InteractiveFly: GeneBrief

Retinal homeobox : Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The possibility that mechanisms of retinal determination may be similar between vertebrates and Drosophila has been supported by the observations that Pax6/eyeless genes are necessary and sufficient for retinal development. These studies suggest that the function of other gene families, operating during early eye development, might also be conserved. One candidate is the retinal homeobox (Rx) family of transcription factors. Vertebrate Rx is expressed in the prospective eye and forebrain and is required for eye morphogenesis, retinal precursor appearance, and normal forebrain development, indicating that it is an essential regulator of early eye and brain formation. The hypothesis that Drosophila Retinal homeobox (Rx) is required for adult and larval eye development was tested. When a Rx null allele was isolated, the mutant compound eye and larval visual system were not detectably abnormal. However, Rx is required for development of a central brain structure, the ellipsoid body, suggesting that Rx function in the brain may be conserved. Finally, a novel anterior head phenotype has been characterized; Rx is required for clypeus development. Thus, the data suggest that Rx may be required for the regulation of genes involved in brain morphogenesis and clypeus precursor development. It is proposed that differences in insect and vertebrate eye development may be explained by changes in gene regulation and/or the tissue of origin for eye precursor cells (Davis, 2003).

The Rx family of transcription factors contains a paired-like homeodomain and has been identified in chordates and invertebrates. Vertebrate Rx is expressed in the anterior neural plate (ANP), which gives rise to the eye and forebrain. Expression in the presumptive eye resolves to the neuroretina, while expression in the prospective forebrain becomes restricted to the hypothalamus, pituitary, and pineal gland. Loss-of-function mutations in mouse and medaka Rx result in severe early eye phenotypes (anophthalmia), in addition to defects in forebrain development. Retinal phenotypes are also observed in chicken and Xenopus embryos expressing dominant-negative Rx constructs. In addition, Rx is required for expression of Pax6 in the optic vesicle, consistent with an early role in establishing retinal precursors. Finally, overexpression of Rx induces ectopic retinal pigmented epithelium and duplications of the retina and neural tube. These studies are consistent with a role for Rx in the specification, patterning, and/or proliferation of eye and brain precursors within the ANP (Davis, 2003 and references therein).

Drosophila Rx (Drx) and vertebrate Rx share greater than 95% amino acid identity within their predicted homeodomains, indicating a high level of structural conservation (Eggert, 1998, Mathers, 1997 and Ohuchi, 1999). Drosophila rx is expressed in the procephalon, a region that gives rise to eye imaginal primordia and brain hemispheres. This pattern partly resembles vertebrate Rx expression in the ANP, suggesting that Drosophila Rx function has been conserved between taxa (Eggert, 1998; Mathers, 1997). However, although Drosophila rx is expressed in the embryonic brain, it has not been detected in eye imaginal primordia of late embryos or larval imaginal discs, suggesting that rx may have no function during eye development or that it functions earlier during the establishment of the eye primordia (Davis, 2003).

P-element-mediated mutagenesis and a local hopping strategy were used to generate Rx mutations. The ammunition element fam^k07505 is located ~85 kb proximal to Rx. An initial screen yielded a viable insertion, act57B^P(F5), located 431 bp upstream of act57B exon1. Using act57B^P(F5) in a second screen, a chromosome was isolated with two viable insertions: Rx^P(3A2), located 2.5 kb upstream of Rx, and the original act57B^P(F5). To generate deletions of the Rx locus, an excision screen was performed, using the double-insertion chromosome and Delta2-3 transposase. Lines were screened for loss of Rx sequences by PCR (Davis, 2003).

Excision line Rx^ex8 was selected for further analysis. In Rx^ex8 fly cultures, the majority of homozygotes fail to eclose and die as pharate adults. In the rare mutants that do eclose, their wings fail to unfold, they become dehydrated, and do not survive beyond 48 h. Unfolding of Rx^ex8 mutant wings with KOH demonstrates that there are no gross structural defects. Complementation analysis using deficiencies in the region demonstrates that the Rx^ex8 culture phenotype maps near the Rx locus. To confirm that loss of Rx function is responsible for the culture phenotype, attempts were made to rescue Rx^ex8 mutants with a Rx minigene, BSKK. While the majority of Rx^ex8 mutants fail to eclose, Rx^ex8BSKK/Rx^ex8 animals can successfully eclose, have straight wings, survive, and reproduce (Davis, 2003).

The clypeus is a part of the cibarium, which acts as a pump to draw food under the labrum, between anterior and posterior cibarial plates and into the esophagus. Suction is created by separation of the plates, due to contraction of muscles attached to the anterior cibarial plate and clypeus. To determine whether other parts of the oral pump were missing, both an external and internal evaluation of the Rx^ex8 cibarium was performed. Scanning electron microscopy reveals that the labrum is present in the mutants. However, analysis of H and E-stained sections from Rx^ex8 mutants reveals an abnormal cibarium. In controls, the anterior and posterior plates run parallel to each other and flank cibarial musculature. These muscles are organized by attachment to an apodeme, which is part of the anterior plate, and the clypeus . In frontal sections of Rx^ex8 mutants, the cibarial plates are present, as well as the anterior apodeme. However, the musculature is disorganized and does not appear to be consistently attached to the apodeme. These data demonstrate that Rx is not required for formation of the labrum or cibarial plates (Davis, 2003).

Transplantation studies have demonstrated that the clypeus, labrum, and cibarial plates are derived from the clypeolabral imaginal disc. Since Rx is required for development of the clypeus and is expressed in the embryonic clypeolabral bud, whether Rx is expressed in the larval clypeolabral imaginal disc was determined. Three criteria were used to identify the disc: (1) the clypeolabral disc is within the cephalopharyngeal skeleton (CPS); (2) imaginal disc cells are evident based on their small size compared with larval cells, and (3) a lacZ enhancer trap within the headcase locus is active in the clypeolabral disc. Using Rx and ß-galactosidase antibodies, immunohistochemistry was performed on the clypeolabral disc from headcase-lacZ larvae. Comparison of the Rx and ß-galactosidease staining patterns reveals that Rx is expressed in a subset of clypeolabral disc cells. This result correlates with a requirement for Rx in clypeus, but not the labrum or cibarial plates development (Davis, 2003).

