Dichaete

InteractiveFly: GeneBrief

Gene name - Dichaete

Synonyms -

Cytological map position - 70D1-2

Function - Transcription factor

Keywords - regulation of pair rule genes, CNS, hindgut, ventral midline

Symbol - D

FlyBase ID: FBgn0000411

Genetic map position - 3-40.7

Classification - HMG-domain protein - SOX-domain protein

Cellular location - nuclear

NCBI links: Entrez Gene

D orthologs: Biolitmine

Recent literature

Carl, S. H. and Russell, S. (2015). Common binding by redundant group B Sox proteins is evolutionarily conserved in Drosophila. BMC Genomics 16: 292. PubMed ID: 25887553
Summary:
Group B Sox proteins are a highly conserved group of transcription factors that act extensively to coordinate nervous system development in higher metazoans while showing both co-expression and functional redundancy across a broad group of taxa. In Drosophila melanogaster, the two group B Sox proteins Dichaete and SoxNeuro show widespread common binding across the genome. While some instances of functional compensation have been observed in Drosophila, the function of common binding and the extent of its evolutionary conservation is not known. This study used DamID-seq to examine the genome-wide binding patterns of Dichaete and SoxNeuro in four species of Drosophila. Through a quantitative comparison of Dichaete binding, the rate of binding site turnover was evaluated across the genome as well as at specific functional sites. The presence of Sox motifs was examined within binding intervals, along with the correlation between sequence conservation and binding conservation. To determine whether common binding between Dichaete and SoxNeuro is conserved, a detailed analysis was performed of the binding patterns of both factors in two species. This study found that, while the regulatory networks driven by Dichaete and SoxNeuro are largely conserved across the drosophilids studied, binding site turnover is widespread and correlated with phylogenetic distance. Nonetheless, binding is preferentially conserved at known cis-regulatory modules and core, independently verified binding sites. The strongest binding conservation was observed at sites that are commonly bound by Dichaete and SoxNeuro, suggesting that these sites are functionally important. This analysis provides insights into the evolution of group B Sox function, highlighting the specific conservation of shared binding sites and suggesting alternative sources of neofunctionalisation between paralogous family members.

Clark, E., Battistara, M. and Benton, M. A. (2022). A timer gene network is spatially regulated by the terminal system in the Drosophila embryo. Elife 11. PubMed ID: 36524728
Summary:
In insect embryos, anteroposterior patterning is coordinated by the sequential expression of the 'timer' genes caudal, Dichaete and odd-paired, whose expression dynamics correlate with the mode of segmentation. In Drosophila, the timer genes are expressed broadly across much of the blastoderm, which segments simultaneously, but their expression is delayed in a small 'tail' region, just anterior to the hindgut, which segments during germband extension. Specification of the tail and the hindgut depends on the terminal gap gene tailless, but beyond this the regulation of the timer genes is poorly understood. This study used a combination of multiplexed imaging, mutant analysis, and gene network modelling to resolve the regulation of the timer genes, identifying 11 new regulatory interactions and clarifying the mechanism of posterior terminal patterning. It is proposed that a dynamic Tailless expression gradient modulates the intrinsic dynamics of a timer gene cross-regulatory module, delineating the tail region and delaying its developmental maturation.

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).

Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size

The regulation of organ size constitutes a major unsolved question in developmental biology. The wing imaginal disc of Drosophila serves as a widely used model system to study this question. Several mechanisms have been proposed to have an impact on final size, but they are either contradicted by experimental data or they cannot explain a number of key experimental observations and may thus be missing crucial elements. This study has modeled a regulatory network that integrates the experimentally confirmed molecular interactions underlying other available models. Furthermore, the network includes hypothetical interactions between mechanical forces and specific growth regulators, leading to a size regulation mechanism that conceptually combines elements of existing models, and can be understood in terms of a compression gradient model. According to this model, compression increases in the center of the disc during growth. Growth stops once compression levels in the disc center reach a certain threshold and the compression gradient drops below a certain level in the rest of the disc. This model can account for growth termination as well as for the paradoxical observation that growth occurs uniformly in the presence of a growth factor gradient and non-uniformly in the presence of a uniform growth factor distribution. Furthermore, it can account for other experimental observations that argue either in favor or against other models. The model also makes specific predictions about the distribution of cell shape and size in the developing disc, which were confirmed experimentally (Aegerter-Wilmsen, 2012).

This paper presents a new model for the regulation of wing disc size. The model contains a rather complex regulatory network, which consists of a considerable number of interactions, receives nonuniform input of protein activities, and interacts with a mechanical stress pattern that emerges over time and space. It is assumed that the regulatory network represents protein activities and interactions that regulate these activities. The model does not distinguish between interactions at the transcriptional and protein activity level, but considers effects on net activities. All protein activities emerge from the network, except for those of Dpp, Wg and N, which are implemented in the model. In the regulatory network, differences in Ds and Fj concentrations between neighboring cells lead to activation of Dichate (D) by changing its intracellular localization. In addition, it is assumed that a weighted average of the area of a cell and its neighbors is a good readout for mechanical stress, that cells do not rearrange when exposed to mechanical tension, and that the planar polarization of D imposes a bias on the direction of the division plane. The interactions are hypothetical and form the main untested assumptions underlying the model. The regulation of ds by mechanical compression is not essential for the principle behind size regulation in the model, but improves the fit of simulation results with experimental data (Aegerter-Wilmsen, 2012).

A qualitative understanding can be gained by considering it in terms of a compression gradient model. During growth, compression increases in the center of the disc. Growth ceases when compression in the center reaches a certain threshold and the gradient of the compression gradient drops below a certain threshold in the rest of the disc. Read-out of the compression gradient is accomplished by a mechanism that involves Vg and the Hippo pathway. Numerical simulations were used to show that the model can account for growth termination and that it reproduces a large range of additional data on growth regulation, including some emergent properties of the system. Based upon the principle underlying the model, predictions can be made with respect to cell shape patterns. In order to take into account the curved surface of the wing pouch, an open source image analysis program was developed. The results showed that the general dynamics of the formation of cell shape patterns is indeed similar to the one predicted by the model. This analysis is, however, based on images from different discs and, especially during the early stages, there is variation among discs. It would therefore be interesting to assess whether the predicted dynamics is also present in the temporal evolution of single discs. However, this first requires the development of experimental methods with which single discs can be followed over time (Aegerter-Wilmsen, 2012).

Even though the development of cell shape patterns constitutes a fundamental prediction of the model, it would be an interesting future experimental challenge to test the model's basic assumptions directly, i.e., the regulation of Yki, Arm and ds by mechanical forces. The regulation of Yki by mechanical compression is most relevant for the model's behavior and appears necessary to obtain growth termination in combination with roughly uniform growth. The regulation of Arm by compression seems to be involved in stabilizing the Vg gradient, which could be relatively unstable if it would be regulated by Vg autoregulation alone. In addition, this interaction smoothens the compression gradient, which might have implications for the 3D structure of the wing disc. Last, the regulation of ds by mechanical forces is not essential for the principle behind size regulation, but improves the modeling results and also contributes to smoothening of the compression gradient. While developing the model, focus was placed on its ability to reproduce specific features of growth dynamics, as well as a number of key experiments that are used to argue in favor and against current models. One of the latter results, the decrease of medial growth upon induction of uniform Dpp signaling, could not be reproduced. In the simulations, these discs grow very fast. It is conceivable that such growth rates cannot be sustained in vivo because of a limited availability of nutrients and oxygen. When imposing a maximum total growth rate on disc growth, it is indeed possible to obtain growth rates in the medial part that are lower than those in wildtype discs, whereas lateral growth rates are higher, in agreement with experiments. Thus, with this additional assumption, the model can reproduce the results it was aimed to reproduce (Aegerter-Wilmsen, 2012).

There are currently no experimental data available on the parameters underlying the model and therefore they were fitted manually. As has become clear from the parameter analysis, there are only a few parameter combinations that can reproduce all results. However, it is not known whether this set is reproduced robustly in vivo and there is no natural selection on reproducing experimental manipulations robustly. Nevertheless, it is entirely possible that a larger set of parameter values should reproduce the results. In addition, even though the model can reproduce the selected set of experimentally observed features, there are related observations it cannot reproduce. For example, the final size reached in the model is too small, the experimentally observed nonautonomous growth induction by clones overexpressing brk is nearly absent in the model, and growth induction along the boundary of ds overexpressing clones extends further inside the clone than measured experimentally. It would be interesting to study whether there are factors missing in the model, which would make the parameter space less strict. For example, the parameter space was strongly restricted by the stipulation to reproduce the absence of Vg-BE activity in ap0 mutants upon ectopic wg expression. If it could be assumed that smaller discs have a different geometry in vivo than larger ones, the number of possible parameter combinations would increase. It will be interesting to assess the geometrical properties of discs in young larvae and evaluate whether the model should be adjusted in this respect (Aegerter-Wilmsen, 2012).

Very recently, another model has been formulated for growth regulation that assumes that growth is regulated by increases of Dpp signaling levels over time. However, growth is increased in wing discs in which Brk and Dpp signaling are removed. This either contradicts this model or the current understanding of Dpp signaling needs to be revised. The current model reproduces increased growth in such mutants, including its non-uniformity (Aegerter-Wilmsen, 2012).

The adult wing is covered by bristles, which point towards the distal part of the wing. This orientation is regulated by planar polarity genes. Regulation of planar polarity seems to be related to growth regulation. For example, Ds and Fj are not only important for growth regulation, but are also required for the development of a proximodistal polarity pattern. It is currently not clear whether Ds and Fj are directly involved in regulating planar polarity. If this were the case, then the model would suggest that planar polarity may, at least in part, arise from an interplay between morphogens and mechanical forces. The model presented in this study was developed for the wing imaginal disc of Drosophila. It would be interesting to see whether a similar model could also reproduce size regulation and additional experimental results in other systems. For other imaginal discs, it has been shown that their centers are also compressed at the end of growth. The precise regulatory networks involved in growth and size regulation are different for the different discs, but it would be interesting to see whether certain principles are conserved. In mammals, mechanical forces regulate growth in many tissues. However, the situation is often very different from that in the wing disc in that most mammalian tissues reach their final size while they perform a biological function. Thus, it would be interesting to study whether principles similar to those described here apply for mammalian organs early during development (Aegerter-Wilmsen, 2012).

Combinatorial temporal patterning in progenitors expands neural diversity

Human outer subventricular zone (OSVZ) neural progenitors and Drosophila type II neuroblasts both generate intermediate neural progenitors (INPs) that populate the adult cerebral cortex or central complex, respectively. It is unknown whether INPs simply expand or also diversify neural cell types. This study shows that Drosophila INPs sequentially generate distinct neural subtypes, that INPs sequentially express Dichaete, Grainy head and Eyeless transcription factors, and that these transcription factors are required for the production of distinct neural subtypes. Moreover, parental type II neuroblasts also sequentially express transcription factors and generate different neuronal/glial progeny over time, providing a second temporal identity axis. It is concluded that neuroblast and INP temporal patterning axes act together to generate increased neural diversity within the adult central complex; OSVZ progenitors may use similar mechanisms to increase neural diversity in the human brain (Bayraktar, 2013).

Tests were carried out to determine whether D, Grh and Ey exhibit cross-regulation in INPs. wor-gal4, ase-gal80 was used to drive UAS-D^RNAi in a Dichaete heterozygous background (subsequently called D RNAi, in which RNAi denotes RNA interference), which removed detectable D from INP lineages. Compared to wild type, D RNAi resulted in a significant loss of early born Grh⁺ Ey⁻ INPs, without altering the number of later-born Grh⁺ Ey⁺ INPs. The same result was observed in D mutant clones. By contrast, misexpression of D did not lead to ectopic Grh expression. Thus, D is necessary for the timely activation of Grh in INP lineages, although D-independent inputs also exist (Bayraktar, 2013).

To test whether Grh regulates D or Ey, R9D11-gal4 was used to drive UAS-grh^RNAi in a grh heterozygous background (subsequently called grh RNAi), which significantly reduced Grh levels in middle-aged INPs. grh RNAi increased the number of D⁺ INPs at the expense of Ey⁺ INPs without altering the total number of INPs. As expected, grh RNAi did not change the numbers of D⁺ and Ey⁺ INPs in the DM1 lineage, which lacks Grh expression, nor did misexpression of Grh lead to ectopic Ey expression. It is concluded that Grh represses D and activates Ey within INP lineages (Bayraktar, 2013).

To determine whether Ey regulates D or Grh, R12E09^D-gal4 UAS-FLP actin-FRT-stop-FRT-gal4 was used to drive permanent expression UAS-ey^RNAi within INPs (subsequently called R12E09^D) act-gal4 or INP-specific ey RNAi. It was confirmed that INP-specific ey RNAi removed Ey expression from INPs, without affecting Ey in the mushroom body or optic lobes. ey RNAi resulted in a notable increase in the number of old D^-Grh⁺ INPs, without affecting the number of young D⁺ INPs. Conversely, Ey misexpression in INPs significantly reduced the number of Grh⁺ INPs without altering the total number of INPs. An increase was observed in D⁺ INPs, consistent with a regulatory hierarchy in which Ey represses Grh, which represses D. This effect was not due to ectopic Ey directly activating D because misexpression of Ey had no effect on D⁺ INP numbers in the DM1 lineage, which lacks Grh expression. It is concluded that Ey is necessary and sufficient to terminate the Grh expression window in INPs. A 'feedforward activation/feedback repression' model is proposed for D-to-Grh-to-Ey cross-regulation (Bayraktar, 2013).

Next, it was asked whether distinct neuronal or glial subtypes were generated during each transcription factor expression window. To determine the cell types produced by young D⁺ INPs or old Ey⁺ INPs, permanent lineage tracing was used. Cells labelled by R12E09^D but not OK107^ey are generated by young INPs, whereas cells labelled by OK107^ey are generated by old INPs. A collection of 60 transcription factor antibodies was screened and two were found that labelled subsets of young INP progeny, and two that labelled subsets of old INP progeny. The transcription factors D and Brain-specific homeobox (Bsh) labelled sparse, non-overlapping subsets of young INP progeny, but not old INP progeny. Thus, young INPs generate Bsh⁺ neurons, D⁺ neurons, and many neurons that express neither gene. By contrast, the glial transcription factor Reverse polarity (Repo) and the neuronal transcription factor Twin of eyeless (Toy) labelled sparse, non-overlapping subsets of old INP progeny, but not young INP progeny. Additional mechanisms must restrict each marker (D, Bsh, Repo and Toy) to small subsets of young or old INP progeny; for example, each population could arise from just early or late born INPs within a type II neuroblast lineage. It is concluded that INPs sequentially express the D, Grh and Ey transcription factors, and they generate distinct neuronal and glial cell types during successive transcription factor expression windows. These data provide the first evidence in any organism that INPs undergo temporal patterning (Bayraktar, 2013).

Experiments were designed to determine whether D, Grh and Ey act as temporal identity factors that specify the identity of INP progeny born during their window of expression. First, the role of Ey in the specification of late born INP progeny was investigated. INP-specific ey RNAi resulted in the complete loss of the late born Toy⁺ neurons and Repo⁺ neuropil glia, but did not alter the number of early born D⁺ and Bsh⁺ neurons. Removal of Toy⁺ neurons (using toy RNAi) does not alter the number of Repo⁺ glia, and conversely removal of Repo⁺ glia (using gcm RNAi) does not alter the number of Toy⁺ neurons; thus Ey is independently required for the formation of both classes of late INP progeny. Conversely, permanent misexpression of Ey in early INPs increased late born Toy⁺neurons and decreased early born Bsh⁺ neurons, consistent with Ey specifying late INP temporal identity. Unexpectedly, ectopic Ey reduced the number of late born Repo⁺ glia. Itis concluded that Ey is an INP temporal identity factor that promotes the independent specification of late born Toy⁺ neurons and Repo⁺ glia (Bayraktar, 2013).

Next tests were performed to see whether D and Grh specify early and mid INP temporal identity. INP-specific D RNAi led to a small but significant reduction in the number of early born Bsh⁺ neurons, whereas INP-specific grh RNAi severely reduced the number of early born Bsh⁺ neurons without impairing INP proliferation or late INP progeny. This is consistent with the Bsh⁺ neurons deriving from the D⁺ Grh⁺ expression window. Interestingly, misexpression of D or Grh did not increase Bsh⁺ neuron numbers; perhaps D/Grh co-misexpression is required to generate Bsh⁺ neurons. It is concluded that both D and Grh are required, but not sufficient, for the production of Bsh⁺ early INP progeny (Bayraktar, 2013).

The function of early or late born INP progeny in adult brain development is unknown. This study determined the role of late born INP neurons and glia in the development and function of the adult central complex (CCX), an evolutionarily conserved insect brain structure containing many type II neuroblast progeny. The CCX consists of four interconnected compartments at the protocerebrum midline: the ellipsoid body, the fan-shaped body, the bilaterally paired noduli, and the protocerebral bridge; each of these compartments is formed by a highly diverse set of neurons. First, permanent lineage tracing was used to map the contribution of late born Ey⁺ INP progeny to the adult CCX. Cell bodies were detected in the dorsoposterior region of the CCX, and their axonal projections extensively innervated the entire ellipsoid body, fan-shaped body, and protocerebral bridge, with much weaker labelling of the paired noduli. It is concluded that old INPs contribute neurons primarily to the ellipsoid body, fan-shaped bod and protocerebral bridge regions of the CCX. Second, INP-specific ey RNAi was used to delete the late born Toy⁺ neurons and Repo⁺ glia. Loss of late born INP progeny generated major neuroanatomical defects throughout the adult CCX: the ellipsoid body and paired noduli were no longer discernible, the fan-shaped body was enlarged, and the protocerebral bridge was fragmented. Subsets of this phenotype were observed after removal of Toy⁺ neurons or Repo⁺ glia, showing that they contribute to distinct aspects of the CCX. Previous studies have described similar or weaker morphological CCX defects in ey hypomorphs, toy mutants, and after broad glia ablation during larval stages. In addition, ey RNAi adults were found to have relatively normal locomotion, but have a significant deficit in negative geotaxis. It is concluded that Ey is a temporal identity factor that specifies late born neuron and glial identity, and that these late born neural cell types are essential for assembly of the adult central complex (Bayraktar, 2013).

Bsh⁺ neurons and Repo⁺ glia were found to be sparse within the total population of young and old INP progeny, respectively, indicating that other mechanisms must help to restrict the formation of these neural subtypes. One mechanism could be temporal patterning within type II neuroblast lineages (Bayraktar, 2013).

To determine whether type II neuroblasts change their transcriptional profiles over time, known temporal transcription factors were examined for expression in type II neuroblasts at five time points in their lineage (24, 48, 72, 96 and 120 h ALH). No type II neuroblast expression for Hunchback, Kruppel, Pdm1/2 and Broad, and Grh was expressed in all type II neuroblasts at all time points. However, three transcription factors were identified with temporal expression in type II neuroblasts. D and Castor (Cas) were specifically detected in early type II neuroblasts: 3-4 neuroblasts at 24 h ALH, 0-1 neuroblast at 48 h ALH, and none later. Although D was never detected simultaneously in all type II neuroblasts at 24 h, permanent lineage tracing with R12E09^D labels all type II neuroblasts, indicating that all transiently express D. The third transcription factor, Seven up (Svp), showed a pulse of expression in a subset of type II neuroblasts at 48 h ALH, but was typically absent from younger or older type II neuroblasts. D, Cas and Svp are all detected in the anterior-most type II neuroblasts (probably corresponding to DM1-DM3), and thus at least these type II neuroblasts must sequentially express D or Cas, and Svp. It is concluded that type II neuroblasts can change gene expression over time (Bayraktar, 2013).

Next, tests were performed to determine whether type II neuroblasts produce different INPs over time. Permanently labelled clones were generated within the type II neuroblast lineages at progressively later time points. If type II neuroblasts change over time to make different INPs, early and late neuroblast clones should contain different neural subtypes. Clones were assayed for Repo⁺ glia and Bsh⁺ neurons, choosing these markers because Repo⁺ neuropil glia have been proposed to be born early in type II neuroblast lineages and Bsh⁺ neurons were positioned far from the Repo⁺ glia consistent with a different birth-order. Bsh⁺ neuron numbers began to decline only in clones induced at the latest time point, showing that they are generated late in the type II neuroblast lineage. By contrast, Repo⁺ glia were detected in clones induced early but not lat. This allows assigning of Repo⁺ glia to an 'early neuroblast, old INP' portion of the lineage, and Bsh⁺ neurons to a 'late neuroblast, young INP' portion of the lineage. It is concluded that type II neuroblasts undergo temporal patterning, and neuroblast temporal patterning was proposed to act together with INP temporal patterning to increase neural diversity in the adult brain (Bayraktar, 2013).

This study has shown that INPs sequentially express three transcription factors (D, Grh and then Ey), and that different neural subtypes are generated from successive transcription factor windows. It is likely that multiple GMCs are born from each of the four known INP gene expression windows; GMCs born from a particular gene expression window may have the same identity, or may be further distinguished by 'subtemporal genes' as in embryonic type I neuroblast lineages. This study also showed that each temporal factor is required for the production of a distinct temporal neural subtype. Loss of D or Grh leads to the loss of Bsh⁺ neurons; loss of Ey leads to loss of Toy⁺ neurons and Repo⁺ glia, although the fate of the missing cells is unknown. An unexpected finding was that Ey limits the lifespan of INPs. Mechanisms that prevent INP de-differentiation have been characterized -- loss of the translational repressor Brain tumour (Brat) or the transcription factor Earmuff (Erm) causes INPs to de-differentiate into tumorigenic type II neuroblasts, but factors that terminate normal INP proliferation have never before been identified (Bayraktar, 2013).

The D-to-Grh-to-Ey INP temporal identity factors are all used in other contexts during Drosophila development. Many embryonic neuroblasts sequentially express D and Grh. Ey is expressed in mushroom body neuroblasts, and is required for development of the adult brain mushroom body. Interestingly, mammalian orthologues of D and Ey (SOX2 and PAX6, respectively) are expressed in neural progenitors, including OSVZ progenitor, but have not been tested for a role in temporal patterning (Bayraktar, 2013).

This study has shown that there are two axes of temporal patterning within type II neuroblast lineages: both neuroblasts and INPs change over time to make different neurons and glia, thereby expanding neural diversity. It will be important to investigate whether INPs generated by OSVZ neural stem cells undergo similar temporal patterning (perhaps using SOX2 and PAX6), and whether combinatorial temporal patterning contributes to the neuronal complexity of the human neocortex (Bayraktar, 2013).

