Table of contents

Orthodenticle homologs in frogs

The most anterior part of the neural plate is fated to give rise to the retina and anterior brain regions. In Xenopus, this territory is initially included within the expression domain of the bicoid-class homeobox gene Xotx2 but very soon, at the beginning of neurulation, it becomes devoid of Xotx2 transcripts in spatiotemporal concomitance with the transcriptional activation of the paired-like homeobox gene Xrx1. By use of gain- and loss-of-function approaches, the role played by Xrx1 in the anterior neural plate and its interactions with other anterior homeobox genes were examined. At early neurula stage Xrx1 is able to repress Xotx2 expression, thus first defining the retina-diencephalon territory in the anterior neural plate. Overexpression studies indicate that Xrx1 possesses a proliferative activity that is coupled with the specification of anterior fate. Expression of a Xrx1 dominant repressor construct (Xrx1-EnR) results in a severe impairment of eye and anterior brain development. Analysis of several brain markers in early Xrx1-EnR-injected embryos reveals that anterior deletions are preceded by a reduction of anterior gene expression domains in the neural plate. Accordingly, expression of anterior markers is abolished or decreased in animal caps coinjected with the neural inducer chordin and the Xrx1-EnR construct. The lack of expansion of mid-hindbrain markers, and the increase of apoptosis in the anterior neural plate after Xrx1-EnR injection, indicate that anterior deletions result from an early loss of anterior neural plate territories rather than posteriorization of the neuroectoderm. Altogether, these data suggest that Xrx1 plays a role in assigning anterior and proliferative properties to the rostralmost part of the neural plate, and therefore Xrx-1 is required for eye and anterior brain development (Andreazzoli, 1999).

One of the earliest genes to be expressed in the presumptive anterior neuroectoderm at the end of gastrulation is the bicoid-class homeobox gene Xotx2. This gene shows a dynamic expression in this region: it is repressed in the anteriormost part of the neural plate at the beginning of neurulation. This anterior repression of Xotx2 coincides spatially and temporally with the first appearance of Xrx1 transcripts and, in fact, a double whole-mount in situ hybridization shows an almost complete complementarity between the expression pattern of these two genes at stage 12.5. At this time, Xrx1 is also expressed in presumptive telencephalon where it overlaps the expression of XBF-1 and partially overlaps the expression of Xotx2. Thus, different combinations of gene expression appear to pattern the anterior neural plate and define specific territories. The area expressing Xrx1 but neither Xotx2 nor XBF-1 is fated to give rise to retina and diencephalon territories. Even if not perfectly overlapping with Xrx1, Xpax6 and Xsix3 are also expressed in this region. The neural plate area where Xrx1, XBF-1 and Xotx2 are coexpressed corresponds to the presumptive telencephalon while, ventral to the Xrx1 expression domain, the cement gland presumptive region is marked by the expression of XAG-1 and, in part, Xotx2. The lack of apparent activation of Xpax6 and Xsix3 by Xrx1 overexpression in stage 13 embryos may suggest that these two genes are not downstream of Xrx1 at least at this stage, although other explanations cannot be formally excluded. Moreover, the inability of injected Xpax6 RNA both to rescue the Xrx1-EnR phenotypes and to modify Xrx1 expression seems to indicate that Xpax6 and Xrx1 play non-redundant functions in early head development, even if the occurrence of such interactions at later stages cannot be rigorously ruled out (Andreazzoli, 1999).

Regions of the anterior neural plate, where Xrx1 is expressed, are also characterized by a prolonged proliferative period, undergoing neurogenesis with a remarkable delay compared to the posterior neural plate. Overexpression experiments have shown that Xrx1 is able to induce hyperproliferation of the neural tube, neural retina and retinal pigmented epithelium, suggesting that Xrx1 may be responsible for some of the proliferative properties of the anterior neural plate. When the expression of various neuroectodermal markers in Xrx1-injected embryos was analyzed at the tailbud stage, the anterior genes Xpax6, Xsix3 and Xotx2 were found to be ectopically activated in the proliferating area. This ectopic activation is not appreciable at early neural stage, suggesting the existence of stage-dependent differences in Xrx1 activity. For example, in a very speculative scheme, the concentration of Xrx1 at early neurula might be above a threshold level required to support the intensive neural plate proliferation, thus rendering Xrx1 overexpression partly ineffective; on the contrary, the subsequent decrease in the proliferation rate could be counteracted by Xrx1 overexpression, as observed at tailbud stage (Andreazzoli, 1999).

