InteractiveFly: GeneBrief

Optix: Biological Overview | Regulation | Developmental Biology | Evolutionary Homologs | References


Gene name - Optix

Synonyms - Six3

Cytological map position - 43F--44A

Function - transcription factor

Keywords - eye development

Symbol - Optix

FlyBase ID: FBgn0025360

Genetic map position -

Classification - Six domain and homeodomain

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Dominguez-Cejudo, M. A. and Casares, F. (2015). Antero-posterior patterning of Drosophila ocelli requires an anti-repressor mechanism within the hh-pathway mediated by the Six3 gene Optix. Development 142(16):2801-9. PubMed ID: 26160900
Summary:
In addition to the compound eyes, most insects possess a set of three dorsal ocelli that develop at the vertices of a triangular cuticle patch, forming the ocellar complex. The wingless and hedgehog signaling pathways, together with the transcription factor encoded by orthodenticle, are known to play major roles in the specification and patterning of the ocellar complex. Specifically, hedgehog is responsible for the choice between ocellus and cuticle fates within the ocellar complex primordium. However, the interaction between signals and transcription factors known to date do not fully explain how this choice is controlled. This study shows that this binary choice depends on dynamic changes in the domains of hedgehog signaling. In this dynamics, the restricted expression of engrailed, a hedgehog-signaling target, is key because it defines a domain within the complex where hh transcription is maintained while the pathway activity is blocked. The Drosophila Six3, Optix, is expressed in and required for the development of the anterior ocellus specifically. Optix would not act as an ocellar selector, but rather as a patterning gene, limiting the en expression domain. These results indicate that, despite their genetic and structural similarity, anterior and posterior ocelli are under different genetic control.
BIOLOGICAL OVERVIEW

Optix is a new Drosophila member of the Six/sine oculis gene family that contains both a Six domain and a homeodomain. Because of its high amino acid sequence similarity with the mouse Six3 gene, Optix is considered to be the orthologous gene from Drosophila, rather than sine oculis as was previously believed. Whereas Sine oculis belongs to the Six1 subclass of the Six/so gene family, Optix belongs to the Six3 subclass. Optix expression is detected in the eye, wing and haltere imaginal discs. Ectopic expression of Optix leads to the formation of ectopic eyes, suggesting that Optix has important functions in eye development. Although Optix and sine oculis both belong to the Six/so gene family and share a high degree of amino acid sequence identity, there are a number of factors that suggest that their developmental roles are different: (1) the expression patterns of Optix and sine oculis are clearly distinct; (2) sine oculis acts downstream of eyeless, whereas Optix is expressed independent of eyeless; (3) sine oculis functions synergistically with eyes absent in eye development whereas Optix does not; (4) ectopic expression of Optix alone, but not of sine oculis, can induce ectopic eyes in the antennal disc. These results suggest that Optix is involved in eye morphogenesis by an eyeless-independent mechanism (Seimiya, 2000).

In order to determine a possible epistatic relationship between Optix and eyeless, Optix expression was examined in the ey2 mutant. In ey2, no ey transcripts can be detected, either in the embryonic eye primordia or in the larval eye disc. In ey2 eye discs, Optix expression is not affected (Seimiya, 2000). In contrast, so expression is no longer observed in the early third instar eye discs of ey2 mutants (Halder, 1998).

Optix is expressed in front of the morphogenetic furrow, strongly suggesting that Optix may play an important role in early eye disc development. Since to date no mutant for Optix has been identified, the potential for Optix to induce the formation of ectopic eyes was examined using a gain-of-function strategy. The GAL4 system was used to target Optix expression to various imaginal discs where Optix is normally not expressed. UAS-optix was crossed to dppblink-GAL4 that expresses GAL4 along the anteroposterior compartment boundary in leg, wing and antennal imaginal discs. Targeted expression of Optix cDNA induces ectopic eye structures just in the antenna and the anterior medial region of the head, but not in the legs or in the wings. The normal eyes are reduced in size and only rarely detected are extra ocelli and interocellar bristles, around the vertex region. The efficiency of induction of ectopic eyes is relatively low (i.e. 20% as compared to 100% in ey). In contrast to Optix, ectopic expression of so alone cannot induce ectopic eyes. UAS-Optix was crossed to E132-GAL4 which can induce ectopic eyes in combination with UAS-ey. However, the UAS-Optix x E132-GAL4 flies die as embryos, whereas the UAS-ey x E132-GAL4 controls survive and formed ectopic eyes (Seimiya, 2000).

Since ectopic expression of eyes absent, dachshund, eya plus sine oculis and eya plus dac requires eyeless to form ectopic eyes, an examination was performed to see whether ey expression is also induced during ectopic eye formation by Optix. In the eye discs of UAS-Optix dpp-GAL4 flies, no ectopic ey expression was detected. Therefore attempts were made to induce ectopic eye formation with Optix in an ey2 mutant background. Targeted expression of the Optix gene in an ey2 background results in ectopic eye formation. The efficiency of occurrence of ectopic eyes does not change from the wild-type background situation, but extra ocelli are induced more often than in a wild-type background. From these results, it is concluded that Optix does not require ey expression for the induction of ectopic eyes (Seimiya, 2000).

Since ey is expressed much earlier in the eye anlagen than Optix, the fact that Optix can induce ectopic eyes only in the eye disc while ey can induce ectopic eyes in other discs as well suggests that ey induces a larger set of target genes than Optix, and that the activity of some of those genes are required for eye induction by Optix. This interpretation is supported by the observation that Optix cannot induce ectopic eyes in a so or eya mutant background. Furthermore, the ectopic expression of ey is sufficient to induce ectopic Optix expression, although in normal eye development Optix transcription is not regulated by ey. Since all these results come from an ectopic situation it will be necessary to analyze the relationship of Optix and ey in an Optix mutant background (Seimiya, 2000).

A Sine oculis/Eyes absent complex regulates multiple steps in eye development and functions within the context of a network of genes to specify eye tissue identity. Ectopic expression of so alone does not induce ectopic eyes, and ectopic expression of eya alone induces ectopic eyes just in the antenna at low frequency (10%), but coexpression of so and eya leads to an increase in the induction of ectopic eyes in the antenna both in frequency (76%) and size. This synergistic effect is probably due to the capability of So and Eya to form a protein complex. The domains required for complex formation are the evolutionarily conserved Six and Eya domains. Since Optix has a Six domain as well, a test was performed to see whether Optix and Eya also synergize and enhance ectopic eye induction. UAS-eya;UAS-Optix was crossed to dpp blink-GAL4 and the frequency of induction of ectopic eyes was examined. Optix can induce ectopic eyes (22%) but so cannot (0%); so has a synergistic function with eya (0% and 10% individually, to 60% when coexpressed), but coexpression of Optix and eya does not lead to an increase in frequency (20%) nor in size of ectopic eyes. Therefore, although Optix has a Six domain, no synergistic interaction with Eya has been demonstrated (Seimiya, 2000).

The isolation and functional analysis of Optix provides new insights into the evolution of the Six/so gene family. [table below]

Subclasses
six3 six1six4
Optix
Six3
Optx2
so
Six1
Six2
Six4
Six5

Optix belongs to the Six3 subclass, whereas so has been assigned to the same subclass as Six1; Six4 and Six5 form a third subclass.

The mouse genes, Six3 and Optx2, which are in the Six3 subclass, the same as Optix, are expressed in the optic vesicles and the lens, i.e. in eye morphogenesis (Oliver, 1995; Toy, 1998). In contrast Six1 and Six2, members of the Six1 subclass, are expressed in phalangeal tendons, skeletal and smooth muscle, i.e. primarily in myogenesis. Although [Six1 and Six2] and [Six4 and Six5] are assigned to different subclasses on the basis of their amino acid sequences, both Six1 and Six5 seem to control early steps of myogenesis, and Six1 and Six4 are able to transactivate a reporter gene containing a myogenin promoter fragment. These Six genes seem to act at a high level in the hierarchical cascade controlling myogenesis. Based on these reports, it is conceivable that genes in subclasses Six1 and Six4 share the same functions and are controlling muscle formation. In contrast, Six3 subclass genes have an important function in eye development. Therefore, it seems that these two groups of Six genes might have diverged to serve different functions. This also applies to the interactions with Eya genes. In the mouse, Six2, Six4 and Six5 induce nuclear translocation of Eya1, Eya2 and Eya3, which are all localized in the cytoplasm, but Six3 does not. Furthermore, Six1/Eya2 and Six2/Eya1 genes are widely coexpressed in many tissues during organogenesis. Moreover, the Pax3 gene is also required for the same steps. These findings suggest the possibility that Pax, Six and Eya proteins, all of which are coexpressed during vertebrate somitogenesis, cooperate during vertebrate muscle development. In addition to their major roles in myogenesis, Six2, Six4 and Six5 are expressed in the retina, but the gene that plays a major role in eye development is Six3. For this reason, it had been thought that so is the Drosophila ortholog of Six3, but this assignment needs to be revised. Optix is the putative Six3 ortholog; so clearly belongs to the Six1 subclass. This phylogenetic relationship is also supported by the fact that so interacts with eya, whereas Optix does not (Seimiya, 2000 and references therein).

Specific expression and function of the Six3 optix in Drosophila serially homologous organs

Organ size and pattern results from the integration of two positional information systems. One global, encoded by the Hox genes, links organ type with position along the main body axis. Within specific organs, local information is conveyed by signaling molecules that regulate organ growth and pattern. The mesothoracic (T2) wing and the metathoracic (T3) haltere of Drosophila represent a paradigmatic example of this coordination. The Hox gene Ultrabithorax (Ubx), expressed in the developing T3, selects haltere identity by, among other processes, modulating the production and signaling efficiency of Dpp, a BMP2-like molecule that acts as a major regulator of size and pattern. Still, the mechanisms of the Hox-signal integration even in this well-studied system are incomplete. This study has investigated this issue by studying the expression and function of the Six3 transcription factor optix during the development of the Drosophila wing and haltere development. In both organs Dpp defines the expression domain of optix through repression, and the specific position of this domain in wing and haltere seems to reflect the differential signaling profile among these organs. optix expression in wing and haltere primordia is conserved beyond Drosophila in other higher diptera. In Drosophila, optix is necessary for the growth of wing and haltere: In the wing, optix is required for the growth of the most anterior/proximal region (the 'marginal cell') and for the correct formation of sensory structures along the proximal anterior wing margin, and the halteres of optix mutants are also significantly reduced. In addition, in the haltere optix is necessary for the suppression of sensory bristles (Al Khatib, 2017).

In the haltere, Ubx modifies the wing developmental program in two ways. First, as a transcription factor, Ubx regulates the expression of some targets. For example, Ubx represses sal expression (Weatherbee, 1998). Second, Ubx modifies the shape of the Dpp-generated signaling gradient indirectly, by controlling the expression of proteoglycans required for Dpp dispersion (Crickmore, 2006; de Navas, 2006). Globally, these modifications of Dpp signaling and target gene activation by Ubx have been related to the size and patterning differences between halteres and wings (Al Khatib, 2017).

Since Dpp signaling generates a signaling gradient that spans the whole wing pouch and its activity is required throughout the wing, it is expected to control the expression of target genes not only in central region of the pouch, but also in more lateral ones. The Six3-type transcription factor optix has been reported to be expressed in the lateral region of the wing pouch, as well as in the haltere (Seimiya, 2000). Functional studies show that optix is required for the normal patterning of the anterior portion of the wing and that its expression is negatively regulated by sal genes (Organista, 2015). The fact that sal genes are Dpp signaling targets in the wing, places optix downstream of Dpp regulation. However, since sal genes are not expressed in haltere discs (Weatherbee, 1998), the mechanism of optix regulation in this organ is still unknown. This study analyzed comparatively the expression, function and regulation of optix in wing and haltere discs. In both discs, optix expression is anteriorly restricted by Dpp signaling, although in the wing the precise expression boundary may be set with the collaboration of wing specific Dpp targets, such as sal. optix shows organ-specific functions: in the wing, previous results were confirmed showing it is necessary for the growth of the anterior/proximal wing ('marginal cell') and the development of wing margin sensory bristles. However, in the haltere optix is required for the suppression of sensory bristle formation. Overexpression of optix in the entire wing pouch affects only anterior wing development, suggesting that other parts of the wing cannot integrate ectopic Optix input. This observation may provide a mechanistic explanation for a widespread re-deployment of optix expression in wing spot formation in various butterfly species (Al Khatib, 2017).

The Dpp signaling gradient is required for the patterning of the whole wing, from the center to its margin. This gradient is translated into a series of contiguous domains expressing distinct transcription factors, each required for the specification of specific features in the adult organ. However, while the transcription factors acting in the central wing were known, the most anterior region of the wing -- the region comprised between the longitudinal vein 2 (L2) and the anterior margin (L1) -- lacked a specific transcription factor. This paper showS that this transcription factor, or at least one of them, is Optix (Al Khatib, 2017).

The results confirm previous findings (Organista, 2015) that optix is expressed in, and required for the growth of this most anterior sector of the wing, the so-called margin cell. This study now shows that optix is also required for the growth of the wing's serially homologous organ: the haltere. This role is in agreement with previous results showing that Six3 regulates cell proliferation in vertebrate systems. This study further shows that Dpp signaling plays a major role in setting the optix expression domain. Although it has been reported before that sal genes are required to set the central limit of this domain, in discs lacking sal function optix does not extend all the way to the AP border (Organista, 2015), suggesting additional mechanisms involved in optix repression. The fact that sal is not expressed in the haltere pouch and still optix does not extend all the way to the AP border, the exclusion of optix expression from intermediate/high Dpp signaling in both wing and haltere, and the requirement of Dpp signaling to repress optix in any position of the anterior wing compartment globally suggested that either Dpp activates a different repressor closer to the AP border, or that Dpp signaling represses directly optix transcription. The current work cannot distinguish between these possibilities. Regarding another well characterized Dpp target, omb, the extensive coexpression of omb and optix in the haltere also seems to exclude omb as a repressor. Therefore, either another unknown repressor exists, or Dpp signaling acts as a direct optix repressor. While in the haltere, the domain of optix would be set directly by Dpp, in the wing sal would be an additional repressor. By intercalating sal, the Dpp positioning system may be able to push the limit of optix expression farther away from the AP border of the wing. The Sal proteins have been previously shown to act as transcriptional repressors of knirps (kni) to position vein L2. Thus, adding sal repression may help to align the optix domain with L2. This additional repression would not be operating in the haltere, which lacks venation (Al Khatib, 2017).

Interestingly, the logic of optix regulation by Dpp is different from that of other Dpp targets. The activation of the sal paralogs (sal-m and sal-r) and aristaless (al), another target required for vein L2 formation, proceeds through a double repression mechanism: In the absence of signal, the Brinker repressor keeps sal and al off. Activation of the pathway leads to the phosphorylation of the nuclear transducer Mad (pMad) which, in turn, represses brk, thus relieving the repression on sal and al. Therefore, optix regulation by Dpp signaling could be more direct similarly to that of brk (Al Khatib, 2017).

One interesting aspect of optix function is that it plays an additional specific role in the haltere. While in the wing optix is required for the development of the anterior-most portion of the wing (including the margin bristles), in the haltere optix serves to suppress the development of sensory bristles, a task known to be carried out by the Hox gene Ubx. A role for optix in regulating Ubx expression has been ruled out, at least when judged from Ubx protein levels. Therefore, optix is required for a subset of Ubx's normal functions. Since optix encodes a Six3-type transcription factor, this interaction could be happening at the level of target enhancers, where the combination of Ubx and Optix would allow the activation or repression of specific sets of genes (Al Khatib, 2017).

Finally, it was observed that the expression of optix in wing and haltere primordia is conserved across higher Diptera. Interestingly, optix is expressed in the developing wings of passion vine butterflies (genus Heliconius). In Heliconius species, optix has been co-opted for red color patterning in wings. However, the ancestral pattern found in basal Heliconiini is in the proximal complex, a region that runs along the base of the forewing costa, the most anterior region of the forewing. This similarity between optix expression patterns in forewings of Diptera and Lepidoptera leads to the hypothesis that an ancestral role of optix might have been 'structural', being required for the development of the anterior wing. Once expressed in the wings, recruitment of red pigmentation genes allowed optix co-option for color pattern diversification through regulatory evolution. It is noted that a pre-requisite for this co-option in wing pigmentation patterning must have been that optix would not interfere with the developmental pathway leading to the formation of a normal wing in the first place. The fact that the effects of optix overexpression throughout the wing primordium in Drosophila are restricted to the anterior/proximal wing -its normal expression domain- indicates that optix cannot engage in promiscuous gene regulation, and that its function depends on other competence factors, which would limit its gene expression regulatory potential (Al Khatib, 2017).


