Dichaete/Sox box protein 70D

DEVELOPMENTAL BIOLOGY

Embryonic

Dichaete transcription is first detected in cycle 13 syncytial blastoderm embryos as a wide circumferential band, corresponding to the entire trunk region. This trunk ectodermal expression rapidly splits into two subdomains and, by early cycle 14, high levels of transcript are present in a narrow stripe at approximately 50% egg length and a wider stripe from about 15-30% egg length (nearer the posterior). At this time, transcripts become detectable in the procephalic region. During cellularization, expression is quickly refined into a series of seven irregular stripes and a strong dorsal 'saddle.' Some stripes stain more intensely than others, and the stripes are not evenly spaced. These stripes both overlap and flank specific fushi tarazu stripes. Thus Dichaete stripe 6 corresponds to parasegment 11, between ftz stripes 5 and 6, while stripe 7 is coincident with ftz stripe 7 in parasegment 14 (Nambu, 1996). During stage 6, the posterior stripe follows the pole cells as the germ band extends and eventually fades as the pole cells are internalized at the amnioproctodeal invagination.

During gastrulation and early germ-band extension the seven ectodermal stripes diminish and are replaced by two longitudinal columns of expression that are approximately 4 cells wide and flank the invaginating mesoderm. Expression is maintained in the developing cephalic neuroectoderm. Later expression is detected in subsets of cells in the brain and CNS midline, the hindgut and segmentally repeated stripes of cells along the ventral epidermis (Nambu, 1996).

During cellularization, the band of Dichaete protein bifurcates; expression is also detected in the cephalic neuroectoderm. This pattern quickly resolves into a series of irregular stripes and a dorsal saddle of cells during cellularization. During germ-band extension the stripes of protein are replaced by broad expression in the developing cephalic and ventral neurogenic ectoderm. Dichaete protein also is expressed in the developing hindgut from about stage 10 onwards. As neurogenesis continues, Dichaete continues to be broadly expressed in the CNS. During germ band retraction, Dichaete expression becomes much more restricted: it is present in a subset of cells in the brain, in small lateral clusters of cells in the thoracic ganglia, and in a single bilaterally paired cell in each of the abdominal ganglia. In abdominal ganglia A8 and A9, Dichaete expression is also detected in small clusters of CNS midline cells. Dichaete continues to be expressed strongly in the hindgut and in a series of ventral epidermal stripes. In stage 16 embryos Dichaete proteins are detected in chordotonal neurons of the peripheral nervous system, as well as in a small set of ventral muscle fiber nuclei. In germ-band extending embryos Dichaete protein is distributed in both nuclear and cytoplamsic compartments of neuroectodermal cells. However, in germ-band retracted embryos, Dichaete is restricted to the nuclei of CNS, PNS, hindgut, and muscle cells (Ma, 1998).

SOX-domain proteins are a class of developmentally important transcriptional regulators related to the mammalian testis determining factor SRY. In common with other SOX-domain genes, the Drosophila Dichaete gene has a dynamic expression profile in the developing central nervous system, including cells of the ventral midline. As expected for a putative transcription factor, the protein is nuclear in most tissues; however, in some cells, particularly in the neuroectoderm, high levels are found cytoplasmically. The significance of this observation is at present unclear. The pattern of Dichaete expression is very similar to that of Dichaete mRNA. An early dynamic phase takes place, similar to that shown by other zygotic transcriptional regulators involved in segmentation. This expression is followed, after gastrulation, by widespread expression in the central nervous system (CNS) of the trunk and head. Dichaete is first detected in the neuroectoderm at late stage 6 to early stage 7. The expression is very dynamic and over the course of the next few hours waves of neuroblasts initiate Dichaete expression. By stage 16, Dichaete is restricted to two clusters of cells in each of the thoracic segments and a single cell in each of the abdominal segments. Dichaete is also localized in the hindgut, brain and the chordotonal organs of peripheral nervous system. Dichaete is localized in the developing midline; it is first detected very weakly at early stage 7 and by stage 9 strongly labels the two rows of midline progenitors . As development proceeds, expression is lost from some midline cells. These Dichaete-expressing cells were mapped with the enhancer trap line AA142, which, from stage 12, expresses lacZ in the anterior midline glia (MGA) and the middle midline glia (MGM) and at much lower levels in the posterior midline glia (MGP). At stage 12, Dichaete colocalises with beta-gal from the AA142 enhancer trap in all three pairs of midline glia and, as with AA142, expression is strongest in the MGA and MGM. By stage 14, Dichaete is now found at high levels in MGP and at much lower levels in MGA and MGM; this is in contrast to AA142, where the MGA and MGM continue to express lacZ at high levels, Thus, Dichaete expression in the midline is dynamically modulated, as in the neuroectoderm (Soriano, 1998).

Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila

The timing mechanisms responsible for terminating cell proliferation toward the end of development remain unclear. In the Drosophila CNS, individual progenitors called neuroblasts are known to express a series of transcription factors endowing daughter neurons with different temporal identities. This study shows that Castor and Seven-Up, members of this temporal series, regulate key events in many different neuroblast lineages during late neurogenesis. First, they schedule a switch in the cell size and identity of neurons involving the targets Chinmo and Broad Complex. Second, they regulate the time at which neuroblasts undergo Prospero-dependent cell-cycle exit or Reaper/Hid/Grim-dependent apoptosis. Both types of progenitor termination require the combined action of a late phase of the temporal series and indirect feedforward via Castor targets such as Grainyhead and Dichaete. These studies identify the timing mechanism ending CNS proliferation and reveal how aging progenitors transduce bursts of transcription factors into long-lasting changes in cell proliferation and cell identity (Maurange, 2008).

Initially investigated was whether distinct temporal subsets of neurons are generated throughout the larval CNS. Chinmo and Broad Complex (Br-C), two BTB-zinc finger proteins known to be expressed in the postembryonic CNS, are distributed in complementary patterns in the central brain, thoracic, and abdominal neuromeres at the larval/prepupal transition stage at 96 hr (timings are relative to larval hatching at 0 hr). Chinmo is expressed by early-born neurons located in a deep layer, whereas Br-C marks later-born neurons in a largely nonoverlapping and more superficial layer. The deep Chinmo⁺ layer comprises most/all neurons born in the embryo plus an early subset of those generated postembryonically. Thoracic postembryonic neuroblasts undergo the Chinmo --> Br-C switch at ~60 hr such that they have each generated an average of 15 Chinmo⁺ cells expressing little or no Br-C and 39 Chinmo^- Br-C⁺ cells by 96 hr. The Chinmo⁺ and Br-C⁺ neuronal identities can be recognized as distinct cell populations on the basis of an ~2-fold difference in cell-body volume. This equates to an average cell-body diameter for Chinmo⁺ neurons of 4.5 μm, compared to only 3.6 μm for Br-C⁺ neurons. Plotting cell diameter versus deep-to-superficial position within postembryonic neuroblast clones reveals an abrupt decrease in neuronal size at the Chinmo --> Br-C transition. Together, these results provide evidence that most, if not all, postembryonic neuroblasts sequentially generate at least two different populations of neurons. First they generate large Chinmo⁺ neurons and then they switch to producing smaller Br-C⁺ neurons (Maurange, 2008).

To begin dissecting the neuronal switching mechanism, the functions of Chinmo and Br-C were investigated, but neither factor was found to be required for the transition in cell identity and cell size. Next it was asked whether a temporal transcription factor series related to the embryonic Hb --> Kr --> Pdm --> Cas sequence might be involved. Cas is known to be expressed in the larval CNS, and this study shows that many different thoracic neuroblasts progress through a transient Cas⁺ phase during the 30-50 hr time window. Thoracic neuroblasts transiently express another member of the embryonic temporal series, Svp, during a somewhat later time window, from ~40 to ~60 hr. These results indicate that postembryonic Cas and Svp bursts are observed in many, but probably not all, thoracic progenitors and that their timing varies from neuroblast to neuroblast (Maurange, 2008).

To determine Svp function, thoracic neuroblast clones were generated homozygous for svp^e22, an amorphic allele. In ~53% of svp^e22 neuroblast clones induced in the early larva (at 12-36 hr), the Br-C⁺ neuronal identity is completely absent, all neurons express Chinmo, and there is no sharp decrease in neuronal size. The proportion of lineages failing to generate Br-C⁺ neurons rises to ~70% when clones are induced in the embryo and falls to only ~7% with late-larval (65-75 hr) induction. This is consistent with a previous finding that Svp bursts are asynchronous from neuroblast to neuroblast. The expression and clonal analyses together demonstrate that a progenitor-specific burst of Svp is required in many lineages for the switch from large Chinmo⁺ to small Br-C⁺ neurons (Maurange, 2008).

