orthodenticle

EVOLUTIONARY HOMOLOGS

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Orthodenticle homologs in fish

There are three zebrafish otd homologs (otx1, otx2 and otx3), all with roles in brain development (Mori, 1994). Xenopus otd homologs are Xotx2, a target of goosecoid (Blitz, 1995). There are two murine homologs involved in head morphogenesis (Otx1 and Otx2). The two murine genes have a role in establishing the boundary between presumptive dorsal and ventral thalamus. They are also expressed in the olfactory, auricolar and ocular systems (Simoni, 1993).

Two zebrafish sequences contain a homeobox related to orthodenticle. One of these, termed otx1 is clearly homologous to Otx1, a homeobox gene expressed in the developing rostral brain of the mouse. The second gene, termed otx3, is not as closely related to Otx1 and is equally divergent from Otx2, a second otd homolog expressed in the developing rostral brain of the mouse. Both genes are expressed in early-gastrula zebrafish embryos in the involuting presumptive anterior mesendoderm. With the extension of the body axis, the expression domain of both genes extends to neuroectodermal regions fated to become fore- and mid-brain. From this stage the expression domains of the two genes differ slightly from each other but both cover the rostral brain with a sharp posterior boundary coinciding with that boundary found between midbrain and hind-brain. This late expression closely corresponds to that of the murine Otx1 gene, whereas the earliest expression of both zebrafish otx genes is different from that of Otx1 and reminiscent of that of Otx2 in the mouse. From this perspective, the zebrafish otx1 and otx3 genes appear to share some expression features of both murine Otx1 and Otx2. The peculiar spatio-temporal pattern of these genes during early zebrafish gastrulation suggests a role of this gene family in interactions between anterior mesendoderm and neuroectoderm (Mercier, 1995).

It has been an intriguing problem to solve: do the polypeptide growth factors belonging to the transforming growth factor-beta (TGF-beta) superfamily function as direct and long-range signaling molecules in pattern formation of the early embryo? In this study, the mechanism of signal propagation of bone morphogenetic protein (BMP) was determined in the ectodermal patterning of zebrafish embryos, in which BMP functions as an epidermal inducer and a neural inhibitor. To estimate the effective range of zbmp-2, whole-mount in situ hybridization analysis was performed. The zbmp-2-expressing domain and the neuroectoderm, marked by otx-2 expression, are complementary, suggesting that BMP has a short-range effect in vivo. Moreover, mosaic experiments using a constitutively active form of a zebrafish BMP type I receptor (CA-BRIA) demonstrate that the cell-fate conversion, revealed by ectopic expression of gata-3 and repression of otx-2, occurs in a cell-autonomous manner, denying the involvement of the relay mechanism. zbmp-2 is induced cell autonomously within the transplanted cells in the host ectoderm, suggesting that BMP cannot influence even the neighboring cells. This result is consistent with the observation that there is no gap between the expression domains of zbmp-2 and otx-2. Taken together, it is proposed that, in ectodermal patterning, BMP exerts a direct and cell-autonomous effect on the fates of uncommitted ectodermal cells, making them become epidermis (Nikaido, 1999).

Retinoid signaling plays an important role in embryonic pattern formation. Excess of retinoic acid during gastrulation results in axial defects in vertebrate embryos, suggesting that retinoids are involved in early anteroposterior patterning. To study retinoid signaling in zebrafish embryos, a novel method was developed to detect endogenous retinoids in situ in embryos, using a fusion protein of the ligand inducible transactivation domain from a retinoic acid receptor and a heterologous DNA binding domain. Using this method, retinoid signaling is shown to be localized in zebrafish embryos in the region of the embryonic shield, and towards the end of gastrulation in a posterior dorsal domain. To investigate the relationships between the spatial distribution of retinoid signaling and the regulation of retinoid target genes, the downregulation by retinoic acid of goodsecoid and otx1, two genes expressed in anterior regions of the embryo, was studied. These experiments show that expression of both genes is strongly downregulated in the anterior neurectoderm of zebrafish embryos treated with retinoic acid, whereas mesendodermal expression is only mildly affected. Interestingly, a significant downregulation of goosecoid expression by retinoic acid is observed only during midgastrulation but not in earlier stages. In agreement with these results, spatial expression of goosecoid and otx1 does not overlap with the region of retinoid signaling in the late gastrula. These data support the hypothesis that a localized retinoid signal is involved in axial patterning during early development, at least in part through the repression of anterior genes in posterior regions of the embryo. The data suggest that the action of retinoids is spatially as well as temporally regulated in the developing embryo (Joore, 1998).