REGULATION

Transcriptional Regulation

The discovery of direct downstream targets of transcription factors (TFs) is necessary for understanding the genetic mechanisms underlying complex, highly regulated processes such as development. In this report, a combinatorial strategy was used to conduct a genome-wide search for novel direct targets of Eyeless (Ey), a key transcription factor controlling early eye development in Drosophila. To overcome the lack of high-quality consensus binding site sequences, phylogenetic shadowing of known Ey binding sites in sine oculis (so) was used to construct a position weight matrix (PWM) of the Ey protein. This PWM was then used for in silico prediction of potential binding sites in the Drosophila melanogaster genome. To reduce the false positive rate, conservation of these potential binding sites was assessed by comparing the genomic sequences from seven Drosophila species. In parallel, microarray analysis of wild-type versus ectopic ey-expressing tissue, followed by microarray-based epistasis experiments in an atonal (ato) mutant background, identified 188 genes induced by ey. Intersection of in silico predicted conserved Ey binding sites with the candidate gene list produced through expression profiling yielded a list of 20 putative ey-induced, eye-enriched, ato-independent, direct targets of Ey. The accuracy of this list of genes was confirmed using both in vitro and in vivo methods. Initial analysis reveals three genes, eyes absent, shifted, and Optix, as novel direct targets of Ey. These results suggest that the integrated strategy of computational biology, genomics, and genetics is a powerful approach to identify direct downstream targets for any transcription factor genome-wide (Ostrin, 2006).

The cis-regulatory code of Hox function in Drosophila

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, binding regions were identified for the Hox TF Deformed (Dfd) in the Drosophila genome. This analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. It was also found that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, these results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo (Sorge, 2012).

In order to quantitatively identify genomic regions bound by the Hox TF Dfd in Drosophila, two complementing approaches were employed: ChIP-seq, which has been successfully applied previously to identify stage- and tissue-specific enhancer activities, and computational detection of clusters of TF binding sequences, which allows the identification of cis-regulatory modules irrespective of temporal and spatial context. To generate genome-wide maps of Dfd binding in vivo, ChIP was performed using stage 10-12 Drosophila embryos and a Dfd-specific antibod. Stage-independent in silico Dfd-specific Hox response elements (HREs) were identified by searching for clusters of conserved Dfd binding motifs, as defined by a position weight matrix (PWM), in the non-coding regions of the genomes of 12 distinct Drosophila species. By applying both approaches, 4526 genomic regions containing clusters of Dfd binding sites and 1079 Dfd ChIP-seq enrichment peaks were identified, including two out of the three well-characterized Dfd-HREs, namely rpr-4S3 and Dfd-EAE. To study the regulatory capacity of novel in silico and ChIP-seq detected HREs, cell culture-based enhancer assays were performed for 11 randomly selected HREs, and it was found that reporter expression driven by the identified genomic regions was in all cases dependent on Dfd binding. In vivo activity was tested of 21 arbitrarily selected enhancers in transgenic reporter lines, revealing that 7 out of 11 ChIP-identified and 5 out of 10 in silico-predicted Dfd-HREs recapitulate the spatio-temporal expression of adjacent genes). Most importantly, it was possible to demonstrate Dfd-dependent regulation of both transgenic reporter expression and endogenous gene expression, suggesting that they are bona fide direct Dfd target genes. Thus, the identified Dfd-HREs represent a data set of biologically relevant regulatory regions and an excellent resource to unravel sequence features within Hox responsive enhancers that might be essential for the highly selective Hox target gene regulation (Sorge, 2012).

Transcriptional regulation in many cases relies on the assembly of regulatory protein complexes mediated by closely spaced TF binding sites within a cis-regulatory module and previous studies have shown that Hox proteins employ this mechanism to control target gene activity in small subsets of cells. The novel HREs were systematically scanned for TF binding motifs appearing in close proximity to Dfd binding sites. Using a statistical test for pair-wise distance distributions, w11 overrepresented DNA motifs for known TFs were found adjoining to Dfd binding sites with 5 of the motifs occurring in both the ChIP-seq and in silico-identified Dfd-HREs. When the expression patterns of six of these transcriptional regulators known to bind to the 11 motifs that were identified were examined, colocalization with Dfd was found in different sub-populations of cells in all cases. Colocalization was already known for two TFs, whose binding sites were coupled to Dfd motifs, including Extradenticle (Exd) , which is known to cooperatively bind with Hox proteins to DNA and thereby increase Hox DNA-binding selectivity. It was next asked whether the short-distance arrangements in Dfd-HREs are of biological relevance and translated into the regulation of similar classes of target genes. To this end, the overrepresentation was statistically tested of expression and biological terms of genes associated with HREs harbouring specific combinations of Dfd and close-by motifs. This analysis revealed that only those Dfd-HREs with short distance intervals between the Dfd and adjacent motifs were coupled to similar gene classes, while random distance intervals did not show any correlation. Strikingly, genes associated with specific short-distance HREs had similar expression and functional annotations as the TFs interacting with the Hox adjoining motifs, suggesting that time and place of Hox action is dictated by spatio-temporally restricted co-regulators. Support for this hypothesis stems from the observation that one of the close-distance partners, Optix, regulates similar processes as Dfd, since Dfd and Optix mutants displayed comparable morphological defects in the head region, such as the absence of mouth hooks, a maxillary segment-derived structure known to be specified by Dfd. In addition, one of the genes associated with a Dfd-Optix HRE, the known Dfd target gene reaper (rpr), is expressed in the ventral epidermis primordium as predicted by its HRE architecture, and regulated by Dfd and Optix in ventral-maxillary cells, which also express these factors. A cell-culture assay using the well-established Dfd responsive module responsible for rpr expression in a few anterior-maxillary cells, the rpr-4S3 Dfd-HRE, with wild-type or mutated Dfd binding sites or reduction of Dfd levels by RNAi confirmed the requirement for simultaneous activity of Dfd and Optix on the rpr-4S3 Dfd-HRE for strong reporter gene induction. Optix binding to the rpr-4S3 Dfd-HRE was additionally confirmed by electrophoretic mobility shift assay (EMSA) experiments. Furthermore, transgenic reporter expression induced by the rpr-4S3 Dfd-HRE was lost in Optix mutant embryos or when the Optix binding sites were mutated. These results demonstrate that Optix, one of the newly identified factors, is a Dfd co-regulator required for proper regulation of the important Hox target gene rpr (Sorge, 2012).

Whether The precise spacing between Hox and adjacent binding sites plays a role for enhancer activity was explored. The rpr-4S3 Dfd HRE, which induces gene expression in a few anterior-maxillary cells, has previously been shown to be under the control of Dfd and Glial cells missing (Gcm), a Dfd co-regulator also identified in this study. Dfd and Gcm as well as Optix binding sites within the rpr-4S3 HRE are directly adjacent to each other, thus a 5- and 10-bp spacer was introduced to interfere with potential interactions of the proteins on the enhancer. In all cases, reporter gene expression was strongly reduced or completely abolished, showing that the close-distance arrangements between Dfd and Gcm as well as Dfd and Optix are required for the in vivo activity of the rpr-4S3 enhancer (Sorge, 2012).

While the results regarding the close-distance arrangement of Dfd and Gcm binding sites suggested the formation of a Dfd-Gcm protein complex, like in the case of Dfd and Exd, only independent binding of the two proteins to the rpr-4S3 enhancer was observed in EMSA experiments , supporting the idea of Hox proteins collaborating with other TFs on target HREs in the absence of physical contact. It has been shown before that Hox proteins together with other TFs that bind in the immediate vicinity recruit non-DNA binding cofactors to HREs. To test if such factors could interact with Dfd and the newly identified short distance binding TFs, the modENCODE data set was scanned and it was found that dCBP/Nej, a member of the CBP/p300 family of transcriptional co-activators bearing acetyltransferase activity, binds to the rpr-4S3 enhancer in vivo. As nej has been previously reported to genetically interact with Dfd, its function was examined in Dfd/Gcm-mediated transcriptional activation. Both factors, Dfd and Gcm, are required for transcriptional activation, since expression of Gcm in Drosophila D.Mel-2 cells, which have basal levels of Dfd activity, resulted in strong induction of reporter gene expression, while abolishing Dfd binding to the rpr-4S3 HRE by mutating all Dfd binding sites or by reducing Dfd protein levels in D.Mel-2 cells using RNAi, strongly reduced reporter gene expression in the presence of Gcm. Strikingly, Dfd- and Gcm-mediated reporter gene expression was strongly reduced in nej dsRNA-treated cells, whereas inhibition of protein deacetylation by Trichostatin A (TSA0) restored reporter gene expression. Consistently, rpr expression was abolished in nej mutant embryos. These results demonstrate that dCBP/Nej-mediated protein acetylation/histone modification is important for the combined activity of Dfd and Gcm on the rpr-4S3 HRE. While it was not possible to demonstrate that nej physically interacts with Dfd protein using various assays, EMSA experiments show that nej interacts with Gcm. Furthermore, acetylation of transiently transfected Gcm was detected in cultured Drosophila cells. Acetylation of Gcm is dependent on Nej, as it was reduced upon RNAi-mediated downregulation of nej. These results are consistent with published work demonstrating that in human cells CBP interacts with Gcma, resulting in its acetylation and stimulation of its transcriptional activity. Since about 10% of all Dfd and nej in vivo genomic binding events during embryonic stages 10-12 overlap, the functional interaction of Dfd and nej observed at the rpr locus does not seem an exception. This finding suggests that the interaction of co-activators (and co-repressors) with Hox proteins and close distance binding TFs on enhancer modules could be a commonly used mechanism to achieve highly specific spatio-temporal control of target gene activity. In this scenario, Hox proteins would control downstream genes by direct transcriptional and/or epigenetic regulation depending on HRE composition and thus cofactor identity and recruitment (Sorge, 2012)

Despite very similar DNA binding behaviour in vitro, Hox proteins regulate distinct morphological features along the anterior-posterior body axis in animal systems. To elucidate the mechanistic basis for the differences in their regulatory properties, Dfd-HREs identified in this study were compared to genomic regions bound by the Hox TF Ultrabithorax (Ubx) at identical developmental stages, as identified by the modENCODE consortium. Searching for overrepresented DNA motifs in both enriched ChIP regions, it was found that Dfd and Ubx bind to identical DNA sequences in vivo, reminiscent to in vitro systems. However, individual binding motifs seem to play only a minor role for Hox binding site selection in vivo, since this analysis revealed that Dfd and Ubx exclusively interact with non-overlapping genomic regions in embryonic stages 9-12. Consequently, Dfd- and Ubx-HREs were found to be associated with distinct classes of genes, revealing that genes with roles in the epidermis are primarily under the control of Dfd at the analysed embryonic stages while genes with mesoderm-related functions are predominantly regulated by Ubx. Consistently, it was found that the expression of tartan (trn), one of the genes associated with a Dfd-HRE, is regulated exclusively by Dfd, but not by Ubx, in epidermal cells, while parcas (pcs), one of the genes linked to a Ubx-HRE is under the selective control of Ubx in mesodermal cells. Furthermore, only Ubx-HREs were found to substantially overlap with cis-regulatory elements stage specifically bound by the mesoderm-specifying TFs Myocyte enhancer factor 2 (Mef2), Twist and Tinman. In contrast, the common ability of both Dfd and Ubx to regulate genes involved in nervous system development was underlined by comparable representations of binding motifs for the neuronal-specifying TFs Asense, Deadpan and Snail in Dfd- and Ubx-HREs (Sorge, 2012).

Strikingly, the basic design principles of Dfd- and Ubx-HREs were found to be similar: like in Dfd-HREs, six binding motifs for known TFs were located adjacent to Ubx binding sites and colocalization studies showed that they are expressed in subsets of Ubx-positive cells. Again, Ubx binding sites and motifs for potential co-regulators occurred most frequently in specific short intervals and only those Ubx-HREs with the preferred distance were associated with specific gene classes. This analysis also revealed that four of the six short-distance motifs were specific for Ubx-HREs, which is consistent with the data showing that Hox proteins interact with different and spatially restricted co-regulators to control target gene expression in selected cells. Importantly, in the cases of the close-distance motifs detected in both HREs, namely the binding sites for the TFs Ladybird early (Lbe) and Cut (Ct), the associated target genes were also expressed in non-overlapping tissues. This raised the question of how different Hox proteins can act on distinct target genes, even when their target HREs exhibit similar binding site compositions including short-distance arrangements. Since Lbe is active in both mesodermal and epidermal cells, one Dfd-Lbe and one Ubx-Lbe HRE was exemplarily analysed, and binding of Lbe protein was confirmed to both HREs by EMSAs. As predicted by the presence of Lbe binding sequences. Complex formation between the Hox protein and Lbe was observed in the case of Ubx and Lbe while Dfd and Lbe interact independently with the Dfd-Lbe HRE, indicating that the two Hox proteins employ different mechanisms for binding to the selected HREs. Lbe interaction with the Dfd-Lbe and Ubx-Lbe HREs is essential for in vivo activity, since in both cases ectopic reporter gene expression was observed when Lbe binding sites were mutated. Even more important, reporter gene expression was specifically changed only in segments in which either Dfd or Ubx is active, meaning in the case of the Dfd-Lbe HRE in maxillary cells and in the case of the Ubx-Lbe HRE in abdominal segments A1-A7. Taken together, these results demonstrate that the combined activity of Lbe and the Hox proteins Dfd or Ubx on selected HREs is critical for the precise spatiotemporal and segment-specific control of HRE activity. It was next asked whether additional (DNA- and non-DNA-binding) factors contribute to the predicted cell type-specific expression of the Dfd-Lbe and Ubx-Lbe HREs. Using the Drosophila Interactions Database (DroID; Murali, 2011) and published genome-wide DNA binding studies a search was carried out for unique Dfd-lbe and Ubx-lbe interactors. It was discovered that almost 20% of all Ubx-Lbe HREs but none of the Dfd-Lbe HREs were found to interact with the mesoderm-specifying factor Mef2 in vivo, while H3K9me3 histone marks, which are mediated by one of the unique Dfd-lbe interactors, Enhancer of zeste E(z), are enriched only within Dfd-Lbe HREs. Interestingly, E(z) modifies chromatin also by trimethylating H3K27 residues, a histone mark highly enriched at the genomic region spanning the ChIP-detected Dfd-Lbe HRE. Consistent with the repressive function of this histone modification, loss of Lbe binding to the Dfd-Lbe HRE results in ectopic reporter gene expression, suggesting that Lbe (and Dfd) recruits E(z) to the Dfd-Lbe HRE for cell type-specific target gene repression (Sorge, 2012).

Taken together, these results demonstrate that Hox proteins interact with different regulatory proteins on HREs, which allows them to differentially regulate their target genes despite their similar DNA binding properties. The fact that these interactions occur only in a few cells for a short period of time is very likely one of the major reasons why the identification of factors conferring regulatory precision and specificity to Hox function has met with little success so far (Sorge, 2012).

This study, has identified crucial features of HREs, which are essential for cell type-specific regulation of Hox target genes in vivo. In addition to motif composition the exact spatial arrangement of TF binding elements is critical to translate Dfd function into transcriptional regulation in vivo. These architectural features of Dfd-HREs alone accurately predict target gene function and expression patterns. Furthermore, it was found that epigenetic regulators bind to HREs on a genome-wide scale, suggesting that they generally collaborate with Hox proteins to achieve stable target gene regulation. This is in line with recent findings showing that chromatin modifications at enhancers strongly correlate with functional enhancer activity and tissue specificity. By comparing HREs regulated by Dfd and Ubx, two different Hox proteins with different embryonic regulatory specificities, this study shows that while similar design principles apply, specificity is encoded by distinct sets of co-occurring DNA motifs. Due to the highly dynamic regulatory output of Hox TFs in space and time, cell type-specific approaches are required in future to elucidate all relevant aspects of Hox-chromatin and Hox-cofactor interactions (Sorge, 2012).

Protein Interactions

Fly SIX-type homeodomain proteins Sine oculis and Optix partner with different cofactors during eye development

Two members from the Six class of homeobox transcription factors, Sine oculis (SO) and Optix, function during development of the fly visual system. Differences in gain-of-function phenotypes and gene expression suggest that these related factors play distinct roles in the formation of the fly eye. However, the molecular nature of their functional differences remains unclear. This study reports the identification of two novel factors that participate in specific partnerships with Sine oculis and Optix during photoreceptor neurons formation and in eye progenitor cells. This work shows that different cofactors likely mediate unique functions of Sine oculis and Optix during the development of the fly eye and that the repeated requirement for SO function at multiple stages of eye development reflects the activity of different SO-cofactor complexes (Kenyon, 2005).

