Hedgehog and neural development
In the ventral nerve cord of Drosophila most axons are organized in a simple, ladder-like pattern. Two
segmental commissures connect the hemisegments along the mediolateral axis and two longitudinal connectives
connect individual neuromeres along the anterior-posterior axis. Cells located at the midline of the
developing CNS first guide commissural growth cones toward and across the midline.
four sequential steps involved in commissure development. Initially, single minded, jaywalker, Egf receptor and slit are involved in the first step in midline formation: the formation of the anlage of the CNS midline. Next the segment polarity genes hedgehog, engrailed, patched and wingless are involved in the specification of midline cell number. It is possible that midline and ectodermal pattern formations occur at the same time. In addition to the segment polarity genes other signaling mechanisms appear important. Notch, for example, is required to specify the different midline lineages.
The third step in commissure formation consists of the formation of commissures. Once the midline cells have been specified, they guide commissural growth cones toward and across the midline. Here, the Netrins, frazzled, commissureless, weniger, schizo, roundabout and karussel play an essential role. The fourth step in commissure development involves the separation of the commissures (Hummel, 1999).
The Hedgehog gene product, secreted from engrailed-expressing neuroectoderm, is required for the formation of post-S1 neuroblasts in rows 2, 5 and 6. The Hedgehog protein functions not only as a paracrine but also as an autocrine factor and its transient action on the neuroectoderm 1-2 hours (at 18¡C) prior to neuroblast delamination is necessary and sufficient to form normal In contrast to epidermal development, hedgehog expression required for neuroblast formation is regulated by neither engrailed nor wingless. hedgehog and wingless, at virtually the same time, bestow composite positional cues on the neuroectodermal regions for S2-S4 neuroblasts and, consequently, post-S1 neuroblasts in different rows can acquire different positional values along the anterior-posterior axis. The average number of proneural cells for each of three eagle-positive S4-S5 neuroblasts (Eagle is a member of the nuclear receptor superfamily and is expressed in late neuroblasts) was found to be 5-9, the same for S1 NBs. As with wingless, huckebein expression in putative proneural regions for certain post-S1 neuroblasts is under the control of hedgehog. hedgehog and wingless are involved in separate, parallel pathways and loss of either is compensated for by the other in NB 7-3 formation. NBs 6-4 and 7-3, arising from the engrailed domain, were also found to be specified by the differential expression of two homeobox genes, gooseberry-distal and engrailed. Loss of eagle, gsb-d, and en, may not be involved in proneural fate acquisition, since, unlike hh and wg mutants, no appreciable gap regions are found in the neuroblast layer in these mutants. These latter genes, however, are involved in determination of neuroblast identity (Matsuzaki, 1996).
In vertebrates (deuterostomes), brain patterning depends on signals from adjacent tissues. For example,
holoprosencephaly, the most common brain anomaly in humans, results from defects in signaling between the
embryonic prechordal plate (consisting of the dorsal foregut endoderm and mesoderm) and the brain. Whether a similar mechanism of brain development occurs in Drosophila (a protostome) has been examined -- the foregut and
mesoderm have been found to act to pattern the fly embryonic brain. When the foregut and mesoderm of Drosophila are ablated, brain
patterning is disrupted. The loss of Hedgehog expressed in the foregut appears to mediate this effect, as it does in vertebrates. One mechanism
whereby these defects occur is a disruption of normal apoptosis in the brain. These data argue that the last common ancestor of protostomes and
deuterostomes had a prototype of the brain present in modern animals, and also suggest that the foregut and mesoderm contributed to the patterning
of this 'proto-brain'. They also argue that the foreguts of protostomes and deuterostomes, which have traditionally been assigned to different germ
layers, are actually homologous (Page, 2002).
As the Drosophila foregut invaginates, it normally becomes ensheathed by visceral mesoderm. Thus, when the foregut is ablated, visceral mesoderm is displaced from its normal position adjacent to the brain. How much does the loss of mesoderm contribute to the brain phenotype seen in foregut ablated animals? Embryos lacking function of the NK-2 class transcription factor Tinman have defects in forming mesoderm around the foregut, as revealed using mesodermal
markers as Fasciclin III expression, but do form foregut ectoderm. In 65% of
Tinman loss-of-function embryos there were excess cells at the dorsal midline of b1; the area occupied by neuronal nuclei was increased when compared
with wild type in this region of the brain, and the preoral brain commissure was abnormally thin (Page, 2002).
Why are there excess cells at the dorsal midline in foregut- and mesoderm-ablated embryos? During normal brain development, more neurons are born than will be present in the adult brain and apoptosis eliminates the excess cells. Defects in apoptosis could contribute to the
observed defects in brain patterning by failing to remove excess cells. To see if apoptosis was perturbed when the foregut and mesoderm were ablated, Acridine
Orange staining, which labels apoptotic cells, was carried out. In forkhead loss-of-function embryos, the pattern of apoptosis in the brain at
the level of the preoral brain commissure was clearly different from wild type at late ES13. In the wild-type b1 neuromere, there were groups of apoptotic cells at the dorsomedial edges of the hemispheres. This correlates with previous observations regarding the expression of the apoptosis regulatory protein Reaper. In forkhead loss-of function embryos, which are defective in foregut development, there was a clear reduction in the number of these cells. Examination of tinman
loss-of-function embryos, which are defective in mesodermal development, showed that removal of mesoderm results in a similar reduction in the number of apoptotic cells at the dorsal midline, thus
suggesting that the mesoderm and possibly the foregut have an influence on the normal pattern of apoptosis in brain development (Page, 2002).
The results of foregut and mesoderm ablation experiments strongly suggest that the brain is patterned by induction from these tissues. Did ablation of these tissues
remove inductive signals required for normal brain development? What molecular signals could be mediating this effect? In vertebrates, Hedgehog signaling originating
from the prechordal plate functions in forebrain patterning. Thus, the Hedgehog pathway in Drosophila seemed a good place to begin to look for inductive signals
involved in brain development. Null mutations in Drosophila Hedgehog result in a phenotype in 70% of embryos that strongly resembles the one seen in the foregut ablation
experiments. In brain segment 1 (b1), the right and left hemispheres of the brain are joined at the midline or separated by an abnormally small space
because of excess cells in this region, and the preoral brain commissure shows abnormal defasciculation. In addition, in b1 the frontal commissure is missing, and there is a significant decrease in the area occupied by neuronal nuclei and the number of glia. In b2-S3 (S3 is ventrolateral to the foregut), the longitudinal connectives are
disrupted, and the area occupied by neuronal nuclei and the number of glia is significantly reduced, and the number of Fasciclin II-expressing
neurons is reduced (Page, 2002).
Mutations in the Drosophila terribly reduced optic lobes (trol) gene cause cell cycle arrest of neuroblasts in the larval brain. trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF (Branchless) and Hedgehog (Hh) signaling. Addition of human FGF-2 to trol mutant brains
in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in
wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are
consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through
both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system (Park, 2003).
Hedgehog has been shown to affect stem cell division of
somatic stem cells in the Drosophila ovaries.
In addition, blocking Sonic Hedgehog activity decreases the
number of neural crest stem cells in the chick, and Sonic hedgehog activity is required
for proliferation of precursor cells in avian somites
and murine spinal cord. Loss of Hh using
a temperature-sensitive mutation results in fewer S-phase
neuroblasts at the time these cells begin to proliferate, while
production of Hh with an inducible hh gene almost doubles
the number of BrdU-labeled neuroblasts. While it
is possible that high levels of Hh actually cause apoptosis,
which is then repaired by increased neuroblast division, this is found to be unlikely given that induction of the hs-hh transgene rapidly produces excess S-phase neuroblasts. These results indicate that Hh is required to activate stem cell division in quiescent larval neuroblasts and that the level of Hh signaling may limit the number of neuroblasts capable of dividing (Park, 2003).
Genetic interaction studies have demonstrated that the weak
lethality of trolb22 is enhanced by mutations in hh or ptc, but
not wg. Thus, while trol is required for more
than one signaling pathway, there is specificity of trol function.
Two independent mutations in hh dominantly enhance
the trol proliferation phenotype. Analysis of
a temperature-sensitive hh allele shows that, like FGF signaling,
Hh signaling is required in first instar for normal
activation of neuroblast division. Biochemical studies
indicate a potential interaction between Perlecan and
Hedgehog proteins in both flies and mammals that is not
competed by added heparin. This contrasts with
FGF-mPerlecan and FGF-Trol interactions that are competed
with heparin (Park, 2003).
Thus, mammalian and Drosophila Perlecan may
form complexes with Hedgehog proteins. Future studies
will investigate the direct association between Perlecans and
Hedgehogs. Genetic analyses and an initial biochemical
study suggest that Trol modulates both Hh and FGF
signaling, perhaps through formation of a signaling complex (Park, 2003).
Some hh21/+ animals (with normal copies of trol and tout-velu)
show extra BrdU-labeled neuroblasts, indicating that the
hh21 mutation results in increased Hh signaling.
The molecular basis for the hh21 mutation is likely an
altered splicing event resulting in an insertion in the portion
of the hh mRNA that encodes the signaling activity. Such a
mutation could affect the ability of the mutant Hh protein to
be processed and released from the cell surface. When
placed in a trolb22 background, hh21/+ brains have almost twice as many BrdU-labeled neuroblasts as controls,
indicating that a mutation in trol increases the hyperactive
phenotype of the hh21 mutation (Park, 2003).
Surprisingly, analysis of trolb22; ttv00681+ brains yielded supernumerary labeled neuroblasts similar to
those observed in hs-hh samples. Analysis of expression of
the Hh target gene ptc in trolb22; ttv00681/+ brains has revealed
a higher level of ptc expression in the portions of the brain
expressing hh, when compared with controls. This
increase in ptc expression near the source of Hh suggests
that the combination of a mutation in trol and decrease of
Ttv activity results in higher levels of Hh signaling near
hh-expressing cells. A possible model to explain this observation
is that when the function of trol is compromised,
movement of Hh away from the source of Hh production
competes with binding of Hh to nearby responding cells,
resulting in stronger signaling nearby and suggesting that trol may function in the diffusion and/or reception of signaling by Hh (Park, 2003).
Taken together with previous studies, these results show
that both Drosophila and mPerlecans participate both as
structural components of basement membranes and as functional
components of the FGF-2 signaling pathway associated
with cellular proliferation. In addition, evidence is provided that Perlecans are also involved in signaling by Hedgehog proteins, suggesting an alternative
interpretation of perlecan knock-out mouse phenotypes previously
attributed to structural defects in extracellular matrix. In mPerlecan null mice, chondrocyte
division is altered, leading to disorganization of the
proliferative zone similar to that seen in the proliferation
zones of the optic lobe in third instar trol mutants. Altered patterns of chondrocyte proliferation could be due to changes in FGF and
Indian hedgehog signaling, correlating with altered neuroblast proliferation
in trol mutants with diminished FGF or Hh signaling (Park, 2003).
The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, a very highly specific marker was used that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. The data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2-A4. Moreover, it was shown that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5-A7, although they undergo apoptosis before expressing Capa. In addition, a targeted genetic screen was performed to identify players involved in ABCA specification. It was found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, it was shown that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third s segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification (Gabilondo, 2011).
The findings strongly suggest that the Capaergic abdominal ABCA neuron arises from NB 5-3. This conclusion is based on the expression in ABCA cells of gsb, wg and unpg, and the absence of the markers lbe(K) and hkb. However, even though gsb expression is known to be maintained specifically in the lineage of rows 5 and 6 NBs, whether expression of the other genetic markers used to identify NBs at stage 11 changes late in embryogenesis remains unanswered. Nonetheless, the specific combination of NB markers found in ABCA cells and their position in the hemineuromere are consistent with their birth from NB 5-3. Previous accounts showed that this NB gives rise to a lineage of 9–15 cells. Additionally, observations derived from studies in which PCD was blocked showed that NB 5-3 can potentially produce a large lineage (ranging from 19 to 27 cells), suggesting that it could generate 13 or 14 GMCs. The lack of a NB 5-3 specific-lineage marker prevented resolution of its complete lineage, and thus determining the birth order of the ABCA cell (Gabilondo, 2011).
Recent findings on the NB 5-6 and NB 5-5 demonstrate that cas and grh act together as critical temporal genes to specify peptidergic cell fates at the end of these lineages. cas mutants lack ABCA cells and Cas is expressed in these cells, while the normal pattern of ABCA cells is found in grh mutants, and Grh is not present in ABCA neurons. These data strongly support the birth of ABCA cells in a cas-only temporal window. This is different from the s Capaergic SECA cells, which while also arising from NB 5-3, show a reduction in cell number in both pdm and cas mutants, demonstrating birth at a mixed pdm/cas temporal window. Previous studies in other lineages have shown that when a temporal gene is mis-expressed, all progeny cells posterior to that temporal window can be transformed to the specific fate born at that particular temporal window. However, cas mis-expression failed at inducing ectopic ABCA cells, suggesting that cas in necessary but not sufficient to specify the ABCA fate (Gabilondo, 2011).
Programmed cell death (PCD) is a basic process in normal development. The results suggest that the ABCA and its sibling are equivalent cells committed to achieve the ABCA fate. First, it was shown that the ABCA-sibling cell dies by apoptosis, but produces an ABCA-like Capaergic neuron if PCD is inhibited. Second, when PCD is blocked, NB 5-3 also produces a GMC generating two ABCA-like Capaergic cells in the A5–A7 segments. These data indicate that a segment-specific mechanism prevents death of the ABCA cells in A2–A4 neuromeres. Segment specific cell death has been previously reported for the NB 5-3 lineage, and detailed studies on segment-specific apoptosis of other lineages have shown that this process is under homeotic control. In addition, the results show a different timing in the PCD undergone by the ABCA sibling and the ABCA cells born in A5–A7. This interpretation is based on the differential effect of p35 expression when cas-Gal4 or elav-Gal4 drivers were used. Although elav-Gal4 is transiently express in NBs and GMCs, robust and maintained driver expression commences in differentiating neurons. In contrast, cas expression starts in the NB and is maintained in the GMC and neuronal progeny. Therefore, the finding that death of the ABCA-sibling cell can be prevented by directing p35 with cas-Gal4, but not with elav-Gal4, suggests that the death of the ABCA sibling occurs earlier in development than the death undergone by the ABCA cells in A5–A7 segments (Gabilondo, 2011).
In the ABLK/LK peptidergic fate (derived from the NB 5-5), activation of Notch (N) signaling in the peptidergic cell prevents its death, while its sibling, NOFF cell undergoes apoptosis. On the contrary, in the EW3/Crz peptidergic fate (derived from the NB 7-3), silencing of N signaling is essential for the neuron survival, and therefore for it proper specification. The current results are in accordance with the last scenario, in which the ABCA cell is NOFF, and it sibling, which undergoes apoptosis, is NON. Therefore, Notch signaling must be switch off for the proper specification of the ABCA neuron (Gabilondo, 2011).
To search for genes involved in specification of the ABCA neural fate, a reduced set of mutants was screened of genes that are expressed in the embryonic CNS at stage 11, a time at which distinctly defined sublineages are being generated from all active NBs. Even though this method will certainly overlook important genes, the results reveal that it is in fact a very effective way to find genes involved in specification of a particular neural fate. Indeed, the ratio of success has been very satisfactory: 33.3% of the genes analyzed display a significant phenotype. Moreover, the set of identified genes could be further expanded by, for example, searching in interactome databases, and performing the subsequent screen on those putative interactors (Gabilondo, 2011).
