precursors differentiated as cone cells (Mavromatakis, 2013).
The sev.N* construct is a very useful activator of the N pathway in
developing eye cells. Since N activity drives sev expression, the
sev.N* transgene feeds back on itself and promotes its own
expression, and by subsequent iterations of this effect the cells are
left with potent N activity. This level is still within the physiological
range, unlike that produced using Gal4/UAS techniques, and is
therefore the choice method for activating the N pathway. The
transgene that was routinely use to knock down N activity
[sev.Su(H)EnR] has the opposite effect; it reduces its own
expression, and mildly compromises N activity. This level of
reduction in N activity is usually sufficient to trigger major effects
without the disadvantage of the severe downregulation that can
accompany the use of Gal4/UAS technology. Since the sev.lz
construct would also be downregulated by sev.Su(H)EnR, it was necessary to ectopically express Lz in the precluster using another enhancer element, and to this end the ro.Gal4 line was generated. When UAS.lz was expressed under ro control, the R3/4 precursors
frequently differentiated as R7s, and crucially, when N activity was
concomitantly reduced [ro.Gal4; UAS.lz; sev.Su(H)EnR] cells
displaying R1/6 molecular features were now detected in the R3/4
precursors (Mavromatakis, 2013).
The cells in the R2/5 positions in ro.G4; UAS.lz developing
ommatidia appear to develop normally; they express Elav and Ro,
but none of the other fate markers. This suggests that R2/5 cells are
insensitive to the presence of Lz, and argues that there is a major
molecular difference between these cells and the R3/4 precursors.
Also noteworthy is the transformation of all lz mutant second wave
cells into R3/4 types characterized by the expression of Svp (a
marker that is not expressed in R2/5 precursors) and Elav. Thus, it
appears that ectopic Lz selectively transforms R3/4 precursors of
the precluster to the second wave fate, and second wave cells
lacking Lz adopt the R3/4 fate. Hence, it is suspected that Lz might not
provide the intrinsic information that distinguishes the second wave
cells from precluster cells per se, but rather distinguishes second
wave cells from R3/4 types. Experiments to evaluate this view are currently being undertaken (Mavromatakis, 2013).
A counter-argument emerges from the fate of the majority of cells
in the R3 positions in sev.lz eyes, which do not switch their fate.
Only when N activity is activated or reduced in these cells is a
change seen in their fates, and to be sure that the R2/5 cells are insensitive
to Lz expression, it would also need to correspondingly vary N
activity in them. Experiments to do this using ro.Gal4 produced
severely disrupted preclusters, presumably as a result of interference
with N function at earlier stages of precluster formation. Since these
clusters were largely uninterpretable, the issue of whether R2/5 cells
are insensitive to Lz expression remains unresolved (Mavromatakis, 2013).
For many years, the role of N in photoreceptor specification was
confusing. In some contexts N appeared to oppose photoreceptor
specification and in others N seemed to promote it, and this
confusion prevented substantial progress in defining the fate codes
that specified the different cell types. In recent work
three distinct roles for N in this process were identified, and with that information
the cell fate codes for R1/6, R7 and the cone cells were inferred.
A major goal of this current work has to been to test this code by its
reiteration in the R3/4 cells of the precluster using Lz expression to
endow them with second wave cell qualities. In these experiments,
each of the cell codes induced the expected cell fates, providing
cogent support for the validity of the code (Mavromatakis, 2013).
DER is assumed to be ubiquitously expressed in the eye disc tissue,
and its ligand, Spitz, diffusing from precluster cells, is thought to
reach more distant cells with time. But the N and Sev signals are
regulated in a different manner. Both their ligands are membrane
bound, and their receptor activations only occur in immediate
neighbors. Dl, the ligand for N, is expressed transiently by
differentiating cells, and, accordingly, activates N in neighboring
cells for only short periods of time (a few hours). sev is an N
response gene and, in consequence, Sev is only expressed in cells
for a short period of time. By contrast, Boss, the Sev ligand, is
expressed for a prolonged period by the R8 precursor. Thus, both the
N and Sev signaling systems are only available to the cells for
restricted periods, with this restriction controlled by ligand
expression in the N system and by receptor expression in the Sev
system (Mavromatakis, 2013).
Although Lz is expressed in the second wave cells in a persistent
manner in the eye disc, the experiments suggest that it, like the
extrinsic signals, is required only for a brief developmental window.
Consider the transformation of sev.lz R3/4 cells to the R7 fate. The
sev enhancer is only active in these cells for a few hours and yet a
complete transformation of the cells is achieved. The expression of
specific cell type markers in the eye disc might erroneously indicate
the transformation of a cell when only a transient effect occurs, but
the presence of ectopic R7s in the adult retina argues otherwise and
suggests that the transformations are potent and permanent. This
view is further validated by the rescue of lz mutant second wave
cells by the sev.lz transgene. This rescue is complete and is evident
by the molecular markers expressed in the disc and by the
morphology of the adult cells. Thus, it is inferred that Lz is only
required during the same time window when the RTK/N signals are
transduced, and it is further inferred that the combined activities of the
RTK and N pathways, in concert with Lz, function in a short-lived
manner to lock in the fate of the cells. How the presence of
ephemeral extrinsic and intrinsic information is molecularly
'remembered' by the cells to allow their appropriate differentiation
over a prolonged developmental period remains an intriguing
question (Mavromatakis, 2013).
Sporadic evidence suggests Notch is involved in cell adhesion. However, the underlying mechanism is unknown. This study has investigated an epithelial remodeling process in the Drosophila eye in which two primary pigment cells (PPCs) with a characteristic 'kidney' shape enwrap and eventually isolate a group of cone cells from inter-ommatidial cells (IOCs). This paper shows that in the developing Drosophila eye the ligand Delta is transcribed in cone cells and Notch is activated in the adjacent PPC precursors. In the absence of Notch, emerging PPCs fail to enwrap cone cells, and hibris (hbs) and sns, two genes coding for adhesion molecules of the Nephrin group that mediate preferential adhesion, are not transcribed in PPC precursors. Conversely, activation of Notch in single IOCs leads to ectopic expression of hbs and sns. By contrast, in a single IOC that normally transcribes rst, a gene coding for an adhesion molecule of the Neph1 group that binds Hbs and Sns, activation of Notch leads to a loss of rst transcription. In addition, in a Notch mutant where two emerging PPCs fail to enwrap cone cells, expression of hbs in PPC precursors restores the ability of these cells to surround cone cells. Further, expression of hbs or rst in a single rst- or hbs-expressing cell, respectively, leads to removal of the counterpart from the membrane within the same cell through cis-interaction and forced expression of Rst in all hbs-expressing PPCs strongly disrupts the remodeling process. Finally, a loss of both hbs and sns in single PPC precursors leads to constriction of the apical surface that compromises the 'kidney' shape of PPCs. Taken together, these results indicate that cone cells utilize Notch signaling to instruct neighboring PPC precursors to surround them and Notch controls the remodeling process by differentially regulating four adhesion genes (Bao, 2014).
The Drosophila optic lobe develops from neuroepithelial cells, which function as symmetrically dividing neural progenitors. This study describes a role for the Fat-Hippo pathway in controlling the growth and differentiation of Drosophila optic neuroepithelia. Mutation of tumor suppressor genes within the pathway, or expression of activated Yorkie, promotes overgrowth of neuroepithelial cells and delays or blocks their differentiation; mutation of yorkie inhibits growth and accelerates differentiation. Neuroblasts and other neural cells, by contrast, appear unaffected by Yorkie activation. Neuroepithelial cells undergo a cell cycle arrest before converting to neuroblasts; this cell cycle arrest is regulated by Fat-Hippo signaling. Combinations of cell cycle regulators, including E2f1 and CyclinD, delay neuroepithelial differentiation, and Fat-Hippo signaling delays differentiation in part through E2f1. Roles for Jak-Stat and Notch signaling were also characterized. These studies establish that the progression of neuroepithelial cells to neuroblasts is regulated by Notch signaling, and suggest a model in which Fat-Hippo and Jak-Stat signaling influence differentiation by their acceleration of cell cycle progression and consequent impairment of Delta accumulation, thereby modulating Notch signaling. This characterization of Fat-Hippo signaling in neuroepithelial growth and differentiation also provides insights into the potential roles of Yes-associated protein in vertebrate neural development and medullablastoma (Reddy, 2010).
Both normal development and homeostasis require that cells transition from proliferating undifferentiated cells to quiescent differentiated cells. Failure to undergo this transition results in tumor formation, whereas premature differentiation results in hypotrophy. Some tissues balance proliferation and differentiation by employing stem cells that divide asymmetrically to yield both a stem cell and a progenitor cell, which will then give rise to differentiated cells. Most of the Drosophila central nervous system develops in this way: individual cells within the embryonic ectoderm become specified as neural stem cells called neuroblasts (NBs), which divide asymmetrically to yield a neuroblast and a progenitor cell called a ganglion mother cell (GMC). By contrast, much of the vertebrate central nervous system initially develops from neuroepithelia (NE), sheets of epithelial neural progenitor cells that function as symmetrically dividing neural stem cells. This provides for rapid expansion of neural tissue, and then, as development proceeds, asymmetrically dividing progenitor cells arise, although the mechanisms that govern their appearance are not well understood. The optic lobe of Drosophila is unlike the rest of the Drosophila nervous system in that, akin to the vertebrate nervous system, it develops from NE. The optic lobe may thus serve as a model in which the powerful experimental approaches available in Drosophila can be used to investigate mechanisms that control the growth and differentiation of NE (Reddy, 2010).
At the end of larval development, the optic lobes comprise the lateral half of each of the two brain hemispheres, and are organized into lamina, medulla and lobula layers. The optic lobes originate from clusters of epithelial cells that invaginate from a small region on the surface of the embryo (the optic placode). During larval development, these cells separate into an inner optic anlagen (IOA), which will give rise to the lobula and inner part of the medulla, and an outer optic anlagen (OOA), which will give rise to the outer part of the medulla and the lamina. Initially, the IOA and OOA are composed entirely of NE cells, but during the third larval instar they begin to differentiate. Along the lateral margin of the OOA, NE cells undergo cell cycle arrest in G1, and then are recruited to differentiate into lamina neurons by signals from the arriving retinal axons. Along the medial margin of the OOA, a wave of differentiation sweeps across the NE from medial to lateral, converting NE cells into medulla NBs. These NBs divide perpendicularly to the plane of the neuroepithelium, and appear to follow a NB developmental program, giving rise to additional self-renewing NBs, and to GMCs, which ultimately give rise to neurons (Reddy, 2010).
The Fat-Hippo signaling pathway encompasses distinct downstream branches that regulate planar cell polarity and gene expression. Transcriptional targets of the pathway include genes that influence cell proliferation and cell survival, and consequently Fat-Hippo signaling is an important regulator of growth from Drosophila to vertebrates. The influence of Fat-Hippo signaling on transcription is mediated by a co-activator protein, called Yorkie (Yki) in Drosophila and Yes-associated protein (YAP) in vertebrates. Warts (Wts)-mediated phosphorylation and binding to cytoplasmic proteins negatively regulate Yki by promoting its retention in the cytoplasm. Wts is regulated in at least two ways: Wts kinase activity is promoted by Hippo; and Wts protein levels are influenced by Dachs. Upstream regulators of the pathway include the large cadherin Fat, and the FERM-domain proteins Merlin (Mer) and Expanded (Ex). Fat acts as a transmembrane receptor, regulated by the cadherin Dachsous (Ds), and the cadherin-domain kinase Four-jointed (Fj). The mechanisms that regulate Ex and Mer are not completely understood, but Ex localization can be influenced by Fat, and, in mammalian cells, Mer mediates an influence of contact inhibition on Hippo signaling. Genetic studies in Drosophila have also revealed that the relative contributions of pathway components can vary among different tissues (Reddy, 2010).