The fly SIX-HD transcription factors SO and Optix function during eye development and are expressed in different but overlapping patterns within the eye epithelium. Targeted expression of SO or Optix results in distinct phenotypes. Optix is able to induce eye development when ectopically expressed in the antenna and blocks eye morphogenesis when overexpressed within the eye epithelium. On the contrary, SO does not induce ectopic eye formation nor does it block neuronal differentiation within eye tissue. These observations strongly suggest that SO and Optix fulfill different roles during eye development. The identification of multiple unique partners for these Six factors clearly supports a model in which differences in cofactor recruitment contribute significantly to their specific function. This paper describes the identification of two novel potential partners of SO and Optix: one that is SO-specific by virtue of its expression pattern, and another likely to be Optix-specific by virtue of its protein binding profile (Kenyon, 2005).

The Sbp gene (Flybase ID FBgn0033654) encodes a novel factor of unknown function. It does, however, contain three highly conserved motifs and a proline-rich region. The high level of similarity from fly to vertebrates suggests that these sequences represent novel protein motifs or domains with specific functions. Since they do not appear related to previously characterized domains, little can be concluded about their potential function. However, Box 1 is not required for binding to the SD of SO, because the clone isolated through the yeast two-hybrid screen does not include this sequence. Its interaction with SO suggests that Sbp may function as a transcriptional regulator. The presence of a proline-rich region supports this hypothesis because proline-rich domains have been implicated in both transcriptional activation and repression. In a manner similar to Gro and unlike Eya, Sbp can interact in yeast with both SO and Optix. However, Sbp is specifically coexpressed with SO and not Optix within the eye epithelium. Hence, its interaction in vivo is restricted to SO by virtue of its expression posterior to the MF. This hypothesis is consistent with the observation that ectopic expression of Sbp in progenitor cells, where Optix is also expressed, interferes with normal eye development. However, alternative explanations are also possible. The effect of Sbp misexpression may be due to its abnormal association with SO itself, or other nuclear factors, in progenitor cells. Similarly, the disruption of eye development due to the misexpression of Optix in the developing neuronal field may result from abnormal interactions with other yet unidentified factors. Nonetheless, restricted expression of Sbp posterior to the MF and Optix anterior to the MF is obviously critical for normal eye development and effectively excludes binding between Optix and Sbp (Kenyon, 2005).

Sbp expression within the developing neuronal array, and not in progenitor cells, indicates that this gene is not required at an early stage but functions at the time of neuronal differentiation. On the contrary, SO function is required at multiple stages of eye development, including during specification of eye progenitor cells, at the time of patterning (in the MF) and during neuronal development. The Eya cofactor has also been implicated in these steps, and both proteins are continuously expressed in eye tissue through these developmental stages. These studies do not provide clues regarding the function of SO at each developmental stage. Based on these data, SO could function simply by driving expression of a few genes required at all times to maintain eye identity. The identification of an SO partner specifically expressed in the differentiating epithelium contradicts this hypothesis and shows that there are significant differences in at least some of the SO-based complexes that function at distinct developmental stages. Thus, posterior to the MF, Sbp may associate with the SO-Eya complex and modify its activity turning it from an activator into a repressor. Evidence of complex interactions between the mouse Six1, Dac, and Eya proteins has been described. Alternatively, Sbp may form a distinct SO-Sbp complex. In either case, however, SO function at various times during eye development would be modulated by its interaction with specific partners (Kenyon, 2005).

The Obp gene (Flybase ID FBgn0050443) encodes a protein characterized by nine putative zinc-finger domains of the C2H2 type. Although most members of this family are thought to regulate transcription through DNA binding, recent reports have also implicated C2H2-type zinc-fingers in protein-protein interactions. Because zinc-fingers 4 through 9 as well as the C-terminal sequence of Obp are present in the fragment isolated in the screen, it cannot be excluded that one or more of these zinc-finger domains mediate the interaction with the Optix transcription factor. Obp is coexpressed with both SO and Optix in eye progenitor cells. However, it behaves as an Optix-specific interactor in yeast two-hybrid tests and does not impair eye development when ectopically expressed in the differentiating neuronal field (where only SO is present). Hence, this factor is likely to be a specific partner of Optix in vivo (Kenyon, 2005).

This work has not identified clear homologues of this factor. This finding is not entirely unexpected, because zinc-finger domains display only low-level conservation at positions other than the C and H residues that bind zinc. In the related proteins from D. pseudoobscura and A. mellifera, conservation is restricted to the region encoding zinc-fingers 4 through 8. Hence, additional criteria (such as expression in eye progenitor cells, interaction with Optix, and/or mutant rescue) are required to assess whether they are indeed homologues. Obp is reported in Flybase to encode a product putatively involved in cell proliferation. In fact, the fourth and sizth zinc-fingers show similarity to zinc-fingers present in the Sfp1 transcription factor. In yeast, Sfp1 functions in cellular growth and proliferation by regulating ribosomal proteins expression. Obp expression, specifically anterior to the MF where undifferentiated progenitors proliferate and then arrest just before the start of eye morphogenesis, is consistent with a potential role in regulating cell proliferation. However, the alignment between Obp and Sfp1 is limited to less than 20% of what is currently defined as the Sfp1 domain. Given the limited conservation and the lack of functional data, a role for Obp in proliferation control is speculative at this time (Kenyon, 2005).


DEVELOPMENTAL BIOLOGY

Embryonic

The expression pattern of Optix during embryonic and larval stages was examined by whole-mount in situ hybridization and compared to the pattern of sine oculis expression. Optix is first expressed in a ring at the anterior end of the blastoderm embryo. This expression is similar to so expression, but lies more anteriorly. During germ band extension, Optix is restricted to the anterior and in contrast to so, is not expressed in the optic lobe primordia. At stage 11, Optix expression is detected in the clypeolabrum and remains limited to the anterior region, whereas so expression is also detected bilaterally at the segmental boundaries in a set of unidentified epidermal cells, and in the optic lobe primordium. At stage 14 Optix is expressed in the ectoderm covering the supraesophageal ganglion, which will give rise to parts of the brain, but Optix is not expressed in Bolwig's organ (Seimiya, 2000).

The origin of islet-like cells in Drosophila identifies parallels to the vertebrate endocrine axis

Single-cell resolution lineage information is a critical key to understanding how the states of gene regulatory networks respond to cell interactions and thereby establish distinct cell fates. This study identified a single pair of neural stem cells (neuroblasts) as progenitors of the brain insulin-producing neurosecretory cells of Drosophila, which are homologous to islet β cells. Likewise, a second pair of neuroblasts was identified as progenitors of the neurosecretory Corpora cardiaca cells, which are homologous to the glucagon-secreting islet α cells. Both progenitors originate as neighboring cells from anterior neuroectoderm, which expresses genes orthologous to those expressed in the vertebrate adenohypophyseal placode, the source of endocrine anterior pituitary and neurosecretory hypothalamic cells. This ontogenic-molecular concordance suggests that a rudimentary brain endocrine axis was present in the common ancestor of humans and flies, where it orchestrated the islet-like endocrine functions of insulin and glucagon biology (Wang, 2007).

The principal insulin producing-cells (IPCs) in higher metazoans, such as flies and mammals, direct organismal growth, metabolism, aging, and reproduction via a conserved signal transduction pathway. Gut- or pancreas-based IPCs, with endodermal origin, emerged as the principal IPC locus with the evolution of lower vertebrates such as the jawless fish. In contrast, the principal IPCs of invertebrates are found in the nervous system and are likely of ectodermal origin. Despite this difference, the possibility that gene regulatory modules may be conserved for cell fate programming the principal IPCs of all higher animals, irrespective of germ layer origin, has led the development of islet-like cells to be addressed in Drosophila (Wang, 2007).

Brain IPCs in Drosophila were first recognized by their expression of insulin (Drosophila insulin-like peptide, Dilp2) at the end of embryonic development. The goal of this work was to understand the developmental origin of these cells. The absence of morphological and vital markers for identifying brain neuroblasts for dye-labeled lineage tracing necessitated the combined use of mosaic analysis to demonstrate lineage relationships and immunohistology to follow cell identities. In this study, 16 molecular lineage markers corresponding to conserved genes were used to follow cells in fixed embryos. To identify genes involved in early IPC lineage development, before the differentiation of IPCs, 650 transposable GAL4-transgene insertions, obtained from public collections, that reported gene enhancer activity (GAL4 enhancer traps) in the CNS, were screened. Enhancer-driven GAL4 activity was used to trigger heritable and irreversible lineage labeling, which was assayed for coexpression with Dilp2 in late larval brains, thereby identifying lineage markers and potential developmental determinants. It was found that enhancers near the genes dachshund (dac), eyeless (ey), optix, and tiptop (tio) each triggered IPC lineage labeling by the time of Dilp2 expression onset just before hatching (late-stage 17). tio enhancer-triggered labeling was highly specific to the IPCs within the pars intercerebrallis (PI), the dorsomedial brain region harboring the IPCs and other neurosecretory cells. Antibody staining of Dac, Ey, and Optix proteins recapitulated enhancer reporter labeling and revealed expression in the tio+ cell cluster in late-stage embryos just after IPC differentiation, and before IPC differentiation at early-stage 17. Thus, a bilateral cluster of 10-12 Dac+ Ey+ cells were identified, 6-8 of which expressed tio before continuing on to express insulin (Dilp2) slightly later in development (Wang, 2007).

The hypothesis was tested that the Dac+ Ey+ cluster is generated by the proliferation of a single neuroblast. The pre-Dilp2 Dac+ Ey+ cluster comprised 10-12 cells at stage 17, but only a single Dac+ cell at stage 12, suggesting that a lineage expanded from a single progenitor beginning at stage 12. The Dac+ cluster maintains a posterior and lateral position within the anterior PI, identified by dChx1 expression, which allows following it during the morphogenetic changes in the developing brain. To mark progenitors and their lineage descendants, stage 11-12 embryos harboring both a heat-shock promoter-flip recombinase (hsp70-flp) transgene and an FRT-mediated flip-out Actin promoter-LacZ reporter were heat-shocked to induce random clone marking events in cell lineages. After aging embryos for 6 h at 25°C to reach stage 16-17, marked clusters of clonally related cells were occasionally recovered that comprised the 10-12 cell Dac+ Ey+ cluster. Clones that partly labeled the Dac+ Ey+ cluster, which were posterior in the cluster, were interpreted as being labeled by a lineage marking event induced after the neuroblast had divided one or more times. It was unlikely that multiple marking events accounted for the apparent clonal labeling of IPCs because the frequency of marked clone induction was extremely low (tens per brain). Clones were also found that labeled neighboring cells, but do not label Dac+ Ey+ cells, suggesting there is a lineage restriction that defined the Dac+ Ey+ cluster. Thus, all data are consistent with a lineage model whereby one neuroblast produced 10-12 Dac+ Ey+ cells, 6-8 of which were IPCs (Wang, 2007).

Whether the single Dac+ cell progenitor of IPCs seen at stage 12 was indeed a neuroblast was further tested by using markers of neuroblast lineage development. Asymmetrically dividing neuroblasts can be identified by nuclear expression of the pan-neuroblast marker Deadpan (Dpn) and Prospero (Pros) localization to the plasma membrane. It was found that the single Dac+ cell expressed Dpn and also showed Pros localization at the plasma membrane, which indicated that it was a neuroblast. As the Dac+ cluster increased in cell number with age, it was found that Pros was present in the nucleus of Dac+ cells anterior to the Dac+ neuroblast, which indicated that these were the neuroblast daughter cells, or ganglion mother cells (GMCs) generated by asymmetric neuroblast divisions. By stage 14, the most anterior Dac+ cells in the cluster lacked Dpn and Pros, suggesting that they were early, undifferentiated neurons or neurosecretory cells generated by GMC cell divisions. It was also found that tio expression occurs in the most anterior Dac+ cells of the lineage group, furthest from the posterior-located Dac+ neuroblast, suggesting that the six to eight IPCs are the products of the first three to four GMCs to be generated by asymmetric neuroblast division. This observation confirmed the interpretation of the marked clone data that showed partial labeling by a clone occupies the posterior, more recently formed region of the Dac+ Ey+ cluster, near the IPC neuroblast. Thus, a histological pattern of cell identities and divisions within the Dac+ IPC lineage group was observed that was consistent with the generic lineage development of a single neuroblast, with the IPCs being produced from the first three to four GMCs formed (Wang, 2007).

Further attempts were made to identify the precise origin of the IPC neuroblast within the neuroectoderm epithelium and the blastoderm embryo to place this lineage in the context of early axial patterning. The IPC neuroblast was first recognized by Dac expression only after neuroblast formation, but before its first division. However, preceding the formation of the IPC neuroblast, the markers Castor (Cas) and dChx1 and the proneural factor Lethal of Scute (L'Sc) showed coexpression in eight nearby cells of the neuroectoderm epithelium. Cas and dChx1 were maintained in all neuroblast lineages that delaminated from this group, as indicated by coexpression of Dpn. The IPC neuroblast was the only neuroblast from this group to express Dac, and it was always the first Dpn+ neuroblast to delaminate, becoming the most posterior in a chain of delaminating Cas+ dChx1+ neuroblasts. The Cas+ dChx1+ L'Sc+ proneural group lies within a 'gap gene' head stripe corresponding to the Bicoid responsive giant head stripe 1 (gt1), which suggested that the IPC neuroblast, or its earliest progenitor, arose from this pattern element of the precellular blastoderm (Wang, 2007).

β Cell and α cell development in mammals shares a largely common pathway. Thus attempts were made to study the origin of the α-like cells in Drosophila and their development relative to the IPC lineage. Corpora cardiaca (CC) cells are analogous in function to islet α cells. These neuroendocrine cells reside in the endocrine ring gland, just dorsal to the brain. CC cells produce and secrete a glucagon-like peptide, adipokinetic hormone, in response to circulating glucose levels, via a conserved Katp sensor. The gene glass (gl) is a marker of CC cells and their precursors that specifically labels the CC lineage beginning at stage 10. The Gl+ group of cells expands in number to form a bilateral pair of six to eight cell clusters, aligned at the border of the brain and the developing foregut (stage 13). The Gl+ clusters then migrated out of the protocerebrum (stage 14), and posterior along the roof of the pharynx, to ultimately coalesce at the midline within the prospective ring gland (stage 16). Remarkably, the first Gl+ cells appeared a single cell diameter apart from the dChx1+ cluster containing the IPC neuroblast, also within the gt1 stripe (Wang, 2007).

These results suggested that the CC cell lineage, like the IPC lineage, is also generated from a progenitor within the gt1+ dorsal neuroectoderm. Indeed, a neuroblast progenitor for CC cells was suggested by expression of a Kruppel reporter (Kr-GFP) found to specifically label the Gl+ cells and an adjacent cell that both was Dpn+ and showed membrane localized Pros, indicating that it was a neuroblast. As for IPCs, tests were made to see if CC cells are derived from a single progenitor, perhaps the Kr-GFP+ neuroblast. Gl+ β-gal+-marked clones were recovered that comprised all or part of a CC cell cluster, after their migration to the prospective ring gland at stage 16. Because labeled CC cells had moved from their point of origin in the developing PI, it could not be determine whether a progenitor also produced other cells besides the CC cells, which did not similarly migrate. Together, these observations suggest that the CC cells are related by lineage to a neuroblast progenitor (Wang, 2007).

Typically, neuroblasts inherit the expression of cell specification factors from their point of origin in the patterned neuroectoderm before the neuroblast forms. It was found that this was the case with the IPC neuroblast, which retains dChx1 and Cas expression from the neuroectoderm. It was therefore hypothesized that this may also be the case for the CC cell neuroblast. CC cell specification was shown to require the function of gt, sine oculis (so), twist (twi), and snail (sna). Indeed, it was found that all of these factors are expressed in the Gl+ CC cell lineage. Moreover, the Kr-GFP+ cell group, containing the neuroblast and CC cell precursors, also expressed Eyes absent (Eya), the cognate protein tyrosine phosphatase of So. It was subsequently found that at stage 10, the time that Gl+ cells are first detected, a region of gt1+ neurectoderm shows expression of So. It was also found that one to two So+ gt1+ neuroblasts can be detected by labeling with Dpn at this stage. Thus, it is proposed that the So+ Eya+ gt1+ neuroectoderm gives rise to the Kr-GFP+ So+ Eya+ gt1+ neuroblast, which is the single progenitor of the CC cells (Wang, 2007).