It is assumed that the specification of a concrete cellular fate requires the combination of several transcription factors, namely a genetic combinatorial code. Recently, a detailed combinatorial code has been reported for three neuropeptidergic fates: ap4/FMRFa, ap1/Nplp1 and ABLK/Lk. However, very little is known about the specification of the rest of the 30 peptidergical fates. This study has identified several genes involved in the specification of the ABCA fate, which fit into three categories. First, genes were found whose loss-of-function produces a relevant increase of the number of ABCA cells. Most remarkable are the klu and rn phenotypes, which consist of duplications of the ABCA cells. These phenotypes suggest that these two transcription factors repress the ABCA fate in other neural cells (or/and NBs/GMCs). Interestingly, the normal phenotype of nab mutants indicates that, contrary to its mode of action in the wing, Rn does not work with the transcription cofactor Nab in this context (Gabilondo, 2011).
Second, genes were found whose loss-of-function produces a significant decrease of the number of ABCA neurons. In this category, the zhf2, ftz and grain phenotypes stand out. The effects of mutations on ftz are in agreement with its early role in segmentation: ftz is a pair-rule segmentation gene that defines even-numbered parasegments in the early embryo, and absence of ABCA cells was found in the A3 segment in ftz mutants. However, zfh2 and grain seem to be part of the specific combinatorial code of the ABCA cells. The Drosophila GATA transcription factor Grain has been reported to be involved in the specification of other cell fates, such as the aCC motoneuron fate. Based on its expression, the zinc finger homeodomain protein zfh2 has been proposed to mediate specification of the serotoninergic fate, but this has not been further demonstrated. Interestingly, during wing formation, zfh2 is required for establishing proximo-distal domains in the wing disc, and it does so partly by repressing gene activation by Rn. The opposite phenotypes that was observed in rn and zfh2 mutants suggest that similar interactions occur during ABCA specification. Analyses aimed to test this hypothesis are currently being performed (Gabilondo, 2011).
Third, two genes were found whose loss-of-function abolishes the ABCA fate: Grunge and hh. Grunge encodes a member of the Atrophin family of transcriptional co-repressors that plays multiple roles during Drosophila development. Taken together, studies from C. elegans to mammals suggest that Atrophin proteins function as transcriptional co-repressors that shuttle between nucleus and cytoplasm to transduce extracellular signals, and that they are part of a complex gene regulatory network that governs cell fate in various developmental contexts. Similarly, Hh is an extracellular signaling molecule essential for the proper patterning and development of tissues in metazoan organisms. It is noteworthy that two genes implicated in extracellular signaling pathways, Grunge and hh, are absolutely required for ABCA fate. Further studies will be needed to identify at which step/s they exert their actions, and to unravel possible interactions between them and with other players of the combinatorial code for ABCA specification (Gabilondo, 2011).
Hedgehog and glial cell migration
In the Drosophila visual system, photoreceptor neurons (R cells) extend axons towards glial cells located at the posterior edge of the eye disc. In gilgamesh (gish) mutants, glial cells invade anterior regions of the eye disc prior to R cell differentiation and R cell axons extend anteriorly along these cells. gish encodes casein kinase Igamma. gish, sine oculis, eyeless, and hedgehog (hh) act in the posterior region of the eye disc to prevent precocious glial cell migration. Targeted expression of Hh in this region rescues the gish phenotype, though the glial cells do not require the canonical Hh signaling pathway to respond. It is proposed that the
spatiotemporal control of glial cell migration plays a critical role in determining the directionality of R cell axon outgrowth (Hummel, 2002).
In the developing Drosophila visual system, photoreceptor cell (R cell) axons contact numerous glial cell types along the pathway to the target and within the target itself. These glial cells originate from
different regions of the developing visual system and migrate to their final destinations where they associate with axons. The first glial cells encountered by extending R cell axons are retinal basal glial (RBG) cells located along the
basal surface of the eye disc epithelium. As R cells differentiate, they extend
growth cones basally where they contact RBG cells and turn posteriorly toward the optic stalk. These glial cells originate in the optic stalk and migrate into the eye disc. Migration is temporally
and spatially linked to R cell development. The extension of axons from R cell clusters occurs in a sequential
fashion that reflects the highly ordered pattern of R cell differentiation in the eye disc. R cells in the posterior region differentiate first and additional R cell clusters form more anteriorly as a wave of differentiation sweeps across the eye disc. The leading edge of this wave is marked by a depression in the apical region of the disc epithelium called the morphogenetic furrow (MF) (Hummel, 2002).
Glial cells start to migrate into the eye disc as R cell differentiation at the posterior margin is initiated. During MF progression, RBG cells migrate along the basal surface up to but not past the youngest R cell axons. RBGs appear to migrate along R cell axons thereby making a guidance role for them unlikely. Glial cells can migrate out of the optic stalk into the eye disc in the absence of axon contact; instead, glial cells are essential for R cell axons to enter the optic stalk. These observations suggest that the precise coordination of glial cell development in the optic stalk and R cell differentiation in the eye disc is important for normal visual system development. In the course of a genetic screen for axon guidance mutants, a loss-of-function mutation was identified that
temporally uncoupled glial cell migration from R cell differentiation. Mutations in the gilgamesh locus lead to precocious glial cell migration from the optic stalk into the eye disc prior to R cell differentiation. As a result, glial cells are misplaced anteriorly in the eye disc as R cell axons extend. R cell
axons frequently project along pathways demarcated by these ectopic glial cells, providing strong evidence that the positioning of glial cells plays an instructive role in regulating the directionality of R cell axon outgrowth in the
eye disc. The gish locus acts in conjunction with the eye
specification genes, eyeless and sine oculis, to prevent precocious entry of glial cells into the eye disc. Loss-of-function hh mutants exhibit precocious glial cell migration and targeted
expression of Hh in the posterior region of the eye disc suppresses the ectopic glial cell migration phenotype in gish mutants (Hummel, 2002).
A set of genes encoding nuclear proteins [e.g., eyeless (ey), eyes absent (eya), sine oculis (so) and secreted factors such as hedgehog (hh)] regulates the initiation of neuronal differentiation in the posterior region of the eye disc. The effect of loss-of-function mutations in these genes on glial cell migration was tested. As in gish mutants, glial cells migrate precociously out of the optic stalk in a hh temperature-sensitive mutation incubated at the nonpermissive temperature during first and second instar. This is an early function of hh, since ectopic glial cells are not observed in hh1; in this allele, the posterior eye field develops normally, but anterior progression of the MF is inhibited. A similar early onset glial cell migration defect is observed in eye-specific alleles of so and ey. In contrast, glial cells did not migrate out from the optic stalk in an eye-specific allele of eya, raising the possibility that eya is required to activate glial cell migration. Since glial cells migrate out of the stalk precociously in eya/gish double mutants, the production of an eya-dependent signal is not necessary to promote anterior migration. Hence, in contrast to their role in R cell development, eye specification genes ey and so seem to function independent of eya to control the onset of glial cell migration (Hummel, 2002).
These observations raise the possibility that gish also contributes to the genetic circuitry regulating eye specification. Indeed, while ey-FLP-induced clones of gish lead to only minor defects in MF progression during third instar stage, gish mutant adult eyes are smaller and frequently contain a reduced number of ommatidia in the anterior region. These phenotypes are frequently seen in weak alleles of eye specification genes. Furthermore, double mutants of ey1 and gish1 , as well as so1 and gish1, reveal strong synergistic effects in R cell development. The glial cell migration phenotypes in double mutants is similar in severity to the single mutants. In summary, these data argue that gish acts in conjunction with eye specification genes to coordinate neuronal development and glial cell migration in the eye disc (Hummel, 2002).
Since Hh is expressed in the right place and time to function as the glial cell repellent, and since glial cells migrate precociously in hh mutants, it became important to assess whether Hh directly regulates glial cell migration. In support of this view, targeted expression of Hh at the posterior region of the second instar eye disc rescues precocious cell migration in gish mutants. In about 80% of the gish mutant larvae carrying Dpp-Gal4 driving UAS-hh, premature glial cell migration was prevented in second instar, and glial cells remain posterior to the MF at third instar (Hummel, 2002).
Does Hh directly regulate glial cell migration? To address this question, whether the canonical Hh signaling pathway is activated in glial cells was assessed and whether it is required to prevent precocious migration. patched (ptc) expression, an indicator of reception of the Hh signal, is not elevated in glial cells prior to migration into the eye disc. It is, however, induced in epithelial cells located at the most posterior edge of the eye disc immediately juxtaposed to the pre-migratory glial cells in the optic stalk. In support of the importance of the posterior margin in signaling glial cells, a significant reduction in the level of ptc-lacZ expression was observed in this region of the second instar eye in gish mutants. Alternatively, this reduction may simply provide a sensitive indicator that the level of Hh is reduced in gish mutants. Since the level of Hh protein is only slightly above background in wild-type, the level of the Hh signal in these mutants could not be critically assess with the available reagents. Further evidence that the canonical Hh pathway is not required in glial cells came from the analysis of mutations in smoothened (smo) and Cubitis interruptus (Ci) genes encoding a Hh receptor component and a downstream transcription factor, respectively. Mosaic clones of smo mutant glial cells do not migrate prematurely at second instar and were found exclusively posterior to the MF. Similarly, targeted expression of a dominant-negative version of Ci in glial cells does not alter early migratory behavior of eye disc glial cells. These findings argue that Hh acts either indirectly to control glial cell migration or acts directly upon glial cells through a novel pathway independent of ptc, smo, and Ci (Hummel, 2002).
Thus, the timing of glial cell migration plays an essential role in regulating axon guidance in the fly visual system. Glial cells in the posterior region of the eye disc epithelium provide an intermediate target for R cell axons as they project from the eye to the brain. In gish mutants, glial cells migrate out of the optic stalk to more anterior regions of the eye disc prior to R cell differentiation, and as a consequence, R cell axons frequently extend anteriorly in gish mutants along the surface of these ectopically located glial cells. gish acts in combination with eye specification genes, ey and so, and the extracellular signaling protein Hh to control glial cell migration. Since these genes, including gish, also regulate neuronal development in the eye disc, it is proposed that they define a signaling center in the posterior region of the eye disc that controls both neurogenesis and glial cell migration to ensure normal patterns of R cell axon outgrowth (Hummel, 2002).
Through genetic mosaic analysis and transgene rescue experiments, it has been established that gish acts within the eye disc epithelium to inhibit glial cell migration. In principle, gish could regulate the production of a repellent preventing migration of glial cells out of the optic stalk, an attractant that promotes their close association to the posterior edge or alternatively an antagonist to an attractive signal produced by cells in the disc. Gish belongs to the casein kinase I family of highly conserved and widely expressed enzymes. These enzymes contain small but varied amino termini and large, highly diverse carboxy-terminal domains. Gish is most similar to mammalian CKIgamma3. CKIgamma3, like gish, is alternatively spliced -- this can result in kinases with different biochemical properties and functions. CKIs act on proteins previously phosphorylated by other kinases. CKIs have been shown to phosphorylate a large number of proteins in vitro. Regrettably, little evidence exists to establish a link between phosphorylation by CKI and specific developmental pathways. Recent studies have shown that casein kinase Iepsilon (CKIepsilon) can regulate ß-catenin in the Wnt pathway in both worms and frogs. Loss- and gain-of-function manipulation of Wg signaling components, however, did not disrupt glial cell migration into the eye disc. This is not surprising given that CKIepsilon and CKIgamma differ significantly in their C-terminal regions and deletion analysis has revealed that the unique C-terminal domain is important for the interaction of CKIepsilon with the axin signaling complex. Since gish does not encode a secreted molecule, it must act indirectly to affect signaling from the epithelium to the glial cells (Hummel, 2002).
Several lines of evidence support a model in which gish regulates Hh signaling: (1) like gish, hh is required in the posterior region of the second instar eye disc to inhibit anterior migration of glial cells; (2) gish mutants display defects in morphogenetic furrow progression similar to those seen for hypomorphic alleles of hh, and (3) the expression of Hh target genes ptc and Ci in the eye disc epithelium is reduced in gish mutants. These data suggest that gish acts upstream of (or in parallel to) hh. Although genetic evidence suggests that the gish mutants studied here are strong loss-of-function alleles, since low levels of gish mRNA can still be detected in homozygous gish1 larvae, the epistatic relationship between gish and hh must be qualified. Indeed, Gish may function downstream from Hh and limiting levels of activity in loss-of-function alleles may be compensated by increasing the level of Hh upstream (Hummel, 2002).
The location of a specific subtype of glial cells is not only necessary for posterior-directed outgrowth of R cell axons, but also sufficient. Indeed, some 50% of gish mutant eye discs at an early stage of development contain R cell fibers that project anteriorly along the surface of misplaced glial cells. During normal development, however, more anterior R cells differentiate as a continuous wave, and glial cells migrate to a region just posterior to differentiating R cells. As a consequence, as these axons extend to the basal surface, they contact glial cells that lie posterior to them and follow them into the optic stalk. Axon-glial cell interactions in the fly eye show interesting parallels to intra-retinal pathfinding in the vertebrate eye. Retinal ganglion cell axons project radially toward the optic disk, their first intermediate target in the center of the eye. Here, axons receive contact-dependent signals from glial cells to exit the retina and enter the optic nerve. In addition, in vertebrates, a barrier at the junction between the optic nerve and the retina has been proposed to inhibit glial cell precursors from migrating into the retina. It will be interesting to assess whether the molecular strategies coordinating axon outgrowth and glial cell migration have been conserved between vertebrate and invertebrate visual systems (Hummel, 2002 and references therein).
Development of complex organs depends on intensive cell-cell interactions, which help coordinate movements of many cell types. In a genetic screen aimed to identify genes controlling midline glia migration in the Drosophila nervous system, mutations in the gene kästchen were identified. During embryogenesis kästchen is also required for the normal migration of longitudinal and peripheral glial cells. During larval development, kästchen non-cell autonomously affects the migration of the subretinal glia into the eye disc. During embryonic development, kästchen not only affects glial cell migration but also controls the migration of muscle cells toward their attachment sites. In all cases, kästchen apparently functions in terminating or restricting cell migration. The molecular nature of the gene was idenfied by performing transgenic rescue experiments and by sequence analysis of mutant alleles. Kästchen corresponds to fear-of-intimacy (foi) that was identified in a screen for genes affecting germ cell migration, suggesting that Foi-Kästchen is more generally involved in regulating cell migration. It encodes a member of an eight-transmembrane domain protein family of putative Zinc transporters or proteases. The topology of the Foi protein was determined by using antisera against luminal and intracellular domains of the protein and evidence is provided that it does not act as a Zinc transporter. Genetic evidence suggests that one of the functions of foi may be associated with hedgehog signaling (Pielage, 2004).
During the development of the nervous system, cell migration can be observed for several different classes of glial cells. Within the embryonic CNS, midline glial cells have to migrate along cell processes of the VUM neurons to generate the regular pattern of anterior and posterior commissures found in every neuromere. The presence of the neuronal substrate is crucially important to initiate and guide glial migration. Similarly, glial cells covering the longitudinal connectives that emerge from laterally located glioblasts have to migrate toward the CNS midline where they interact with neuronal growth cones that will eventually pioneer the longitudinal tracts. The Slit-Roundabout ligand-receptor system that controls axonal pathfinding across the CNS midline also regulates some aspects of glial migration (Pielage, 2004).
In the larval PNS, the axons of the developing photoreceptors project through the optic stalk to the brain. Glial cells are born in the optic lobes and migrate through the optic stalk toward photoreceptor cells. Recent analysis has shown that these glial cells are guided by a signal released from developing photoreceptor cells in the eye disc. Part of the signaling mechanism may be conveyed by the signaling molecule Hedgehog and the casein kinase Igamma Gilgamesh that prevents precocious glial cell migration (Hummel, 2002). Glial cell migration also plays an important role within the developing optic lobes. However, the molecules guiding this migration have not yet been identified (Pielage, 2004).