Temporal patterning of Drosophila medulla neuroblasts controls neural fates

In the Drosophila optic lobes, the medulla processes visual information coming from inner photoreceptors R7 and R8 and from lamina neurons. It contains approximately 40,000 neurons belonging to more than 70 different types. This study describes how precise temporal patterning of neural progenitors generates these different neural types. Five transcription factors - Homothorax, Eyeless, Sloppy paired, Dichaete and Tailless - are sequentially expressed in a temporal cascade in each of the medulla neuroblasts as they age. Loss of Eyeless, Sloppy paired or Dichaete blocks further progression of the temporal sequence. Evidence is provided that this temporal sequence in neuroblasts, together with Notch-dependent binary fate choice, controls the diversification of the neuronal progeny. Although a temporal sequence of transcription factors had been identified in Drosophila embryonic neuroblasts, this work illustrates the generality of this strategy, with different sequences of transcription factors being used in different contexts (Li, 2013).

In the developing medulla, the wave of conversion of neuroepithelium into neuroblasts makes it possible to visualize neuroblasts at different temporal stages in one snapshot, with newly generated neuroblasts on the lateral edge and the oldest neuroblasts on the medial edge of the expanding crescent shaped neuroblast region. An antibody screen was conducted for transcription factors expressed in the developing medulla and five transcription factors, Hth, Ey, Slp1, D and Tll, were identified that are expressed in five consecutive stripes in neuroblasts of increasing ages, with Hth expressed in newly differentiated neuroblasts, and Tll in the oldest neuroblasts. This suggests that these transcription factors are sequentially expressed in medulla neuroblasts as they age. Neighbouring transcription factor stripes show partial overlap in neuroblasts with the exception of the D and Tll stripes, which abut each other. Previous studies have reported that Hth and Ey< were expressed in medulla neuroblasts, but they had not been implicated in controlling neuroblast temporal identities. Hth and Tll also show expression in the neuroepithelium (Li, 2013).

To address whether each neuroblast sequentially expresses the five transcription factors, their expression was examined in the neuroblast progeny. Hth, Ey and Slp1 are expressed in three different layers of neurons that correlate with birth order, that is, Hth in the first-born neurons of each lineage in the deepest layers; Ey or Slp1 in correspondingly more superficial layers, closer to the neuroblasts. This suggests that they are born sequentially in each lineage. D is expressed in two distinct populations of neurons. The more superficial population inherit D from D⁺ neuroblasts. D⁺ neurons in deeper layers (corresponding to the Hth and Ey layers) turn on D expression independently and will be discussed later. Single neuroblast clones were generated, and the expression of the transcription factors was examined in the neuroblast and its progeny. Single neuroblast clones in which the neuroblast is at the Ey⁺ stage include Ey⁺ GMCs/neurons as well as Hth⁺ neurons. This indicates that Ey⁺ neuroblasts have transited through the Hth⁺ stage and generated Hth⁺ neurons. Clones in which the neuroblast is at the D⁺ stage contain Slp1⁺ GMCs and Ey⁺ neurons, suggesting that D⁺ neuroblasts have already transited through the Slp⁺ and Ey⁺ stages. This supports the model that each medulla neuroblast sequentially expresses Hth, Ey, Slp1 and D as it ages, and sequentially produces neurons that inherit and maintain expression of the transcription factor (Li, 2013).

slp1 and slp2 are two homologous genes arranged in tandem and function redundantly in embryonic and eye development. Slp2 is expressed in the same set of medulla neuroblasts as Slp1. Slp1 and Slp2 are referred to collectively as Slp (Li, 2013).

Tll is expressed in the oldest medulla neuroblasts. The oldest Tll⁺ neuroblasts show nuclear localization of Prospero (Pros), suggesting that they undergo Pros-dependent cell-cycle exit at the end of their life, as in larval nerve cord and central brain neuroblasts. Tll⁺ neuroblasts and their progeny express glial cells missing (gcm), and the progeny gradually turn off Tll and turn on Repo, a glial-specific marker. These cells migrate towards deeper neuronal layers and take their final position as glial cells around the medulla neuropil. Thus, Tll⁺ neuroblasts correspond to previously identified glioblasts between the optic lobe and central brain that express gcm and generate medulla neuropil glia. Clones in which the neuroblast is at the Tll⁺ stage contain Hth⁺ neurons and Ey⁺ neurons, among others, confirming that Tll⁺ neuroblasts represent the final temporal stage of medulla neuroblasts rather than a separate population of glioblasts. Therefore, these data clearly show that medulla neuroblasts sequentially express five transcription factors as they age. The four earlier temporal stages generate neurons that inherit and maintain the temporal transcription factor present at their birth, although a subset of neurons born during the Ey, Slp or D neuroblast stages lose expression of the neuroblast transcription factor. At the final temporal stage, neuroblasts switch to glioblasts and then exit the cell cycle (Li, 2013).

Whether cross-regulation among transcription factors of the neuroblast temporal sequence contributes to the transition from one transcription factor to the next was examined. Loss of hth or its cofactor, extradenticle (exd), does not affect the expression of Ey and subsequent progression of the neuroblast temporal sequence (Li, 2013).

ey-null mutant clones were generated using a bacterial artificial chromosome (BAC) rescue construct recombined on a chromosome containing a Flip recombinase target (FRT) site in an ey^J5.71 null background. ey^J5.71 homozygous mutant larvae were also tested. In both cases, Slp expression is lost in neuroblasts, along with neuronal progeny produced by Slp⁺ neuroblasts, marked by the transcription factor Twin of eyeless (Toy, see below). However, neuroblast division is not affected, and Hth remains expressed in only the youngest neuroblasts and first-born neurons. Targeted ey RNA interference (RNAi) using a Vsx-Gal4 driver that is expressed in the central region of the neuroepithelium and neuroblasts gives the same phenotype. This suggests that Ey is required to turn on the next transcription factor, Slp, but is not required to repress Hth (Li, 2013).

In clones of a deficiency mutation, slp^S37A, that deletes both slp1 and slp2, neuroblasts normally transit from Hth⁺ to Ey⁺, but older neuroblasts maintain the expression of Ey and do not progress to express D or Tll, suggesting that Slp is required to repress ey and activate D (Li, 2013).

Similarly, in D mutant clones, neuroblasts are also blocked at the Slp⁺ stage, and do not turn on Tll, indicating that D is required to repress slp and activate tll. Finally, in tll mutant clones, D expression is not expanded into oldest neuroblasts, suggesting that tll is not required for neuroblasts to turn off D. Thus, in the medulla neuroblast temporal sequence, ey, slp and D are each required for turning on the next transcription factor. slp and D are also required for turning off the preceding transcription factor (Li, 2013).

Gain-of-function phenotypes of each gene were studied. However, misexpression of Hth, Ey, Slp1 or Slp2, or D in all neuroblasts or in large neuroblast clones is not sufficient to activate the next transcription factor or repress the previous transcription factor in neuroblasts. Only misexpressing tll in all neuroblasts is sufficient to repress D expression (Li, 2013).

In summary, cross-regulation among transcription factors is required for at least some of the transitions. No cross-regulation was observed between hth and ey. Because ey is already expressed at low levels in the neuroepithelium and in Hth⁺ neuroblasts, an as yet unidentified factor might gradually upregulate ey and repress hth to achieve the first transition. As tll is sufficient but not required to repress D expression, additional factors must act redundantly with Tll to repress D (Li, 2013).

The temporal sequence of neuroblasts described above could specify at least four neuron types plus glia (in fact more than ten neuron types plus glia considering that neuroblasts divide several times at each stage with overlaps between neighbouring temporal transcription factors). As this is not sufficient to generate the 70 medulla neuron types, it was asked whether another process increases diversity in the progeny neurons born from a neuroblast at a specific temporal stage. Apterous (Ap) is known to mark about half of the 70 medulla neuron types. In the larval medulla, Ap is expressed in a salt-and-pepper manner in subsets of neurons born from all temporal stages. In the progeny from Hth⁺ neuroblasts, all neurons seem to maintain Hth, with a subset also expressing Ap. However, only half of the neurons born from neuroblasts at other transcription factor stages maintain expression of the neuroblast transcription factor. For instance, in the progeny of Ey⁺ neuroblasts, Ey⁺ neurons are intermingled with about an equal number of Ey⁻ neurons that instead express Ap. Neuroblast clones contain intermingled Ey⁺ and Ap⁺ neurons. This is also true for the progeny of Slp⁺ neuroblasts: Slp1⁺ neurons are intermingled with Slp1⁻ Ap⁺ neurons. In the progeny of D⁺ neuroblasts, D and Ap are co-expressed in the same neurons, and they are intermingled with neurons that express neither D nor Ap. Neurons in deeper neuronal layers (corresponding to the Ey⁺ and Hth⁺ neuron layers) also express D independently, and these neurons are Ap⁻. The expression of Ap is stable from larval to adult stages (Li, 2013).

The intermingling of Ap⁺ and Ap⁻ neurons raised the possibility that asymmetric division of GMCs gives rise to one Ap⁺ and one Ap⁻ neuron. Two-cell clones were generated to visualize the two daughters of a GMC. In every case, one neuron is Ap⁺ and the other is Ap^-, suggesting that asymmetric division of GMCs diversifies medulla neuron fates by controlling Ap expression (Li, 2013).

Asymmetric division of GMCs in Drosophila involves Notch (N)-dependent binary fate choice. In the developing medulla, the N pathway is involved in the transition from neuroepithelium to neuroblast, and loss of Su(H), the transcriptional effector of N signalling, leads to faster progression of neurogenesis and neuroblast formation. However, Su(H) mutant neuroblasts still follow the same transcription factor sequence and generate GMCs and neuronal progeny, allowing analysis of the effect of loss of N function on GMC progeny diversification. Notably, neurons completely lose Ap expression in Su(H) mutant clones. All mutant neurons born during the Hth⁺ stage still express Hth, but not Ap, suggesting that the N^ON daughters of Hth⁺ GMCs are the neurons expressing both Ap and Hth. In contrast to wild-type clones, all Su(H) mutant neurons born during the Ey⁺ neuroblast stage express Ey and none express Ap. Similarly, all mutant neurons born during the Slp⁺ neuroblast stage express Slp1 but lose Ap. These data suggest that, for Ey⁺ or Slp⁺ GMCs, the N^OFF daughter maintains the neuroblast transcription factor expression, whereas the N^ON daughter loses this expression but expresses Ap. In the wild-type progeny born during the D⁺ neuroblast stage, Ap⁺ neurons co-express D. Both D and Ap are lost in Su(H) mutant clones in the D⁺ neuroblast progeny, confirming that D is transmitted to the Ap⁺N^ON daughter of D⁺ GMCs. By contrast, the D⁺Ap⁻ neurons in the deeper layers (corresponding to the N^OFF progeny born during the Ey⁺ and Hth⁺ neuroblast stages, see above) are expanded in Su(H) mutant clones at the expense of Ap⁺ neurons. Therefore, the deeper layer of D expression is turned on independently in the N^OFF daughters of Hth⁺ and Ey⁺ GMCs (Li, 2013).

Finally, in wild type, a considerable amount of apoptotic cells were observed dispersed among neurons, suggesting that one daughter of certain GMCs undergoes apoptosis in some of the lineages. Together these data suggest that Notch-dependent asymmetric division of GMCs further diversifies neuronal identities generated by the temporal sequence of transcription factors (Li, 2013).

How does the neuroblast transcription factor temporal sequence, together with the Notch-dependent binary fate choice, control neuronal identities in the medulla? Transcription factor markers specifically expressed in subsets of medulla neurons, but not in neuroblasts, were examined including Brain-specific homeobox (Bsh) and Drifter (Dfr), as well as other transcription factors identified in the antibody screen, for example, Lim3 and Toy. Bsh is required and sufficient for the Mi1 cell fate, and Dfr is required for the morphogenesis of nine types of medulla neurons, including Mi10, Tm3, TmY3, Tm27 and Tm27Y (Hasegawa, 2011). Investigation were carried out to identify at which neuroblast temporal stage these neurons were born by examining co-expression with the inherited neuroblast transcription factors. Then whether the neuroblast transcription factors regulate expression of these markers and neuron fates was investigated. The results for each neuroblast stage are described below (Li, 2013).

Bsh is expressed in a subset of Hth⁺ neurons, suggesting that Bsh is in the N^ON daughter of Hth⁺ GMCs. Indeed, Bsh expression is lost in both Su(H) and hth mutant clones. Thus, both Notch activity and Hth are required for specifying the Mi1 fate, consistent with the previous report that Hth is required for the Mi1 fate. Ectopic expression of Hth in older neuroblasts is also sufficient to generate ectopic Bsh⁺ neurons, although the phenotype becomes less pronounced in later parts of the lineage. These data suggest that Hth is necessary and sufficient to specify early born neurons, but the competence to do so in response to sustained expression of Hth decreases over time. This is similar to embryonic CNS neuroblasts, where ectopic Hb is only able to specify early born neurons during a specific time window (Li, 2013).

Lim3 is expressed in all Ap⁻ progeny of both Hth⁺ and Ey⁺ neuroblasts. Toy and Dfr are expressed in subsets of neurons born from Ey⁺ neuroblasts, as indicated by their expression in the Ey⁺ neuron progeny layer. The most superficial row of Ey⁺Ap⁻ neurons express Toy (and Lim3), suggesting that they are the N^OFF progeny of the last-born Ey⁺ GMCs. Dfr is co-expressed with Ap in two or three rows of neurons that are intermingled with Ey⁺ neurons, suggesting that they are the N^ON progeny from Ey⁺ GMCs. In addition to these Ap⁺ Dfr⁺ neurons, Dfr is also expressed in some later-born neurons that are Ap⁻ but express another transcription factor: Dachshund (Dac), in specific sub-regions of the medulla crescent (Li, 2013).

Whether Ey in neuroblasts regulates Dfr expression in neurons was tested. As expected, Dfr-expressing neurons are lost in ey-null mutant clones, suggesting that they require Ey activity in neuroblasts, even though Ey is not maintained in Ap⁺ Dfr⁺ neurons. Furthermore, in slp mutant clones in which neuroblasts remain blocked in the Ey⁺ state, the Ap⁺ Dfr⁺ neuron population is expanded into later-born neurons, suggesting that the transition from Ey⁺ to Slp⁺ in neuroblasts is required for shutting off the production of Ap⁺ Dfr⁺ neurons. In addition, Ap⁺ Dfr⁺ neurons are lost in Su(H) mutant clones. Thus, Ey expression in neuroblasts and the Notch pathway together control the generation of Ap⁺ Dfr⁺ neurons (Li, 2013).

In addition to its expression with Ey in the N^OFF progeny of the last-born Ey⁺ GMCs, Toy is also expressed in Ap⁺ (N^ON) neurons in more superficial layers generated by Slp⁺ and D⁺ neuroblasts. Consistently, in Su(H) mutant clones, an expansion of Toy⁺ Ey⁺ neurons is seen in the Ey progeny layer, followed by loss of Toy in the Slp and D progeny layer (Li, 2013).

Tests were performed to see whether Slp is required for the neuroblasts to switch from generating Toy⁺Ap⁻ neurons, progeny of Ey⁺ neuroblasts, to generating Toy⁺ Ap⁺ neurons. Indeed, in slp mutant clones, the Toy⁺Ap⁺ neurons largely disappear, whereas Toy⁺ Ap⁻ neurons expand (Li, 2013).

WAp and Toy expression was examined in specific adult neurons. OrtC1-gal4 primarily labels Tm20 and Tm5 plus a few TmY10 neurons, and these neurons express both Ap and Toy. To examine whether Slp is required for the specification of these neuron types, wild-type or slp mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique by heat-shocking for 1 h at early larval stage, and the number of OrtC1-gal4-marked neurons in the adult medulla was examined. In wild-type clones, OrtC1-gal4 marks ~100 neurons per medulla. By contrast, very few neurons are marked by OrtC1-gal4 in slp mutant clones. Slp is unlikely to directly regulate the Ort promoter because Slp expression is not maintained in Ap⁺ Toy⁺ neurons. Furthermore, the expression level of OrtC1-gal4 in lamina L3 neurons is not affected by slp mutation. These data suggest that loss of Slp expression in neuroblasts strongly affects the generation of Tm20 and Tm5 neurons (Li, 2013).

In summary, these data show that the sequential expression of transcription factors in medulla neuroblasts controls the birth-order-dependent expression of different neuronal transcription factor markers, and thus the sequential generation of different neuron types (Li, 2013).

Although a temporal transcription factor sequence that patterns Drosophila nerve cord neuroblasts was reported more than a decade ago, it was not clear whether the same or a similar transcription factor sequence patterns neural progenitors in other contexts. The current identification of a novel temporal transcription factor sequence patterning the Drosophila medulla suggests that temporal patterning of neural progenitors is a common theme for generating neuronal diversity, and that different transcription factor sequences might be recruited in different contexts (Li, 2013).

There are both similarities and differences between the two neuroblast temporal sequences. In the Hb-Kr-Pdm-Cas-Grh sequence, ectopically expressing one gene is sufficient to activate the next gene, and repress the previous gene, but these cross-regulations are not necessary for the transitions, with the exception of Castor. In the Hth-Ey-Slp-D-Tll sequence, removal of Ey, Slp or D does disrupt cross-regulations necessary for temporal transitions (except the Hth-Ey transition). However, in most cases these cross-regulations are not sufficient to ensure temporal transitions, suggesting that additional timing mechanisms or factors are required (Li, 2013).

For simplicity, the medulla neuroblasts are represented as transiting through five transcription factor stages, whereas in fact the number of stages is clearly larger than five. First, neuroblasts divide more than once while expressing a given temporal transcription factor, and each GMC can have different sub-temporal identities. Furthermore, there is considerable overlap between subsequent temporal neuroblast transcription factors: neuroblasts expressing two transcription factors are likely to generate different neuron types from neuroblasts expressing either one alone (Li, 2013).

Although the complete lineage of medulla neuroblasts is still being investigated, this study shows how a novel temporal sequence of transcription factors is required to generate sequentially the diverse neurons that compose the medulla. The requirement for transcription factor sequences in the medulla and in embryonic neuroblasts suggests that this is a general mechanism for the generation of neuronal diversity. Interestingly, the mammalian orthologue of Slp1, FOXG1, acts in cortical progenitors to suppress early born cortical cell fates. Thus, transcription-factor-dependent temporal patterning of neural progenitors might be a common theme in both vertebrate and invertebrate systems (Li, 2013).

A temporal mechanism that produces neuronal diversity in the Drosophila visual center

The brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf/Vvl), correspond to types of medulla neurons. This study examined the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, transcription factors were sought that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates [Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)]. The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. The results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequential ly expressed in NBs (Suzuki, 2013).

In the embryonic central nervous system, the heterochronic transcription factors suchas Hb, Kr, Pdm, Cas and Grh are expressed in NBs to regulate the temporal specification of neuronal identity. They regulate each other to achieve sequential changes in their expression in NBs without cell-extrinsic factors. However, expression of the embryonic heterochronic genes was not detected in the medulla NBs.Instead this study found that Hth, Klu, Ey, Slp and D are transiently and sequentially expressed in medulla NBs. The expression of Hth and Klu was observed in lateral NBs, while that of Ey/Slp and D was observed in intermediate and medial NBs, respectively. These observations suggest that the expression of heterochronic transcription factors changes sequentially as each NB ages, as observed in the development of the embryonic central nervous system (Suzuki, 2013).

This study demonstrates that at least three of the temporal factors Ey, Slp and D regulate each other to form a genetic cascade that ensures the transition from Ey expression to D expression in the medulla NBs. Ey expression in NBs activates Slp, while Slp inactivates Ey expression. Similarly, Slp expression in NBs activates D expression, while D inactivates Slp expression. In fact, the expression of Slp is not strong in newer NBs in which Ey is strongly expressed, but is up regulated in older NBs in which Ey is weakly expressed in the wildtype medulla. A similar relationship is found between Slp and D, supporting the idea that Ey, Slp and D regulate each other's expression to control the transition from Ey-expression to D-expression. In the embryonic central nervous system, similar interaction is mainly observed between adjacent genes of the cascade hb-Kr-pdm-cas-grh, and this concept may also be applied to the medulla primordium. The expression pattern and function of Ey, Slp and D suggest that they are adjacent to each other in the cascade of transcription factor expression in medulla NBs (Suzuki, 2013).

However, no such relationship was found between Hth, Klu and the other temporal factors.The sequential expression of Hth and Klu could be regulated by an unidentified mechanism that is totally different from the genetic cascade that controls the transition through Ey-Slp-D. Or, there might be unidentified temporal factors that are expressed in lateral NBs which act upstream of Hth and Klu to regulate their expression. It is necessary to identify additional transcription factors that are transiently expressed in medulla NBs (Suzuki, 2013).

The expression of concentric transcription factors in the medulla neurons correlates with the temporal sequence of neuron production from the medulla NBs (Hasegawa, 2011). In the larval medulla primordium, the neurons are located in the order of Hth/Bsh-, Run- and Drf-positive cells from inside to outside, and these domains are adjacent to each other (Hasegawa, 2011). Given that NBs generate neurons toward the center of the developing medulla, Hth/Bsh-positive neurons are produced at first, and then Run-positive and Drf-positive neurons. Thus Hth/Bsh, Run and Drf were used as markers to examine roles of Hth, Klu, Ey, Slp and D expressed in NBs in specifying types of medulla neurons. The continuous expression of Hth and Ey from NBs to neurons and the results of clonal analyses that visualize the progeny of NBs expressing each one of the temporal transcription factors suggest that the temporal windows of NBs expressing Hth, Klu and Ey approximately correspond to the production of Hth/Bsh-, Run- and Drf- positive neurons, respectively. Indeed, the results of the genetic study suggest that Hth and Ey are necessary and sufficient to induce the production of Hth/Bsh- and Drf-positive neurons,respectively (Hasegawa, 2011, 2013). Ectopic Klu expression at least induces the produc tion of Run-positive neurons (Suzuki, 2013).

Slp and D expression in NBs may correspond to the temporal windows that produce medulla neurons in the outer domains of the concentric zones, which are most likely produced after the production of Drf-positive neurons. The results at least suggest that Slp is necessary and sufficient and D is sufficient to repress the production of Drf-positive neurons. Identification of additional markers that are expressed in the outer concentric zones compared to the Drf-positive domain would be needed to elucidate the roles of Slp and D in specification of medulla neuron types (Suzuki, 2013).