Optic NE cells proliferate during larval development, but aside from a requirement for the transcription factor DVSX1 (Erclik, 2008), how this proliferation is regulated is not understood. The progression of NE cells to medulla NBs in the OOA is antagonized by Jak-Stat signaling (Yasugi, 2008), but, aside from this, the regulation of this differentiation wave is not understood. This study demonstrates that Fat-Hippo signaling regulates the proliferation and differentiation of NE cells in the optic lobe. By contrast, Fat-Hippo signaling does not detectably influence the proliferation or differentiation of NBs or their progeny. A role is identified for Notch signaling in controlling the progression of NE cells to medulla NBs, and relationships are characterized between the Fat-Hippo, Jak-Stat and Notch signaling pathways. The results indicate that a transient pause in the cell cycle is needed for cells to transition from NE cells to NBs, and suggest a model in which a cell cycle arrest modulates Notch signaling by contributing to accumulation of Delta expression. The insights these results provide into the role of Fat-Hippo signaling in NE growth and differentiation in Drosophila are likely to be relevant to recently described roles of YAP in vertebrate neural development and medulloblastoma (Reddy, 2010).
The Fat-Hippo pathway has emerged as an important regulator of growth, but has not previously been implicated in neural development in Drosophila. The observation that expression of an activated form of Yki, or mutation of tumor suppressors in the pathway (i.e. fat, ex or wts), promotes growth, whereas mutation of yki impairs growth, identify a crucial role for Fat-Hippo signaling in regulating the proliferation of optic neural progenitor cells (i.e. NE). Indeed, expression of activated Yki can result in massive overgrowths that are taken up in folded sheets of NE, which push into the central brain, forming tumors of undifferentiated NE cells. Although the influence of Fat-Hippo signaling on NE growth parallels its influence on imaginal discs, the influence of Fat-Hippo signaling on NE differentiation does not, as clones of cells mutant for tumor suppressors in the pathway can differentiate cuticle in the head, thorax and abdomen (Reddy, 2010).
In contrast to the extensive overgrowth and suppressed differentiation of NE, NBs and their more differentiated progeny appear refractory to Fat-Hippo signaling. Developing tissues that are unaffected by Fat-Hippo signaling have not been well characterized. The restriction of Fat-Hippo signaling to the NE is matched by the preferential expression of several pathway components, but even when a constitutively activated form of Yki was expressed outside of the NE, neural development in the central brain was not obviously perturbed. Given the emerging importance of Hippo signaling in cancer, determination of what makes different cell types sensitive or resistant to activated Yki is an important direction for future studies (Reddy, 2010).
The progressive nature of NE to NB differentiation in the optic lobe, with different stages displayed in a spatial pattern, make it a sensitive system for investigating differentiation. The extent of delay associated with Fat-Hippo pathway tumor suppressors varied depending on strength of the mutations, which suggests that progression of NE to NB involves a balance of positive and negative influences. The silencing of Yki expression as cells differentiate further suggests that there is negative feedback of differentiation signals onto Yki, which might normally help to ensure a sharp transition between NE and NBs. When Yki activity is further elevated, by overexpression of activated Yki, a complete block in differentiation could be achieved. The observation that a complete block in differentiation could also be achieved by combining overexpression of wild-type Yki with a mutation that influences Yki phosphorylation (wts) is intriguing in light of observations that several human cancers are associated with an increase in levels of Yki expression, rather than a simple change in its localization or phosphorylation. Thus, it is suggested that the two-hit scenario observed in the optic lobe, in which both Yki activity and Yki levels need to be affected in order to transform cells permanently, could also be relevant to human tumors (Reddy, 2010).
This analysis of optic lobe development and the influence of Fat-Hippo signaling implies that a transient pause in the cell cycle is required for cells to transition from NE to medulla NBs, and that Fat-Hippo signaling influences differentiation via an effect on the cell cycle. This model is supported by several observations: there is normally a cell cycle pause along the edge of the outer optic anlagen NE; inhibition of Fat-Hippo signaling, or activation of Yki, impairs both this cell cycle pause and differentiation; and direct manipulation of multiple cell cycle regulators can delay NE differentiation. Although multiple cell cycle regulators appear to be involved in this cell cycle pause, this analysis implicates E2f1 as a key player. PCNA-GFP is downregulated at the edge of the NE, which indicates that E2f1 activity is low there. As E2f1 activity is negatively regulated by association with Rb, and Rb is negatively regulated by phosphorylation by Cdks, expression of CycD+Cdk4 is expected to increase E2f1 activity. Thus, the significant delay in differentiation observed when CycD+Cdk4 were co-expressed with E2F1+DP could all be due to increased E2f1 activity. Importantly, E2f1 is normally regulated by Fat-Hippo signaling in the optic NE, and E2f1 is functionally important for the influence of Fat-Hippo signaling on NE differentiation, because mutation of E2f1 suppressed the wts-mediated differentiation delay. A cell cycle pause also occurs in conjunction with a wave of differentiation that sweeps across the developing eye imaginal disc; however, direct manipulation of cell cycle progression did not affect the differentiation wave in the eye disc, nor does mutation of wts, hpo or sav affect differentiation of photoreceptor cells, even though it does prevent the normal cell cycle pause in the eye disc (Reddy, 2010).
The transition from NE to NB is regulated by Notch signaling, and the results of this study suggest a model in which high level expression of Dl at the edge of the NE autonomously inhibits Notch activation, resulting in upregulation of L(1)sc, which promotes NB fate. This model is supported by the observations that activation of Notch or mutation of Dl can inhibit NE differentiation. At the same time, high-level expression of Dl should enhance Notch activation in neighboring cells, which, as Dl is upregulated by Notch activation, would contribute to the progressive spread of elevated Dl expression across the NE. This simple model allows for the input of other pathways into NE to NB progression via effects on Dl expression, and indeed this appears to be the point at which Fat-Hippo and Jak-Stat signaling intersect with Notch. As a unifying model, it is proposed that a cell cycle pause facilitates the accumulation of the high levels of Dl expression needed to autonomously block Notch signaling, and thereby to upregulate the expression of proneural genes like L(1)sc. A possible mechanism for this hypothesized effect on Delta is suggested by the recent observation in vertebrate NE that Delta1 transcripts are unstable during S-phase. The hypothesis that the influence of Fat-Hippo signaling on differentiation is due to its effect on Dl expression also provides an explanation for the specificity of this phenotype, as Dl is not generally required for the differentiation of imaginal disc cells (Reddy, 2010).
Studies of homologues of Yki, Sd, Hpo and Wts in the chick neural tube identified influences on proliferation and differentiation (Cao, 2008). These studies identified effects on Sox2-expressing neural progenitor cells, but could not distinguish between effects on NE cells versus other neural progenitor cells. A recent study has also implicated YAP in Hedgehog-associated medulloblastoma. Vertebrate NE cells give rise to progenitor cells (e.g. radial glial cells and basal progenitors) that share with neuroblasts the ability to divide asymmetrically to give rise to both another progenitor cell and a more differentiated cell. Since this analysis of the Drosophila optic lobe indicates that Fat-Hippo signaling functions specifically to regulate the proliferation and differentiation of NE, it is suggested that YAP might also function specifically within NE cells in vertebrates. Notably, the observation that depending on the level of expression, Yki can delay rather than block differentiation, provides for the possibility that YAP-dependent tumors could nonetheless contain a mixture of NE cells and more differentiated cells. In Drosophila, each of the three upstream branches of the pathway (i.e. Fat-dependent, Ex-dependent and Mer-dependent, contribute to Yki regulation in NE. Studies in vertebrates have not addressed how the pathway is normally regulated, but Fat-, Ds- and Fj-related genes are all normally expressed in vertebrate NE, consistent with the possibility that they function there (Reddy, 2010).
Artificially slowing the cell cycle can promote precocious differentiation in the cortex, although in this context increasing cell cycle length was associated with a transition from proliferative to differentiative divisions of basal progenitors, which appear functionally similar to NBs rather than to NE cells. The differentiation of optic lobe NE cells into medulla NBs also differs from the general model of increasing cell cycle length causing differentiation, because NBs proliferate even more rapidly than NE cells, and thus this step is not associated with a general lengthening of the cell cycle, but rather a transient pause. Nonetheless, it is intriguing that, in the spinal cord, overexpression of CyclinD did not block differentiation, but did appear to transiently delay it, reminiscent of the delay in NE to NB progression that this study identified in the optic lobe. Moreover, CyclinD expression is regulated by Hippo signaling in the chick neural tube, and overexpression of CyclinD inhibits differentiation there. Although further studies are required to identify the CyclinD-sensitive mechanism in the vertebrate nervous system, the reported instability of Delta1 transcripts during S phase, together with the role of Notch signaling in maintaining NE progenitors in vertebrates and the analysis of NE differentiation and Dl expression in the Drosophila optic lobe, suggest that the possibility of a general influence of cell cycle progression on Notch signaling warrants further investigation as a contributor to the link between cell cycle progression and differentiation in the nervous system across different phyla (Reddy, 2010).
The proper balance between symmetric and asymmetric stem cell division is crucial both to maintain a population of stem cells and to prevent tumorous overgrowth. Neural stem cells in the Drosophila optic lobe originate within a polarised neuroepithelium, where they divide symmetrically. Neuroepithelial cells are transformed into asymmetrically dividing neuroblasts in a precisely regulated fashion. This cell fate transition is highly reminiscent of the switch from neuroepithelial cells to radial glial cells in the developing mammalian cerebral cortex. To identify the molecules that mediate the transition, neuroepithelial cells were microdissected, and their transcriptional profile was compared with similarly obtained optic lobe neuroblasts. Genes encoding members of the Notch pathway were found expressed in neuroepithelial cells. Notch mutant clones are extruded from the neuroepithelium and undergo premature neurogenesis. A wave of proneural gene expression is thought to regulate the timing of the transition from neuroepithelium to neuroblast. The proneural wave transiently suppresses Notch activity in neuroepithelial cells, and inhibition of Notch triggers the switch from symmetric, proliferative division, to asymmetric, differentiative division (Egger, 2010).
In the developing mammalian cortex, neural stem cells initially divide symmetrically to produce two neural stem cells, thereby increasing the neural precursor pool. The radial glial cells subsequently divide asymmetrically to produce a neural stem cell and either a basal progenitor cell or an immature neuron. Most basal progenitor cells divide once more to generate two postmitotic neuron. The Notch signalling pathway is thought to play a role in maintaining the undifferentiated state of neuroepithelial cells, radial glia and basal progenitors, but the downstream signalling cascades activated in these cells might be differentially regulated. Neurogenesis is initiated by proneural genes, such as Mash1 and Neurogenin2 (Ngn2) (Egger, 2010).
This study shows that this sequence of neurogenic events is remarkably similar to that seen in the development of the optic lobe in Drosophila. Notch is activated in the neuroepithelial cells, which remain undifferentiated. The proneural gene l'sc is expressed within the transition zone, and levels of Delta are increased, while Notch activity is decreased. Thus neuroepithelial cells ultimately give rise to a variety of differentiated neurons, but only after they have passed through the transition zone (Egger, 2010).