The induction of large foi mutant eye clones results in a prominent adult compound eye phenotype. Anterior eye structures are affected and those remaining have a rough appearance. In addition, the formation of the dorsal head case is severely affected. The formation of the bristles is impaired and the ocelli are often missing. The eye phenotype is reminiscent of some aspects of a hedgehog mutant phenotype. Interestingly, hedgehog has been shown to regulate glial migration in the developing eye disc. Phenotypic analyses have suggested that foi and hedgehog might be interacting during eye development. Homozygous hedgehogbar3 (hh1) flies are viable and show an eye phenotype reminiscent of mutant foi eyes. The anterior portion of the eye is missing due to a premature stop of morphogenetic furrow progression. To analyze a possible genetic interaction between hedgehog and foi, the effects of a 50% reduction of foi was examined in a hedgehog mutant background. hh1/hh1 mutant eyes develop only 9-10 rows of photoreceptor cells. When one copy of foi was removed in this genetic background, the phenotype was enhanced so that only 6-7 rows of photoreceptor cells form, suggesting that the two genes may interact during eye development (Pielage, 2004).
When foi and hedgehog indeed interact, one may expect further phenotypic similarities. To address this question in more detail, germ line clones were generated using the FRT ovoD technique and the development of the embryonic nervous system was analyzed as well as the pattern of the larval cuticle. It was possible to induce a sufficient number of germline clones only when using the hypomorphic O1-41 mutation; for the strong mutation B1-89, only very few embryos were obtained, indicating some requirements of foi during oogenesis. In both cases, however, paternal rescue was noted. Maternal and zygotic mutant foi embryos displayed a wide range of mutant phenotypes. In about 50% of the cases, a severely disrupted neural development resembling a mutant hedgehog phenotype was noted. In one quarter of the embryos, there was a very strong foi phenotype. In the remaining embryos, no development was observed. To compare foi and hedgehog phenotypes, cuticle preparations were also analyzed. Wild-type larvae show denticle belts in every segment and have a characteristic head skeleton. Mutant hedgehog larvae lack a well-differentiated head skeleton and are characterized by a lawn of denticles. Maternal and zygotic mutant foi embryos display a range of phenotypes. In the most extreme situation, the animals lack the head skeleton and have expanded ventral denticle belts. The other embryos showed a reduced degree of severity, which can be judged by defects in the head skeleton. Thus, during embryogenesis, foi and hedgehog mutants share at least some phenotypic traits supporting the notion that both genes may in part act in a common pathway (Pielage, 2004).
How could foi affect migration? foi mutations were initially isolated due to their fused commissure phenotype, which is indicative for defective midline glial cell migration. Among the collection of mutants identified in this large scale mutagenesis, foi appeared unique, since it did not completely block midline glia migration as do, for example, mutations in pointed. Rather foi is required to control and stop cell migration. This phenotypic trait is also found in other migratory cells. Termination of migration may be a cell autonomous property but is very likely to be regulated by extracellular signals. The genetic data are consistent with a possible interaction of foi with components of the hedgehog signaling cascade. Loss of foi function leads to phenotypes resembling aspects of the hedgehog mutant phenotype. This is particularly evident during head development, where both ommatidial development as well as head capsule formation is affect by foi and hedgehog. Furthermore, gene dose experiments support the interaction of the two genes (Pielage, 2004).
Hedgehog is an evolutionary well-conserved signaling molecule that controls a wide range of cellular interactions. Within the eye disc, Hedgehog not only controls the differentiation of neuronal cells units but also directly regulates the migration of glial cells that invade the eye disc epithelium from the optic stalk. In addition, Hedgehog is required for germ cell migration (Pielage, 2004).
During development of the vertebrate nervous system, Hedgehog controls neuronal migration as well as growth cone navigation. In the peripheral nervous system, ectopic Hedgehog blocks migration of trigeminal precursors whereas loss of hedgehog function leads to a migration of the trigeminal precursor cells to ectopic targets. In vitro experiments show that this might be in part a consequence of changes in cell adhesion. Interestingly, Hedgehog affects migration of neural crest cells independent of the canonical Patched-Smoothened signaling pathway. To assay whether Hedgehog affects glial migration through the canonical pathway, a dominant negative version of the Patched receptor was expressed in developing glial cells but no glial migration phenotype was obtained. Similarly, the loss of components of the canonical Hedgehog pathway in glial cells does not lead to a migration phenotype. Thus, as observed for neural crest cells, Hedgehog may not be acting through Patched to control glial migration in Drosophila (Pielage, 2004).
How could Foi interact with Hedgehog? Foi is predicted to encode a multiple pass transmembrane protein containing eight transmembrane domains. It belongs to the so-called ZIP family (Zrt, Irt-like proteins), a large group of at least 86 members that bind or transport zinc. The ZIP family can be further subdivided into four subgroups. The largest group has recently been termed LZT subfamily (LIV-1 subfamily of ZIP transporters) and currently comprises 36 members (Taylor, 2003). However, all LZT members are also characterized by a so-called HELP motif that defines the active site of matrix metalloproteases (MMP). Because this catalytic zinc-binding motif is predicted to be in the transmembrane domain V, Foi may be a new transmembrane protease (Pielage, 2004).
Intramembrane proteolysis has recently been recognized as an important control mechanism found throughout evolution. Bacteria use it to generate extracellular pheromones or to liberate transcription factors within the cell. Similarly, eukaryotic cells utilize intramembrane proteolysis in diverse cellular processes such as lipid metabolism, response to unfolded proteins, and cell differentiation (Pielage, 2004).
The experiments demonstrate that it is unlikely that Foi is regulating Zn2+ homeostasis. The genetic studies identified the Hedgehog signaling cascade as a possible candidate of Foi function. The analysis of foi mutant eye clones demonstrates that Foi affects eye development in a non-cell autonomous manner. This suggests that Foi is required for the generation of the signaling molecule and not for the perception of the signal. The Hedgehog protein is generated as a 45-kDa precursor that undergoes autocatalytic processing to generate an active 22-kDa N-terminal protein. It has been reported that Zinc ions can influence autocatalytic properties of inteins, which exhibit similar autocatalytic processing as Hedgehog. However, no changes were detected in the zinc ion concentration following Foi overexpression. Alternatively, Foi may regulate the activity of a yet unidentified signaling pathway, since not all mutant phenotypes can easily be explained by a lack of hedgehog signaling. Future experiments will allow a determination of whether Foi activates parts of the Hedgehog signal transduction cascade or other signaling systems by functioning as an intramembrane protease (Pielage, 2004).
The Drosophila CNS contains a variety of glia, including highly specialized glia that reside at the CNS midline and functionally resemble the midline floor plate glia of the vertebrate spinal cord. Both insect and vertebrate midline glia play important roles in ensheathing axons that cross the midline and secreting signals that control a variety of developmental processes. The Drosophila midline glia consist of two spatially and functionally distinct populations. The anterior midline glia (AMG) are ensheathing glia that migrate, surround and send processes into the axon commissures. By contrast, the posterior midline glia (PMG) are non-ensheathing glia. Together, the Notch and hedgehog signaling pathways generate AMG and PMG from midline neural precursors. Notch signaling is required for midline glial formation and for transcription of a core set of midline glial-expressed genes. The Hedgehog morphogen is secreted from ectodermal cells adjacent to the CNS midline and directs a subset of midline glia to become PMG. Two transcription factor genes, runt and engrailed, play important roles in AMG and PMG development. The runt gene is expressed in AMG, represses engrailed and maintains AMG gene expression. The engrailed gene is expressed in PMG, represses runt and maintains PMG gene expression. In addition, engrailed can direct midline glia to a PMG-like non-ensheathing fate. Thus, two signaling pathways and runt-engrailed mutual repression initiate and maintain two distinct populations of midline glia that differ functionally in gene expression, glial migration, axon ensheathment, process extension and patterns of apoptosis (Watson, 2011).
This paper describes how the Hh morphogen patterns the midline cells to generate two populations of MG with distinct functional properties. The key output of this signaling is the expression of en that imparts PMG cell fate, in part, by repressing runt. In turn, the runt gene maintains AMG fate by repressing en. Thus, morphogenetic signaling and transcriptional regulation lead to AMG and PMG with divergent molecular, morphological and functional differences (Watson, 2011).
At stage 10, the 16 midline cells per segment consist of three equivalence groups of neural precursors, four to six cells each. Notch signaling directs ten of these 16 cells to become MG; the remainder become MPs and the MNB. Thus, Notch represses neuronal development in MG and activates a core set of MG-expressed genes (e.g., wrapper). MG in the anterior of the segment become AMG; those in the posterior of the segment become PMG. Notch signaling by itself is unlikely to influence different MG fates, as expression of activated Suppressor of Hairless in midline cells drives all cells into a MG fate but does not affect their AMG or PMG patterns of gene expression. Thus, additional factors that can direct AMG and PMG cell fates were sought (Watson, 2011).
Previous work demonstrated that hh can pattern midline cells along the A/P axis, and, indeed, this study demonstrates that hh is required for PMG cell fate. The source of Hh is not in the midline, but in the lateral ectoderm in a stripe of cells, collinear with the pair of midline early en+ cells. Hh signals to midline cells posterior to the early en+ cells, inducing en in an additional six to seven cells. These late en+ cells plus the early en+ cells become about four PMG, as well as MP4-6 and the MNB. Misexpression and mutant analyses indicate that hh is required for all PMG gene expression and for repressing AMG expression. hh signaling probably has multiple target genes because hh is required for en and l(1)sc expression, but en does not regulate l(1)sc. Misexpression of hh can activate en expression in anterior MG, and both hh and en misexpression convert these cells functionally into non-ensheathing MG that resemble PMG, results also consistent with observations that ectopic expression of hh and en in midline cells affects AMG differentiation. However, neither hh nor en can activate all PMG gene expression in anterior MG, because neither activates masquerade (mas) expression in anterior MG. The mas gene is expressed transiently at stage 12 in a subset of PMG, suggesting that functionally distinct classes of PMG might exist. Expression of mas might require other signals in addition to hh that are absent in anterior MG (Watson, 2011).
runt is present in AMG and represses en and PMG-specific gene expression. In runt mutant cells that are runt- en+, expression of three genes expressed in only AMG (CG33275, Fhos and nemy) are absent and wrapper is reduced. This could be due to runt repression of en, repression of other genes or activation by runt. In runt mutant cells that are runt- en-, Fhos and nemy are present, wrapper is at high levels, but CG33275 expression is absent. This suggests that runt does not activate expression of Fhos, nemy and wrapper in AMG, but maintains their AMG levels by repressing en. By contrast, runt is required for expression of CG33275, possibly indicating a positive role for runt in AMG differentiation in addition to its repressive role in AMG maintenance. However, CG33275 is most prominently expressed in a subset of AMG closest to the commissures, and this AMG expression could be dependent on additional signals, perhaps from the developing axon commissure. Thus, absence of CG33275 expression in runt mutant embryos could alternatively be due to an effect of runt on developing axons or CNS development (Watson, 2011).
As most AMG gene expression is not dependent on runt, it is proposed that Notch signaling initially induces an AMG pattern of gene expression in all glia and, either simultaneously or soon after, Hh signaling in the posterior of the segment generates PMG. One important downstream target of Notch signaling is likely to be the sim gene, which encodes a bHLH-PAS protein that functions as a DNA-binding heterodimer with the Tango (Tgo) bHLH-PAS protein. During early development, sim is expressed in all midline primordia and is required for midline cell development. However, later in development, sim is restricted to MG and a subset of midline neurons. Genetically, sim expression is absent in embryos mutant for Notch signaling. The sim gene is likely to be an important aspect of MG transcription, because mutation of Sim-Tgo binding sites in the slit and wrapper MG enhancers results in loss of MG expression, and Sim-Tgo binding sites are present in other identified MG enhancers. The Hh morphogen transforms only posterior MG into PMG. It is unknown why hh does not affect anterior MG, but it is likely to be owing to the presence of unknown factors in these cells that inhibit
hh signaling. Since Notch signaling, rather than runt, is primarily required for AMG gene expression, the key role of runt is probably to maintain AMG gene expression by repressing en. Similarly, en functions to maintain PMG gene expression by repressing runt, but also contributes positively to PMG cell fate, as en misexpression confers PMG-like function to AMG (Watson, 2011).
The most striking features of AMG are their ability to migrate around the commissures, ensheath them and extend processes into the axons. The function of PMG is unknown, but they are unable to ensheath the commissures, even though they are in close proximity. One of the major factors influencing AMG-axon interactions is Nrx-IV-Wrapper adhesion. Levels of wrapper expression in AMG are higher than in PMG, and this is likely to be a key determinant of why AMG ensheath commissures, and PMG do not, because loss of wrapper expression results in incomplete migration and ensheathment. Recent work has demonstrated that sim directly regulates wrapper expression, and spitz signaling from axons might also form a positive feedback loop to control wrapper levels and strengthen Nrx-IV-Wrapper interactions. As en genetically reduces wrapper levels in PMG, it will be interesting to determine if this regulation is direct or indirect. Although the control of wrapper levels is likely to be a major factor in AMG-PMG differences and the ability of glia to ensheath axons, other genes whose levels differ between AMG and PMG might also contribute. This illustrates why it will be important to identify target genes and understand better the roles that Notch//Suppressor of Hairless, sim, hh, Ci, en, runt and other MG transcription factors play in regulating MG gene expression and function (Watson, 2011).
Hedgehog in wing development
To clarify possible hh functions in adult appendage formation, a mutant was isolated with ectopic expression of hh in the anterior edge of the wing pouch in the wing disc. This abnormal hh expression results in a mirror-image duplication and ectopic outgrowth in the anterior wing compartment, and also the
ectopic expression of patched and decapentaplegic. This evidence strongly suggests that the HH product serves as a morphogen or an inducer essential for wing development, including the proximal/distal axis formation (Kojima, 1994).
A dominant gain-of-function allele of hedgehog causes ectopic expression of HH mRNA in the anterior compartment of wing discs, leading to overgrowth of tissue in the anterior of the wing and partial duplication of distal wing structures. The posterior compartment of the wing remains unaffected. Other imaginal derivatives are affected, resulting in duplications of legs and antennae and malformations of eyes. In mutant imaginal wing discs, expression of the decapentaplegic gene (implicated in the Hedgehog signaling pathway) is also perturbed. Therefore, Hedgehog protein may act in the wing as a signal to instruct neighboring cells to adopt fates appropriate to the region of the wing just anterior to the compartmental boundary (Felsenfeld, 1995).
The secreted protein Hedgehog (Hh) transmits a signal from posterior to anterior cells that is essential for limb development in insects and vertebrates. In Drosophila, Hh has been thought to act primarily to induce localized expression of Decapentaplegic and Wingless, which in turn relay patterning cues at
long range. Hh plays an additional role in patterning the wing. Engrailed is expressed in the posterior compartment and in anterior cells close to the AP boundary. Anterior En levels decrease rapidly with distance from the posterior comparment. Expression of En in anterior cells is thought to be regulated by Hh. Consistent with this, anterior clones of smoothened fail to express En, whereas posterior smo clones express En normally. Likewise, anterior expression of Ptc is regulated by Hh. As Hh acts directly to induce Dpp, to upregulate Ptc in a broad domain of 8-10 cell diameters, and to induce En in a narrow band of cells close to the AP boundary, it is possible that the broad spatial domains of Ptc and Dpp and the narrow domain of En reflect requirements for different levels of Hh activity. Use of a temperature sensitive allele of hh shows that dpp can be activated by levels of Hh activity that are below the minimal levels required to activate En (Strigini, 1997).
By replacing endogenous Hh activity with that of a membrane-tethered form of Hh, it has been shown that Hh acts directly to pattern the central region of the wing, in addition to its role as an inducer of Dpp. Comparing the biological activities of secreted and membrane-tethered Hh provides evidence that Hh forms a local
concentration gradient and functions as a concentration-dependent morphogen in the fly wing. Such tethered Hh can only induce En in immediately adjacent cells. Tethered Hh expressing cells also act on adjacent cells to induce dpp. It is noted that dpp expression is reduced in cells that express En at high levels, suggesting that En represses dpp. Membrane-tethered Hh flies develop to pharate adults that completely lack wings. Restoring Hh during development allows flies to form wings that show substantial rescue of anterior and posterior structures while missing structures from the cental region. These results suggest that Hh plays an important role in directly patterning the central region of the wing, while Dpp is primarily responsible for patterning at long range (Strigini, 1997).