D mutant clones did not produce any significant phenotype except for derepression of Slp expression in NBs. Drf expression in neurons was not affected either. Since D is a Sox family transcription factor, SoxN, another Sox family transcription factor, is a potential candidate molecule that acts together with D in the medulla NBs. However, its expression was found in neuroepithelia cells and lateral NBs that overlap with Hth-positive cells but not with D-positive cells. All the potential heterochronic transcription factors examined in this study are expressed in three to five cell rows of NBs. Nevertheless, one NB has been observed to produce one Bsh- positive and one Run-positive neuron (Hasegawa, 2011). Therefore, the expression pattern of the heterochronic transcription factors is not sufficient to explain the stable production of one Bsh-positive and one Run-positive neuron from a single NB.The combinatorial action of multiple temporal factors expressed in NBs may play important roles in the specification of Bsh- and Run- positive neurons (Suzuki, 2013).

Another possible mechanism that guarantees the production of a limited number of the same neuronal type from multiple rows of NBs expressing a temporal transcription factor could be a mutual repression between concentric transcription factors expressed in medulla neurons. For example, Hth/Bsh, Run and Drf may repress each other to restrict the number of neurons that express either of these transcription factors. However, expression of Run and Drf was not essentially affected in hth mutant clones and in clones expressing Hth (Hasegawa, 2011). Similarly, expression of Hth and Drf was not essentially affected in clones expressing run RNAi under the control of AyGal4, in which Run expression is eliminated. Hth and Run expression was not affected in drf mutant clones (Hasegawa, 2011). These results suggest that Hth/Bsh, Run and Drf do not essentially regulate each other during the formation of concentric zones in the medulla (Suzuki, 2013).

During embryonic development, the heterochronic genes that are expressed in NBs (hb-Kr-pdm-cas-grh) are maintained and act in GMCs to specify neuronal type. Similarly, Hth and Ey are continuously expressed from NBs to neurons, suggesting that their expression may also be inherited through GMCs (Hasegawa, 2011). However, this type of regulatory mechanism may be somewhat modified in the case of Klu, Slp and D (Suzuki, 2013).

Klu is expressed in NBs and GMCs, but not in neurons. Slp and D are predominantly detected in NBs and neurons visualized by Dpn and Elav, respectively. Occasionally, however, expression of D was found in putative GMCs, which are situated between NBs and neurons. Additionally, both D-positive and D-negative cells were found among Miranda-positive GMCs. Slp expression was not found in Miranda-positive GMCs. Finally, D is expressed in medulla neurons forming a concentric zone in addition to its expression in medial NBs. However, D expression was abolished in slp mutant NBs but remained in the mutant neurons, suggesting that D expression in medulla neurons is not inherited from the NBs. These results suggest that Slp and D expression are not maintained from NBs to neurons and that not all the temporal transcription factors expressed in NBs are inherited through GMCs. However, it is possible to speculate that Klu, Slp and D regulate expression of unidentified transcription factors in NBs that are inherited from NBs to neurons through GMCs (Suzuki, 2013).

A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system

Brain areas each generate specific neuron subtypes during development. However, underlying regional variations in neurogenesis strategies and regulatory mechanisms remain poorly understood. In Drosophila, neurons in four optic lobe ganglia originate from two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers. Using genetic manipulations, this study found that one IPC neuroepithelial domain progressively transformed into migratory progenitors that matured into neural stem cells (neuroblasts) in a second domain. Progenitors emerged by an epithelial-mesenchymal transition-like mechanism that required the Snail-family member Escargot and, in subdomains, Decapentaplegic signaling. The proneural factors Lethal of scute and Asense differentially controlled progenitor supply and maturation into neuroblasts. These switched expression from Asense to a third proneural protein, Atonal. Dichaete and Tailless mediated this transition, which was essential for generating two neuron populations at defined positions. It is proposed that this neurogenesis mode is central for setting up a new proliferative zone to facilitate spatio-temporal matching of neurogenesis and connectivity across ganglia. (Apitz, 2014).

Recent studies have distinguished three neurogenesis modes in the Drosophila CNS. First, type I neuroblasts arise from neuroepithelia and generate GMCs, which produce neuronal and glial progeny. Second, Dpn+ type II neuroblasts in the dorsomedial central brain go through a transit-amplifying Dpn+, Ase+ population, called intermediate neural precursors, which generate GMCs and postmitotic offspring. Third, lateral OPC neuroepithelial cells bypass the neuroblast stage and generate lamina precursor cells (LPCs) that divide once to produce lamina neurons. The current results provide evidence for a fourth strategy: p-IPC neuroepithelial cells give rise to progenitors that migrate to a second neurogenic domain, where they mature into type I neuroblasts. These progenitors are distinct, as they originate from the neuroepithelium, do not express markers for neuroblasts, intermediate neural precursors, GMCs or postmitotic neurons, and acquire NSC properties after completing their migration (Apitz, 2014).

Migratory progenitors arise from the p-IPC by a mechanism that shares cellular and molecular characteristics with EMT. On the basis of data on gastrulation and neural crest formation, EMT is commonly associated with cells adopting a mesenchymal state, enabling them to leave their epithelial tissue and migrate through the extracellular matrix to new locations. A recent study also reported an EMT-like process in the mammalian neocortex, whereby newborn neurons and intermediate progenitors delaminate from the ventricular neuroepithelium and radially migrate to the pial surface. This study observed that neuroepithelial cells at the p-IPC margins and migratory progenitors upregulated the Snail homolog Esg, whereas E-cad levels were decreased. Moreover, esg knockdown caused the formation of ectopic E-cad-expressing clusters adjacent to the p-IPC. Although this is a previously uncharacterized role of Drosophila esg, these findings are consistent with the requirement of two Snail transcription factors, Scratch1 and 2, and downregulation of E-cad in cortical EMT migration (Apitz, 2014).

Although TGFβ signaling is well known to induce EMT, it was unclear whether it could have such a role in the brain. Two lines of evidence are consistent with a requirement of the Drosophila family member Dpp. First, it is expressed and downstream signaling is activated in dorsal and ventral p-IPC subdomains and emerging cell streams. Second, tkv mutant cells form small neuroepithelial clusters in p-IPC vicinity. Similar to the neural crest, where distinct molecular cascades control delamination in the head and trunk, region-specific regulators may also be required in p-IPC subdomains. Because neuroblasts derived from Dpp-dependent cell streams map to defined areas in the d-IPC, this pathway could potentially couple EMT and neuron subtype specification (Apitz, 2014).

Cell migration is an essential feature of vertebrate brain development. Commonly, postmitotic immature neurons migrate from their proliferation zones to distant regions, where they further differentiate and integrate into local circuits. Examples include the radial migration of projection neurons and tangential migration of interneurons in the embryonic cortex, as well as migration of interneuron precursors in the rostral migratory stream to the olfactory bulb in adults. In contrast, IPC progenitors develop into NSCs (neuroblasts) after they migrated. A recent study found that NSCs relocating from the embryonic ventral hippocampus to the dentate gyrus act as source for adult NSCs in the subgranular zone. In addition, cerebellar granule cell precursors migrate from the rhombic lip to the external granule layer, where they proliferate during early postnatal development. The migration of neural cell types that become proliferative in a new niche could therefore constitute a more general strategy. IPC progenitors form streams of elongated, closely associated cells. Despite their different developmental state, their organization is notably similar to the neuronal chain network in the lateral walls of the subventricular zone and the rostral migratory stream in mammals, or of migratory trunk neural crest cells in chick. Further studies will need to identify the determinants directing migratory progenitors into the d-IPC (Apitz, 2014).

Several constraints could shape a neurogenesis mode that requires migratory progenitors in the larval optic lobe. The OPC is located superficially and the IPC is positioned centrally. If medulla and lobula neurons arose by neuroepithelial duplications, these new populations would need to be integrated into an ancestral visual circuit consisting of lamina and lobula plate neurons. Cellular migration may therefore be a derived feature and serve as an essential spatial adjustment of the IPC to the newly added medulla. In principle, the migratory population could consist of immature neurons. However, migratory progenitors help to establish a new superficial proliferative niche, and to align OPC and d-IPC neuroblast positions. This in turn enables the OPC and IPC to use spatially matching birth order-driven neurogenesis patterns for establishing functionally coherent connections across ganglia (Apitz, 2014).

IPC progenitors were primed to mature into neuroblasts, but were prevented to do so in cell streams. Consistently, progenitors showed weak cytoplasmic Mira expression and prematurely differentiated into neuroblasts following loss of Pcl. Although Dichaete has been shown to repress ase to maintain embryonic neuroectodermal cells in an undifferentiated state, this study did not identify such a role in the IPC. Future studies are therefore required to distinguish whether this block in neuroblast maturation is released in the d-IPC by cell-intrinsic mechanisms or locally acting signals (Apitz, 2014).

The p-IPC and d-IPC consecutively expressed three proneural factors. esg-positive p-IPC neuroepithelial cells transiently expressed L'sc as they converted into progenitors. Following arrival in the d-IPC, progenitors matured into neuroblasts, which switched bHLH protein expression from Ase to Ato. This correlated with a change in cell division orientations from toward the lamina to the optic lobe surface and the generation of two lineages, distal cells and lobula plate neurons. The progression of neuroblasts through two stages is supported by the observations that progenitors solely entered the lower d-IPC, all neuroblasts were labeled with Ase in this area, and idpp reporter gene expression in a progenitor subset persisted in both lower and upper d-IPC neuroblasts and their progeny (Apitz, 2014).

Late l'sc knockdown reduced the number of d-IPC neuroblasts and both neuron classes, whereas p-IPC formation and EMT of progenitors appeared to be unaffected. This supports the idea that l'sc promotes neuroblast formation by controlling the rate of conversion and the progenitor supply. In contrast, ase loss severely decreased the amount of lower d-IPC neuroblasts and distal cells. This revealed a central role in the maturation of progenitors into neuroblasts, endowing them with the potential to proliferate and generate a specific lineage. Although these functions are the opposite of those observed in the OPC, they align with the role of a murine Ase homolog, Achaete-scute homolog 1 (Ascl1), in the embryonic telencephalon. Ase^- neuroblasts with type I proliferation patterns have not previously been described. Further underscoring the context-dependent activities of proneural bHLH factors, ato does not have the equivalent role of ase in conferring neurogenic properties to upper d-IPC neuroblasts, but acts upstream of differentiation programs controlling the projections of lobula plate neurons (Apitz, 2014).

Although Ase and Ato each regulated distinct aspects of d-IPC development, they were not required for either the transition or the extent of their expression domains. These functions were fulfilled by Dichaete and tll, whose cross-regulatory interactions were essential for the transition from Ase⁺ to Ato⁺, Dac⁺ expression. To link birth order and fate, temporal identity transcription factors are sequentially expressed by neuroblasts and inherited by GMCs and their progeny born during a given developmental window. Acting as the final two members of the OPC-specific series of temporal identity factors, Dichaete is required for Tll expression, whereas tll is sufficient, but not required, to inhibit Dichaete Although OPC and d-IPC neuroblasts shared the sequential expression of Dichaete and Tll, key differences include the fact that d-IPC progeny did not maintain Dichaete, that Tll was transiently expressed in newborn progeny of the upper d-IPC and was not maintained in older lineages, that Dichaete in the lower d-IPC was not required in its own expression domain for neurogenesis, and that Dichaete was required to activate tll, and tll to repress Dichaete and ase, as well as to independently upregulate Ato and Dac. Although the mechanisms that trigger the timing of the switch require further analysis, these observations support the notion that, in the d-IPC, Dichaete and tll do not function as temporal identity factors, but as switching factors between two sequential neuroblast stages. The vertebrate homologs of Dichaete and tll, Sox2 and Tlx, are essential for adult NSC maintenance and Sox2 positively regulates Tlx expression, suiggesting that core regulatory interactions between Dichaete and tll family members may be conserved (Apitz, 2014).

These studies uncovered molecular signatures for generating a migratory neural population by EMT and subsequent NSC development that are in part shared between the fly optic lobe and vertebrate cortical neurogenesis. The unexpected parallels suggest that ancestral gene regulatory cassettes imparting specific cellular properties may have been re-employed during vertebrate brain development. Analysis of p-IPC and d-IPC neurogenesis in the Drosophila optic lobe therefore opens new possibilities for systematically identifying genes regulating EMT, cell migration and sequential NSC specification (Apitz, 2014).

EFFECTS OF MUTATION

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 lsc 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).

The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. This study found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete-expressing local interneurons in development of the adult olfactory circuitry (Melnattur, 2012).

During embryonic and larval stages, Dichaete expression is observed in a highly dynamic and diverse pattern that includes many different cell types and tissues. Indeed, the Dichaete gene has pleiotropic functions in these stages and influences a wide range of developmental processes. Similarly, Dichaete expression in the adult CNS is also complex. Within the central brain, strong Dichaete expression was detected in several prominent paired clusters of neurons. This includes the LAAL cells as well as other clusters located medially and dorsally near the optic lobes, and along the dorsal margin of the protocerebrum. The ~225 LAAL cells minimally consist of a heterogeneous mixture of GABAergic and cholinergic LNs, as well as ring neurons of the central complex ellipsoid body. Among the Dichaete-expressing LNs are descendants of the lateral neuroblast (lNb) that gives rise to LNs, PNs and other neuronal types. However Dichaete expression was not detected in any PN progeny of the INb. Strong Dichaete expression was also observed in both neurons and glia in the medulla of the adult optic lobes. Many of these Dichaete-expressing cells also express Eyeless, which has highly conserved, essential functions in eye development. In vertebrates, the Dichaete homolog SOX2 is important for expression of lens crystallin genes and proper differentiation of eye tissues, and human SOX2 mutations are associated with haploinsufficient bilaterial anopthalmia. Furthermore, SOX2 forms a functional complex with the Eyeless homolog, Pax6 that initiates lens placode development. These data suggests that the semi-lethal Dichaete alleles described in this study may also be useful to examine conserved functions of Sox genes in visual system development (Melnattur, 2012).

While Dichaete null alleles exhibit complete embryonic lethality associated with major disruptions in segmentation and nervous system formation, Dichaete mutants were first identified based on an adult phenotype affecting wing posture. These dominant gain-of-function phenotypes correspond to ectopic Dichaete expression in the wing hinge region that results from inversion breakpoints within Dichaete gene regulatory regions. The semi-lethal D¹⁰⁷, D⁸⁹, and D¹⁷⁵ mutations described in this study appear to be hypomorphic regulatory alleles. Thus, each excision mutant contains only internal deletion(s) within the PZ P element of P[rJ375]; none contain a detectable deletion of the Dichaete coding region or intron. Analysis of Dichaete mutant adult brains revealed a mosaic reduction of Dichaete protein expression levels. Both D⁸⁹ and D¹⁰⁷ exhibited strongly reduced Dichaete expression in the optic lobes and D⁸⁹ mutants also exhibited an overall reduction of Dichaete expression in most sites within the central brain, including the LAAL cells. In contrast, Dichaete expression in D¹⁰⁷ brains is not as strongly diminished in LAAL cells, but is reduced in other brain regions. The milder disruption of Dichaete expression in D¹⁰⁷ compared to D⁸⁹ is consistent with the greater viability of this allele (Melnattur, 2012).

The incomplete penetrance of PN defects in Dichaete mutants could be a consequence of alterations in Dichaete expression in a specific subset of LNs essential for PN targeting: Dichaete expression may not be uniformly disrupted in every individual mutant. Alternatively, the incomplete penetrance of Dichaete mutant phenotypes might reflect a specific biochemical role for Dichaete protein where reduced levels of Dichaete function may only incompletely disrupt expression of specific Dichaete target genes (Melnattur, 2012).

Overall, the data suggest that the loss of internal P element sequences in the D⁸⁹ and D¹⁰⁷ alleles results in disruption of proper transcriptional regulation of the native Dichaete gene. As the original P element insertion associated with P[rJ375] enhancer trap strain does not appear to alter Dichaete gene function or expression, the internal deletions in the viable excision mutants may result in the formation of novel sequences within the P element that impact Dichaete gene transcription. These sequences could correspond to ectopic repressor elements that disrupt the actions of brain enhancer elements in the native Dichaete gene. Alternately, in all three Dichaete mutants the 5' end of the lacZ gene is intact; thus, it is possible an aberrant RNA transcript is generated that disrupts Dichaete gene transcription in the adult brain. While limited data is available for comparison, internal P element excisions have been shown to influence expression of the vestigial and yellow genes (Melnattur, 2012).

Significantly, while viable Dichaete mutants exhibited alterations in PN dendritic and axonal processes, Dichaete expression was not detected in any mature or developing PNs, and analysis of D⁸⁷ mutant clones indicated that loss of Dichaete function in developing GH146-GAL4 PNs did not result in detectable PN abnormalities. In addition, Dichaete was not identified in a search for PN transcription factors important for PN dendritic targeting. These observations suggest that Dichaete influences PN differentiation via non-cell-autonomous mechanisms. In this case, what are the relevant sites of Dichaete expression for PN differentiation? Within the glomeruli, PNs interact with both ORNs and LNs. No Dichaete expression was detected in developing antennal ORNs, strongly suggesting that they are not the relevant cell type. However, prominent Dichaete expression was observed in multiple LN types present in LAAL clusters that are in close proximity to PNs. While any of these LAAL cells could interact with neighboring PNs, given their axonal projections into the AL, the Dichaete-expressing LNs are particularly attractive candidates. This suggests two potential, nonmutually exclusive models. One possibility is that a loss of specific LNs in Dichaete mutants might deprive PN dendrites of a physical substrate necessary to guide proper growth into and targeting within the AL. However, while Dichaete mutant brains did exhibit slight displacement of some LN cell bodies, there did not appear to be significant alterations in LN numbers or projection patterns. LN organization was largely unaltered. Thus, it seems unlikely that LN neurites serve merely a physical substrate for PN dendrite outgrowth. A distinct possibility is that some or all LNs participate in a signalling pathway that influences PN dendritic elaboration. Interestingly, it has recently demonstrated that Hedgehog protein secreted by AL neurons into the developing AL is required for the targeting of many ORN classes. Thus there is significant precedent to suggest that cell non-autonomous signals direct the targeting of olfactory neural processes. Given the close proximity of LN and PN processes, such a signal could be mediated via direct cell/cell contact or over short distances. Identification of specific LN and PN classes that may be involved in this signalling pathway remains to be determined. GH146-GAL4 lPN targeting and differentiation were normal in D⁸⁷ null mutant lNb clones where Dichaete function is lost in lNb-derived LNs. This result indicates that Dichaete function is not required in lNb-derived LNs for differentiation of GH146-GAL4 (and therefore, Mz19-GAL4) lPNs. However, it remains possible that Dichaete function is required in lNb-derived LNs for the targeting and differentiation of Mz19-GAL4 adPNs. Careful examination of the Mz19-GAL4 PN defects in Dichaete mutants appears to support of this idea. Thus, in all instances of Dichaete mutants where Mz19-GAL4 PN dendrites innervated a single glomerulus, that glomerulus appeared to be DA1, normally a target of lPNs. This phenotype was frequently accompanied by a loss of Mz19-GAL4 adPNs, supporting the notion that innervation of Mz19-GAL4 adPN target glomeruli is selectively lost in Dichaete mutants. Alternatively, Dichaete function could be required in a novel subset of LNs that do not derive from the lNb (Melnattur, 2012).

Similarly, proper midline crossing of axons from specific ORN classes may also require non-cell autonomous functions of Dichaete. Thus, the contralateral but not ipsilateral projections of OR88a-Gal4, UAS-mCD8GFP and OR47b-Gal4, UASmCD8GFP expressing neurons lack GFP expression in Dichaete mutants, even though Dichaete is not expressed in these ORNs. This effect is cell-type specific as both the ipsilateral and contralateral projections of ORD67d-GAL4; UAS-mCD8GFP expressing neurons are unaffected in Dichaete mutant brains and exhibit the same GFP expression pattern as observed in a wild type background. While the basis for this ORN axon defect is uncertain and could reflect disruptions in GFP localization as opposed to ORN axon guidance, the midline crossing of ORN axons has been found to require Robo2 in the ORNs themselves and Slit in an unidentified cell type. It is possible that Dichaete may influence Slit expression in relevant adult brain cell types similarly to its regulation of Slit expression in the embryonic CNS midline. Another potential explanation for the ORN axon defects in Dichaete mutants is that the OR88a and OR47b axons may require guidance cues from a set of LN axons that project across the midline. Dichaete mutants could disrupt the functions of these LNs, thereby altering midline guidance signals utilized by some ORNs. At this point there is no compelling data supporting either explanation and the unaffected midline crossing of OR67d axons in Dichaete mutants suggests highly specific guidance defects (Melnattur, 2012).

In summary, this study identifies a role for the Dichaete Sox protein in the organization of the adult Drosophila olfactory circuit. Dichaete expression was observed in discrete clusters of neurons within the Drosophila adult brain that were shown to include excitatory and inhibitory LNs as well as several central complex ring neurons. No Dichaete expression was detected in PNs or ORNs. Analysis of novel viable Dichaete mutant alleles revealed functions for Dichaete in the proper elaboration of dendritic and axonal processes of specific PNs. Normal differentiation of PNs thus appears to require input from distinct Dichaete-expressing cells. It is of interest to ultimately identify the nature and source of this input and characterize the Dichaete-dependent processes that contribute to olfactory circuit formation (Melnattur, 2012).

EVOLUTIONARY HOMOLOGS Phylogeny of Sox factors

Members of the SOX family of transcription factors are found throughout the animal kingdom, are characterized by the presence of a DNA-binding HMG domain, and are involved in a diverse range of developmental processes. Support is found for subdivision of the family into groups A-H, and for the assignment of two new groups, I and J. For vertebrate genes, it appears that relatedness as suggested by HMG domain sequence is congruent with relatedness as indicated by overall structure of the full-length protein and intron-exon structure of the genes. Most of the SOX groups identified in vertebrates are represented by a single SOX sequence in each invertebrate species studied. An HMG domain signature motif has been identifed which may be considered representative of the SOX family. Based on this data, a robust phylogeny of SOX genes is presented that reflects their evolutionary history in metazoans (Bowles, 2000).