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).

The embryonic progenitors that give rise to the vertebrate retina acquire their cell fate identity through a series of transitions that ultimately determine their final, differentiated retinal cell fates. In Xenopus, these transitions have been broadly defined as competence, specification, and determination. The expression of several transcription factors within the anterior neural plate at the time when the presumptive eye field separates from other neural derivatives suggests that these genes function to specify competent embryonic progenitors toward a retinal fate. In support of this, some transcription factors expressed in the anterior neural ectoderm and/or presumptive eye field (otx2, pax6, and rx1) change the fate of competent, ventral progenitors, which normally do not contribute to the retina, from an epidermal to a retinal fate. Furthermore, the expression of these factors changes the morphogenetic movements of progenitors during gastrulation, causing ventral cells to populate the native anterior neural plate. In addition, the efficacy of pax6 to specify retinal cells depends on the position of the affected cell relative to the field of neural induction. Thereby, otx2, pax6, and rx1 mediate early steps of retinal specification, including the regulation of morphogenetic cell movements, that are dependent on the level of neural-inductive signaling (Kenyon, 2001).

In the zebrafish embryo, cells fated to give rise to the rostral brain move in a concerted fashion and retain tissue coherence during morphogenesis. Otx proteins have a dramatic effect on cell- cell interactions when expressed ectopically in the zebrafish embryo. Injection of zebrafish Otx1 or Drosophila OTD mRNAs into a single cell at the 16-cell stage results in aggregation of descendants of the injected cell. The Otx/Otd homeodomain is necessary for aggregation and appears to be sufficient for the effect when substituted for the homeodomain of an unrelated homeodomain protein. When cells containing injected zOtx1 RNA are limited to the area that is normally fated to become the anterior brain and neural retina, the induced aggregates contribute to anterior brain and retina tissues. In many other embryonic regions, which do not express endogenous zOtx1, the aggregates appear to be incompatible with normal development and do not integrate into developing tissues. By using an activatable Otx1-glutocorticoid receptor fusion protein that results in the stimulation of cell association, it has been demonstrated that cell aggregates can form as a result of Otx1 activity even after gastrulation is completed. Time-lapse analysis of cell movements show that cell aggregation occurs with only a slight inhibition of the rate of convergence. These results suggest that promotion of cell adhesion or mediation of cell repulsion may be one of the normal functions of the Otx proteins in the establishment of the anterior brain (Bellipanni, 2000).