Rx expression in the embryonic, larval and adult brain suggests that Rx may be required for brain development (Eggert, 1998). H and E-stained sections of Rx^ex8 mutant adult brains were examined. At the level of the central complex, analysis reveals an abnormal ellipsoid body (EB) in the mutants. In wild-type brains, the H and E-stained EB neuropile appears as a midline ring, dorsal to the esophagus. In contrast, the EB in Rx^ex8 mutants is disrupted. To confirm and extend these findings, immunohistochemistry was performed on frozen sections using FasII antibodies. In controls, the FasII-stained EB appears as concentric rings flanked by the mushroom body peduncle. In contrast, the FasII-stained EB rings are missing and are replaced by an elongated neuropile. Analysis using Leonardo antibodies, which also stain the EB, confirms that the Rx^ex8 mutant EB is malformed. These findings indicate that, in Rx^ex8 mutants, the EB is abnormally structured (Davis, 2003).

The GAL4/UAS system has been used to characterize the EB. Based on studies using multiple GAL4 drivers, the EB consists of large and small field neural processes that form distinct rings. Four main types of large field neurons, R1, R2, R3, and R4, have been identified. To examine the Rx^ex8 EB phenotype in more detail, the P{GAL4^c232} line (c232) was used; this line expresses GAL4 in R3 and R4 neurons, which contribute to the inner and middle ring, and outer ring, respectively. To directly visualize cell bodies and projections, a P{UAS-mCD8::GFP} GAL4 reporter gene (UCG), which expresses membrane-bound GFP, was used. In controls, the c232 cell bodies are located near the anterior surface of the brain. These neurons send projections toward the midline, through the lateral triangle and arborize into two concentric EB rings. This pattern does not vary significantly between control males, females, pharate, newly eclosed, or older adults. In contrast, in Rx^ex8 homozygotes, the pattern of EB arborization is severely disrupted. Although the mutant c232 cells project axons, which form a partial inner and outer ring, the rings fail to assemble at the midline (Davis, 2003).

During analysis of FasII-stained brain sections, variation was observed in the EB phenotype. To confirm this observation, c232 neurons of 47 mutant female brains were examined and compared. The results demonstrate 3 categories of EB phenotypes. The most severe 'unfused' phenotype, where termini fail to intersect at the midline, is observed in over half of the mutants (57%). A less severe 'elongated' phenotype is seen in one-third of the mutants, where the EB processes cross the midline, but are abnormal in width (28%). Finally, the least severe 'ventral' phenotype, where the EB width is normal, but the ventral processes fail to close the rings, is also the least frequent (15%). These phenotypes are observed in both sexes, in pharate adults and rare escapers (Davis, 2003).

To determine the role of Rx in brain development, the ability of a Rx minigene BSKK to rescue the c232 EB phenotype was tested. The Rx minigene is functional, since it can rescue the Rx^ex8 eclosion and clypeus phenotypes. The BSKK minigene can, at low frequency, completely rescue the EB phenotype. In addition, there is a significant reduction in the percentage of animals with the most severe 'unfused' phenotype (from 57% to 16%), and an increase in the percentage of animals with less severe 'elongated' (28% to 47%) and 'ventral' defective (15% to 33%) phenotypes. These data indicate an essential role for Rx in correctly forming and positioning the c232 EB (Davis, 2003).

In conclusion, the ellipsoid body in Rx mutants is abnormally formed, suggesting a conserved role for Rx during brain development. In addition, Rx mutants lack an anterior head structure, the clypeus, which is a component of the feeding apparatus. In general, these phenotypes correlate with the embryonic and larval expression patterns of Rx: while not detectably expressed in direct eye precursor cells, Rx is expressed in the developing brain and a derivative of the clypeolabral bud (Eggert, 1998; Davis, 2003).

Vertebrate Rx genes are expressed in strikingly similar patterns and have similar functions. In early vertebrate embryos, Rx is expressed in the anterior neural plate, including the prospective eye and forebrain. As might be expected, loss-of-function mutations in mouse and medaka Rx result in anophthalmia and severe forebrain defects. Similarly, expression of dominant-negative Rx constructs in chicken and Xenopus embryos results in eye and brain phenotypes. In addition, Rx is required for Pax6 expression in optic vesicle progenitors. Moreover, overexpression studies demonstrate Rx can induce ectopic retinal development. These studies indicate a role for Rx in establishing the early eye field in such diverse species as fish, amphibians, birds, and mammals (Davis, 2003 and references therein).

Although Rx and vertebrate Rx share some similarities, these genes differ in their requirements during establishment of the eye. The Drosophila Rx homeodomain shares greater than 95% amino acid identity with vertebrate Rx homeodomains, indicating a high level of structural conservation. In addition, Rx expression in the embryonic procephalic region bears a resemblance to vertebrate Rx expression in the ANP. However, the visual system in Drosophila Rx mutants is normal and gross abnormalities were detected. Since Rx^ex8 is a null allele and no other Rx homologs are in the sequenced fly genome, there is no residual Rx function in the Rx^ex8 flies. Recently, planarian Rx homologs from Dugesia japonica and Girardia tigrina have been cloned, but they are not detectably expressed in the eye (Salo, 2002). These results suggest that the utilization of Rx function in the development of light-sensing organs of some invertebrates has not been required since the divergence of vertebrata from a common bilaterian ancestor. However, it is still possible that Rx plays some role in eye function or may be required for aspects of eye development that have not been detected by the methods that were used to examine the mutant phenotype (Davis, 2003).

Evidence has been forwarded that the topology map of the embryonic brain/eye anlage in Drosophila is similar to the fate map of the vertebrate ANP. This map is based on the effect of hedgehog and decapentaplegic mutations on patterning the expression of conserved genes implicated in brain and eye development, including orthodenticle, tailless, eyeless, eyes absent, and sine oculis. However, comparison of vertebrate Rx and Rx expression patterns indicates a level where the topology map is not conserved. While vertebrate Rx expression in the ANP encompasses forebrain and retinal anlage, Rx expression appears within the brain anlage but probably not in the region giving rise to the visual system. Moreover, the data confirm the absence of a requirement for Rx in the gross development of the adult and larval visual systems. Thus, the lists of genes required for establishment of the fly and vertebrate eye anlage are not identical, despite the otherwise striking parallels in embryonic head patterning (Davis, 2003 and references therein).

There are many possible explanations for a 'breakdown' within the conserved topology map, including differences related to the development of the eye between insects and vertebrates and/or changes in Rx regulation during evolution. The vertebrate retina is derived from the neuroepithelium, while the Drosophila compound eye develops from embryonic surface ectoderm, which is set aside as an imaginal disc and develops as retinal tissue during larval stages. In this sense, the insect eye shares more character with the vertebrate lens, which is also derived from the surface ectoderm adjacent to the neuralized epithelium. It is interesting to note that expression of vertebrate Rx genes has not been detected in the developing lens. One possibility is that the tissue-specific regulation of Rx has remained the same in insects and vertebrates, but that the pool of cells recruited to become the primary light-sensing organ differs between animals. Alternatively, some aspects of Rx regulation may have changed since the divergence of ancestral bilaterians or Rx may not have been recruited for function in the invertebrate visual system due to compensatory involvement of other genes (Davis, 2003).