By convention, SOX proteins are more than 50% identical to SRY in the HMG domain. This definition is now inaccurate, with the identification of new SOX genes that do not conform to this rule. For instance, the group H sequence hu-SOX30 is 48% and ce-SOXJ is only 46% identical to hu-SRY in the HMG domain. By comparison, mo-LEF1 is 24% identical to hu-SRY. Based on these figures, it seems that classification based on a strict 50% identity to SRY may not be a suitable indicator of SOX family membership. Reference to the SRY sequence was a historical, arbitrary, and, in retrospect, poor choice for such SOX family comparisons, since SRY has arisen only in the mammalian lineage and is clearly very divergent. It may be more appropriate to compare identity to another SOX sequence or to the SOX consensus sequence. However, even if this is done, a 50% cutoff value may be too stringent (e.g., human SOX30, 46% identity to the SOX consensus). The results provide an alternative criterion to define SOX genes using the conservation of key motifs within the HMG domain. The HMG domain sequence RPMNAF (positions 5-10) appears to be conserved for all SOX sequences, including those of groups H, I, and J, but not for the most closely related outgroups fu-MATA1, mo-LEF1, and mo-TCF1. However, this sequence is also present in a recently defined SOX-like gene in Drosophila, capicua (cic), which has apparent orthologs in C. elegans and humans, suggesting that this 6-amino-acid motif is insufficient to strictly define SOX genes. The extended version, common to all non-SRY SOX members (RPMNAFMVW), appears to be the most reliable signature of the SOX family (Bowles, 2000).

Invertebrate representatives have been discovered for most of the SOX groups thus far identified. C. elegans proteins ce-SOXB1, ce-SOXB2, ce-SOXC, and ce-SOXD (known as COG-2) are associated with subgroups B1 and B2 and groups C and D, respectively. No C. elegans genes encoding proteins with homology to groups E, F, G, H, or I have been detected. An additional C. elegans SOX protein which cannot be assigned to any of the existing groups has been provisionally allocated to a new group J. A putative C. elegans ortholog of LEF/TCF (ce-LEF/TCF) has also been identified. Drosophila SOX proteins dr-SOXB1 (CG18024 or SoxNeuro), dr-SOXB2.1 (dichaete), dr-SOXB2.2, dr-SOXB2.3, dr-SOXC (Sox box protein 14 or CG17263), dr-SOXD, dr-SOXE (Sox100B or CG12098), and dr-SOXF (Sox box protein 15 or CG8404) are associated with groups B1, B2, C, D, E, and F as indicated by their names. No Drosophila sequences have been found for groups G, H, I, or J. Similarly, sea urchin sequences se-SOXB1, se-SOXB2, and se-SOXD1 are associated with subgroups B1 and B2 and group D. It is entirely likely that additional sea urchin SOX genes remain to be identified -- this genome is not yet completely sequenced. For each invertebrate species examined, only one representative sequence has been identified for each group -- the exception to this is group B. This suggests that for each of the currently recognizable SOX groups, a single ancestral form existed before the origin of the vertebrate lineage. In contrast to this general trend, four group B SOX sequences have been identified in Drosophila. These include a single group B1 representative (SoxB1) along with three group B2 genes (SoxB2.1, SoxB2.2, and SoxB2.3). The three SoxB2 genes are physically linked and it is possible that lineage-specific duplication and diversification have occurred in this case. In support of this possibility, the HMG boxes of SoxB2.2 and SoxB2.3 are approximately 50 and 70 kb downstream of the HMG box of SoxB2.1 (dichaete) on chromosome 3. This relatively recent divergence is confirmed by maximum likelihood analysis: the four group B sequences clustered in 100% of analyses (Bowles, 2000).

Based on phylogenetic considerations, it is not possible to define the invertebrate orthologs of specific mammalian genes. It has been suggested that dichaete (here called SOXB2.1) is the Drosophila equivalent of mammalian SOX2. Although mouse SOX2 can functionally substitute for dichaete, the analysis suggests that the Drosophila protein might more reasonably be considered to represent an ancestral form of the entire SOXB or SOXB2 group. dichaete is similar to vertebrate SOX2 sequences only in the HMG domain and a short C-terminal region that does not appear to be essential for the gene's function, suggesting that rescue of the dichaete mutant might be possible also with other vertebrate group B proteins (Bowles, 2000).

Parallel expansions of Sox transcription factor group B predating the diversifications of the arthropods and jawed vertebrates

Group B of the Sox transcription factor family is crucial in embryo development in the insects and vertebrates. Sox group B, unlike the other Sox groups, has an unusually enlarged functional repertoire in insects, but the timing and mechanism of the expansion of this group were unclear. Data for Sox group B was collected and analyzed from 36 species of 12 phyla representing the major metazoan clades, with an emphasis on arthropods, to reconstruct the evolutionary history of SoxB in bilaterians and to date the expansion of Sox group B in insects. It was found that the genome of the bilaterian last common ancestor probably contained one SoxB1 and one SoxB2 gene only and that tandem duplications of SoxB2 occurred before the arthropod diversification but after the arthropod-nematode divergence, resulting in the basal repertoire of Sox group B in diverse arthropod lineages. The arthropod Sox group B repertoire expanded differently from the vertebrate repertoire, which resulted from genome duplications. The parallel increases in the Sox group B repertoires of the arthropods and vertebrates are consistent with the parallel increases in the complexity and diversification of these two important organismal groups (Zhong, 2011).

Previous studies have suggested two incompatible models for the expansion of Sox group B in Drosophila. One of these models places one of the four SoxB members into subgroup B1, and the other three into subgroup B2. Although Sox21a (SoxB2a) into subgroup B2, the other model maintains that Dichaete (SoxB2b1) and Sox21b (SoxB2b2) are both co-orthologous to both vertebrate Sox1 and Sox2 rather than to the vertebrate SoxB2 members, and that the Protostome-Deuterostome LCA had a three-member complement of Sox group B proteins. The resolution of this dispute lies in the correct orthology assignments of the Drosophila SoxB members with the vertebrate ones, and a valid reconstruction of the ancestral SoxB repertoire at key phylogenetic nodes. A related and interesting question concerns the phylogenetic timing of the expansion of Sox group B in Drosophila. Initially, this expansion was attributed to relatively recent duplications, but later research involving more insect taxa indicated that the four-member SoxB inventory is phylogenetically old, and was at least present in the LCA of the Hymenoptera and Diptera. However, whether this expansion is even older remained an open question at that time (Zhong, 2011).

A comparison of the two models of Sox group B evolution is presented. The first model is for Sox group B evolution proposed by a previous study. In this model, an ancestral SoxB generate original Dichaete and SoxNeuro by an ancient genome duplication, a subsequent tandem duplication generate original Sox21a before the Deuterostome/Protostome split. After the Deuterostome/Protostome split, a further tandem duplication generated Sox21b in insects and an independent genome duplication event increased the copy number of SoxB in vertebrates. The second model shows this study's proposal for Sox group B evolution. In this model, the Protostome-Deuterostome last common ancester had one SoxB1 and one SoxB2 generated by an ancient tandem duplication of an ancestral SoxB. After the Deuterostome/Protostome split, two further tandem duplications gave rise to the additional two copies of SoxB2 in arthropods, and a linkage break between SoxB1 and SoxB2s occurred in the ancestor of Drosophila, resulting in the different chromosome locations of SoxB1 and SoxB2s in Drosophila; independently, the vertebrates increased their copy number of SoxB through the two rounds of genome duplication. Forks on the rectangles indicate pseudogenization leading to gene loss. SoxB2b1, SoxB2b2, and SoxB2a are the preferred synonyms for Dichaete, Sox21a, and Sox21b, respectively. Sry is currently considered to have evolved from allele Sox3 on the Y chromosome, and is therefore not shown in the models. The second model, described in this work, shows Sox neuro as belonging to subgroup SoxB1, homolgous to Sox1 and Sox2 of vertebrates. The SoxB2 subgroup in vertebrates is represented by Sox21 and Sox14 (Zhong, 2011).

Invertebrate Sox factors

In C. elegans, the anchor cell (AC) plays multiple, essential roles during vulval cell development. Among other roles, the AC signals six of twelve granddaughters of the ventral uterine cells, causing them to adopt a fate, pi, different from the default fate of their six sisters and cousins. The six pi precursors (three per side) divide to form twelve pi cells. Eight of the pi cells (four per side) fuse to form the large multinucleate uterine seam cell that lies over the top of the vulva orifice and underlies the developing uterus. The remaining four pi cells (two per side) adopt the uv1 fate and also make connections with vulval cells. In screens for mutants defective in vulval morphogenesis, multiple mutants were isolated in which the uterus and the vulva fail to make a proper connection. Five alleles are described that define the gene cog-2 (connection of gonad defective-2). To form a functional connection between the vulva and the uterus, the AC must fuse with the multinucleate uterine seam cell, derived from uterine cells that adopt a pi lineage. In cog-2 mutants, the anchor cell does not fuse to the uterine seam cell and remains instead at the apex of the vulva, blocking the connection between the vulval and uterine lumens, resulting in an egg-laying defective phenotype. According to lineage analysis and expression assays for two pi-cell-specific markers, induction of the pi fate occurs normally in cog-2 mutants. cog-2 is shown to encode a Sox family transcription factor that is expressed in the pi lineage. Thus, it appears that COG-2 is a transcription factor that regulates a late-stage aspect of uterine seam cell differentiation that specifically affects anchor cell-uterine seam cell fusion (Hanna-Rose, 1999).

Amphioxus (Cephalochordata within the phylum Chordata) , as the closest living invertebrate relative of the vertebrates, can provide insights into the evolutionary origin of the vertebrate body plan. Therefore, to investigate the evolution of genetic mechanisms for establishing and patterning the neuroectoderm, two amphioxus transcription factors, AmphiSox1/2/3 and AmphiNeurogenin were cloned and characterized. These genes are the earliest known markers for presumptive neuroectoderm in amphioxus. Genes in the Sox1/2/3 group are Sry-related HMG box transcription factors most closely related to Drosophila Dichaete. Until the discovery of target of Pox-n (tap), it was thought that Drosophila had no close relative of neurogenin and NeuroD, the nearest relative being atonal, most closely related to vertebrate MATH-1. However, Drosophila tap has sequence affinities with neurogenins, not with NeuroD, suggesting that neurogenin and NeuroD arose by gene duplication before the deuterostome-protostome split. Thus, both amphioxus and Drosophila may have homologs of NeuroD that are yet to be discovered (Holland, 2000).

By the early neurula stage, AmphiNeurogenin expression becomes restricted to two bilateral columns of segmentally arranged neural plate cells, which probably include precursors of motor neurons. This is the earliest indication of segmentation in the amphioxus nerve cord. Later, expression extends to dorsal cells in the nerve cord, which may include precursors of sensory neurons. By the midneurula, AmphiSox1/2/3 expression becomes limited to the dorsal part of the forming neural tube. These patterns resemble those of their vertebrate and Drosophila homologs. Taken together with the evolutionarily conserved expression of the dorsoventral patterning genes of chordates and Drosophila, BMP2/4 and chordin, respectively in nonneural and neural ectoderm, these results are consistent with the evolution of the chordate dorsal nerve cord and the insect ventral nerve cord from a longitudinal nerve cord in a common bilaterian ancestor. However, AmphiSox1/2/3 differs from its vertebrate homologs in not being expressed outside the CNS, suggesting that additional roles for this gene have evolved in connection with gene duplication in the vertebrate lineage. In contrast, expression in the midgut of AmphiNeurogenin, together with the gene encoding the insulin-like peptide, suggest that amphioxus may have homologs of vertebrate pancreatic islet cells, which express neurogenin3. In addition, AmphiNeurogenin, like its vertebrate and Drosophila homologs, is expressed in apparent precursors of epidermal chemosensory and possibly mechanosensory cells, suggesting a common origin for protostome and deuterostome epidermal sensory cells in the ancestral bilaterian (Holland 2000).

AmphiSox1/2/3 is a highly specific marker for presumptive neuroectoderm. Its expression is first detectable by in situ hybridization in the early gastrula (cap-shaped stage) in the dorsal epiblast, including the presumptive neuroectoderm. Expression remains uniformly strong in the presumptive neuroectoderm throughout gastrulation. Whether the expression domain includes any cells in adjacent nonneural ectoderm at these early stages is not clear because of the lack of anatomical markers. However, at the onset of neurulation when the neural plate flattens and becomes distinct from nonneural ectoderm, it is evident that expression is limited to neural ectoderm and is excluded from nonneural ectoderm. Since the edges of the neural plate curl dorsally, expression initially remains panneural. Later, before the edges of the neural plate have fused dorsally, expression becomes down-regulated along the midline and in the anterior part of the CNS. At the midneurula stage, expression is limited to the extreme posterior-dorsal portion of CNS and then ceases entirely. However, in much later larvae at the one- and two-gill-slit stages (2-5 days), prolonged staining reveals extremely weak expression in a few cells scattered in the CNS, rostral to the pigment spot at the level of somite 5. Expression is not detected in tissues other than neuroectoderm (Holland, 2000).

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Although neurons are dispersed throughout the epidermis in the adult, it has not been demonstrated that neurogenesis in the embryo is uniform. To determine the site of neurogenesis, the domains of expression were localized for three orthologs of pan-neural genes of chordates and Drosophila -- nrp/ musashi, sox1/2/3/ soxneuro, and hu/elav. The first two are markers of proliferating neuron precursors, whereas the third is a marker of differentiating neurons. All are expressed in the neural plate of various chordates, but not in the epidermis. nrp/musashi and sox1/2/3 /soxneuro are expressed in the entire ectoderm of the early S. kowalevskii embryo (except for the ciliated band, which all probes except emx fail to stain). In later stages, the expression remains strong in the prosome and declines in the metasome, correlating with Bullock's observation of decreasing neuron density posteriorly. In sections, weak expression of nrp/musashi can be detected in the posterior endoderm, possibly correlated with a sparse endodermal nerve net. Hu/elav exhibits similar diffuse staining throughout the ectoderm in early stages. Additionally, Hu/elav staining remains strong along the posterior dorsal midline at later stages, in a punctate pattern perhaps reflecting a concentration of early-differentiating nerves at this site. In sagittal sections of embryos, hu/elav expression appears localized toward the basal side of the ectoderm (basiepithelial); it is absent from the mesoderm. Thus, S. kowalevskii shows pervasive neurogenesis with no large, contiguous nonneurogenic subregion, as occurs in chordates (Lowe, 2003).

Tfap2 and Sox1/2/3 cooperatively specify ectodermal fates in ascidian embryos

Epidermis and neural tissues differentiate from the ectoderm in animal embryos. While epidermal fate is thought to be induced in vertebrate embryos, embryological evidence has indicated that no intercellular interactions during early stages are required for epidermal fate in ascidian embryos. To test this hypothesis, the gene regulatory circuits were determined for epidermal and neural specification in the ascidian embryo. These circuits started with Tfap2-r.b (AP-2-like2; see Drosophila AP-2) and Sox1/2/3 (see Drosophila Dichaete), which are expressed in the ectodermal lineage immediately after zygotic genome activation. Tfap2-r.b expression was diminished in the neural lineages upon of fibroblast growth factor signaling, which is known to induce neural fate, and sustained only in the epidermal lineage. Tfap2-r.b specified the epidermal fate cooperatively with Dlx.b (see Drosophila Dll), which was activated by Sox1/2/3. This Sox1/2/3-Dlx.b circuit was also required for specification of the anterior neural fate. In the posterior neural lineage, Sox1/2/3 activated Nodal, which is required for specification of the posterior neural fate. These findings support the hypothesis that the epidermal fate is specified autonomously in ascidian embryos (Satou, 2016).

Fish and Xenopus Sox factors

Waardenburg-Shah syndrome combines the reduced enteric nervous system characteristic of Hirschsprung's disease with reduced pigment cell number, although the cell biological basis of the disease is unclear. A zebrafish Waardenburg-Shah syndrome model has been analyzed. The colourless gene encodes a sox10 homolog, sox10 lesions have been identified in mutant alleles and the mutant phenotype has been rescued by ectopic sox10 expression. Using iontophoretic labelling of neural crest cells, it has been demonstrated that colourless mutant neural crest cells form ectomesenchymal fates. By contrast, neural crest cells which in wild types form non-ectomesenchymal fates generally fail to migrate and do not overtly differentiate. These cells die by apoptosis between 35 and 45 hours post fertilization. Evidence is provided that melanophore defects in colourless mutants can be largely explained by disruption of nacre/mitf expression. It is proposed that all defects of affected crest derivatives are consistent with a primary role for colourless/sox10 in specification of non-ectomesenchymal crest derivatives. This suggests a novel mechanism for the etiology of Waardenburg-Shah syndrome in which affected neural crest derivatives fail to be generated from the neural crest (Dutton, 2001).

From early stages of development, Sox2-class transcription factors (Sox1, Sox2 and Sox3) are expressed in neural tissues and sensory epithelia. Sox2 function is required for neural differentiation of early Xenopus ectoderm. Microinjection of dominant-negative forms of Sox2 (dnSox2) mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Expression of dnSox2 in developing embryos suppresses expression of N-CAM and regional neural markers. Temporal requirements of Sox2-mediated signaling were analyzed by using an inducible dnSox2 construct fused to the ligand-binding domain of the glucocorticoid receptor. Attenuation of Sox2 function both from the late blastula stage and from the late gastrula stage onwards causes an inhibition of neural differentiation in animal caps and in whole embryos. Additionally, dnSox2-injected cells that fail to differentiate into neural tissues are not able to adopt epidermal cell fate. These data suggest that Sox2-class genes are essential for early neuroectoderm cells to consolidate their neural identity during secondary steps of neural differentiation (Kishi, 2000).

Sox2 signaling is required during secondary stages of neural differentiation, starting from late gastrula. However, it remains unclear whether Sox2 function is necessary for the initial step of neural induction, which occurs around stage 10. Overexpression of Sox2 mRNA per se has little effects on animal cap ectoderm. When combined with FGF, Sox2 can modify the responsiveness of animal cap cells to FGF neuralizing signals. Sox2 alone is not sufficient to direct cells to neural fate but rather plays a role in changing the competence of the ectoderm. When 100 pg of Sox2 mRNA is injected into all the animal cells of 8-cell embryos, overexpression of Sox2 had very weak effects, if any, on the expression of neural markers at neurula stages. Higher doses of Sox2 mRNA causes non-specific effects such as exogastrulation. Therefore, as of yet, there is no particular evidence for instructive roles of Sox2 in early neural development. Sox2 and/or its close relatives are necessary for the ectoderm to develop into neural tissues during secondary steps of neural differentiation. However, it remains to be elucidated exactly which members of the Sox2-subfamily are responsible for this role. Being so closely related in structure and in expression pattern, it is likely that Sox2-subfamily genes have largely redundant functions. Additionally, it also remains to be clarified precisely which signaling steps in the downstream cascade of neural induction require the presence of Sox2-class factors. The transgenic frog technique may prove useful in the study of detailed gene interaction between early regulators of neural differentiation and analysis of the promoters of early neural marker genes. This new technique should provide information about the mode of action of Sox2 on neural-specific transcription (Kishi, 2000).

Cranial placodes, which give rise to sensory organs in the vertebrate head, are important embryonic structures whose development has not been well studied because of their transient nature and paucity of molecular markers. Markers of pre-placodal ectoderm (PPE) (six1, eya1) have been used to determine that gradients of both neural inducers and anteroposterior signals are necessary to induce and appropriately position the PPE. Overexpression of six1 expands the PPE at the expense of neural crest and epidermis, whereas knock-down of Six1 results in reduction of the PPE domain and expansion of the neural plate, neural crest and epidermis. Using expression of activator and repressor constructs of six1 or co-expression of wild-type six1 with activating or repressing co-factors (eya1 and groucho, respectively), it has been demonstrated that Six1 inhibits neural crest and epidermal genes via transcriptional repression and enhances PPE genes via transcriptional activation. Ectopic expression of neural plate, neural crest and epidermal genes in the PPE demonstrates that these factors mutually influence each other to establish the appropriate boundaries between these ectodermal domains (Brugmann, 2004).

These studies predict complex in vivo interactions between six1 and other ectodermal genes. sox2 and sox3 are both induced in presumptive neural ectoderm by neural inductive signaling, and promote stabilization of a neural fate. Later, they are both expressed in neural stem cells. Endogenous six1 expression in the LNE precedes sox2/3 placodal expression, indicating that sox2/3 are not upstream of six1. six1 overexpression in the lateral neurogenic ectoderm (LNE) has no significant effect on sox2/3 neural plate expression, indicating that its effects in this border domain are cell autonomous and not due to intermediate signaling. However, when six1 is reduced in the lateral ectoderm, sox2/3 expression expands laterally. This could be a secondary result of the expansion of foxD3, which in turn expands sox2/3 and/or a mutual antagonism between six1 and sox2/3. The latter possibility is supported by the observations that six1 expression in the neural plate dramatically represses sox2/3 and that expression of sox2 in the LNE represses six1. At later stages sox2/3 are expressed in placodal domains that presumably overlap with six1 expression. How these genes interact at this later phase of placode development remains to be determined (Brugmann, 2004).

Formation of the organizer is one of the most central patterning events in vertebrate development. Organizer-derived signals are responsible for establishing the CNS and patterning the dorsal ventral axis. The mechanisms promoting organizer formation are known to involve cooperation between Nodal and Wnt signalling. However, the organizer forms in a very restricted region, suggesting the presence of mechanisms that repress its formation. This study shows in zebrafish that the transcription factor Sox3 represses multiple steps in the signalling events that lead to organizer formation. Although beta-catenin, Bozozok and Squint are known to play major roles in establishing the dorsal organizer in vertebrate embryos, overexpression of any of these is insufficient to induce robust expression of markers of the organizer in ectopic positions in the animal pole, where Sox3 is strongly expressed. A dominant-negative nuclear localisation mutant of Sox3 can cause ectopic expression of organizer genes via a mechanism that activates all of these earlier factors, resulting in later axis duplication including major bifurcations of the CNS. It was also found that the related SoxB1 factor, Sox19b, can act redundantly with Sox3 in these effects. It therefore seems that the broad expression of these SoxB1 genes throughout the early epiblast and their subsequent restriction to the ectoderm is a primary regulator of when and where the organizer forms (Shih, 2010).

