Table of contents

Vertebrate Eyeless homologs

The Pax6 gene plays a developmental role in various metazoans as the master regulatory gene for eye patterning. Pax6 is also spatially regulated in particular regions of the neural tube. Because the amphioxus has no neuromeres, an understanding of Pax6 expression in the agnathans is crucial for an insight into the origin of neuromerism in the vertebrates. A single cognate cDNA of the Pax6 gene, LjPax6, has been isolated from a Lampetra japonica cDNA library and the pattern of its expression has been observed using in situ hybridization. Phylogenetic analysis has revealed that LjPax6 occurs as an sister group of gnathostome Pax6. In lamprey embryos, LjPax6 is expressed in the eye, the nasohypophysial plate, the oral ectoderm and the brain. In the central nervous system, LjPax6 is expressed in clearly delineated domains in the hindbrain, midbrain and forebrain. The pattern of LjPax6 expression was compared with that of other brain-specific regulatory genes, including LjOtxA, LjPax2/5/8, LjDlx1/6, LjEmx and LjTTF1. Most of the gene expression domains show a conserved pattern, which reflects the situation in the gnathostomes, conforming partly to the neuromeric patterns proposed for the gnathostomes. It is concluded that most of the segmented domains of the vertebrate brain were already established in the ancestor common to all vertebrates. Major evolutionary changes in the vertebrate brain may have involved local restriction of cell lineages, leading to the establishment of neuromeres (Murakami, 2001).

In the evolutionary context, the crucial questions are, therefore, how many segments are arranged in which pattern in the lamprey brain, and which of these patterns are shared between the lamprey and the gnathostomes? In gnathostomes, the positions of the nerve tracts are conserved between species. Such anatomical conservation is known to be associated with compartmentalization of the neural tube: P1 is characterized by the posterior commissure and P2 by the habenular commissure. Caudal to the optic chiasm, tuberal and mammillary hypothalamic territories are clearly identifiable. In the present study, the posterior commissure, the habenular commissure and the optic chiasm were found to have homologous topography, implying the presence of P1 and P2 in all vertebrate brains. This is also consistent with the expression of regulatory genes. In the stage 26 lamprey brain, the rostral domain of LjOtxA expression overlaps the caudal part of the LjPax6-expressing domain. Considering the positions of the epiphysis, the posterior commissure and the habenular commissure, and comparing the pattern with that known for the embryonic mouse brain, the LjOtxA-LjPax6 co-expressing domain most probably corresponds to the dorsal thalamus plus the pretectum (P1+P2). Gene expression patterns and anatomical structures are the only clue to the boundaries of the more rostral segments (Murakami, 2001).

The LjOtxA-expressing domain terminates rostrally at the presumptive P2/P3 boundary (zona limitans), which is assumed from the position of the epiphysis. Rostral to this, LjPax6 and LjDlx1/6 are co-expressed in the dorsal diencephalon, and no expression is seen in the ventral diencephalon (hypothalamus). The expression of LjTTF1 appears to be complementary to the latter (in the ventral diencephalon or hypothalamus), and no transverse segmental boundaries in the forebrain can be detected for the telencephalon. Furthermore, the boundary between the LjPax6- LjDlx1/6 co-expressing domain and the LjTTF1-expressing domain may correspond to the alar-basal plate boundary (Murakami, 2001).