Thoracic neuroblast lineages homozygous for a strong cas allele, cas²⁴, show no obvious defects in the Chinmo --> Br-C transition when induced at 12-36 hr. However, since Cas is expressed in many postembryonic neuroblasts before their first larval division, it can only be removed by inducing clones during embryonic neurogenesis. Such cas²⁴ clones generate supernumerary Chinmo⁺ neurons and completely lack Br-C⁺ neurons at 96 hr, although this switching phenotype is restricted to only ~16% of thoracic neuroblasts. Constitutively expressing Cas blocks the Chinmo --> Br-C switch in a similar manner, with a frequency dependent upon whether thoracic UAS-cas clones are induced during embryogenesis (~47%), at early-larval (~10%) or at late-larval (0%) stages. This indicates that the response to Cas misexpression decreases as neuroblasts age. Together, the expression and loss- and gain-of-function analyses demonstrate that Chinmo and Br-C are negative and positive targets, respectively, of Cas and Svp. They also strongly suggest that progression through transient Cas⁺ and Svp⁺ states permits many postembryonic neuroblasts to switch from generating large to small neurons (Maurange, 2008).

To investigate whether Cas and Svp regulate neural proliferation as well as neuronal fates, the effector mechanism ending neurogenesis in the central brain and thorax was identified. In these regions, most neuroblasts cease dividing in the pupa at ~120 hr. Correspondingly, neurogenesis in all regions of the wild-type CNS ceases before the adult fly ecloses such that no adult neuroblasts are detected. In contrast to the central abdomen, blocking cell death by removing Reaper, Hid, and/or Grim (RHG) activity in the central brain and thorax does not prevent or delay pupal neuroblast disappearance. However, time-lapse movies of individual thoracic neuroblasts at ~120 hr reveal an atypical mitosis that is much slower than at ~96 hr, producing two daughters of almost equal size. This is largely accounted for by a reduction in the average diameter of neuroblasts from 10.4 μm at 96 hr to 7 μm at 120 hr, as GMC size does not vary significantly during this time window. The end of this atypical progenitor mitosis temporally correlates with reduced numbers of Mira⁺ cells and disappearance of the M phase marker phosphorylated-Histone H3 (PH3), indicating that it marks the terminal division of the neuroblast (Maurange, 2008).

Next whether late changes in basal complex components might underlie loss of neuroblast self-renewal was addressed. At 120 hr, it was found that Mira becomes delocalized from the cortex to the cytoplasm and nucleus of many interphase neuroblasts. In metaphase neuroblasts, Mira fails to localize to the basal side of the cortex, although it does selectively partition into one daughter during telophase. This late basal restoration resembles the 'telophase rescue' associated with several apical complex mutations. Pros, the Mira-binding transcription factor and GMC-determinant, is not detectable in the neuroblast nucleus at 96 hr, but at 120 hr a burst of Pros was observe in the nucleus of many Mira⁺ cells of intermediate size indicative of neuroblasts in the interphase preceding the terminal mitosis. Clones lacking Pros activity contain multiple Mira⁺ neuroblast-like cells. It was found that they do not respect the ~120 hr proliferation endpoint and even retain numerous dividing Mira⁺ progenitors into adulthood. In addition, GAL80^ts induction was used to induce transiently the expression of a YFP-Pros fusion protein well before the normal ~120 hr endpoint. Under these conditions, YFP-Pros can be observed in the nucleus of neuroblasts, most Mira⁺ progenitors disappear prematurely, and neural proliferation ceases much earlier than normal. Together, these results provide evidence that most neuroblasts terminate activity in the pupa via a nuclear burst of Pros that induces cell-cycle exit. These progenitors are referred to as type I neuroblasts to distinguish them from the much smaller population of type II neuroblasts that terminate via RHG-dependent apoptosis (Maurange, 2008).

Whether the postembryonic pulses of Cas and Svp in type I neuroblasts are implicated in scheduling their subsequent cell-cycle exit was tested. Remarkably, it was observed that many svp^e22 clones induced at early-larval stages retain a single Mira⁺ neuroblast at 7 days into adulthood. The persistent adult neuroblasts in a proportion of these clones also express the M phase marker, PH3, indicating that they remain engaged in the cell cycle and, accordingly, they generate approximately twice the normal number of cells by 3 days into adulthood. Furthermore, superficial last-born neurons in these over proliferating svp^e22 adult clones are Chinmo⁺ Br-C^- indicating a blocked Chinmo --> Br-C transition. Similar phenotypes are obtained in some UAS-cas and cas²⁴ clones. This analysis demonstrates that stalling the temporal series not only inhibits the late-larval switch to Br-C⁺ neuronal identity but also prevents the pupal cell-cycle exit of type I neuroblasts (Maurange, 2008).

To test the regulatory relationship between Pros and the temporal series, svp^e22 clones were examined at pupal stages. It was found that mutant interphase neuroblasts fail to switch on nuclear Pros at 120 hr, although svp^e22 GMCs express nuclear Pros as normal. This likely accounts for why adult clones lacking Svp retain only a single neuroblast, whereas those lacking Pros contain multiple neuroblast-like progenitors. Importantly, these results demonstrate that nuclear Pros acts downstream of the temporal series in type I neuroblasts. Together, the genetic and expression analyses of Svp and Pros show that the temporal series triggers a burst of nuclear Pros in type I neuroblasts, thus inducing their cell-cycle exit (Maurange, 2008).

To determine how the temporal series is linked to the cessation of progenitor divisions, two transcription factors expressed in neuroblasts in a temporally restricted manner were examined. Dichaete (D), a member of the SoxB family, is dynamically expressed in the early embryo and is required for neuroblast formation. Consistent with the previous studies, it was observed that most or all embryonic neuroblasts progress through a transient D⁺ phase, but those in the lateral column of the ventral nerve cord initiate expression after their medial and intermediate counterparts. Dichaete subsequently becomes repressed in ~85% of neuroblasts during late-embryonic and postembryonic stages. Grainyhead (Grh) is first activated in neuroblasts in the late embryo and is required for regulating their mitotic activity during larval stages. Blocking early temporal series progression in the embryo, either by persistent Hb or loss of Cas activity, prevents most neuroblasts from downregulating D and also from activating Grh at late-embryonic and postembryonic stages. As forcing premature Cas expression leads to precocious D repression and Grh activation, both factors are likely to be regulated by Cas rather than by a later member of the temporal series. These results demonstrate that transient embryonic Cas activity permanently switches the expression of Grh on and D off. They also identify Grh and D as positive and negative targets, respectively, of the temporal series in many neuroblasts (Maurange, 2008).

Loss of Grh activity in thoracic neuroblasts (here defined as type I neuroblasts) leads to their reduced cell-cycle speed and disappearance during larval stages. At 96 hr, it was observed that 65% of grh³⁷⁰ type I neuroblasts are smaller than normal (~6.4 μm in diameter), delocalize Mira from the cortex to the cytoplasm and nucleus, and strongly express Pros in the nucleus. These events, reminiscent of the 120 hr terminal cell cycle of wild-type progenitors, show that Grh is required to prevent the premature cell-cycle exit of type I neuroblasts. Given this finding, and that Cas is required to activate Grh, the question arises as to how some neuroblasts lacking embryonic Cas activity are able to continue dividing into adulthood. However, cas²⁴ neuroblasts persisting in adults all retain Grh, suggesting that they may derive from clones that lacked only the last of the two Cas pulses observed in some embryonic neuroblasts, perhaps retaining enough Cas activity to support Grh activation but not later progression of the temporal series (Maurange, 2008).

To determine if late temporal series inputs, after embryonic Cas, are also required to maintain the long-lasting postembryonic expression of Grh, svp^e22 clones were induced in early larvae. Although type I neuroblasts in these mutant clones have a stalled temporal series, they retain postembryonic Grh expression through to adult stages. Thus, two sequential inputs from the temporal series are required for type I neuroblasts to undergo timely Pros-dependent cell-cycle exit. First, embryonic Cas activity switches on sustained Grh expression, inhibiting premature nuclear Pros and permitting continued mitotic activity. Second, a late postembryonic input, requiring Svp, counteracts this activity of Grh by triggering a pupal burst of nuclear Pros (Maurange, 2008).

Despite undergoing premature cell-cycle exit, it was noticed that grh mutant neuroblasts can still generate both Chinmo⁺ and Br-C⁺ neurons. Thus, Grh is not required for the neuronal Chinmo --> Br-C switch. Conversely, neither Chinmo nor Br-C appears to be required postembryonically for neuroblast cell-cycle exit. In summary, the properties of both neuroblasts and neurons are regulated by downstream targets of the temporal series (Maurange, 2008).