Progenitors in the developing central nervous system acquire neural potential and proliferate to expand the pool of precursors competent to undergo neuronal differentiation. Both the formation and maintenance of neural-competent precursors are regulated by SoxB1 transcription factors, and evidence that their expression is regionally regulated suggests that specific signals regulate neural potential in subdomains of the developing nervous system. The frizzled (Fz) transmembrane receptor Xfz5 selectively governs neural potential in the developing Xenopus retina by regulating the expression of Sox2. Blocking either Xfz5 or canonical Wnt signaling within the developing retina inhibits Sox2 expression, reduces cell proliferation, inhibits the onset of proneural gene expression, and biases individual progenitors toward a nonneural fate, without altering the expression of multiple progenitor markers. Blocking Sox2 function mimics these effects. Rescue experiments indicate that Sox2 is downstream of Xfz5. Thus, Fz signaling can regulate the neural potential of progenitors in the developing nervous system (Van Raay, 2005).

Progenitor cells in the central nervous system must leave the cell cycle to become neurons and glia, but the signals that coordinate this transition remain largely unknown. Wnt signaling, acting through Sox2, promotes neural competence in the Xenopus retina by activating proneural gene expression. This study reports that Wnt and Sox2 inhibit neural differentiation through Notch activation. Independently of Sox2, Wnt stimulates retinal progenitor proliferation and this, when combined with the block on differentiation, maintains retinal progenitor fates. Feedback inhibition by Sox2 on Wnt signaling and by the proneural transcription factors on Sox2 mean that each element of the core pathway activates the next element and inhibits the previous one, providing a directional network that ensures retinal cells make the transition from progenitors to neurons and glia (Agathocleous, 2009).

Wnt/β-catenin signaling acting through Sox2 activates proneural gene expression in the frog retina. This study shows that Wnt and Sox2 inhibit proneural action through Notch, thereby blocking neuronal differentiation. In addition, Wnt signaling stimulates proliferation independently of Sox2, maintaining the progenitor fate, while Sox2 pushes retinal progenitors to Müller glial fates. Concurrent activation of Sox2 and the cell cycle can recapitulate the effects of Wnt in maintaining the retinal precursor cell (RPC) fate. Finally, inhibition of Wnt signaling by Sox2, and of Sox2 by the proneural transcription factors, facilitates a transition from proliferation to differentiation, thereby ensuring that progenitors progress forwards to a differentiated state (Agathocleous, 2009).

These results tie together disparate strands in the function of Wnt/β-catenin and Sox2 signaling as investigated in various vertebrate models. Sox2 both sets up neural potential and inhibits terminal neuronal differentiation. The present study shows that Sox2 plays a central role in suppressing retinal neurogenesis downstream of Wnt/β-catenin signaling, but it enhances Müller glial differentiation and does not maintain progenitors. Similarly, Sox2 overexpression increases Müller cells in mouse retinal explants and promotes the in vitro differentiation of neocortical progenitors into astroglial cells. Notch activation by Sox2 may be involved in this gliogenic effect, as activated Notch signaling promotes gliogenesis. Therefore, either the absence of proneural gene expression or the suppression of proneural activity allows retinal progenitors to adopt the glial fate (Agathocleous, 2009).

The Wnt pathway is activated in the peripheral retina near the ciliary marginal zone in other species besides Xenopus. Yet, Wnt activation in the chick causes cells to be blocked in a proneural-negative progenitor state and in the mouse they assume non-neuronal peripheral fates. In chick and mouse, Wnt signaling does not appear to regulate Sox gene expression; however, the suppression of neurogenesis via activation of Wnt/β-catenin is common to the frog, chick and mammalian retina (Agathocleous, 2009).

There is strong evidence for connections between Wnt/β-catenin, SoxB1 and proneural genes in the regulation of neural differentiation in other tissues. In the zebrafish hypothalamus, canonical Wnt signaling, acting via Sox3, is necessary for the expression of proneural and neurogenic genes. LRP mutant mice exhibit dramatic hypoplasia of the developing neocortex owing to a reduction in neurogenesis as well as in proliferation. Similarly, in the adult hippocampus, Wnt activation promotes both neurogenesis and stem cell proliferation in a dissociable manner, which fits with the explanation that Wnt/β-catenin signaling sets up neuronal potential but then suppresses differentiation and maintains progenitor cells (Agathocleous, 2009).

The results suggest two parallel aspects of the progenitor cell fate: the suppression of neuronal differentiation and the maintenance of proliferative ability, controlled by two branches of Wnt signaling, one of which is Sox2 dependent. This model fits with findings in the spinal cord that Wnt activates proliferation, whereas Sox2 does not. The parallel control of differentiation and proliferation might be a more general feature of Wnt signaling; for example, in the developing limb, Wnt/β-catenin signaling and Sox9 interact to couple proliferation and chondrocyte differentiation (Agathocleous, 2009).

If Sox2 is not mediating the proliferative effects of Wnt/β-catenin signaling, other effectors must be involved. Although exogenous Cyclin E1 was able to cooperate with Sox2 in progenitor maintenance, little or no change was detected in Cyclin E1 retinal expression after Wnt signaling perturbations, nor in the expression of other cell cycle activators including Cyclin D1, Cyclin A2, n-Myc and c-Myc, suggesting that these genes might not be transcriptional targets in the frog retina. Perhaps other genes might function as Wnt-dependent effectors of proliferation here, or perhaps proliferation is regulated through post-transcriptional mechanisms or by changing the mode of progenitor division (Agathocleous, 2009).

Müller cells are transcriptionally very similar to neuroepithelial progenitor cells. They can divide after injury or provision of growth factors, at which point they may return to a neuroepithelial-like state, perhaps through a Wnt-dependent mechanism. These and results therefore suggest that a crucial distinction between RPCs and Müller cells is a Wnt-mediated capacity to proliferate (Agathocleous, 2009).

For the progression from a progenitor to a neuronal fate, both Wnt/β-catenin signaling and Sox2 must be switched off, relieving the inhibition of proneural activity and stopping proliferation. The inhibition of Wnt by Sox2 is likely to take place during retinogenesis, as Sox2 injections do not result in early defects in the specification of retinal progenitor identity. This therefore suggests a negative-feedback mechanism of Sox2 on Wnt signaling. Interestingly, mutations in human SOX2 associated with anophthalmia have been mapped to the C-terminal domain, which normally interacts with β-catenin, resulting in an inability of Sox2 to inhibit canonical Wnt signaling in vitro (Agathocleous, 2009).

For neuronal differentiation to proceed, Sox2 must also be switched off to relieve the inhibition of proneural activity. In the Xenopus retina, it was found that the proneural bHLH transcription factor Xath5 induced a dramatic reduction of the Sox2 protein. In the cortex, a serine protease cleaves Sox2 specifically in neuronal but not glial precursors, thus relieving the block on neurogenesis. It will be interesting to see whether in the retina, proneural genes feed back on Sox2 through this mechanism or through transcriptional repression (Agathocleous, 2009).

Wnt, Sox2 and the proneural genes appear to form a modular circuit in which each step activates the subsequent step and is in turn inactivated by it, driving cells towards differentiation, while limiting the ability of an external proliferation signal, such as Wnt, to continue signaling indefinitely. The relative levels of Wnt, Sox2 and proneural genes determine where a cell lies along the pathway from proliferation to differentiation and whether it assumes a progenitor, glial or neuronal fate. Understanding fully the function of each interaction in the cascade must await a more quantitative analysis of the relationship between the participating factors. This mechanism of transition from one cell state to another by the integration of directional interactions and feedback loops resembles that reported in diverse systems; for example, during sporulation of Bacillus subtilis, where a circuit with five basic nodes displays successive hierarchical gene activations, coupled with negative-feedback loops that switch off the previous state. Further investigations will reveal whether general aspects of the mechanism that is described here are at work in other neural and non-neural tissues, and how this directional pathway integrates with other factors that help to coordinate neuronal proliferation and differentiation (Agathocleous, 2009).

Chicken Sox factors

The epibranchial placodes are ectodermal thickenings that generate sensory neurons of the distal ganglia of the branchial nerves. Although significant advances in understanding of neurogenesis from the placodes have recently been made, the events prior to the onset of neurogenesis remain unclear. Chick Sox3 (cSox3) shows a highly dynamic pattern of expression before becoming confined to the final placodes: one pre-otic (geniculate) and three post-otic (one petrosal and two nodose) placodes. A fate-mapping study using lipophilic dyes has revealed that all post-otic placodes arise within a single broad cSox3-positive domain, where cSox3 expression and epithelial thickness are retained only in much smaller final neurogenic placodes. The data presented here suggest that post-otic placodes are remnants of a common primordium defined as a discrete domain of cSox3 expression (Ishii, 2001).

Sox2 expression marks neural and sensory primordia at various stages of development. A 50 kb genomic region of chicken Sox2 was isolated and scanned for enhancer activity utilizing embryo electroporation, resulting in identification of a battery of enhancers. Although Sox2 expression in the early embryonic CNS appears uniform, it is actually pieced together by five separate enhancers with distinct spatio-temporal specificities, including the one activated by the neural induction signals emanating from Hensen's node. Enhancers for Sox2 expression in the lens and nasal/otic placodes and in the neural crest were also determined. These functionally identified Sox2 enhancers exactly correspond to the extragenic sequence blocks conspicuously conserved between chicken and mammals; these blocks are not discernible by sequence comparison among mammals (Uchikawa, 2003).

The nucleotide sequences of the Sox2-flanking regions between chicken and mouse, and between chicken and human were compared using a stringent criterion to assess similarity (>60% identity in a stretch longer than 100 bp). The analysis revealed 25 blocks of sequence highly conserved between chicken and mammals and distributed in the region of analysis. Most remarkably, all ten identified neural and placodal enhancers (except for one) matched these blocks. The sequence alignment of the neural enhancers N-1 to N-5 derived from the three animal species confirms the high degree of conservation, including the putative transcription factor binding sites (Uchikawa, 2003).

Mammalian Sox factors

The mammalian genome contains a family of genes that are related to SRY, the mammalian sex determining gene. The homology is restricted to the region of SRY that encodes a DNA binding motif of the HMG-box class. These genes have been named SOX genes (SRY-related HMG-box genes). SOX3, a member of the human SOX gene family, maps to the X chromosome in the region Xq26-27. A mentally retarded male patient with hemophilia B is deleted for both the Factor IX gene and SOX3. This suggests that SOX3 is not essential for testis formation. The phenotype of the patient and the expression of SOX3 gene in neuronal tissues raises the possibility that this gene is a candidate gene for Borjeson-Forssman-Lehmann, an X-linked mental retardation syndrome (Stevanovic, 1993).

SRY and SOX9, members of the family of high-mobility group (HMG) domain transcription factors, are both essential for testis formation during human embryonic development. The HMG domain is a DNA-binding and DNA-bending motif comprising about 80 amino acid residues. It has been shown that SRY and SOX9 are nuclear proteins. Using normal or mutant SRY-beta-galactosidase and SOX9-beta-galactosidase fusion proteins in transfection studies involving COS-7 cells, two nuclear localization signals (NLSs) have been located within the HMG domains of both proteins that can independently direct the fusion proteins into the nucleus. Only mutational inactivation of both NLS motifs result in complete exclusion of the fusion proteins from the nucleus. The NLS sequences are located at the N and C termini of the HMG domain and are a bipartite NLS motif and a basic cluster NLS motif, respectively. Both NLS motifs are conserved in the HMG domains of other transcription factors. Implications of these results include (1) the apparent dual function of certain basic amino acid residues in the HMG domain of SRY in both DNA binding and in nuclear localization and (2) the possible control of SOX9 in early gonadal differentiation at the level of nuclear translocation (Südbeck, 1997).

cSox21 is a novel member of the Sox gene family of transcription factors. This gene is a member of the subgroup B, which includes Sox1, Sox2 and Sox3. Although all of these genes are expressed predominantly in the nervous system, only cSox21 expression is positionally restricted within the CNS. Longitudinal stripes are seen in the spinal cord; a more complex pattern is seen in the brain. The timing and position of cSox21 expression stripes provide further insight into dorsoventral patterning in the CNS. The expression of cSox21 as well as other genes (such as Delta, Serrate and Pax genes) may play a part in defining the developmental fate of cells along the dorsoventral axis (Rex, 1997).

Sox factors, sex determination and gonad development

Fibroblast growth factor 4 (FGF-4) is a signaling molecule whose expression is essential for postimplantation mouse development and, at later embryonic stages, for limb patterning and growth. The FGF-4 gene is expressed in the blastocyst inner cell mass, and later in distinct embryonic tissues. In tissue culture FGF-4 expression is restricted to undifferentiated embryonic stem (ES) cells and embryonal carcinoma (EC) cell lines. EC cell-specific transcriptional activation of the FGF-4 gene depends on a synergistic interaction between octamer-binding proteins Sox2, a member of the Sry-related Sox factors family. Sox2 can form a ternary complex with either the ubiquitous Oct-1 or the embryonic-specific Oct-3 protein on FGF-4 enhancer DNA sequences. However, only the Sox2/Oct-3 complex is able to promote transcriptional activation. These findings identify FGF-4 as the first known embryonic target gene for Oct-3 as well as for any of the Sox factors, and offers insights into the mechanisms of selective gene activation by Sox and octamer-binding proteins during embryogenesis (Yuan, 1995).

The Sry gene regulates sex determination in rodents and humans. Sry of mouse is expressed by germ cells in the adult testis and by somatic cells in the genital ridge. Transcripts in the former exist as circular RNA molecules of 1.23 kb, which are unlikely to be efficiently translated. SRY mRNA begins in the unique region 5' of the protein coding region and extends several kilobases into the 3' arm of the large inverted repeat that bounds the Sry genomic locus. This transcript, which is very different from that of the human SRY gene, reveals several features which may be involved in translational control. There appears to be two promoters for the Sry gene: a proximal one gives functional transcripts in the genital ridge, and a distal promoter is used in germ cells in the adult testis. Sry transcripts are first detectable just after 10.5 days post coitum, they reach a peak at 11.5 days and then decline sharply so that none are detected 24 hours later. This was compared with anti-Mullerian hormone gene expression, an early marker of Sertoli cells and the first known downstream gene of Sry. Amh expression begins 20 hours after the onset of Sry expression at a time when Sry transcripts are at their peak. While this result does not prove a direct interaction between the two genes, it defines the critical period during which Sry must act to initiate Sertoli cell differentiation (Hacker, 1995).

Murine Sox-3, located on the X chromosome, is most closely related to Sry. The main site of expression of Sox-3 is in the developing CNS, suggesting a role for Sox-3 in neural development. Sox-3, as well as Sox-1 and Sox-2 are expressed in the urogenital ridge, and their protein products are able to bind the same DNA sequence motif as Sry in vitro, but with different affinities. At 11.5 days of development, when Sry is thought to act, the indifferent gonad consists of primordial germ cells that are not required for testis determination, and two bipotential somatic cell lineages that give rise to supporting and steroidigenic cell types. Sry acts within the supporting cell precursors that produce Sertoli cells in the testis and granulosa cells in the ovary. Sox-3 is expressed in the somatic cells of genital ridges at a level equivalent to or greater than Sry. These findings raise two questions: what is the relationship between Sox-3 and Sry, and could Sox-3 also function in sex determination. One simple hypothesis would be that the action of SOX-3 protein on its gene target(s) is a critical step in the normal genetic pathway leading to differentiation of an ovary, and that in a male, SRY protein competes for the same target site(s) (Collignon, 1996).

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

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

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

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

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

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

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

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

Sox is a large family of genes related to the sex-determining region Y gene (designated as the SRY gene), In mammals, Sry expression in the bipotential, undifferentiated gonad directs the support cell precursors to differentiate as Sertoli cells, thus initiating the testis differentiation pathway. In the absence of Sry, or if Sry is expressed at insufficient levels, the support cell precursors differentiate as granulosa cells, thus initiating the ovarian pathway. The molecular mechanisms upstream and downstream of Sry are not well understood. The transcription factor GATA4 and its co-factor FOG2 are required for gonadal differentiation. Mouse fetuses homozygous for a null allele of Fog2 or homozygous for a targeted mutation in Gata4 (Gata4^ki) that abrogates the interaction of GATA4 with FOG co-factors exhibit abnormalities in gonadogenesis. Sry transcript levels are significantly reduced in XY Fog2^–/– gonads at E11.5, which is the time when Sry expression normally reaches its peak. In addition, three genes crucial for normal Sertoli cell function (Sox9, Mis and Dhh) and three Leydig cell steroid biosynthetic enzymes (p450scc, 3ßHSD and p450c17) are not expressed in XY Fog2^–/– and Gata^ki/ki gonads, whereas Wnt4, a gene required for normal ovarian development, is expressed ectopically. By contrast, Wt1 and Sf1, which are expressed prior to Sry and necessary for gonad development in both sexes, are expressed normally in both types of mutant XY gonads. These results indicate that GATA4 and FOG2 and their physical interaction are required for normal gonadal development (Tevosian, 2002).

Sox factors: protein interactions and interaction with DNA

The HMG box domain of the testis determining factor, SRY, includes a basic amphiphilic sequence common to calmodulin (CaM) binding proteins. SRY exhibits calcium-dependent binding to CaM. Binding occurs via the HMG box, and an SRY peptide of residues 57-80 binds CaM like the intact domain. SRY/CaM complex formation is specifically inhibited by the SRY DNA binding site sequence AACAAT. The binding exhibits a 1:1 stoichiometry and is accompanied by a conformational change in SRY. The A domain of HMG1 also binds CaM and it is proposed that CaM binding is a property of the wider HMG box family, including SOX and TCF/LEF proteins. These results suggest that CaM may regulate the DNA binding activity of HMG box transcription factors (Harley, 1996).

A PDZ domain protein (see Drosophila Discs large), termed SIP-1, interacts with human SRY. Interacting domains map to the C-terminal seven amino acids of SRY and to the PDZ domains of SIP-1. SIP-1, possessing two PDZ domains, could connect SRY to other transcription factors providing for SRY, trans-regulation function, as SRY itself has no trans-regulation domain (Poulat, 1997).

SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. The DNA binding domains (HMG-boxes) of mutant SRY proteins have been analyzed from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination (Pontiggia, 1994).

The HMG domain of the SRY protein represents a DNA binding motif that displays rather unusually weak evolutionary conservation of amino acids between human and mouse sequences. The human (h) SRY gene is unable to induce a male phenotype in genetically female transgenic mice. The DNA binding and bending properties of the HMG domains of murine (m) SRY and hSRY differ from each other. In comparison, mSRY shows more-extensive major-groove contacts with DNA and a higher specificity of sequence recognition than hSRY. Moreover, the extent of protein-induced DNA bending differs from the HMG domains of hSRY and mSRY. These differences in DNA binding by hSRY and mSRY may, in part, account for the functional differences observed with these gene products (Giese, 1995).

SOX proteins bind similar DNA motifs through their high-mobility-group (HMG) domains, but their action is highly specific with respect to target genes and cell type. The mechanism of target selection was examined by comparing SOX1/2/3, which activates delta-crystallin minimal enhancer DC5, with SOX9, which activates Col2a1 minimal enhancer COL2C2. These enhancers depend on both the SOX binding site and the binding site of a putative partner factor. The DC5 site is equally bound and bent by the HMG domains of SOX1/2 and SOX9. The activation domains of these SOX proteins mapped at the distal portions of the C-terminal domains are not cell specific and are independent of the partner factor. Chimeric proteins produced between SOX1 and SOX9 show that to activate the DC5 enhancer, the C-terminal domain must be that of SOX1, although the HMG domains are replaceable. The SOX2-VP16 fusion protein, in which the activation domain of SOX2 was replaced by that of VP16, activates the DC5 enhancer still in a partner factor-dependent manner. The results argue that the proximal portion of the C-terminal domain of SOX1/2 specifically interacts with the partner factor, and this interaction determines the specificity of the SOX1/2 action. Essentially the same results were obtained in the converse experiments in which COL2C2 activation by SOX9 was analyzed, except that specificity of SOX9-partner factor interaction also involves the SOX9 HMG domain. The highly selective SOX-partner factor interactions presumably stabilize the DNA binding of the SOX proteins and provide the mechanism for regulatory target selection (Kamachi, 1999).

The Pax6 gene plays crucial roles in eye development and encodes a transcription factor containing both a paired domain and a homeodomain. During embryogenesis, Pax6 is expressed in restricted tissues under the direction of distinct cis-regulatory regions. The head surface ectoderm-specific enhancer of mouse Pax6 directs reporter expression in the derivatives of the ectoderm in the eye, such as lens and cornea, but the molecular mechanism of its control remains largely unknown. A Pax6 protein-responsive element termed LE9 (52 bp in length) has been identified within the head surface ectoderm-specific enhancer. LE9, a sequence well conserved across vertebrates, acted as a highly effective enhancer in reporter analyses. Pax6 protein forms in vitro a complex with the distal half of LE9 in a manner dependent on the paired domain. The proximal half of the LE9 sequence contains three plausible sites of HMG domain recognition, and HMG domain-containing transcription factors Sox2 and Sox3 activate LE9 synergistically with Pax6. A scanning mutagenesis experiment indicates that the central site is most important among the three presumptive HMG domain recognition sites. Furthermore, Pax6 and Sox2 proteins form a complex when they are expressed together. Based on these findings, a model is proposed in which Pax6 protein directly and positively regulates its own gene expression, and Sox2 and Sox3 proteins interact with Pax6 protein, resulting in modification of the transcriptional activation by Pax6 protein (Aota, 2003).

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

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

Transcriptional targets of Sox factors

Fibroblast growth factor 4 (FGF-4) has been shown to be a signaling molecule whose expression is essential for postimplantation mouse development and, at later embryonic stages, for limb patterning and growth. The FGF-4 gene is expressed in the blastocyst inner cell mass and later in distinct embryonic tissues but is transcriptionally silent in the adult. In tissue culture, FGF-4 expression is restricted to undifferentiated embryonic stem cells and embryonal carcinoma (EC) cell lines. EC cell-specific transcriptional activation of the FGF-4 gene depends on a synergistic interaction between octamer-binding proteins and an EC-specific factor, Fx, that binds adjacent sites on the FGF-4 enhancer. This latter activity is carried out by Sox2, a member of the Sry-related Sox factors family. Sox2 can form a ternary complex with either the ubiquitous Oct-1 or the embryonic-specific Oct-3 protein on FGF-4 enhancer DNA sequences. However, only the Sox2/Oct-3 complex is able to promote transcriptional activation. These findings identify FGF-4 as the first known embryonic target gene for Oct-3 and for any of the Sox factors, and offer insights into the mechanisms of selective gene activation by Sox and octamer-binding proteins during embryogenesis (Yuan, 1995).