The model of a dorsal neurectoderm origin for CC cells is in disagreement with another extant model. The anterior ventral furrow (AVF) epithelium was suggested to be the CC cell origin based on gene expression and function studies implicating So, Gt, Twi, and Sna in CC cell formation. To distinguish between the AVF and dorsal neuroectoderm as possible origins of CC cells, two newly available gt promoter fragment reporters were used whose expression persists late enough in development, beyond endogenous protein and transcript expression, to serve as a coarse-grain lineage marker of CC cells. The AVF is marked by the gt23 reporter, whose expression is limited to the two gt head stripes posterior to gt1 at the blastoderm stage. This reporter does not label the Gl+ cells. However, as has been shown, the Gl+ cells arise in the context of the most anterior gt head stripe, gt1, which reaffirms the proposed origin from the gt1+ neuroectoderm (Wang, 2007).

The organization of this gt1+ segment-derived proendocrine neuroectoderm was investigated with respect to the conserved factors Optix, So, Eya, and dChx1. Optix and Eya expression aligned with the gt1 reporter expression domain. The D-six4 gene also shows expression specific to this domain. Labeling studies showed that this domain is subdivided into several small compartments of 2-12 cells with discrete gene expression profiles. The data indicate that the IPC neuroblast was derived from compartment B (Optix+, dChx1+, Cas+, So-, low-level Eya) and the CC cell neuroblast arose from the adjacent compartment C (Optix+, So+, Eya+, dChx1-). This somewhat surprising finding suggests that the largely common developmental pathway of β and α cells may be partly conserved in Drosophila, perhaps with respect to a domain of Sine oculis/Six family and Eya gene expression (Wang, 2007).

The early expression of the mouse ortholog of the Drosophila homeodomain gene optix, Six6, demarcates the hypophyseal placode and infundibular region, which give rise to the anterior pituitary and neurosecretory hypothalamus, respectively. Mutation of the Six6 gene leads to reduction of the pituitary in mice and humans. The hypophyseal placode and adjacent ectoderm also expresses the other so-called 'placode genes,' Six1, Six4, and Eya, and this coexpression pattern is conserved in amphibians, fish, and lower chordates such as ascidians. In mice, the anterior pituitary is reduced in size in the double mutant of Eya1 and Six1, and in zebrafish, Eya1 is essential for differentiation of all pituitary cell types except for prolactin-expressing cells. In Drosophila, So and Eya are essential for CC cell formation. Thus, there is a striking conservation of the molecular signature of tissues that give rise to elements of the brain endocrine axis in flies, mammals, lower vertebrates, and lower chordates (Wang, 2007).

There are also parallels between vertebrate and fly with respect to tissue morphogenesis within the developing brain endocrine system and adjacent oral ectoderm, although there appears to be considerable variation on a general theme. For example, in mouse, the progenitors of the anterior pituitary and neurosecretory hypothalamus appear to arise respectively from Rathke's pouch, an invagination of the oral ectoderm, and the neurectoderm, which do not start as neighboring regions, but come into direct contact only after neurulation. However, in the zebrafish, which does not form a Rathke's pouch, the progenitors of the anterior pituitary and neurosecretory hypothalamic cells (GnRH1+) arise from neighboring regions of the hypohyseal placode, which is situated directly dorsal to the stomodeal ectoderm. In Drosophila, the ventral cells of the gt1+ Optix+ Eya+ ectoderm invaginate to form the roof of the pharynx, the fly's oral ectoderm, whereas the dorsal cells contribute to the endocrine axis. Therefore, there is considerable evidence for evolutionarily conservation of the close relationship between the oral ectoderm and the developing compartments of the endocrine axis, all of which express the hypophyseal placode genes. The gene expression profile and specification of endocrine cell functions from the anterior ectoderm appears to be more 'fixed' across the bilateria, whereas the pattern of accompanying tissue morphogenesis and diversity of cell types is more variable, just as has been demonstrated for the specification of the bilaterian CNS, eye, gut, and heart (Wang, 2007).

The model proposed in this study contrasts with the prior suggestion, based on the proximity of developing CC cells to the posterior foregut in the moth, Manduca, that CC cells originate from neurogenic placodes of the foregut that engender the stomatogastric nervous system. Because CC cell progenitors were not identified in those studies, and subsequent mutational analysis in Drosophila demonstrated that the CC cells develop independently of the stomatogastric nervous system and posterior foregut, it is suggested that the current model of CC cell origin is the most strongly supported (Wang, 2007).

It is proposed that the brain endocrine systems of invertebrates and vertebrates are derived from a common ancestry because they both develop from a domain of Eya and sine oculis/Six family gene expression that comprises the anterior neuroectoderm and adjacent oral ectoderm. Indeed, these results extend prior observations that the neurosecretory cells of the PI and ring gland show other aspects of homology to the hypothalamic-pituitary axis. The specification of islet-like cells within a conserved brain endocrine axis raises the intriguing possibility that islet organogenesis, which is a derived feature of vertebrates, may have coopted brain endocrine cis-regulatory modules for specification of islet fates in endoderm. Indeed, the ectopic expression of the nominal rat insulin promoter reporter in anterior pituitary and hypothalamus underscores the similar gene regulatory state of these endocrine tissues. It is expected that further genetic analysis of endocrine cell fate specification within the gt1 domain of Drosophila will lead to insights into the patterning and organogenesis of endocrine compartments and provide the basis for identifying conserved pan-IPC regulatory modules with relevance to mammalian systems (Wang, 2007).

Larval

In wild-type eye imaginal discs, Optix RNA is detected in front of the morphogenetic furrow (MF), in the head region and just anterior to the vertex region. In late second instar larvae, before the MF forms, Optix expression covers the entire eye disc but later the expression becomes restricted to a region anterior to the MF. This expression pattern is similar to the pattern of eyeless and twin of eyeless (toy) (Quiring, 1994; Czerny, 1999) and it suggests that Optix may play an important role in early eye disc development as do ey and toy. In contrast, so starts to be expressed at early third instar just before MF initiation and later becomes restricted to a zone just anterior, inside and posterior to the MF. In addition, Optix is expressed in wing and haltere discs, but not in leg discs. This expression continues throughout the third instar larval stage, whereas so is not expressed in wing and haltere discs, but in the leg discs (Seimiya, 2000).

Functional genomics identifies neural stem cell sub-type expression profiles and genes regulating neuroblast homeostasis

The Drosophila larval central brain contains about 10,000 differentiated neurons and 200 scattered neural progenitors (neuroblasts), which can be further subdivided into ~95 type I neuroblasts and eight type II neuroblasts per brain lobe. Only type II neuroblasts generate self-renewing intermediate neural progenitors (INPs), and consequently each contributes more neurons to the brain, including much of the central complex. Six different mutant genotypes were characterized that lead to expansion of neuroblast numbers; some preferentially expand type II or type I neuroblasts. Transcriptional profiling of larval brains from these mutant genotypes versus wild-type allowed identification of small clusters of transcripts enriched in type II or type I neuroblast,s, and these clusters were validated by gene expression analysis. Unexpectedly, only a few genes were found to be differentially expressed between type I/II neuroblasts, suggesting that these genes play a large role in establishing the different cell types. A large group of genes predicted to be expressed in all neuroblasts but not in neurons were identified. A neuroblast-specific, RNAi-based functional screen was performed and 84 genes were identified that are required to maintain proper neuroblast numbers; all have conserved mammalian orthologs. These genes are excellent candidates for regulating neural progenitor self-renewal in Drosophila and mammals (Carney, 2012).

To identify genes expressed differentially between type I and type II neuroblasts, genes were sought clustered with pros and ase, the only two genes known to be differentially expressed in type II neuroblasts. It was found that pros and ase reside together in a small sub-cluster of only 11 genes within group B. This sub-cluster as a whole exhibits reduced expression in brat, lgl, and lgl lgd mutants and enrichment in aur, aPKCCAAX, and lgl pins; remarkably, no other sub-cluster exhibits such a pattern. This suggests that the other nine genes in the cluster may also be specifically expressed in type I neuroblasts, like pros and ase, and that these are potentially the only genes that exhibit this unique pattern (Carney, 2011).

To test whether other genes in the small pros/ase cluster are also expressed in type I neuroblasts but not type II neuroblasts, an antibody was obtained to a candidate from this cluster, Retinal homeobox (Rx), a homeodomain-containing transcription factor. It was found that Rx is completely absent from type II neuroblasts, similar to Pros and Ase; Rx is detected in several type I neuroblasts as well as in a subset of differentiated type II progeny. Consistent with this expression pattern, it was found that brat mutants, which overproduce type II neuroblasts, show a loss of Rx staining. In contrast, lgl pins mutants, which have ectopic type I neuroblasts, show territories of strong Rx expression which is confined to Pros+ (likely type I-originating) cells. The fact that only a small patch of lgl pins mutant brain tissue is Rx+ is probably because Rx is normally expressed in a subset of type I neuroblasts. It is concluded that Rx, like Pros and Ase, is expressed in type I but not type II neuroblasts. Thus, most or all of the 11 genes in the pros/ase sub-cluster may be expressed in type I but not type II neuroblasts (Carney, 2011).

Genes expressed in type II neuroblasts but not type I neuroblasts were sought, as there are currently no known markers specifically expressed in type II neuroblasts. It was reasoned that transcripts expressed in type II neuroblasts should be enriched in genotypes that overproduce type II neuroblasts: brat, lgl and lgl lgd. One small cluster was found enriched in two of the three mutants (brat and lgl lgd). This cluster contains just 10 genes, seven encoding transcription factors. To verify the expression pattern of this gene cluster, the expression of one gene product, Optix, was examined. Optix is a conserved homeodomain-containing transcription factor required for eye development. Consistent with the microarray data, it was found that most of the Optix expression in the brain is indeed restricted to type II lineages; four of the six dorso-medial type II neuroblasts (DM1, 2, 3, and 6) express Optix, as do most of the INPs, GMCs, and neurons in these lineages. In addtion, recent work has shown that another gene in this cluster, pointedP1, is also preferentially expressed in type II neuroblasts (Sijun Zhu and Y.N. Jan, personal communication to Carney, 2011). The other two dorso-medial type II lineages (DM4 and 5) exhibit some expression of Optix in a subset of neuronal progeny, but it is absent from the neuroblasts and INPs in these lineages. In addition, a single dorsal type I neuroblast expresses Optix. Inspection of mutant brains further confirmed the type II-biased expression of Optix, in that brat mutant brains exhibit a marked increase in Optix+ neuroblasts, and in lgl pins, the increase in Optix is almost exclusively in a Pros (type II-originating) region of the brain. These results indicate that the clustering relationships can be used to predict type I/type II expression bias with good accuracy. It is concluded that Optix is primarily expressed in type II but not type I neuroblasts, and that Optix and the other five genes in this cluster are excellent candidates for regulators of type II neuroblast identity (Carney, 2011).

It has previously been shown that co-clustering of genes in expression profiling data is likely to reflect physical or genetic interactions and participation in the same pathway. The current results are consistent with these conclusions. For example, a small group of 11 genes was identified containing the only two genes known to be expressed in type I but not type II neuroblasts, and a third gene was shown to have a similar pattern of expression — thus all genes in this cluster are likely to be expressed in type I but not type II neuroblasts. Furthermore, the strong enrichment of GO terms in small sub-clusters within both group A and group C indicates that genes within these clusters are likely to share similar functions or processes (Carney, 2011).

At the outset of this study, a large group of genes was expected to be differentially expressed in type II versus type I neuroblasts, because these neuroblasts have such strikingly different cell lineages. However, only a few gene clusters were identified that were differentially regulated in such a type I/type II consistent manner — the 11 genes in the pros/ase cluster depleted in type II neuroblasts and the 10 genes enriched in type II neuroblasts. This suggests that the small number of genes identified may play a disproportionately large role in generating differences between type I and type II neuroblasts. Might pros and ase be the only genes regulating type I/type II differences? Both Ase and Pros can promote cell cycle exit, which may result in the Ase+ Pros+ type I progeny taking a GMC identity and undergoing just one terminal division and the Ase Pros type II progeny taking an INP identity and continuing to proliferate. Indeed, the misexpression of either Ase or low levels of Pros in type II neuroblasts is sufficient to cause the loss of INPs and/or their premature cell cycle exit, thereby decreasing lineage size toward the size of type I neuroblasts. However, it is unclear what is required to fully transform these cells into type I neuroblasts; addressing this question will require additional molecular markers and tracing the axon projections of the progeny of these 'transformed' neuroblasts (e.g., do they now fail to make intrinsic neurons of the adult central complex?). The fact that mutants in ase and pros do not transform type I neuroblasts into type II neuroblasts indicates that other genes, perhaps some in the pros/ase cluster described here, are also important for specification of type I neuroblast identity (Carney, 2011).

It was found that the neuroblasts in each mutant have remarkably similar expression profiles, as shown by the extensive list of similarly expressed genes in group A and by the list of genes with depleted expression in mutant brains, represented by group C. It is believed that these categories provide lists of genes that are representative of those expressed in neuroblasts and neurons, respectively, based on all known neuroblast-specific genes showing up in group A and all known neuron- or glial-specific genes being excluded from group A (Carney, 2011).

Group B genes apparently are not expressed in all neuroblasts like the group A genes, nor in all neurons or glia like group C genes. However, group B genes are more likely to be expressed in subsets of neurons, not neuroblasts, because group B genes as a whole have an over-representation of GO terms more similar to group C than to group A. Why then are group B genes excluded from group C, the neuron cluster? One possible explanation is that different neuroblast lineages are affected in each mutant, and thus different subsets of neurons are missing in each mutant. If different neuroblast lineages express different genes (which seems likely), then each mutant would be missing a unique subset of neural differentiation genes, leading to the cluster being excluded from group C. This model raises the intriguing possibility that group B sub-clusters may represent lineage-specific genes (Carney, 2011).

It is also possible that the mutant genotypes themselves may cause unique transcriptional differences, leading to a cluster of genes in group B. For example, several small sub-clusters in group B are expressed differently only in aPKCCAAX brains. These transcriptional differences are not correlated with the number of type I or type II neuroblasts. Instead, these genes appear to be differentially expressed in response to elevated aPKC. Drosophila aPKC has been best studied as a component of the apical complex in mitotic neuroblasts, and its capacity for causing ectopic self-renewal has been shown to be reliant on both its catalytic activity and its membrane localizatio. However, aPKC has been ascribed a role in neuroblast proliferation as well as in polarit, and a vertebrate homolog, PKC-zeta, was shown to possess a nuclear role in both proliferation of neural progenitors and neuronal cell fate specification. These observations are consistent with a role of aPKC in causing transcriptional differences (Carney, 2011).

The findings of this study highlight the importance of expression profiling of multiple genotypes. This method gave a more reliable picture of the group A genes expressed in neuroblasts, because genes with lineage-specific or genetic background-specific changes in expression appeared to be focused into group B, where they do not interfere with the clustering of groups A and C. In addition, two small sub-clusters of genes were identified in group B that are excellent candidates for being preferentially expressed in type I or type II neuroblasts, for which there have been few examples to date. Finally, it is concluded that group A genes are likely to be expressed in neuroblasts, and functional studies have identified 84 genes that are conserved in mammals and required for regulating neuroblast numbers in Drosophila. Future phenotypic analysis in Drosophila will determine whether these genes regulate neuroblast survival, quiescence, asymmetric cell division, and/or self-renewal. Future studies on the expression and function of orthologous genes in mouse neural progenitors and human stem cells (IP or neural) will reveal whether they have conserved roles from flies to mammals (Carney, 2011).

Optix defines a neuroepithelial compartment in the optic lobe of the Drosophila brain

During early brain development, the organisation of neural progenitors into a neuroepithelial sheet maintains tissue integrity during growth. Neuroepithelial cohesion and patterning is essential for orderly proliferation and neural fate specification. Neuroepithelia are regionalised by the expression of transcription factors and signalling molecules, resulting in the formation of distinct developmental, and ultimately functional, domains. This study discovered that the Six3/6 family orthologue Optix is an essential regulator of neuroepithelial maintenance and patterning in the Drosophila brain. Six3 and Six6 are required for mammalian eye and forebrain development, and mutations in humans are associated with severe eye and brain malformation. In Drosophila, Optix is expressed in a sharply defined region of the larval optic lobe, and its expression is reciprocal to that of the transcription factor Vsx1. Optix gain- and loss-of-function affects neuroepithelial adhesion, integrity and polarity. Restricted cell lineage boundaries were found that correspond to transcription factor expression domains. It is proposed that the optic lobe is compartmentalised by expression of Optix and Vsx1. These findings provide insight into the spatial patterning of a complex region of the brain, and suggest an evolutionarily conserved principle of visual system development (Gold, 2014).