Next whether the temporal series and its targets also function in type II neuroblasts, which terminate via RHG-dependent apoptosis rather than Pros-dependent cell-cycle exit, was tested. Focus was placed on one identified type II neuroblast in the central abdomen, called dl, which undergoes apoptosis at 70-75 hr and produces only a small postembryonic lineage of ~10 neurons. It was observed that the dl neuroblast expresses bursts of Cas (~45 to ~60 hr) and Svp (~62 to ~65 hr) and sequentially generates Chinmo⁺ (~7 deep) and Br-C⁺ (~3 superficial) neurons. Loss of Cas or Svp activity, or prevention of temporal series progression in several other ways all lead to a blocked Chinmo --> Br-C transition, a failure to die at 70-75 hr, and the subsequent generation of many supernumerary progeny. These results show that the temporal series performs similar functions in type I and type II neuroblast lineages, regulating both the Chinmo --> Br-C neuronal switch and the cessation of progenitor activity. Next, the mechanism linking the temporal series to RHG-dependent death of type II neuroblasts was tested. As for type I neuroblasts, dl progenitors in cas²⁴ clones, induced in the embryo, fail to activate Grh and repress D. Moreover, if grh activity is reduced, or if D is continuously misexpressed, dl progenitors persist long after 75 hr. Therefore, both of these early Cas-dependent events are essential for subsequent type-II neuroblast apoptosis. However, in contrast to members of the temporal series themselves, persistent misexpression of their target D does not block the Chinmo --> Br-C switch in the dl lineage. Thus, the D^- Grh⁺ state of type I and type II neuroblasts, installed via an embryonic Cas pulse, appears to be necessary for progenitor termination but not for Chinmo --> Br-C switching. Nevertheless, dl neuroblasts stalled at a postembryonic stage in svp^e22 and UAS-cas clones retain the D^- Grh⁺ code yet still fail to undergo apoptosis. Therefore, as with type I cell-cycle exit, timely type II apoptosis requires both embryonic Cas-dependent and postembryonic Svp-dependent inputs from the temporal series (Maurange, 2008).

Finally, the regulatory relationship between the temporal series and AbdA, a Hox protein transiently expressed in postembryonic type II (but not type I) neuroblasts and required for their apoptosis was dissected. dl neuroblasts lacking postembryonic Svp activity or persistently expressing Cas still retain AbdA expression yet do not die. This suggests that AbdA is unable to kill neuroblasts unless they progress, in a Svp-dependent manner, to a late Cas^- temporal state. To test this prediction directly, use was made of the previous finding that ectopic AbdA is sufficient to induce neuroblast apoptosis, albeit only within a late time window. Constitutive AbdA-induced apoptosis is efficiently suppressed by persistent Cas, not only in type II but also in type I neuroblasts. This result demonstrates that, in order to terminate, type II neuroblasts must progress to a late Cas^- state, thus acquiring a D^- Grh⁺ Cas^- AbdA⁺ code. It also suggests that AbdA is sufficient to intercept progression of the temporal series in type I neuroblasts, inducing an early type II-like termination (Maurange, 2008).

This study has found that the Drosophila CNS contains two distinct types of self-renewing progenitors: type I neuroblasts terminate divisions by cell-cycle withdrawal and type II neuroblasts via apoptosis. Despite these different exit strategies, both progenitor types use a similar molecular timer, the temporal series, to shut down proliferation and thus prevent CNS overgrowth. These findings demonstrate that the temporal series does considerably more than just modifying neurons; it also has multiple inputs into neural proliferation. The identification and analysis of several pan-lineage targets of the temporal series also begins to shed light on the mechanism by which developmental age modifies the properties of neuroblasts and neurons. Two targets, Chinmo and Br-C, are part of a downstream pathway temporally regulating the size and identity of neurons. Two other temporal series targets, Grh and D, function in neuroblasts to regulate Prospero/RHG activity, thereby setting the time at which proliferation ends. The temporal series regulates both cell proliferation and cell identity; a feedforward mechanism is proposed for generating combinatorial transcription factor codes during progenitor aging (Maurange, 2008).

This study has found that the temporal series regulates a widespread postembryonic switch in neuronal identity. Most, if not all, type I and type II neuroblasts first generate a deep layer of large Chinmo⁺ neurons and then switch to producing a superficial layer of small Br-C⁺ neurons. Two lines of evidence argue that this postembryonic neuronal switch is likely to be regulated by a continuation of the same temporal series controlling early/late neuronal identities in the embryo. First, the postembryonic Chinmo --> Br-C neuronal switch is promoted by the transient redeployment of two known components of the embryonic temporal series, Cas and Svp. Second, this switch remains inhibitable by misexpression of the other embryonic temporal factors such as Hb. Since both Cas and Svp are expressed somewhat earlier than the neuronal-size transition, it is likely that they promote bursts of later, as yet unknown, members of the temporal series that more directly regulate Chinmo and Br-C. Although neuronal functions for both BTB zinc-finger targets have yet to be characterized, a progressive early-to-late decrease in postmitotic Chinmo levels is known to regulate the temporal identities of mushroom-body neurons. The current results now suggest that this postmitotic gradient mechanism may be linked to, rather than independent from, the temporal series (Maurange, 2008).

Type I neuroblasts in clones lacking postembryonic Cas/Svp activity or retaining an early temporal factor, fail to express nuclear Pros during pupal stages and thus continue dividing long into adulthood. These overproliferating adult clones each contain only a single neuroblast, sharply contrasting with adult clones lacking Brat or Pros, in which there are multiple neuroblast-like progenitors. Hence, manipulations of the temporal series and its progenitor targets offer the prospect of immortalizing neural precursors in a controlled manner, without disrupting their self-renewing asymmetric divisions (Maurange, 2008).

This study demonstrates that type I and type II neuroblasts must progress through at least two critical phases of the temporal series in order to acquire the D^- Grh⁺ Cas^- combinatorial transcription factor code that precedes Pros/RHG activation. The early phase corresponds to embryonic Cas activity switching neuroblasts from D⁺ Grh^- to D^- Grh⁺ status. The equally essential, but less well-defined, late postembryonic phase of the temporal series requires transition to a Cas^- state and a late Svp burst. For type I neuroblasts, Grh and a late Cas^- temporal identity are both required for timely expression of nuclear Pros and subsequent cell-cycle withdrawal. For type II neuroblasts, these two inputs are also necessary for RHG-dependent apoptosis, with the additional requirement that D must remain repressed. Although the temporal series and its targets are similarly expressed in type I and type II neuroblasts, only the latter progenitors undergo a larval burst of AbdA. This AbdA expression is likely to be the final event required to convert the D^- Grh⁺ Cas^- state, installed by the temporal series, into the D^- Grh⁺ Cas^- AbdA⁺ combinatorial code for RHG-dependent apoptosis. This code prevents type II neuroblasts in the abdomen from reaching the end of the temporal series and accounts for why they generate fewer progeny and terminate earlier than their type I counterparts in the central brain and thorax (Maurange, 2008).

The data in this study support an indirect feedforward model for neuroblast aging. Key to this model is the finding that, although members of the temporal series are only expressed very transiently, some of their targets can be activated or repressed in a sustained manner, as observed for Chinmo/Br-C in neurons and also for Grh/D in neuroblasts. In principle, this indirect feedforward allows aging progenitors to acquire step-wise the combinatorial transcription factor codes modulating cell-cycle speed, growth-factor dependence, competence states, and neural potential. Like Drosophila neuroblasts, isolated mammalian cortical progenitors can sequentially generate neuronal fates in the correct in vivo order. These studies suggest that it will be important to investigate whether the transcription factors controlling this process also regulate cortical proliferation and whether their targets include BTB-zinc finger, Grh, SoxB, Prox, or proapoptotic proteins. Some insect/mammalian parallels seem likely, since it is known that Sox2 downregulation and Prox1 upregulation can both promote the cell-cycle exit of certain types of vertebrate neural progenitors (Dyer, 2003, Graham, 2003). Thus, although insect and mammalian neural progenitors do not appear to use the same sequence of temporal transcription factors, at least some of the more downstream components identified in this study might be functionally conserved (Maurange, 2008).

Effects of Mutation or Deletion

The embryonic phenotype of lethal alleles was examined in cuticle preparations. There are variable segmentation defects, which include deletions that remove half of the segments, as well as weaker partial deletions and also segment fusions; in all cases, the even numbered metameres are more often affected. There are also variable defects in head development. It is important to emphasize the variability in the phenotype. This may indicate that the gene may be acting as a supporting factor in segmentation, rather than as a specific factor (Russell, 1996 and Nambu, 1996).

The strong neuroectodermal expression of Dichaete suggests a potential role in nervous system development. Mutants exhibit severe and variable defects in CNS organization, with fusions between several adjacent neuromeres resulting in 3-4 fewer ganglia as compared to wild type. In some segments there is a narrowing of the longitudinal axon connectives, and fusion of the anterior and posterior axon commissures. During germ-band retraction nearly all segments begin to exhibit loss and/or fusion of midline and lateral engrailed-expressing CNS cells. Loss of CNS midline cells, including the median neuroblast and the VUM neurons, is likely responsible for at least some of the axon scaffold defects observed (Nambu, 1996).

The strong segmentation phenotype of Dichaete null alleles hampers an assessment of the role of Dichaete in neurogenesis. To circumvent this problem, analysis focused on the thoracic segments, since segmentation defects are largely restricted to the abdominal segments. One hypomorphic mutation encodes protein with a glycine-to-serine substitution in the second helix of the DNA-binding domain. This mutant protein can still bind DNA but with reduced sequence specificity suggesting it may be defective in target site recognition. Dichaete mutants show complete or partial fusion of commissures, thinning of longitudinals and a collapse of longitudinals towards the midline. In hemizygotes for the hypomorphic allele, 80% of the mutant embryos have neuropile defects and 30% have disruptions throughout the length of the nerve cord. The neuropile defects are due to a requirement for Dichaete in the nervous system and are not a consequence of defects in segmentation (Soriano, 1998).