Octamer binding and Sox factors are thought to play important roles in development by potentiating the transcriptional activation of specific gene subsets. The proteins within these factor families are related by the presence of highly conserved DNA binding domains, the octamer binding protein POU domain or the Sox factors HMG domain. Fibroblast growth factor 4 (FGF-4) gene expression in embryonal carcinoma cells requires a synergistic interaction between Oct-3 and Sox2 on the FGF-4 enhancer. Sox2 and Oct-3 bind to adjacent sites within this enhancer to form a ternary protein-DNA complex (Oct-3*) whose assembly correlates with enhancer activity. Increasing the distance between the octamer and Sox binding sites by base pair insertion results in a loss of enhancer function. Significantly, those enhancer "spacing mutants" which fail to activate transcription are also compromised in their ability to form the Oct* complexes even though they can still bind both Sox2 and the octamer binding proteins, suggesting that a direct interaction between Sox2 and Oct-3 is necessary for enhancer function. Consistent with this hypothesis, Oct-3 and Sox2 can participate in a direct protein-protein interaction in vitro in the absence of DNA, and both this interaction and assembly of the ternary Oct* complexes require only the octamer protein POU and Sox2 HMG domains. Assembly of the ternary complex by these two protein domains occurs in a cooperative manner on FGF-4 enhancer DNA, and the loss of this cooperative interaction contributes to the defect in Oct-3* formation observed for the enhancer spacing mutants. These observations indicate that Oct-3* assembly results from protein-protein interactions between the domains of Sox2 and Oct-3 that mediate their binding to DNA, but it also requires a specific arrangement of the binding sites within the FGF-4 enhancer DNA. Thus, these results define one parameter that is fundamental to synergistic activation by Sox2 and Oct-3 and further emphasize the critical role of enhancer DNA sequences in the proper assembly of functional activation complexes (Ambrosetti, 1997).

Embryonic development requires a complex program of events that are directed by a number of signaling molecules whose expression must be rigorously regulated. Expression of Fgf4, which plays an important role in postimplantation development and growth and patterning of the limb, is regulated in embryonal carcinoma (EC) cells by the synergistic interaction of Sox2 and Oct-3 with the Fgf4 EC cell-specific enhancer. To verify whether this mechanism is also operating in vivo, and to identify new elements controlling Fgf4 gene expression in distinct developmental stages, the expression of LacZ reporter plasmids containing different fragments of the Fgf4 gene have been analyzed in transgenic mouse embryos. Utilizing these transgenic constructs Fgf4 gene expression could be recapitulated, for the most part, during embryonic development. Most of the cis-acting regulatory elements determining Fgf4 embryonic expression are located in conserved regions within the 3' UTR of the gene. The EC cell-specific enhancer is required to drive gene expression in the ICM of the blastocyst, and its activity requires the Sox and Oct-proteins binding sites. Specific and distinct enhancer elements could be identified that govern postimplantation expression in the somitic myotomes and the limb bud AER. The myotome-specific elements contain binding sites for bHLH myogenic regulatory factors, which appear to be essential for myotome expression. Evidence is also presented that the very restricted pattern of expression of Fgf4 transcripts in the AER results from the combined action of positive and negative regulatory elements located 3' to the Fgf4 coding sequences. Thus the Fgf4 gene relies on multiple and distinct regulatory elements to achieve stage- and tissue-specific embryonic expression (Fraidenraich, 1998).

Delta 1-crystallin gene activation occurs early in lens cell differentiation. An essential element of the delta 1-crystallin enhancer is bound by chicken SOX-2 protein (cSOX-2), which is structurally related to the sex-determining factor SRY. Sox-2 is expressed at high levels in the early developing lens in both chicken and mouse embryos. Overexpression of delta cSOX-2 increased delta 1-crystallin enhancer activity to a plateau in lens cells, but not in fibroblasts, consistent with the previously drawn conclusion that SOX-2 activates transcription only in concert with another factor present in the lens. This result supports the model that SOX proteins act as architectural components in the activating complex formed on an enhancer, as indicated for lymphoid enhancer binding factor 1 (LEF-1), another HMG domain protein. SOX protein binding is essential for lens-specific promoter activity of the mouse gamma F-crystallin gene. This work is the first to show delta- and gamma-crystallin genes as examples of direct regulatory targets of SOX proteins and provides evidence that diversified crystallin genes are regulated, at least in part, by a common mechanism (Kamachi, 1995).

Gamma-crystallins are major structural components of the lens fiber cells in amphibians and mammals. Many dominant inherited cataracts in humans and mice have been shown to map within the gamma-crystallin gene cluster. Several transcription factors, including PAX6 and SOX proteins, have been suggested as candidates for crystallin gene regulation. The targeted deletion of Sox1 in mice causes microphthalmia and cataract. Mutant lens fiber cells fail to elongate, probably as a result of an almost complete absence of gamma-crystallins. It appears that the direct interaction of the SOX1 protein with a promoter element conserved in all gamma-crystallin genes is responsible for their expression (Nishiguchi, 1998).

The POU transcription factor Oct-4, which has no known Drosophila homolog, is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin subtypes and modulate cell adhesion/migration. Oct-4 and OPN are coexpressed in the preimplantation mouse embryo and during differentiation of embryonal cell lines. Immunoprecipitation of the first intron of OPN (i-opn) from covalently fixed chromatin of embryonal stem cells by Oct-4-specific antibodies indicates that Oct-4 binds to this fragment in vivo. The i-opn fragment functions as an enhancer in cell lines that resemble cells of the preimplantation embryo. It contains a novel palindromic Oct factor recognition element (PORE) that is composed of an inverted pair of homeodomain-binding sites separated by exactly 5 bp (ATTTG +5 CAAAT). POU proteins can homo- and hetero-dimerize on the PORE in a configuration that has not been described previously. Strong transcriptional activation of the OPN element requires an intact PORE. In contrast, the canonical octamer overlapping with the downstream half of the PORE is not essential. Sox-2 is a transcription factor that contains an HMG box and is coexpressed with Oct-4 in the early mouse embryo. Sox-2 represses Oct-4 mediated activation of i-opn by way of a canonical Sox element that is located close to the PORE. Repression depends on a carboxy-terminal region of Sox-2 that is outside of the HMG box. Expression, DNA binding, and transactivation data are consistent with the hypothesis that OPN expression is regulated by Oct-4 and Sox-2 in preimplantation development (Botquin, 1998).

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

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

Conserved POU binding DNA sites in the Sox2 upstream enhancer regulate gene expression in embryonic and neural stem cells

The Sox2 transcription factor is expressed early in the stem cells of the blastocyst inner cell mass and, later, in neural stem cells. Previous work has identified a Sox2 5'-regulatory region directing transgene expression to the inner cell mass and, later, to neural stem cells and precursors of the forebrain. This study identified a core enhancer element able to specify transgene expression in forebrain neural precursors of mouse embryos, and the same core element was shown to efficiently activate transcription in inner cell mass-derived embryonic stem (ES) cells. Mutation of POU factor binding sites, able to recognize the neural factors Brn1 and Brn2, shows that these sites contribute to transgene activity in neural cells. The same sites are also essential for activity in ES cells, where they bind different members of the POU family, including Oct4, as shown by gel shift assays and chromatin immunoprecipitation with anti-Oct4 antibodies. These findings indicate a role for the same POU binding motifs in Sox2 transgene regulation in both ES and neural precursor cells. Oct4 might play a role in the regulation of Sox2 in ES (inner cell mass) cells and, possibly, at the transition between inner cell mass and neural cells, before recruitment of neural POU factors such as Brn1 and Brn2 (Catena, 2004).

Characterization of enhancers active in the mouse embryonic cerebral cortex suggests Sox/Pou cis-regulatory logics and heterogeneity of cortical progenitors

This study aimed to identify cis-regulatory elements that control gene expression in progenitors of the cerebral cortex. A list of 975 putative enhancers were retrieved from a ChIP-Seq experiment performed in NS5 mouse stem cells with antibodies to Sox2, Brn2/Pou3f2, or Brn1/Pou3f3. Through a selection pipeline including gene ontology and expression pattern, the number of candidate enhancer sequences was reduced to 20. Ex vivo electroporation of green fluorescent pProtein (GFP) reporter constructs in the telencephalon of mouse embryos showed that 35% of the 20 selected candidate sequences displayed enhancer activity in the developing cortex at E13.5. In silico transcription factor binding site (TFBS) searches and mutagenesis experiments showed that enhancer activity is related to the presence of Sox/Pou TFBS pairs in the sequence. Comparative genomic analyses showed that enhancer activity is not related to the evolutionary conservation of the sequence. Finally, the combination of in utero electroporation of GFP reporter constructs with immunostaining for Tbr2 (basal progenitor marker) and phospho-histoneH3 (mitotic activity marker) demonstrated that each enhancer is specifically active in precise subpopulations of progenitors in the cortical germinal zone, highlighting the heterogeneity of these progenitors in terms of cis-regulation (Retaux, 2013).

Sox factors and development

In vertebrates, the delineation of the neural plate from a region of the primitive ectoderm is accompanied by the onset of specific gene expression, which in turn promotes the formation of the nervous system. SOX1, an HMG-box protein related to SRY, is one of the earliest transcription factors to be expressed in ectodermal cells committed to the neural fate: the onset of expression of SOX1 appears to coincide with the induction of neural ectoderm. In the mouse, expression of SOX1 is first detected at 7.5 days of development in the anterior half of the late-streak egg cylinder. Cross sections through the embryo at this stage reveal expression in columnar ectodermal cells, which appear to define the neural plate, while cells located more laterally are negative. SOX1 is maintained in all neuroepithelial cells along the entire anteroposterior axis as the neural plate bends and fuses to form the neural tube. The expression of SOX1 throughout the neural plate and early neural tube implies a similarity among these cells. After neural tube closure, neuroepithelial cells begin to differentiate into defined classes of neurons at specific dorsoventral (D/V) positions within the spinal cord. As development proceeds, SOX1 is downregulated in a stereotyped manner in cells along the D/V axis of the neural tube (Pevny, 1998).

In the spinal cord, expression is first downregulated in cells that occupy the ventral midline, then in the ventral motor horns and subsequently the dorsal regions. These regions appear to correlate with floor plate, motor neurons and sensory relay interneurons, respectively. SOX1 is expressed by early neural cells in the CNS and is downregulated in the developing neural tube coincident with neural differentiation. SOX1 expression marks proliferating cells within the embryonic neural tube. Using a series of antigenic markers that identify early neural cell types in combination with BrdU labeling, a temporal and spatial correlation has been demonstrated between the differentiation of cell types along the dorsoventral axis of the neural tube and the downregulation of SOX1 expression. Downregulation of SOX1 in the neural tube correlates with exit from mitosis. A role for SOX1 has been demonstrated in neural determination and differentiation using an inducible expression P19 cell system as an in vitro model of neurogenesis. Misexpression of SOX1 can substitute for the requirement of retinoic acid to impart neural fate to competent ectodermal P19 cells. In vitro SOX1 expression is initiated within 24 hours of the addition of retinoic acid to P19 aggregates coincident with the induction of neuropithelial markers such as NESTIN, Mash1 and Wnt1. SOX1, therefore, defines the dividing neural precursors of the embryonic central nervous system (Pevny, 1998).

A number of vertebrate transcription factors have previously been shown to impart neural fate on uncommitted ectodermal cells. Examples of these include XASH-3 and CASH-4 (the ASH-3 Xenopus and chicken homologs of the Drosophila AS-C genes); Neurogenin and NeuroD (vertebrate homologs of the Drosophila atonal genes), and MyT1, a Xenopus C2HC zinc finger protein. In animal cap assays, CASH-4, Neurogenin and NeuroD alone, as well as the combination of XASH-3 and MyT1, can induce neural differentiation in naive ectoderm in the absence of additional neural inducing molecules. This is analogous to the manner in which SOX1 promotes neural differentiation in P19 ectodermal cells in the absence of retinoic acid. However, the relatively late and restricted onset of expression of Neurogenin, NeuroD and MyT1, as well as the phenotype elicited by their misexpression in Xenopus embryos has led to the suggestion that these genes function in neuronal determination and differentiation rather than in neuroepithelial precursor determination. Only CASH-4, whose early expression in neural precursors is initiated by neural inducing signals and whose ectopic expression leads to the expansion of the neural plate, seems to function to promote the formation of neuroepithelial precursors. However, in vivo, the expression of CASH-4 is restricted to the posterior neural plate and therefore may play a role in specifying just posterior neural fate. The early expression of SOX1 throughout the anteroposterior axis of the neural plate and its ability to elicit a neural response in P19 cells implicates a general role for SOX1 in neuroepithelial cell fate determination (Pevny, 1998).

An important question is at what cellular stage does CNS patterning arise in development. Is patterning already established in stem cells, generating regional heterogeneity among these cells, or in precursors downstream to the stem cell? Further, once established, how is patterning maintained during the multiple cell divisions occurring in embryogenesis and to what extent is it reversible in response to progressive modifications of the environment? Sox2 is one of the earliest known transcription factors expressed in the developing neural tube. Although it is expressed throughout the early neuroepithelium, its later expression must depend on the activity of more than one regionally restricted enhancer element. Thus, by using transgenic assays and by homologous recombination-mediated deletion, a region upstream of Sox2 (-5.7 to -3.3 kb) has been identified that can not only drive expression of a beta-geo transgene to the developing dorsal telencephalon, but is also required to do so in the context of the endogenous gene. The critical enhancer can be further delimited to an 800 bp fragment of DNA surrounding a nuclease hypersensitive site within this region, as this is sufficient to confer telencephalic expression to a 3.3 kb fragment including the Sox2 promoter, which is otherwise inactive in the CNS. Expression of the 5.7 kb Sox2 beta-geo transgene localizes to the neural plate and later to the telencephalic ventricular zone. Transgene-expressing (and thus resistent to the antibiotic G418) ventricular zone cells include cells displaying functional properties of stem cells, i.e. self-renewal and multipotentiality. The majority of telencephalic stem cells express the transgene, and this expression is largely maintained over two months in culture (more than 40 cell divisions) in the absence of G418 selective pressure. In contrast, stem cells grown in parallel from the spinal cord never express the transgene, and die in G418. Expression of endogenous telencephalic genes has been similarly observed in long-term cultures derived from the dorsal telencephalon, but not in spinal cord-derived cultures. Thus, neural stem cells of the midgestation embryo are endowed with region-specific gene expression (at least with respect to some networks of transcription factors, such as those driving telencephalic expression of the Sox2 transgene), which can be inherited through multiple divisions outside the embryonic environment (Zappone, 2000).

In a differential screen for downstream genes of the neural inducers, two extremely early neural genes induced by Chordin and suppressed by BMP-4 have been identified: Zic-related-1 (Zic-r1), a zinc finger factor related to the Drosophila pair-rule gene odd-paired, and Sox-2, a Sry-related HMG factor. Expression of the two genes is first detected widely in the prospective neuroectoderm at the beginning of gastrulation, following the onset of Chordin expression and preceding that of Neurogenin (Xngnr-1). Zic-r1 mRNA injection activates the proneural gene Xngnr-1, and initiates neural and neuronal differentiation in isolated animal caps and in vivo. In contrast, Sox-2 alone is not sufficient to cause neural differentiation, but can work synergistically with FGF signaling to initiate neural induction. Thus, Zic-r1 acts in the pathway bridging the neural inducer with the downstream proneural genes, while Sox-2 makes the ectoderm responsive to extracellular signals, demonstrating that the early phase of neural induction involves simultaneous activation of multiple functions (Mizuseki, 1998).

Three chicken Sox (SRY-like box) genes have been identified that show an interactive pattern of expression in the developing embryonic nervous system. cSox2 and cSox3 code for related proteins and both are predominantly expressed in the immature neural epithelium of the entire CNS of HH stage 10 to 34 embryos. cSox11 is related to cSox2 and cSox3 only by virtue of containing an SRY-like HMG-box sequence but shows extensive homology with Sox-4 at its C-terminus. cSox11 is expressed in the neural epithelium but is transiently upregulated in maturing neurons after they leave the neural epithelium. These patterns of expression suggest that Sox genes play a role in neural development and that the developmental program from immature to mature neurons may involve switching of Sox gene expression. cSox11 also exhibits a lineage restricted pattern of expression in the peripheral nervous system (Uwanogho, 1995).

Sox genes and lens development

Several stages in the lens determination process have been defined, though it is not known which gene products control these events. At mid-gastrula stages in Xenopus, ectoderm is transiently competent to respond to lens-inducing signals. Between late gastrula and neural tube stages, the presumptive lens ectoderm acquires a lens-forming bias, becomes specified to form lens and begins differentiation. Several genes have been identified, either by expression pattern, mutant phenotype or involvement in crystallin gene regulation, that may play a role in lens bias and specification. Fate mapping shows that the transcriptional regulators Otx-2, Pax-6 and Sox-3 are expressed in the presumptive lens ectoderm prior to lens differentiation. Otx-2 appears first, followed by Pax-6, during the stages of lens bias (late neural plate stages); expression of Sox-3 follows neural tube closure and lens specification. The expression of these genes is demonstrated in competent ectoderm transplanted to the lens-forming region. Expression of these genes is maintained or activated preferentially in ectoderm in response to the anterior head environment. Activation of these genes is examined in response to early and late lens-inducing signals. Activation of Otx-2, Pax-6 and Sox-3 in competent ectoderm occurs in response to the early inducing tissue, the anterior neural plate. Since Sox-3 is activated following neural tube closure, an examination was carried out of its dependence on the later inducing tissue, the optic vesicle, which contacts lens ectoderm at this later stage. Sox-3 is not expressed in lens ectoderm, nor does a lens form, when the optic vesicle anlage is removed at late neural plate stages. Expression of these genes demarcates patterning events preceding differentiation and is tightly coupled to particular phases of lens induction (Zygar, 1998).

Activation of the first lens-specific gene of the chicken, (delta)1-crystallin, is dependent on a group of lens nuclear factors, (delta)EF2, interacting with the (delta)1-crystallin minimal enhancer, DC5. One of the (delta)EF2 factors was previously identified as SOX2. Two related SOX proteins, SOX1 and SOX3, are shown to account for the remaining members of (delta)EF2. Activation of the DC5 enhancer is dependent on the C-terminal domains of these proteins. Expression of Sox1-3 in the eye region during lens induction was studied in comparison with Pax6 and (delta)1-crystallin. Pax6, known to be required for the inductive response of the ectoderm, is broadly expressed in the lateral head ectoderm from before lens induction. After tight association of the optic vesicle (around stage 10-11, 40 hours after egg incubation), expression of Sox2 and Sox3 is activated in the vesicle-facing ectoderm at stage 12 (44 hours). These cells, expressing together Pax6 and Sox2/3, subsequently give rise to the lens, beginning with formation of the lens placode and expression of (delta)-crystallin at stage 13 (48 hours). Sox1 then starts to be expressed in the lens-forming cells at stage 14. When the prospective retina area of the neural plate is unilaterally ablated at stage 7, expression of Sox2/3 is lost in the side of lateral head ectoderm lacking the optic cup, implying that an inductive signal from the optic cup activates Sox2/3 expression. In the mouse embryonic lens, this subfamily of Sox genes is expressed in an analogous fashion, although Sox3 transcripts have not been detected and Sox2 expression is down-regulated when Sox1 is activated. In ectodermal tissues of the chicken embryo, (delta)-crystallin expression occurs in a few ectopic sites. These are always characterized by overlapping expression of Sox2/3 and Pax6. Thus, an essential molecular event in lens induction is the 'turning on' of the transcriptional regulators SOX2/3 in the Pax6-expressing ectoderm and these SOX proteins activate crystallin gene expression. Continued activity, especially of SOX1, is then essential for further development of the lens (Kamachi, 1998).

Pax6 is a key transcription factor in eye development, particularly in lens development, but its molecular action has not been clarified. Pax6 initiates lens development by forming a molecular complex with SOX2 (most closely related to Drosophila Sox Neuro) on the lens-specific enhancer elements, e.g., the delta-crystallin minimal enhancer DC5. DC5 shows a limited similarity to the binding consensus sequence of Pax6 and is bound poorly by Pax6 alone. However, Pax6 binds cooperatively with SOX2 to the DC5 sequence, resulting in formation of a high-mobility form of ternary complex in vitro, which correlates with the enhancer activation in vivo. Pax6 and SOX2-interdependent factor occupancy of DC5 is observed in a chromatin environment in vivo, providing the molecular basis of synergistic activation by Pax6 and SOX2. Subtle alterations of the Pax6-binding-site sequence of DC5 or of the inter-binding-sites distance diminish the cooperative binding and causes formation of a non-functional low-mobility form complex, suggesting DNA sequence-guided and protein interaction-induced conformation change of the Pax6 protein. When ectopically expressed in embryo ectoderm, Pax6 and SOX2 in combination activate delta-crystallin gene and elicit lens placode development, indicating that the complex of Pax6 and SOX2 formed on specific DNA sequences is the genetic switch for initiation of lens differentiation (Kamachi, 2001).

Group B Sox genes, Sox1, -2 and -3 are known to activate crystallin genes and to be involved in differentiation of lens and neural tissues. Screening of chicken genomic sequences for more Group B Sox genes has identified two additional genes, Sox14 and Sox21. Proteins encoded by Sox14 and Sox21 genes are similar to each other but distinct from those coded by Sox1-3 (subgroup B1) except for the HMG domain and Group B homology immediately C-proximal to the HMG domain. C-terminal domains of SOX21 and SOX14 proteins function as strong and weak repression domains, respectively, when linked to the GAL4 DNA binding domain. These SOX proteins strongly (SOX21) or moderately (SOX14) inhibit activation of delta1-crystallin DC5 enhancer by SOX1 or SOX2, establishing that Sox14 and Sox21 constitute a repressing subgroup (B2) of Group B Sox genes. This provides the first evidence for the occurrence of repressor SOX proteins. Activating (B1) and repressing (B2) subgroups of Group B Sox genes display interesting overlaps of expression domains in developing tissues (e.g. optic tectum, spinal cord, inner ear, alimentary tract, branchial arches). Within each subgroup, most expression domains of Sox1 and -3 are included in those of Sox2 (e.g. CNS, PNS, inner ear), while co-expression of Sox14 and Sox21 occurs in highly restricted sites of the CNS, with the likely temporal order of Sox21 preceding Sox14 (e.g. interneurons of the spinal cord). These expression patterns suggest that target genes of Group B SOX proteins are finely regulated by the counterbalance of activating and repressing SOX proteins (Uchikawa, 1999).