Low levels of Delta expression were found thoughout the optic lobe neuroepithelium, with increased expression in the transition zone. Several Brd genes were found within the neuroepithelium. Negative regulation of Delta activity by the Brd proteins would be expected to further reduce the level of Delta signalling. This situation might be analogous to the oscillations in Delta and Ngn2 levels observed in vertebrates, and it will be interesting to assess whether the expression of Delta, HLHm5 or proneural genes also oscillate in flies. Strikingly, when Delta activity is inhibited throughout the epithelium, the premature transformation of the entire neuroepithelium into neuroblasts is observed. This suggests that neuroepithelial cells might both send and receive the Notch signal (Egger, 2010).
Higher levels of Delta were observed in the L'sc positive transition zone. High levels of Delta or Serrate can inhibit Notch signalling through cis-inhibition, suggesting one possible mechanism for the downregulation of Notch signalling at the transition zone. Interestingly, very recent results suggest that cis-inhibition can create sharp boundaries and this could be the role of the high levels of Delta that were observe in the transition zone (Egger, 2010).
Epithelial integrity might be important to maintain proliferative cell division. This study shows that Notch mutant clones are extruded from the neuroepithelium. Furthermore, expression in the optic lobe neuroepithelial cells was found of a number of genes involved in cell adhesion. Notch could regulate cell adhesion molecules at the transcriptional level, or might itself form a complex with adhesion molecules. In either case, Notch loss of function would disrupt cell adhesion and lead to the extrusion of epithelial cells. Subtypes of cadherins, such as DE-Cad, Cad99C, Fat, which were found preferentially expressed in the neuroepithelium, might be activated by Notch to maintain the neuroepithelium, and repressed by L'sc to promote neurogenesis. Notch mutant clones also upregulate expression of the neuroblast transcription factor Dpn (but not of L'sc), and divide asymmetrically only once they have delaminated from the epithelium. In contrast to Notch mutant clones, L'sc misexpression clones upregulate Dpn and switch to asymmetric division whilst still embedded within the neuroepithelium. L'sc acts, at least in part, through repression of Notch signalling, but might also induce neuroblast-specific genes directly (Egger, 2010).
JAK/STAT signalling negatively regulates the progression of the proneural wave and neurogenesis in the optic lobe. Interestingly, the ability of Notch to maintain radial glial cell fate appears to be largely dependent on functional JAK/STAT signalling. It remains to be seen whether the Notch pathway interacts with JAK/STAT in the Drosophila optic lobe (Egger, 2010).
This study has shown that the development of the Drosophila optic lobe parallels that of the vertebrate cerebral cortex, suggesting that the pathways regulating the transition from symmetric to asymmetric division might be conserved from flies to mammals. Identifying the effector genes that are regulated by Notch and L'sc, and the links between JAK/STAT and Notch signalling, will yield further insights into the molecular mechanisms that maintain an expanding neural stem cell pool and regulate the timely transition to differentiation (Egger, 2010).
The first step in the development of the Drosophila optic medullar primordia is the expansion of symmetrically dividing neuroepithelial cells (NEs); this step is then followed by the appearance of asymmetrically dividing neuroblasts (NBs). However, the mechanisms responsible for the change from NEs to NBs remain unclear. In this study a detailed analyses was performed demonstrating that individual NEs are converted into NBs. This transition occurs during an elongated G1 phase. During this G1 phase, the morphological features and gene expressions of each columnar NE changed dynamically. Once the NE-to-NB transition was completed, the former NE changed its cell-cycling behavior, commencing asymmetric division. It was also found that Notch signaling pathway was activated just before the transition and was rapidly downregulated. Furthermore, the clonal loss of the Notch wild copy in the NE region near the medial edge caused the ectopic accumulation of Delta, leading to the precocious onset of transition. Taken together, these findings indicate that the activation of Notch signaling during a finite window coordinates the proper timing of the NE-to-NB transition (Orihara-Ono, 2011).
This study provides a detailed description of the differentiation of optic medullar progenitors in Drosophila larval brains. Cells with features that were intermediate between NEs and NBs were identified and whose locations were also intermediate, forming a band between the NE and NB regions. Considering the fact that NBs are derived from NEs and that all NEs divide in an exclusively symmetric manner, only the transition of existing individual NEs into NBs without mitosis can explain the observed division patterns and increases in cell populations accounting for the expansion of the medullar primordia. In fact, it was demonstrated that the intermediate cells between the NEs and the NBs were in a transitional state and were suspended in a prolonged G1 phase (Orihara-Ono, 2011).
Genetic cell lineage analyses have shown that outer optic anlagen/outer proliferation center (OOA/OPC) NBs are derived from NEs. Consistent with this observation, a distinct population of cells was identified intermediate between NEs and NBs that did not incorporate BrdU during a labeling period of around 8 h, indicating that the BrdU-negative cells never passed through S phase during the labeling period. The BrdU-negative clusters present at the medial edge of the NE region were probably identical to a previously reported [3H]-thymidine-free band of cells, which was also reported recently (Reddy, 2010). The cell cycle of the BrdU-negative cells was investigated using Mcm2 antibody, which is specific for the pre-replication complex that begins to form during the late M phase and increases until it reaches a peak during S phase. The BrdU-negative NEs were found to be in G1 phase, since they expressed Mcm2. Furthermore, a BrdU-negative cell population was retained even after 16 h of labeling, indicating that the G1 phase of BrdU-negative NEs intermediate between the BrdU-positive NEs and NBs is extremely prolonged. Taken together, these results indicate that the NEs at the medial edge are converted into NBs during a prolonged G1 phase (Orihara-Ono, 2011).
Cell cycle progression patterns differ from cell to cell. For example, syncytial blastodermal cells lack the G1 and G2 phases entirely. The G1 phase is also reportedly absent in some neural lineages of the Drosophila peripheral nervous system. The so-called label-retaining cells in the germinal centers of the adult brain also have an extremely long cell-cycle period. Therefore, the current study may have revealed a common mechanism by which stem cells can retain their capability to reenter the cell cycle despite long periods of quiescence (Orihara-Ono, 2011).
Analogous with the optic medullar anlagen, the formation of the morphogenetic furrow of the Drosophila larval eye disc requires several processes, including the conversion of the cytoskeletal scaffold to a fluid, mobile state and the side-by-side synchrony of the cell-cycle progression. As a result, the G1 cells in the morphogenetic furrow adopt a drop-like shape with a narrow apical 'footprint' when viewed from the surface, whereas the cells in the S-G2-M phases are spherical and exhibit a larger apical surface area. Other evidence supporting this analogy was recently reported in a study examining the proneural wave, in which EGF signaling is involved. The similarities between the maturation of the optic medullar progenitors and the cells in the morphogenetic furrow strongly suggest the existence of a common mechanism regulated by Notch. Notch has also been reported to act in combination with Wg to arrest posterior compartment wing margin cells in the G1 phase and anterior compartment side cells in the G2 phase, supporting the idea that Notch regulates the cell cycle under a number of different circumstances. These mechanisms would explain our current results quite well (Orihara-Ono, 2011).
Notch signaling has been shown to mediate a wide array of cell fate decisions during development. In a recent study of fly brain homozygous for the Notch ts allele, the fate specification was perturbed and the precocious determination may have involved the malformation of the larval optic lobes (Ngo, 2010). Similar evidence has been provided by an overexpression study using a dominant-negative form of the Notch-Delta transduction cascade and mitotic mosaic production of the Notch null allele. These results are consistent with the present findings showing that Notch downregulation forced the precocious transition of NEs. Moreover, this study provides evidence that this effect occurred as a result of a cell non-autonomous process. Since Delta was used as a sensitive effector of the reception of Notch-Delta signaling, attempts were made to identify the Delta pattern as precisely as possible, especially regarding the morphological changes. Previous work demonstrated that the downregulation of Notch signaling by the ectopic expression of Numb in NEs caused a reduction in the progenies derived from a single NE, demonstrating that Notch signaling is required for the proliferation of neural cells in the OOA/OPC (Orihara-Ono, 2011).
Focus was placed on the role of Notch signaling in the NE-to-NB transition at the mid-third instar larval OOA/OPC, which was early enough to examine the acute effects of Notch-depletion. Notch signaling was found to be activated just before the NE-to-NB transition at the medial edge, although Notch was expressed throughout the entire NE population. Both the activation and downregulation of Notch signaling occurred during a finite window for the proper timing of the NE-to-NB transition. However, if Notch signaling was depleted from the NEs at a much earlier stage and NEs devoid of Notch signaling were allowed to develop for a much longer period, the number of NBs that transitioned from the NEs did not change noticeably, compared with the wild-type situation. One plausible explanation is that NEs at the medial edge region cannot be sustained without Notch signaling. Thus, NEs at the medial edge were converted into NBs via the downregulation of Notch. Even if the Notch-deficient clonal cells at the medial edge region are converted into NBs earlier than the surrounding wild-type cells, the final number of NBs generated from the clonal region should not change, since the NEs in the medial edge are supposedly not yet able to divide. In addition, if the fates of the NBs are further specified depending on the timing of their determination from the NEs, precocious transition may cause changes in the fates of the NBs. Further analyses of NB subtypes in Notch-mutant clones might provide intriguing findings (Orihara-Ono, 2011).
Notch's involvement in neural fate decisions has been described in almost all the animals in which this topic has been studied. In a study examining the embryonic Drosophila optic primordium, it has been implied that the loss of Notch causes a precocious epithelial-to-neuroblast transition typically seen in wild-type development during late embryonic stages. The findings of the present paper further explored this precocious transition during the larval stage. Furthermore, evidence is provided that additional factors, such as Delta and Su(H)-dependent downstream genes, might be involved in this process. The downstream effects of Notch are broad and include the regulation of G1/S progression via myc and translational control via Hes proteins, resulting in cell differentiation. In addition, cross-talk between the EGF and the Wnt/βcatenin pathways can alter the morphological features, which may in turn alter the signal strengths of various pathways, including Notch (Orihara-Ono, 2011).
Notch-signal transducing components include numerous ligands, modifiers, and mediators, and each step in Notch-mediated fate changes appears to be controlled by the trafficking and localization of these components. The specific localizations of these components contribute to the efficiency of downstream signaling. This study has presented evidence that a reduction in Notch, which activates the expression and trafficking of Delta, forces a cell-fate change (Orihara-Ono, 2011).
During the course of the NE-to-NB transition of optic medullar progenitors, two peaks in Notch expression were typically observed: the first peak, which occurred early during the NE-to-NB transition, was followed by a period of reduced expression during the cell-type transition, after which Notch expression once again increased as the new NBs resumed division. After several divisions, Notch was downregulated and ultimately became undetectable. Interestingly, similar changes have been reported in the vertebrate spinal cord primordium, suggesting that this pattern of Notch expression may be conserved during vertebrate neurogenesis (Orihara-Ono, 2011).
The frequent apical constriction of single-layered epithelial cells and the subsequent downregulation of E-cad are often coupled to dynamic morphogenetic movements involving the invagination of bottle-shaped cells during gastrulation, which is one of the most important examples of the epithelial mesenchymal transition (EMT). The polarity of cells is sometimes maintained throughout development, but sometimes the polarity is progressively lost and the cells' morphology is remodeled. Typically, the cell cycle is slowed (mostly by a prolonged G1 phase) during this remodeling phase. In other words, a morphogenetically active zone often corresponds to a mitotically inactive one, in which the cells are arrested in the G1 phase. Examples include bottle cells in gastrulating Xenopus embryos, the Drosophila eye morphogenetic furrow, and the laminar furrow of the optic lobes. In most cases, these events are associated with JAK signaling pathways as well as Wnt signaling and TGF-β signaling pathways. In addition, in view of the similarity between this phenomenon and the EMT, cross talk between the Notch and Delta signaling pathways must be considered. Confirming this notion, the upregulation of both Snail and Worniu, Drosophila homologs of the EMT promoting factor Snail1, has been reported in embryonic NBs. Although the relative contribution of various factors to the cell-fate decision remains unclear, it has been reported that the Jak/Stat signal is strong during the neuroepithelial phase but weakens after the NEs commit to a progenitor fate, suggesting that the Jak/Stat signal maintains the cell in its undifferentiated state. Developmental stage-dependent signaling from the environment might change the signal balance and push the cell toward differentiation (Orihara-Ono, 2011).