The Otx2 gene, containing a highly conserved paired-type homeobox, plays a pivotal role in the development of the rostral head throughout vertebrates. Precise regulation of the temporal and spatial expression of Otx2 is likely to be crucial for proper head specification. However, regulatory mechanisms of Otx2 expression remain largely unknown. In this study, the Otx2 genome of the puffer fish Fugu rubripes, which has been proposed as a model vertebrate owing to its highly compact genome, was cloned. Consistently, Fugu Otx2 possesses introns threefold smaller in size than those of the mouse Otx2 gene. Otx2 mRNA was transcribed after MBT, and expressed in the rostral head region throughout the segmentation and pharyngula periods of wild-type Fugu embryos. To elucidate regulatory mechanisms of Otx2 expression, the expression of Otx2-lacZ reporter genes [nearly covering the Fugu Otx2 locus, from -30.5 to +38.5 kb] was analyzed by generating transgenic mice. Subsequently, seven independent cis-regulators were identified over an expanse of 60 kb; these regulators are involved in the mediation of spatiotemporally distinct subdomains of Otx2 expression. Additionally, these expression domains appear to coincide with local signaling centers and developing sense organs. Interestingly, most domains do not overlap with one another, which implies that cis-regulators for redundant expression may be abolished exclusively in the pufferfish so as to reduce its genome size. Moreover, these cis-regions were also able to direct expression in zebrafish embryos equivalent to that observed in transgenic mice. Further comparative sequence analysis of mouse and pufferfish intergenic regions revealed eight highly conserved elements within these cis-regulators. Therefore, it is proposed that, in vertebrate evolution, the Otx2 promoter acquires multiple, spatiotemporally specific cis-regulators in order to precisely control highly coordinated processes in head development (Kimura-Yoshida, 2004).

The F3, F5 and F8 cis-regulatory regions identified in this study direct transgene expression that nearly coincides with local signaling centers in the rostral brain, as defined by the expression Bmps, Shh and Wnts, respectively. The F3 cis-acting region governed expression in the most medial and caudal aspects of the telencephalon, and in the roof of the diencephalon, coincident with the prospective site of choroid plexus development. The expression domains afforded by the F3 cis-region are strikingly colocalized with Bmps, noggin, Msx1 and transthyretin (Ttr). Indeed, Otx2 is expressed in the dorsocaudal telencephalon and in the dorsal diencphalon, including in the prospective choroid plexus and the 'cortical hem' in mouse embryos. In order to establish whether the F3 cis-region is controlled directly by Bmp signaling, experiments were performed involving neural plate explants with BMP beads. The neuroectoderm was isolated at the level of the forebrain from F3placZ transgenic embryos at 10.5 dpc. Recombinant BMP2-coated beads were transplanted into these forebrain explants, and the explants were then cultured for 24 hours. However, no ectopic lacZ expression induced by the BMP2-coated beads was detected. These data suggest that the F3 cis-regulator may not be regulated by BMP signaling. Alternatively, the Otx2 expression from this F3 cis-regulator might participate in the expression of Bmp molecules in the prospective choroid plexus. By contrast, the F8 cis-acting region directed transgene expression in the mediocaudal telencephalon where Wnt molecules are co-expressed, defining a zone termed 'cortical hem.' Therefore, Wnt signals might control the Otx2 expression mediated by the F8 cis-region in the cortical hem and dorsal telencephalon. Additional transgenic zebrafish studies have indicated that F3 and F8 cis-regions directed considerably conserved expression in the forebrain. Concomitant with this finding, zebrafish Wnt8b is expressed in the dorsal forebrain. These data suggest that local signaling centers play an essential role in forebrain development in zebrafish embryos and are evolutionarily conserved among vertebrates (Kimura-Yoshida, 2004).

The F5 cis-acting region directs expression in the zona limitans intrathalamica (ZLI), and in ventral portions of the diencephalon and the lateral mesencephalon that may be related to longitudinal columns termed 'midbrain arcs.' Notably, mouse Otx2 mRNA and Otx2 protein are expressed in the ZLI, and in the ventral diencephalon and mesencephalon in mouse embryos. Importantly, expression in the ZLI, ventral diencephalon and midbrain arcs are proposed to be closely related to Shh signaling, which is essential for the dorsoventral patterning of the ventral neural tube. Therefore, these expression patterns suggest the possibility that Shh signaling controls the Otx2 expression mediated by the F5 cis-acting region (Kimura-Yoshida, 2004).