Integration of temporal and spatial patterning generates neural diversity

In the Drosophila optic lobes, 800 retinotopically organized columns in the medulla act as functional units for processing visual information. The medulla contains over 80 types of neuron, which belong to two classes: uni-columnar neurons have a stoichiometry of one per column, while multi-columnar neurons contact multiple columns. This study shows that combinatorial inputs from temporal and spatial axes generate this neuronal diversity: all neuroblasts switch fates over time to produce different neurons; the neuroepithelium that generates neuroblasts is also subdivided into six compartments by the expression of specific factors (see The OPC neuroepithelium is patterned along its dorsal-ventral axis). Uni-columnar neurons are produced in all spatial compartments independently of spatial input; they innervate the neuropil where they are generated. Multi-columnar neurons are generated in smaller numbers in restricted compartments and require spatial input; the majority of their cell bodies subsequently move to cover the entire medulla. The selective integration of spatial inputs by a fixed temporal neuroblast cascade thus acts as a powerful mechanism for generating neural diversity, regulating stoichiometry and the formation of retinotopy (Erclik, 2017).

The optic lobes, composed of the lamina, medulla and the lobula complex, are the visual processing centres of the Drosophila brain. The lamina and medulla receive input from photoreceptors in the compound eye, process information and relay it to the lobula complex and central brain. The medulla, composed of ~40,000 cells, is the largest compartment in the optic lobe and is responsible for processing both motion and colour information. It receives direct synaptic input from the two colour-detecting photoreceptors, R7 and R8. It also receives input from five types of lamina neuron that are contacted directly or indirectly by the outer photoreceptors involved in motion detection (Erclik, 2017).

Associated with each of the ~800 sets of R7/R8 and lamina neuron projections are 800 medulla columns defined as fixed cassettes of cells that process information from one point in space. Columns represent the functional units in the medulla and propagate the retinotopic map established in the compound eye. Each column is contributed to by more than 80 neuronal types, which can be categorized into two broad classes. Uni-columnar neurons have arborizations principally limited to one medulla column and there are thus 800 cells of each uni-columnar type. Multi-columnar neurons possess wider arborizations, spreading over multiple columns. They compare information covering larger receptor fields. Although they are fewer in number, their arborizations cover the entire visual field (Erclik, 2017).

The medulla develops from a crescent-shaped neuroepithelium, the outer proliferation centre (OPC). During the third larval instar, the OPC neuroepithelium is converted into lamina on its lateral side and into medulla neuroblasts on its medial side. A wave of neurogenesis moves through the neuroepithelial cells, transforming them into neuroblasts; the youngest neuroblasts are closest to the neuroepithelium while the oldest are adjacent to the central brain. Neuroblasts divide asymmetrically multiple times to regenerate themselves and produce a ganglion mother cell that divides once more to generate medulla neurons. Recent studies have shown that six transcription factors are expressed sequentially in neuroblasts as they age: neuroblasts first express Homothorax (Hth), then Klumpfuss (Klu), Eyeless (Ey), Sloppy-paired 1 (Slp1), Dichaete (D) and Tailless (Tll). This temporal series is reminiscent of the Hb --> Kr --> Pdm --> Cas --> Grh series observed in Drosophila ventral nerve cord neuroblasts that generates neuronal diversity in the embryo. Indeed, distinct neurons are generated by medulla neuroblasts in each temporal window. Further neuronal diversification occurs through Notch-based asymmetric division of ganglion mother cells. In total, over 20 neuronal types can theoretically be generated using combinations of temporal factors and Notch patterning mechanisms. However, little is known about how the OPC specifies the additional ~60 neuronal cell types that constitute the medulla (Erclik, 2017).

To understand the logic underlying medulla development, late larval brains were stained with 215 antibodies generated against transcription factors and 35 genes were identifiied that are expressed in subsets of medulla progenitors and neurons. The OPC neuroepithelial crescent can be subdivided along the dorsal-ventral axis by the mutually exclusive expression of three homeodomain-containing transcription factors: Vsx1 is expressed in the central OPC (cOPC), Rx in the dorsal and ventral posterior arms of the crescent (pOPC), and Optix in the two intervening 'main arms' (mOPC). These three proteins are regionally expressed as early as the embryonic optic anlage and together mark the entire OPC neuroepithelium with sharp, non-overlapping boundaries. Indeed, these three regions grow as classic compartments: lineage trace experiments show that cells permanently marked in the early larva in one OPC region do not intermingle at later stages with cells from adjacent compartments. Of note, Vsx1 is expressed in cOPC progenitor cells and is maintained in a subset of their neuronal progeny whereas Optix and Rx are not expressed in post-mitotic medulla neurons. The OPC can be further subdivided into dorsal (D) and ventral (V) halves: a lineage trace with hedgehog-Gal4 (hh-Gal4) marks only the ventral half of the OPC, bisecting the cOPC compartment. As hedgehog is not expressed in the larval OPC, this dorsal-ventral boundary is set up in the embryo. Thus, six compartments (ventral cOPC, mOPC and pOPC and their dorsal counterparts) exist in the OPC. The pOPC compartment can be further subdivided by the expression of the wingless and dpp signalling genes. Cells in the wingless domain behave in a very distinct manner from the rest of the OPC, and have been described elsewhere (Erclik, 2017).

The Hth --> Klu --> Ey --> Slp1 --> D --> Tll temporal progression is not affected by the compartmentalization of the OPC epithelium; the same neuroblast progression throughout the entire OPC. Thus, in the developing medulla, neuroblasts expressing the same temporal factors are generated by developmentally distinct epithelial compartments (Erclik, 2017).

To test whether the intersection of the dorsal-ventral and temporal neuroblast axes leads to the production of distinct neural cell types, focus was placed on the progeny of Hth neuroblasts, which maintain Hth expression. In the late third instar, Hth neurons are found in a crescent that mirrors the OPC (see Distinct neuronal cell types are generated along the dorsal-ventral axis of the OPC). The NotchON (NON) progeny of Hth+ ganglion mother cells express Bsh and Ap, and they are distributed throughout the entire medulla crescent. In contrast, the NotchOFF (NOFF) progeny, which are BshHth+ neurons, express different combinations of transcription factors, and can be subdivided into three domains along the dorsal-ventral axis: (1) in the cOPC, NOFFHth+ neurons express Vsx1, Seven-Up (Svp) and Lim3; (2) in the pOPC, these neurons also express Svp and Lim3, but not Vsx1; (3) in the ventral pOPC exclusively, these neurons additionally express Teashirt (Tsh). NOFFHth+ cells are not observed in the mOPC. Rather, Cleaved-Caspase-3+ cells are intermingled with Bsh+ neurons. When cell death is prevented, Bsh+Hth+ cells become intermingled with neurons that express the NOFF marker Lim3, confirming that the NOFFHth+ progeny undergo apoptosis in the mOPC (Erclik, 2017).

It was therefore possible to distinguish three regional populations of Hth neurons (plus one that is eliminated by apoptosis) and a fourth population that is generated throughout the OPC. The neuronal identity of each of these populations was identified, as follows. (1) Bsh is a specific marker of Mi1 uni-columnar interneurons that are generated in all regions of the OPC. (2) To determine the identity of Hth+NOFF cOPC-derived neurons, Hth+ single cell flip-out clones were generated (using hth-Gal4) in the adult medulla. The only Hth+ neurons that are also Vsx1+Svp+ are Pm3 multi-columnar local neurons. (3) For Hth+NOFF pOPC-derived neurons, 27b-Gal4 was used; it drives expression in larval pOPC Hth+NOFF neurons and is maintained to adulthood. Flip-out clones with 27b-Gal4 mark Pm1 and Pm2 neurons, as well as Hth- Tm1 uni-columnar neurons that come from a different temporal window. Both Pm1 and Pm2 neurons (but not Tm1) express Hth and Svp. Pm1 neurons also express Tsh, which only labels larval ventral pOPC neurons (Erclik, 2017).

Thus, in addition to uni-columnar Mi1 neurons generated throughout the OPC, Hth neuroblasts generate three region-specific neuronal types: multi-columnar Pm3 neurons in the cOPC; multi-columnar Pm1 neurons in the ventral pOPC; and multi-columnar Pm2 neurons in the dorsal pOPC (Erclik, 2017).

To determine the contribution of the temporal and spatial factors to the generation of the different neuronal fates, the factors were mutated them and whether neuronal identity was lost was examined. To test the temporal axis, hth was mutated. As previously reported, Bsh expression is lost in hth mutant clones. Loss of hth in clones also leads to the loss of the Pm3 marker Svp without affecting expression of Vsx1, indicating that Vsx1 is not sufficient to activate Svp and can only do so in the context of an Hth+ neuroblast. Hth is also required for the specification of Pm1 and Pm2 in the pOPC as Svp and Tsh expression is lost in hth mutant larval clones. Ectopic expression of Hth in older neuroblasts is not able to expand Pm1, 2 or 3 fates (on the basis of the expression of Svp) into later born neurons, although it is able to expand Bsh expression. Thus, temporal input is necessary for the specification of all Hth+ neuronal fates but only sufficient for the generation of Mi1 neurons (see Temporal and spatial inputs are required for neuronal specification in the medulla. ) (Erclik, 2017).

Next, whether regional inputs are necessary and/or sufficient to specify neuronal fates in the progeny of Hth+ neuroblasts was determined. In Vsx1 RNA interference (RNAi) clones, Svp expression is lost in the cOPC but Bsh is unaffected. Additionally, Hth+Lim3+ cells are absent, suggesting that NOFF cells undergo apoptosis in these clones. Conversely, ectopic expression of Vsx1 leads to the expression of Svp in mOPC Hth+ neurons but does not affect Bsh expression. Therefore, Vsx1 is both necessary and sufficient for the specification of Pm3 fates in the larva. However, unlike the temporal factor Hth, Vsx1 does not affect the generation of Mi1 neurons (Erclik, 2017).

In Rx whole mutant larvae and in mutant clones, Svp+Lim3+Hth+ larval neurons (that is, Pm1 and Pm2 neurons) in the pOPC are lost. Additionally, the Pm1 marker Tsh is lost in ventral pOPC Hth+ cells. Consistent with the Vsx1 mutant data, larval Bsh expression is not affected by the loss of Rx. In adults, the Pm1/Pm2 markers (Svp, Tsh and 27b-Gal4) are lost in the medulla (Erclik, 2017).

Ectopic expression of Rx leads to the activation of Svp in mOPC Hth+ neurons, but does not affect the expression of Bsh. It also leads to the activation of Tsh, but only in the ventral half of mOPC Hth+ neurons, suggesting that a ventral factor acts together with Rx to specify ventral fates. Taken together, the above data show that Rx is both necessary and sufficient for the specification of Pm1/2 neurons but (like Vsx1) does not affect the generation of Mi1 neurons (Erclik, 2017).

Finally, the role of the mOPC marker Optix in neuronal specification was examined. In Optix mutant clones, Svp is ectopically expressed in the mOPC, but Bsh expression is not affected. Of note, these ectopic Svp+ neurons fail to express the region-specific Pm markers Vsx1 or Tsh (in ventral clones), which suggests that they assume a generic Pm fate. Conversely, ectopic expression of Optix leads to the loss of Svp expressing neurons in both the cOPC and pOPC but does not affect Bsh. These NOFF neurons die by apoptosis as no Lim3+ neurons are found intermingled with Bsh+Hth+NON neurons. When apoptosis is prevented in mOPC-derived neurons, Svp is not derepressed in the persisting Hth+NOFF neurons, which suggests that Optix both represses Svp expression and promotes cell death in Hth+NOFF neurons (Erclik, 2017).

The above data demonstrate that input from both the temporal and regional axes is required to specify neuronal fates. The temporal factor Hth is required for both Mi1 and Pm1/2/3 specification. The spatial genes are not required for the specification of NON Mi1 neurons, consistent with the observation that Mi1 is generated in all OPC compartments. The spatial genes, however, are both necessary and sufficient for the activation (Vsx1 and Rx) or repression (Optix) of the NOFF Pm1/2/3 neurons. Thus, Hth+ neuroblasts generate two types of progeny: NOFF neurons that are sensitive to spatial input (Pm1/2/3) and NON neurons that are refractory to spatial input (Mi1). Vsx1 expression in the cOPC is only maintained in Hth+NOFF neurons, suggesting that spatial information may be 'erased' in Mi1, thus allowing the same neural type to be produced throughout the OPC (Erclik, 2017).

Do spatial genes regulate each other in the neuroepithelium? In Vsx1 mutant clones, Optix (but not Rx) is derepressed in the cOPC epithelium. Conversely, ectopic Vsx1 is sufficient to repress Optix in the mOPC and Rx in the pOPC. Similarly, Optix, but not Vsx1, is derepressed in Rx mutant clones in the pOPC epithelium and ectopic Rx is sufficient to repress Optix in the mOPC (but not Vsx1 in the cOPC). In Optix mutant clones, neither Vsx1 nor Rx are derepressed in the mOPC epithelium, but ectopic Optix is sufficient to repress both Vsx1 in the cOPC and Rx in the pOPC. The observation that Optix is not necessary to suppress Vsx1 or Rx in the mOPC neuroepithelium is surprising because Svp is activated in a subset of Hth+ neurons in the mOPC in Optix mutant clones. Nevertheless, when cell death in the mOPC is abolished, the ectopic undead NOFF neurons express Lim3 but not Svp, which confirms that Optix represses Svp expression in mOPC neurons. Taken together, these results support a model in which Optix is sufficient to repress Vsx1 and Rx, to promote the death of Hth+NOFF neurons and to repress Pm1/2/3 fates (see Spatial genes cross-regulate each other in the OPC neuroepithelium). Vsx1 and Rx act to promote Pm3 (Vsx1) or Pm1/2 (Rx) fates but can only do so in the absence of Optix (Erclik, 2017).

These results suggest that multi-columnar neurons are generated at specific locations in the medulla crescent. However, since these neurons are required to process visual information from the entire retina in the adult medulla, how does the doral-ventral position of neuronal birth in the larval crescent correlate with their final position in the adult? Lineage-tracing experiments were performed with Vsx1-Gal4 to permanently mark neurons generated in the cOPC and with Optix-Gal4 for mOPC neurons, and the position of the cell bodies of these neurons was analyzed. In larvae, neurons from the cOPC or from the mOPC remain located in the same dorsal-ventral position where they were born. However, in adults, both populations have moved to populate the entire medulla cortex along the dorsal-ventral axis (see Neuronal movement during medulla development is restricted to multi-columnar cell types). The kinetics of cell movement during development was analyzed by following cOPC neurons. Neurons born in the cOPC remain tightly clustered until 20 h after puparium formation (P20), after which point the cell bodies spread throughout the medulla cortex. By P30 the neurons are distributed over the entire dorsal-ventral axis of the medulla cortex. In the adult, most neurons derived from the cOPC neuroepithelium are located throughout the cortex although there is an enrichment of neurons in the central region of the cortex (Erclik, 2017).

To determine whether these observed movements involve the entire neuron or just the cell body, the initial targeting of cOPC or mOPC-derived neurons in larvae was examined before the onset of cell movement. In larvae, both populations send processes that target the entire dorsal-ventral axis of the medulla neuropi. Therefore, medulla neurons first send projections to reach their target columns throughout the entire medulla. Later, remodelling of the medulla results in extensive movement of cell bodies along the dorsal-ventral axis, leading to their even distribution in the cortex (Erclik, 2017).

What is the underlying logic behind why some neurons move while others do not? Markers were studied for the Mi1 (Bsh), Pm2 (Hth+Svp+), Pm1 (Hth+Svp+Tsh+), and Pm3 (Vsx1+Svp+Hth+) populations of neurons through pupal stages and up to the adult. Mi1 neurons are generated evenly throughout the larval OPC and remain regularly distributed across the dorsal-ventral axis at all stages. The lineage-tracing experiment was repeated with Vsx1-Gal4 to follow Mi1 neurons produced by the cOPC. These Mi1 neurons remain exclusively in the centre of the adult medulla cortex, demonstrating that they do not move. In contrast, Pm3 neurons remain tightly clustered in the central region until P20, at which point they move to occupy the entire cortex (Erclik, 2017).

However, not all multi-columnar neurons have cell bodies that move to occupy the entire medulla cortex. Unlike Mi1 and Pm3, adult Pm1 and Pm2 cell bodies are not located in the adult medulla cortex but instead in the medulla rim, at the edges of the cortex. Pm1 and Pm2 markers remain clustered at the ventral (Pm1) or dorsal (Pm2) posterior edges of the medulla cortex throughout all pupal stages. In the adult, both populations occupy the medulla rim from where they send long horizontal projections that reach the entire dorsal-ventral axis of the medulla neuropil. The pOPC may be a specialized region where many of the medulla rim cell types are generated. Even though most of cOPC-derived neurons move during development, a cOPC-derived multi-columnar neuron (TmY14) was identified that sends processes targeting the entire dorsal-ventral length of the medulla neuropil but whose cell bodies remain in the central medulla cortex in the adult (Erclik, 2017).