The morphology of the telencephalon is also problematic. Based on the expression patterns of the regulatory genes, the gnathostome telencephalon is assumed to be composed of three major components: the pallium (medial, dorsal, and lateral pallium), the intermediate zone (ventral pallium) and the subpallium (striatum). Emx and Pax6 are expressed in the pallium, and Dlx in the subpallium. In the stage 26 lampery forebrain, a transverse (morphologically horizontal) furrow is found, designated as the sulcus intraencephalicus anterior. Characteristic gene expression is observed in the part of the brain that is rostral and dorsal to this sulcus. LjPax6 is expressed in the dorsal part and LjDlx1/6 in the ventral part, possibly corresponding to the pallium and striatum in the lamprey, respectively. Furthermore LjEmx is restricted to a small dorsal domain that expresses this gene plus LjPax6, and resembles the dorsal pallium of the gnathostomes. These patterns of gene expression in this part of the lamprey brain are extremely reminiscent of the gnathostome telencephalon. Although this may also imply the presence of the P3/P4 boundary (pallio-subpallial boundary), it could not be followed into the dorsal diencephalic and hypothalamic regions. Finally, there is a region in the gnathostome telencephalon that includes the pallidum, in which Dlx and TTF1 are both expressed. The loss of TTF1 expression in the ventral telencephalic region of the lamprey forebrain may be related to the apparent absence of a pallidum in this animal (Murakami, 2001).

In conclusion, the present study of the lamprey brain primordium suggests the presence of the P1 and P2 segments, a longitudinally extending sulcus limitans that terminates rostrally, close to the optic chiasm, a hypothalamus and a tripartite telencephalon-like domain. All these features are directly comparable with those in the model established in the mouse. These results have not further clarified the number of segments in the rostralmost part of the brain. The shared morphological patterns described above are assumed to be very old in origin, possibly dating to the divergence of the lampreys and the gnathostomes. Since recent analyses based on several molecules place hagfishes as the sister group of the lamprey, the segments listed above were already present in the common ancestor of all the vertebrates. The recent discovery of the earliest fossil vertebrates in the early Cambrian period (490-545 million years ago) suggests that the segmental plan underlying vertebrate brain development may have an even longer history. The absence of compartments and the presence of similar anteroposterior regulation by various regulatory genes in cephalochordates imply that the vertebrate-specific compartments listed above were acquired by rough regionalization of the neurectoderm already present in the cephalochordates. It may have been proliferation of neurectodermal cells, as well as the restriction of local cell lineages to form boundaries, that facilitated this most curious evolutionary transition (Murakami, 2001).

Pax6 of zebrafish is initially distributed contiguously throughout a large domain of the anterior neural plate including the presumptive eye field and the dorsal diencephalon. After evagination of the optic vesicle, Pax6 becomes restricted to all proliferating cells of the pigment epithelial and neural layers of the retina. Pax6 is downregulated in most cells concomitant with differentiation. However, it remains present in several mature cell types of the eye, including amacrine cells and the lens and corneal epithelia (Macdonald, 1997).

Development of the vertebrate eye requires a series of steps that include specification of the anterior neural plate, evagination of the optic vesicles from the ventral forebrain, and the cellular differentiation of the lens and retina. Homeobox-containing genes, especially the transcription regulator Pax6, play a critical role in vertebrate and invertebrate eye formation. Mutations in Pax6 function result in eye malformations known as Aniridia in humans and Small eye syndrome in mice. The Drosophila homolog of Pax6, eyeless, is also necessary for correct invertebrate eye development, and its misexpression leads to formation of ectopic eyes in Drosophila. Here it is shown that a conserved vertebrate homeobox gene, Rx, a Pax 6 homeodomain homolog isolated from Xenopus animal-cap ectoderm, is essential for normal eye development, and that its misexpression has profound effects on eye morphology. Xenopus embryos injected with synthetic Rx RNA develop ectopic retinal tissue and display hyperproliferation in the neuroretina. Mouse embryos carrying a null allele of this gene do not form optic cups and therefore do not develop eyes. The Rx gene family plays an important role in the establishment and/or proliferation of retinal progenitor cells (Mathers, 1997).