Expression in midbrain-hindbrain boundary of both Xpax2 and En2 as well as rhomboencephalic expression of Krox20 are found to be repressed in Xrx1-injected tailbud embryos. These data suggest that the anteriorizing activity of Xrx1 antagonizes with posteriorizing signals acting in caudal brain regions. This leads to speculate that, during normal development, Xrx1 might contribute to exclude the most anterior regions of the neural plate, where it is expressed, from the range of action of posteriorizing signals. Since posteriorizing signals have also been shown to trigger neuronal differentiation, their repression in the anterior neural plate could represent a basic requirement to allow cell proliferation. Altogether these results indicate that the proliferative activity of Xrx1 is linked to the promotion of the anterior fate, in agreement with other lines of evidence suggesting that mechanisms regulating cell proliferation-neuronal differentiation interact with those that control the anterior-posterior patterning (Andreazzoli, 1999).

Analyses using amphibian embryos have proposed that induction and anteroposterior patterning of the central nervous system is initiated by signals that are produced by the organizer and organizer-derived axial mesoderm. However, here it is shown that the initial anteroposterior pattern of the zebrafish central nervous system depends on the differential competence of the epiblast and is not imposed by organizer-derived signals. This anteroposterior information is present throughout the epiblast in ectodermal cells that normally give rise both to neural and non-neural derivatives. Because of this information, organizer tissues transplanted to the ventral side of the embryo induce neural tissue but the anteroposterior identity of the induced neural tissue is dependent on the position of the induced tissue within the epiblast. Thus, otx2, an anterior neural marker, was only induced in the anterior regions of the embryo, irrespective of the position of the grafts. Similarly, hoxa-1, a posterior neural marker is induced only in the posterior regions. The boundary of each ectopic expression domain on the ventral side is always at an equivalent latitude to that of the endogenous expression of the dorsal side of the embryo. The anteroposterior specification of the epiblast is independent of the dorsoventral specification of the embryo because neural tissues induced in the ventralized embryos also show anteroposterior polarity. Cell transplantation and RNA injection experiments show that non-axial marginal mesoderm and FGF signaling is required for anteroposterior specification of the epiblast. However, the requirement for FGF signaling is indirect because cells with compromised ability to respond to FGF can still respond to anteroposterior positional information (Koshida, 1998).

Previous studies suggested that the Otx2 gene plays an essential role in the development of cranial skeletons and nerves of mesencephalic neural crest origin. To clarify this role, the cis-acting elements in mouse and pufferfish Otx2 genes have been identified as those responsible for the expression in the crest cells. In mouse, 49 bp sequences in the proximal 5' region upstream are essential and sufficient to direct the transgene expression in the cephalic mesenchyme. In pufferfish, the 1.1 kb distal region, located far downstream (from +14.4 to +15.5 kb), has an almost identical activity. Between them, several DNA sequences are conserved, and mutational analyses indicate that motif A is critical for the transgene expression in the premandibular region while motif B is critical in both premandibular and mandibular regions. Motif B, CTAATTA, contains the core motif for binding of homeodomain proteins while motif A, TAAATCTG, does not match any known consensus binding sequences for transcriptional factors. The 5' TA dinucleotide preceding that ATTA is known to bind with a higher affinity to the Gln residue at position 50 in the recognition helix of such homeodomains as those of engrailed, MHox and Msx1 products. The region of cephalic mesenchyme where Otx2 expression is driven by these cis-elements will most likely correspond to mesencephalic crest cells. Thus the molecular machinery regulating Otx2 expression in these cells appears to be conserved between mouse and fish, implying a crucial role for the Otx2 gene in development of the neural-crest-derived structures of the gnathostome rostral head (Kimura, 1997).