Thus, the four populations of Hth neurons follow different kinetics: Mi1 neurons are born throughout the OPC and do not move; Pm3 neurons are born centrally and then move to distribute throughout the entire cortex; and Pm1/Pm2 neurons are born at the ventral or dorsal posterior edges of the OPC and occupy the medulla rim in adults (Erclik, 2017).

It is noted that uni-columnar Mi1 neurons, whose cell bodies do not move, reside in the distal cortex whereas multi-columnar Pm3 neurons, which move, reside in the proximal cortex. The hypothesis was thus tested that neurons whose cell bodies are located distally in the medulla cortex represent uni-columnar neurons generated homogeneously throughout the OPC that do not move. In contrast, proximal neurons, which are fewer in number and are generated in specific subregions of the medulla OPC, would be multi-columnar and move to their final position (Erclik, 2017).

It was first confirmed that neurons that move have their cell bodies predominantly in the proximal medulla cortex. The cell body position of neurons born ventrally that have moved dorsally was analyzed using the hh-Gal4 lineage trace: in the adult, the cell bodies found dorsally are mostly in the proximal medulla cortex, whereas the cell bodies in the ventral region are evenly distributed throughout the distal-proximal axis of the ventral cortex. They probably represent both distal uni-columnar neurons that did not move as well as proximal multi-columnar neurons that remained in the ventral region (Erclik, 2017).

Next the pattern of movement of Tm2 uni-columnar neurons from the ventral and dorsal halves of the OPC was analyzed using the hh-based lineage-trace. The cell bodies of Tm2 neurons are located throughout the dorsal-ventral axis in the adult medulla cortex but are co-labelled with the hh lineage marker only in the ventral half. Thus, like Mi1, Tm2 uni-columnar neurons do not move. Furthermore, uni-columnar Tm1 neurons, labelled by 27b-Gal4, are born throughout the dorsal-ventral axis of the OPC crescent with distal cell bodies in the adult, suggesting that they also remain where they were born (Erclik, 2017).

Conversely, it was asked whether neurons that are specified in only one region, such as the Vsx+ neurons of the cOPC, are multi-columnar in morphology. By sparsely labelling cOPC-derived neurons using the Vsx1-Gal4 driver, 13 distinct cell types were characterized that retain Vsx1 expression in the adult medulla. Strikingly, all are multi-columnar in morphology, further supporting the model that it is the multi-columnar neurons that move during pupal development (Erclik, 2017).

Finally, MARCM clones were generated in the OPC neuroepithelium and visualized using cell-type-specific Gal4 drivers in the adult medulla. Two classes of adult clone distribution were observed: clones in which neurons are tightly clustered, and clones in which neurons are dispersed. Consistent with the model, the clustered clones are those labelled with uni-columnar neuronal drivers, whereas the dispersed clones are those labelled with a multi-columnar driver (Erclik, 2017).

Taken together, these data demonstrate that neurons that do not move are uni-columnar (with cell bodies in the distal cortex), whereas most multi-columnar neurons (with cell bodies in the proximal cortex) move (Erclik, 2017).

This study shows that combinatorial inputs from the temporal and spatial axes act together to promote neural diversity in the medulla. Previous work has shown that a temporal series of transcription factors expressed in medulla neuroblasts allows for a diversification of the cell types generated by the neuroblasts as they age. This study now shows that input from the dorsal-ventral axis leads to further diversification of the neurons made by neuroblasts; at a given temporal stage, neuroblasts produce the same uni-columnar neuronal type globally as well as smaller numbers of multi-columnar cell types regionally. This situation is reminiscent of the mode of neurogenesis in the Drosophila ventral nerve cord, in which each neuroblast also expresses a (different) temporal series of transcription factors that specifies multiple neuronal types in the lineage. Spatial cues from segment polarity, dorsal-ventral and Hox genes then intersect to impart unique identities to each of the lineages. However, neuroblasts from the different segments give rise to distinct lineages to accommodate the specific function of each segment. In contrast, in the medulla, the entire OPC contributes to framing the repeating units that form the retinotopic map. It is therefore likely that each neuroblast produces a common set of neurons that connect to each pair of incoming R7 and R8 cells, or L1-L5 lamina neurons. This serves to produce 800 medulla columns with a 1:1 stoichiometry of medulla neurons to photoreceptors. The medulla neurons that are produced by neuroblasts throughout the dorsal-ventral axis of the OPC are thus uni-columnar The production of the same neuronal type along the entire OPC could be achieved by selectively 'erasing' spatial information in uni-columnar neurons, as observed in Mi1 neurons (Erclik, 2017).

Regional differences in the OPC confer further spatial identities to neuroblasts with the same temporal identity, and lead to specific differences in the lineages produced in the compartments along the dorsal-ventral axis of the medulla. These differences produce smaller numbers of multi-columnar neurons whose stoichiometry is much lower than 1:1. The majority of these neurons move during development to be uniformly distributed in the adult medulla cortex. This combination of regional and global neuronal specification in the medulla presents a powerful mechanism to produce the proper diversity and stoichiometry of neuronal types and generate the retinotopic map (Erclik, 2017).


EVOLUTIONARY HOMOLOGS

The Six/Sine oculus family of genes

Five cDNA clones of the Six gene family have been identified, all of which are expressed in retina. They are Six2, Six3 alpha and Six3 beta (which are derived from alternative splicing forms), Six5, and AREC3/Six4. All of these Six family genes possess extensive sequence similarity between one another in the Sina oculis-homologous region (Six domain and homeodomain) but they differ greatly in structure in some other regions. The amino acid sequence similarity of the Sina oculis-homologous region to the previously identified AREC3/Six4 is 70.1% for Six2; 57.3% for Six3 alpha and Six3 beta, and 70.3% for Six5. The expression of these genes is observed in the inner and outer nuclear layer, ganglion cell layer, and pigment epithelium of mouse retina. The So-homologous region of each Six family protein has specific DNA binding activity. Six5 and Six2 bind to the same sequence as does AREC3/Six4, while Six3 does not. These observations suggest that some of the Six family genes can regulate the same target genes (Kawakami, 1996).

The Six3 subclass of the Six/Sine oculus family of genes

The murine homeobox gene Six3 carries out regulatory functions in eye development. Two isolated and characterized zebrafish genes, six3 and six6, are closely related to the murine Six3 gene. Zebrafish six3 may be the structural ortholog, while the six6 gene (identical to Kobayashi's [1998] six3) is more similar with respect to embryonic expression. Transcripts of both zebrafish six genes are first detected in involuting axial mesendoderm and subsequently in the overlying anterior neural plate from which the optic vesicles and the forebrain will develop. Direct correspondence between six3/six6 expression boundaries and the optic vesicles indicate essential roles in defining the eye primordia. During later stages only the six6 gene displays similar features of expression in the eyes and rostral brain, as reported previously for murine Six3 (Seo, 1998a).

In zebrafish, in addition to two previously reported homologs of murine Six3, a related gene (six7) has been identified. Although the deduced Six7 protein shares less than 68% sequence identity with the other known zebrafish Six3-like proteins, the embryonic expression patterns have highly conserved features. The six7 transcripts are first detected in involuting axial mesendoderm and, subsequently, in the overlying neurectoderm from which the forebrain and optic primordia develop. Similar to the two other zebrafish Six3 homologs, the expression boundaries of six7 correspond quite closely with the edges of the optic vesicles. Hence, the partially overlapping expression domains of these three six genes probably contribute to anteroposterior specification and in defining the eye primordia (Seo, 1998b).

Zebrafish six3 is the apparent ortholog of the mouse Six3 gene. Zebrafish six3 transcripts are first seen in hypoblast cells in early gastrula embryos and are found in the anterior axial mesendoderm through gastrulation. six3 expression in the head ectoderm begins at late gastrula. Throughout the segmentation period, six3 is expressed in the rostral region of the prospective forebrain. Overexpression of six3 in zebrafish embryos induces enlargement of the rostral forebrain, enhances expression of pax2 in the optic stalk and leads to a general disorganization of the brain. Disruption of either the Six domain or the homeodomain abolishes these effects, implying that these domains are essential for six3 gene function. These results suggest that the vertebrate Six3 genes are involved in the formation of the rostral forebrain (Kobayashi, 1998).

Six3 is expressed in the anterior neural plate and optic vesicles, lens, olfactory placodes and ventral forebrain. Overexpression of mouse Six3 gene in medaka fish embryos (Orvzias latipes) results in the formation of an ectopic lens, indicating that Six3 activity can trigger the genetic pathway leading to lens formation. The medaka Six3 homolog has now been isolated and its expression pattern analyzed in the medaka embryo. Medaka Six3 is phylogenetically quite distantly related to zebrafish six3/six6 (similar to zebrafish six7) (Seo, 1999). It is expressed initially in the anterior embryonic shield and later in the developing eye and prosencephalon. The early localized expression of medaka Six3 suggests a role in the regionalization of the rostral head (Loosli, 1998).

Xenopus XOptx2 (a Six3 subclass member) is a new member of the Six/sine oculis family. A characteristic distinction between the Six3 and Optx2 proteins is the length of the pre-Six domain region. All known Optx2 proteins are smaller at the amino terminus than all known Six3 proteins. XOptx mRNA expression is first detected in stage 14 embryos. At stage 15, XOptx2 is detected as a single band of expression at the most anterior edge of the developing neural plate. At approximately stage 17, expression extends laterally. By neural groove stage, this single band of expression separates into two distinct regions consistent with the location of the eye fields. As the protrusion of the eyes begins to become distinct (stage 20 to 22), XOptx2 expression appears restricted to the eyes. At stage 25, XOptx2 is also detected in the pineal gland primordia and the ventral forebrain. Expression continues to be detected in the eyes, the maturing pineal gland, and the ventral forebrain of tailbud embryos (Zuber, 1999).

Overexpression of XOptx2, a member of the family, in the Xenopus embryonic eye field results in a dramatic increase in eye size. A hybrid protein containing the repressor domain of Engrailed (XOptx2-Engrailed repressor) gives a similar phenotype, while a XOptx2-VP16 activator hybrid protein reduces eye size. XOptx2 stimulates bromodeoxyuridine incorporation, and XOptx2-induced eye enlargement is dependent on cellular proliferation. Moreover, retinoblasts transfected with XOptx2 produce clones of cells approximately twice as large as control clones. Pax6, which does not increase eye size on its own, acts synergistically with XOptx2. These results suggest that XOptx2, in combination with other genes expressed in the eye field, is crucially involved in the proliferative state of retinoblasts and thereby the size of the eye (Zuber, 1999).

Two distinct but nonexclusive mechanisms could explain the XOptx2-dependent increase in eye size. (1) Uncommitted cells not destined to be eye cells might be induced to change their fate to the eye cell lineage. This kind of cell fate conversion is common with overexpression-induced enlargements of the nervous system in Xenopus embryos. (2) Cells already destined to form an eye may have increased mitotic activity and therefore an increase in the number of cells in the optic vesicle and eye proper. One way to determine if XOptx2 causes extra cell divisions in eye field cells is the analysis of cell number in clones overexpressing this gene. Retinal cells of stage 17 embryos were cotransfected in vivo with either XOptx2 DNA and the tracer GFP or vector only and GFP. Transfected cells were detected by fluorescence in cryostat-sectioned eyes of stage 41 embryos. Although it could not be confirmed that single cells were initially transfected, there is reason to suspect that such clusters have clonal origins: (1) the clusters tended to be compact, oriented in vertical columns, and contain all cell types, as is found when single cells are injected with fluorescent or enzymatic lineage tracers; and (2) when low doses of DNA are injected, about half the eyes have no transfected cells, implying a Poisson distribution of hits, yet the average number of cells in a cluster does not decrease when the hit frequency is very low. To increase the probability that true clones were examined, the amount of DNA lipofected was minimalized so that most retinas contained either no transfected cells or single 'clones'. In addition, when more than one cluster was detected in transfected retinas, they were scored only if the clusters were well separated. Interestingly, XOptx2 has no effect on the relative proportion of cell types generated from transfected cells in these clones. This result implies that XOptx2 does not influence retinal cell fate, per se. However, XOptx2 nearly doubles the number of GFP-positive cells in retinal clones. The average clone size in XOptx2-transfected retinas is 16.1 cells per retinal section, while the cell number observed in control-expressing clones is 10.4. These results indicate that XOptx2 induces proliferation (Zuber, 1999).

Reported here is the cloning and developmental pattern of expression of xSix3, the Xenopus laevis homolog of Six3. In addition, the known sequences of vertebrate Six3 genes have been compared. xSix3 is very homologous to Six3 in other vertebrates in terms of amino acid sequence. The reported developmental pattern of expression of Six3 in chick and mouse includes not only the developing eyes and the ventral diencephalic tissue between them, but also a large, sagittally-oriented telencephalic region. The distribution of xSix3, however, is virtually restricted to the eyes and ventral diencephalon, showing only a very small territory of expression in the telencephalon (Zhou, 2000).

cSix3 is a chick homolog of the murine Six3. cSix3 transcripts are expressed from presomitic stages in the most anterior portion of the neural plate. As the neural tube folds and the optic vesicles evaginate, cSix3 is expressed in the optic vesicle and the rostroventral forebrain. At later stages, cSix3 is found in most of the structures derived from the anterior neural plate, i.e. olfactory epithelium, septum, adenohypophysis, hypothalamus and preoptic areas. During eye development, cSix3 expression is first found in the entire optic vesicle and the overlying ectoderm but soon becomes restricted to the prospective neural retina and to the lens placode. In the developing neural retina, cSix3 is expressed in the entire undifferentiated neuroepithelium but is rapidly downregulated, first in the postmitotic photoreceptors and later in the majority of retinal ganglion cells (Bovolenta, 1998).

A detailed expression analysis in chick and mouse of Six9 (Optx2), genes of the Six/sine oculis family closely related to Six3, is reported. Six9 (Optx2) is first expressed at presomitic stages in the head-fold, both in the neural plate and in the underlying axial mesoderm. Thereafter, Six9 (Optx2) is strongly expressed in the presumptive and differentiating neural retina and ventral optic stalk, in the olfactory placodes, in the hypothalamus and in the pituitary gland. This expression pattern largely overlaps with that of Six3, but several differences exist between the expression domain of the two genes. At presomitic stages, the posterior boundary of Six3 expression is at the same axial level both in the prechordal plate and in the overlying neural plate. In contrast, Six9 (Optx2) expression in the prechordal plate extends more caudal to that of the neural plate, occupying a more restricted V-shaped territory. Similarly, during the early events of eye patterning, Six3 is first expressed in the entire optic vesicle and lens placode. Only later does its expression become confined to the prospective and differentiating neural retina. Conversely, Six9 (Optx2) is never observed in the lens placode of either chick and mouse, and from early stages of optic vesicle development, Six9 (Optx2) transcripts are restrained to the prospective ventral neural retina and optic stalks (L pez-Rios, 1999).

A vertebrate member of the so/Six gene family, Six3, is expressed in the developing eye and forebrain. Injection of Six3 RNA into medaka fish embryos causes ectopic Pax6 and Rx2 expression in midbrain and cerebellum, resulting in the formation of retinal primordia at ectopic locations in the midbrain and prospective cerebellum, involving a regulatory interaction of Six3 and Pax6. Similar to the wild-type situation in the developing eye, Pax6 and Six3 are expressed in the region where the ectopic retinal primordia will subsequently form. These ectopic retinal primordia have the potential to develop into optic cups, as visualized by morphology and marker gene expression. The higher frequency of ectopic retinal primordia at early somitogenesis stages, as compared to ectopic optic cups formed at the 34-somite stage, indicates that not all ectopic retinal primordia develop into an optic cup. Thus, Six3 initiates, but does not fully implement, later stages of retinal development. Injected mouse Six3 RNA initiates ectopic expression of endogenous medaka Six3, uncovering a feedback control of Six3 expression. Initiation of ectopic retina formation reveals a pivotal role for Six3 in vertebrate retina development and hints at a conserved regulatory network underlying vertebrate and invertebrate eye development (Loosli, 1999).

A murine homeobox-containing gene, Six6 (Optx2), has been isolated that shows extended identity in its coding region with Six3, the only member of the mammalian Six gene family known to be expressed in the optic primordium. Phylogenetic analysis demonstrates that Six6 and Six3 belong to a separate group of homeobox-genes that are closely related to the recently identified Drosophila Optix. Earliest Six6 expression is detected in the floor of the diencephalic portion of the primitive forebrain, a region predicted to give rise to the neurohypophysis and to the hypothalamus. Later on, Six6 mRNA is found in the primordial tissues giving rise to the mature pituitary: the Rathke's pouch and the infundibular recess. In the optic primordium, Six6 demarcates the presumptive ventral optic stalk and the ventral portion of the future neural retina. In the developing eye, Six6 expression is detected in the neural retina, the optic chiasma and optic stalk, but not in the lens. When compared to Six6, Six3 expression pattern is highly similar, but with a generally broader transcript distribution in the brain and in the visual system. Six6 does not require Pax6 for its expression in the optic primordium, suggesting that Six6 acts on a parallel and/or independent pathway with Pax6 in the genetic cascade governing early development of the eye (Jean, 1999).