Midline cells, especially the midline glia (MGL), are known to be essential for normal neuropile formation. Since Dichaete is expressed in midline progenitors and later in MGL, the fate of these cells was examined in Dichaete mutants. In wild-type embryos, at the end of stage 12 when the commissures are separating, 5-6 MGL in each segment strongly express lacZ from the AA142 enhancer trap line, specific for midline glia. In wild-type stage 16 embryos, the AA142-positive MGL are reduced to 4 cells per segment with 65% of the labelled nuclei located dorsal to the commissures, either over the anterior commissure or over the space separating anterior and posterior commissure. The remainder of the nuclei are ventral to and between both commissures. To examine the fate of these cells in Dichaete mutants, a recombinant chromosome carrying AA142 and a Dichaete deficiency was constructed. In null mutants at stage 16, an average of 2-3 AA142-positive cells are found per segment, a clear reduction compared to wild type. The remaining cells are almost always found ventral to the commissures and only very rarely dorsal to them. In addition to these changes in number and dorsoventral distribution, there are also changes in the anterior-posterior distribution of MGL. As an additional MGL marker, the expression of Slit protein was examined. In wild-type stage 16 embryos, Slit is localized on the surface of MGL and shows the glia completely ensheathing the anterior and posterior commissures. In Dichaete null mutants, there are few slit-expressing cells at stage 16, and those remaining are mostly located ventral to the commissures (Soriano, 1998).

The midline phenotypes of Dichaete mutations are rescued by expression of mouse SOX2. Vertebrate SOX2 proteins are the closest known relatives to Dichaete. The HMG-domains of Dichaete and SOX2 are 88% identical and both proteins bind to very similar sequences. To test for functional conservation, transgenic flies were generated that express the mouse SOX2 gene ectopically. SOX2 can efficiently substitute for Dichaete function in the midline. Outside of the DNA-binding domain the only other region of similarity between Dichaete and SOX2 is at the C terminus: this region is required for SOX2 function in a cell culture assay. A truncated form of Dichaete was generated, that lacks the C-terminal 75 amino acids. Truncated Dichaete driven by simGAL4 efficiently rescues the midline phenotypes. In the wing hinge, where ectopic expression of wild-type Dichaete induces structural deletions, truncated Dichaete is not functional. This suggests that the C-terminal domain has a context-dependent function that is not required in the midline (Soriano, 1998).

Since SOX2 can rescue Dichaete mutant phenotypes and is known to interact with the POU-domain transcription factor OCT-3, the possibility of a genetic interaction between Dichaete and the Drosophila POU-domain gene ventral veinless was examined. vvl is expressed in the midline and is required for correct MGL development. In embryos homozygous for the vvl ZM allele, which has reduced but detectable levels of Vvl, anterior and posterior commissures fail to separate correctly, the longitudinals are thinner and there are regions where they collapse towards the midline. As with Dichaete hypomorph, only a small number of neuromeres are affected. However, in Dichaete;vvl double mutants all phenotypes are far more pronounced and occur in almost every hemisegment demonstrating a strong synergistic effect on the development of the nerve cord. Staining with anti-Fasciclin II shows that most of the longitudinal axons cross the midline many times with a roundabout-like phenotype. The midline of the double mutant embryos was tested with anti-Slit and very few cells were found at stage 16. The few remaining cells stain very weakly and are found ventral to the commissures. Similar results are obtained with Argos mRNA. In single Dichaete and vvl mutants, Slit expression is only weakly affected. However, glial cells with reduced expression and aberrant morphology have been found. To support the contention that Dichaete and vvl interact, the consequences of ectopic expression of Dichaete and Vvl were examined. Ectopic expression of Vvl in segregating neuroblasts alone does not disrupt the neuropile. Expression of Dichaete alone causes weak defects in the commissures mainly thinning of posterior commissures and thickening of the anterior commissure. When Dichaete and Vvl are expressed together in neuroblasts, however, the neuropile phenotypes are far more severe. The longitudinals collapse toward the midline throughout the neuropile and, in some segments, commissures appear fused. In this case, unlike the Dichaete;vvl double mutant, anti-Fasciclin II staining shows collapse of the longitudinals toward the midline but they do not cross the midline. Taken together, these data suggest that, as in the mouse, SOX and POU domain transcription factors interact to regulate the expression of target genes (Soriano, 1998).

The Dichaete gene of Drosophila encodes a group B Sox protein related to mammalian Sox1, -2, and -3. Like these proteins, Dichate is widely and dynamically expressed throughout embryogenesis. In order to unravel new Dichaete functions, the organization of the Dichaete gene was characterized using a combination of regulatory mutant alleles and reporter gene constructs. Dichaete expression is tightly controlled during embryonic development by a complex of regulatory elements distributed over 25 kb downstream and 3 kb upstream of the transcription unit. A series of regulatory alleles which affect tissue-specific domains of Dichaete were used to demonstrate that Dichaete has functions in addition to those during segmentation and midline development that have been previously described. (1) Dichaete has functions in the developing brain. A specific group of neural cells in the tritocerebrum fails to develop correctly in the absence of Dichaete, as revealed by reduced expression of labial, Zn finger homeodomain 2, wingless, and engrailed. (2) Dichaete is required for the correct differentiation of the hindgut. The Dichaete requirement in hindgut morphogenesis is achieved, in part, via regulation of dpp, since ectopically supplied dpp can rescue Dichaete phenotypes in the hindgut. Taken together, there are now four distinct in vivo functions described for Dichaete that can be used as models for context-dependent comparative studies of Sox function (Sanchez-Soriano, 2000).

In the developing CNS there are three prominent sites of Dichaete expression: the ventral midline; the CNS of the trunk outside the midline (here referred to as the 'paraxial nerve cord'), and the cephalic CNS. Expression appears to be independently regulated in each of these domains. In wild type, Dichaete is expressed in the midline from stages 7 to 14. Two groups of alleles were identified that have opposite effects on midline expression: the 5' mutation, D^r321 lacks midline expression until stage 12; thereafter, it appears to be wild type. This suggests that regulatory elements for early midline expression are located close to the 5' end of Dichaete. Accordingly, all of the 3' alleles show normal midline expression up to stage 12. However, subsequently midline expression in all of the 3' alleles declines prematurely, indicating regulatory elements for late midline expression distal to minus 25 kb. In the paraxial nerve cord of wild-type embryos, Dichaete is detected in the neuroectoderm from stage 7 and subsequently in a complex and dynamic pattern in many segregating neural precursors and their progeny. All the 3' breakpoint alleles retain substantial (though less than wild type) early neurectodermal expression until stage 10. By stage 11 this expression is affected; the alleles with break-points proximal to minus 12 kb show a strong reduction in paraxial nerve cord expression. In mutant embryos with breakpoints distal to minus 12 kb Dichaete product can be detected in segregated neuroblasts and their progeny; in D⁷ only a single strongly staining cell and a few weakly staining cells are found in each hemisegment; and in the more distally mapping D¹⁰ allele more cells are stained, though still less than in wild type. Taken together, these data suggest several discrete regulatory regions for paraxial nerve cord CNS expression: (1) an early neurectodermal element proximal to the D^r8 breakpoint; (2) elements in the area distal to the D^r8 breakpoint up to beyond the D¹⁰ break-point, which drive expression in different sets of segregated neuroblasts and progeny; (3) sequences distal to the D¹⁰ breakpoint, which drive late neurectodermal expression together with expression in further neuroblasts and progeny (Sanchez-Soriano, 2000).

At the anterior end of the wild-type embryo Dichaete is expressed at the blastoderm stage in a domain in the procephalic region. This expression is normal in D^r321, but lacking in all of the mutant alleles with 3' break-points, indicating that regulatory elements for procephalic blastoderm expression lie distal to minus 25 kb. Subsequently, Dichaete is expressed, again in a complex and dynamic pattern, in the developing brain. Much of the postblastoderm brain expression is lost in the 3' alleles (but not in D^r321). The exception is a patch of expression in the dorsal region of the brain that increases in intensity in alleles with increasing portions of 3' DNA. This suggests that there may be regulatory elements required for brain expression dispersed throughout the 3' region. Finally, in wild-type embryos, Dichaete is expressed in the developing hindgut from stage 10. In Dichaete mutant embryos there is a sharp demarcation between D⁴, which has wild-type expression, and the alleles with breakpoints proximal to this (D⁶ and D^r8) showing no discernible expression. This indicates that a hindgut regulatory element is located proximal to minus 10 kb (Sanchez-Soriano, 2000).