This paper has focused on the Notch-mediated NE-to-NB transition within a specific, transitional phase of the cell cycle. The work highlights the usefulness of this model system for studying early neurogenesis in the neural plate stage, and these findings may be applicable to general neural stem cell biology (Orihara-Ono, 2011).
The role of scabrous in the evenly spaced bristle pattern of Drosophila has been explored. Loss-of-function of sca results in development of an excess of
bristles. Segregation of alternately spaced bristle precursors and epidermal
cells from a group of equipotential cells relies on lateral inhibition mediated
by Notch and Delta (Dl). In this process, presumptive bristle precursors inhibit
the neural fate of neighboring cells, causing them to adopt the epidermal fate.
Dl, a membrane-bound ligand for Notch, can inhibit adjacent cells, in direct contact with the precursor, in the absence of Sca. In contrast, inhibition of cells not adjacent to the precursor requires, in addition, Sca, a secreted molecule with a fibrinogen-related domain. Over-expression of Sca in a wild-type background, leads to increased spacing between bristles, suggesting that the range of signaling has been increased. scabrous acts nonautonomously, and evidence is presented that, during bristle precursor segregation, Sca is required to maintain the normal adhesive properties of epithelial cells. The possible effects of such changes on the range of signaling are discussed. It is also shown that the sensory organ precursors extend numerous fine cytoplasmic extensions bearing Dl molecules, and these structures may play an active role during signaling (Renaud, 2001).
sca functions in the inhibition of the neural fate, since in its absence an excess of neural precursors form. The sca mutant phenotype is very similar to that of hypomorphic N and Dl mutants and indeed Notch and Dl are known to be the main components of the signaling pathway regulating the spaced pattern of bristles. Thus, Sca is likely to positively modulate N signaling. During bristle precursor selection, like Dl, sca acts nonautonomously and is not required for reception of the signal that places its activity upstream of N. What are the respective roles of Dl and sca in the segregation of bristle precursors? Both genes are expressed in proneural domains and then at high levels in the bristle precursors, and their products act nonautonomously on neighboring presumptive epidermal cells. Both proteins associate with N, but so far only Dl has been shown to be an activating ligand. Scabrous is a secreted molecule, whereas data accumulated to date suggest that active Dl is membrane-bound. In the complete absence of Dl, all cells adopt the neural fate and thus bristle precursors arise adjacent to one another. In the complete absence of Sca, there is an excess of bristle precursors but they are never adjacent and are always separated by at least one epidermal cell. This indicates that sca is not needed for the bristles to be spaced apart by a short distance, and that Dl, which is expressed in sca mutants, is able to inhibit cells immediately adjacent to the precursors without any help from Sca. In contrast, in the absence of Sca, Dl is unable to inhibit those cells not in direct contact with the precursor. This suggests a role for Sca in the inhibition of cells not adjacent to the precursor. These two, possibly separable events, are referred to as 'short' and 'long' range signaling (Renaud, 2001).
Formally, there are three possible mechanisms for 'long' range signaling. The first would be that sca is part of a relay mechanism whereby cells immediately adjacent to the precursor are inhibited by Dl and then relay the signal farther out by means of Sca. This hypothesis is very unlikely, however, because one would expect sca to be expressed in cells adjacent to the precursor following activation of N by Dl. In fact, sca protein is only detectable in the precursor itself, although sca is earlier expressed at low levels in the proneural domain. Furthermore, sca is expressed in the absence of Dl and therefore does not require a prior signaling event mediated by Dl (Renaud, 2001).
A second possible mechanism is that there may be two independent signals, one acting at a 'short' range (Dl) and another at a 'long' range (Sca). Two observations suggest that this, too, is unlikely: (1) in the absence of Dl, all cells adopt the neural fate and so the process of bristle spacing is completely abolished. scabrous is expressed in cells mutant for Dl so this result indicates that Sca alone is unable to repress the neural fate; (2) the results indicate that sca is not involved in the process of lateral inhibition whereby a pattern of alternating neural and epidermal cells is generated. Along a border between wild-type cells and cells mutant for Dl, the precursors are nearly always selected from the pool of wild-type cells, rather than from the mutant cells. This is thought to be because the mutant cells produce little or no signal and are inhibited by the Dl-producing wild-type cells. Along sca mosaic borders, the mutant cells can adopt the neural fate, indicating that they are not defective in the production of the activating ligand Dl. Furthermore, since wild-type precursors also form along the mosaic border, neural precursors can be chosen from cells of either genotype. Thus, cells choose the neural or epidermal fate regardless of whether or not they express sca (Renaud, 2001).
This would explain the fact that precursors are never adjacent in sca mutants and is not inconsistent with the observed sca phenotype of excess bristles. During segregation of both the normal component of precursors, as well as the additional precursors, adjacent cells have to choose between epidermal and neural fates. Any failure of this process would result in the presence of adjacent bristle precursors, a phenotype characteristic of N and Dl mutants, but not sca mutants. Segregation of single neural precursors surrounded by epidermal neighbors is thus probably mediated by Dl alone, which would explain why bristle spacing is completely abolished in the absence of Dl, even though Sca is present (Renaud, 2001).
The third possibility is that 'long' range signaling requires both Dl and Sca. Under this hypothesis, Dl would be the activating ligand in 'long' range signaling, but would require Sca in order to inhibit cells not directly in contact with the precursor. This could be the case regardless of whether the signal originates exclusively in the precursor or additionally in proneural cells. One observation in favour of this hypothesis is afforded by examination of flies mosaic for Dl. Along the edges of Dl mutant clones, mutant cells are able to differentiate as epidermis under the influence of an inhibitory signal from neighboring wild-type cells. This 'rescue,' due to expression of Dl in the wild-type neighbours, can extend up to four cell diameters. If there is no relay mechanism and no other signal, then Dl must somehow be able to activate N several cell diameters away from the cell in which it is produced. Scabrous is of course present in both the Dl+ and the Dl- cells, but is unable to effect any rescue of cells mutant for Dl that are situated more than about four cells away from the wild-type Dl-expressing cells. Examination of Dl mutant clones in a mutant sca background would indicate the range of Dl signaling in the absence of Sca (Renaud, 2001). Clones doubly mutant for sca and Dl, however, fail to differentiate the cuticular components of the bristles (for all allelic combinations tested) and so are uninformative (Renaud, 2001).
It is suggested that bristle spacing may be the result of two signaling events. The first step of lateral signaling involves Dl alone and allows a group of cells to choose a single neural precursor that will inhibit adjacent cells. This step proceeds normally in the absence of Sca. The second step would act to inhibit cells that are not adjacent to the precursor and would require the activity of both Dl and sca. This step is impaired in both sca and Dl mutants. In Dl mutants, in the absence of the inhibitory signal, lateral inhibition fails, leading to adjacent precursors and a loss of epidermal cells. In sca mutants, an excess of precursors form, but they segregate singly and are spaced by at least one epidermal cell through the activity of Dl. Examination of the nascent precursors with neu-lacZ, fail, to reveal two temporally separate waves of precursor formation in sca mutants. Staining of wild-type pupal nota with DE-cadherin, however, may provide a visual correlate of the two groups of target cells. A rosette-like ring of wedge-shaped cells surrounds the bristle precursor; it is reminiscent of the cell preclusters that precede segregation of the R8 photoreceptor during development of ommatidia. These supra-cellular structures may allow more cells to enter into direct contact with the precursor during the first step of inhibitory signaling (Renaud, 2001).
The results demonstrate a requirement for local discontinuities in the levels of Sca between cells during inhibitory signaling. This was also reported to be the case for eye development. Uniform expression of Sca under experimental conditions is unable to rescue the sca mutant phenotype. So the relative quantitative differences in the level of Sca between neighbouring cells is an essential feature of the spacing mechanism. Over-expression of Sca in a wild-type fly causes a phenotype opposite that of the loss-of-function phenotype. The distance between bristles actually increases and there are correspondingly fewer bristles in the domain of over-expression. In these flies, although exogenous Sca is uniformly distributed, regulation of the endogenous gene will provide local differences in levels of the protein. The greater distance between bristles suggests an extension in the range of the inhibitory signal. Scabrous is likely to be present in a gradient of decreasing concentration around each precursor. The molecule has been shown to have a very short half-life. Thus, it would decay rapidly after secretion and this would help maintain a graded distribution from the source. It is postulated that such a gradient could define the range of the inhibitory signal (Renaud, 2001).
A rescue of up to four cell diameters has been observed at the edges of clones of cells mutant for Dl. This suggests a signaling range that exceeds that required in wild-type flies, where bristles are usually only four to five cells apart. It is possible, however, that the signal range is longer than usual in this experimental situation because very large amounts of Sca are likely to be present in these clones due to the hyperplasia of precursors. It is, however, noteworthy that in other drosophilid species, such as D. ararama, the bristles are spaced by eight or nine cells, suggesting a possibly greater signaling range (Renaud, 2001).
One property of Sca is to modulate adhesive parameters of the epithelial cells surrounding the precursor. Discrete changes in the distribution of DE-cadherin, Discs large, and other junctional proteins are seen in the epidermis of sca mutants, that are associated with mislocalization of the adherens and septate junctions and some disruption of epithelial organization. The monolayered nature of the epithelium is retained, consistent with the fact that the morphogenetic changes of metamorphosis are not impaired in sca mutants. This phenotype is only observed in late third instar discs and early pupae, when bristle precursors are forming. If ac-sc activity is removed, the late third instar disc epithelium is wild type. In ac-sc mutants, the absence of Ac and Sc entails a loss of Sca, whose expression is dependent on Ac-Sc (Renaud, 2001).
The epithelial defects resulting from a lack of Sca protein thus coincide with ac-sc expression and the process of lateral inhibition. Scabrous may therefore be required to maintain normal epithelial integrity by counteracting the effects of a protein(s) activated during precursor segregation. It is therefore likely to act through association with a protein(s) whose expression is regulated by Ac and Sc. Notch is ubiquitously expressed in the epithelium, but is activated and probably up-regulated in future epidermal cells surrounding the precursors. Powell (2001) demonstrated that Sca binds N and as a result N is stabilized at the cell surface in S2 cells. Interestingly, in Nts1 mutants at 29°C, where the activity of N is strongly reduced, the distribution of DE-cadherin in the disc epithelium is also discretely altered and the epithelium appears similar, but not identical to that of sca. This suggests that the epithelial defects seen in sca mutants may be the result of a failure to stabilize N protein in epithelial cells. These results are consistent with the idea that Sca acts through N, and with earlier observations linking N to epithelial cell adhesion (Renaud, 2001).
scabrous is not required for inhibition of cells that are adjacent to the precursor. Furthermore, the requirement for sca in the fly is quite restricted: it is not expressed in many other tissues where Notch signaling takes place in its absence. While it is expressed in neural precursors in the embryo, loss of the protein there seems to be without consequence, perhaps because the interactions involve adjacent cells. This leads to the hypothesisis that the effects of Sca on cell adhesion and the stabilization of N may be specifically required when the levels of Dl are limiting (Renaud, 2001).