A mutagenesis screen was performed for mutations that dominantly suppress
Hedgehog overexpression phenotypes in the Drosophila wing. An existing dominant hh allele, hhMoonrat, induces overgrowth and repatterning in the wing. The hhMrt
phenotype is mild and thus can be used as a background to isolate suppressors. Four mutations were isolated that influence
Hedgehog signaling. These were analyzed in the amenable wing system using genetic and molecular techniques. One of these four
mutations affects the stability of the Hedgehog expression domain boundary, also known as the organizer, in the developing wing. Another mutation affects a possible
Hedgehog autoregulation mechanism, which stabilizes the same boundary (Haines, 2000).
One mutant, Su(hh) I, suppresses the phenotypes caused by overexpression of hh (or shh), and this mutant suppresses the induction of ectopic target gene expression (dpp) by Hh signaling. It maps to the left arm of the second chromosome at map position 31-33. No mutants that affect Hh signaling have been characterized in this region. This mutant could thus represent a new gene that functions in the Hh pathway. Interestingly, a homolog of the ptc gene can be found here in the annotated genome database (accession no. CG5722). This ptc homolog shows significant homology to the human NPC 1 gene; this gene was isolated in humans as the cause of the Nieman-Pick C1 disease (Haines, 2000).
In contrast, in Su(hh) II discs, the expression domain of the Hh target gene dpp expands. This locus was isolated as the strongest suppressor of Hh overexpression phenotypes; it also suppresses (albeit weakly) ci and dpp overexpression phenotypes. Upon further analysis of the induction of dpp in this mutant background, it was found that the gain of dpp in the discs is due to a loss of ectopically induced En. Ectopic shh induces endogenous En expression in the anterior compartment. In Su(hh) II discs, En expression is lost in the areas where high levels of shh ectopically induced it. En suppresses dpp expression as a transcriptional repressor, thus loss of En will lead to gain of dpp expression, as seen in this mutant. However, in the 30AGAL4 UAS shh wing imaginal discs, in addition to En expression in the cells exposed to high levels of shh, these cells also show endogenous hh expression. It is not known if this ectopic hh is directly induced by ectopic shh or is dependent on the ectopic En. The expression of endogenous hh in these cells does, however, point to a possible change of cell identity of these cells, from anterior (non-hh-expressing cells) to posterior (hh-expressing) cells. The interpretation of the loss of dpp expression in these cells might thus be due to the fact that these cells are now 'true' posterior cells. These results indicate, though, that the Su(hh) II acts in a pathway that leads to concomitant En and hh expression in the anterior compartment induced by Hh signaling. It is thought that the signaling leading to En expression in the anterior compartment proceeds through the normal Hh pathway. Since Su(hh) II also suppresses ectopic ci phenotypes, a position for Su(hh) II downstream of ci function but upstream of the induction of hh and En expression is most likely. It would thus function in a Hh autoregulatory pathway, upstream of hh and En expression but downstream of the Hh signaling pathway. Su(hh) II maps to the right arm of the second chromosome at map position 53-57 and no genes previously characterized as interacting with the Hh signal transduction pathway are known to map to this position (Haines, 2000).
Both Su(hh) III and IV are embryonic lethal but neither shows any distinct abnormal cuticle phenotype. If the lethality of these mutants is associated with the suppression of the dominant hh mutation, and if these mutants are absolutely required for Hh signaling, perhaps embryonic segmentation phenotypes would be expected. The apparent lack of these might indicate a maternal contribution for the gene. This would be consistent with the fact that these loci have not been found in mutagenesis screens for zygotic embryonic phenotypes that resemble the hh phenotype. No germline clones have been generated to remove maternal contribution since the use of radiation as a mutagen might have caused mutations in more than one gene in these mutants. A role in embryonic patterning for these mutants is thus possible but one cannot exclude an exclusive role for these genes in wing/imaginal Hh patterning (Haines, 2000).
Su(hh) III maps to a region where several loci of interest to wing patterning driven by Hh or Dpp signaling have been placed (map position 59-60). This mutant is not allelic to gbb, a dpp homolog in this region that has been shown to cooperate with Dpp signaling in disc patterning. Another gene situated close by is the G-protein alpha-subunit encoding gene (Galphas). Since the Hh signaling pathway contains a putative G-protein-coupled receptor, smoothened, a G-protein might influence Hh signaling. However Su(hh) III does not appear to map to Galphas either, since a combination of a deficiency covering Su(hh) III and Galphas combined with a translocation containing Galphas to the X chromosome still suppresses the test phenotypes. Upon further analysis of the expression of dpp in discs heterozygous mutant for Su(hh) III, differences in the normal expression domain have been observed. The normally sharp boundary between the posterior and anterior compartments is less defined, leading up to (in extreme cases) a very disrupted and unclear compartment boundary. Indeed, this observation has been confirmed using other markers for the boundary: Ci and En. It is known that Hh signaling is required for proper establishment of the boundary but little is known of the cell biology underlying the clonal and affinity restrictions at the boundary. It is possible that Su(hh) III is in some way involved downstream of Hh in establishing the boundary. Consistent with its being downstream of Hh but upstream of Dpp signaling is that Su(hh) III suppresses ectopic ci but not ectopic dpp. There are many loci in this region of the chromosome and further analysis will be needed to determine which genes are affected in Su(hh) III (Haines, 2000).
Su (hh) IV is localized on the third chromosome to map position 68-69. It suppresses well the phenotypes generated by overexpression of shh. It also weakly suppresses those generated by dpp ectopic expression. One observation indicates a particular role for this locus. When ci is overexpressed to a low level overlying the boundary between hh-expressing and nonexpressing cells, rescue of the resulting phenotype is observed but only in the domain of the wing that does not express hh. This result indicates that this locus plays a role in Hh signaling at the level of Ci or directly downstream but only in anterior compartment cells since here the ci-driven phenotypes are rescued. There are no genes in this area that show homology to genes known to act in the Hh signaling pathway (Haines, 2000).
Hedgehog promotes Bowl protein accumulation by promoting drm
expression, while Wingless antagonizes Hedgehog function and Bowl
accumulation by repressing drm expression
The operation of the Drm/Lines/Bowl regulatory pathway was examined in
the context of the epidermal organizer. Across the dorsal embryonic epidermis,
Hedgehog and Wingless are the key pattern-organizing signals. Hedgehog
specifies cell fate in half the PS (the 1°-3° cell fates), while
Wingless specifies the remaining cell fate (the 4° cell fate) in the
complementary half. To investigate whether Hedgehog and Wingless engage the
Drm/Lines/Bowl regulatory pathway, drm gene expression and
Bowl protein accumulation were examined under conditions of loss or excess of Hedgehog or Wingless signaling. Expression of drm was found to be decreased in hedgehog mutants, and expanded posteriorly
in embryos expressing the secreted form of Hedgehog in
Engrailed/Hedgehog-expressing cells. Two points
are noteworthy here: (1) while Hedgehog can directly control drm
expression posterior to the Hedgehog domain, control within the Hedgehog domain
is likely indirect since these cells cannot themselves respond to Hedgehog
signaling; (2) the fact that
excess Hedgehog does not induce drm expression in anterior cells suggests
that Wingless signaling represses drm expression in this region.
Consistent with this prospect, it was found that drm expression is
ectopically activated in wingless mutants and repressed upon ectopic
activation of the Wingless pathway. It was
also found that changes in drm expression due to manipulations of
Hedgehog and Wingless signaling largely led to the expected changes in Bowl
protein accumulation. For instance, broadened drm expression caused by
excess Hedgehog leads to a broadened Bowl domain,
while the ectopic stripe of drm expression in wingless mutants
also leads to increased Bowl accumulation, although
Bowl accumulates rather more broadly than the narrow drm stripe would
suggest. These changes in Bowl accumulation correlate nicely with the
patterning changes observed with inactivation or activation of Hedgehog or
Wingless signaling. It is concluded
that the asymmetric response of drm to Hedgehog underlies the pattern of
epidermal cell differentiation since drm promotes the accumulation of Bowl
in drm-expressing cells and consequent cellular responses elicited by
Bowl. Note that Bowl accumulates in two rows of cells but apparently is required
for patterning across a broader region. This observation implies that Bowl
controls expression of a new signal that further elaborates epidermal pattern (Hatini, 2005).
Hedgehog and Eye Development
Patterning of the compound eye begins at the posterior edge of the eye imaginal disc and progresses anteriorly toward the disc margin (see Progression of the morphogenetic furrow across the eye disc). The advancing front of ommatidial differentiation is marked by the morphogenetic furrow (MF). Hedgehog (Hh), secreted from two distinct populations of cells has two distinct functions: (1) MF propagation and (2) MF initiation. There is in addition a third function of Hh neural patterning. MF propagation: It is well documented that Hh expression in the differentiating photoreceptor cells drives the morphogenetic furrow. Only in the center of large hh mutant clones is the progression of the MF retarded relative to the adjacent tissue. Because neuronal differentiation and MF propagation can proceed normally through and beyond mutant hh clones situated entirely within the eye field, it appears that Hh secreted from the neighboring wild-type ommatidia rescues, in a nonautonomous manner, the loss of hh (Dominguez, 1997).
MF initiation: Hh also has an early essential role in the initiation of the MF, in addition to its role in MF propagation. Loss of hh from the disc margin, where it is expressed prior to the onset of eye patterning, impedes growth of the disc and prevents all aspects of MF initiation. These results are in conflict with previous reports suggesting that Hh is not required for MF initiation; these reports showed that MF initiation is normal in the hypomorphic hh1 allele. However, it is likely that MF initiation in hh1 initiates normally because the early expression of hh in the margin is normal in the hh1 mutant. In contrast to the direct role of Hh in MF initiation, it appears that the control of MF initiation by Dpp is indirect; it acts by repressing wg. The primary function of Dpp in MF initiation is the repression of wg, which prevents ommatidial differentiation. As in the leg disc, the early expression of dpp and wg may be induced by Hh. It is likely that Hh directly induces early expression of wg and dpp by antagonizing pka activity at the eye disc margin (Dominguez, 1997).
In addition to a requirement for Hh in MF initiation and propagation, Hh, secreted from cells at the posterior disc margin, is absolutely required for the initiation of patterning and predisposes ommatidial precursor cells to enter ommatidial assembly later. Loss of hh activity in the internal region of the disc has very little consequences, presumably because of the presence of Hh secreting tissue in sufficient quantity to rescue the lack of hh function over a relatively long distance (about three ommatidial units). In contrast, the loss of hh expression at the margin, even in small clones, is not completely rescued by the adjacent Hh-producing ommatidia. This constitutes the evidence that Hh is required for ommatidial differentiation. As the MF progresses in a mosiac disc carrying a marginal hh cone, only a single ommatidial unit adjacent to Hh-secreting ommatidia is rescued. Hh induces ommatidial development in the absence of its secondary signals Wingless (Wg) and Dpp (Dominguez, 1997).
These two functions of Hh in eye patterning (initiation of patterning followed later by differentiation of neurons) are similar to the biphasic requirement for Sonic Hh in patterning of the ventral neural tube in vertebrates (signaling from notochord to ventralize the neural tube followed later by specification of motor neurons by Sonic hedgehog secreted from the floor plate). These results show that the regulatory relationships between Hh, Dpp, and Wg in the eye are similar to those found in other imaginal discs, such as the leg disc, despite obvious differences in their modes of development (Dominguez, 1997).
The adult eye of Drosophila is a highly ordered structure. It is composed of about 800 ommatidia, each displaying precise polarity. The ommatidia are arranged about an axis of mirror image symmetry, an equator along the dorsoventral midline of the eye. hedgehog pathway mutants were used to induce ectopic morphogenetic furrows and these were used as a tool to investigate the establishment of ommatidial polarity. The results show that ommatidial clusters are self-organising units whose polarity in one axis is determined by the direction of furrow progression. They can independently define the position of an equator without reference to the global coordinates of the eye disc (Strutt, 1995b).
Neuronal differentiation in the Drosophila retinal primordium (the eye imaginal disc) begins at the posterior tip of the disc and progresses anteriorly as a wave. The morphogenetic furrow (MF) marks the boundary between undifferentiated anterior cells and differentiating posterior cells. Anterior progression of differentiation is driven by Hedgehog, synthesized by cells located posterior to the MF. hedgehog , which is expressed prior to the start of differentiation along the disc's posterior margin, also plays a crucial role in the initiation of differentiation. Using a temperature-sensitive allele it has been shown that hh is normally required at the posterior margin to maintain the expression of both decapentaplegic (dpp) and the proneural gene atonal. In addition, ectopic differentiation driven by ectopic dpp expression or loss of wingless function requires hh. Consistent with this is the observation that ectopic dpp induces the expression of hh along the anterior margin even in the absence of differentiation. Taken together, these data reveal a novel positive regulatory loop between dpp and hh that is essential for the initiation of differentiation in the eye disc (Borod, 1998).
Genetic analysis reveals an interaction between dachshund and hedgehog. hh is required for the progression of the morphogenetic furrow. hh loss-of-function mutants dominantly enhance the recessive eye phenotype of weak dac alleles. These genetic results suggest that dac and hh act in the same or in parallel pathways during eye development. hh is not expressed in dac mutant eye discs, but hh expression in other imaginal discs, including the antennal, leg and wing discs, is not affected by loss of dac function. hh expression is normal in dac clones. Thus, dac function is not cell autonomously required for hh expression (Mardon, 1994).
A new segment polarity gene of Drosophila melanogaster, oroshigane (oro) was identified as a dominant enhancer of Bar (B). The B1 allele of the Bar locus is associated with a tandem duplication of the division 16A of the X chromosome and causes the overexpression of the Bar (B) gene. decapentaplegic expression in the morphogenetic furrow is abolished in the B background, and therefore morphogenetic furrow progression prematurely ceases resulting in the characteristic bar-shapped compound eye. hedgehog expression also fades 8 hours after heat induction of the Bar protein, indicating that hh is another target for inhibition by B. Since the Bar protein is required for R1, R6 and primary pigment cell differentiion behind the furrow, the overexpressed B protein apparently interferes with furrow progression by inhibiting dpp expression in the furrow and hedgehog expression just behind the furrow. Overexpression of the Bar protein has little deleterious effect on differentiation of photoreceptor clusters that have already started to develop behind the morphogenetic furrow. Failure of morphogenetic furrow progression likely triggers programmed cell death in the undifferentiated cells ahead of the morphogenetic furrow (Epps, 1997).
oro is a recessive embryonic lethal, and homozygous oro embryos show variable substitution of denticles for naked cuticle. These patterns are distinctly similar to those of hedgehog and wingless mutant embryos, which indicates that oro functions in determining embryonic segment polarity. oro works downstream of hedgehog but upstream of dpp to enhance the Bar phenotypes. Although dpp expression is reduced in oro heterozygotes, hh expression remains the same as that found in wild-type discs. Evidence that oro function is involved in Hh signal transduction during embryogenesis is provided by its genetic interactions with the segment polarity genes patched and fused. ptcIN is a dominant suppressor of the oro embryonic lethal phenotype, suggesting a close and dose-dependent relationship between oro and ptc in Hh signal transduction. oro function is also required in imaginal development. The oro1 allele significantly reduces decapentaplegic (but not hh) expression in the eye imaginal disc. oro enhances the fused1 wing phenotype in a dominant manner. Based upon the interactions of oro with hh, ptc, and fu, it is proposed that the oro gene plays important roles in Hh signal transduction (Epps, 1997).