Other aspects of Rx expression suggest conservation of Rx function in the brain. In particular, Rx expression in the brain anlage is in close proximity to, or contained within, orthodenticle and tailless expression domains, which is similar to the expression profiles of vertebrate Rx, Otx2, and Tlx in the prospective forebrain. This suggests that some modes of Rx/drx regulation by neuralizing factors may have originated in ancestral bilaterians and remain mostly unchanged (Davis, 2003 and references therein).

Analysis of the Rx^ex8 allele reveals ellipsoid body (EB) abnormalities within the mutant adult brain. The EB is a circular neuropile that is part of the central complex, which includes the mushroom body, fan-shaped body, noduli, and protocerebral bridge. The EB has been implicated in the control of specific locomotor skills, since mutations that affect EB structure are associated with changes in walking and flight activities. No EB embryonic precursor has been identified, but an EB-specific GAL4 line has been used to demonstrate that ring development begins around 32 h after puparation formation (APF) and is completed by midpupal stage (~48 h APF). GAL4 drivers have been used to analyze R2, R4m, and R4d EB neurons in ellipsoid body open (ebo), central body defect (cbd), and central complex deranged (ccd) mutants. ebo mutant EB neuropiles are fused at the midline, but lack variable aspects of the ventral ring. cbd mutant EBs frequently fail to join at the midline and display ventral defects. Finally, ccb mutants EB exhibit a range of phenotypes, from duplication at the midline to ventral defects (Davis, 2003).

R3 and R4d EB neuropiles were analyzed in Rx^ex8 mutants, using the c232 GAL4 driver, and phenotypes similar to ebo, cbd, and ccd: unfused, elongated, and incompletely fused EBs, were found. In the most severe phenotype, Rx^ex8 mutant EBs fail to fuse at the midline, which is similar to the cbd phenotype, and consistently lack a ventral aspect similar to the ebo and ccd mutant EBs. However, Rx^ex8 mutants also exhibit an 'elongated' EB, which has not been documented in ebo or cbd mutants. In each instance, the EB ring is incomplete along the ventral aspect, suggesting that ring completion is more sensitive to genetic perturbation than ring positioning. These EB phenotypes may be due to differences in the ability of growth cones to respond to midline cues. Alternatively, brains with mutant EBs may be missing pioneering tracts or supporting glia that are required for ring positioning. Finally, loss-of-function mutations in these genes may affect similar, but nonidentical sets of neurons that constitute the EB (Davis, 2003).

Rescue analysis indicates that both Rx and act57B transgenes reverse the Rx^ex8 EB phenotype in a similar manner. This picture contrasts with activities of these transgenes in other assays. While the Rx transgene rescues the Rx^ex8 culture and clypeus phenotypes, the act57B transgenes fail to do so. Conversely, the act57B transgene can rescue the prepupal lethality in Rx^ex8/E2 mutants, but the Rx transgene cannot rescue this phenotype. Taken together, these data indicate that Rx and act57B are both required for EB development. When the transgenes are combined in the same fly, their effect on EB rescue is additive but not synergistic, consistent with the possibility that the two genes act independently (Davis, 2003).

At this early stage of analysis, it is difficult to precisely define the mechanisms by which Rx and act57B function in EB development. However, some predictions can be made based on the expected properties of these proteins. act57B encodes an actin isoform expressed in embryos, larvae, and adults, and no mutations in this gene have previously been described. Since cell motility depends on actin polymerization dynamics, it is possible that the Rx^ex8 EB phenotype is partly due to abnormal act57B levels; this results in a cell population that fails to migrate/project axons correctly within the brain. Indeed, the actin binding protein Ciboulot is important in regulating cytoskeleton dynamics and is required for normal EB development. Of interest, the ciboulot mutant EB phenotype is similar to the ventrally defective Rx^ex8 EB phenotype, suggesting that regulation of act57B polymerization by Ciboulot may be required for axons closing the EB ring (Davis, 2003 and references therein).

In contrast, Rx encodes a putative transcription factor, suggesting a role regulating gene expression in the developing brain. It is possible that, in Rx^ex8 mutant brains, the absence of Rx results in changes in the specification, differentiation, and/or growth of cells that are required for normal EB development. Other transcription factors, such as AP-2, Dachshund, and Eyeless, are also required for normal EB development. Since Rx and act57B appear to act in different pathways, it is unlikely that Rx directly regulates act57B. The isolation of gene-specific mutations is required to characterize the individual roles of Rx and act57B in the formation of the EB. Once gene-specific mutations are generated, it will be possible to precisely assign other genes to either the Rx or act57B pathways (Davis, 2003).

Larval transplantation studies have demonstrated that the clypeolabral imaginal disc lies within the cephalopharyngeal skeleton (CPS) and gives rise to the adult clypeus, labrum, and cibarial plates, which form components of the oral pump (cibarium). Histological analysis using an imaginal disc marker, headcase-lacZ, has confirmed the presence of a single pair of discs inside the CPS. Beyond this, however, little information about the disc or its derivatives is available. No fate mapping studies or developmental characterization of the disc has been conducted, most likely a result of a lack of useful adult markers and the disc's intractable location. Finally, no mutations have previously been described that specifically affect the adult structures derived from the disc (Davis, 2003).

This work represents an initial step toward understanding this obscure aspect of fly development. In Rx^ex8 mutants, the clypeus is missing, while the labrum and cibarial plates are present. The findings of a requirement for Rx in clypeus development and Rx expression in a subset of the disc suggest that Rx is required for clypeus precursor cell development. There are two possible functions of clypeus cells. Since the clypeus is an exoskeleton element, they may secrete cuticle to form this structure. In addition, since the clypeus is an attachment site for the underlying cibarial musculature, the cells may act as 'tendon cells' by forming attachments with myotubes. The absence of an external clypeus and abnormal cibarial musculature in Rx^ex8 mutants suggests that Rx is required for both of these functions. Since the anatomy of the clypeus has not been characterized in detail, it is not clear whether these functions are mediated by a single cell type or multiple cell populations (Davis, 2003).