pax-6 is thought to be a master control gene for eye development in species ranging from insects to mammals. A pax-6 cDNA homolog of the newt, Cynops pyrrhogaster, has been isolated. RT-PCR and sequence analyses predicts four alternatively spliced forms derived from inclusion or exclusion of the region corresponding to exons 5a and 12 in the human pax-6 ortholog. This gene shares extensive sequence identitiy and similar expression patterns with those of mouse and zebrafish. pax-6 signal is first detected at the anterior ridge of the neural plate, and later at the eye and nasal primordium and in the central nervous system (except for the midbrain). The injection of sonic hedgehog (shh) mRNA inhibits the expression of pax-6 within the optic vesicle and disturbs eye cup formation. A similar suppressive effect of shh is also observed in the conjugation of the animal caps (preloaded with exogenous shh and noggin mRNA), which are used as an inducer of pax-6. In contrast, shh injection has no effect on the expression of pax-6 in the surface ectoderm overlying the optic cup, suggesting that the expression of pax-6 in the surface ectoderm is not regulated by shh in vivo. Transient activation of pax-6 is found in animal cap explants at the sibling stage of mid-late gastrula. This observation raises the possibility that the ectoderm is competent to the lens-inducing signal at a stage as early as mid gastrula (Mizuno, 1997).

Members of the paired box (Pax) gene family are expressed in discrete regions of the developing central nervous system, suggesting a role in neural patterning. In this study, the chicken homologs of Pax-3 (Drosophila homolog: Paired) and Pax-6 have been isolated: both genes are very highly conserved and share extensive homology with the mouse Pax-3 and Pax-6 genes. Pax-3 is expressed in the primitive streak and in two bands of cells at the lateral extremity of the neural plate. In the spinal cord, Pax-6 is expressed later than Pax-3, with the first detectable expression preceding closure of the neural tube. Once the neural tube closes, transcripts of both genes become dorsoventrally restricted in the undifferentiated mitotic neuroepithelium. The removal of the notochord, or implantation of an additional notochord, dramatically alters the dorsoventral (DV) expression patterns of Pax-3 and Pax-6. These manipulations suggest that signals from the notochord and floor plate regulate the establishment of the dorsoventrally restricted expression domains of Pax-3 and Pax-6 in the spinal cord. The rapid changes to Pax gene expression that occur in neural progenitor cells following the grafting of an ectopic notochord suggest that changes to Pax gene expression are an early effect of the notochord on spinal cord patterning (Goulding, 1993).

Chicken PAX6 and PAX7 are members of the PAX gene family expressed in late stages of chick nervous system development. Their protein distributions in the CNS, neural crest cells and muscle precursor cells were analyzed by generating monoclonal antibodies against these PAX molecules. Several new features of PAX expression patterns. PAX6 and PAX7 are expressed in restricted regions in the CNS during early stages. However, at later stages, the expression becomes localized to subsets of postmitotic cells in further restricted regions. Such a transition from region-specific expression to cell-specific expression suggests two different roles for these PAX molecules in development: the regionalization and subdivision of the nervous system during early stages, and the differentiation of specific cell populations during late stages (Kawakami, 1997).

Members of the PAX family of transcription factors are candidates for controlling cell identity in the spinal cord. Cells have been morphologically analyzed that express one of these transcription factors, PAX2, demonstrating that multiple interneuron cell types express PAX2. Two ventral populations of PAX2-expressing interneurons in the spinal cord are marked by coexpression of the transcription factors EN1 and EVX1. Interestingly, the expression domains of PAX2, EN1 and EVX1 in postmitotic neurons correlate closely with those of Pax6 and Pax7 in the ventricular zone, implicating these patterning genes in the regulation of PAX2, EN1 and EVX1. PAX2 is first expressed in newly postmitotic cells in the process of migrating laterally from the ventricular zone into the mantle zone. One patterning genes, Pax6, is required for the correct specification of ventral PAX2+ interneurons that coexpress EN1. These results demonstrate that the early activity of patterning genes in the ventricular zone determines interneuron identity in the spinal cord (Burrill, 1997).