Cyclic GMP-dependent protein kinase II is a molecular switch from proliferation to hypertrophic differentiation of chondrocytes

The Komeda miniature rat Ishikawa (KMI) is a naturally occurring mutant caused by an autosomal recessive mutation mri, that exhibits longitudinal growth retardation. The mri mutation has been identified as a deletion in the rat gene encoding cGMP-dependent protein kinase type II (cGKII). KMIs show an expanded growth plate and impaired bone healing with abnormal accumulation of postmitotic but nonhypertrophic chondrocytes. Ex vivo culture of KMI chondrocytes reproduce the differentiation impairment, which was restored by introducing the adenovirus-mediated cGKII gene. The expression of Sox9, an inhibitory regulator of hypertrophic differentiation, persists in the nuclei of postmitotic chondrocytes of the KMI growth plate. Transfection experiments in culture systems reveal that cGKII attenuates the Sox9 functions to induce the chondrogenic differentiation and to inhibit the hypertrophic differentiation of chondrocytes. This attenuation of Sox9 is due to the cGKII inhibition of nuclear entry of Sox9. The impaired differentiation of cultured KMI chondrocytes is restored by the silencing of Sox9 through RNA interference. Hence, the present study for the first time shed light on a novel role of cGKII as a molecular switch, coupling the cessation of proliferation and the start of hypertrophic differentiation of chondrocytes through attenuation of Sox9 function (Chikuda, 2004).

Sox 2 is required for induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures

Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. This study demonstrates induction of pluripotent stem cells from mouse embryonic or adult tail tip fibroblasts (TTFs) by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which have been designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors (Takahashi, 2007).

Oct3/4, Sox2, and Nanog have been shown to function as core transcription factors in maintaining pluripotency. Among the three, it was found that Oct3/4 and Sox2 are essential for the generation of iPS cells. c-Myc and Klf4 were also identified as essential factors. These two tumor-related factors could not be replaced by other oncogenes including E-Ras, Tcl1, β-catenin, and Stat3 (Takahashi, 2007).

The c-Myc protein has many downstream targets that enhance proliferation and transformation, many of which may have roles in the generation of iPS cells. Of note, c-Myc associates with histone acetyltransferase (HAT) complexes, including TRRAP, which is a core subunit of the TIP60 and GCN5 HAT complexes, CREB binding protein (CBP), and p300. Within the mammalian genome, there may be up to 25,000 c-Myc binding sites, many more than the predicted number of Oct3/4 and Sox2 binding sites. c-Myc protein may induce global histone acetylation, thus allowing Oct3/4 and Sox2 to bind to their specific target loci. Klf4 has been shown to repress p53 directly, and p53 protein has been shown to suppress Nanog during ES cell differentiation. iPS cells showed levels of p53 protein lower than those in MEFs. Thus, Klf4 might contribute to activation of Nanog and other ES cell-specific genes through p53 repression. Alternatively, Klf4 might function as an inhibitor of Myc-induced apoptosis through the repression of p53 in this system. In contrast, Klf4 activates p21CIP1, thereby suppressing cell proliferation. This antiproliferation function of Klf4 might be inhibited by c-Myc, which suppresses the expression of p21CIP1. The balance between c-Myc and Klf4 may be important for the generation of iPS cells (Takahashi, 2007).

One question that remains concerns the origin of the iPS cells. With the retroviral expression system, it is estimated that only a small portion of cells expressing the four factors become iPS cells. The low frequency suggests that rare tissue stem/progenitor cells that coexisted in the fibroblast cultures might have given rise to the iPS cells. Indeed, multipotent stem cells have been isolated from skin. These studies showed that ~0.067% of mouse skin cells are stem cells. One explanation for the low frequency of iPS cell derivation is that the four factors transform tissue stem cells. However, it was found that the four factors induced iPS cells with comparably low efficiency even from bone marrow stroma, which should be more enriched in mesenchymal stem cells and other multipotent cells. Furthermore, cells induced by the three factors were nullipotent. DNA microarray analyses suggested that iPS-MEF4 cells and iPS-MEF3 cells have the same origin. These results do not favor multipotent tissue stem cells as the origin of iPS cells (Takahashi, 2007).

There are several other possibilities for the low frequency of iPS cell derivation. First, the levels of the four factors required for generation of pluripotent cells may have narrow ranges, and only a small portion of cells expressing all four of the factors at the right levels can acquire ES cell-like properties. Consistent with this idea, a mere 50% increase or decrease in Oct3/4 proteins induces differentiation of ES cells. iPS clones overexpressed the four factors when RNA levels were analyzed, but their protein levels were comparable to those in ES cells, suggesting that the iPS clones possess a mechanism (or mechanisms) that tightly regulates the protein levels of the four factors. It is speculated that high amounts of the four factors are required in the initial stage of iPS cell generation, but, once they acquire ES cell-like status, too much of the factors are detrimental for self-renewal. Only a small portion of transduced cells show such appropriate transgene expression. Second, generation of pluripotent cells may require additional chromosomal alterations, which take place spontaneously during culture or are induced by some of the four factors. Although the iPS-TTFgfp4 clones had largely normal karyotypes, the existence of minor chromosomal alterations cannot be ruled out. Site-specific retroviral insertion may also play a role. Southern blot analyses showed that each iPS clone has ~20 retroviral integrations. Some of these may have caused silencing or fusion with endogenous genes. Further studies will be required to determine the origin of iPS cells (Takahashi, 2007).

Another unsolved question is whether the four factors identified play roles in reprogramming induced by fusion with ES cells or nuclear transfer into oocytes. Since the four factors are expressed in ES cells at high levels, it is reasonable to speculate that they are involved in the reprogramming machinery that exists in ES cells. These result is also consistent with the finding that the reprogramming activity resides in the nucleus, but not in the cytoplasm, of ES cells. However, iPS cells were not identical to ES cells, as shown by the global gene-expression patterns and DNA methylation status. It is possible that additional important factors have been missed. One such candidate is ECAT1, although its forced expression in iPS cells did not consistently upregulate ES cell marker genes (Takahashi, 2007).

More obscure are the roles of the four factors, especially Klf4 and c-Myc, in the reprogramming observed in oocytes. Both Klf4 and c-Myc are dispensable for preimplantation mouse development. Furthermore, c-myc is not detected in oocytes. In contrast, L-myc is expressed maternally in oocytes. Klf17 and Klf7, but not Klf4, are found in expressed sequence-tag libraries derived from unfertilized mouse eggs. Klf4 and c-Myc might be compensated by these related proteins. It is highly likely that other factors are also required to induce complete reprogramming and totipotency in oocytes (Takahashi, 2007).

Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells

It has been shown that defined sets of transcription factors are sufficient to convert mouse and human fibroblasts directly into cells resembling functional neurons, referred to as 'induced neuronal' (iN) cells. For some applications however, it would be desirable to convert fibroblasts into proliferative neural precursor cells (NPCs) instead of neurons. It was hypothesized that NPC-like cells may be induced using the same principal approach used for generating iN cells. Toward this goal, mouse embryonic fibroblasts derived from Sox2-EGFP mice were transfected with a set of 11 transcription factors highly expressed in NPCs. Twenty-four days after transgene induction, Sox2-EGFP(+) colonies emerged that expressed NPC-specific genes and differentiated into neuronal and astrocytic cells. Using stepwise elimination, it was found that Sox2 and FoxG1 are capable of generating clonal self-renewing, bipotent induced NPCs that gave rise to astrocytes and functional neurons. When the Pou and Homeobox domain-containing transcription factor Brn2 to was added Sox2 and FoxG1, it was possible to induce tripotent NPCs that could be differentiated not only into neurons and astrocytes but also into oligodendrocytes. The transcription factors FoxG1 and Brn2 alone also were capable of inducing NPC-like cells; however, these cells generated less mature neurons, although they did produce astrocytes and even oligodendrocytes capable of integration into dysmyelinated Shiverer brain. These data demonstrate that direct lineage reprogramming using target cell-type-specific transcription factors can be used to induce NPC-like cells that potentially could be used for autologous cell transplantation-based therapies in the brain or spinal cord (Lujan, 2012).

Single-molecule dynamics of enhanceosome assembly in embryonic stem cells

Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. This study combined in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. Enhanceosome assembly was found to be hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. These findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters (Chen, 2014).

Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm

Sox2 is expressed in developing foregut endoderm, with highest levels in the future esophagus and anterior stomach. By contrast, Nkx2.1 (Titf1) is expressed ventrally, in the future trachea. In humans, heterozygosity for SOX2 is associated with anopthalmia-esophageal-genital syndrome (OMIM 600992), a condition including esophageal atresia (EA) and tracheoesophageal fistula (TEF), in which the trachea and esophagus fail to separate. Mouse embryos heterozygous for the null allele, Sox2^EGFP, appear normal. However, further reductions in Sox2, using Sox2^LP and Sox2^COND hypomorphic alleles, result in multiple abnormalities. Approximately 60% of Sox2^EGFP/COND embryos have EA with distal TEF in which Sox2 is undetectable by immunohistochemistry or western blot. The mutant esophagus morphologically resembles the trachea, with ectopic expression of Nkx2.1, a columnar, ciliated epithelium, and very few p63⁺ basal cells. By contrast, the abnormal foregut of Nkx2.1-null embryos expresses elevated Sox2 and p63, suggesting reciprocal regulation of Sox2 and Nkx2.1 during early dorsal/ventral foregut patterning. Organ culture experiments further suggest that FGF signaling from the ventral mesenchyme regulates Sox2 expression in the endoderm. In the 40% Sox2^EGFP/COND embryos in which Sox2 levels are ~18% of wild type there is no TEF. However, the esophagus is still abnormal, with luminal mucus-producing cells, fewer p63⁺ cells, and ectopic expression of genes normally expressed in glandular stomach and intestine. In all hypomorphic embryos the forestomach has an abnormal phenotype, with reduced keratinization, ectopic mucus cells and columnar epithelium. These findings suggest that Sox2 plays a second role in establishing the boundary between the keratinized, squamous esophagus/forestomach and glandular hindstomach (Que, 2007).

Multiple roles for Sox2 in the developing and adult mouse trachea

The esophagus, trachea and lung develop from the embryonic foregut, yet acquire and maintain distinct tissue phenotypes. Sox2 is necessary for foregut morphogenesis and esophagus development. Sox2 is also required for the normal development of the trachea and lung. In both the embryo and adult, Sox2 is exclusively expressed in the epithelium of the trachea and airways. An Nkx2.5-Cre transgene and a Sox2 floxed allele were used to conditionally delete Sox2 in the ventral epithelial domain of the early anterior foregut, which gives rise to the future trachea and lung buds. All conditional mutants die of respiratory distress at birth, probably due to abnormal differentiation of the laryngeal and tracheal cartilage as a result of defective epithelial-mesenchymal interaction. About 60% of the mutants have a short trachea, suggesting that the primary budding site of the lung shifts anteriorly. In the tracheal epithelium of all conditional mutants there are significantly more mucus-producing cells compared with wild type, and fewer basal stem cells, ciliated and Clara cells. Differentiation of the epithelium lining the conducting airways in the lung is abnormal, suggesting that Sox2 also plays a role in the differentiation of embryonic airway progenitors into specific lineages. Conditional deletion of Sox2 was then used to test its role in adult epithelium maintenance. It was found that epithelial cells, including basal stem cells, lacking Sox2 show a reduced capacity to proliferate in culture and to repair after injury in vivo. Taken together, these results define multiple roles for Sox2 in the developing and adult trachea (Que, 2009).

The hippo pathway effector yap controls patterning and differentiation of airway epithelial progenitors

How epithelial progenitor cells integrate local signals to balance expansion with differentiation during organogenesis is still little understood. This study provides evidence that the Hippo pathway effector Yap is a key regulator of this process in the developing lung. When epithelial tubules are forming and branching, a nucleocytoplasmic shift in Yap localization marks the boundary between the airway and the distal lung compartments. At this transition zone, Yap specifies a transcriptional program that controls Sox2 expression and ultimately generates the airway epithelium. Without Yap, epithelial progenitors are unable to properly respond to local TGF-beta-induced cues and control levels and distribution of Sox2 to form airways. Yap levels and subcellular localization also markedly influence Sox2 expression and differentiation of adult airway progenitors. These data reveal a role for the Hippo-Yap pathway in integrating growth-factor-induced cues in the developing and adult lung potentially key for homeostasis and regeneration repair (Mahoney, 2014).

Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment

The mammalian hair represents an unparalleled model system to understand both developmental processes and stem cell biology. The hair follicle consists of several concentric epithelial sheaths with the outer root sheath (ORS) forming the outermost layer. Functionally, the ORS has been implicated in the migration of hair stem cells from the stem cell niche toward the hair bulb. However, factors required for the differentiation of this critical cell lineage remain to be identified. This study describes an unexpected role of the HMG-box-containing gene Sox9 in hair development. Sox9 expression can be first detected in the epithelial component of the hair placode but then becomes restricted to the outer root sheath (ORS) and the hair stem cell compartment (bulge). Using tissue-specific inactivation of Sox9, it was demonstrated that this gene serves a crucial role in hair differentiation and that skin deleted for Sox9 lacks external hair. Strikingly, the ORS acquires epidermal characteristics with ectopic expression of GATA3. Moreover, Sox9 knock hair show severe proliferative defects and the stem cell niche never forms. Finally, this study shows that Sox9 expression depends on sonic hedgehog (Shh) signaling and demonstrates overexpression in skin tumors in mouse and man. It is concluded that although Sox9 is dispensable for hair induction, it directs differentiation of the ORS and is required for the formation of the hair stem cell compartment. Genetic analysis places Sox9 in a molecular cascade downstream of sonic hedgehog and suggests that this gene is involved in basal cell carcinoma (Vidal, 2005).

Sox2-positive dermal papilla cells specify hair follicle type in mammalian epidermis

The dermal papilla comprises the specialised mesenchymal cells at the base of the hair follicle. Communication between dermal papilla cells and the overlying epithelium is essential for differentiation of the hair follicle lineages. Sox2 is expressed in all dermal papillae at E16.5, but from E18.5 onwards expression is confined to a subset of dermal papillae. In postnatal skin, Sox2 is only expressed in the dermal papillae of guard/awl/auchene follicles, whereas CD133 is expressed both in guard/awl/auchene and in zigzag dermal papillae. Using transgenic mice that express GFP under the control of the Sox2 promoter, Sox2⁺ (GFP⁺) CD133⁺ cells were isolated and compared with Sox2^- (GFP^-) CD133⁺ dermal papilla cells. In addition to the 'core' dermal papilla gene signature, each subpopulation expressed distinct sets of genes. GFP⁺ CD133⁺ cells had upregulated Wnt, FGF and BMP pathways and expressed neural crest markers. In GFP^- CD133⁺ cells, the hedgehog, IGF, Notch and integrin pathways were prominent. In skin reconstitution assays, hair follicles failed to form when dermis was depleted of both GFP⁺ CD133⁺ and GFP^- CD133⁺ cells. In the absence of GFP⁺ CD133⁺ cells, awl/auchene hairs failed to form and only zigzag hairs were found. This study has thus demonstrated a previously unrecognised heterogeneity in dermal papilla cells and shown that Sox2-positive cells specify particular hair follicle types (Driskell, 2009).

Identification and characterisation of the early differentiating cells in neural differentiation of human embryonic stem cells

One of the challenges in studying early differentiation of human embryonic stem cells (hESCs) is being able to discriminate the initial differentiated cells from the original pluripotent stem cells and their committed progenies. It remains unclear how a pluripotent stem cell becomes a lineage-specific cell type during early development, and how, or if, pluripotent genes, such as Oct4 and Sox2, play a role in this transition. Here, by studying the dynamic changes in the expression of embryonic surface antigens, this study identified the sequential loss of the markers Tra-1-81 and SSEA4 during hESC neural differentiation and isolated a transient Tra-1-81(-)/SSEA4(+) (TR-/S4+) cell population in the early stage of neural differentiation. These cells are distinct from both undifferentiated hESCs and their committed neural progenitor cells (NPCs) in their gene expression profiles and response to extracellular signalling; they co-express both the pluripotent gene Oct4 and the neural marker Pax6. Furthermore, these TR-/S4+ cells are able to produce cells of both neural and non-neural lineages, depending on their environmental cues. The results demonstrate that expression of the pluripotent factor Oct4 is progressively downregulated and is accompanied by the gradual upregulation of neural genes, whereas the pluripotent factor Sox2 is consistently expressed at high levels, indicating that these pluripotent factors may play different roles in the regulation of neural differentiation. The identification of TR-S4+ cells provides a cell model for further elucidation of the molecular mechanisms underlying hESC neural differentiation (Noisa, 2012).

SOX2 co-occupies distal enhancer elements with distinct POU factors in ESCs and NPCs to specify cell state

SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, there is no detailed understanding of how SOX2 controls these distinct stem cell populations. This study shows by genome-wide analysis that, while SOX2 binds to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 binds the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. Similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupies a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs leads to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. These findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors (Lodato, 2013).

Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain

In many species, the Sox2 transcription factor is a marker of the nervous system from the beginning of its development, and Sox2 is expressed in embryonic neural stem cells. It is also expressed in, and is essential for, totipotent inner cell mass stem cells and other multipotent cell lineages, and its ablation causes early embryonic lethality. To investigate the role of Sox2 in the nervous system, different mouse mutant alleles were generated: a null allele (Sox2^ß-geo `knock-in'), and a regulatory mutant allele (Sox2^DeltaENH), in which a neural cell-specific enhancer is deleted. Sox2 is expressed in embryonic early neural precursors of the ventricular zone and, in the adult, in ependyma (a descendant of the ventricular zone). It is also expressed in the vast majority of dividing precursors in the neurogenic regions, and in a small proportion of differentiated neurons, particularly in the thalamus, striatum and septum. Compound Sox2^{ß-geo/DeltaENH} heterozygotes show important cerebral malformations, with parenchymal loss and ventricle enlargement, and L-dopa-rescuable circling behaviour and epilepsy. Striking abnormalities were observed in neurons; degeneration and cytoplasmic protein aggregates, a feature common to diverse human neurodegenerative diseases, are observed in thalamus, striatum and septum. Furthermore, ependymal cells show ciliary loss and pathological lipid inclusions. Finally, precursor cell proliferation and the generation of new neurons in adult neurogenic regions are greatly decreased, and GFAP/nestin-positive hippocampal cells, which include the earliest neurogenic precursors, are strikingly diminished. These findings highlight a crucial and unexpected role for Sox2 in the maintenance of neurons in selected brain areas, and suggest a contribution of neural cell proliferative defects to the pathological phenotype (Ferri, 2004).

Stem cell factor Sox2 and its close relative Sox3 have differentiation functions in oligodendrocytes

Neural precursor cells of the ventricular zone give rise to all neurons and glia of the central nervous system and rely for maintenance of their precursor characteristics on the closely related SoxB1 transcription factors Sox1, Sox2 and Sox3. This study shows in mouse spinal cord that, whereas SoxB1 proteins are usually downregulated upon neuronal specification, they continue to be expressed in glial precursors. In the oligodendrocyte lineage, Sox2 and Sox3 remain present into the early phases of terminal differentiation. Surprisingly, their deletion does not alter precursor characteristics but interferes with proper differentiation. Although a direct influence on myelin gene expression may be part of their function, evidence is provided for another mode of action. SoxB1 proteins promote oligodendrocyte differentiation in part by negatively controlling miR145 and thereby preventing this microRNA from inhibiting several pro-differentiation factors. This study presents one of the few cases in which SoxB1 proteins, including the stem cell factor Sox2, are associated with differentiation rather than precursor functions (Hoffmann, 2014).

A Sox10 enhancer element common to the otic placode and neural crest is activated by tissue-specific paralogs

The otic placode, a specialized region of ectoderm, gives rise to components of the inner ear and shares many characteristics with the neural crest, including expression of the key transcription factor Sox10. This study shows that in avian embryos, a highly conserved cranial neural crest enhancer, Sox10E2, also controls the onset of Sox10 expression in the otic placode. Interestingly, this study showed that different combinations of paralogous transcription factors (Sox8, Pea3 and cMyb versus Sox9, Ets1 and cMyb) are required to mediate Sox10E2 activity in the ear and neural crest, respectively. Mutating their binding motifs within Sox10E2 greatly reduces enhancer activity in the ear. Moreover, simultaneous knockdown of Sox8, Pea3 and cMyb eliminates not only the enhancer-driven reporter expression, but also the onset of endogenous Sox10 expression in the ear. Rescue experiments confirm that the specific combination of Myb together with Sox8 and Pea3 is responsible for the onset of Sox10 expression in the otic placode, as opposed to Myb plus Sox9 and Ets1 for neural crest Sox10 expression. Whereas SUMOylation of Sox8 is not required for the initial onset of Sox10 expression, it is necessary for later otic vesicle formation. This new role of Sox8, Pea3 and cMyb in controlling Sox10 expression via a common otic/neural crest enhancer suggests an evolutionarily conserved function for the combination of paralogous transcription factors in these tissues of distinct embryological origin (Betancur, 2011).

Sequential requirement of Sox4 and Sox11 during development of the sympathetic nervous system

The highly related transcription factors Sox4 and Sox11 are expressed in the developing sympathetic nervous system. In the mouse, Sox11 appears first, whereas Sox4 is prevalent later. Using mouse mutagenesis and overexpression strategies in chicken, the role of both SoxC proteins was studed in this tissue. Neither Sox4 nor Sox11 predominantly functioned by promoting pan-neuronal or noradrenergic differentiation of sympathetic neurons as might have been expected from studies in neuronal precursors of the central nervous system. The transcriptional network that regulates the differentiation of sympathetic neurons remained intact and expression of noradrenergic markers showed only minor alterations. Instead, Sox11 was required in early sympathetic ganglia for proliferation of tyrosine hydroxylase-expressing cells, whereas Sox4 ensured the survival of these cells at later stages. In the absence of both Sox4 and Sox11, sympathetic ganglia remained hypoplastic throughout embryogenesis because of consecutive proliferation and survival defects. As a consequence, sympathetic ganglia were rudimentary in the adult and sympathetic innervation of target tissues was impaired leading to severe dysautonomia (Potzner, 2010).