The absence of a clypeus in Rx^ex8 mutants likely results in an ineffective cibarium, which may explain three aspects of the culture phenotype: failure of pharate adults to eclose, and in rare escapers, folded-up wings and dehydration. The frontal ganglion innervates the cibarial muscles of manduca sexta and is required for the cibarial motor program, feeding, eclosion, and wing expansion. Surgical ablation of the frontal ganglion in pupae results in the majority of pharate moths failing to eclose, and among those that do eclose, their wings fail to expand. It is believed that the motor activity of the cibarium is required for swallowing fluids before eclosion and swallowing air afterward, which may be important in generating abdominal pressure necessary either for escape from the pupal case or forcing hemocoel into the wing veins, respectively. The Rx genomic transgene rescues both the clypeus and culture phenotype, suggesting that Rx function in the clypeus is sufficient to restore cibarial function and reverse the culture phenotype. However, it is possible that other Rx functions, not associated with the clypeus, are required for eclosion. In the future, the generation of a clypeus-specific GAL4 driver may be useful in testing this hypothesis. These studies have now generated reagents and a conceptual foundation to begin to explore the underlying mechanisms of Rx function in brain and clypeolabral development (Davis, 2003).

Integration of temporal and spatial patterning generates neural diversity

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

DEVELOPMENTAL BIOLOGY

Embryonic

Drosophila Rx is expressed in the embryo in the procephalic region and in the clypeolabrum from stage 8 on and later in the brain and the central nervous system. Compared with eyeless, Rx expression in the embryo starts earlier, similar to the pattern in vertebrates, where Rx expression precedes Pax-6 expression. Because the vertebrate Rx genes have a function during brain and eye development, it was proposed that Drosophila Rx has a similar function. The Rx expression pattern argues for a conserved function, at least during brain development, but no expression was detected in the embryonic eye primordia or in the larval eye imaginal discs. Therefore Rx could be considered as a homolog of vertebrate Rx genes. The Rx genes might be involved in brain patterning processes and specify eye fields in different phyla (Eggert, 1998).

Drosophila embryos were examined for Rx expression by whole mount in situ hybridization. During the early stages of embryonic development, the syncitial and cellular blastoderm stage, no signals were detected. With the onset of gastrulation and germ-band extension at early stage 8, the first expression is seen in two dorsolateral spots in the procephalic region. At the end of stage 8, an additional signal is visible in a dorsal region that later on will gives rise to the clypeolabrum. The Rx expression becomes more pronounced at stage 9, when the dorsolateral spots are increasing in size. During extended germ-band stage, when the clypeolabrum becomes a distinct structure of the procephalon, cells expressing DRx are moving closer to the midline, and an additional expression in cells of the central nervous system is detected. During stage 12, when the germ-band retracts and metamerization is clearly visible, the optic lobe starts to invaginate. The cells expressing Rx in the procephalon move even closer together, and the expression pattern splits at this stage and the clypeolabrum expression extends more laterally. Because of the morphogenetic movements during head involution, DRx-positive cells in the clypeolabrum move inside the embryo. At this stage expression is observed in the antennomaxillary complex. Staining in the medial edges of the two brain lobes, in the clypeolabrum, and in the antennomaxillary complex is then seen until the end of embryogenesis. Rx expression in the brain is similar to that of eyeless, but the expression patterns are not completely overlapping. However, in contrast to eyeless, no staining of the eye disc primordia per se is observed, when they become distinct structures during stage 16, nor is DRx expressed in imaginal discs of third-instar larvae (Eggert, 1998).

EVOLUTIONARY HOMOLOGS

The 57B cluster of homeobox genes

The homeobrain (hbn) gene is a new paired-like homeobox gene that is expressed in the embryonic brain and the ventral nerve cord. Expression of homeobrain initiates during the blastoderm stage in the anterior dorsal head primordia and the gene is persistently expressed in these cells that form parts of the brain during later embryonic stages. An additional weaker expression pattern is detected in cells of the ventral nerve cord from stage 11 on. The homeodomain in the Homeobrain protein is most similar to the Drosophila proteins Rx, Aristaless and Munster. In addition, the localized brain expression patterns of homeobrain and Rx resemble each other. Two other homeobox genes, orthopedia and Rx are clustered in the 57B region along with homeobrain. The current evidence indicates that homeobrain, Rx and orthopedia form a homeobox gene cluster in which all the members are expressed in specific embryonic brain subregions (Walldorf, 2000).

In contrast to paired-type homeodomains that have serine at position 50, the genes similar to homeobrain have paired-like homeodomains of the subclass that possess a glutamine (Rx, Al, Mu, Repo, Otp) or lysine (D-Gsc) at position 50. Another common motif that is encoded by Rx, homeobrain and the Gsc genes is the octapeptide/GEHdomain, a transcriptional repression domain. The OAR domain, an activation domain found only in some homeoproteins of the paired class is not present in the Homeobrain protein sequence. It is interesting that nearly all of the genes in this subclass (with the exception of repo) reside in two gene complexes at 21C (al, mu and Gsc) and 57B (otp, Rx and homeobrain) (Walldorf, 2000).

A phylogenetic tree indicates that Hbn is only slightly more closely related to Mu and Al proteins than to Rx, Gsc and Otp in the paired-like homeobox family. These data do not support the idea that there was one ancestral complex of brain/tail homeobox genes that then duplicated and diverged to form the two clusters of hbn related genes in the current Drosophila genome. A comparison of the chromosomal location of vertebrate orthologs might give hints concerning the evolution of the two gene clusters. Although Otp, Gsc and Rx have obvious vertebrate orthologs, there is not yet strong evidence that there are distinct orthologs for Hbn, Mu and Al proteins in vertebrate genomes (Walldorf, 2000).

Homeobrain transcript expression starts at the syncytial blastoderm stage (stage 4) as a horseshoe-like pattern in the dorsal head region. During the cellular blastoderm stage (stage 5) the expression domain retracts from the ventrolateral side. A second more laterally located expression domain becomes visible during early gastrulation. In addition, the horseshoe pattern resolves into two distinct domains, a dorsal domain showing weaker staining and dorsal±lateral domains with stronger staining. During embryonic stages 9 and 10, transcript expression is detected in three domains on either side of the dorsal posterior head and in a dorsal-anterior spot. When the germband is fully extended at stage 11, a few cells in each neuromere of the CNS express homeobrain transcripts and shortly afterwards each of the three dorsal±lateral expression domains in the head split, resulting in six dorsal±lateral patches of expression. Another expression domain in the posterior region of the embryo (hindgut/midgut boundary) appears during germband retraction. Discrete localized expression domains in the brain and (at lower levels) in the ventral nerve cord are detected until the end of embryogenesis, whereas the expression in the posterior (gut) region is absent at the terminal stages of embryonic development (Walldorf, 2000).