A binding site for the paired domain of Pax proteins (designated PBS) has been identified in the mouse N-CAM (See Drosophila Fasciclin 2) promoter. A transcription factor known to be important for development of the central nervous system, Pax-6, binds to the N-CAM PBS; the PBS can also influence N-CAM expression in vivo. Pax-6, produced in COS-1 cells, binds to the PBS through two half-sites, PBS-1 and PBS-2; mutations in both of these sites completely disrupted binding. Moreover, nuclear extracts from embryonic day (E) 11.5 mouse embryos bind to the PBS, and this binding is inhibited by antibodies to Pax-6. To determine the role of the PBS in vivo, transgenic mice were generated with N-CAM promoter/lacZ gene constructs containing either a wild-type or a mutated PBS. Mutations in PBS-1 and PBS-2 decrease the extent of beta-galactosidase expression in the mantle layer of the spinal cord limiting it to ventral regions at E11.5. At E14.5, these mutations eliminated most of the expression that was seen in the wild-type spinal cord. Taken together with previous observations that the PBS binds multiple Pax proteins, the data indicate that such binding contributes to the regulation of N-CAM gene expression during neural development (Holst, 1997).

Three members of a new family of vertebrate genes, designated Eya1, Eya2 and Eya3 have been identified that share high sequence similarity with the Drosophila eyes absent gene. Comparison of all three murine Eya gene products with that encoded by the Drosophila eya gene defines a 271 amino acid carboxyl terminal Eya domain, apparently one that has been highly conserved during evolution. Eya1 and Eya2, which are closely related, are extensively expressed in cranial placodes, in the branchial arches and CNS and in complementary or overlapping patterns during organogenesis. Eya3 is also expressed in the branchial arches and CNS, but lacks cranial placode expression. All three Eya genes are expressed in the developing eye. Eya1 and Eya2 expressions in the lens and nasal placode overlap with and depend upon expression of Pax6. The high sequence similarity with Drosophila eya, the conserved developmental expression of Eya genes in the eye and the Pax6 dependence of Eya expression in the lens and nasal placode indicates that these genes likely represent functional homologs of the Drosophila eya gene (Xu, 1997).

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. The PST domain was dissected in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. In both systems all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act synergistically to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but surprisingly, they do not produce much activity in the context of native PAX6, although the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. The data suggest that these mutants may antagonize the wild-type PAX6 activity by competing for target DNA-binding sites. It is concluded that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree and that its full transactivation activity relies on the synergistic action of the four exon fragments (Tang, 1998).

A short segment of the 5' flanking region of mouse Pax-6 gene has been identified that is necessary and sufficient for reporter construct expression in components of the eye derived from non-neural ectoderm. This transcriptional control element has a highly conserved nucleotide sequence over 341 bp and is located approximately 3.5 kb upstream of the start-point for transcription from the most proximal promoter of the Pax-6 gene. The level of identity between the human and mouse Pax-6 genes in this region is 93%. When combined either with its natural promoter or a heterologous minimal promoter, this element directs reporter construct expression to a region of surface ectoderm overlying the optic cup beginning at E8.5-9.0 (12-14 somites). Subsequently, expression is restricted to the lens (primarily the lens epithelium) and the corneal epithelium. This element will provide an important tool in future transgenic analyses of lens formation and will allow identification of transcription factors that carry out central functions in lens development (Williams, 1998).

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

A survey of ancient conserved non-coding elements in the PAX6 locus reveals a landscape of interdigitated cis-regulatory archipelagos

Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck. This study employed a strategy that combines gnathostome sequence conservation with transgenic mouse and zebrafish reporter assays to survey the genomic locus of the developmental control gene PAX6 for the presence of novel cis-regulatory elements. Sequence comparison between human and the cartilaginous elephant shark (Callorhinchus milii) revealed several ancient gnathostome conserved non-coding elements (agCNEs) dispersed widely throughout the PAX6 locus, extending the range of the known PAX6 cis-regulatory landscape to contain the full upstream PAX6-RCN1 intergenic region. These data indicates that ancient conserved regulatory sequences can be tested effectively in transgenic zebrafish even when not conserved in zebrafish themselves. The strategy also allows efficient dissection of compound regulatory regions previously assessed in transgenic mice. Remarkable overlap in expression patterns driven by sets of agCNEs indicates that PAX6 resides in a landscape of multiple tissue-specific regulatory archipelagos (Bhatia, 2014).