Delta is expressed in proneural domains and can still be detected in presumptive epidermal cells after precursor segregation. Nevertheless the amount of Dl remaining in presumptive epidermal cells appears to be insufficient, by itself, to repress the neural fate. In the absence of Sca, or in flies carrying hypomorphic alleles of Dl, the space between bristles is decreased. Furthermore, in epidermal cells, the transcription of Dl progressively declines due to repression of its regulators ac and sc following N activation, whereas in the bristle precursors the levels of Dl increase as the levels of Ac and Sc rise. This leaves two possibilities. One is that all of the signal originates in the precursor cell, in which case Dl must be transported, by some as yet unknown means, from the precursor to cells not in direct contact with the latter. The other, is that the concentration of Dl molecules remaining on the presumptive epidermal cells is insufficient to inhibit by itself, but can do so if helped by Sca. Dl from both groups of cells may participate in the wild type. In either case, stabilization of N may therefore be a means to increase the chances of receptor activation in the presence of limiting amounts of Dl (Renaud, 2001).
Changes in cell adhesion could also be the cause of the abnormal bristle organs seen in sca mutants, where two or more cells of the bristle organ lineage adopt the same fate at the expense of the others. In the wild type, spatial arrangements of the cells of the bristle organs are stereotyped as a result of the nonrandom orientation of the mitotic spindles at each division. In sca mutants, the cells are often randomly arranged and in some cases appear to drift apart from one another. This would be likely to prevent the precise cell-cell interactions, mediated by the N signaling pathway, necessary for the assignment of the correct cell fates (Renaud, 2001).
The precursor cells have a quite distinctive shape, reminiscent of neurons with a number of filopodial-like extensions that fan out in a planar orientation. It is not known whether, during bristle precursor segregation, the epidermal cells of the notum extend similar filopodia. Oriented epidermal outgrowths have been described in the epidermis of other insects and also in the wing pouch and peripodial membrane of Drosophila imaginal discs, where it has been suggested that they may function during signaling. Some of these structures project basally and others extend long, straight and polarized structures. Their morphology differs from that of the extensions observed (Renaud, 2001).
It is not known whether Dl from the precursor cell is able to reach cells that are not adjacent to the Dl-expressing precursor, but one means by which this could occur is via cytoplasmic extensions. This has been suggested for Lag-2 signaling in the nematode germ line. Indeed, the presence of Dl molecules can be detected on the filopodia. Although formation of filopodia will depend on properties of the neural precursor itself, subtle changes in junctional contacts and adhesion between surrounding epithelial cells may help to orient or stabilize these structures. The changes in the bristle density of both wild-type and sca flies that are seen after expression of a dominant negative form of DE-cadherin, suggest a role for adhesion molecules in bristle spacing. Preliminary observations indicate that Sca is not required for the extension of filopods. This is consistent with the nonautonomy of sca mutant cells. If filopodia are the means whereby nonadjacent cells are inhibited, and if Sca were to be required for extension of filopodia from the Sca-producing bristle precursors, then sca would be expected to behave autonomously (Renaud, 2001).
Further studies are necessary to determine the molecular basis of Sca function, but one possibility is that binding of Sca to N leads to discrete modifications in epithelial structure that allow Dl molecules on the cytoplasmic extensions to form stable ligand-receptor complexes. The colocalisation of Sca and Dl in cytoplasmic vesicles may indicate cellular trafficking of protein complexes that include Dl, N, and Sca (Renaud, 2001).
The organization of bristles on the Drosophila notum has long served as a popular model of robust tissue patterning. During this process, membrane-tethered Delta activates intracellular Notch signaling in neighboring epithelial cells, which inhibits Delta expression. This induces lateral inhibition, yielding a pattern in which each Delta-expressing mechanosensory organ precursor cell in the epithelium is surrounded on all sides by cells with active Notch signaling. This study shows that conventional models of Delta-Notch signaling cannot account for bristle spacing or the gradual refinement of this pattern. Instead, the pattern refinement observed using live imaging is dependent upon dynamic, basal actin-based filopodia and can be quantitatively reproduced by simulations of lateral inhibition incorporating Delta-Notch signaling by transient filopodial contacts between nonneighboring cells. Significantly, the intermittent signaling induced by these filopodial dynamics generates a type of structured noise that is uniquely suited to the generation of well-ordered, tissue-wide epithelial patterns (Cohen, 2010).
The analysis of lateral inhibition in the notum described in this study reveals an extended period of plasticity during pupal development in which a gradual process of pattern refinement takes place, after which cells take on an epithelial fate or undergo an asymmetric division to initiate the development of the mechanosensory organ. Importantly, the path of this pattern formation process and the final spacing observed can be quantitatively reproduced using a model of Delta-Notch signaling in which cells are able to exchange signals via a population of dynamic, basal filopodia (parameterized based on in vivo observations) that interact over a distance of several cell diameters. As quantitatively predicted by a model of filopodia to filopodia signaling, changes in protrusion length and dynamics lead to corresponding changes in bristle spacing, implying the involvement of filopodia in both signal sending and receiving cells. Moreover, the model suggests that protrusions are likely to determine the spacing of bristle precursors even in instances in which they transmit the minority of the total Delta-Notch signal (Cohen, 2010).
Although previous authors have suggested roles for long, stable protrusions in cell-cell signaling events, this analysis shows that the generation of a robust, well-ordered, and well-spaced pattern of bristle precursors across an entire tissue requires filopodia that are dynamic. In the case of the notum, these protrusions are actin based, and appear to be generated through the action of Rac and the SCAR complex (Georgiou, 2010). Although this is unexpected, given the well-described involvement of the SCAR complex in the generation of lamellipodia, there are precedents for the involvement of the SCAR complex in the generation of filopodia in Drosophila cells. Moreover, filopodia have previously been seen predominating in a three-dimensional (3D) tissue context. It is also noted that the role described here for SCAR-dependent protrusions in determining precursor cell spacing is strongly supported by the recent identification of the full complement of components of the SCAR complex (SCAR/WAVE, Abi, Sra1, Hem/Kette, and HSPC300/Sip1) in a recent genome-wide RNAi screen as genes required to maintain bristle spacing within the notum without affecting subsequent mechanosensory organ development (Mummery-Widmer, 2009). Loss of SCAR function leads to the specific loss of F-actin and dynamic protrusions within the basolateral domain without affecting cell morphology, cell size, cell polarity, or endocytosis (Georgiou, 2008; Georgiou, 2010), making it less likely that SCAR functions by directly altering the Notch signaling pathway. Additionally, it was found that scar mutant cells within the external sensory organ (but not RacN17 expressing cells) differentiate correctly despite having an altered morphology. Conversely, as recently reported by Rajan (2009) and as seen in the Mummery-Widmer screen, the Arp2/3 complex and WASp appear to function in Notch signaling following precursor cell division, leading to a loss of bristles from the nota of adult flies (Cohen, 2010).
Since physical tension has been shown to enhance Notch cleavage and hence Delta-Notch signaling, it is possible that forces generated through the retraction of actin-based protrusions engaged in signaling could function to enhance Notch activation. Testing this hypothesis, however, will likely have to await the future development of tools enabling Notch cleavage to be imaged as it occurs in vivo (Cohen, 2010).
Significantly, theoretical analysis suggests that the dynamics of these actin-based filopodia, as measured in vivo, could induce intermittent Delta-Notch signaling to drive a process defined as 'pattern refinement' -- whereby the self-organizing pattern improves steadily over an extended period of developmental time as cells undergo switches in their gene expression profile. This theoretical prediction fits with the observed shift in bristle-precursor cell patterns observed during development of the notum, and the extended period of time over which the bristle precursor pattern remains labile, as measured using laser ablation and a temperature-sensitive Notch allele. In theory, since intermittent signaling could be generated in the absence of protrusions using cell autonomous oscillations in Notch-Delta signaling, the oscillations seen in some other systems could serve a similar function-aiding gradual pattern refinement. Dynamic filopodia, however, have several features that make them uniquely suited to a role in pattern refinement. A network of filopodia can be quickly established and eliminated, and the length and direction of filopodia tuned in order to define a precise gradient of signaling over a distance of several cell diameters. Thus, dynamic filopodial signaling appears to be an ideal alternative to morphogen diffusion as a mediator of developmental signaling at a distance (Cohen, 2010).
The Drosophila protein kinase Par-1 is expressed throughout Drosophila development, but its function has not been extensively characterized because of oocyte lethality of null mutants. This report characterizes the function of Par-1 in embryonic and post-embryonic epithelia. Par-1 protein is dynamically localized during embryonic cell polarization, transiently restricted to the lateral membrane domain, followed by apicolateral localization. Maternal and zygotic par-1 was depleated by RNAi and a requirement was revealed for Par-1 in establishing cell polarity. Par-1 restricts the coalescing adherens junction to an apicolateral position and prevents its widespread formation along the lateral domain. Par-1 also promotes the localization of lateral membrane proteins such as Delta. These activities are important for the further development of cell polarity during gastrulation. By contrast, Par-1 is not essential to maintain epithelial polarity once it has been established. However, it still has a maintenance role since overexpression causes severe polarity disruption. Additionally, a novel role is found for Par-1 in Notch signal transduction during embryonic neurogenesis and retina determination. Epistasis analysis indicates that Par-1 functions upstream of Notch and is critical for proper localization of the Notch ligand Delta (Bayraktar, 2006).
It might not be that Par-1 simply defines the limits of the AJ. Rather, it might also act positively to specify the basolateral domain. Par-1 is required for localization of Delta to the basolateral region. This positive effect of Par-1 on Delta is not due to preventing the AJ from inhibiting Delta. Delta co-localizes with the AJ both normally and when Par-1 is depleted. So how does Par-1 guide membrane regionalization during cellularization? Par-1 protein is distributed along the lateral membrane immediately basal to the incipient AJ. Based on this, three models suggest themselves. Par-1 could assemble a diffusion barrier that physically blocks movement of SAJs into the lateral region and limits movement of Delta into the apicolateral region. If par-1(RNAi) disrupts such a barrier, then other mechanisms must maintain apical Crb restriction from the lateral region. An alternative is that Par-1 has a role in the polarized targeting of transport vesicles carrying SAJ and Delta proteins. In this model, Par-1 might interact with the 'exocyst', a secretory targeting apparatus involved in polarized segregation of transmembrane proteins. Data from yeast Par-1 indicate that it directly associates with a t-SNARE, a membrane-bound component of the exocyst. Moreover, Par-1 phosphorylation of the t-SNARE protein triggers its release from the cell membrane. If Drosophila Par-1 also interacts with the exocyst, then it might selectively block the fusion of SAJ exocytic vesicles to the basolateral membrane. In support of this model, punctate intracellular staining of Par-1 can be seen during cellularization and is reminiscent of vesicles. Par-1 might also stimulate targeting of other cargo, such as Delta-loaded vesicles, to the basolateral membrane. Consistent with this notion, Par-1-depleted ectoderm cells accumulate Delta-positive cytoplasmic vesicles. Finally, Par-1 could differentially affect the stability of proteins in the lateral domain, by de-stabilizing some and stabilizing others. This could occur through degradation or rapid recycling via endocytosis (Bayraktar, 2006).