Rx homologs in invertebrates

Planarians are the free-living members (order Tricladida) of the phylum Platyhelminthes. They are triploblastic, acoelomate, unsegmented and located at the base of the Lophotrochozoa clade. Besides their huge regenerative capacity, planarians have simple eyes, considered similar to the prototypic eye suggested by Charles Darwin in his book 'On the Origin of Species'. The conserved genetic network that determines the initial steps of eye development across metazoans supports a monophyletic origin of the various eye types present in the animal kingdom. This study summarizes the pattern of expression of certain genes involved in the eye network that have been isolated in planarians, such as Otx, Pax-6, Six, Rax and opsin. The effects of RNA interference-mediated loss of function on eye regeneration are described. Finally, the relevance of these findings for the evolution of the eye gene network is discussed (Salo, 2002).

Rx homologs in fish

The homeobox Ol-Rx3 gene is a medaka gene homologous to the mouse, Xenopus, zebrafish and Drosophila Rx genes. Ol-Rx3 starts to be expressed, at late gastrula stages, in the presumptive territories of the anterior brain. Subsequently, transcripts are localized in an antero-ventral region of the prosencephalon and in the primordia of the optic vesicles. During organogenesis, distribution of Ol-Rx3 transcripts are gradually restricted to the floor of the diencephalon, the prospective territory of the hypothalamus and the neurohypophysis. During late development and in adult, Ol-Rx3 expression is maintained in hypothalamic nuclei bordering the third ventricle. In the optic vesicles, Ol-Rx3 expression is temporarily switched off when the eye cup morphogenesis is complete, but it is turned on again in the inner nuclear layer of the retina. Thus, the early expression pattern of Ol-Rx3 is in agreement with a conserved role in the specification of the ventral forebrain and eye field. Putative functions linked to late expression domains are discussed in light of the different hypothesis concerning the involvement of vertebrate Rx genes in the maintenance of particular cell fate (Deschet, 1999).

The teleost Astyanax mexicanus exhibits eyed surface dwelling (surface fish) and blind cave dwelling (cavefish) forms. Despite the lack of functional eyes as adults, cavefish embryos form eye primordia, which later arrest in development, degenerate and sink into the orbit. The expression patterns of various eye regulatory genes during surfacefish and cavefish development have been compared to determine the cause of eye degeneration. Rx and Chx/Vsx family homeobox genes, which have a major role in cell proliferation in the vertebrate retina, have been examined in this study. A full-length RxcDNA clone (As-Rx1) and part of a Chx/Vsx(As-Vsx2) gene, which appear to be most closely related to the zebrafish Rx1 and Alx/Vsx2 genes respectively, were isolated and sequenced. In situ hybridization shows that these genes have similar but non-identical expression patterns during Astyanax eye development. Expression is first detected in the optic vesicle, then throughout the presumptive retina of the optic cup, and finally in the ciliary marginal zone (CMZ), the region of the growing retina where most new retinoblasts are formed. In addition, As-Rx1 is expressed in the outer nuclear layer (ONL) of the retina, which contains the photoreceptor cells, and As-Vsx2 is expressed in the inner nuclear layer, probably in the bipolar cells. With the exception of reduced As-Rx-1 expression in the ONL, the As-Rx1 and As-Vsx2 expression patterns were unchanged in the developing retina of two different cavefish populations, suggesting that cell proliferation is not inhibited. These results were confirmed by using PCNA and BrdU markers for retinal cell division. It is concluded that the CMZ is active in cell proliferation long after eye growth is diminished and is therefore not the major cause of eye degeneration (Strickler, 2000).

In early vertebrate eye development, the retinal anlage is specified in the anterior neuroectoderm. During neurulation, the optic vesicles evaginate from the lateral wall of the prosencephalon. The temperature-sensitive mutation eyeless is described in the Japanese medakafish. Marker gene analysis indicates that, whereas specification of two retinal primordia and proximodistal patterning takes place in the mutant embryo, optic vesicle evagination does not occur and subsequent differentiation of the retinal primordia is not observed. The mutation eyeless thus uncouples patterning and morphogenesis at early steps of retinal development. Temperature-shift experiments indicate a requirement for eyeless activity prior to optic vesicle evagination. Cell transplantation shows that eyeless acts cell autonomously (Winkler, 2000).

Eye development and brain structures of a mutant teleost fish were investigated. The el (eyeless) mutation in medaka (Oryzias atipes) is recessive and affects eye formation; in the most severe cases, it results in the absence of eyes. Developmental studies reveal that normal eyeballs are not formed in the el mutant embryos, but small optic cup-like structures differentiate in situ in the walls of the prosencephalon without evagination. The anophthalmic el homozygous fish hatch normally, although they do not respond behaviorally to visual stimuli. A small fraction of these fish grow to adulthood. In the adult anophthalmic el homozygous fish, the brain exhibits abnormalities in several subdivisions. A pair of small abnormal protrusions is observed on the surface of the ventral telencephalon and preoptic area. Immunocytochemistry using a rhodopsin monoclonal antibody shows that opsin-positive cells are present in the abnormal structures. Bodian staining showed that the optic nerves are present near the abnormal structures, although the number of optic nerve fibers is extremely small. The optic tectum is extremely small, and the thickness of the stratum opticum and stratum fibrosum et griseum superficiale is reduced. These behavioral and morphological observations suggest that the adult anophthalmic el homozygous fish are functionally blind, although small retina-like structures are partially differentiated and persist in the adult fish brain. Moreover, the adult anophthalmic el homozygous fish are infertile, and the sizes of the hypophysis and the hypothalamus are reduced. Thus, the el mutation affects not only the brain structures that are related to the visual system but also those related to the reproductive system (Ishikawa, 2001).

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, by that regulating organ size. Thus, Rx3 acts at a key position coupling the determination with subsequent morphogenesis and differentiation of the developing eye (Loosli, 2001).

The paired-class homeobox gene, Rx, is important in eye development. Expression patterns of three zebrafish Rx genes (Zrx1, 2, 3) has been studied in embryos and adults. All three genes show dynamic spatiotemporal patterns of expression. Zrx3 is expressed earliest, in the anteriormost region of the neural plate, in regions that give rise to ventral diencephalon and retinae. As development proceeds, Zrx3 expression is reduced in the lateral optic primordia, and is absent in the optic cup, but is retained at the ventral midline of the diencephalon, and is expressed in hypothalamus in the adult. As the neural retina begins to differentiate, Zrx3 is re-expressed in a subset of cells in the inner nuclear layer, presumably bipolar cells, and this expression is retained in the adult. In contrast, Zrx1/2 have a slightly later onset of expression, are initially coincident with Zrx3, but then become complementary, remaining on in the optic primordia but disappearing from the ventral midline of the diencephalon. Zrx1/2 are down-regulated as the retina differentiates, except in the outer nuclear layer where they continue to be expressed at high levels in cone, but not rod, photoreceptors. This is the first transcription factor described that distinguishes between cone and rod photoreceptors (Chuang, 1999).