Xotx1 and Xotx2 are two Xenopus homologs of the Drosophila orthodenticle gene that are specifically expressed in presumptive head regions that do not undergo convergent extension movements during gastrulation. The function of Xotx1 was compared to that of Xotx2. Ectopic expression of each of the two genes has similar effects in impairing trunk and tail development. Experimental evidence suggests that posterior deficiencies observed in microinjected embryos are due to negative interference with convergent extension movements, a function required for the formation of the tail. Transplantations of putative tail-forming regions show that, while Xotx1 overexpression inhibits tail organizer activity, Xotx2 overexpression is able to turn a tail organizer into a head organizer. Xotx1 and Xotx2 are activated by factors involved in head formation and repressed by a posteriorizing signal like retinoic acid. Taken together, these data suggest that Xotx genes are involved in head-organizing activity. They also suggest that the head organizer may act not only by stimulating the formation of anterior regions, but also by repressing the formation of posterior structures (Andreazzoli, 1997).

Gain-of-function assays in Xenopus have demonstrated that Xwnt-3a (see Drosophila Wingless) can pattern neural tissue by reducing the expression of anterior neural genes, and elevating the expression of posterior neural genes. To date, no loss-of-function studies have been conducted in Xenopus to show a requirement of endogenous Wnt signaling for patterning of the neural ectoderm along the anteroposterior axis. Expression of a dominant negative Wnt in Xenopus embryos causes a reduction in the expression of posterior neural genes, and an elevation in the expression of anterior neural genes, thereby confirming the involvement of endogenous Wnt signaling in patterning the neural axis. The ability of Xwnt-3a to decrease expression of anterior neural genes in noggin-treated explants (noggin is a neural inducer) is dependent on a functional FGF signaling pathway, while the elevation of expression of posterior neural genes does not require FGF signaling. In Xenopus, eGFG, FGF3 and XFGF-9 are expressed in the posterior dorsal mesoderm during gastrulation, consistent with potential roles in neural patterning. The previously reported ability of FGF to elevate the expression of posterior neural genes in noggin-treated explants is found to be dependent on endogenous Wnt signaling. It is concluded that neural induction occurs initially in a Wnt-independent manner, but that generation of complete anteroposterior neural pattern requires the cooperative actions of Wnt and FGF pathways. Noggin induces the anterior markers Xanf-1 and Otx-2 in animal cap explants but in the presence of Xwnt-3a, expression of both markers is reduced. At the same time there is an elevation in expression of the posterior neural markers En-2 and Krox-2, although not the spinal cord marker Hox B9. In the presence of FGF, noggin (in contrast) does not reduce the expression of Xanf-1 or Otx-2, while there is a concurrent induction of posterior genes, including Hox B9. Thus Wnts and FGF can both pattern neural tissues but these factors exhibit differences in their neural patterning activities. Xwnt-3a cannot suppress anterior neural genes in the absence of FGF signaling, indicating that the two pathways work together in neural patterning (McGrew, 1997).

The cement gland of Xenopus forms at early neurula from extreme anterior ectoderm and corresponds to the chin primordium of mammals. Previous studies have shown that misexpressed otx2 can activate cement gland formation. Regionally restricted factors regulate otx2 activity since otx2 is able to activate the cement gland markers XCG and XAG only in ventrolateral ectoderm, and never in the neural plate. Temporal responsiveness of the ectoderm to otx2 is limited, beginning only at mid-gastrula but continuing as late as tailbud stages. otx2 activates expression of the cement gland differentiation marker XCG in the absence of protein synthesis, identifying a direct target of otx2. otx2 can also activate expression of the endogenous otx2 gene, defining an autoregulatory loop. otx2 is sufficient to overcome the inhibitory effects of retinoic acid on cement gland formation, indicating that this effect is caused by failure to express otx2. otx2 autoactivation is prevented by retinoic acid. Together, these findings suggest that otx2 directly controls cement gland differentiation, and that spatial and temporal modulation of otx2 activity limits cement gland formation to the front of the embryo (Gammill, 1997).