How directly would Par-1 participate in these mechanisms? This is unclear at present. None of the known substrates for Drosophila Par-1 kinase include Delta or AJ components. In ovarian follicle cells, Par-1 phosphorylation of Baz prevents Baz association with Par-6-aPKC. Baz and Par-6 are among the earliest acting proteins in polarization of blastoderm. During cellularization, Baz associates with the apicolateral membrane, whereas Par-6 is localized to the apical cortex. If either Baz or Par-6 is mutated, the apical AJ proteins do not coalesce but disperse along the lateral membrane. Thus, Baz could be a candidate for mediating the effects of Par-1 in the blastoderm. However, several observations indicate that Par-1 phosphorylation of Baz is not necessarily essential to establish AJ localization. First, Par-1 does not have a polarized distribution during early cellularization and is detected in the apicolateral regions where Baz and Par-6 are already localized. A similar co-distribution is seen by the time the ectoderm has reached mid-gastrula stage. Second, although Baz is required for apical localization of Crb and Patj, Par-1 has no significant effect on their apical localization. Third, establishment of the AJ in follicle cells is not dependent upon Par-1 phosphorylation of Baz (Bayraktar, 2006).
Par-1 plays a curious role in maintenance of polarity of imaginal disc epithelia that derive directly from ectoderm. Par-1 is localized to the apical and marginal zones of imaginal disc cells but is not essential for their polarity. Possibly, redundant mechanisms operate in the absence of Par-1. This idea is supported by overexpression experiments. When Par-1 is overexpressed, the AJ and apical domain are disorganized, and cells are compromised for differentiation, growth and death. This result argues that Par-1 normally plays a role in maintaining cell polarity that is sensitive to its activity level. By contrast, Par-1 is essential to maintain polarity in follicle cell epithelia surrounding adult egg chambers, suggesting that redundancy is restricted to imaginal discs (Bayraktar, 2006).
Par-1 also regulates Notch signaling and it acts upstream of Notch as determined by epistasis analysis. Two different Notch signaling decisions regulated by Par-1 are detected. The first was in the embryonic ectoderm where Par-1 depletion disables Notch-mediated lateral inhibition. The second is in the eye imaginal disc where Par-1 overexpression disables Notch-mediated eye cell determination. Since Notch is disabled when Par-1 is missing or overactive, it suggests that Par-1 is not playing an instructive role in Notch signaling. Rather, it is probably a permissive effect that is related to cell polarity regulation. Indeed, localization of Delta is dramatically reduced along the basolateral domains of blastoderm and ectoderm cells of par-1(RNAi) embryos. It is reasonable to think that Par-1 acts in Notch signaling by localizing Delta to a region of the membrane where it can make a productive interaction with Notch. This permissive model of Notch signaling is nevertheless specific; other regulators of ectoderm polarity do not affect Notch signaling. Moreover, other signaling pathways active in the ectoderm are unaffected by Par-1. Interestingly, a synergistic interaction between Par-1 and Notch was found in the eye imaginal disc. Disc growth significantly increased when Par-1 was overexpressed with ligand-independent Notch. The extra eye tissue developed photoreceptors, indicating the ectopic cells are properly specified. Since loss of cell polarity is associated with hyperplasia in the eye disc, this supports the notion that Par-1 exerts this effect through perturbation of eye disc cell polarity. The synergistic interaction with Notch may be useful in the future for screening of genes involved in tumor formation or progression to a cancerous state (Bayraktar, 2006).
Wing development in Drosophila requires the activation of Wingless (Wg) in a small stripe along the boundary of Fringe (Fng) expressing and non-expressing cells (FB), which coincides with the dorso-ventral (D/V) boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser) and Delta (Dl) respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, the function of Fng, Ser and Dl was analyzed during wing development at an ectopic FB and at the D/V boundary. Notch signalling was found to be initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, the analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB (Troost, 2012).
During wing development, the activity of the Notch pathway is required to establish a stripe-like domain of expression of several genes along the D/V boundary that control wing growth and patterning, chief among them are wg and vg. The D/V boundary is a FB, which provides an interface that is crucial for the activation of Notch and establishment of this organising centre. The current understanding is that Fng promotes Dl signalling and prevents Ser signalling through the modification of Notch. As a result, Dl signals strongly from ventral to dorsal and Ser from dorsal to ventral cells boundary cells. The simultaneous signalling of the ligands in opposite direction establishes the expression of Notch target genes at both sides of the FB. It is essential for this model to work at the D/V boundary that induction of expression of Ser is restricted to dorsal and that of Dl to ventral cells. If e.g. Dl expression could also be induced in dorsal cells by the Notch pathway, the activity of Notch would immediately spread throughout the dorsal half of the wing anlage. In agreement with this requirement, it has been observed that expression of Ser is restricted to dorsal cells upon expression of activated Notch in dorsal and ventral cells. The combination of spatially restricted expression of the ligands and the Dl/Ser loop restricts the activation of Notch to the D/V boundary during early stages of wing development. At the middle of the third larval instar stage the Dl/Ser/Wg loop takes over to maintain the activity of Notch. Thus, a critical step is the establishment of expression of Wg in boundary cells. Once this is achieved the second feedback-loop assures expression of Wg and Notch signalling throughout wing development (Troost, 2012).
While this model can explain the events at the D/V boundary, it cannot explain the events at an ectopic FB, since differential expression of the ligands is unlikely to occur there. Nevertheless, the expression of Wg is also restricted at the ectopic FB. The presented work provides further evidence for the current model of Fng action, but adds new details that enable it to explain also the events at an ectopic FB. One addition is the initial sequential establishment of the expression domain of Wg through asymmetric Notch signalling during early stages of wing development. Asymmetric expression of Wg was observed only in ventral boundary cells (VBCs) at the D/V boundary, indicating that these cells achieve sufficiently high activation of Notch to initiate expression of Wg. The existence of the early asymmetric phase of Notch activation was surprising given the fact that activation of Notch results in the activation of the expression of Dl and Ser. Consequently, the activation of Notch in ventral cells should immediately lead to up-regulation of expression of Dl in ventral cells and back-signalling to dorsal cells. It was therefore expected that if an asymmetric phase exists, it would be too short in time to be detected. Importantly, the initial asymmetric phase appears to be a general property for Notch signalling at a FB, since it was observed on both analysed FBs. The existence of the asymmetric phase also indicates that a FB can be used to generate activity domains of the Notch pathway where the feedback-loop that regulates the expression of the ligands through Notch activation does not occur. So far the loop has been found only in the wing pouch. In the absence of the loop the asymmetric state would remain and thus, a defined stripe of high Notch activation would be generated in a field of cells that uniformly express Dl even in the absence of Ser (Troost, 2012).
At the ectopic FB, it was found that eliminating the activity of the Notch pathway in PBCs does not result in the loss of expression of Wg in ABCs. It only prevents the late symmetric phase. This indicates that establishment of a Dl/Ser feedback loop in A/P boundary cells is not essential for reaching sufficient high levels of Notch signalling to induce Wg expression during the asymmetric phase. The same was observed for the D/V boundary: Depletion of Su(H) function or over-expression of H causes a restriction of expression of Wg to VBCs, but not its abolishment. Thus, the Dl/Ser loop is probably mainly required for the later occurring patterning of the future wing margin, but not for the establishment of the wing primordium (Troost, 2012).
A further addition is that the basic expression of Dl is independent of Ser signalling. This is indicated by the observation that 1) Dl is expressed throughout the wing anlage in early discs and 2) Dl signals to DBCs at the D/V boundary in absence of Ser function. This holds true also for the ectopic FB: here Dl signals to the anterior boundary cells (ABCs) in the absence of Ser. However, in both cases Dl signalling is not strong enough to initiate Wg expression, which is a crucial event for wing development (Troost, 2012).
The results also reveal an unanticipated requirement of Ser in ABCs for the expression of Wg. This requirement could be explained through Ser signalling from the ABCs to PBCs to up-regulate Dl there, which in turn signals back to ABCs (Ser/Dl loop). This explanation would imply that the levels of Notch activation required for the induction of Dl expression are lower than that for Wg. Otherwise, the expression of Wg would not stay asymmetric as it is observed. However, it was found that Notch signalling is not required in PBCs during the early asymmetric phase. This excludes the mentioned explanation and suggests that Ser must activate Notch in the Fng expressing ABCs. This assumedly weak activation contributes to the total activity of Notch in these cells and guarantees levels of Notch signalling above the threshold required for expression of Wg. In the absence of Ser the activation by Dl from PBCs appears to fail to reach the threshold level in a fraction of discs. In agreement with this notion it has been shown that expression of Ser is broadly induced upon ectopic expression of Fng (Troost, 2012).
It was further observed that concomitant loss of Ser and Dl function in ABCs always abolishes the expression of Wg at the ectopic FB. Hence, Dl must also signal in Fng expressing ABCs and contribute to Notch signalling in these cells. The results suggest that the total amount of Notch activity in ABCs is the sum of signalling by Dl and Ser in the Fng domain and Dl signalling from PBCs to ABCs. Whereby the signal from posteriors to anterior is the more important one, since its loss always abolishes expression of Wg. A similar requirement at on both sides of the boundary for Dl had been described for the D/V boundary. Interestingly, the data indicate that Dl plays a similar role there as Ser at the ectopic FB (Troost, 2012).
Another addition to the current model is that Fng has two antagonistic effects on each ligand. The modification of Notch by Fringe is known to polarise signalling at the D/V-boundary by enhancing Dl- and suppressing Ser-signalling. Loss of function of fng during early stages of wing development abolishes expression of Wg at a time where it is solely dependent on Ser signalling. This observation indicates that Fng has a positive effect on Ser signalling in addition to its known negative one: it enhances Ser-signalling from Fng expressing dorsal to non-expressing ventral cells. This enhancement is required to activate expression of Wg in cells across the boundary. This finding is in good agreement with previous work that reports that Fng can enhance the ability of Ser to induce ectopic wing margins upon their co-overexpression. It is a possibility that this positive influence on Ser is indirect: The modification of Notch mediated by Fng results in a decrease of binding Ser to Notch. As a consequence high levels of free Ser are available in Fng expressing boundary cells at the FB that can bind in trans to the unmodified Notch on the adjacent Fng non-expressing boundary cells. At the analysed ectopic FB, it was found that Dl is required in Fng non-expressing PBCs to raise the activity of Notch signalling to a level that is sufficient for expression of Wg in Fng expressing anterior boundary cells, although Dl is expressed ubiquitously in early discs and thus, present in both cell populations at the same levels. This finding indicates that although Dl can induce activity of Notch in the Fng domain, this activity is insufficient to initiate the expression of Wg. The activity rises beyond the threshold only if the cells receive an additional Fng-enhanced Dl signal from non-expressing. This suggests that Fng has a suppressing effect on Dl signalling within its domain. The opposing effects on the activity of each ligand have important implications for restricted Notch-signalling at the FB. Restricted expression of the ligands on opposite sides of the FB is not required to restrict Notch activation to the FB. Only Dl outside the Fng domain is sufficiently active to induce Wg expression in Fng expressing cells. In turn only Ser in Fng expressing cells is sufficiently active to induce Wg expression in Fng non-expressing cells. These conditions are only met at the FB. These properties assure that expression of Wg is restricted to a FB even in a tissue where Dl or Ser are initially expressed uniformly, as it is the case for the ectopic FB (Troost, 2012).