lacZ activity directed by the F9, F11 and F12 cis-regions appears to be closely related to the developing organs. Notably, it has been suggested that Otx2 is required for the development and specification of these specific organs. The F9 cis-region governs expression in the cephalic mesenchyme. Consistently, Otx2 is expressed in the cephalic mesenchyme, and functions in the formation of premandibular and mandibular skulls. The F11 region governs expression in the dorsal pretectum and mesencephalon, including in the posterior commissure, the mesencephalic trigeminal neurons, the oculomotor nerve, the first and second branches of the trigeminal nerve, and the trigeminal ganglions. In actuality, Otx2 mRNA is expressed in the dorsal pretectum and mesencephalon, in the trigeminal ganglions and opthalmic branch. Furthermore, as Otx2 heterozygous mutant mice display anomalies of these neurons and cranial nerves, Otx2 may be required for the proper development of these tissues cell-autonomously. The F12 cis-region drives expression in the developing sense organs, the olfactory epithelium, the pigment layer of the retina, and the developing inner and outer ears. Otx2 expression is consistently found in these developing sense organs. Moreover, Otx2 is required for normal development of the inner ear and pigment epithelium, cooperatively with the Otx1 gene (Kimura-Yoshida, 2004).

Positioning of the zebrafish midbrain-hindbrain boundary organizer through global posteriorization of the neuroectoderm mediated by Wnt8 signaling

The organizing center located at the midbrain-hindbrain boundary (MHB) patterns the midbrain and hindbrain primordia of the neural plate. Studies in several vertebrates have shown that the interface between cells expressing Otx and Gbx transcription factors marks the location in the neural plate where the organizer forms, but it is unclear how this location is set up. Using mutant analyses and shield ablation experiments in zebrafish, it has been found that axial mesendoderm, as a candidate tissue, has only a minor role in positioning the MHB. Instead, the blastoderm margin of the gastrula embryo acts as a source of signal(s) involved in this process. Positioning of the MHB organizer is tightly linked to overall neuroectodermal posteriorization, and specifically depends on Wnt8 signaling emanating from lateral mesendodermal precursors. Wnt8 is required for the initial subdivision of the neuroectoderm, including onset of posterior gbx1 expression and establishment of the posterior border of otx2 expression. Cell transplantation experiments further show that Wnt8 signaling acts directly and non-cell-autonomously. Consistent with these findings, a GFP-Wnt8 fusion protein travels from donor cells through early neural plate tissue. These findings argue that graded Wnt8 activity mediates overall neuroectodermal posteriorization and thus determines the location of the MHB organize (Rhinn, 2005).

How does Wnt8 participate in positioning of the MHB organizer? wnt8 is expressed in the marginal cells and hypoblast and two receptors, fz8c and fz9, are detected in both hypoblast and epiblast. Conceivably, Wnt8 is transmitted in a planar fashion through the neuroectoderm. This idea is supported by the clonal analysis of wnt8 overexpressing cells: gbx1 is activated in the host tissue one or two cells distant from the transplanted cells, and otx2 is repressed four or five cells distant from the transplanted cells. In unmanipulated neuroectoderm, the onset of gbx1 expression occurs close to the wnt8 domain with little or no overlap, and the otx2 expression domain is situated eight to ten cell diameters away from the wnt8 domain at 60% of epiboly. Thus, the wnt8 expression domain is appropriately located to generate a graded morphogenetic Wnt8 signal that regulates the expression of gbx1 and otx2 genes in vivo. This finding is more generally consistent with the ability of Wnt molecules to form gradients and to activate target genes in a concentration-dependent manner, as in the Drosophila wing imaginal disc, where expression of wingless target genes like neuralized, distalless and vestigial depends on the distance from wingless-expressing cells. Similarly, in the unmanipulated zebrafish neuroectoderm, the otx2 and the gbx1 domains are located at different distances from the Wnt8 source at the lateral blastoderm margin. Following global misexpression experiments, different Wnt8 doses can differentially regulate otx2 and gbx1 expression: wnt8 ectopic expression can induce gbx1 expression at low/intermediate doses, but represses at high doses. Conversely, otx2 is increasingly repressed with increasing wnt8 concentration. Similarly, around wnt8-expressing clones, gbx1 is induced at a distance of one or two cells around the clone, whereas otx2 is repressed at a distance of four or five cells. This suggests that a lower Wnt8 concentration is needed to repress otx2 than to induce gbx1. Altogether, these observations suggest that Wnt8 has properties of a morphogen whose activity is required to correctly position the otx2/gbx1 interface, and probably other target genes in the forming neural plate. The observation of secreted Wnt8-GFP protein emanating from clones of producing cells is generally consistent with this possibility. Distribution of another signaling molecule in the early neural plate, Fgf8, is carefully controlled by endocytosis. It will be interesting to determine if Wnt8 protein is indeed distributed in a graded fashion, and which mechanisms control this distribution. In mice, Wnt8 is expressed in the posterior epiblast of early primitive streak-stage embryos; although its function is unknown, Wnt8 may therefore serve a similar function as proposed in this study (Rhinn, 2005).