The homeobox gene otx2 is a key regulator for specifying the rostral part of the vertebrate head. In Xenopus, otx2 directly controls the differentiation of the cement gland, the anterior-most organ formed in the tadpole. Since embryos of a direct developing frog, Eleutherodactylus coqui, lack a cement gland, it was of interest to see whether altered expression of the otx2 gene is involved in this evolutionary change. The E. coqui homolog of otx2, Ecotx2, was cloned. The homeodomain of the Ecotx2 protein is identical to the mouse and zebrafish Otx2 proteins and differs by a single amino acid from the Xenopus Otx2 protein. Study of the spatiotemporal expression pattern shows that Ecotx2 RNA is progressively restricted to the anterior region of the embryo during gastrulation and becomes further restricted to the future forebrain and midbrain during neural development. In Xenopus, in addition to the conserved expression in the anterior neuroectoderm, the expression in ectoderm expands more anteriorly to the cement gland primordium. This anterior expansion of otx2 expression is not found in E. coqui, correlating with the loss of a cement gland. When misexpressed in Xenopus laevis ectoderm, Ecotx2 can activate expression of the cement-gland-specific genes XCG and XAG1, indicating that the function of activating the pathway of cement gland formation is retained by the Ecotx2 protein. These results indicate that there are modifications in the pathway of cement gland formation, upstream of otx2 expression, in the development of E. coqui (Fang, 1999).

From the onset of neurectoderm differentiation, homeobox genes of the Anf class are expressed within a region corresponding to the presumptive telencephalic and rostral diencephalic primordia. A Xenopus representative of Anf, Xanf-1, is able to control not only early patterning of the forebrain primordium, but also the initial steps of neural commitment of embryonic ectoderm. These data indicate that neurogenesis in vertebrates is regionally specified from the very beginning by the genetic system responsible for the neurectoderm patterning. Ectopic Xanf-1 can expand the neural plate at expense of adjacent non-neural ectoderm. In tadpoles, the expanded regions of the plate develop into abnormal brain outgrowths. At the same time, Xanf-1 can inhibit terminal differentiation of primary neurons. During gastrula/ neurula stages, the exogenous Xanf-1 can downregulate four transcription regulators, XBF-1, Otx-2, Pax-6 and the endogenous Xanf-1, that are expressed in the anterior neurectoderm. However, during further development, when the exogenous Xanf-1 is presumably degraded, re-activation of XBF-1, Otx-2 and Pax-6 is observed in the abnormal outgrowths developed from blastomeres microinjected with Xanf-1 mRNA. Other effects of the ectopic Xanf-1 include cyclopic phenotype and inhibition of the cement gland, both by Otx-2-dependent and -independent mechanisms. Using fusions of Xanf-1 with the repressor domain of Drosophila engrailed or activator domain of herpes virus VP16 protein, it has been shown that most of the observed effects of Xanf-1 are probably elicited by its functioning as a transcription repressor. Altogether, these data indicate that the repressor function of Xanf-1 may be necessary for regulation of both neural differentiation and patterning in the presumptive anterior neurectoderm (Ermakova, 1999).

otx2 activates ectopic formation of the Xenopus cement gland only in ventrolateral ectoderm, defining a region of the embryo that is permissive for cement gland formation. The molecular identity of this permissive area has been explored. One candidate permissive factor is BMP4: putative graded inhibition of BMP4 by factors such as noggin has been proposed to activate both cement gland and neural fates. Several lines of evidence are presented to suggest that BMP signaling and otx2 work together to activate cement gland formation.(1) BMP4 is highly expressed in the cement gland primordium together with otx2; (2) cement gland formation in isolated ectoderm is always accompanied by coexpression of otx2 and BMP4 mRNA, whether cement gland is induced by otx2 or by the BMP protein inhibitor noggin; (3) BMP signaling can modulate otx2 activity, such that increasing BMP signaling preferentially inhibits neural induction by otx2, while decreasing BMP signaling prevents cement gland formation. A hormone-inducible otx2 activates both ectopic neural and cement gland formation within the cement gland permissive region, in a pattern reminiscent of that found in the embryo (Gammill, 2000).