Since a set of Dichaete mutant alleles affects tissue-specific aspects of Dichaete expression, these alleles can be used to study Dichaete requirements in different Dichaete-expressing tissues. Dichaete is required for the correct specification and differentiation of midline glia, and loss of Dichaete in the midline results in characteristic defects in the mature axon scaffold. In agreement with this, Dr³²¹ mutant embryos, which lack midline expression before stage 12/13, have defects in the development of thoracic and posterior abdominal mid-line glia and of the neuropile (fusion of commissures, thinning and collapse of longitudinal connectives toward the midline). The 3' alleles, which lack only late midline expression of Dichaete, show no defects. This finding confirms previous work on the requirements of Dichaete in the midline and demonstrates that Dichaete function is required before stage 12/13. The effects were examined of loss of Dichaete expression in the paraxial nerve cord, in the cephalic CNS, and in the hindgut primordium. Of the regulatory alleles characterized, D^r8 has the strongest effect on Dichaete expression in the paraxial nerve cord. From late stage 11 there is no expression in segregated neuroblasts and their progeny; however, earlier neurectodermal expression is prominent. The nervous system of D^r8 hemizygous embryos was examined using global neuronal markers, such as BP102, and the specific lineage markers Even skipped and Engrailed. In all cases the nervous system appears to be wild type: similar results were obtained with D⁶ and D¹⁰. In contrast, embryos carrying Dichaete null alleles show defects in Eve lineages (an increase in the number of cells in the position of the EL cluster, the RP2, aCC, and pCC neurons and a loss of En-expressing cells, a phenotype previously reported for Dichaete null alleles. The discrepancy between the null and regulatory alleles may reflect the fact that the regulatory alleles retain early neurectodermal expression and it is only this early expression which is important for the phenotypes observed. Alternatively, defects in the paraxial nerve cord of null mutant embryos may be caused secondarily by early segmentation defects. In this case, Dichaete has no easily detectable primary function in the CNS or its function is masked by other redundant neural Sox genes, as is believed to be the case in vertebrates (Sanchez-Soriano, 2000).

Dichaete is expressed in the procephalic neurectoderm before neuroblast formation and subsequently in the developing brain. Since the expression of Dichaete in the developing brain is widespread, complex, and dynamic, including neurectodermal cells, neuroblasts, and differentiated neuronal cells, it is difficult to precisely map with respect to the existing neuroblast maps. Some of the Dichaete-positive cells were mapped by double labeling with Dichaete, Wingless (Wg), and Engrailed (En) antibodies. At embryonic stage 11, Wg and En are expressed in adjacent domains called wg/en intercalary spot, wg/en antennal stripe, and wg/en head blob or spot. These domains of expression contribute to the tritocerebrum, deuterocerebrum, and protocerebrum, respectively, and, in particular, En expression can be used as a landmark to delimit these three brain neuromeres. At stage 11, Dichaete coexpresses with these markers in the intercalary spot, a portion of the antennal stripe and in the 'head blobs'. However, it is apparent that within the three brain neuromeres, very many brain cells express Dichaete at high levels. All of the 3' regulatory alleles lack procephalic neurectodermal expression at the blastoderm stage in addition to most staining in the developing brain until late stage 10. After this D^r8 mutant embryos show very little staining whereas D¹⁰ mutant embryos have more extensive expression. To look for defects in the development of Dichaete mutant brains the expression of Zfh-2, a transcription factor expressed in many neuronal lineages, was examined. In comparison to the wild type, in D^r8 stage 16 mutant embryos, a reduction in Zfh-2-positive is seen in cells in the posterior part of the brain, most likely the tritocerebrum. Similarly, ming-LacZ expression, another marker abundant in the CNS, is reduced in this region at stage 12 and 16 when analyzed in null and D^r8 mutant backgrounds. However, anti-Fasciclin II stainings of wild-type and mutant embryos reveal no detectable differences in the location or arrangement of the major brain commissures and longitudinal connectives. Thus, despite widespread expression, Dichaete requirement is mainly restricted to a specific region of the embryonic brain and has no apparent function in the development of the major identified axon tracts in the brain (Sanchez-Soriano, 2000).

The tritocerebrum originates from neuroblasts that segregate from the intercalary segment during stages 10 and 11. The intercalary segment is marked by the expression of the homoeotic gene labial (lab) and, as development proceeds, labial expression is maintained in the tritocerebrum. In D^r8 hemizygotes at stage 16 a reduction in the number of lab-expressing cells was seen and compared to the wild type; D¹⁰ and the null allele D³ shows similar phenotypes. In the wild-type brain, Dichaete is coexpressed in a subset of lab-expressing cells until stage 11 but not thereafter. Therefore, Dichaete appears to be required early for the correct development of this fraction of labial-expressing cells. Earlier stages of brain development were examined. Preliminary analysis of lethal of scute (l'sc)-expressing cells in stages 10-11 D^r8 mutant embryos, reveals widespread defects in the trito-cerebrum anlage. This is in contrast to other regions of the brain which appear unaffected or show subtle defects (e.g., deutocerebrum). Since the l‘sc pattern of expression is complex and dynamic, the more restricted markers Wg and En were used. At stage 10 in the wild-type embryo, there are one to two large Wg-positive cells (most likely neuroblasts) in the intercalary segment. Wg expression in these cells is undetectable or severely reduced in D r⁸ hemizygous embryos, whereas other Wg domains in the head, which normally express Dichaete, appear unaffected. Similar data were obtained in embryos expressing lacZ under the control of the wingless promoter. In the wild type, at stage 11, En is expressed in at least 10 neuroblasts and/or neuronal cells in the intercalary segment. In D^r8 mutant embryos En expression in the intercalary segment is strongly reduced since four or fewer positive cells are detected. As for Wg, the other En domains in the developing brain appear to be unaffected. Taken together these data indicate that Dichaete is required for the development of a subpopulation of cells, including neuroblasts, within the anlage of the tritocerebrum at stage 10/11 when the neural precursors of the tritocerebrum form. Therefore, loss of Dichaete might affect the correct specification or differentiation of specific neuroblast lineages, leading to loss of Zfh-2, ming-lacZ, and lab expression (Sanchez-Soriano, 2000).

The Sox-domain-containing gene Dichaete/fish-hook plays a crucial role in patterning the neuroectoderm along the DV axis. Dichaete is expressed in the medial and intermediate columns of the neuroectoderm, and mutant analysis indicates that Dichaete regulates cell fate and neuroblast formation in these domains. Molecular epistasis tests, double mutant analysis and dosage-sensitive interactions demonstrate that during these processes, Dichaete functions in parallel with ventral nerve cord defective and intermediate neuroblasts defective, and downstream of EGF receptor signaling to mediate its effect on development. These results identify Dichaete as an important regulator of dorsoventral pattern in the neuroectoderm, and indicate that Dichaete acts in concert with ventral nerve cord defective and intermediate neuroblasts defective to regulate pattern and cell fate in the neuroectoderm (Zhao, 2002).

vnd, ind and Egfr are key factors that regulate pattern and cell fate along the DV axis of the neuroectoderm. To ask if Egfr pathway activity depends on vnd or ind, MAPK activity was assayed in homozygous vnd or ind single mutant embryos. In both backgrounds, the initial activation of Egfr signaling in the medial and intermediate columns is normal. Thus, in the early neuroectoderm, Egfr acts either upstream or in parallel to vnd and ind. To investigate whether Egfr acts upstream of vnd or ind, vnd and ind expression was assayed in embryos homozygous mutant for the Egfr null allele flb^IK35 (referred to as Egfr mutant embryos). ind expression is absent in Egfr mutant embryos, indicating that Egfr activates ind expression in the intermediate column. By contrast, vnd expression in Egfr mutant embryos appears normal through the onset of stage 8. However, during stage 8, vnd expression begins to dissipate in medial column cells, and by early stage 10 these cells no longer express vnd. Conversely, medial column NBs that form in Egfr mutant embryos express vnd normally and retain vnd expression throughout embryogenesis. Thus, Egfr functions to maintain vnd expression in the neuroectoderm but is dispensable for vnd expression in NBs. These data indicate that Egfr resides atop the genetic hierarchy known to subdivide the neuroectoderm along the DV axis (Zhao, 2002).

These results suggest that Egfr patterns the neuroectoderm, at least in part, through its regulation of vnd and ind. To determine if additional genes act downstream of Egfr in this process, the phenotypes of embryos singly mutant for Egfr and ind were compared. It was reasoned that if Egfr patterns the intermediate column solely through regulation of ind, then Egfr and ind mutant embryos should exhibit identical intermediate column phenotypes. To compare the early CNS phenotypes of Egfr and ind, a precise analysis of msh expression and the NB pattern was carried out. In both cases, Egfr exhibits a more severe phenotype than ind. msh expression expands more medially in Egfr mutant embryos than in ind mutant embryos. In addition, lateral NBs are most often separated from medial NBs by a gap in ind mutant embryos, while lateral NBs develop immediately adjacent to medial NBs in Egfr mutant embryos. These data indicate a greater disruption to the intermediate column in Egfr mutant embryos than in ind mutant embryos. These phenotypic differences are consistent with the presence of additional genes acting downstream of Egfr and in parallel to ind to control cell fate in the intermediate column. However, Egfr maintains vnd expression in the neuroectoderm; thus, these data do not exclude the possibility that the differences in phenotype between Egfr and ind arise due to the late regulation of vnd expression by Egfr (Zhao, 2002).