Neuron-glia interactions are crucial for the establishment of normal connectivity in the nervous system during development, but the molecular signals involved in these interactions are largely unknown. Differentiating photoreceptors in the developing Drosophila eye influence the proliferative and migratory behavior of the subretinal glia through the diffusible factors Decapentaplegic (Dpp) and Hedgehog (Hh). Subretinal glial precursors originate in the optic stalk and migrate into the eye imaginal disc concomitant with the onset of differentiation of photoreceptors. Their presence in the eye disc, in turn, is necessary for guiding photoreceptor axons from the eye disc into the optic stalk. Proliferation and migration of the glia are separable processes, and Dpp promotes both the proliferation and motility of the glia, whereas Hh appears to promote only their motility; neither specifies the direction of migration. Evidence is presented that Dpp and Hh act on the glia in parallel and through the regulation of transcription. Ectopic migration of subretinal glia can result in the ectopic projection of photoreceptor axons. This study suggests a novel function for Hh in regulating migratory behavior and provides further evidence for a complex mutual dependence between glial and neuronal cells during development (Rangarajan, 2001).
In the developing Drosophila visual system, the eye disc
is connected to the optic lobe by the optic stalk. Photoreceptor
cells are generated in the eye disc in a posterior-to-anterior
progression; the wave of differentiation is marked at
its front by an indentation of the disc epithelium called the
morphogenetic furrow. The photoreceptors extend axons
into the basal layer of the eye disc, where they turn and
proceed posteriorly to exit the disc through the optic stalk. The subretinal glia (also known as retinal basal glia) originate from precursor cells in the optic stalk; the glial precursors in the stalk already express the glial-specific
marker Repo. The glia begin to migrate into the eye disc with the onset of the differentiation of photoreceptors, but do not require
the presence of axons in the stalk to find their way into the
eye disc. Once in the eye disc, the glia migrate anteriorly on the basal surface of the eye epithelium; the anterior border of their migration lies
typically 2-4 rows of ommatidia posterior to the morphogenetic
furrow, which is where axons sent out by the differentiating
photoreceptors begin to turn posteriorly. Thus, glia are present only in the axonal portion of the eye disc. The glial cells associate closely with the photoreceptor axons and develop extensive processes that
surround the axons and fill the intervening space. The development of the subretinal glia thus involves proliferation, migration, and terminal differentiation events, and it is closely tied, both spatially and
temporally, to the development of the photoreceptors (Rangarajan, 2001).
The coordination of neuronal and glial development is
crucial for the establishment of functional circuitry in the
visual system. Normal photoreceptor axon projections
depend on the proper positioning of the subretinal glia:
Glia have to be present in the eye disc near the entrance
to the optic stalk for photoreceptor axons to find their way
into the stalk; if glia are induced to mismigrate to ectopic positions in
the anterior, undifferentiated portion of the eye disc, axons
frequently follow. To prevent anterior misprojection of
axons it is therefore imperative that the migration of the
subretinal glia be restricted to the differentiating portion
of the eye disc. In addition, the number of glia in the eye
disc has to be roughly proportional to the number of ommatidia
to ensure proper wrapping and insulation of the photoreceptor
axons. This means that the glial population has to
expand quickly in response to the growing number of
ommatidia progressively forming in the eye disc. This
matching of cell numbers requires the appropriate modulation
of glial proliferation and migration (Rangarajan, 2001).
Neuron-glia interactions have also been shown to play a
role in oligodendrocyte and astrocyte development in the
vertebrate visual system. Retinal ganglion cell axons signal
to the glia in various ways to promote their proliferation and
survival, thereby ensuring precise matching of the
two cell populations. However, there has been no evidence
to date for neuronal regulation of glial migration other than
through haptotaxis (Rangarajan, 2001).
For Hh loss of function data show only a
mild effect on the accumulation of glia in the eye disc, and
the gain of function results confirm that there is no significant
effect of Hh on glial proliferation. This is somewhat
surprising, given that Hh is required for the growth of the
Drosophila eye disc and the proliferation of the neurons in
the lamina and in view of its mitogenic effects on many cell types of the
vertebrate nervous system, including astrocytes in the optic
nerve of rodents (Rangarajan, 2001).
Subretinal glia migrate from the optic stalk into eye discs
only when photoreceptors have begun to differentiate in the
eye disc, but they do not require axons as a substrate for their migration. They also migrate towards ectopic islands of differentiating
photoreceptors, again without axonal substrate.
These findings suggest that differentiating photoreceptors
regulate the migration of the subretinal glia over a distance,
either by secreting diffusible molecules or by preferentially
stabilizing far-reaching glial filopodia. The present study demonstrates that the diffusible factors Dpp and Hh are among the signals the differentiating
photoreceptors send to promote subretinal glial motility. Gain-of-function experiments show that overactivity of Dpp as well as of Hh signaling leads subretinal glial cells to overshoot their normal anterior boundary, either in a relatively narrow stream or in a broad front. In the case of Dpp,
this ectopic migration of the glia is accompanied by severe
overproliferation, and therefore the possibility
that the changes in migratory behavior are merely a
secondary consequence of the increase in cell number had to be considered.
However, severe overproliferation of glia in
otherwise wild type eye discs does not lead to ectopic migration.
This is true not only when overproliferation is induced
by acceleration of the cell cycle, but also when it is caused by
Ras overactivity, which presumably mimics the effects of a wide range
of growth factors. Similarly, overproliferation of glia in the
optic stalk of eya mutants, which lack photoreceptors, does
not induce migration into the eye disc. Conversely, ectopic migration of glia is observed in eye discs with Hh overactivity,
i.e., in discs in which the number of glial cells is
largely normal. These results demonstrate that overproliferation
in itself is not sufficient to induce abnormal migration of
glial cells and that proliferation and migration are in fact
independently regulated processes (Rangarajan, 2001).
The ectopic migrations of glial cells that was observed are
not preferentially directed towards the sources of Dpp or
Hh. Moreover, similar migratory phenotypes can be induced
by expression of constitutively active components of the
Dpp and Hh pathways within the glial cells. Such cell-autonomous
activation of the pathway precludes the use of information about ligand distribution as a positional cue. Both findings therefore argue that Dpp and Hh exert their effect on the glia not by providing positional information, but by stimulating motility. This motogenic effect may be due to an enhanced ability to migrate or to an impaired ability to respond to an inhibitory signal. In any case, both Dpp and Hh appear to act by regulating transcription within the glial cells, through their canonical signal transduction machinery (Rangarajan, 2001).
One of the best understood motogens is scatter factor (SF), which
disperses cohesive colonies of epithelial cells by stimulating
random motility. However, it can also act as a mitogen
(hepatocyte growth factor), morphogen, trophic factor, or
chemoattractant depending on the cellular context; in all
cases, its effects are mediated by the receptor tyrosine
kinase c-Met. The actual scattering induced by SF occurs
only 4-6 h after addition of SF and is inhibited by cycloheximide,
suggesting that regulation of gene expression is
involved. Thus, Dpp and Hh resemble SF both in terms of phenotypic effect and with regard to their mechanism of action (Rangarajan, 2001).
The function of Hh as a motogen is novel. Although the
molecule is being studied in many contexts, a role in cell
motility has not been attributed to it to date. Dpp and its
vertebrate counterparts, in contrast, have been implicated
in the control of migratory processes during development.
Dpp plays a role in tracheal cell migration along the
dorso-ventral body axis and in the
dorsal migration of the ectoderm during dorsal closure of
the Drosophila embryo. During wound healing, the migration of various
cell types, including astrocytes, depends on TGFß (Rangarajan, 2001).
Why are not more clear-cut effects on the migratory behavior of the subretinal glia observed in loss of function experiments? Mosaic data confirm at least for Dpp that the molecule is required for the normal accumulation of
glia in the eye disc. But because of the limitation of the
mosaic technique it is difficult to distinguish between effects
on proliferation and on motility and to discern specific
migratory effects: the failure of ombGAL4 to drive recombination
to completion prior to entry of the glia into the eye
disc means that at least some glia arrive in the eye disc and
migrate anteriorly as heterozygotes, i.e. ,before undergoing
recombination to become homozygous mutant. Another major reason for the lack of a more pronounced loss of function effect is most likely that the Dpp and Hh
signal transduction pathways are (partially) functionally redundant with other pathways in regulating glial cell development (Rangarajan, 2001).
Ras signaling, possibly triggered by diffusible RTK ligands, is very likely to
play a role in regulating both the proliferative and migratory
behavior of the subretinal glia. Generally, Dpp and Hh must be part of a more complex network of signals regulating glial development in the eye
disc. For example, it is not yet known how the sharp anterior
boundary of glial migration behind the morphogenetic
furrow is established. Possible mechanisms include an
attractive substrate or signal in the posterior, or an inhibitory
signal in the anterior. Since in wild type the distribution of
both Dpp and Hh extends further to the anterior than the
glial cells migrate, this mechanism also has to be able to
counteract the motogenic effects of Dpp and Hh (Rangarajan, 2001).
How do Dpp and Hh exert their effects on subretinal glial
migration? Since Hh and Dpp are known to regulate each
other's expression in other tissues, it would
be conceivable that they act in sequence. However, neither
dpp nor hh is expressed in the subretinal glial cells, which
rules out the possibility that they mutually regulate each
others transcription and implies that Dpp and Hh both act
in a paracrine fashion. It therefore seems likely that Dpp and
Hh signaling converge to control the transcription of genes
required for cell motility (or possibly for reading the stop
signal). These targets could include genes coding for cytoskeletal
components, proteins regulating cell adhesion, or
enzymes to degrade the extracellular matrix. The idea that
such genes could be transcriptionally controlled by Dpp or Hh is supported by the recent finding that in the Drosophila wing compartment, cell-sorting at the anterior/posterior boundary is under the opposing transcriptional control of Hh and En, suggesting that these molecules regulate the expression of a single cell adhesion molecule. The identification of transcriptional targets of Dpp and Hh signaling will provide important insights into the mechanism by which these signals regulate glial cell motility (Rangarajan, 2001).
In the Drosophila visual system, photoreceptor neurons (R cells) extend axons towards glial cells located at the posterior edge of the eye disc. In gilgamesh (gish) mutants, glial cells invade anterior regions of the eye disc prior to R cell differentiation and R cell axons extend anteriorly along these cells. gish encodes casein kinase Igamma. gish, sine oculis, eyeless, and hedgehog (hh) act in the posterior region of the eye disc to prevent precocious glial cell migration. Targeted expression of Hh in this region rescues the gish phenotype, though the glial cells do not require the canonical Hh signaling pathway to respond. It is proposed that the
spatiotemporal control of glial cell migration plays a critical role in determining the directionality of R cell axon outgrowth (Hummel, 2002).
In the developing Drosophila visual system, photoreceptor cell (R cell) axons contact numerous glial cell types along the pathway to the target and within the target itself. These glial cells originate from different regions of the developing visual system and migrate to their final destinations where they associate with axons. The first glial cells encountered by extending R cell axons are retinal basal glial (RBG) cells located along the basal surface of the eye disc epithelium. As R cells differentiate, they extend growth cones basally where they contact RBG cells and turn posteriorly toward the optic stalk. These glial cells originate in the optic stalk and migrate into the eye disc. Migration is temporally
and spatially linked to R cell development. The extension of axons from R cell clusters occurs in a sequential
fashion that reflects the highly ordered pattern of R cell differentiation in the eye disc. R cells in the posterior region differentiate first and additional R cell clusters form more anteriorly as a wave of differentiation sweeps across the eye disc. The leading edge of this wave is marked by a depression in the apical region of the disc epithelium called the morphogenetic furrow (MF) (Hummel, 2002).
Glial cells start to migrate into the eye disc as R cell differentiation at the posterior margin is initiated. During MF progression, RBG cells migrate along the basal surface up to but not past the youngest R cell axons. RBGs appear to migrate along R cell axons thereby making a guidance role for them unlikely. Glial cells can migrate out of the optic stalk into the eye disc in the absence of axon contact; instead, glial cells are essential for R cell axons to enter the optic stalk. These observations suggest that the precise coordination of glial cell development in the optic stalk and R cell differentiation in the eye disc is important for normal visual system development. In the course of a genetic screen for axon guidance mutants, a loss-of-function mutation was identified that
temporally uncoupled glial cell migration from R cell differentiation. Mutations in the gilgamesh locus lead to precocious glial cell migration from the optic stalk into the eye disc prior to R cell differentiation. As a result, glial cells are misplaced anteriorly in the eye disc as R cell axons extend. R cell
axons frequently project along pathways demarcated by these ectopic glial cells, providing strong evidence that the positioning of glial cells plays an instructive role in regulating the directionality of R cell axon outgrowth in the
eye disc. The gish locus acts in conjunction with the eye
specification genes, eyeless and sine oculis, to prevent precocious entry of glial cells into the eye disc. Loss-of-function hh mutants exhibit precocious glial cell migration and targeted expression of Hh in the posterior region of the eye disc suppresses the ectopic glial cell migration phenotype in gish mutants (Hummel, 2002).
A set of genes encoding nuclear proteins [e.g., eyeless (ey), eyes absent (eya), sine oculis (so) and secreted factors such as hedgehog (hh)] regulates the initiation of neuronal differentiation in the posterior region of the eye disc. The effect of loss-of-function mutations in these genes on glial cell migration was tested. As in gish mutants, glial cells migrate precociously out of the optic stalk in a hh temperature-sensitive mutation incubated at the nonpermissive temperature during first and second instar. This is an early function of hh, since ectopic glial cells are not observed in hh1; in this allele, the posterior eye field develops normally, but anterior progression of the MF is inhibited. A similar early onset glial cell migration defect is observed in eye-specific alleles of so and ey. In contrast, glial cells did not migrate out from the optic stalk in an eye-specific allele of eya, raising the possibility that eya is required to activate glial cell migration. Since glial cells migrate out of the stalk precociously in eya/gish double mutants, the production of an eya-dependent signal is not necessary to promote anterior migration. Hence, in contrast to their role in R cell development, eye specification genes ey and so seem to function independent of eya to control the onset of glial cell migration (Hummel, 2002).
These observations raise the possibility that gish also contributes to the genetic circuitry regulating eye specification. Indeed, while ey-FLP-induced clones of gish lead to only minor defects in MF progression during third instar stage, gish mutant adult eyes are smaller and frequently contain a reduced number of ommatidia in the anterior region. These phenotypes are frequently seen in weak alleles of eye specification genes. Furthermore, double mutants of ey1 and gish1 , as well as so1 and gish1, reveal strong synergistic effects in R cell development. The glial cell migration phenotypes in double mutants is similar in severity to the single mutants. In summary, these data argue that gish acts in conjunction with eye specification genes to coordinate neuronal development and glial cell migration in the eye disc (Hummel, 2002).
Since Hh is expressed in the right place and time to function as the glial cell repellent, and since glial cells migrate precociously in hh mutants, it became important to assess whether Hh directly regulates glial cell migration. In support of this view, targeted expression of Hh at the posterior region of the second instar eye disc rescues precocious cell migration in gish mutants. In about 80% of the gish mutant larvae carrying Dpp-Gal4 driving UAS-hh, premature glial cell migration was prevented in second instar, and glial cells remain posterior to the MF at third instar (Hummel, 2002).