A difference between the ectopic FB and the D/V boundary is that the roles of the ligands are reversed: at the ectopic FB, Dl signalling is required for the establishment of the initial asymmetric phase and Ser to establish the symmetric phase and to maintain expression of Notch activity at a high level. In contrast, Ser signalling is required for the initial asymmetric phase at the D/V boundary and Dl for the establishment of the later symmetric phase and probably maintenance during later stages. Thus, activation of Notch-signalling at a FB can be initiated by both ligands. Another difference is the location of the asymmetric stripe of wg expression, which is located in Fng expressing cells at the ectopic FB, but in non-expressing cells at the DV boundary. It is believed that the events observed at the ectopic FB represents the more general mode of interactions, since the interactions occur solely in the ventral pouch where the cells differ mainly with respect to the expression of Fng. In contrast, at the D/V boundary dorsal cells differ from ventral cells by expression the selector Ap, which might modify the outcome of the interactions (Troost, 2012).
On the basis of these results, the events at the ectopic boundary can be summarized. During early stages of wing development, Dl is ubiquitously expressed throughout the wing anlage. Ectopic expression of fng with ptcGal4, creates a band-like Fng domain in the ventral pouch. During early stages Dl and Ser (probably induced by Dl) activate Notch signalling throughout the Fng domain at low levels that are not sufficient for activation of Wg. At the sharp posterior FB, Dl signalling from PBCs to ABCs, enhanced by Fng, raises the levels of Notch in ABCs beyond the threshold required for expression of Wg. The asymmetric phase is established. Dl signalling also up-regulates expression of Ser in ABCs. Over time Ser signalling from ABCs to PBCs (enhanced by Fng), induces Wg expression in PBCs. After the solid induction of symmetric expression of Wg, the Dl/Ser/Wg loop takes over and maintains Notch signalling at the D/V boundary. It was observed that the expression of Gbe+Su(H)-lacZ is broader upon ectopic expression of Fng in Ser mutant early third instar discs. Moreover, Wg is ectopically expressed throughout the ventral ptc domain upon ectopic expression of Fng by with ptcGal4 in Ser H, but not in H mutant discs. Thus, Ser probably contributes to keeping the Notch activity in the Fng domain at low level. It is known that the expression of Ser can contribute to the suppression of Notch activity in a cell-autonomous manner through cis-inhibition. Cis-inhibition has been discovered during analysis of wing development and appears to be involved in regulation and directional Notch signalling in several processes. This mechanism causes strong Ser signalling only from Ser expressing to non-expressing cells. Since Ser expression is induced in the Fng domain, strong signalling occurs from ABCs to PBCs. Thus, it is likely that cis-inhibition contributes to the directional signalling of from ABCs to PBCs to induce the later symmetric phase (Troost, 2012).
At the D/V boundary signalling is initiated differently. This study has shown that loss of the activity of the Notch pathway in dorsal cells does not prevent the establishment of expression of Wg along the D/V boundary, but restricts it to VBCs and allows wing development to proceed. Thus, the dorsal to ventral signal, which is mediated by Ser is most important. This notion is also in agreement with the null phenotype of Ser. Ser and Fng are initially expressed in all dorsal cells. Fng enhances Ser signalling to VBCs, but suppresses signalling among dorsal cells. This strong polarised signalling results in the activation of Wg expression and up-regulation of Dl expression in VBCs. It is likely, that the cis-inhibitory effect of Ser contributes to the suppression of the activation of Notch in dorsal cells through the initial phase of wing development, since the expansion of the expression of Gbe+Su(H)–lacZ over the whole dorsal wing anlage in early Ser mutant discs. This is probably induced by the weak ubiquitous expression of Dl that was observed in early wing discs. The analysis of the Ser null and Ser H double mutants indicates that Dl can activate Notch signalling in absence of Ser function, but not strong enough to induce Wg expression, despite the presence of the FB. It appears that at the D/V boundary, Ser has to up-regulate the expression of Dl in VBCs over time beyond the threshold that is required to induce Wg expression in DBCs. Fng contributes to induction of Wg expression by enhancing Dl signalling from Fng non-expressing VBCs to expressing DBCs. The requirement for accumulation of Dl could contribute to the observed delay of the establishment of the symmetric phase of expression. After the initial asymmetric phase, the Ser/Dl/Wg loop is established to maintain Notch signalling and symmetric expression of Wg. These results indicate that the initial Fng enhanced Ser signal is sufficiently strong to induce Wg in VBCs in a manner that enables also the establishment of the Ser/Dl/Wg loop. This is indicated by the observation that suppression of Notch activity in all dorsal cells throughout wing development does not prevent maintenance of expression of Wg in late stages of the third instar and even allows the development of adult wings with only minor patterning problems. Thus, the later signal from VBCs to DBCs is mainly required for positioning and patterning of the wing margin (Troost, 2012).
Asymmetric signalling through the Hh pathway has been shown to establish the organising centre for the A/P axis at the anterior side of the boundary. It has been assumed that one difference in the establishment of the D/V organising centre is its symmetric placement on the D/V boundary. The current results suggest that at least during initial phases, the signalling at the D/V boundary is also asymmetric and that the established organising centre can also work if it is displaced ventrally. Thus, it appears that the signalling events at both boundaries are more similar than previously anticipated (Troost, 2012).
Organisers control the patterning and growth of many tissues and organs. Correctly regulating the size of these organisers is crucial for proper differentiation to occur. Organiser activity in the epithelium of the Drosophila ovarian follicle resides in a pair of cells called polar cells. It is known that these two cells are selected from a cluster of equivalent cells. However, the mechanisms responsible for this selection are still unclear. This study presents evidence that the selection of the two cells is not random but, by contrast, depends on an atypical two-step Notch-dependent mechanism. This sequential process begins when one cell becomes refractory to Notch activation and is selected as the initial polar cell. This cell then produces a Delta signal that induces a high level of Notch activation in one other cell within the cluster. This Notch activity prevents elimination by apoptosis, allowing its selection as the second polar cell. Therefore, the mechanism used to select precisely two cells from among an equivalence group involves an inductive Delta signal that originates from one cell, itself unable to respond to Notch activation, and results in one other cell being selected to adopt the same fate. Given its properties, this two-step Notch-dependent mechanism represents a novel aspect of Notch action (Vachias, 2010).
Controlling the number of final polar cells (PCs) is essential in limiting the activity of the organizer, which triggers the differentiation of the neighbouring cells. Indeed, increasing or decreasing the PC number affects the number of border cells (population of about eight cells in wild type immediately adjacent to the PC). As supernumerary PC are quite rare in wild type, a robust mechanism must exist to guarantee the selection of only two cells from among a group of cells with similar developmental histories and similar developmental potential. At least three models can be invoked to explain such a selection: (1) the two cells are chosen randomly; (2) two cells acquire similar selective properties; or (3) two cells acquire selective properties that are different from each other, as well as from the others. The data rule out the first two, as it was demonstrated in several ways that the polar-fated cells (pfcs) are no longer equivalent at the time of selection, and that the two selected cells display different properties. By contrast, the results fit and even improve upon, the third model by revealing that the selection of the two cells with the same eventual fate occurs sequentially, and that the selection of the second depends on a signal from the first. It is proposed that among the cluster, one pfc becomes unable to respond to the N pathway and also resistant to apoptosis by a mechanism that remains to be determined. In turn, this pfc produces a Dl signal that activates the N pathway in a second pfc and prevents it from being eliminated by apoptosis. In parallel, this signalling also promotes an efficient apoptotic process that eliminates all of the other pfc. Cells other than the N-refractory pfc (follicle cells, stalk cells, germline cells and/or other pfc) could participate to some extent in the specific N activation in the second selected pfc. Nevertheless, the data show that the role of the N-refractory cell in producing Dl is preponderant. This is in agreement with multiple studies in which it has been shown that cells that do not respond to N activation are more efficient in producing the ligands. One important aspect of the mechanism described is that the selection of the second pfc is tightly linked to the elimination of the other pfc, as the same signal is required for both. This renders the mechanism of PC pair selection highly efficient. The implication of the N pathway in the protection of cells from apoptosis or in the induction of apoptosis has already been described in several developmental contexts. In the process of PC pair selection, the regulation of DIAP1 expression by Notch possibly provides a direct link that merits further exploration (Vachias, 2010).
The mechanisms by which one cell becomes more efficient in sending the signal remain unknown. Recently, it has been shown that the first anterior and posterior pfc are specified in region IIb of the germarium and that additional pfc are formed later during stages 1 and 2 (Nystul, 2010). One possibility is that this difference in timing is necessary for the first specified pfc to become refractory to N activation and thus more efficient in producing Dl. Moreover, the difficulty in detecting neur mRNA or protein prevents the authors from rejecting the possibility that neur is unequally distributed among the pfc. Similarly, it would also be interesting to determine why one pfc is more efficient at receiving the signal. The simplest explanation is that this depends upon the extent of contact between the sending and the receiving cells (Vachias, 2010).
Three modes of action -- lateral inhibition, lineage decisions and boundary formation -- have been proposed to describe how N activity regulates cell differentiation. Two of the main criteria used to define these modes are the state of the sending and receiving cells (equivalent or not), and the outcomes induced (positive or negative). In lateral inhibition, N signalling occurs between equivalent cells with roughly equivalent developmental properties. The signal-sending activity increases in one cell, which in turn increases the signal-receiving activity in the others, preventing them from adopting the fate of the signal-sending cell (negative outcome). In lineage decisions, the signalling happens between daughter cells and depends on the asymmetrical inheritance of some N regulators, such as Numb or Partner of Numb. Their presence in one cell autonomously antagonises N activation, whereas the activation does occur in the sibling cell, preventing it from taking on the same identity (negative outcome). During boundary formation, N signalling takes place at the interface between two compartments of differentially fated cells. The cells of the first row of each compartment are both the sending and the receiving cells and acquire identical new characteristics (positive outcome). In the mechanism described in this study, PC pair selection commences within a group of equivalent cells, with one cell acquiring specific properties and then signalling to another one to induce an identical fate (positive outcome). Based on the criteria mentioned above, this mechanism cannot be classed with either of the first two modes. First, it differs from the lineage decision mode, as the outcome of this mode is necessarily negative and because the two selected polar cells are not always sibling cells. Second, although the mechanisms used in PC pair selection and in the lateral inhibition mode both act upon a set of equivalent cells and generate Dl-expressing cells and N-expressing cells, the outcomes established are different. The mechanism described in this study is more closely related to the inductive signalling mode, as both generate positive outcomes and promote the acquisition of organizing activities by the Dl-sending and -receiving cells. But the fact that, in the signalling used to select the PC pair, the Dl-sending cell might be in part refractory to N activation because of differentially regulated endocytic trafficking possibly reveals an atypical N mechanism. Support for the existence of such a mechanism comes from recent data about the role of EGFR signalling and phyllopod during Drosophila eye formation. In that case, EGFR signalling suppresses some Nact phenotypes by activating phyllopod, which acts to regulate the residence time of the N components in early endocytic vesicles. In light of these observations, analysis of known trafficking regulators during PC pair formation could help to resolve the mechanisms that allow one pfc to become refractory to N activation. Thus, the data might define a novel aspect of the inductive signalling mode or, alternatively, might be representative of a fourth mode of N action. The discovery of other, similar examples would allow clarification of this point (Vachias, 2010).
The follicle cells of the Drosophila egg chamber provide an excellent model in which to study modulation of the cell cycle. During mid-oogenesis, the follicle cells undergo a variation of the cell cycle, endocycle, in which the cells replicate their DNA, but do not go through mitosis. Previously, it was shown that Notch signaling is required for the mitotic-to-endocycle transition, through downregulating String/Cdc25, and Dacapo/p21 and upregulating Fizzy-related/Cdh1.