To test whether the phenotypic differences between ind and Egfr mutant embryos are an indirect result of the regulation of vnd expression by Egfr, it was asked whether these differences are equalized in double mutants where vnd function is also removed. In vnd;ind mutant embryos, msh is expressed throughout the neuroectoderm, although its expression is higher in the lateral column relative to the medial column. By contrast, msh is expressed at uniformly strong levels throughout the neuroectoderm in vnd;Egfr mutant embryos. Thus, removal of vnd and Egfr causes a stronger derepression of msh in the neuroectoderm than loss of vnd and ind. These results suggest that additional gene(s) act downstream of Egfr and in parallel to vnd and ind to regulate DV pattern in the neuroectoderm. They also suggest that in the absence of vnd and Egfr function, the entire neuroectoderm acquires a lateral column fate (Zhao, 2002).

Based on its restricted expression pattern in the ventral region of the neuroectoderm, the Sox-domain-containing gene Dichaete was identified as a likely candidate to act downstream of Egfr to regulate DV pattern in the neuroectoderm. To investigate whether Dichaete contributes to neuroectodermal patterning, the precise limits of Dichaete expression in the neuroectoderm were determined using the expression of msh and achaete (ac) to mark different longitudinal columns. msh is expressed in the lateral column; ac is expressed in neural equivalence groups (proneural clusters) in the medial and lateral columns of rows 3 and 7. Within the neuroectoderm, Dichaete expression begins during stage 7. Dichaete is expressed uniformly in the ventral region of the neuroectoderm with a lateral expression boundary that precisely abuts the medial limit of msh and ac expression in the lateral column. Within the neuroectoderm, Dichaete expression is restricted to the medial and intermediate columns through late stage 12, at which point Dichaete expression expands to include the entire neuroectoderm. Thus, in contrast to the transient presence of Egfr and ind activity in the intermediate column, Dichaete is expressed in the intermediate and medial columns throughout all waves of NB formation (Zhao, 2002).

During analysis of Dichaete expression in the neuroectoderm, it was noticed that most medial and intermediate NBs do not express Dichaete at detectable levels. To determine the pattern of Dichaete expression in neuroblasts, wild-type embryos were co-labeled for Dichaete and hunchback, a marker for all neuroblasts. Newly formed medial and intermediate column NBs express weak levels of Dichaete but most older NBs in these domains do not express Dichaete. Exceptions to this exist, since two conspicuous NBs express Dichaete -- one in the medial column of row 4 and one in the intermediate column of row 3. These data suggest that newly formed medial and intermediate NBs retain residual Dichaete expression from the neuroectoderm but that Dichaete expression is downregulated in most medial and intermediate NBs once they form (Zhao, 2002).

In contrast to medial and intermediate NBs, many lateral NBs activate Dichaete at specific points in their lineages. NB 7-4 is the first lateral NB to activate Dichaete expression during late stage 10. Dichaete expression in lateral NBs is dynamic. NBs 5-6, NB 2-5 and eventually NB 3-5 express Dichaete. Thus, all medial and intermediate column neuroectodermal cells express Dichaete but most medial and intermediate NBs do not express Dichaete. Conversely, lateral NBs but not neuroectodermal cells express Dichaete. These data are consistent with Dichaete regulating cell fate in the medial and intermediate neuroectodermal columns, and at specific points in the lineage of lateral NBs (Zhao, 2002).

The restricted expression of Dichaete in the medial and intermediate columns suggests that Dichaete regulates cell fate and NB formation in this region. However, Dichaete mutant embryos exhibit AP patterning defects, owing to an early requirement in segmentation. The segmental defects are largely restricted to the abdominal segments; thoracic segments appear largely normal. These segmentation defects could obscure a role for Dichaete during neuroectodermal patterning. Thus, analysis of Dichaete function in the neuroectoderm was restricted to thoracic segments (Zhao, 2002).

To investigate whether Dichaete patterns the neuroectoderm, early neural development was followed in embryos mutant for the Dichaete⁸⁷ and Dichaete⁹⁶ null alleles. Whether Dchaete regulates gene expression in the neuroectoderm was tested by following ac and msh expression. Normally, ac is expressed in the medial and lateral, but not intermediate, proneural clusters of rows 3 and 7 during the first wave of NB formation. In Dichaete mutant embryos, a partial derepression of ac expression was observed in the intermediate column. Roughly 50% of the cells within the intermediate column of rows 3 and 7 express ac. ac expression in the medial column appears normal, as do the AP limits of ac expression in the thoracic segments. In contrast to ac, no obvious alterations to msh expression were detected in the neuroectoderm. Since ac is a key determinant of neural fate, its derepression in the intermediate column is interpreted to indicate that Dichaete regulates cell fate in this column. However, the msh results indicate that lateral fates are specified normally in Dichaete mutant embryos (Zhao, 2002).

ind normally represses ac expression in the intermediate column, because in ind mutant embryos, ac expression is completely derepressed within rows 3 and 7 of the intermediate column. The Dichaete and ind phenotypes demonstrate that both genes are necessary for intermediate column fates. To determine if Dichaete and ind function in a linear pathway to regulate intermediate cell fates, ind expression was followed in Dichaete mutant embryos and Dichaete expression in ind mutant embryos. ind expression is normal in Dichaete mutant embryos and Dichaete expression is normal in ind mutant embryos. Thus, ind and Dichaete are regulated independently of each other (Zhao, 2002).

Double labeling Dichaete mutant embryos for ac and ind, and double labeling ind mutant embryos for ac and Dichaete reveals an interdependent relationship between Dichaete and ind. In Dichaete mutant embryos, a significant number of row 3 and 7 intermediate column cells and NBs co-express ac and ind -- an occurrence never observed in wild-type embryos. Thus, the ability of ind to repress ac in the intermediate column requires Dichaete activity. Reciprocally, in ind mutant embryos, all row 3 and 7 intermediate column cells co-express ac and Dichaete. Thus, the ability of Dichaete to repress ac in the intermediate column requires ind activity (Zhao, 2002).

Whether Dichaete regulates NB formation in the medial and intermediate columns was examined. To do this, the development of individual NBs was followed using a panel of molecular markers that identify specific NBs or their progeny. Svp-lacZ was used to label the medial column SI NBs 5-2 and 7-1, as well as SIII NB 4-1; castor expression was used to label the medial column SIII NB 6-1, and eve expression to label the first-born progeny of SI medial column NBs 1-1 and 7-1, and of SII intermediate column NB 4-2. In Dichaete mutant embryos, NBs 1-1 (99% formation), 5-2 (99.6%) and 7-1 (97.8%) develop normally. Thus, SI medial column NBs form normally in the absence of Dichaete function. By contrast, defects were observed in the formation of SII and SIII NBs in Dichaete mutants. For example, no Svp-lacZ-positive NB 4-1 was found in 30.9% of thoracic hemisegments and no Castor-positive NB 6-1 was found in 7.9% of hemisegments. In addition, no Eve-positive RP2 neuron was found in 12.9% of hemisegments, suggesting the absence of NB 4-2 in these hemisegments. Together with expression analyses, these phenotypic studies demonstrate that Dichaete acts in the neuroectoderm to promote the formation of late-forming NBs in the medial and intermediate columns (Zhao, 2002).

These loss of function analyses have identified Dichaete as a regulator of DV pattern and cell fate in the neuroectoderm. To place Dichaete within the known genetic regulatory hierarchy that governs DV pattern in the neuroectoderm, systematic molecular epistasis tests were performed for Dichaete, ind, vnd and Egfr. Initially, vnd and ind expression, as well as Egfr activity was assayed in Dichaete mutant embryos. Dichaete mutant embryos exhibit no obvious defects to the expression of vnd or ind, or the activity of Egfr. Thus, Egfr, vnd and ind function upstream or in parallel to Dichaete (Zhao, 2002).

To investigate whether Egfr, vnd or ind regulate Dichaete, Dichaete expression was assayed in embryos mutant for each gene. No alterations were observed to the initial pattern of Dichaete expression in vnd or ind mutants, or in embryos doubly mutant for vnd and ind. Dichaete expression remains normal in ind mutant embryos throughout embryogenesis. However, by stage 11 in vnd and vnd; ind mutant embryos, Dichaete expression narrows inappropriately to an irregularly patterned stripe two-to-four cells wide immediately adjacent to the ventral midline. These results show that Dichaete is regulated independently of ind and is activated independently of vnd, but that vnd helps maintain Dichaete expression in the neuroectoderm (Zhao, 2002).

In contrast to vnd and ind, the initial pattern of Dichaete in Egfr mutant embryos is greatly reduced in the intermediate column and moderately reduced in the medial column during early neurogenesis. By stage 11, Dichaete expression narrows inappropriately to a thin and irregular stripe zero-to-three cells wide immediately adjacent to the ventral midline; Dichaete expression in the ventral midline is normal. These data identify Egfr as a key positive regulator of Dichaete in the neuroectoderm, and indicate that at least one other gene acts with Egfr to activate Dichaete expression in the medial column (Zhao, 2002).