Does Hh directly regulate glial cell migration? To address this question, whether the canonical Hh signaling pathway is activated in glial cells was assessed and whether it is required to prevent precocious migration. patched (ptc) expression, an indicator of reception of the Hh signal, is not elevated in glial cells prior to migration into the eye disc. It is, however, induced in epithelial cells located at the most posterior edge of the eye disc immediately juxtaposed to the pre-migratory glial cells in the optic stalk. In support of the importance of the posterior margin in signaling glial cells, a significant reduction in the level of ptc-lacZ expression was observed in this region of the second instar eye in gish mutants. Alternatively, this reduction may simply provide a sensitive indicator that the level of Hh is reduced in gish mutants. Since the level of Hh protein is only slightly above background in wild-type, the level of the Hh signal in these mutants could not be critically assess with the available reagents. Further evidence that the canonical Hh pathway is not required in glial cells came from the analysis of mutations in smoothened (smo) and Cubitis interruptus (Ci) genes encoding a Hh receptor component and a downstream transcription factor, respectively. Mosaic clones of smo mutant glial cells do not migrate prematurely at second instar and were found exclusively posterior to the MF. Similarly, targeted expression of a dominant-negative version of Ci in glial cells does not alter early migratory behavior of eye disc glial cells. These findings argue that Hh acts either indirectly to control glial cell migration or acts directly upon glial cells through a novel pathway independent of ptc, smo, and Ci (Hummel, 2002).
Thus, the timing of glial cell migration plays an essential role in regulating axon guidance in the fly visual system. Glial cells in the posterior region of the eye disc epithelium provide an intermediate target for R cell axons as they project from the eye to the brain. In gish mutants, glial cells migrate out of the optic stalk to more anterior regions of the eye disc prior to R cell differentiation, and as a consequence, R cell axons frequently extend anteriorly in gish mutants along the surface of these ectopically located glial cells. gish acts in combination with eye specification genes, ey and so, and the extracellular signaling protein Hh to control glial cell migration. Since these genes, including gish, also regulate neuronal development in the eye disc, it is proposed that they define a signaling center in the posterior region of the eye disc that controls both neurogenesis and glial cell migration to ensure normal patterns of R cell axon outgrowth (Hummel, 2002).
Through genetic mosaic analysis and transgene rescue experiments, it has been established that gish acts within the eye disc epithelium to inhibit glial cell migration. In principle, gish could regulate the production of a repellent preventing migration of glial cells out of the optic stalk, an attractant that promotes their close association to the posterior edge or alternatively an antagonist to an attractive signal produced by cells in the disc. Gish belongs to the casein kinase I family of highly conserved and widely expressed enzymes. These enzymes contain small but varied amino termini and large, highly diverse carboxy-terminal domains. Gish is most similar to mammalian CKIgamma3. CKIgamma3, like gish, is alternatively spliced -- this can result in kinases with different biochemical properties and functions. CKIs act on proteins previously phosphorylated by other kinases. CKIs have been shown to phosphorylate a large number of proteins in vitro. Regrettably, little evidence exists to establish a link between phosphorylation by CKI and specific developmental pathways. Recent studies have shown that casein kinase Iepsilon (CKIepsilon) can regulate ß-catenin in the Wnt pathway in both worms and frogs. Loss- and gain-of-function manipulation of Wg signaling components, however, did not disrupt glial cell migration into the eye disc. This is not surprising given that CKIepsilon and CKIgamma differ significantly in their C-terminal regions and deletion analysis has revealed that the unique C-terminal domain is important for the interaction of CKIepsilon with the axin signaling complex. Since gish does not encode a secreted molecule, it must act indirectly to affect signaling from the epithelium to the glial cells (Hummel, 2002).
Several lines of evidence support a model in which gish regulates Hh signaling: (1) like gish, hh is required in the posterior region of the second instar eye disc to inhibit anterior migration of glial cells; (2) gish mutants display defects in morphogenetic furrow progression similar to those seen for hypomorphic alleles of hh, and (3) the expression of Hh target genes ptc and Ci in the eye disc epithelium is reduced in gish mutants. These data suggest that gish acts upstream of (or in parallel to) hh. Although genetic evidence suggests that the gish mutants studied here are strong loss-of-function alleles, since low levels of gish mRNA can still be detected in homozygous gish1 larvae, the epistatic relationship between gish and hh must be qualified. Indeed, Gish may function downstream from Hh and limiting levels of activity in loss-of-function alleles may be compensated by increasing the level of Hh upstream (Hummel, 2002).
The location of a specific subtype of glial cells is not only necessary for posterior-directed outgrowth of R cell axons, but also sufficient. Indeed, some 50% of gish mutant eye discs at an early stage of development contain R cell fibers that project anteriorly along the surface of misplaced glial cells. During normal development, however, more anterior R cells differentiate as a continuous wave, and glial cells migrate to a region just posterior to differentiating R cells. As a consequence, as these axons extend to the basal surface, they contact glial cells that lie posterior to them and follow them into the optic stalk. Axon-glial cell interactions in the fly eye show interesting parallels to intra-retinal pathfinding in the vertebrate eye. Retinal ganglion cell axons project radially toward the optic disk, their first intermediate target in the center of the eye. Here, axons receive contact-dependent signals from glial cells to exit the retina and enter the optic nerve. In addition, in vertebrates, a barrier at the junction between the optic nerve and the retina has been proposed to inhibit glial cell precursors from migrating into the retina. It will be interesting to assess whether the molecular strategies coordinating axon outgrowth and glial cell migration have been conserved between vertebrate and invertebrate visual systems (Hummel, 2002 and references therein).
In the developing Drosophila eye, the morphogenetic furrow is a developmental organizing center for patterning and cell proliferation. The furrow acts both to limit eye size and to coordinate the number of cells to the number of facets. This study reports the molecular and functional characterization of Drosophila mini-me (mnm), a potential regulator of cell proliferation and survival in the developing eye. mnm was identified as a dominant modifier of hedgehog loss-of-function in the developing eye. mnm encodes a conserved protein with zinc knuckle and RING finger domains. mnm is dispensable for patterning of the eye disc, but required in the eye for normal cell proliferation and survival. mnm null mutant cells exhibit altered cell cycle profiles and contain excess nucleic acid. Moreover, mnm overexpression can induce cells to proliferate and incorporate BrdU. Thus, these data implicate mnm as a regulator of mitotic progression during the proliferative phase of eye development, possibly through the control of nucleic acid metabolism (Jones, 2006).
mnm gene encodes a conserved protein with a novel N-terminus
and Zinc knuckle, RING finger, proline-rich and coiled coil domains. mnm is
expressed everywhere in the developing eye disc and is enriched ahead of the
morphogenetic furrow. The expression of mnm is dependent upon Hedgehog signaling (perhaps indirectly), as loss of Hedgehog signaling through an inactivating mutation in hedgehog greatly reduces its expression (Jones, 2006).
From a timed analysis of mutant clones, it appears that mnm null cells in
proliferative regions of the developing eye (and wing) can replicate for two or three
times over 48 hours, but between 48 and 72 hours after clone induction, they suffer
some crisis and die. It may be that this delayed defect is due to perdurance of the Mnm protein. If during that time window, null cells receive developmental signals to cease proliferation and differentiate, they can then survive. The morphogenetic furrow and subsequent events do provide such differentiation signals so that mnm null clones can persist in the retina, if they are induced late enough. The data show that if the furrow passes over mnm null cells in the first 24-48 hours after they become homozygous, they can persist to the adult eye, and many differentiate as morphologically normal photoreceptors and accessory cells. Taken together, these data suggest that Mnm is required for some function in proliferative cells, but not in post-mitotic cells (Jones, 2006).
Because the mnm mutant clones posterior to the furrow can survive and differentiate as apparently perfect, yet tiny copies of their wild-type twin-spots, the gene was name 'mini-me' (after a character in 'Austin Powers: Goldmember') (Jones, 2006).
FACS analysis of mnm null cells suggests that mnm null cells have abnormal nucleic acid content. This could reflect changes in nuclear DNA, mitochondrial DNA, and/or RNA content. It could be that the mnm mutant cells have lost the correct coupling of DNA synthesis to cell division and accumulate DNA beyond 4N. It may be that these cells over-replicate DNA during S-phase, mis-segregate DNA during mitosis, or fail to divide and become aneuploid. mnm overexpression is sufficient to induce proliferation; and this excessive proliferation is toxic and leads to cell death. An increase in nucleic acid content associated with mnm loss-of-function and the overproliferation of mnm-overexpressing cells are consistent with a role for mnm as a regulator of mitotic progression, although whether Mnm plays a role in DNA replication, the DNA damage checkpoints, or mitotic entry/exit is not clear (Jones, 2006).
The closest human and murine homologs of Drosophila Mnm are RBBP6 and
PACT/P2P-R. These proteins have been shown to associate with Retinoblastoma
protein (Rb) and p53 proteins in vitro, which are potent regulators of the cell cycle, including regulating entry into S-phase and the monitoring of DNA integrity. This could be consistent with the suggestion that loss of
Mnm may lead to aberrant DNA metabolism. Furthermore P2P-R is down regulated in
differentiating cells, consistent with the observation of a lack of
Mnm function in post-mitotic territories in the developing eye. RNAi knockdown of P2PR in mouse 3T3 cells affects nocodazole-induced arrest and UV-induced apoptosis,
also possibly consistent with a disturbance in DNA metabolism (Jones, 2006).
Hedgehog signaling has been implicated in cell-cycle regulation in both flies and
vertebrates. The link between hedgehog and mnm may be a new mechanism for this control. However, the interaction between hedgehog and mnm could be indirect: the small phenotype of mnm clones is quite dissimilar to that of smoothened clones, which are not small (lacking the Hedgehog receptor). Phenotypic effects of mnm loss-of-function outside of the territories where the Hedgehog signal is received. Thus it is suggested that while mnm may be controlled in part by hedgehog, it has much more general functions and is likely, also, to be regulated by other pathways (Jones, 2006).
It is interesting that loss of mnm function strongly interacts genetically with the Dpp and Notch pathways in opposite ways. Both pathways have recently been
characterized to have significant roles in regulating cell cycle progression in the
developing eye. Dpp signaling promotes G1 arrest, while Notch signaling regulates S
phase entry in the second mitotic wave. It could be that mnm is interacting directly with these pathways to regulate cell cycle progression. However, the precise mechanism remains to be resolved (Jones, 2006).
Hedgehog and Gonad Development
The primitive gonad of the Drosophila embryo is formed from two cell types, the somatic gonad precursor cells (SGPs) and the germ cells, which originate at distant sites. To reach the SGPs the germ cells must undergo a complex series of cell movements. While there is evidence that attractive and repulsive signals guide germ cell migration through the embryo, the molecular identity of these instructive molecules has remained elusive. Evidence is presented suggesting that hedgehog (hh) may serve as such an attractive guidance cue. Misexpression of hh in the soma induces germ cells to migrate to inappropriate locations. Conversely, cell-autonomous components of the hh pathway appear to be required in the germline for proper germ cell migration (Deshpande, 2001).
Previous studies have implicated hh in the specification of the SGP cells; however, while it is known that hh is expressed in the overlying ectoderm, it has not been established whether hh is also expressed in SGP cells themselves or in their neighboring mesodermal cells. To address this question, embryos carrying a transgene in which hh promoter drives LacZ expression were immunostained with antibodies against ß-galactosidase and the SGP marker Clift. ß-galactosidase can be detected in the Clift-positive mesodermal cells in parasegments 10, 11, and 12 in germ band extended embryos. Assuming that the expression pattern of the hh-lacZ transgene faithfully mimics the endogenous hh gene, this finding indicates that hh is expressed in the SGPs. ß-galactosidase can also be detected at this stage in Clift-negative mesodermal cells in parasegments anterior to PS10. Based on their homologous position in the mesoderm, these cells are likely to be fat body precursors. This is consistent with the finding that hh is required not only for the formation of the somatic gonad but also for the specification of the fat body (Deshpande, 2001).
Since hh seems to be expressed in SGPs just at the time when the germ cells begin to exit the midgut, this signaling molecule might have an additional function in orchestrating the migration of germ cells through the embryo and/or their association with the SGP cells. If hh does play a role in germ cell movement, then it should be possible to alter the migration pattern of these cells by expressing the Hh protein at ectopic sites in the embryo. For this purpose, advantage was taken of the UAS-Gal4 misexpression system (Deshpande, 2001).
In the first experiment, the expression of Hh protein was induced from the UAS-hh transgene in alternate segments using a hairy-Gal4 driver and then the effects on germ cell migration were examined. The UAS-hh transgene by itself has no effect on the migration behavior of germ cells, and the typical elongated cluster of Vasa positive germ cells is observed in the mesoderm near the posterior end of stage 13 embryos. While a germ cell cluster is observed at this same position in stage 13 hairy-Gal4/UAS-hh embryos, germ cells are also seen at a variety of abnormal locations in the posterior ectoderm. Migration defects are also evident at earlier stages just after the germ cells leave the posterior midgut. Instead of migrating dorsally, they move toward the overlying mesoderm (Deshpande, 2001).
Ectopic expression of Hh protein in the CNS using an elav-Gal4 driver also induces aberrant migration patterns. In stage 13 wild-type or control UAS-hh transgene embryos, the germ cells are typically arranged in two elongated but tight clusters on either side of the ventral midline. While two clusters can usually be discerned in elav-GAL4/UAS-hh stage 13 embryos, most of the cells in the clusters are not tightly associated with each other and some are dispersed at distant sites in the posterior mesoderm. In addition, a subset of the germ cells are typically found near the ventral midline in the region just underlying the CNS where expression of Hh from the elav-GAL4 driver is expected to be highest at this stage of development. Perhaps because of differences in timing and/or tissue specificity, Hh expression directed by the elav-GAL4 driver does not noticeably perturb the early steps in migration when the germ cells have just exited the gut (Deshpande, 2001).
Even more dramatic alterations in germ cell migration are observed when hh expression is driven in the mesoderm by a twist-GAL4 driver. In stage 12-13 embryos, germ cells are found in several small clusters of 3-6 cells scattered in different segments along the A-P axis. Since misexpression of another signaling molecule, wg, under the control of the same twist-GAL4 driver, has no obvious effects on germ cell behavior, it would appear that the aberrant migration pattern seen in twist-GAL4/UAS-hh embryos is due to ectopic hh expression (Deshpande, 2001).
Known cell-autonomous components of the Hh signaling pathway also appear to be required in germ cells for normal migration behavior. Germline clones were used to test four different hh pathway genes -- ptc, pka, smo, and fu. For all four, abnormalities in germ cell migration were observed in the progeny. In the case of both the ptc and smo germline clones, eggs fertilized by wild-type sperm developed into completely normal adults. Moreover, there are no apparent defects in the formation of the somatic gonad or in the pattern of Clift expression. These findings would support the view that the migration defects seen in ptcmat-zyg+ and smomat-zyg+ embryos arise from cell-autonomous deficiencies in the response to Hh by the germ cells. However, it should be pointed out that there could be some undetected nonautonomous problem in somatic hh signaling in these embryos that induces abnormalities in germ cell behavior (Deshpande, 2001).
As would be expected from the known properties of these four genes in other well characterized hh pathways, the phenotypes produced by ptc and pka germline clones are similar and quite distinct from those observed for smo and fu. Moreover, the migration defects observed in ptc/pka and smo/fu germline clones can be explained by the antagonistic role of these genes in the hh signaling pathway. In the absence of maternal ptc or pka, smo and its downstream effectors in the hh pathway are activated in the germ cells independent of the Hh ligand. As a consequence, many of the germ cells clump together as they begin passing through the midgut, and then remain in place instead of migrating toward the SGP cells. Additionally, the mitotic cycle in ptcmat- (and to a lesser extent pkamat-) germ cells is inappropriately activated. Up regulation of cell division has been observed in somatic tumors that lack ptc function and in ptc mutant C. elegans germ cells. In the case of smo and fu, the germ cells can't respond to the Hh ligand, and they are unable to detect or associate with the SGP cells, and instead migrate randomly through the mesoderm (Deshpande, 2001).