Thus there is a tight correlation between otx2 and BMP4 RNA expression and endogenous and experimentally induced cement gland formation. It is curious that these two factors are coexpressed in the cement gland primordium, since BMP4 inhibits otx2 expression and high levels of otx2 inhibit BMP4 RNA expression, and may help clear BMP4 RNA from the neural plate. How then is the overlap of otx2 and BMP4 RNA expression in the cement gland primordium achieved? Noggin and chordin are likely to be endogenous factors that activate otx2. While at high levels these factors inhibit BMP4 RNA expression, intermediate noggin concentrations (and therefore intermediate BMP signaling levels) activate otx2 and permit BMP4 RNA expression, leading to overlap of BMP4 and otx2 RNA expression. Thus, BMP4 RNA may be expressed in the cement gland primordium simply because its expression is not inhibited there. Alternatively, BMP4 may be actively induced in this region, a possibility suggested by higher levels of BMP4 RNA in presumptive cement gland than in adjacent ventral ectoderm. Within the cement gland, it is possible that otx2 and BMP4 are expressed in the same cells at levels where each does not efficiently inhibit the other. Alternately, since BMP4 is secreted and can act non-cell autonomously, otx2 and BMP4 need only be expressed in neighboring cells in order to functionally interact (Gammill, 2000).

otx2 regulates Xenopus cement gland formation in the ectoderm. otx2 is sufficient to direct anterior neural gene expression, and its activity is required for cement gland and anterior neural determination. otx2 activity at midgastrula activates anterior and prevents expression of posterior and ventral gene expression in whole embryos and ectodermal explants. These data suggest that part of the mechanism by which otx2 promotes anterior determination involves repression of posterior and ventral fates. A dominant negative otx2-engrailed repressor fusion protein (otx2-En) ablates endogenous cement gland formation, and inhibits expression of the mid/hindbrain boundary marker engrailed-2. Ectoderm expressing otx2-En is not able to respond to signals from the mesoderm to form cement gland, and is impaired in its ability to form anterior neural tissue. These results compliment analyses in otx2 mutant mice, indicating a role for otx2 in the ectoderm during anterior neural patterning (Gammill, 2001).

The Rel/NF-kappaB gene family encodes a large group of transcriptional activators involved in myriad differentiation events, including embryonic development. Xrel3, a Xenopus Rel/NF-kappaB-related gene, is expressed in the forebrain, dorsal aspect of the mid- and hind-brain, the otocysts and notochord of neurula and larval stage embryos. Overexpression of Xrel3 causes formation of embryonic tumors. Xrel3-induced tumors and animal caps from embryos injected with Xrel3 RNA express Otx2, Shh and Gli1. Heterodimerization of a C-terminally deleted mutant of Xrel3 with wild-type Xrel3 inhibits in vitro binding of wild-type Xrel3 to Rel/NF-kappaB consensus DNA sequences. This dominant interference mutant disrupts Shh, Gli1 and Otx2 mRNA patterning and inhibits anterior development when expressed in the dorsal side of zygotes: anterior development is rescued by co-injecting wild-type Xrel3 mRNA. In chick development, Rel activates Shh signaling, which is required for normal limb formation -- Shh, Gli1 and Otx2 encode important neural patterning elements in vertebrates. The activation of these genes in tumors by Xrel3 overexpression and the inhibition of their expression and head development by a dominant interference mutant of Xrel3 indicates that Rel/NF-kappaB is required for activation of these genes and for anterior neural patterning in Xenopus (Lake, 2001).