To investigate whether vnd acts with Egfr to promote Dichaete expression in the medial column, Dichaete expression was followed in vnd; Egfr mutant embryos. The initial pattern of Dichaete in these embryos is the same as that observed in Egfr mutant embryos. However, by stage 11, Dichaete expression is completely absent from the neuroectoderm, although Dichaete expression is normal in the ventral midline. These results indicate that vnd and Egfr collaborate to maintain Dichaete expression in the neuroectoderm (Zhao, 2002).

To determine if Egfr activity is sufficient to activate Dichaete expression, the GAL4/UAS system system was used to activate Egfr signaling throughout the early Drosophila embryo. Ubiquitous Egfr signaling activates Dichaete expression throughout the neuroectoderm but not in the dorsal ectoderm. Thus, Egfr is necessary and sufficient to activate Dichaete in the neuroectoderm. However, in the dorsal ectoderm, either factors exist that inhibit the ability of Egfr to activate Dichaete or this domain lacks co-factors required for Egfr to activate Dichaete. Molecular epistasis tests place Egfr upstream of Dichaete and indicate that vnd, ind and Dichaete function largely in parallel to regulate pattern and cell fate in the neuroectoderm (Zhao, 2002).

The parallel genetic activities of Dichaete, vnd and ind, the co-expression of Dichaete with vnd and ind, and the similarity of the early Dichaete CNS phenotype to those of vnd and ind led to a test of whether Dichaete interacts genetically with vnd and ind. To ascertain whether Dichaete interacts with vnd, the double mutant vnd;Dichaete was made and the formation of medial column SIII NBs 4-1 and 6-1 was assayed. In Dichaete mutant embryos, NBs 4-1 and 6-1 formed in 69.1% and in 92.1% of hemisegments, respectively. In vnd mutant embryos it was found that NBs 4-1 and 6-1 formed in 39.3% and 35.5 of hemisegments, respectively. In vnd; Dichaete mutant embryos NBs 4-1 and 6-1 formed in 10.8% and 9.1% of hemisegments, respectively. The increased defects in NB formation in vnd; Dichaete mutant embryos relative to either single mutant confirms that Dichaete and vnd do not act in a linear pathway to regulate NB formation -- rather, they demonstrate that Dichaete and vnd function in parallel to control NB formation in the medial column (Zhao, 2002).

Defects in NB formation in vnd; Dichaete mutant embryos are more severe than would be expected if these genes functioned independently. For example, if two genes act independently to promote NB formation, then the frequency of NB formation in the double mutant would be the product of the individual probabilities that the indicated NB will form in each single mutant. Thus, if vnd and Dichaete function independently, it would be expected that NB 4-1 would form 27.2% of the time (0.393 x 0.691=0.272) and NB 6-1 to form 32.7% of the time (0.355 x 0.921=0.327) in vnd; Dichaete mutant embryos. However, NBs 4-1 and 6-1 form ~10% of the time in vnd; Dichaete mutant embryos -- roughly threefold more severe than predicted for independently acting genes. These results reveal a genetic interaction between Dichaete and vnd. Furthermore, these results are interpreted to suggest that the activities of vnd and Dichaete are more convergent than parallel with respect to NB formation (Zhao, 2002).

Next, genetic interactions between Dichaete and ind were tested. The partial derepression of ac expression and the incomplete loss of an Eve-positive RP2 neuron are the most sensitive assays for Dichaete function in the intermediate column. However, strong alleles of ind cause a complete derepression of ac expression, and a complete loss of RP2 neurons in this domain. Thus, an analysis of Dichaete ind double mutant embryos using these markers would be uninformative. To circumvent this problem, a test was performed to see whether ind dominantly enhances the Dichaete intermediate column ac and RP2 phenotypes. Embryos heterozygous for ind exhibit wild-type ac expression and RP2 formation. However, Dichaete ind/Dichaete + mutant embryos exhibit enhanced derepression of ac expression and an approximately threefold enhancement of the RP2 loss phenotype relative to Dichaete mutant embryos. The dominant enhancement of the Dichaete phenotype by ind reveals a genetic interaction between Dichaete and ind (Zhao, 2002).

Initial interest in Dichaete arose from the observation that vnd; Egfr mutant embryos exhibit a more severe neuroectodermal phenotype than vnd; ind mutant embryos. This suggests that at least one other gene acts downstream of Egfr, and in parallel to vnd and ind to pattern the early neuroectoderm: this led to the analysis of Dichaete. To determine if the continued function of Dichaete in vnd; ind mutant embryos can explain the phenotypic differences between vnd; ind and vnd; Egfr mutant embryos, msh expression was followed in vnd;Dichaete;ind triple mutant embryos. In this background, a complete and uniform derepression of msh expression was observed throughout the neuroectoderm. The msh phenotype of vnd; Dichaete; ind embryos is essentially identical to that of vnd; Egfr embryos, and more severe than that of vnd; ind embryos. Thus, with respect to msh expression the difference between the vnd; ind and vnd; Egfr mutant phenotypes appears to result from the persistent function of Dichaete in vnd; ind mutant embryos (Zhao, 2002).

The results in this paper indicate that Dichaete is a key regulator of DV pattern in the neuroectoderm. Dichaete is expressed in the medial and intermediate columns and regulates cell fate and NB formation in these domains. Within the neuroectoderm, Dichaete acts downstream of Egfr and in parallel to vnd and ind. Together with biochemical research on Sox-domain-containing genes in vertebrates this work supports a model in which Dichaete protein physically associates with Vnd and Ind to regulate target gene expression and NB formation in distinct neuroectodermal columns (Zhao, 2002).

Interest in Dichaete arose owing to the observation that removal of vnd and Egfr function caused a stronger derepression of msh expression in the neuroectoderm than removal of vnd and ind function. These results contrast slightly with previous research that did not identify a phenotypic difference between vnd; ind and vnd; Egfr mutant embryos. This work analyzed msh expression in the neuroectoderm at a later stage (late stage 9) than the current work. At late stage 9, identical alterations to msh expression were also observed in vnd; ind mutant embryos relative to vnd; Egfr mutant embryos. However, the msh expression pattern is dynamic -- rapidly changing from uniform expression in the lateral column during stage 8 to a segmentally modulated pattern of cell clusters located within the lateral half of the neuroectoderm by stage 10. The differences in these observations are attributed to the different stages used to assay the effects of vnd, ind and Egfr on neuroectodermal development in the two studies (Zhao, 2002 and references therein).

Dichaete is expressed and regulates cell fate in the medial and intermediate neuroectodermal columns. However, Dichaete carries out distinct functions in each domain: Dichaete represses ac expression in the intermediate column but has no effect on ac expression in the medial column where Dichaete and ac are co-expressed (Zhao, 2002).

How might Dichaete exhibit region specific effects on putative target genes? Work from vertebrate systems suggests that individual Sox-domain-containing proteins exhibit a widespread ability to partner with different transcription factors. Thus, Dichaete protein could exhibit column-specific functions via its association with different transcription factors in different domains. The formation of distinct protein complexes containing Fish could alter the output of Fish activity in at least two ways. Different protein complexes that contain Fish could exhibit different effects on transcription: repression versus activation. Alternatively, different Fish-containing protein complexes could exhibit distinct DNA-binding properties and therefore bind distinct recognition sites. These two possibilities are not mutually exclusive, and different Fish-containing protein complexes may both bind different recognition sites and exert different transcriptional effects on target genes (Zhao, 2002).

Examples of both forms of regulation are known. In the early Drosophila embryo, the transcription factor Dorsal activates one set of target genes ventrally and represses a distinct set dorsally. On its own, Dorsal functions as a transcriptional activator. However, in the dorsal region of the embryo, the interaction of Dorsal with a co-factor that binds to adjacent sites on target promoters converts Dorsal to a repressor. Although less well-defined mechanistically, the vertebrate Sox2 protein appears capable of activating or repressing target gene expression depending on cell-type and the target promoter (Botquin, 1998). In addition, work on vertebrate Sox domain proteins indicates that the composition of Sox-protein containing complexes modulates the DNA-binding specificity of these complexes. For example, in lens cells, Sox2 interacts with the DNA-binding factor deltaEF3 and binds to a bipartite recognition site on the delta-crystallin enhancer (Kamachi, 1998; Kamachi, 1999). In embryonic stem cells, Sox2 interacts with Oct3/4 and binds to a different recognition site in the Fgf4 minimal enhancer (Ambrosetti, 1997). In both enhancers, Sox2 binds to the same individual sequence. However, the specificity for the entire recognition site in one enhancer over the other arises as a consequence of the interaction of Sox2 with different transcription factors in different cell types and the distinct DNA-binding preferences of the entire complex (Zhao, 2002).