Six3 from mice is now included in the new Six/sine oculis subclass of homeobox genes. Early in development Six3 expression is restricted to the anterior neural plate including areas that will later give rise to ectodermal and neural derivatives. Later, once the longitudinal axis of the brain bends, Six3 mRNA is also found in structures derived from the anterior neural plate: the ectoderm of the nasal cavity, the olfactory placode, Rathke's pouch, and also the ventral forebrain, including the region of the optic recess, hypothalamus and optic vesicles. Based on this expression pattern, Six3 appears to be one of the most anterior homeobox genes reported to date. The high sequence similarity of Six3 with Drosophila sine oculis, and its expression during eye development, suggests that this gene is the likely murine homolog. Mammals and insects share control genes such as eyeless/Pax6 and also possibly other members of the regulatory cascade required for eye morphogenesis. In Small eye (Pax6) mouse mutants, Six3 expression is not affected (Oliver, 1995).

Murine Six3 is expressed in the anterior neural plate, a region involved in lens induction in Xenopus. To examine whether Six3 participates in the process of eye formation, mouse Six3 was ectopically expressed in fish embryos. The results show that Six3 is sufficient to promote ectopic lens formation in the area of the otic vesicle and that retinal tissue is not a prerequisite for ectopic lens differentiation. These findings suggest a conserved function for Six3 in metazoan eye development (Oliver, 1996).

Otx2 is required first in the visceral endoderm for induction of forebrain and midbrain, and subsequently in the neurectoderm for its regional specification. Otx2 functions both cell autonomously and non-cell autonomously in neurectoderm cells of the forebrain and midbrain to regulate expression of region-specific homeobox and cell adhesion genes. Using chimeras containing both Otx2 mutant and wild-type (WT) cells in the brain, the effects of Otx on gene expression were analyzed (Rhinn, 1999).

Mutant cells result in a reduction or loss of expression of Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2, while expression of En2 and Six3 is rescued by surrounding wild-type cells. Forebrain Otx2 mutant cells subsequently undergo apoptosis. In the forebrain, Otx2 is required to activate the expression of the homeobox gene Rpx and maintain the expression of another homeobox gene, Six3. To determine if Otx2 is required cell autonomously or non-cell autonomously to regulate expression of these genes, the forebrain of moderate chimeric embryos was analyzed in double-labelling experiments, using histochemical staining for beta-galactosidase activity to distinguish WT from Otx2 mutant cells, and whole-mount RNA in situ hybridization to characterize Rpx or Six3 expression. Rpx is expressed in the forebrain of control embryos at E8.5. In moderate chimeras, Rpx expression is absent from the patches of Otx2 mutant cells, but is present in the surrounding WT forebrain cells. At the border of the mutant cell patches, Otx2 mutant cells fail to express Rpx while neighboring WT forebrain cells maintain expression of the gene. The strict correlation at the cellular level between lack of Otx2 activity and loss of Rpx expression demonstrates that Otx2 is required cell autonomously for expression of this gene in the forebrain. In contrast, Six3, another homeobox gene expressed in the forebrain, is expressed in groups of Otx2 mutant cells as in surrounding WT cells in moderate chimeras at E8.5, indicating that Otx2 is required non-cell autonomously for maintenance of Six3 expression. Thus, Otx2 regulates expression of different regulatory genes in the forebrain through distinct pathways. Similar results were obtained for the regulation of gene expression in the mid-hindbrain region. Otx2 is required for the activation of expression of the signaling molecule Wnt1 and for the maintenance of expression of the homeobox gene En2. Wnt1 expression is observed in WT midbrain cells in control embryos and moderate chimeras but is not detected in any Otx2 mutant cells in the midbrain of moderate chimeras, including those in contact with WT cells. This result demonstrates that Otx2 is required cell autonomously in midbrain cells to activate Wnt1 expression. In contrast, En2 expression in Otx2 mutant cells in the mid-hindbrain of moderate chimeras is rescued by the presence of surrounding WT cells, demonstrating a non-cell autonomous function for Otx2 in regulating En2 expression. Therefore, Otx2 also regulates the expression of mid-hindbrain genes through different mechanisms. Altogether, this study demonstrates that Otx2 is an important regulator of brain patterning and morphogenesis, through its regulation of candidate target genes such as Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2 (Rhinn, 1999).

Holoprosencephaly (HPE) is a common, severe malformation of the brain that involves separation of the central nervous system into left and right halves. Mild HPE can consist of signs such as a single central incisor, hypotelorism, microcephaly, or other craniofacial findings that can be present with or without associated brain malformations. The etiology of HPE is extremely heterogeneous, with the proposed participation of a minimum of 12 HPE-associated genetic loci as well as the causal involvement of specific teratogens acting at the earliest stages of neurulation. The HPE2 locus has recently been characterized as a 1-Mb interval on human chromosome 2p21 that contains a gene associated with HPE. A minimal critical region is defined by a set of six overlapping deletions and three clustered translocations in HPE patients. The isolation and characterization of the human homeobox-containing SIX3 gene from the HPE2 minimal critical region (MCR) is described. At least 2 of the HPE-associated translocation breakpoints in 2p21 are less than 200 kb from the 5' end of SIX3. Mutational analysis has identified four different mutations in the homeodomain of SIX3 that are predicted to interfere with transcriptional activation and are associated with HPE. It is proposed that SIX3 is the HPE2 gene, essential for the development of the anterior neural plate and eye in humans (Wallis, 1999).

The Drosophila gene sine oculis (so), a nuclear homeoprotein that is required for eye development, has several vertebrate homologs (the SIX gene family). Among them, SIX3 is considered to be the functional ortholog of so because it is strongly expressed in the developing eye. However, embryonic SIX3 expression is not limited to the eye field, and SIX3 has been found to be mutated in some patients with holoprosencephaly type 2 (HPE2), suggesting that SIX3 has wide implications for head development. The cloning and characterization of SIX6, a novel human SIX gene that is the homolog of the chick Six6(Optx2) gene, is reported. SIX6 is closely related to SIX3 and is expressed in the developing and adult human retina. Data from chick and mouse suggest that the human SIX6 gene is also expressed in the hypothalamic and the pituitary regions. SIX6 spans 2567 bp of genomic DNA and is split in two exons that are transcribed into a 1393-nucleotide-long mRNA. Chromosomal mapping of SIX6 reveals that it is closely linked to SIX1 and SIX4 in human chromosome 14q22.3-q23, which provides clues about the origin and evolution of the vertebrate SIX family. Recently three independent reports have associated interstitial deletions at 14q22.3-q23 with bilateral anophthalmia and pituitary anomalies. Genomic analyses of one of these cases demonstrates SIX6 hemizygosity, strongly suggesting that SIX6 haploinsufficiency is responsible for these developmental disorders (Gallardo, 1999).

Holoprosencephaly (HPE) is the most common congenital malformation of the forebrain in human. Several genes with essential roles during forebrain development have been identified because they cause HPE when mutated. Among these are genes that encode the secreted growth factor Sonic hedgehog (Shh) and the transcription factors Six3 and Zic2. In the mouse, Six3 and Shh activate each other's transcription, but a role for Zic2 in this interaction has not been tested. This study demonstrates that in zebrafish, as in mouse, Hh signaling activates transcription of six3b in the developing forebrain. zic2a is also activated by Hh signaling, and represses six3b non-cell-autonomously, i.e. outside of its own expression domain, probably through limiting Hh signaling. Zic2a repression of six3b is essential for the correct formation of the prethalamus. The diencephalon-derived optic stalk (OS) and neural retina are also patterned in response to Hh signaling. This study shows that zebrafish Zic2a limits transcription of the Hh targets pax2a and fgf8a in the OS and retina. The effects of Zic2a depletion in the forebrain and in the OS and retina are rescued by blocking Hh signaling or by increasing levels of the Hh antagonist Hhip, suggesting that in both tissues Zic2a acts to attenuate the effects of Hh signaling. These data uncover a novel, essential role for Zic2a as a modulator of Hh-activated gene expression in the developing forebrain and advance understanding of a key gene regulatory network that, when disrupted, causes HPE (Sanek, 2009).

Candidate gene screen in the red flour beetle Tribolium reveals six3 as ancient regulator of anterior median head and central complex development

Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, this study used the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and several genes were identified that had not been implicated in this process before. Furthermore, it was shown that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, it was demonstrated that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly (Posnien, 2011).

Tc-six3 is expressed in an anterior median domain from earliest stages on and that it acts as an upstream component of anterior median patterning. Drosophila optix/six3 is expressed in an anterior blastodermal ring anterior to otd, which persists at the dorsal side. Its ring like expression does not support an involvement in median patterning but relevant genetic interactions remain to be studied. The later expression in the labrum and in bilateral dorsal domains, however, is similar in both species (Posnien, 2011).

Interestingly, aspects of dorsal median head patterning are controlled by dpp in Drosophila. Shortly before gastrulation, the action of dpp and its downstream target zen at the dorsal midline separate the neuroectoderm into paired anlagen by medial repression of genes and by promoting median cell death. This results in the establishment of bilateral expression of marker genes of the respective brain parts [e.g. Dchx (pars intercerebralis); Fas2 and Drx (pars lateralis); sine oculis and eyes-absent (visual system)]. Actually, many other anterior patterning genes initiate their expression as unpaired domains across the dorsal midline that are subsequently medially subdivided in Drosophila (e.g. otd, fezf, Dsix4. In contrast, the Tribolium orthologs of most of these genes are initiated as separate bilateral domains (Tc-rx and Tc-fez, Tc-chx and Tc-Fas2, Tc-tll, Tc-six4, Tc-sine oculis, Tc-eyes-absent. Tc-otd1 starts out with ubiquitous expression related to axis formation but then resolves into paired head lobe domains which are separate as with the aforementioned genes (Posnien, 2011).

Due to differences in topology of the head anlagen, median repression of anterior patterning genes by Tc-dpp is not required in Tribolium. Nevertheless, it is expressed along the rim of the head anlagen at blastoderm stages, some parts of which will become the site of dorsal fusion. However, Tc-Dpp activity (detected by antibodies against pMad) does not occur at the site of expression and is clearly distant from the arising Tc-rx, Tc-chx, Tc-six4, Tc-sine oculis or Tc-fas2 domains. Also the Tc-dpp RNAi phenotypes differ from Drosophila mutants in that the head anlagen are expanded and appear to have lost their dorso-ventral orientation (shown by expansion of Tc-otd1 and the proneural gene Tc-ASH) in an overall ventralized embryo. Hence, the early expression of dpp at the future dorsal midline might be ancestral, but its function with respect to medially repressing gene expression has probably evolved in Drosophila (Posnien, 2011).

The difference in generation of paired dorsal domains in these two insect species reflects the different location of the head anlagen. In the long germ insect Drosophila, extraembryonic tissues are reduced to the dorsally located amnioserosa while the head anlagen are situated in the anterior dorsal blastoderm from earliest stages on. Consequently, the head lobes are never separated along the midline. In contrast, in the short germ insect Tribolium, the anterior blastoderm gives rise to extraembryonic amnion and serosa, which eventually ensheath the embryo. In contrast to Drosophila, the Tribolium head anlagen are located in the ventral median blastoderm from where they move towards anteriorly and bend dorsally. The head lobes are separate from the beginning but fuse at late stages at the dorsal midline forming the dorsal head (bend and zipper model). During these morphogenetic movements, the initially separate expression domains of the head lobes eventually come into close proximity at the dorsal midline like in Drosophila. Both the anterior dorsal location of extraembryonic tissue anlagen and the ventral location of the head anlagen are found in most insects and in the hemimetabolous milkweed bug Oncopeltus fasciatus, gene expression data show a clear separated origin of the head lobes in the blastoderm. Hence, Tribolium is likely to represent the ancestral state in insects (Posnien, 2011).

In striking analogy to Drosophila, the expressions of vertebrate eye field patterning genes start out as one midline spanning domain (e.g., Rx and Pax6. Later, these domains split medially, which is the prerequisite for the formation of bilateral eye anlagen. shh as well as six3 are involved in medial repression of Pax6 and Rx2 with six3 acting upstream of shh. This appears to be more similar to the derived Drosophila situation than to the ancestral split of head lobe anlagen. However, the molecules involved in median split are different (dpp in Drosophila versus six3 and shh in vertebrates) and involvement of Tribolium six3 but not dpp is found in median patterning. Hence, the molecular data actually suggest a higher degree of conservation between Tribolium and vertebrates and convergent evolution of the similarity between Drosophila and vertebrates (Posnien, 2011).

Regarding the likely difference to Drosophila, it is striking that the role of vertebrate six3 in median separation of anterior expression domains is similar to what was found in Tribolium. In vertebrates, six3 represses midbrain derived Wnt signaling, which was also found in Tribolium. In vertebrates, six3 and its paralog six6 are involved in pituitary and hypothalamus development. Based on its expression, six3 has been predicted to contribute to neuroendocrine brain parts in annelids and Drosophila. More generally, the similarity of bilaterian neuroendocrine systems and their common origin from placode like precursors have been noted. This study has added functional data showing that Tc-six3 is indeed required for the expression of neuroendocrine markers for the pars intercerebralis (Tc-chx) and pars lateralis (Tc-fas2) placing it high in the hierarchy of neuroendocrine development in bilaterians (Posnien, 2011).

In mouse embryos with reduced levels of six3 and shh expression, median head and brain structures are affected (e.g., median nasal prominence) or absent (e.g., nasal septum, the septum, corpus callosum). Such holoprosencephaly phenotypes are also seen in some human six3 mutations. Very similarly, loss of median brain structures is seen in Tribolium after RNAi for Tc-six3 Overall, these similarities functionally confirm that the ancestral role of six3 orthologs was in the anterior median patterning of the Urbilateria (Posnien, 2011).

Six3, a medaka homologue of the Drosophila homeobox gene sine oculis is expressed in the anterior embryonic shield and the developing eye

The complete absence of eyes in the medaka fish mutation eyeless is the result of defective optic vesicle evagination. The eyeless mutation is caused by an intronic insertion in the Rx3 homeobox gene resulting in a transcriptional repression of the locus that is rescued by injection of plasmid DNA containing the wild-type locus. Functional analysis reveals that Six3- and Pax6- dependent retina determination does not require Rx3. However, gain- and loss-of-function phenotypes show that Rx3 is indispensable to initiate optic vesicle evagination and to control vesicle proliferation, and consequently organ size. Thus, Rx3 acts at a key position coupling the determination with subsequent morphogenesis and differentiation of the developing eye (Loosli, 2001).

The following model is proposed for early vertebrate retina development. Patterning of the anterior neural plate culminates in defined expression patterns of Six3 and Pax6. This anterior neural plate relies on the repression of wnt and BMP signaling, and requires the activity of the Otx transcription factors. In the region where Six3 and Pax6 expression overlap, retinal fate is specified. An Rx3-independent regulatory feedback loop of these genes then ensures the maintenance of the retinal fate. Six3 overexpression in el-mutant embryos results in dramatically enlarged retinal primordia. This expansion does not occur at the expense of forebrain tissue, suggesting that Six3 also affects cell proliferation independently of Rx3 and thereby regulates the size of the retina anlage. Consistent with the suggested role of Six3 in cell proliferation, the closely related Xenopus Optx2 gene controls the size of the optic vesicles by regulating proliferation. Under the influence of midline signaling, the retinal anlage is split into two retinal primordia. Mutations in Six3 cause holoprosencephaly in humans, indicating a requirement for Six3 in this process. The two retinal primordia then become localized to the lateral wall of the prosencephalon during neurulation (Loosli, 2001 and references therein).

Subsequent evagination of the primordia results in the formation of the optic vesicles. For this process, Rx3 function is essential. Functional studies consistently argue for a regulatory role of vertebrate Rx genes in proliferation of retinal progenitor cells in the optic vesicle, thus regulating its growth. In the absence of Rx3 function, there is no sign of morphogenesis and the specified retinal precursors do not proliferate and eventually die. Rx3 acts downstream of Six3 and Pax6, which determine the retina anlage. However, it is possible that Rx3 initially also receives input from neural plate patterning genes. Subsequent development divides the optic vesicle into specific regions that then give rise to neural retina (NR), retinal pigmented epithelium (RPE) and optic stalk. Several genes that are expressed during these later steps of retinal development require Rx3 function directly or indirectly. Interestingly, the expression of Tbx2 and Tbx3 is specifically affected in the retinal primordium, but not in the hypothalamus, where they are also co-expressed. This indicates a differential regulation of Tbx2 and Tbx3 in these tissues (Loosli, 2001 and references therein).