Pax-6 in fish

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 patterning 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 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).

The homeobox gene mbx is first activated at the end of gastrulation in zebrafish in the presumptive forebrain and midbrain region. During somitogenesis stages, the anterior expression of mbx, which partly overlaps the future eye field, gradually decreases, while midbrain expression intensifies and becomes restricted to the presumptive tectum. Knockdown of mbx expression by morpholino antisense oligonucleotides (mbx-MO) leads to a reduction in the size of the eyes and tectum. Expression domains of rx1 and pax6 in the eye field and of mab21l2 in the eye field and tectum anlage are reduced in size in mbx-MO-injected embryos by somitogenesis stages. Further, induction of islet1 and lim3 expression in the eye at 2 days postfertilization (dpf) is suppressed in mbx-MO-injected embryos. In mbx-MO-injected embryos at 2-5 dpf, the lamination of the eye is disorganized and the number of retinal axons is substantially reduced, but the few remaining axons navigate appropriately to the contralateral tectum. A chimeric protein composed of the Mbx DNA-binding domain and the VP16 activation domain affects eye and tectum development similarly to mbx-MO knockdown, suggesting that Mbx acts as a transcriptional repressor in the zebrafish embryo. Based on these data, it is proposed that the mbx homeobox gene is required for the development of the eyes and tectum (Kawahara, 2002).

The Mbx homeodomain is identical among zebrafish, mouse, and human and possesses some similarity to that of Aristaless and Pax family proteins (about 70% identity in the homeodomain). Aristaless and Pax contain glutamine and serine, respectively, at the critical position 50 in their homeodomains, whereas Mbx and Ptx have lysine residues at this position. Indeed, the entire Mbx protein is more similar to Ptx2 than to Cart1 and Alx4. There is no counterpart to Mbx in Drosophila genome sequences or an additional mbx-related gene in the human genome sequence. In addition, no conserved motif other than the homeodomain was detected in Mbx. Thus, sequence analysis indicates that Mbx is a novel homeoprotein highly conserved from zebrafish to human (Kawahara, 2002).

Modifications in Pax6 homeogene expression produce strong eye phenotypes. This suggested that eye development might be an appropriate model to verify if homeoprotein intercellular passage has important functions in early development. Similar to other homeoproteins, Pax6 has two domains that enable secretion and internalization by live cells and, thus, intercellular passage. In principle, a straightforward way to test the hypothesis would be to mutate one of the two sequences to produce a 'cell autonomous only' Pax6. However, this was not possible because these sequences are in the homeodomain and their modification would affect Pax6 transcriptional properties. An approach was developed aimed at blocking Pax6 only in the extracellular milieu of developing zebrafish embryos (Lesaffre, 2007).

A first strategy was to inject a one-cell embryo with a mRNA encoding a secreted single-chain anti-Pax6 antibody. A second, complementary, strategy was to inject a Pax6 antibody in the blastula extracellular milieu. In both cases, 'dissymmetric eyes', 'one eye only' and 'no eye' phenotypes were produced. In most cases, lens phenotypes paralleled retina malformations. Although eye phenotypes were analyzed 30 hours post-fertilization, there was a strong correlation between early eye field asymmetry, early asymmetry in Pax6 expression and later-occurring eye malformations. Several controls were introduced, demonstrating that the effect is specific to Pax6 and cannot be explained by intracellular antibody activities. This study therefore supports the hypothesis that the Pax6 transcription factor is also a signaling molecule with direct non-cell autonomous activity (Lesaffre, 2007; full text of article).