The circadian cycle is a simple, universal molecular mechanism for imposing cyclical control on cellular processes. The regulation of one of the key circadian genes, Clock, was examined in early Xenopus development. Xclk expression is found initially in the organizer region and overlying ectoderm, coincident with the neural inducer noggin. Further, noggin can induce Xclk expression in ectodermal explants along with markers of neural plate, including the cell adhesion molecule NCAM. Interestingly, NCAM is required for photic resetting of the circadian clock in mice; a mutation in NCAM that blocks its conjugation to polysialic acid results in the gradual running down of the circadian cycle in the SCN. The expression of Clock is dependent on developmental stage, not on time per se, and is mostly restricted to the anterior neural plate. It's expression can be induced by the secreted polypeptide noggin, and subsequently upregulated by Otx2, a transcription factor required for the determination of anterior fate (Green, 2001).

Development and differentiation of the vertebrate caudal midbrain and anterior hindbrain are dependent on the isthmic organizer signals at the midbrain/hindbrain boundary (MHB). The future MHB forms at the boundary between the Otx2 and Gbx2 expression domains. Recent studies in mice and chick have suggested that the apposition of Otx2- and Gbx2-expressing cells is instrumental for the positioning and early induction of the MHB genetic cascade. Otx2 and Gbx2 perform different roles in this process. Ectopically expressed Otx2 on its own can induce a substantial part of the MHB genetic network, namely En2, Wnt1, Pax-2, Fgf8 and Gbx2, in a concentration-dependent manner. This induction does not require protein synthesis and ends during neurulation. In contrast, Gbx2 is a negative regulator of Otx2 and the MHB genes. Based on the temporal patterns of expression of the genes involved, it is proposed that Otx2 might be the early inducer of the isthmic organizer genetic network while Gbx2 restricts Otx2 expression along the anterior-posterior axis and establishes an Otx2 gradient (Tour, 2002a).

Anterior-posterior patterning of the embryo requires the activity of multiple homeobox genes, among them Hox, caudal (Cdx, Xcad) and Otx2. During early gastrulation, Otx2 and Xcad2 establish a cross-regulatory network, which is an early event in the anterior-posterior patterning of the embryo. As gastrulation proceeds and the embryo elongates, a new domain forms, which expresses neither Otx2 nor Xcad2 genes. Early transcription of the Xenopus Gbx2 homolog, Xgbx2a, is spatially restricted between Otx2 and Xcad2. When overexpressed, Otx2 and Xcad2 repress Xgbx2a transcription, suggesting their role in setting the early Xgbx2a expression domain. Homeobox genes have been shown to play crucial roles in the specification of the vertebrate brain. The border between the transcription domains of Otx2 and Gbx2 is the earliest known marker of the region where the midbrain/hindbrain boundary (MHB) organizer will develop. Xgbx2a is a negative regulator of Otx2 and a weak positive regulator of Xcad2. Using obligatory activator and repressor versions of Xgbx2a, it has been demonstrated that during early embryogenesis, Xgbx2a acts as a transcriptional repressor. In addition, taking advantage of hormone-inducible versions of Xgbx2a and its antimorph, it has been shown that the ability of Xgbx2a to induce head malformations is restricted to gastrula stages and correlates with its ability to repress Otx2 during the same developmental stages. It is therefore suggested that the earliest known step of the MHB formation, the establishment of Otx2/Gbx2 boundary, takes place via mutual inhibitory interactions between these two genes and this process begins as early as midgastrulation (Tour, 2002b).