The sea urchin animal pole domain is a Six3-dependent neurogenic patterning center

Two major signaling centers have been shown to control patterning of sea urchin embryos. Canonical Wnt signaling in vegetal blastomeres and Nodal signaling in presumptive oral ectoderm are necessary and sufficient to initiate patterning along the primary and secondary axes, respectively. This study defines and characterizes a third patterning center, the animal pole domain (APD), which contains neurogenic ectoderm, and can oppose Wnt and Nodal signaling. The regulatory influence of the APD is normally restricted to the animal pole region, but can operate in most cells of the embryo because, in the absence of Wnt and Nodal, the APD expands throughout the embryo. Many constituent APD regulatory genes expressed in the early blastula have been identified; expression of most of them requires Six3 function. Furthermore, Six3 is necessary for the differentiation of diverse cell types in the APD, including the neurogenic animal plate and immediately flanking ectoderm, indicating that it functions at or near the top of several APD gene regulatory networks. Remarkably, it is also sufficient to respecify the fates of cells in the rest of the embryo, generating an embryo consisting of a greatly expanded, but correctly patterned, APD. A fraction of the large group of Six3-dependent regulatory proteins are orthologous to those expressed in the vertebrate forebrain, suggesting that they controlled formation of the early neurogenic domain in the common deuterostome ancestor of echinoderms and vertebrates (Wei, 2009).

Six3 inactivation reveals its essential role for the formation and patterning of the vertebrate eye

The establishment of retinal identity and the subsequent patterning of the optic vesicle are the key steps in early vertebrate eye development. To date little is known about the nature and interaction of the genes controlling these steps. So far few genes have been identified that, when over-expressed, can initiate ectopic eye formation. Of note is Six3, which is expressed exclusively in the anterior neural plate. However, 'loss of function' analysis has not been reported. Using medaka fish, it has been shown that vertebrate Six3 is necessary for patterning of the anterior neuroectoderm including the retina anlage. Inactivation of Six3 function by morpholino knock-down results in the lack of forebrain and eyes. Corroborated by gain-of-function experiments, graded interference reveals an additional role of Six3 in the proximodistal patterning of the optic vesicle. During both processes of vertebrate eye formation, Six3 cooperates with Pax6 (Carl, 2002).

These experiments demonstrate that Six3 is essential for the formation of a discrete domain within the anterior neuroectoderm. In the absence of Six3 function, cells in the Six3 expression domain undergo apoptosis resulting in the absence of forebrain and eye. Conversely, overexpression of Six3 results in retinal hyperplasia, indicating that one function of Six3 is the control of proliferation in the presumptive retinal cells. In addition, Six3 functions in the determination of the naive anterior neuroectoderm as loss of function results in the absence of the respective structures, while ectopic Six3 expression leads to their ectopic formation. Gene knockdown data show that Six3 and Pax6 interact genetically at early stages of eye development. However, the morphological and molecular consequences of the loss of Six3 function are more severe. Pax6 acts on gene expression including its own only in cooperation with Six3. As seen for Pax6 morphants, small eye Pax6-/- mouse embryos initially form optic vesicles, indicating that also in the mouse Pax6 is not required for the formation of the retina anlage in the neuroectoderm (Carl, 2002).

Following its role in the determination and formation of the retina anlage, Six3 functions in proximodistal patterning of the optic vesicle. Six3 activity regulates the expression of the regional specification gene Vax1, which is required for the formation of ventral forebrain and proximal eye structures. Graded loss of Six3 function shows that the distally expressed genes Rx2 and Pax6 are less sensitive than Vax1, underscoring the proximodistal patterning activity of Six3. In addition, at this stage Six3 controls proliferation and morphogenesis by regulating the expression of Rx3, which is essential for these processes in the developing optic vesicle. Future experiments will aim to identify factors mediating the differential activity of Six3 along the proximodistal axis (Carl, 2002).

A screen for co-factors of Six3

The vertebrate Six3 gene, a homeobox gene of the Six-family, plays a crucial role in early eye and forebrain development. Candidate factors have been isolate that interact with Six3 in a yeast two-hybrid screen. Among these are two basic helix loop helix (bHLH) domain containing proteins. Biochemical analysis reveals that the bHLH proteins ATH5, ATH3, NEUROD as well as ASH1 interact specifically with XSix3. By defining the interacting domains it has been shown that the bHLH domain of NEUROD interacts with the SIX domain of XSix3. The co-expression of the interacting molecules during late retina determination/differentiation suggests a new role for Six3 and the respective interaction partner also in these late steps of eye development (Tessmar, 2002).

Co-expression of XNeuroD or Xath5 and XSix3 in the eye starts around the time when cell fate determination is still in progress, but differentiation is initiated. At the end of differentiation, their coexpression vanishes, suggesting that their interaction with SIX3 is important for the determination and/or differentiation of distinct cell types in the retina. This finding is in accordance with the notion that in a conditional inactivation of murine Pax6 in retinal progenitor cells, co-expression of Six3 and NeuroD coincides with the exclusive generation of amacrine cells. Therefore, Six3 might permit amacrine cell fate in the presence of NeuroD (Tessmar, 2002).

Xath3 and Xath5 show expression patterns similar to XNeuroD in the developing neuroretina, suggesting that these proteins likewise form part of the determination/differentiation network of the eye. The interaction of SIX3 with a specific combination of ARPs thus may specify distinct cell types of the neuroretina. In contrast, differentiation requires both a stop of proliferation and the expression of cell type specific differentiation genes. Therefore, the ARP/XSIX3 interaction should initiate, directly or indirectly, a proliferation-stop signal. Since Six3 on its own stimulates proliferations, it is tempting to speculate that the interaction of SIX3 with XNEUROD, XATH3 or XATH5 (any of which abolish Six3's proliferative activity) promotes differentiation in those cells of the retina that co-express these atonal-related protein family members (Tessmar, 2002).

One further aspect of this study is the question of evolutionary conservation of these interactions. By performing a cross-species screening experiment, interaction partners have been selected that are presumably conserved between two different vertebrates. Domain-mapping experiments further support this conservation. Since the conserved bHLH domain of XNEUROD interacts with XSIX3, and XSIX3 interacts with XNEUROD via the conserved SIX domain, it is reasonable to speculate that similar interactions might take place in other, non-vertebrate organisms. However, the interaction between the N-terminus of NEUROD and SIX3 appears to be a specific feature of the NEUROD subfamily, since no conserved domain could be detected at the amino acid sequence level (Tessmar, 2002).

The interaction of XSIX3 with the non-Atonal related bHLH protein XASH1, but not with XESR1 or the more distantly related protein XMAX2, clearly indicates specific interactions with other non-atonal-related bHLH transcription factors, the relevance of which will be addressed in future experiments (Tessmar, 2002).

The homeobox protein Six3 interacts with the groucho corepressor and acts as a transcriptional repressor in eye and forebrain formation

Six3 is a vertebrate homeobox gene that is expressed in the anterior neural plate and eye anlage. Dominant transcriptional activator or repressor forms of Six3 were overexpressed in zebrafish embryos to analyze their effect on eye and forebrain formation. RNA injection of the activator form of Six3 into zebrafish embryos causes reduction of the expression domains for rx2, pax2, and emx1 in the anterior neural plate, resulting in eye and forebrain hypoplasia. However, overexpression of the repressor form of Six3 or wild-type Six3 shows phenotypes the opposite of those of the activator form. Six3 has eh1-related motifs, motifs crucial for transcriptional repression function of Drosophila Engrailed which plays a role in tethering the Groucho corepressor to the promoters. One of the zebrafish Groucho family genes, grg3, has been isolated and an interaction between Six3 and Grg3 has been demonstrated using yeast two-hybrid analysis. Point-mutations in the eh1-related motifs in Six3 reduce both its eye and forebrain enlarging activities and its interaction with Grg3. These results strongly argue that Six3 functions as a Groucho-dependent repressor in eye and forebrain formation. Furthermore, zebrafish Six2 and Six4 also interact with Grg3, implying a conserved function among the Six family proteins as transcriptional repressors (Kobayashi, 2001).

Six3-mediated auto repression and eye development requires its interaction with members of the Groucho-related family of co-repressors

Recent findings suggest that Six3, a member of the evolutionarily conserved So/Six homeodomain family (Drosophila homolog: Optix), plays an important role in vertebrate visual system development. However, little is known about the molecular mechanisms by which this function is accomplished. Although several members of the So/Six gene family interact with members of the Eyes absent (Eya) gene family and function as transcriptional activators, Six3 does not interact with any known member of the Eya family. Grg4 and Grg5, mouse counterparts of the Drosophila transcriptional co-repressor Groucho, interact with mouse Six3 and its closely related member Six6 (Drosophila homolog: Sine Oculis), which may also be involved in vertebrate eye development. The specificity of the interaction was validated by co-immunoprecipitation of Six3 and Grg4 complexes from cell lines. The interaction between Six3 and Grg5 requires the Q domain of Grg5 and a conserved phenylalanine residue present in an eh1-like motif located in the Six domain of Six3. The pattern of Grg5 expression in the mouse ventral forebrain and developing optic vesicles overlapped that previously reported for Six3 and Six6. Using PCR, a specific DNA motif has been identified that is bound by Six3 and it has been demonstrated that Six3 acts as a potent transcriptional repressor upon its interaction with Groucho-related members. This interaction is required for Six3 auto repression. The biological significance of this interaction in the retina and lens was assessed by overexpression experiments using either wild type full-length Six3 cDNA or a mutated form of this gene in which the interaction with Groucho proteins was disrupted. Overexpression of wild type Six3 by in vivo retroviral infection of newborn rat retinae leads to an altered photoreceptor phenotype, while the in ovo electroporation of chicken embryos results in failure of lens placode invagination and production of delta-crystallin-negative cells within the placode. These specific alterations were not seen when the mutated form of Six3 cDNA was used in similar experimental approaches, indicating that Six3 interaction with Groucho proteins plays an essential role in vertebrate eye development (Zhu, 2002).

Six3 and Six6 activity is modulated by members of the groucho family

Six3 and Six6 are two genes required for the specification and proliferation of the eye field in vertebrate embryos, suggesting that they might be the functional counterparts of the Drosophila genes sine oculis (so) and/or optix. Phylogenetic and functional analysis have however challenged this idea, raising the possibility that the molecular network in which Six3 and Six6 act may be different from that described for SO. To address this, yeast two-hybrid screens were performed, using either Six3 or Six6 as a bait. The results of the screen using Six6 is described that led to the identification of TLE1 (a transcriptional repressor of the groucho family) and AES (a potential dominant negative form of TLE proteins) as cofactors for both SIX6 and SIX3. Biochemical and mutational analysis shows that the Six domains of both SIX3 and SIX6 strongly interact with the QD domain of TLE1 and AES, but that SIX3 also interacts with TLE proteins via the WDR domain. Tle1 and Aes are expressed in the developing eye of medaka fish (Oryzias latipes) embryos, overlapping with the distribution of both Six3 and Six6. Gain-of-function studies in medaka show a clear synergistic activity between SIX3/SIX6 and TLE1, which, on its own, can expand the eye field. Conversely, AES alone decreases the eye size and abrogates the phenotypic consequences of SIX3/6 over-expression. These data indicate that both Tle1 and Aes participate in the molecular network that controls eye development and are consistent with the view that both Six3 and Six6 act in combination with either Tle1 and/or Aes. Interestingly, Drosophila Optix shows similar interactions with Groucho as well as with TLE1 and AES (López-Ríos, 2003).

Six3 functions in anterior neural plate specification by promoting cell proliferation and inhibiting Bmp4 expression

Although it is well established that Six3 is a crucial regulator of vertebrate eye and forebrain development, it is unknown whether this homeodomain protein has a role in the initial specification of the anterior neural plate. Exogenous Six3 can expand the anterior neural plate in both Xenopus and zebrafish, and this occurs in part through Six3-dependent transcriptional regulation of the cell cycle regulators cyclinD1 and p27Xic1, as well as the anti-neurogenic genes Zic2 and Xhairy2. However, Six3 can still expand the neural plate in the presence of cell cycle inhibitors and this is likely to be due to its ability to repress the expression of Bmp4 in ectoderm adjacent to the anterior neural plate. Furthermore, exogenous Six3 is able to restore the size of the anterior neural plate in chordino mutant zebrafish, indicating that it has the ability to promote anterior neural development by antagonising the activity of the BMP pathway. On its own, Six3 is unable to induce neural tissue in animal caps, but it can do so in combination with Otx2. These results suggest a very early role for Six3 in specification of the anterior neural plate, through the regulation of cell proliferation and the inhibition of BMP signalling (Gestri, 2005).

To elucidate whether Bmp4 and Xsix3 might antagonise each other, the effects that the overexpression of each of these genes exert on the other were analyzed. Bmp4 overexpression leads to a strong reduction of Xsix3 expression. Conversely, interfering with BMP signalling by injection of either tBR, a dominant-negative BMP receptor, or chordin mRNA induces a strong activation of Xsix3 both in animal caps and in the anterior neural plate of the embryo. Conversely, both VP16-Xsix3 and MoXsix3 injection leads to expansion of Bmp4 expression in the presumptive anterior neural plate. Additionally, TUNEL analysis shows that both Bmp4- and VP16-Xsix3-injected embryos display an anterior accumulation of apoptotic nuclei (Gestri, 2005).

To analyse whether the effects of Xsix3 loss of function are a consequence of BMP4 expansion in the anterior neural plate, whether interfering with BMP signalling can counteract the reduction of the anterior neural plate in MoXsix3-injected embryos was examined. To achieve this, the expression of Zic2 (a gene expressed both in the anterior and posterior neural plate that is strongly modulated by Xsix3), was examined in MoXsix3/tBR co-injected embryos. Injection of MoXsix3 alone represses anterior Zic2 expression. Conversely, MoXsix3/tBR co-injected embryos showed a complete or partial rescue of the Zic2 expression domain. None of the co-injected embryos showed the strong expansion of Zic2 seen for tBR alone. As a control, a similar rescue is observed when MoXsix3 is co-injected with Xsix3. Taken together, these results indicate a mutual antagonism between Xsix3 and Bmp4 (Gestri, 2005).

Six3 represses nodal activity to establish early brain asymmetry in zebrafish

The vertebrate brain is anatomically and functionally asymmetric; however, the molecular mechanisms that establish left-right brain patterning are largely unknown. In zebrafish, asymmetric left-sided Nodal signaling within the developing dorsal diencephalon is required for determining the direction of epithalamic asymmetries. Six3, a transcription factor essential for forebrain formation and associated with holoprosencephaly in humans, regulates diencephalic Nodal activity during initial establishment of brain asymmetry. Reduction of Six3 function causes brain-specific deregulation of Nodal pathway activity, resulting in epithalamic laterality defects. Based on misexpression and genetic epistasis experiments, it is proposed that Six3 acts in the neuroectoderm to establish a prepattern of bilateral repression of Nodal activity. Subsequently, Nodal signaling from the left lateral plate mesoderm alleviates this repression ipsilaterally. These data reveal a Six3-dependent mechanism for establishment of correct brain laterality and provide an entry point to understanding the genetic regulation of Nodal signaling in the brain (Inbal, 2007).

Proper differentiation of photoreceptors and amacrine cells depends on a regulatory loop between NeuroD and Six6

Timely generation of distinct neural cell types in appropriate numbers is fundamental for the generation of a functional retina. In vertebrates, the transcription factor Six6 is initially expressed in multipotent retina progenitors and then becomes restricted to differentiated retinal ganglion and amacrine cells. How Six6 expression in the retina is controlled and what are its precise functions are still unclear. To address this issue, bioinformatic searches and transgenic approaches were used in medaka fish (Oryzias latipes) to characterise highly conserved regulatory enhancers responsible for Six6 expression. One of the enhancers drove gene expression in the differentiating and adult retina. A search for transcription factor binding sites, together with luciferase, ChIP assays and gain-of-function studies, indicated that NeuroD, a bHLH transcription factor, directly binds an 'E-box' sequence present in this enhancer and specifically regulates Six6 expression in the retina. NeuroD-induced Six6 overexpression in medaka embryos promoted unorganized retinal progenitor proliferation and, most notably, impaired photoreceptor differentiation, with no apparent changes in other retinal cell types. Conversely, Six6 gain- and loss-of-function changed NeuroD expression levels and altered the expression of the photoreceptor differentiation marker Rhodopsin. In addition, knockdown of Six6 interfered with amacrine cell generation. Together, these results indicate that Six6 and NeuroD control the expression of each other and their functions coordinate amacrine cell generation and photoreceptor terminal differentiation (Conte, 2010).


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