Morphogenesis and cytodifferentiation of the avian retinal pigmented epithelium require downregulation of Group B1 Sox genes

The optic vesicle is a multipotential primordium of the retina, which becomes subdivided into the neural retina and retinal pigmented epithelium domains. Although the roles of several paracrine factors in patterning the optic vesicle have been studied extensively, little is known about cell-autonomous mechanisms that regulate coordinated cell morphogenesis and cytodifferentiation of the retinal pigmented epithelium. This study demonstrates that members of the SoxB1 gene family, Sox1, Sox2 and Sox3, are all downregulated in the presumptive retinal pigmented epithelium. Constitutive maintenance of SoxB1 expression in the presumptive retinal pigmented epithelium both in vivo and in vitro resulted in the absence of cuboidal morphology and pigmentation, and in concomitant induction of neural differentiation markers. It was also demonstrated that exogenous Fgf4 inhibits downregulation of all SoxB1 family members in the presumptive retinal pigment epithelium. These results suggest that retinal pigment epithelium morphogenesis and cytodifferentiation requires SoxB1 downregulation, which depends on the absence of exposure to an FGF-like signal (Ishii, 2009).

Proliferative and transcriptional identity of distinct classes of neural precursors in the mammalian olfactory epithelium

Neural precursors in the developing olfactory epithelium (OE) give rise to three major neuronal classes - olfactory receptor (ORNs), vomeronasal (VRNs) and gonadotropin releasing hormone (GnRH) neurons. Nevertheless, the molecular and proliferative identities of these precursors are largely unknown. Two precursor classes were characterized in the olfactory epithelium (OE) shortly after it becomes a distinct tissue at midgestation in the mouse: slowly dividing self-renewing precursors that express Meis1/2 at high levels, and rapidly dividing neurogenic precursors that express high levels of Sox2 and Ascl1. Precursors expressing high levels of Meis genes primarily reside in the lateral OE, whereas precursors expressing high levels of Sox2 and Ascl1 primarily reside in the medial OE. Fgf8 maintains these expression signatures and proliferative identities. Using electroporation in the wild-type embryonic OE in vitro as well as Fgf8, Sox2 and Ascl1 mutant mice in vivo, it was found that Sox2 dose and Meis1 -- independent of Pbx co-factors -- regulate Ascl1 expression and the transition from lateral to medial precursor state. Thus, proliferative characteristics and a dose-dependent transcriptional network were characterized that define distinct OE precursors: medial precursors that are most probably transit amplifying neurogenic progenitors for ORNs, VRNs and GnRH neurons, and lateral precursors that include multi-potent self-renewing OE neural stem cells (Tucker, 2010).

SOX2 functions to maintain neural progenitor identity

Neural progenitors of the vertebrate CNS are defined by generic cellular characteristics, including their pseudoepithelial morphology and their ability to divide and differentiate. SOXB1 transcription factors, including the three closely related genes Sox1, Sox2, and Sox3, universally mark neural progenitor and stem cells throughout the vertebrate CNS. Constitutive expression of SOX2 inhibits neuronal differentiation and results in the maintenance of progenitor characteristics. Conversely, inhibition of SOX2 signaling results in the delamination of neural progenitor cells from the ventricular zone and exit from cell cycle, which is associated with a loss of progenitor markers and the onset of early neuronal differentiation markers. The phenotype elicited by inhibition of SOX2 signaling can be rescued by coexpression of SOX1, providing evidence for redundant SOXB1 function in CNS progenitors. Taken together, these data indicate that SOXB1 signaling is both necessary and sufficient to maintain panneural properties of neural progenitor cells (Graham, 2003).

Collectively, several lines of evidence suggest that the members of the SOXB1 subfamily are functionally redundant. (1) Microinjection of dominant-negative forms of Sox2 mRNA in Xenopus that inhibit neural differentiation of animal caps can be rescued by injection of Sox3 but not divergent Sox genes such as Sox9 and SoxD. (2) Midline glial defects in Drosophila Dichaete mutants can be rescued by directed expression of SOX1 and SOX2 proteins. (3) The elimination of both members of the Drosophila SOXB subfamily, SoxNeuro and Dichaete, simultaneously results in much more severe phenotypes in the neuroectoderm than the single mutants. Thus, functional redundancy appears to be confined to SOXB1 subfamily and does not extend to more divergent SOX family members. The data provides further evidence that the members of the SOXB1 subfamily are functionally redundant: the phenotypic consequences of the inhibition of SOX2 signaling in chick neural progenitors can be rescued by the coexpression of SOXB1 subfamily member SOX1, and the forced expression of SOX1 in CNS cells phenocopies forced expression of SOX2 (Graham, 2003).

There is now increasing evidence that SOX factors may play a global role in maintaining progenitor/stem cell fates in a variety of tissues including the nervous system. Members of the SOX gene family are expressed in a variety of embryonic and adult tissues where their expression, and in some cases function, is associated with the specification and/or maintenance of progenitor identity. For example, SRY is transiently expressed in the progenitor of Sertoli cells of the XY genital ridge and is responsible for triggering development of the male phenotype. SOX9 is expressed in immature chondrocytes and plays a role in their proliferation and differentiation. Intriguingly, a recent report describing the function of SOX10 in the PNS reveals many functional parallels between the role of SOX10 in the PNS stem/progenitor cells and those described here for SOXB1 factors in CNS progenitor cells. SOX10 is expressed in multipotent neural crest stem cells and is downregulated during their neuronal differentiation. Forced expression of SOX10 is able to override both antigliogenic activity of BMP2 and antineurogenic (antiproliferative) activity of TGFbeta and thus maintain multipotential differentiation capacity of NCSCs. Furthermore, by directly inhibiting terminal neuronal differentiation, SOX10 appears to provide a permissive environment for glial differentiation. It will be interesting to determine if any of the molecular pathways by which SOX10 maintains neural crest stem cell fate in the PNS are also used in the CNS (Graham, 2003 and references therein).

Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells

Purified rat oligodendrocyte precursor cells (OPCs) can be induced by extracellular signals to convert to multipotent neural stem-like cells (NSLCs), which can then generate both neurons and glial cells. Because the conversion of precursor cells to stem-like cells is of both intellectual and practical interest, it is important to understand its molecular basis. The conversion of OPCs to NSLCs depends on the reactivation of the sox2 gene, which in turn depends on the recruitment of the tumor suppressor protein Brca1 and the chromatin-remodeling protein Brahma (Brm) to an enhancer in the sox2 promoter. Moreover, the conversion is associated with the modification of Lys 4 and Lys 9 of histone H3 at the same enhancer. These findings suggest that the conversion of OPCs to NSLCs depends on progressive chromatin remodeling, mediated in part by Brca1 and Brm (Kondo, 2004).

Sequentially acting Sox transcription factors in neural lineage development

Pluripotent embryonic stem (ES) cells can generate all cell types, but how cell lineages are initially specified and maintained during development remains largely unknown. Different classes of Sox transcription factors are expressed during neurogenesis and have been assigned important roles from early lineage specification to neuronal differentiation. This study characterize the genome-wide binding for Sox2, Sox3, and Sox11, which have vital functions in ES cells, neural precursor cells (NPCs), and maturing neurons, respectively. The data demonstrate that Sox factor binding depends on developmental stage-specific constraints and reveal a remarkable sequential binding of Sox proteins to a common set of neural genes. Interestingly, in ES cells, Sox2 preselects for neural lineage-specific genes destined to be bound and activated by Sox3 in NPCs. In NPCs, Sox3 binds genes that are later bound and activated by Sox11 in differentiating neurons. Genes prebound by Sox proteins are associated with a bivalent chromatin signature, which is resolved into a permissive monovalent state upon binding of activating Sox factors. These data indicate that a single key transcription factor family acts sequentially to coordinate neural gene expression from the early lineage specification in pluripotent cells to later stages of neuronal development (Bergsland, 2011).

During development of the CNS, neurons and glia are generated from self-renewing neural progenitor cells (NPCs) that are directed to leave the cell cycle, down-regulate progenitor identities, and activate neuronal or glial gene expression in a spatially and temporally defined manner. The mechanisms regulating gene expression in NPCs and their differentiated progeny have been extensively characterized, but it is largely unknown how neural lineage-specific gene expression programs are initially specified and activated during the course of differentiation (Bergsland, 2011).

An important property of pluripotent stem cells is their capacity to induce gene programs characteristic of all cell lineages. Previous studies in embryonic stem (ES) cells have demonstrated that many genes destined to become activated at later stages of development are already bound by ES cell regulatory transcription factors, including Sox2, Oct4, Nanog, and FoxD3. Moreover, genes poised for activation are often associated with bivalent histone domains consisting of repressive histone modifications combined with modifications associated with transcriptional activation (H3K27me3 and H3K4me3). Bivalent histone marks are subsequently resolved as genes become activated or terminally repressed during development. Together, these findings indicate that many silent genes in ES cells are prebound by transcription factors and epigenetically prepared for activation, but they do not demonstrate how lineage-specific gene expression programs are initially selected and later activated. Insights into these questions come from studies of the liver-specific enhancer Alb1. In ES cells, the Alb1 enhancer is prebound by FoxD3, which ensures the assembly of permissive chromatin. Interestingly, upon endodermal differentiation, FoxD3 binding is replaced by FoxA1, which helps to induce Alb1 expression in a liver-specific manner. Studies of the B-cell-specific lambda5-VpreB1 locus constitute an additional example of how a transcription factor prepares the enhancer for later activation by an alternative member of the same transcription factor family. This locus contains an intergenic enhancer to which Sox2 binds and adds an epigenetic active mark in ES cells. In pro-B cells, Sox2 binding is replaced by Sox4, which leads to the activation of lambda5 expression. Although these studies indicate the importance of pioneering functions of transcription factors, experiments are focused on specific enhancers in individual genes, and it remains unclear whether the sequential regulatory strategy is a more general requirement for activation of larger sets of gene batteries in differentiating cell lineages (Bergsland, 2011).

Apart from the above-mentioned gene regulatory functions in ES cells and roles in early B-cell development, transcription factors of the Sox gene family have important sequential roles in regulating the maintenance and differentiation of progenitor cells from early pluripotent stages to late steps of neurogenesis. Sox2 is necessary for the establishment and maintenance of ES cells. All three SoxB1 proteins (Sox1, Sox2, and Sox3) are expressed in most neural precursors in both the developing and adult CNS, and studies conducted in chick and mouse embryos demonstrate that they act redundantly to maintain neural cells in a progenitor state and counteract neuronal differentiation. The SoxC proteins (Sox4, Sox11, and Sox12) are expressed complementary to Sox1-3 in the developing CNS and can mostly be detected in post-mitotic differentiating neurons. Misexpression experiments in chicks demonstrate that SoxC proteins have the opposite function compared with Sox1-3 and can induce the expression of neuronal proteins, whereas deletion of the SoxC proteins in the embryonic mouse spinal cord leads to a significant decrease in differentiated neurons and an associated increased cell death (Bergsland, 2011).

Despite the importance of Sox factors during the course of neural development, there is very limited information concerning the control of appropriate gene expression programs that are activated in CNS progenitors and their differentiated progeny. This is partly due to the limited number of identified Sox target genes. This study analyzed Sox transcription factors during neural lineage development by generating and comparing genome-wide binding data for Sox2, Sox3, and Sox11 from early lineage specification stages with the onset of neuronal gene expression. The data indicate that sequentially acting Sox transcription factors control neural lineage-specific gene expression by predisposing gene programs to become activated in NPCs and during neuronal and glial differentiation (Bergsland, 2011).

SOX2 expression levels distinguish between neural progenitor populations of the developing dorsal telencephalon

The HMG-Box transcription factor SOX2 is expressed in neural progenitor populations throughout the developing and adult central nervous system and is necessary to maintain their progenitor identity. However, it is unclear whether SOX2 levels are uniformly expressed across all neural progenitor populations. In the developing dorsal telencephalon, two distinct populations of neural progenitors, radial glia and intermediate progenitor cells, are responsible for generating a majority of excitatory neurons found in the adult neocortex. This study demonstrates, using both cellular and molecular analyses, that SOX2 is differentially expressed between radial glial and intermediate progenitor populations. Moreover, utilizing a SOX2(EGFP) mouse line, this differential expression can be used to prospectively isolate distinct, viable populations of radial glia and intermediate cells for in vitro analysis. Given the limited repertoire of cell-surface markers currently available for neural progenitor cells, this provides an invaluable tool for prospectively identifying and isolating distinct classes of neural progenitor cells from the central nervous system (Button, 2011).

Sox2-mediated differential activation of Six3.2 contributes to forebrain patterning

The vertebrate forebrain is patterned during gastrulation into telencephalic, retinal, hypothalamic and diencephalic primordia. Specification of each of these domains requires the concerted activity of combinations of transcription factors (TFs). Paradoxically, some of these factors are widely expressed in the forebrain, which raises the question of how they can mediate regional differences. To address this issue, focus was placed on the homeobox TF Six3.2. With genomic and functional approaches it was demonstrated that, in medaka fish, Six3.2 regulates, in a concentration-dependent manner, telencephalic and retinal specification under the direct control of Sox2. Six3.2 and Sox2 have antagonistic functions in hypothalamic development. These activities are, in part, executed by Foxg1 and Rx3, which seem to be differentially and directly regulated by Six3.2 and Sox2. Together, these data delineate the mechanisms by which Six3.2 diversifies its activity in the forebrain and highlight a novel function for Sox2 as one of the main regulators of anterior forebrain development. They also demonstrate that graded levels of the same TF, probably operating in partially independent transcriptional networks, pattern the vertebrate forebrain along the anterior-posterior axis (Beccari, 2012).

Evidence for the temporal regulation of insect segmentation by a conserved sequence of transcription factors

Long-germ insects, such as the fruit fly Drosophila melanogaster, pattern their segments simultaneously, whereas short-germ insects, such as the beetle Tribolium castaneum, pattern their segments sequentially, from anterior to posterior. While the two modes of segmentation at first appear quite distinct, much of this difference might simply reflect developmental heterochrony. This study now shows that, in both Drosophila and Tribolium, segment patterning occurs within a common framework of sequential Caudal, Dichaete, and Odd-paired expression (see Comparison of long-germ and short-germ segmentation). In Drosophila these transcription factors are expressed like simple timers within the blastoderm, while in Tribolium they form wavefronts that sweep from anterior to posterior across the germband. In Drosophila, all three are known to regulate pair-rule gene expression and influence the temporal progression of segmentation. It is proposed that these regulatory roles are conserved in short-germ embryos, and that therefore the changing expression profiles of these genes across insects provide a mechanistic explanation for observed differences in the timing of segmentation. In support of this hypothesis it was demonstrated that Odd-paired is essential for segmentation in Tribolium, contrary to previous reports (Clark, 2018).

This study has found that segment patterning in both Drosophila and Tribolium occurs within a conserved framework of sequential Caudal, Dichaete and Odd-paired expression. In the case of Opa, there is also evidence for conserved function. However, although the sequence itself is conserved between the two insects, its spatiotemporal deployment across the embryo is divergent. In Drosophila, the factors are expressed ubiquitously within the main trunk, and each turns on or off almost simultaneously, correlating with the temporal progression of a near simultaneous segmentation process. In Tribolium, their expression domains are staggered in space, with developmentally more advanced anterior regions always subjected to a 'later' regulatory signature than more-posterior tissue. These expression domains retract over the course of germband extension, correlating with the temporal progression of a sequential segmentation process built around a segmentation clock (Clark, 2018).

Pair-rule patterning involves several distinct phases of gene expression, each requiring specific regulatory logic. It is proposed that, in both long-germ and short-germ species, the whole process is orchestrated by a series of key regulators, expressed sequentially over time, three of which are the focus of this paper. By rewiring the regulatory connections between other genes, factors such as Dichaete and Opa allow a small set of pair-rule factors to carry out multiple different roles, each specific to a particular spatiotemporal regulatory context. This kind of control logic makes for a flexible, modular regulatory network, and may therefore turn out to be a hallmark of other complex patterning systems (Clark, 2018).

Having highlighted the significance of these 'timing factors' in this paper, the next steps will be to investigate their precise regulatory roles and modes of action. It will be interesting to dissect how genetic interactions with pair-rule factors are implemented at the molecular level. Dichaete is known to act both as a repressive co-factor and as a transcriptional activator; therefore, a number of different mechanisms are plausible. The Odd-paired protein is also likely to possess both these kinds of regulatory activities (Clark, 2018).

Given the phylogenetic distance between beetles and flies (separated by at least 300 million years), it is expected that the similarities seen between Drosophila and Tribolium segmentation are likely to hold true for other insects, and perhaps for many non-insect arthropods as well. It is proposed that these similarities, which argue for the homology of long-germ and short-germ segmentation processes, result from conserved roles of Cad, Dichaete and Opa in the temporal regulation of pair-rule and segment-polarity gene expression during segment patterning. This hypothesis can be tested by detailed comparative studies in various arthropod model organisms (Clark, 2018).

This study provides evidence that a segmentation role for Opa is conserved between Drosophila and Tribolium; clear segmentation phenotypes have also been found for Cad in Nasonia, and for Dichaete in Bombyx. However, as the Tc-opa experiments reveal, functional manipulations in short-germ insects will need to be designed carefully in order to bypass the early roles of these pleiotropic genes. For example, cad knockdowns cause severe axis truncations in many arthropods, whereas Dichaete knockdown in Tribolium yields mainly empty eggs (Clark, 2018).

It was previously thought that Tc-opa was not required for segmentation, and that the segmentation role of Opa may have been recently acquired, in the lineage leading to Drosophila. However, the current analysis reveals that Tc-opa is indeed a segmentation gene, and also has other important roles, including head patterning and blastoderm formation. Given that a similar developmental profile of opa expression is seen in the millipede Glomeris, and even in the onychophoran Euperipatoides, the segmentation role of Opa may actually be ancient (Clark, 2018).

Head phenotypes following Tc-opa RNAi were unexpected, but both the blastoderm expression pattern and cuticle phenotypes that were observed are strikingly similar to those reported for Tc-otd and Tc-ems (Tribolium orthologues of the Drosophila head 'gap' genes orthodenticle and empty spiracles), suggesting that the three genes function together in a gene network that controls early head patterning. This function of Tc-opa might be homologous to the head patterning role for Opa discovered in the spider Parasteatoda, where it interacts with both Otd and Hedgehog (Hh) expression. Opa/Zic is known to modulate Hh signalling, and a role for Hh in head patterning appears to be conserved across arthropods, including Tribolium (Clark, 2018).

Finally, Opa/Zic is also known to modulate Wnt signalling. In chordates, Zic expression tends to overlap with sites of Hh and/or Wnt signalling, suggesting that one of its key roles in development is to ensure cells respond appropriately to these signals. The expression domains of Tc-opa that were observed in Tribolium (e.g. in the head, in the SAZ and between parasegment boundaries) accord well with this idea (Clark, 2018).

Similar embryonic expression patterns of Cad, Dichaete and Opa orthologues are observed in other bilaterian clades, including vertebrates. Cdx genes are expressed in the posterior of vertebrate embryos, where they play crucial roles in axial extension and Hox gene regulation. Sox2 (a Dichaete orthologue) has conserved expression in the nervous system, but is also expressed in a posterior domain, where it is a key determinant of neuromesodermal progenitor (posterior stem cell) fate. Finally, Zic2 and Zic3 (Opa orthologues) are expressed in presomitic mesoderm and nascent somites, and have been functionally implicated in somitogenesis and convergent extension. All three factors thus have important functions in posterior elongation, roles that may well be conserved across Bilateria (Clark, 2018).

In Tribolium, all three factors may be integrated into an ancient gene regulatory network downstream of posterior Wnt signalling, which generates their sequential expression and helps regulate posterior proliferation and/or differentiation. The mutually exclusive patterns of Tc-wg and Tc-Dichaete in the posterior germband are particularly suggestive: Wnt signalling and Sox gene expression are known to interact in many developmental contexts and these interactions may form parts of temporal cascades) (Clark, 2018).

The following outline is suggested as a plausible scenario for the evolution of arthropod segmentation; In non-segmented bilaterian ancestors of the arthropods, Cad, Dichaete and Opa were expressed broadly similarly to how they are expressed in Tribolium today, mediating conserved roles in posterior elongation, while gap and pair-rule genes may have had functions in the nervous system. At some point, segmentation genes came under the regulatory control of these factors, which provided a pre-existing source of spatiotemporal information in the developing embryo. Pair-rule genes began oscillating in the posterior, perhaps under the control of Cad and/or Dichaete, while the posteriorly retracting expression boundaries of the timing factors provided smooth wavefronts that effectively translated these oscillations into periodic patterning of the AP axis, analogous to the roles of the opposing retinoic acid and FGF gradients in vertebrate somitogenesis. Much later, in certain lineages of holometabolous insects, the transition to long-germ segmentation occurred. This would have involved two main modifications of the short-germ segmentation process: (1) changes to the expression of the timing factors, away from the situation seen in Tribolium, and towards the situation seen in Drosophila, causing a heterochronic shift in the deployment of the segmentation machinery from SAZ to blastoderm; and (2) recruitment of gap genes to pattern pair-rule stripes, via the ad hoc evolution of stripe-specific elements (Clark, 2018).

The appeal of this model is that the co-option of existing developmental features at each stage reduces the number of regulatory changes required to evolve de novo, facilitating the evolutionary process. In this scenario, arthropod segmentation would not be homologous to segmentation in other phyla, but would probably have been fashioned from common parts (Clark, 2018).

SOX1 Is Required for the Specification of Rostral Hindbrain Neural Progenitor Cells from Human Embryonic Stem Cells

Region-specific neural progenitor cells (NPCs) can be generated from human embryonic stem cells (hESCs) by modulating signaling pathways. However, how intrinsic transcriptional factors contribute to the neural regionalization is not well characterized. This study generated region-specific NPCs from hESCs and found that SOX1 (see Drosophila Dichaete) is highly expressed in NPCs with the rostral hindbrain identity. Moreover, it was found that OTX2 (see Drosophila Ocelliless) inhibits SOX1 expression, displaying exclusive expression between the two factors. Furthermore, SOX1 knockout (KO) leads to the upregulation of midbrain genes and downregulation of rostral hindbrain genes, indicating that SOX1 is required for specification of rostral hindbrain NPCs. SOX1 chromatin immunoprecipitation sequencing analysis reveals that SOX1 binds to the distal region of GBX2 (see Drosophila unplugged to activate its expression. Overexpression of GBX2 largely abrogates SOX1-KO-induced aberrant gene expression. Taken together, this study uncovers previously unappreciated role of SOX1 in early neural regionalization and provides new information for the precise control of the OTX2/GBX2 interface (Liu, 2020).

REFERENCES

Search PubMed for articles about Drosophila Dichaete

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Biological Overview

date revised: 20 February 2017