In patterning, the Hh ligand can function as both a short and long distance signal. This raises the question of what role Hh plays in germ cell migration. It is possible that Hh acts only over a short distance, attracting germ cells when they are in close proximity to the SGP cells and/or mediating the direct interaction between the two cell types. Alternatively, the Hh ligand could act over a long distance and begin orchestrating germ cell migration as soon as the cells pass through the midgut. While not conclusive, several observations seem to favor this second alternative. (1) Migration defects in the absence of both maternal ptc and pka are evident at the time germ cells begin exiting the midgut. This argues that the machinery required to process and respond to the hh signal is most probably already present before the germ cells come in firm contact with the SGP cells. (2) hh expression driven by the hairy-Gal4 driver induces aberrant germ cell migration at this same point in development, directing germ cells exiting the midgut to move ventrally rather than dorsally. The idea that Hh can act over long distances is also supported by the effects of ectopic Hh on the distribution of germ cells at later stages, when they would normally be associated with the SGP cells. Both the elav-GAL4 and twist-GAL4 drivers are able to attract germ cells to sites quite distant from the SGP cells in PS9-12. Moreover, the ability of ectopic Hh sources to attract germ cells seems to depend upon a competition with Hh protein produced by the endogenous hh gene. This is suggested by the finding that the migration defects induced by GAL4-UAS-driven expression of Hh protein are exacerbated when the embryos are heterozygous for a hh mutation (Deshpande, 2001).
The idea that Hh functions as a long distance signal for germ cell migration poses a number of problems. One is the question of specificity. Is the combination of an attractive Hh signal and a Wunen-dependent repulsive signal sufficient to guide the complex movements of the germ cells after they exit the posterior midgut? They first move to the dorsal surface of the endoderm and then split into two groups which migrate toward the lateral mesoderm on either side of the embryo, where they find and associate with the SGP cells. The specificity problem is compounded by the fact that there are many potential sources of Hh protein in the embryo. In the ectoderm, Hh is expressed in a stripe pattern in each parasegment, while in the mesoderm, it is expressed not only in SGP cells, but also in the fat body precursor cells. If Hh protein emanating from these different sources is able to reach the germ cells at any point in their migration toward the SGP, the germ cells could be diverted toward inappropriate targets. In this case, one would have to suppose that some other signal, superimposed upon the Hh signal, is required to attract germ cells to the SGPs, and prevent them from being directed toward extraneous sources of Hh (Deshpande, 2001).
It is also possible that there are mechanisms which restrict or promote the movement of the Hh ligand within the embryo. For example, directional secretion of Hh protein coupled with a diffusion or transport barrier (such as the extracellular matrix) that seals the ectoderm off from the mesoderm could ensure that the Hh protein expressed in ectodermal cells only effectively signals other cells in the ectoderm. Within the mesoderm, there could be differences in the activity/mobility of Hh protein expressed in fat body and somatic gonadal precursor cells. In this context, it is interesting to note that expression of columbus (clb) in the mesoderm, which is known to have an intimate connection with directing germ cell migration, becomes progressively restricted to the SGP cells as the germ cells begin their migration. Although it is not known how clb functions in germ cell migration, an intriguing possibility is that it plays some role in the production or relay of the hh signal. The Hh protein is palmitoylated at the N terminus and has a cholesterol modification at the C terminus. Moreover, both of these modifications are believed to be important in its function as a long distance signaling molecule. Because clb encodes an enzyme which produces an intermediate for lipid biosynthesis, it would be reasonable to suppose that it might have a role in Hh modification. If this were the case, Hh protein expressed in SGP cells would be appropriately modified to function in long distance signaling, while Hh expressed in the fat body precursor cells would not (Deshpande, 2001).
Another important question is how the Hh signal actually promotes germ cell movement. In somatic cells, Hh signaling activates the Cubitus interruptus (Ci) transcription factor. At this point, it is not clear whether Ci is the target of the Hh ligand in the germ cells. Moreover, if it is, how would the transcription of Ci target genes direct either germ cell migration or the association with SGP cells? Further studies will be required to resolve this and other issues (Deshpande, 2001).
The genital disc consists of three primordia: moving from anterior to posterior they are the female genital primordium, the male genital primordium and the anal primordia. Only one of the two genital primordia develops, depending on the individual's sex, whereas the anal primordium develops in both sexes. It is proposed here that the genital disc, which is of ventral origin, is organized in a manner similar to the antennal and leg discs: the expression domains of decapentaplegic and wingless are mostly complementary and abut engrailed expression. An analysis was made of the roles of the genes hedgehog, patched, dpp and wg in the development of the three primordia that form the genital disc. The morphogenetic alterations produced by ectopic expression of hh mimic a lack of ptc function. Both genetic conditions cause derepression of dpp and wg. Ectopic expression of either of these genes causes non-autonomous duplications and/or reductions of genital and anal structures. Some of these alterations are explained by the mutual repression of wg and dpp. In the development of the genital disc, the functional relationships between these genes seem to be analogous to those described for leg and antennal discs: dpp and wg are induced in the anterior compartment by Hh protein blocking the repressive effect of Ptc, and the mutual repression of dpp and wg restrict one another to their respective domains. It may be concluded that dpp and wg act as general organizers for development of the genital disc (Sanchez, 1997).
Hedgehog and Oogenesis
hedgehog is expressed in terminal filament cells and associated somatic cells at the extreme apical end of fly ovarioles. hh activity stimulates the proliferation of pre-follicular somatic cells, and promotes the specification of polar follicle cells. During egg chamber assembly, hh signaling appears to be associated with the pathways involving neurogenic genes. Egg chamber production involves the specification of several somatic cell types, including polar cells and stalk cells. Polar cells (somatic cells usually present at both anterior and posterior poles of the oocyte) appear ectopically throughout egg chambers exposed to elevated levels of HH. Reduced activity of Notch and Delta also causes the production of an increased number of polar cells at the ends of egg chambers, as well as the loss of stalk cell fate. Prior to egg chamber formation, hh signaling specifies the proper anterior-posterior orientation of polar cells, while cell-cell interactions, mediated by N and DL, ensure that only two cells maintain this fate (Forbes, 1996).
Two small subgroups of follicle cells have been central to several genetic investigations: the polar cells
and the stalk cells. Polar cells are sets of follicle cells located at the anterior and posterior tips of an egg
chamber; stalk cells are a linear group of follicle cells that separate the egg chambers.
Differentiation of these subgroups occurs when follicle cell intercalation separates egg chambers from
the germarium, where germ line division takes place. The fates of these two follicle cell subgroups appear to be linked: mutations in Notch,
Delta, fs(1)Yb, or hedgehog cause simultaneous defects in the specification of stalk cells and polar
cells. Both of these subgroups are determined in the germarium, and both cease division early in
oogenesis. To test the possibility that these subgroups are related by lineage, dominantly
marked mitotic clones in ovaries were generated. Small, restricted clones in stalk cells and polar cells are found
adjacent to each other at a frequency much too high to be explained by independent induction. A model is therefore proposed in which stalk cells and polar cells are derived from a precursor population
that is distinct from the precursors for other follicle cells. This model is supported and extended by
characterization of mutants that affect stalk and polar cell formation. Ectopic expression
of Hedgehog can induce both polar and stalk cell fate, presumably by acting on the precursor stage. In
contrast, stall affects neither the induction of the precursors nor the decision between the
stalk cell and polar cell fate but, rather, some later differentiation step of stalk cells. In addition, ectopic polar and stalk cells disturb the anterior-posterior polarity of the underlying oocyte (Tworoger, 1999).
A group of mutants that share a morphological defect in stalk formation have been identified.
Loss of
function of Notch, Delta, fs(1)Yb, daughterless, hedgehog, toucan, or stall results in large germaria
that accumulate cysts and do not form stalks; overexpression of constitutively active Notch (caN),
Delta, toucan, or hh results in long, stalk-like structures. Having now shown that stalk cells and polar
cells share a distinct precursor, it was asked if the lineage decision between these two groups is affected
in each of the mutants bearing a morphological defect in stalk formation. Lack of Notch activity is known to result in an excess of polar cells and loss of stalk cells because of a defect
in a precursor stage. Ectopic expression of Hedgehog in the germarium prolongs the precursor stage for stalk
cells and polar cells. In oogenesis, hypomorphic alleles of hh produce large germaria that
accumulate germline cells. Transient ectopic expression of hh results in
long, stalk-like structures and an excess of polar cells in ectopic positions. The cell fates were analyzed in these situations
in more detail using a persistent expression system. Receipt of the Hh signal was followed by a ptc enhancer trap line. Two
days after Hh induction, long, stalk-like structures expressing the ptc marker were detected. To analyze the identity of the cells in these long, stalk-like structures, they were stained with Big
Brain antibody. Bib marks the precursors for stalk cells and polar cells, but not the cells that surround
the rest of the egg chamber. The long, stalk-like structures generated by overproduction of Hh do not
contain differentiated polar cells or stalk cells. Instead, the follicle cells between 16 cell cysts resemble wild-type precursor
cells for these populations in morphology and expression of Bib and FasIII proteins. However, not all of these cells remain in a precursor stage: at later time points, some of the
cells in the long stalks 'leak through' the Hh-induced precursor block and differentiate to form extra
stalk or polar cells, further evidence that these cells are precursors for polar and stalk cells (Tworoger, 1999). Hedgehog can induce ectopic stalk cells and polar cells that coincide with a defective oocyte
anterior-posterior axis:
Three days after persistent Hh induction, patchy patched expression, indicative of Hh action, extends
to later-stage egg chambers. Interestingly, in addition to previously observed ectopic
polar cells,
ectopic stalk cells were observed in these egg chambers, suggesting that ectopic Hh can induce both
fates. Therefore, ectopic Hh affects not only the stalk cell and polar cell fates in their normal location,
but it can also induce these fates in ectopic locations, probably by transiently inducing the stalk and
polar cell precursor stage. Clonal analysis has shown that stalk and polar cells have a common
precursor that is separate from a precursor for egg chamber follicle cells. These data show that ectopic
Hh has a capacity to induce this precursor fate (Tworogera, 1999). Coinciding with the ectopic polar cells, an anterior-posterior axis defect is detected in the underlying
oocyte. The typical migration of the oocyte nucleus from the posterior to a dorsal-anterior location
fails to occur. In addition, Kin:ßgal fusion protein fails to localize posteriorly, and the
oocyte microtubule network is defective. These phenotypes have been
previously observed in mutants that compromise the Epidermal growth factor receptor or Notch
pathways in posterior follicle cells, thereby altering the follicle cell-oocyte signaling that is required for
proper anterior-posterior axis formation. stall mutants display defects in egg chamber separation from the germarium that are
morphologically comparable to the Notch loss-of-function defect. It was asked whether this defect is caused by
the elimination of stalk cell fate, as in Notchts mutants. The expression of the
enhancer trap line 93F, which marks stalk
cells, was examined in stall mutants. Although no stalk structures are detected, groups of
93F-positive cells are observed in the mutant germaria, suggesting that cells continue to acquire stalk
cell fate. The ratio of 93F-positive cells to germline cysts found in stall germaria is
consistent with the number of cells found in wild-type stalks. Because 93F marks the
terminal filament cells as well as the stalk cells in wild-type ovaries, it is formally possible that the
93F-positive cells in stall germaria represent terminal filament cells. To rule out this possibility, stall mutant ovaries were examined with an independent terminal filament marker. Visualization of stall mutant germaria displays only the wild-type terminal filament expression pattern. In
addition, some cells that express an exclusive stalk cell marker were detected in stall
mutant germaria. It was concluded that the 93F-positive
cells in stall mutant germaria are stalk cells. Furthermore, no obvious defect in the polar cell population
is detected in stall mutant germaria. These data suggest that the early lineage decision between polar cell and stalk cell fate
occurs normally in the stall mutant. It is concluded that stalk cells are defective in a subsequent
differentiation or migration step (Tworoger, 1999).
The localized expression of Hedgehog (Hh) at the extreme
anterior of Drosophila ovarioles suggests that it might
provide an asymmetric cue that patterns developing egg
chambers along the anteroposterior axis. Ectopic or
excessive Hh signaling disrupts egg chamber patterning
dramatically through primary effects at two developmental
stages. (1) Excess Hh signaling in somatic stem cells
stimulates somatic cell over-proliferation. This likely
disrupts the earliest interactions between somatic and
germline cells and may account for the frequent mis-positioning
of oocytes within egg chambers. (2) The
initiation of the developmental programs of follicle cell
lineages appears to be delayed by ectopic Hh signaling. This
may account for the formation of ectopic polar cells, the
extended proliferation of follicle cells and the defective
differentiation of posterior follicle cells, which, in turn,
disrupts polarity within the oocyte. Somatic cells in the
ovary cannot proliferate normally in the absence of Hh or
Smoothened activity. Loss of protein kinase A activity
restores the proliferation of somatic cells in the absence of
Hh activity and allows the formation of normally patterned
ovarioles. Hence, localized Hh is not essential to direct egg
chamber patterning (Zhang, 2000b).
Hh signaling in Drosophila generally regulates the abundance
and activity of Ci proteins without altering CI mRNA levels. By contrast, vertebrate Hh homologs
frequently regulate transcription of the Ci-related GLI family
of transcriptional effectors. The induction
of CI RNA in ptc mutant follicle cells provides the first evidence
that this circuitry can also be found in Drosophila.
Other consequences of altering the activity of Hh signaling
components in ovarian somatic cells substantiate the
hypothesis that Hh signaling activates at least two distinct
intracellular pathways. One pathway, involving protection of Ci-155
from proteolysis and perhaps also release from
cytoplasmic anchoring, is phenocopied by PKA and cos2
mutations. In the ovary, cos2 mutations elicit stronger
phenotypes than PKA mutations, perhaps because cos2
mutations preferentially disrupt cytoplasmic anchoring of Ci-155.
The second pathway increases the specific activity of Ci-155
in opposition to the inhibitory effects of Su(fu). This pathway is elicited by ptc, but not
by PKA mutations and requires Fu kinase activity. In
accordance with this model, PKA Su(fu) double mutant cells
produce phenotypes almost as strong as for ptc mutants in
ovaries, whereas ptc fu double mutant cells exhibit minimal
phenotypes and PKA mutant phenotypes are not greatly
altered by additional loss of Fu kinase activity.
In imaginal discs high level Hh signaling to nearby cells is
phenocopied by ptc mutations and requires Fu kinase activity,
whereas only low level Hh signaling to more distant cells can
be phenocopied by PKA mutations and does not require Fu
kinase activity. PKA mutations in somatic
ovarian cells can effectively substitute for Hh activity: Fu
kinase activity is not essential for somatic cell proliferation and ptc mutations engender excessive
Hh signaling phenotypes even in the absence of Hh activity.
Hence, it is surmised that ovarian somatic cells normally undergo
only low levels of Hh signaling, in keeping with the
observation that the source of Hh in the germarium is separated
from its target cells by several cell diameters (Zhang, 2000b).
The rescue of apparently normal oogenesis in hhts animals at
the restrictive temperature by PKA mutations in somatic stem
cells implies that there is no essential role for spatially graded
Hh levels in the germarium. However, the level of Hh signaling
must fall within certain bounds for oogenesis to proceed
normally. Normal rates of somatic cell proliferation require
some Hh signaling but also require that Ptc limits Hh signaling. Ptc must also restrain Hh signaling in
order to allow somatic cells to enter the developmental
program appropriate to their lineage in a timely fashion.
It is not clear at this stage whether Hh signaling has any
essential function in oogenesis other than stimulating cell
proliferation. In one case, normal egg chambers can include
smo mutant cells in a variety of positions. In particular, polar
cells can form in normal numbers and at the correct position
from within a group of smo mutant cells, which are presumed
to be unable to transduce any Hh signal. Alternatively,
in smo mutant ovarioles, egg chamber budding
is sometimes arrested or defective, and
normal egg chambers completely enveloped by smo mutant
follicle cells have never been seen. These phenotypes might derive solely from an
insufficient supply of somatic cells, resulting directly from
impaired proliferation of smo mutant cells. However, the possibility cannot be dismissed that Hh signaling has a more
direct role in germline cyst encapsulation, promoting egg
chamber budding, or delaying somatic cell lineage decisions
until the appropriate developmental stage (Zhang, 2000b).