Zebrafish retinal homeobox genes rx1 and rx2 are expressed exclusively in the optic primordia and then in cone photoreceptors of the differentiated neural retina. The rx expression domain is coextensive with the region identified as the retinal field in published fate maps of the neural plate in zebrafish embryos. Analysis of the spatiotemporal relationships between retinal and forebrain precursors suggests that lateral movement of retinal precursors is responsible for evagination of the optic primordia. Overexpression of either rx1 or rx2 results in the loss of forebrain tissue and the ectopic formation of retinal tissue. It was asked whether the deletion of forebrain and expansion of retinal tissue could be explained by the death of telencephalic precursors and enhanced proliferation of retinal precursors, and it was found that it could not. Instead, the data are consistent with a change in cell fate of forebrain precursors associated with reduced expression of telencephalic markers (emx1 and BF-1) and ectopic expression of retinal markers (rx1/2/3, pax6, six6, and vsx2) at the neural keel stage. The rx homeodomain alone is sufficient to induce ectopic retinal tissue, although weakly so, and this observation, together with results from deletion constructs, suggests that interactions with unidentified transcriptional regulators are important for rx1 and rx2 function during early eye development. It is concluded that regulated expression of zebrafish rx1 and rx2 helps to define the region of the forebrain fated to give rise to retinal tissue and may be involved in the cellular migrations that lead to splitting of the retinal field and formation of the optic primordia (Chuang, 2001).

Hedgehog (Hh) signaling is required for eye development in vertebrates; known roles in the zebrafish include regulation of eye morphogenesis and ganglion cell and photoreceptor differentiation. A temporally selective Hh signaling knockdown strategy was used (either antisense morpholino oligonucleotides or the teratogenic alkaloid cyclopamine) in order to dissect the separate roles of Hh signaling arising from specific sources. Also, the eye phenotype was examined of zebrafish slow muscle-omitted (smu) mutants, which lack a functional smoothened gene which encodes a component of the Hh signal transduction pathway. Hh signaling from extraretinal sources is found to be required for the initiation of retinal differentiation, but this involvement may be independent of the effects of Hh signaling on optic stalk development. Hh signals from ganglion cells participate in propagating expression of ath5. It is suggested that the effects of Hh signals from the retinal pigmented epithelium on photoreceptor differentiation may be mediated by the transcription factor rx1 (Stenkamp, 2003).

The results of this study suggest that the effects of Hh signaling on photoreceptor development may involve the transcription factor rx1, and further confirm that Hh signaling from the retinal pigment epithelium (RPE) is primarily implicated. Antisense injections delivered at 51 hpf generated some of the same retinal phenotypes as antisense injections delivered at earlier time points, indicating that interference with Hh signaling rather late in development is sufficient to interfere with photoreceptor differentiation. Knockdown of Hh signaling with antisense-MO consistently resulted in failed rx1 expression in the outer nuclear layer (ONL), while crx expression was unaffected, further supporting the hypothesis that Hh signaling may influence photoreceptor differentiation via the transcription factor rx1. The rx gene product has been shown to participate in regulating photoreceptor-specific gene expression in cell-free systems. The chicken homolog of zebrafish rx1/2, RaxL, is involved in the early stages of photoreceptor differentiation. To confirm the proposed interaction in zebrafish it will be important to demonstrate that rx1 expression regulates photoreceptor differentiation (opsin expression) in vivo. One alternative to this hypothesis is that effects of reduced Hh signaling on rx and opsin genes are related manifestations of a photoreceptor maturation defect (Stenkamp, 2003).

The smu^-/- embryos similarly show reduced expression of photoreceptor markers, and lack of rx1 in the photoreceptor layer. Interestingly, many of the smu^-/- embryos develop normally laminated retinas. It is suspected that these mutants are those that had sufficient maternal smoothened expression to initiate retinal retinal differentiation, but lacked functional (zygotic) Smoothened at the time of Hh signaling from the RPE. In these mutants, it would be predicted that the only notable retinal defects would be those related to Hh signaling from ganglion cells and RPE. A fraction of the mutants showed a small patch of rx1 and rod opsin expression in the ventronasal ONL, suggesting that this region of retina may have requirements for cell differentiation that are distinct from the rest of the retina. This is consistent with the proposal that the ventral retina of the embryonic zebrafish comprises a discrete domain, influenced primarily by signals originating outside the eye, while the differentiation of the remainder of the retina requires the propagation of additional Hh, and other signals, from within the eye (Stenkamp, 2003).

Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis

During forebrain morphogenesis, there is extensive reorganisation of the cells destined to form the eyes, telencephalon and diencephalon. Little is known about the molecular mechanisms that regulate region-specific behaviours and that maintain the coherence of cell populations undergoing specific morphogenetic processes. his study shows that the activity of the Eph/Ephrin signalling pathway maintains segregation between the prospective eyes and adjacent regions of the anterior neural plate during the early stages of forebrain morphogenesis in zebrafish. Several Ephrins and Ephs are expressed in complementary domains in the prospective forebrain and combinatorial abrogation of their activity results in incomplete segregation of the eyes and telencephalon and in defective evagination of the optic vesicles. Conversely, expression of exogenous Ephs or Ephrins in regions of the prospective forebrain where they are not usually expressed changes the adhesion properties of the cells, resulting in segregation to the wrong domain without changing their regional fate. The failure of eye morphogenesis in rx³ mutants is accompanied by a loss of complementary expression of Ephs and Ephrins, suggesting that this pathway is activated downstream of the regional fate specification machinery to establish boundaries between domains undergoing different programmes of morphogenesis (Cavodeassi, 2013).

Rx homologs in frogs

A novel Xenopus homeobox gene, Xrx1, belonging to the paired-like class of homeobox genes, has been isolated. Xrx1 is expressed in the anterior neural plate, and subsequently in the neural structures of the developing eye (neural retina and pigmented epithelium), and in other forebrain structures deriving from the anterior neural plate: in the pineal gland, throughout its development; in the diencephalon floor, and in the hypophysis. Xrx1's rostral limit of expression corresponds to the chiasmatic ridge, which some authors consider as the anteriormost limit of the neural tube: thus, Xrx1 may represent one of the most anteriorly expressed homeobox genes reported to date. Moreover, its expression in organs implicated in the establishment of circadian rhythms, may suggest for Xrx1 a role in the genetic control of this function. Finally, analysis of Xrx1 expression in embryos subjected to various treatments, or microinjected with different dorsalizing agents (noggin, Xwnt-8), suggests that vertical inductive signals leading to head morphogenesis are required to activate Xrx1 (Casarosa, 1997).

The anteriormost 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 studied. 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, thus being required for eye and anterior brain development (Andreazzoli, 1999).