In this paper, it is shown that Notch signaling is modulated by Shaggy and temporally induced by the ligand Delta, at the mitotic-to-endocycle transition. In addition, a downstream target of Notch, tramtrack, acts at the mitotic-to-endocycle transition. It is also demonstrated that the JNK pathway is required to promote mitosis prior to the transition, independent of the cell cycle components acted on by the Notch pathway. This work reveals new insights into the regulation of Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).
Notch controls the mitotic-to-endocycle transition in follicle epithelial cells; Notch pathway activity arrests mitotic cell cycle and promotes endocycles by downregulating string/cdc25 and dacapo/p21, and upregulating fzr/Cdh1. This study identified components regulating this transition, Delta, Shaggy, and Tramtrack. Shaggy and Delta are required for the activation of Notch protein. However, Delta is sufficient to activate Notch in this process, since premature expression of Delta in the germline stops mitotic division of the follicle cells. This study identified Tramtrack as a connection between Notch and the cell cycle regulators stg, fzr, and dap. Loss of Tramtrack function phenocopies the Notch and Su(H) phenotypes; overproliferation and misregulation of cell cycle components. However, high FAS3 expression, indicative of differentiation defects in Notch clones, is not observed in ttk clones, suggesting that Tramtrack might regulate a branch of the Notch pathway specific for cell cycle control. It was also shown that the JNK-pathway is a critical mitosis promoting pathway in follicle cells. Loss of JNK(bsk) or JNKK(hep) activities stop follicle cell mitotic cycles, while loss of JNK promotes premature endocycles. In addition, loss of the negative regulator of the pathway, the phosphatase Puckered, results in a lack of endocycles. However, the Notch-responsive cell cycle targets that, in combination, can induce the mitotic-to-endocycle transition, stg, fzr, and dap, are not regulated by the JNK-pathway (Jordan, 2006).
Notch signaling is highly regulated throughout development. The Notch receptor can be regulated by glycosylation of the extracellular domain, as well as by endocytosis and degradation of the intracellular domain, thus affecting the activity of the pathway. Shaggy has been shown to phosphorylate and thus affect the stability of Notch protein. Normal processing and clearing of Notch protein from the apical surface of follicle cells upon Notch activation does not occur in shaggy clones, indicating that Notch is not normally activated and therefore regulation of the downstream targets does not take place (Jordan, 2006).
In many organisms and tissues the Notch ligands are ubiquitously expressed and thus not likely to regulate Notch pathway activation. However, at the mitotic to endocycle transition, Delta is upregulated in the germline, making ligand expression a likely candidate for regulation of Notch activity. Premature expression of Delta in the germline can cause mitotic division to stop at least one stage earlier than in control ovarioles. Nonetheless, this effect is seen in only half of the ovarioles. Therefore, it is possible that yet another process is regulating Notch activity at the transition in addition to Delta expression. Further testing will determine if endocytosis of Notch might also regulate Notch activity at the mitotic-to-endocycle transition. One possible protein is Numb, which regulates Notch in human mammary carcinomas, indicating that Numb may have a more general role in cell cycle control than just the division of the sensory organ precursors (Jordan, 2006).
The fact that Notch overrides the mitotic activity of the JNK pathway by acting on cell cycle regulators that can induce the mitotic-to-endocycle transition puts further demand on understanding the connection between Su(H) and cell cycle regulators. One such component, the transcription factor Tramtrack, has been identified. Two Tramtrack proteins exist, Ttk69 and Ttk88, both of which are affected by the allele used in these studies. However, staining with antibodies specific to the two forms reveals that only Ttk69 is detectable in the follicle cells and downregulated in Notch clones (Jordan, 2006).
Ttk69 can control proliferation in glial cells, strengthening its candidacy for a critical component between Notch and cell cycle controllers in follicle epithelial cells. In addition, the Ttk-like BTB/POZ-domain zinc-finger transcription repressor in humans is Bcl-6, a protein associated with B-cell lymphomas (Jordan, 2006).
Ttk function in the follicle cell mitotic-to-endocycle transition was analyzed and it has been shown that the Notch-responsive cell cycle components stg, dap, and fzr are responsive to Ttk function. Interestingly, Ttk69 controls the string promoter in the Drosophila eye discs. In the future, it will be important to determine whether Ttk DNA binding sites are found in the Notch-responsive stg promoter as well. In addition, the binding sites of transcription factors that can interact with Ttk will be of interest, since Ttk can act as a DNA binding or non-binding repressor (Jordan, 2006).
Previous work revealed that the JNK pathway is closely connected to cell cycle control. For example, in fibroblasts the JNK pathway is critical for cdc2 expression and G2/M cell cycle progression. In the case of the follicle cell mitotic-to-endocycle transition, it was shown that the JNK pathway is a critical positive controller of the mitotic cycles. Lack of JNK activity leads to a block in mitosis and initiation of premature endocycles. Conversely, lack of the negative regulator of the JNK-pathway, the phosphatase Puckered, results in a loss of endocycles. However, puc mutant clones do not consistently support extra divisions but might induce apoptosis as shown recently in disc clones (Jordan, 2006).
These data are interesting in light of the results showing that the JNK pathway does not control the same cell cycle targets as the Notch pathway, and could be explained by the following hypothesis: the JNK-pathway positively regulates the mitotic cycles prior to stage 6 in follicle epithelial cells. This positive action on mitotic cycles is negatively short-circuited by the direct control of cell cycle regulators by the Notch pathway at stage 6 in oogenesis, resulting in the mitotic-to-endocycle transition. Premature termination of the JNK pathway is sufficient to induce mitotic-to-endocycle transition. However, prolonged JNK activity, while disrupting endocycles, cannot maintain mitotic cycling efficiently, due to Notch action on string, dacapo, and fzr (Jordan, 2006).
What then terminates JNK-pathway activity at stage 6 in oogenesis? Prolonged JNK activity (puc mutant clones) affects endocycles and the expression of pJNK and Puc subsides at stages 6-7; results that both suggest the downregulation of JNK activity at the mitotic-to-endocycle transition. One possibility is that Notch activity downregulates the JNK pathway. However, at least Su(H)-dependent Notch activity does not regulate the JNK pathway, since no effect on puckered expression was observed in Su(H) mutant clones. It is plausible that Su(H)-independent Notch activity regulates the JNK pathway in this context, as has been shown to be the case in dorsal closure. Interestingly, Deltex might play a role in this Su(H)-independent Notch activity (Jordan, 2006).
An important question in analyzing the developmental control of cell cycle is whether the same signaling pathways control both differentiation and cell cycle, and if so, how the labor is divided. The Notch-dependent mitotic-to-endocycle transition is an example of such a question; Notch action in stage 6 follicle cells is critical for the cell cycle switch and for at least some aspects of differentiation. This work reports the first component that separates Notch dependent cell cycle regulation from Fas3 marked differentiation; Ttk. In the ttk mutant clones, upregulation of FAS3, characteristic for Notch clones, is not observed. Therefore, Ttk constitutes a branch of Notch activity that might be solely required for cell cycle control in this context. However, Ttk's independent function cannot yet be rule out. In the future, it will be important to understand whether signaling pathways in general show a clear separation of differentiation and cell cycle control on the level of downstream transcription factors. Importantly, these and previous results have revealed the essential cell cycle regulators and their roles in controlling the Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).
Planar cell polarity (PCP) is a common feature in many epithelia, reflected in cellular organization within the plane of an epithelium. In the Drosophila eye, Frizzled (Fz)/PCP signaling induces cell-fate specification of the R3/R4 photoreceptors through regulation of Notch activation in R4. Except for Dl upregulation in R3, the mechanism of how Fz/PCP signaling regulates Notch in this context is not understood. The E3-ubiquitin ligase Neuralized (Neur), required for Dl-N signaling, is asymmetrically expressed within the R3/R4 pair. It is required in R3, where it is also upregulated in a Fz/PCP-dependent manner. As is the case for Dl, N activity in R4 further represses neur expression, thus, reinforcing the asymmetry. Neur asymmetry is show to be instructive in correct R3/R4 specification. These data indicate that Fz/PCP-dependent Neur expression in R3 ensures the proper directionality of Dl-N signaling during R3/R4 specification (del Alamo, 2006).
PCP establishment in the eye depends on the specification of photoreceptors R3 and R4 in two steps. First, Fz signaling occurs at higher levels in R3, and second, as a consequence, Dl signaling is directed from R3 to the R4 precursor, where N specifies R4 fate. This study shows that neur is required for proper Dl-N signaling directionality in the R3/R4 pair. In the absence of neur, defects occur in R3/R4 cell-fate specification and PCP. Importantly, neur expression is upregulated in R3 in a Fz/PCP-dependent manner. Finally, this study shows that the asymmetry in neur expression is required for PCP specification (del Alamo, 2006).
Neur is an E3-ubiquitin ligase known to enhance Dl signaling in a variety of Dl-N mediated processes, including lateral inhibition or lateral specification events (e.g., pIIb to pIIa specification in sensory organ development). This study shows that neur is required for lateral specification in R3 for Dl to signal to R4. Analysis of R3/R4 Dl mosaics revealed that the Dl mutant cell always acquires R4 fate, while the wt cell acquires R3 fate. This is consistent with neur analysis showing that in 94.2% of the cases, ommatidia mutant only in R3 showed a PCP defect, indicating that neur is required only in R3, the signal-sending cell (del Alamo, 2006).
There is, nevertheless, a difference between the PCP phenotypes of Dl and neur mosaic ommatidia: Dl mosaics show reversed polarity (chirality flips) when R3 is mutant, while the equivalent neurIF65 mosaics show mostly a symmetric phenotype (89.5% of ommatidia displaying chirality defects). It is likely that the cold-sensitive neurIF65 allele is not null and it is not clear if remaining Dl activity is present in the absence of Neur, accounting for the difference (del Alamo, 2006).
Mib1, another E3-ubiquitin ligase-regulating signaling by Dl and Serrate (Ser, the other N ligand in flies), has no effect on PCP specification. These results are in agreement with data showing that Neur and Mib1 have complementary functions. Taken together, the data indicate that neur but not mib1 is required for R3/R4 specification (del Alamo, 2006).
Previous studies suggested that neur has a permissive role in Dl-N signaling. In lateral inhibition processes, neur is expressed in proneural clusters, whereas in asymmetric cell division, Neur is selectively inherited by one of the daughter cells. In either case, Neur makes the cell in which it is expressed competent for Dl signaling. In the eye, Dl is enriched in R3 as a result of Fz signaling, and this study provides evidence that Neur is enriched in R3 and that this enrichment is also regulated by Fz/PCP signaling. While neur is initially expressed in both cells, the data indicate that Fz/PCP-dependent R3 upregulation of neur is necessary and sufficient for Dl signaling directionality. Since Neur affects Dl activity posttranslationally, Dl is still upregulated in R3 when Neur is misexpressed. This implies that the elimination of the difference in Neur levels between the R3/R4 precursors affects the direction of Dl-N signaling. These data indicate that the Neur expression asymmetry, mediated by Fz/PCP signaling, is instructive for R3/R4 specification (del Alamo, 2006).
The phenotypes resulting from Neur misexpression are relatively mild. Only when both Dl and Neur are coexpressed, chirality defects are induced, suggesting that differential expression of both factors in the R3/R4 cell pair is instructive for cell fate. Furthermore, other factors could also be present in R3/R4 precursors to ensure robustness of the cell-fate decision. These observations suggest a complex network of molecular interactions between Fz/PCP and Notch signaling (del Alamo, 2006).
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