To elucidate the molecular basis of organizer functions in Xenopus, target genes were sought of the LIM homeodomain protein Xlim-1, which is one of the organizer-specific transcriptional activators. An activated form of Xlim-1, Xlim-1/3m, initiates ectopic expression of the head-inducing organizer factor BMP antagonist gene cerberus in animal caps. Thus, the cerberus promoter was analyzed using reporter assays. Three consecutive TAAT motifs of the homeodomain-binding sites between positions -141 and -118, collectively designated the '3×TAAT element', are crucial for the response of the cerberus promoter to Xlim-1/3m, and for its activation in the dorsal region of the embryo. Because cooperative activation of the cerberus promoter by Xnr1 and Xwnt8 also requires the 3×TAAT element, focus was place on homeodomain transcriptional activators downstream from either Nodal or Wnt signaling. Wild-type Xlim-1 was found to synergistically activate the cerberus promoter with paired-like homeodomain transcription factor Mix.1 and paired-type homeodomain protein Siamois through the 3×TAAT element, and this synergy requires the LIM domains of Xlim-1. In contrast, Xotx2 acts synergistically with Mix.1, and Siamois through the TAATCT sequence at -95. Electrophoretic mobility shift assays reveal that Xlim-1, Siamois, and Mix.1 are likely to bind as a complex, in a LIM domain-dependent manner, to the region containing the 3×TAAT element. These data suggest that cerberus is a direct target for Xlim-1, Mix.1, Siamois, and Xotx2. Therefore, a model is proposed for the molecular link in the inductive sequence from the formation of the organizer to anterior neural induction (Yamamoto, 2003).

Several eye-field transcription factors (EFTFs) are expressed in the anterior region of the vertebrate neural plate and are essential for eye formation. The Xenopus EFTFs ET, Rx1, Pax6, Six3, Lhx2, tll and Optx2 are expressed in a dynamic, overlapping pattern in the presumptive eye field. Expression of an EFTF cocktail with Otx2 is sufficient to induce ectopic eyes outside the nervous system at high frequency. Using both cocktail subsets and functional (inductive) analysis of individual EFTFs, a genetic network regulating vertebrate eye field specification has been revealed. The results support a model of progressive tissue specification in which neural induction then Otx2-driven neural patterning primes the anterior neural plate for eye field formation. Next, the EFTFs form a self-regulating feedback network that specifies the vertebrate eye field. Striking similarities and differences are found in the network of homologous Drosophila genes that specify the eye imaginal disc, a finding that is consistent with the idea of a partial evolutionary conservation of eye formation (Zuber, 2003).

These remarkable similarities in general developmental design are perhaps logically predicated based on the functional and structural homologies between the Drosophila eye genes and the vertebrate EFTFs. orthodenticle (otd), the Drosophila homolog of Otx genes, is required for development of the eye, antenna and anterior brain, and is normally expressed in a wide domain that spans the dorsal midline and encompasses the entire dorsal head ectoderm. Its expression is turned off in the head midline during development and in the part of the visual primordium that forms the posterior optic lobe and the larval eye. This is strikingly similar to the changes seen in the Xenopus Otx2 expression pattern. The optomotor-blind (omb) gene is a member of the Tbx2 T-box subfamily. ET shares more sequence homology with omb than any other gene in the fly genome. omb expression is first detected in the optic lob anlagen, later expanding to a larger part of the developing larval brain. In the eye imaginal disc, omb is detected in glial precursors, posterior to the morphogenetic furrow and in the optic stalk. Null omb mutants die in pupal stage and show severe optic lobe defects. The Drosophila Rx homolog is not expressed in the larval eye imaginal discs nor the embryonic eye primordia. However, it is expressed prior to ey in the procephalic region from which the eye primordia originates, suggesting a role for Drosophila Rx prior to ey during eye formation in the fly. It has therefore been suggested that Drosophila Rx may only be required for early brain development. Finally, the results showing Pax6 as the most critical component of the Xenopus EFTF cocktail with respect to the induction of ectopic eyes, meshes well with the general prominence given to Pax6 and its Drosophila homologs ey and toy as transcription factors centrally involved in early eye development (Zuber, 2003).