Chicken Rx homologs

Chick rax/rx cDNAs, cRaxL (chick Rax/Rx-like) and cRax, (chick Rax) have been isolated and their expression patterns have been examined during early eye and brain development. The cRaxL cDNA encodes a 228 amino acid protein that is most closely related to the zebrafish Rx1 and Rx2. The cRax cDNA encodes a 317 amino acid protein, which shares higher homology with the Xenopus Rx. In addition to the homeodomain, the octapeptide and paired tail domains are conserved between the cRax and other vertebrate Rax/Rx, while cRaxL lacks the octapeptide containing N-terminal region, which is conserved among all other members of the rax/rx gene family identified so far. The chick rax/rx genes are expressed in overlapping domains in the anterior neural ectoderm, which corresponds to the forebrain and retina field, and later in the optic vesicle. cRax mRNA can be detected earlier than cRaxL prior to the formation of the notochord and its expression domain appears broader than that of cRaxL (Ohuchi, 1999).

Mammalian Rx homologs

A conserved vertebrate homeobox gene, Rx, is essential for normal eye development, and 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 so do not develop eyes. The Rx gene family plays an important role in the establishment and/or proliferation of retinal progenitor cells (Mathers, 1997).

Development of the vertebrate eye has been found to require the activity of several genes encoding homeodomain proteins. Some of these genes, or portions thereof, are highly conserved across phyla. A novel homeobox gene, rax (retina and anterior neural fold homeobox), has been identified; rax expression pattern suggests an important role in eye development. The predicted amino acid sequence of Rax comprises a protein with a paired-type homeobox, as well as the octapeptide that is found in many paired-type homeobox genes. In addition, in the C terminus of Rax, a 15-aa domain was found that has been termed the OAR domain. This domain is also found in several other homeobox genes. In the early mouse embryo, rax is expressed in the anterior neural fold, including areas that will give rise to the ventral forebrain and optic vesicles. Once the optic vesicles form, rax expression is restricted to the ventral diencephalon and the optic vesicles. At later stages, rax expression is found only in the developing retina. After birth, the expression of rax is restricted to the zone of proliferating cells within the retina, and expression gradually decreases as proliferation declines. These findings suggest that rax is one of the molecules that define the eye field during early development and that it has a role in the proliferation and/or differentiation of retinal cells (Furukawa, 1997).

RX, a homeodomain-containing protein essential for proper eye development, binds to the photoreceptor conserved element-1 (PCE-1/Ret 1) in the photoreceptor cell-specific arrestin promoter and stimulates gene expression. RX is found in many retinal cell types including photoreceptor cells. Another homeodomain-containing protein, CRX, which binds to the OTX element to stimulate promoter activity, is found exclusively in photoreceptor cells. Binding assay and cell culture studies indicate that both PCE-1 and OTX elements and at least two different regulatory factors RX and CRX are necessary for high level, photoreceptor cell-restricted gene expression. Thus, photoreceptor specificity can be achieved by multiple promoter elements interacting with a combination of both photoreceptor-specific regulatory factors and factors present in closely related cell lineages (Kimura, 2000).

The eyeless inbred mouse strain ZRDCT has long served as a spontaneous model for human anophthalmia and the evolutionary reduction of eyes that occurs in some naturally blind mammals. ZRDCT mice have orbits but lack eyes and optic tracts and have hypothalamic abnormalities. Segregation data suggest that a small number of interacting genes are responsible, including at least one major recessive locus, ey1. Although predicted since the 1940s, these loci were never identified. ey1 was mapped to chromosome 18 using an F2 genome scan and there a Met10-->Leu mutation was found in Rx/rax, a homeobox gene that is expressed in the anterior headfold, developing retina, pineal, and hypothalamus and is translated via a leaky scanning mechanism. The mutation affects a conserved AUG codon that functions as an alternative translation initiation site and consequently reduces the abundance of Rx protein. In contrast to a targeted Rx null allele, which causes anophthalmia, central nervous system defects, and neonatal death, the hypomorphic M10L allele is fully viable (Tucker, 2000).

Rx plays a critical role in eye formation. Targeted elimination of Rx results in embryos that do not develop eyes. In this study, the expression of Otx2, Six3, and Pax6 was examined in Rx deficient embryos. These genes show normal activation in the anterior neural plate in Rx-/- embryos, but they are not upregulated in the area of the neural plate that would form the primordium of the optic vesicle. In contrast, in homozygous Small eye embryos that lack Pax6 function, Rx shows normal activation in the anterior neural plate and normal upregulation in the optic vesicle/retinal progenitor cells. This suggests that neither Rx expression nor the formation of retinal progenitor cells is dependent on a functional copy of the Pax6 gene, but that Pax6 expression and the formation of the progenitor cells of the optic cup is dependent on a functional copy of the Rx gene (Zhang, 2000).

Despite the growing information concerning the developmental importance of the Prx2 protein, the structural determinants of Prx2 function are poorly understood. To gain insight into the transcription regulatory regions of the Prx2 protein, a series of truncation mutants were generated. Both the Prx2 response element (PRE) and a portion of the tenascin promoter, a downstream target of Prx2, were used as reporters in transient transfection assays. This analysis shows that a conserved domain (PRX), found in both Prx1 and Prx2, activates transcription in NIH 3T3 cells. This PRX domain, as well as other functional regions of Prx2, demonstrate both cell-specific and promoter-dependent transcriptional regulation. A second important region, the OAR (aristaless) domain, which is conserved among 35 Paired-type homeodomain proteins, is observed to inhibit transcription. Deletion of this element results in a 20-fold increase of transcription from the tenascin reporter in NIH 3T3 cells but not in C2C12 cells. The OAR domain does not function as a repressor in chimeric fusions with the Gal4 DNA binding domain in either cell type, characterizing it as an inhibitor instead of a repressor. These results give insight into the function of the Prx2 transcription factor while establishing the framework for comparison with the two isoforms of Prx1 (Norris, 2001).

Rx and gliogenesis

Mechanisms of glial cell development in the vertebrate central nervous system have been examined. Genes have been identified that can direct the formation of glia in the retina. rax, a homeobox gene, Hes1, a basic helix-loop-helix gene, and notch1, a transmembrane receptor gene, are expressed in retinal progenitor cells, downregulated in differentiated neurons, and expressed in Muller glia. Retroviral transduction of any of these genes results in expression of glial markers. In contrast, misexpression of a dominant-negative Hes1 gene reduces the number of glia. Cotransfection of rax with reporter constructs containing the Hes1 or notch1 regulatory regions leads to the upregulation of reporter transcription. These data suggest a regulatory hierarchy that controls the formation of glia at the expense of neurons (Furukawa, 2000).