Studies in mouse, Xenopus and chicken have shown that Otx2 and Gbx2 expression domains are fundamental for positioning the midbrain-hindbrain boundary (MHB) organizer. Of the two zebrafish gbx genes, gbx1 is a likely candidate to participate in this event because its early expression is similar to that reported for Gbx2 in other species. Zebrafish gbx2, in contrast, acts relatively late at the MHB. To investigate the function of zebrafish gbx1 within the early neural plate, a combination of gain- and loss-of-function experiments was used. Ectopic gbx1 expression in the anterior neural plate reduces forebrain and midbrain, represses otx2 expression and repositions the MHB to a more anterior position at the new gbx1/otx2 border. In the case of gbx1 loss-of-function, the initially robust otx2 domain shifts slightly posterior at a given stage (70% epiboly), as does MHB marker expression. Ectopic juxtaposition of otx2 and gbx1 leads to ectopic activation of MHB markers fgf8, pax2.1 and eng2. This indicates that, in zebrafish, an interaction between otx2 and gbx1 determines the site of MHB development. This work also highlights a novel requirement for gbx1 in hindbrain development. Using cell-tracing experiments, gbx1 was found to cell-autonomously transform anterior neural tissue into posterior. Previous studies have shown that gbx1 is a target of Wnt8 graded activity in the early neural plate. Consistent with this, it was shown that gbx1 can partially restore hindbrain patterning in cases of Wnt8 loss-of-function. It is proposed that in addition to its role at the MHB, gbx1 acts at the transcriptional level to mediate Wnt8 posteriorizing signals that pattern the developing hindbrain. These results provide evidence that zebrafish gbx1 is involved in positioning the MHB in the early neural plate by refining the otx2 expression domain. In addition to its role in MHB formation, gbx1 is a novel mediator of Wnt8 signaling during hindbrain patterning (Rhinn, 2009).

In non-mammalian vertebrates, the pineal gland is photoreceptive and contains an intrinsic circadian oscillator that drives rhythmic production and secretion of melatonin. These features require an accurate spatiotemporal expression of an array of specific genes in the pineal gland. Among these is the arylalkylamine N-acetyltransferase, a key enzyme in the melatonin production pathway. In zebrafish, pineal specificity of zfaanat2 is determined by a region designated the pineal-restrictive downstream module (PRDM), which contains three photoreceptor conserved elements (PCEs) and an E-box, elements that are generally associated with photoreceptor-specific and rhythmic expression, respectively. By using in vivo and in vitro approaches, it has been found that the PCEs and E-box of the PRDM mediate a synergistic effect of the photoreceptor-specific homeobox OTX5 and rhythmically expressed clock protein heterodimer, BMAL/CLOCK, on zfaanat2 expression. Furthermore, the distance between the PCEs and the E-box was found to be critical for PRDM function, suggesting a possible physical feature of this synergistic interaction. OTX5-BMAL/CLOCK may act through this mechanism to simultaneously control pineal-specific and rhythmic expression of zfaanat2 and possibly also other pineal and retinal genes (Appelbaum, 2005).

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