Based on these data, Dichaete is expected to associate with different transcription factors in the medial and intermediate columns to carry out its column-specific effects on target genes. The results in this paper identify Vnd and Ind as excellent candidates to be column-specific factors that associate with Dichaete and enable Dichaete to regulate transcription in a region specific manner: (1) Dichaete is co-expressed with Vnd in the medial column and Ind in the intermediate column; (2) the neuroectodermal Dichaete mutant phenotype is similar to those of vnd and ind; (3) Dichaete functions in parallel to vnd and ind in the neuroectoderm; (4) Dichaete exhibits dose-sensitive interactions with ind and genetic interactions with vnd, consistent with these proteins interacting physically. Based on these data, it is speculated that physical interactions between Dichaete and Vnd in the medial column and Dichaete and Ind in the intermediate column mediate the ability of distinct Dichaete protein complexes to bind to and either activate or repress distinct target genes. Validation of this model awaits the determination of whether Dichaete associates with Vnd or Ind, and how these proteins regulate target gene activity. However, recent results provide precedence for the model since genetic interactions between Dichaete, single-minded and drifter during midline development in the Drosophila CNS have led to experiments that show Dichaete physically associates with the Single-minded and Drifter proteins (Zhao, 2002).

These results place Dichaete within the known genetic regulatory hierarchy that controls pattern and cell fate along the DV extent of the neuroectoderm. In the future, it is expected that many additional genes will be joined into this pathway. For example, the Sox-domain-containing gene sox-neuro is expressed throughout the entire neuroectoderm and it may exhibit region-specific effects in the neuroectoderm in a manner similar to that proposed for Dichaete. In addition, the Ras-pathway antagonist yan is expressed in the lateral half of the neuroectoderm during early neurogenesis and may help regulate pattern and cell fate in this domain. A complete understanding of the genetic and molecular mechanisms that pattern the neuroectoderm requires the identification of all such genes and the elucidation of how these genes interact to regulate cell fate along the DV axis of the neuroectoderm (Zhao, 2002).

Sox proteins form a family of HMG-box transcription factors related to SRY, the mammalian testis determining factor. Sox-mediated modulation of gene expression plays an important role in various developmental contexts. Drosophila SoxNeuro, a putative ortholog of the vertebrate Sox1, Sox2 and Sox3 proteins, is one of the earliest transcription factors to be expressed pan-neuroectodermally. SoxNeuro is essential for the formation of the neural progenitor cells in the central nervous system. Loss of function mutations of SoxNeuro are associated with a spatially restricted hypoplasia: neuroblast formation is severely affected in the lateral and intermediate regions of the central nervous system, whereas ventral neuroblast formation is almost normal. Evidence is presented that a requirement for SoxNeuro in ventral neuroblast formation is masked by a functional redundancy with Dichaete, a second Sox protein whose expression partially overlaps that of SoxNeuro. SoxNeuro/Dichaete double mutant embryos show a severe neural hypoplasia throughout the central nervous system, as well as a dramatic loss of achaete expressing proneural clusters and medially derived neuroblasts. Genetic interactions of SoxNeuro and the dorsoventral patterning genes ventral nerve chord defective (vnd) and intermediate neuroblasts defective (ind) underlie ventral and intermediate neuroblast formation. Expression of the Achaete-Scute gene complex suggests that SoxNeuro acts upstream and in parallel with the proneural genes. The finding that Dichaete and SoxN exhibit opposite effects on achaete expression within the intermediate neuroectoderm demonstrates that each protein also has region-specific unique functions during early CNS development in the Drosophila embryo (Buescher, 2002 and Overton, 2002).

The SoxN mutant phenotype shows a strong spatial aspect with respect to the DV axis: loss of SoxN severely affects the formation of NBs that derive from the lateral and intermediate regions of the NE but have little effect on ventral NB formation. This DV effect of SoxN mutations is not mirrored in a corresponding DV SoxN expression pattern. Thus, the mutant phenotype rather reflects a differential requirement for SoxN in different regions. Analysis of ventral NB formation in SoxN;Dichaete double mutant embryos provides at least a partial explanation for these regional differences because the concomitant loss of SoxN and Dichaete results in a strong loss of ventral NBs. This suggests that SoxN and Dichaete may functionally substitute for each other. A functional redundancy of SoxN and Dichaete is not unexpected since the proteins have structural similarities and overlapping expression patterns. Like SoxN, Dichaete has also been classified as a group B Sox protein; the HMG domains of these two proteins show 87% amino acid identity. Since the ability of sequence-specific DNA binding resides within the HMG domain, it is likely that SoxN and Dichaete bind to the same DNA motif present in an identical set of target genes. This is supported by studies that have shown that various vertebrate Sox proteins can bind to the same DNA sequence. Neuroectodermal Dichaete and SoxN expression overlaps in the ventral and intermediate region and therefore a functional redundancy would be expected to occur in ventral and intermediate NB formation. However, the severe phenotype of SoxN single mutants in intermediate NB formation suggests that Dichaete cannot always substitute for SoxN function. Additional evidence that SoxN and Dichaete function is not equivalent stems from the observation that loss of Dichaete or SoxN has different effects on Ac expression in the intermediate region of the NE: in Dichaete, but not in SoxN mutant embryos, Ac expression is partially derepressed in the intermediate column (Zhao, 2002; Buescher, 2002).

SoxN and Dichaete are both expressed early in the neuroectoderm. Dichaete is restricted to the ventral region, extending from the midline to the position of the intermediate column (Zhao, 2002), and SoxN is excluded from the midline and extends more dorsally to encompass the entire neuroectoderm. Dichaete mutants show strong phenotypes in the midline, where Dichaete is uniquely expressed, and SoxN mutants exhibit strong phenotypes in the lateral half of the CNS where SoxN is uniquely expressed. In Dichaete mutants, SI medial NBs are not affected but there is a loss of later delaminating SII and SIII NBs from both medial and intermediate columns. SoxN and Dichaete overlap in the medial and intermediate neuroectodermal columns and in the medial column, SoxN phenotypes are weaker than those observed in the lateral columns. These data are consistent with the idea that the genes may be able to compensate functionally in the medial column neuroectoderm. To examine the consequences of removing group B Sox function from the early CNS, a double mutant combination was constructed, using null alleles for both Dichaete and SoxN. The overall structure of the CNS was examined as well as markers for specific NBs and/or progeny in the double mutant embryos (Overton, 2002 and references therein).

Staining the double mutants with BP102 reveals a severe disruption in the organization and structure of the CNS. A complete loss of longitudinal axons is observed in many segments with frequent gaps in the neuropil. Commissures are often absent, and those that do form are virtually never separated. The phenotype of the double mutants is far more severe than observed with either single mutant and supports the idea that the genes can act redundantly or in related pathways. If this is the case then an enhanced effect is expected on medial NBs and their progeny when both SoxN and Dichaete function are removed, compared with each of the single mutants, since this is the region in which they are extensively co-expressed. In line with this expectation it has been observed that in the SI medial lineages of NB1-1 and NB7-1, identified by eve expression, there is a rather severe reduction in the number of aCC/pCC and CQ cells in double mutants compared with each of the single mutants. Note that these lineages are virtually unaffected in either of the single mutants. Additionally, in the intermediate column, the Hb expressing neuroblast 5-3 is absent at a higher frequency in double mutant embryos than in SoxN or D mutants (79% compared with 52% and 2%, respectively), indicating that Dichaete is to some extent able to compensate for a loss of SoxNeuro within this lineage. Although it is impossible to determine accurately the identity of the remaining Hb expressing SI NBs in the double mutants, the total number of cells in thoracic segments was counted, and in SoxN^U6–35 homozygotes 30% of Hb expressing NBs are missing; in Dichaete mutants less than 1% are missing, whereas 56% are missing in the double mutants. Taken together, it is concluded that in the cases of overall CNS structure as well as medial and intermediate column SI NBs, evidence is seen for functional redundancy between related Group B Sox genes (Overton, 2002).

Both SoxN and Dichaete are expressed early in the neuroectoderm, SoxN expression being initiated slightly before that of Dichaete. It is therefore possible that SoxN regulates the expression of Dichaete and this possibility was examined by staining SoxN^U6–35 mutant embryos for Dichaete. A rather unexpected phenotype was observed; in around half of the mutant embryos, Dichaete levels were apparently normal. However, in the remaining half Dichaete levels were reduced, but only in the anterior half of the neuroectoderm; the posterior appeared to be normal. This is not due to a staining artifact because in the affected embryos Dichaete is expressed normally in the midline all along the AP axis. Thus, it appears that SoxN does have an effect on Dichaete expression, but that this effect is variable and restricted along the AP axis. In any case the SoxN phenotypes cannot be explained by a loss of Dichaete expression in the neuroectoderm because ectopic expression of ac would be expected to be seen in SoxN^U6–35 as is seen in Dichaete and the double mutants (Overton, 2002).

Therefore, it is concluded that in the neuroectoderm the two group B Sox proteins, SoxN and Dichaete, can functionally compensate but they also have antagonistic functions, particularly within the intermediate neuroectoderm (Overton, 2002).

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date revised: 20 February 2011

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