Using the ectodermal explant assay, functional epistatic interactions among the vertebrate EFTFs were examined. There are some striking similarities with the functional interactions among the fly EFTFs. For example, induction of Six3 and Optx2 by Pax6 and induction of Pax6 by Six3 in ectodermal explants are seen. In Drosophila, ey can induce ectopic so and optix expression and ectopic eye formation induced by co-expression of so with eya results in the activation of the ey gene (Zuber, 2003).

Some differences between fly and vertebrate eye formation are also evident. tll is able to induce the expression of Pax6, Six3 and Lhx2, and Pax6 and Six3 induce tll expression. Drosophila tll does not require ey or so in the embryonic visual system. Lhx2 is induced by all the EFTFs investigated in this report with the exception of Optx2. The gene apterous (ap) is the most homologous Drosophila gene to Lhx2; however, apterous loss-of-function mutants have no reported defect in eye formation (Zuber, 2003).

Despite the obvious anatomical differences between the fly and the vertebrate body plans, several genes involved in their development are largely conserved. Evidence is provided that overexpression of the Drosophila orthodenticle (otd) gene in Xenopus laevis has a similar effect to that of its homolog Xotx2. Injections of otd mRNA in whole embryos lead to posterior truncations and to induction of ectopic cement glands, similar to Xotx2 injections. In animal cap assays, otd, like Xotx2, is able to activate the cement gland marker XAG and to suppress the expression of the epidermal marker XK81. otd, like Xotx2, is able to respecify a tail/trunk organizer to a head organizer. In this work it was also shown that Xotx2 and otd share molecular functions that regulate early regional specification of the Xenopus anterior neural plate. Gain-of-function experiment targeting low doses of either otd or Xotx2 mRNAs in the neural plate promote reduction of Xrx1 and Xbf1 expression domain; no changes are observed for the anterior mesodermal marker Xgsc, the dorsal diencephalic marker Xbh1, and the midbrain/hindbrain marker Xen2. otd/Xotx2 inhibition activity of Xrx1 and Xbf1 expression is consistent with the strong inhibition of Xfgf8 expression in the anterior neural ridge observed upon otd/Xotx2 mRNA injection (Lunardi, 2006).

Otx genes, orthologues of the Drosophila orthodenticle, play crucial roles in vertebrate brain development. In the Xenopus eye, Xotx2 and Xotx5b promote bipolar and photoreceptor cell fates, respectively. The molecular basis of their differential action is not completely understood, though the carboxyl termini of the two proteins seem to be crucial. To define the molecular domains that make the action of these proteins so different, and to determine whether their retinal abilities are shared by Drosophila OTD, an in vivo molecular dissection of their activity was performed by transfecting retinal progenitors with several wild-type, deletion and chimeric constructs of Xotx2, Xotx5b and otd. A small 8-10 amino acid divergent region, directly downstream of the homeodomain, was identified that is crucial for the respective activities of XOTX2 and XOTX5b. In lipofection experiments, the exchange of this 'specificity box' completely switches the retinal activity of XOTX5b into that of XOTX2 and vice versa. Moreover, the insertion of this box into Drosophila OTD, which has no effect on retinal cell fate, endows it with the specific activity of either XOTX protein. Significantly, in cell transfection experiments, the diverse ability of XOTX2 and XOTX5b to synergize with NRL, a cofactor essential for vertebrate rod development, to transactivate the rhodopsin promoter is also switched depending on the box. GST-pull down shows that XOTX2 and XOTX5b differentially interact with NRL, though this property is not strictly dependent on the box. These data provide molecular evidence on how closely related homeodomain gene products can differentiate their functions to regulate distinct cell fates. A small 'specificity box' is both necessary and sufficient to confer on XOTX2 and XOTX5b their distinct activities in the developing frog retina and to convert the neutral orthologous OTD protein of Drosophila into a positive and specific XOTX-like retinal regulator. Relatively little is known of what gives developmental specificity to homeodomain regulators. It is proposed that this box is a major domain of XOTX proteins that provides them with the appropriate developmental specificity in retinal histogenesis (Onorati, 2007).

Table of contents

orthodenticle: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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