Pax-6 in Xenopus

Xenopus expression of Pax-6 results in lens formation in a cell autonomous manner. In animal cap experiments, Pax-6 induces expression of the lens-specific marker beta B1-crystallin without inducing the general neural marker NCAM. Ectopic Pax-6 expression also results in the formation of ectopic lenses in whole embryos as well as in animal cap explants, indicating that in vertebrates as well as in Drosophila, Pax-6 can direct the development of major components of the eye. Interestingly, ectopic lenses form in whole embryos without association with neural tissue. Treatments giving rise to anterior neural tissue in animal cap explants result in the expression of both beta B1-crystallin and Pax-6. Given the ability of Pax-6 to direct lens formation, it is proposed that the establishment of Pax-6 expression in the presumptive lens ectoderm during normal development is likely to be a critical response of lens-competent ectoderm to early lens inducers. The uncoupling of lens induction from neural induction, suggests that there is no obligatory association between ectopic lenses and neural tissue. In 60% of ectopic lenses in whole embryos there was direct contact with ectopic neural tissue. All neuralizing treatment, including expression of a type I dominant negative bone morphogenetic protein receptor, follistatin, noggin, chordin as well as cell dissociation, results in the induction of both Pax-6 and ßB1-crystallin. These observations are consistent with studies showing that in some species lenses can form independently of the optic cup (Altmann, 1997).

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).

Loss of Pax 6 function leads to an eyeless phenotype in both mammals and insects, and ectopic expression of both the Drosophila and the mouse gene leads to the induction of ectopic eyes in Drosophila, which suggests that Pax 6 might be a universal master control gene for eye morphogenesis. This study reports the reciprocal experiment in which the RNAs of the Drosophila Pax 6 homologs, eyeless and twin of eyeless, are transferred into a vertebrate embryo; i.e., early Xenopus embryos at the 2- and 16-cell stages. In both cases, ectopic eye structures are formed. To understand the genetic program specifying eye morphogenesis, the regulatory mechanisms of Pax 6 expression that initiates eye development have been examined. Notch signaling regulates the expression of eyeless and twin of eyeless in Drosophila. In Xenopus, activation of Notch signaling also induces eye-related gene expression, including Pax 6, in isolated animal caps. In Xenopus embryos, the activation of Notch signaling causes eye duplications and proximal eye defects, which are also induced by overexpression of eyeless and twin of eyeless. These findings indicate that the gene regulatory cascade is similar in vertebrates and invertebrates (Onuma, 2002).

Both Pax 6 homologs, eyeless and twin of eyeless, induce ectopic eye structures in Xenopus embryo. However, judging from the frequency of the ectopic induction of eye-related structures, eyeless is a stronger activator of induction than twin of eyeless. Eyeless is capable of inducing ectopic lenses, whereas twin of eyeless fails to do so, even at higher concentrations. Corresponding to these phenotypical analyses, in the animal cap assays eyeless also shows stronger activity for induction of eye-related gene expression than twin of eyeless. Particularly, alpha-crystallin expression is only induced by misexpression of eyeless and not by twin of eyeless. Overexpression of vertebrate Pax 6 induces ectopic eye structures with lens and ectopic expression of alpha-crystallin in Xenopus embryos. These observations indicate that eyeless is more closely related to vertebrate Pax 6 with respect to eye induction than is twin of eyeless. Because two Pax 6 genes are only found in holometabolous insects and not in hemimetabolous or apterygote insects, the functional divergence of these genes seems to have occurred during insect evolution. The twin of eyeless protein is more similar to the vertebrate Pax 6 proteins than to the eyeless protein, particularly in its overall length and at the C-terminal region. However, the Eyeless protein is more closely related in the paired domain to vertebrate Pax 6 proteins than the twin of eyeless protein. In Drosophila the paired domain of eyeless is essential for its ectopic eye induction, and the expression of a truncated eyeless protein lacking the homeodomain is sufficient to induce ectopic eyes. Therefore, the paired domain of Pax 6 seems to be critical for its conserved function in eye induction (Onuma, 2002).

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).

Table of contents

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

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