Although Hedgehog proteins most commonly affect cell fate, they can also stimulate cell proliferation. In humans several distinctive cancers,
including basal-cell carcinoma, result from mutations that aberrantly activate Hh signal transduction. In Drosophila, Hh directly stimulates proliferation of
ovarian somatic cells. Hh acts specifically on stem cells in the Drosophila ovary. These cells cannot proliferate as stem cells in the absence
of Hh signaling, whereas excessive Hh signaling produces supernumerary stem cells. It is deduced that Hh is a stem-cell factor and it is suggested that human cancers due to excessive Hh signaling might result from aberrant expansion of stem cell pools (Y. Zhang, 2001).
In adult Drosophila females, egg chambers are produced continuously in the germarium of each of the 15-18 ovarioles that are bundled together to form an ovary. In regions 1 and 2a of the germarium, 16-cell germline cysts develop from germline stem cells. Each cyst is enveloped by a monolayer of follicle cells in region 2b and separated from the next cyst by a short stalk as it buds from region 3 to form an egg chamber. Follicle and stalk cells derive from somatic stem cells that reside at the region 2a/2b border. When a somatic stem cell divides, one daughter retains a stem cell identity and continues to divide as a stem cell for several days. The other 'pre-follicle cell' daughter proliferates for about eight cycles as its progeny associate with germline cysts, pass posteriorly down the ovariole over a five to six day period, and differentiate into multiple specialized cell types. Hedgehog (Hh) is expressed selectively in specialized non-proliferating, somatic 'terminal filament' and 'cap' cells at the anterior tip of the germarium. Inactivation of Hh, using conditional hh alleles, arrests egg chamber budding, and causes germline cysts to accumulate in swollen germaria, suggesting that too few follicle cells are being produced. Conversely, excessive Hh signaling in germarial region 2 can be induced by temporally controlled activation of an hh transgene or by inactivation of the Hh receptor Patched (Ptc), and causes marked overproliferation of somatic cells, which accumulate between egg chambers (Y. Zhang, 2001).
The proliferative response to excessive Hh signal transduction was investigated further by using antibodies against phospho-histone H3, which stains cells only during mitosis. High levels of Hh signal transduction were induced by inactivating patched (ptc) in marked somatic cell clones generated by heat-shock induced mitotic recombination. Ovaries were examined 8 d later to ensure that all proliferating somatic cells assayed derived from stem cells that were present at the time of clone induction. As each ovariole contains more than one somatic stem cell, this procedure generates some ovarioles containing only ptc mutant somatic cells and others that are mosaic for wild-type and ptc mutant cells. The number of mitotic somatic cells in germarial region 2 in ovarioles containing only ptc mutant somatic cells was twice that in wild-type ovarioles. A similar ratio was observed in region 3 of the germarium and in newly budded (stage 1-2) egg chambers, suggesting an early increase in proliferation of ptc mutant cells followed by wild-type rates of proliferation of a twofold enlarged cell population. Accordingly, the follicular monolayers surrounding stage 6 egg chambers in wild-type and ptc mutant ovarioles contained almost identical numbers of mitotic cells within numerically equivalent cell populations. In ovarioles mosaic for ptc mutant and wild-type somatic cells, the number of cells in mitosis was increased roughly 1.5-fold in region 2; again, this ratio was maintained in region 3 and in the earliest egg chambers. This is consistent with a cell-autonomous effect of ptc inactivation, affecting roughly half of the somatic cells in a mosaic germarium (Y. Zhang, 2001).
No definitive positive marker for ovarian somatic stem cells has been described. However, when a single wild-type lineage of proliferating somatic cells is labelled by a stem cell recombination event, only one cell at the most anterior position fails to stain with Profilin or Fasciclin III, suggesting that this cell is a somatic stem cell. Ovarioles were therefore double-stained with antibodies against Fasciclin III (Fas III) and phospho-histone H3, and counted the number of proliferating cells at the border of region 2a/2b that failed to stain with Fas III. The number of such putative stem cells in mitosis in pure ptc mutant ovarioles was roughly twice (0.17/0.08) that in wild-type ovarioles. This shows that excessive Hh signal transduction promotes somatic stem cell proliferation and that this might account entirely for the ensuing somatic cell overproliferation (Y. Zhang, 2001).
To establish whether excessive Hh signaling accelerates somatic stem cell cycles or increases the number of stem cells in an ovariole, stem cells were counted by using mitotic recombination. Thus, in almost every ovariole examined, a loss of ptc activity led to a cell-autonomous doubling of somatic stem cell number. In 19 of these instances, the two ptc mutant stem cells were directly adjacent, suggesting that excessive Hh signal transduction allowed local expansion of a stem cell niche. In the remaining 12 cases the additional ptc mutant stem cell had migrated away from its presumed sister cell (Y. Zhang, 2001).
Whether Hh is required for somatic stem cell maintenance or proliferation was investigated by using conditional hh alleles and by generating somatic cell clones lacking smoothened (smo) activity. Inactivation of smo universally blocks Hh signal transduction cell-autonomously. The results demonstrate that a cell that is unable to transduce an Hh signal cannot proliferate as a somatic stem cell. It is suspected that smo mutant somatic stem cells remain abnormally quiescent for up to 7-8 d and at some point during this period acquire the characteristics of a pre-follicle cell, proliferating normally in that capacity to produce a clone occupying roughly one-third of an egg chamber (Y. Zhang, 2001).
In hhts2 animals shifted to the restrictive temperature of 29°C a statistically significant reduction in the number of mitotic cells labelled by phospho-histone H3 antibody was seen in region 2b by 72 h, and in germarial region 3 by 96 h. Double-staining with Fas III antibody shows that the number of stem cells undergoing mitosis is already reduced by 48 h and is undetectable by 72 h. These reductions are not statistically significant because wild-type ovarioles sampled at these time points collectively contained only two or three somatic stem cells. Nevertheless, these mitotic labelling data are the first direct evidence that Hh inactivation reduces somatic cell proliferation and are consistent with a primary effect on stem cell proliferation (Y. Zhang, 2001).
The effects of Hh signaling on cell fate determination in Drosophila are mediated largely by altering the activity of the transcription factor Cubitus interruptus (Ci). The role of Ci in somatic stem cell proliferation was examined by inducing somatic clones lacking ci activity. As with smo, very few clones were recovered 8-10 d after clone induction and these clones occupied only a small proportion of the ovariole, indicating that stem cells cannot proliferate normally in the absence of ci activity. When the expression of a constitutively active derivative of Ci was induced by heat-shock-induced excision of a transcriptional terminator, ovarioles were recovered showing massive overproliferation of somatic cells, which accumulated between egg chambers as observed for ptc mutant ovarioles. Thus, the activity of Hh as a stem cell factor seems to depend on Ci-mediated regulation of transcription (Y. Zhang, 2001).
Somatic stem cells seem to have a limited lifespan. The longevity of wild-type ovarian somatic stem cells was examined by inducing marked clones in third-instar larvae and examining adult ovaries 8-14 d later. The proportion of mosaic ovarioles declined from 56% (in 3-day-old adults) to 32% (in 9-day-old adults) during this period, indicating a loss of some of the stem cells present at the time of clone induction. To see whether these stem cells were being replaced, a complementary experiment was performed in which clones were induced in adults of various ages and then assayed 8 d later. The frequency of mosaic ovarioles was unchanged in adults from zero to 12 d after eclosion, suggesting that the total number of somatic stem cells was constant over this period. It is therefore deduced that, on average, wild-type stem cells are lost roughly every 8-9 d but are subsequently replaced by division of another stem cell in the same ovariole to produce two sister stem cells (Y. Zhang, 2001).
All of these observations can most easily be rationalized by postulating that there are only a limited number of positions (literally three) within a germarium where somatic cells receive the correct combination of extracellular signals to instruct them to behave as stem cells. In a wild-type ovariole one of the two daughters of a stem cell division necessarily leaves this environment and therefore does not adopt a stem cell fate. Occasionally, a stem cell might leave its niche and proliferate as a pre-follicle cell. The vacant niche becomes occupied shortly afterwards by the daughter of another stem cell. In this model the division of stem cells is not intrinsically asymmetric. Rather, the different behavior of the two daughters is dictated by their physical environment, so that both daughters can become stem cells if two stem cell niches are vacant. Hh seems to be one of the key environmental signals for directing somatic stem cell behavior. Proliferation of a stem cell requires Hh signal transduction; excessive Hh signal transduction allows expansion of the stem cell niche. These results suggest that each stem cell niche can accommodate two stem cells if these cells are undergoing excessive Hh signal transduction (Y. Zhang, 2001).
Because Hh signaling, like all major signaling pathways, is used in a large number of contexts, there is no compelling reason to expect that the proliferative effects of Hh proteins are always targeted to stem cells. However, several characteristics of normal and deregulated development of vertebrate epidermis and hair follicles prompt the suggestion that Sonic hedgehog (Shh) might also act on stem cells in this context. Shh is required for hair follicle development in mice, and Shh mutant phenotypes include reduced proliferation of ectodermal stem cell derivatives. Conversely, inappropriate mutational activation of the Shh signal transduction pathway is universally observed in human familial and sporadic basal-cell carcinomas (BCCs). Several observations suggest that activation of this pathway might be obligatory in the etiology of BCC and can suffice to initiate this cancer. The cellular origin of BCC has not been defined, but outer root sheath cells of hair follicles, which include epidermal and follicular stem cells in a specialized bulged region, are strongly implicated. In the light of the present studies, it is suggested that proliferation of follicular stem cells might normally require Shh, among other factors, and that mutational activation of the Shh signaling pathway in BCC acts specifically on these stem cells to increase their cycling frequency or their cell number, or perhaps even to expand their niche into or towards the surrounding epithelium. Any of these changes could produce a sustainable large increase in a population of transiently proliferating cells, which would constitute the bulk of the carcinoma. At present these hypotheses concerning Hh-associated cancers are speculative and remain to be tested (Y. Zhang, 2001).
The sex determination master switch, Sex-lethal, regulates the mitosis of early germ cells in Drosophila. Sex-lethal is an RNA binding protein that regulates splicing and translation of specific targets in the soma, but the germline targets are
unknown. In an immunoprecipitation experiment aimed at identifying targets of Sex-lethal in early germ cells, the RNA encoded by gutfeeling, the Drosophila homolog of ornithine decarboxylase (ODC) antizyme, was isolated (Vied, 2003).
Mammalian Antizyme negatively regulates ODC catalytically as well by directing the inactivated enzyme to the proteasome for degradation. This negative regulation of ODC is part of a feedback loop that controls the levels of polyamines within the cell. Translation of Antizyme is dependent on ribosomal frameshifting, which is promoted by high levels of polyamines. As polyamine levels in the cell rise, more Antizyme is synthesized, leading to the turnover of ODC. Polyamines have been implicated in many processes, including cell growth, transcription, and differentiation. In mammals Antizyme and ubiquitin are thought to be respectively two types of proteasome targeting devices that mark proteins for both ubiquitin-independent and ubiquitin-dependent degradation by the 26 S proteasome (Vied, 2003 and references therein).
Drosophila gutfeeling interacts genetically with Sex-lethal. It is not only a target of Sex-lethal, but also appears to regulate the nuclear entry and overall levels of Sex-lethal in early germ cells. This regulation of Sex-lethal by gutfeeling appears to occur downstream of the Hedgehog signal. Gutfeeling appears to regulate the nuclear entry of Cyclin B as well. Hedgehog, Gutfeeling, and Sex-lethal function to regulate Cyclin B, providing a link between Sex-lethal and mitosis (Vied, 2003).
In Drosophila, Cyclin B is not essential for viability but
is necessary for female fertility. Since Sxl has been shown to be
important for the mitosis of early germ cells,
a correlation between Sxl and Cyclin B was sought. In early germ cells,
Cyclin B colocalizes with Sxl and is downregulated concomitantly
with Sxl. Since Sxl and Cyclin B colocalize in early germ cells, the effect of Hh on Cyclin B was examined. Overexpression of
Hh results in fewer Cyclin B-expressing early germ cells,
as seen for Sxl. Furthermore, the germ cells that
express Cyclin B show reduced levels of the protein. This
similarity in response prompted a test to see whether Hh also
regulates the nuclear entry of Cyclin B. Overexpression of
Hh and treatment with LMB results in higher levels of
nuclear Cyclin B than in wild-type early germ cells treated
with LMB only. This observation suggests that
Hh promotes the nuclear entry of Cyclin B (Vied, 2003).
Since Guf appears to act downstream of Hh to regulate Sxl,
whether Cyclin B would also respond to Guf was also examined.
Ectopic expression of Guf has
the same effect on Cyclin B as on Sxl. Fewer early germ
cells with cytoplasmic Cyclin B were observed, and Sxl and
Cyclin B continued to colocalize. LMB treatment
with Guf overexpression increases the nuclear levels
of Cyclin B. As for Sxl, Cyclin B responds
more strongly to Guf than Hh (Vied, 2003).
To determine whether Guf also acts downstream of Hh in
affecting Cyclin B nuclear entry, hs-hh; guf118-3/+ ovaries treated with LMB were examined. Under these conditions,
Cyclin B was predominantly cytoplasmic,
suggesting that Guf also functions downstream of Hh in the
regulation of Cyclin B (Vied, 2003).
In vertebrates, the intracellular localization of Cyclin B is
critical to its function. An NES within the cytoplasmic
retention signal (CRS) allows Cyclin B to rapidly shuttle out
of the nucleus. Phosphorylation of the CRS results in nuclear
accumulation of Cyclin B and its associated Cyclin-dependent kinase with which Cyclin B initiates mitosis. Since Sxl and Cyclin B appear to undergo similar changes in response to Hh and
Guf, tests were made to determine whether Cyclin B is affected by changes in
Sxl.
Cyclin B was examined in ovaries with mutant Sxl
protein. Like Sxl, Cyclin B is localized to the cytoplasm
of Sxlf4 germ cells. Treatment of Sxlf4 ovaries with LMB
caused the Sxl, but little Cyclin B, protein to accumulate
in the germ cell nuclei. As overexpression of
Hh with LMB treatment increases the nuclear levels of
both Sxl and Cyclin B in wild-type ovaries,
the intracellular localization of both proteins was examined in Sxlf4
ovaries after similar treatment. The mutant Sxl protein
accumulates in the nuclei, but surprisingly, Cyclin B
remains in the cytoplasm. Consistent with
this observation, ovaries doubly homozygous for Sxlf4
and Su(fu) treated with LMB show nuclear accumulation
of Sxlf4 protein, but not Cyclin B. These
results suggest that, normally, Sxl affects the rate of
nuclear entry of Cyclin B. Interestingly, the mutations in
the Sxlfs alleles alter a region of the protein that is
proline-rich. Proline-rich domains
have been described as being involved in protein-protein
interactions (Vied, 2003).
Sxl can enter the nucleus without the concomitant
entry of Cyclin B. Cyclin B mutant ovaries were examined
to test the assumption that Sxl does not require Cyclin B
for nuclear exiting. A null mutation for cyclin B derived
from the imprecise excision of a P-element in the 5' UTR
(cycB3) is not lethal except when combined with other
cell cycle regulators. The
cycB3 mutation results in female sterility with many agametic
ovarioles. When germ cells were
present, the localization of Sxl was primarily cytoplasmic.
Treatment with LMB shows that Sxl is able to
enter the nucleus at levels comparable to those observed
in wild-type ovaries\. These results suggest
that Sxl does not require Cyclin B for nuclear exiting. Otherwise, Sxl would be primarily nuclear in the absence of Cyclin B (Vied, 2003).
hedgehog
Biological Overview | Evolutionary Homologs | Regulation
| Targets of Activity | Protein Interactions | Developmental Biology | References
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