Expression of Rx in the adult hypothalamus

Homeobox genes are important regulators of cellular identity. Several homeobox genes are known to be specifically expressed in subsets of neurons in the forebrain, exclusively, or in distinct combinations. This study explores the expression of Homeobox genes in the forebrain of the adult rat, using a degenerate polymerase chain reaction cloning strategy. The expression of 12 homeobox genes was identified, several of which display a remarkable restricted expression pattern in the adult brain. The expression of goosecoid is demonstrated in a very small set of neurons in the hypothalamus. By using Otp as a marker, these goosecoid-positive cells were found to constitute a small area just beside the paraventricular nucleus. Furthermore, expression of Rx was found in the pineal gland, along with Alx4. Rx was additionally found in the posterior pituitary and in cells aligning the bottom of the third ventricle. These findings form a starting point to reveal functions of the described homeobox genes in the forebrain (Asbreuk, 2002).

REFERENCES

Search PubMed for articles about Drosophila Retinal homeobox

Andreazzoli, M., et al. (1999). Role of Xrx1 in Xenopus eye and anterior brain development. Development 126(11): 2451-60. 10226004

Asbreuk, C. H., et al. (2002). Survey for paired-like homeodomain gene expression in the hypothalamus: restricted expression patterns of Rx, Alx4 and goosecoid. Neuroscience. 114(4): 883-9. 12379244

Casarosa, S., Andreazzoli, M., Simeone, A. and Barsacchi, G. (1997). Xrx1, a novel Xenopus homeobox gene expressed during eye and pineal gland development. Mech. Dev. 61(1-2): 187-98. 9076688

Cavodeassi, F., Ivanovitch, K. and Wilson, S. W. (2013). Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis. Development 140: 4193-4202. PubMed ID: 24026122

Chuang, J. C., Mathers, P. H. and Raymond, P. A. (1999). Expression of three Rx homeobox genes in embryonic and adult zebrafish. Mech. Dev. 84(1-2): 195-8. 10473141

Chuang, J. C. and Raymond, P. A. (2001). Zebrafish genes rx1 and rx2 help define the region of forebrain that gives rise to retina. Dev. Biol. 231(1): 13-30. 11180949

Davis, R. J., Tavsanli, B. C., Dittrich, C., Walldorf, U. and Mardon, G. (2003). Drosophila retinal homeobox (Rx) is not required for establishment of the visual system, but is required for brain and clypeus development. Dev. Bio. 259: 272-287. 12871701

Deschet, K., et al. (1999). Expression of the medaka (Oryzias latipes) Ol-Rx3 paired-like gene in two diencephalic derivatives, the eye and the hypothalamus. Mech. Dev. 83(1-2): 179-82. 10381578

Eggert, T., Hauck, B., Hildebrandt, N., Gehring, W. J. and Walldorf, U. (1998). Isolation of a Drosophila homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development. Proc. Natl. Acad. Sci. 95(5): 2343-8. 9482887

Erclik, T., Li, X., Courgeon, M., Bertet, C., Chen, Z., Baumert, R., Ng, J., Koo, C., Arain, U., Behnia, R., Rodriguez, A. D., Senderowicz, L., Negre, N., White, K. P. and Desplan, C. (2017). Integration of temporal and spatial patterning generates neural diversity. Nature [Epub ahead of print]. PubMed ID: 28077877

Furukawa, T., Kozak, C. A. and Cepko, C. L. (1997). rax, a novel paired-type homeobox gene, shows expression in the anterior neural fold and developing retina. Proc. Natl. Acad. Sci. 94(7): 3088-93. 9096350

Furukawa, T., Mukherjee, S., Bao, Z. Z., Morrow, E. M. and Cepko, C. L. (2000). rax, Hes1, and notch1 promote the formation of Muller glia by postnatal retinal progenitor cells. Neuron. 26(2): 383-94. 10839357

Ishikawa, Y., et al. (2001). Brain structures of a medaka mutant, el (eyeless), in which eye vesicles do not evaginate. Brain Behav. Evol. 58(3): 173-84. 11910174

Kalionis, B. and O'Farrell, P. H. (1993). A universal target sequence is bound in vitro by diverse homeodomains. Mech. Dev. 43(1): 57-70. 7902124

Kimura, A., et al. (2000). Both PCE-1/RX and OTX/CRX interactions are necessary for photoreceptor-specific gene expression. J. Biol. Chem. 275(2): 1152-60. 10625658

Loosli, F., et al. (2001). Medaka eyeless is the key factor linking retinal determination and eye growth. Development. 128(20): 4035-44. 11641226

Mathers, P. H., et al. (1997). The Rx homeobox gene is essential for vertebrate eye development. Nature 387(6633): 603-7. 9177348

Norris, R. A. and Kern, M. J. (2001). Identification of domains mediating transcription activation, repression, and inhibition in the paired-related homeobox protein, Prx2 (S8). DNA Cell Biol. 20(2): 89-99. 11244566

Ohuchi, H., Tomonari, S., Itoh, H., Mikawa, T. and Noji, S. (1999). Identification of chick rax/rx genes with overlapping patterns of expression during early eye and brain development. Mech. Dev. 85(1-2): 193-5. 10415362

Salo, E., et al. (2002). Genetic network of the eye in Platyhelminthes: expression and functional analysis of some players during planarian regeneration. Gene 287(1-2): 67-74. 11992724

Stenkamp, D. L. and Frey, R. A. (2003). Extraretinal and retinal hedgehog signaling sequentially regulate retinal differentiation in zebrafish. Dev. Biol. 258: 349-363. 12798293

Strickler, A. G., Famuditimi, K. and Jeffery, W. R. (2000). Retinal homeobox genes and the role of cell proliferation in cavefish eye degeneration. Int. J. Dev. Biol. 46(3): 285-94. 12068949

Tucker, P., et al. (2000). The eyeless mouse mutation (ey1) removes an alternative start codon from the Rx/rax homeobox gene. Genesis 31(1): 43-53. 11668677

Walldorf, U., Kiewe, A., Wickert, M., Ronshaugen, M. and McGinnis, W. (2000). Homeobrain, a novel paired-like homeobox gene is expressed in the Drosophila brain. Mech Dev. 96(1): 141-4. 10940637 =

Winkler, S., et al. (2000). The conditional medaka mutation eyeless uncouples patterning and morphogenesis of the eye. Development127(9): 1911-9. 10751179

Zhang, L., Mathers, P. H. and Jamrich, M. (2000). Function of Rx, but not Pax6, is essential for the formation of retinal progenitor cells in mice. Genesis 28(3-4): 135-42. 11105055

date revised: 22 February 2022

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