Interactive Fly, Drosophila

eyeless


EVOLUTIONARY HOMOLOGS


Table of contents

Pax domain proteins

There are seven Pax genes in Drosophila and nine Pax genes known in mouse and human. Different Pax proteins use multiple combinations of the HTH motifs to recognize several types of target sites. Drosophila Paired protein can bind, in vitro exclusively through its PAI domain (the N-terminal portion of the bipartite paired domain), or through a dimer of its Homeodomain, or through cooperative interaction between PAI domain and HD. However, paired function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD (such as Pax-5) appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein Lune, may rely on the RED domain and HD. Thus Pax protein appear to recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire (Jun, 1996).

Pax proteins are a family of transcription factors with a highly conserved paired domain; many members also contain a paired-type homeodomain and/or an octapeptide. Nine mammalian Pax genes are known and classified into four subgroups: Pax-1/9, Pax-2/5/8, Pax-3/7, and Pax-4/6. Most of these genes are involved in nervous system development. In particular, Pax-6 is a key regulator that controls eye development in vertebrates and Drosophila. Although the Pax-4/6 subgroup seems to be more closely related to Pax-2/5/8 than to Pax-3/7 or Pax-1/9, its evolutionary origin is unknown. A Pax-6 homolog and related genes were sought in Cnidaria, the least complex phylum of animals known to possess a nervous system and eyes. A sea nettle (a jellyfish) genomic library was constructed and two pax genes (Pax-A and -B) were isolated and partially sequenced. Surprisingly, unlike most known Pax genes, the paired box in these two genes contains no intron. In addition, the complete cDNA sequences of hydra Pax-A and -B were obtained. Hydra Pax-B contains both the homeodomain and the octapeptide, whereas hydra Pax-A contains neither. DNA binding assays showed that sea nettle Pax-A and -B and hydra Pax-A paired domains bind to a Pax-5/6 site and a Pax-5 site, although hydra Pax-B paired domain bind neither. An alignment of all available paired domain sequences reveal two highly conserved regions that cover the DNA binding contact positions. Phylogenetic analysis shows that Pax-A and especially Pax-B are more closely related to Pax-4/6 and Pax-2/5/8 (Drosophila homolog: Sparkling) and than to either Pax-1/9 (Drosophila homolog: Pox meso) or Pax-3/7 (Drosophila homolog: Paired and the Gooseberrys). The Pax genes were then classified into two supergroups: Pax-A/Pax-B/Pax-2/5/8/4/6 and Pax-1/9/3/7. From this analysis and the gene structure, it is proposed that modern Pax-4/6 and Pax-2/5/8 genes evolved from an ancestral gene similar to cnidarian Pax-B, having both the homeodomain and the octapeptide (Sun, 1997).

Whereas Pax6 is expressed in the visual system and is restricted to regions fated to constitute the eye deriving from the distal optic vesicle and the ectodermal lens placode, expression of the paired box containing Pax2 is initially confined to the ventral optic vesicle, without proximodistal restriction, later being confined mostly to the proximal regions destined to contribute to the optic nerve and optic chiasm. During gestation, Pax2 is expressed in the mid-hindbrain area, developing eye and inner ear. Pax2 null mutant mice show the requirement of Pax2 for the establishment of axonal pathways along the optic stalks and ventral diencephalon. In mutant brains, the optic tracts remain totally ipsilateral due to agenesis of the optic chiasma. Furthermore, Pax2 mutants show extension of the pigmented retina into the optic stalks and failure of the optic fissure to close resulting in coloboma. In the inner ear, Pax2 mutants show agenesis of the cochlea and the spiral ganglion, i.e., the parts of the organ responsible for auditory function and in whose primordium Pax2 is expressed. These results identify Pax2 as a major regulator of patterning during organogenesis of the eye and inner ear and indicate its function in morphogenetic events required for closure of the optic fissure and neural tube (Torres, 1996).

Pax proteins play a diverse role in early animal development and contain the characteristic paired domain, consisting of two conserved helix-turn-helix motifs. In many Pax proteins the paired domain is fused to a second DNA binding domain of the paired-like homeobox family. By amino acid sequence alignments, secondary structure prediction, 3D-structure comparison, and phylogenetic reconstruction, the relationship between Pax proteins and members of the Tc1 family of transposases, which possibly share a common ancestor with Pax proteins, has been examined. It is suggested that the DNA binding domain of an ancestral transposase (proto-Pax transposase) was fused to a homeodomain shortly after the emergence of metazoans about one billion years ago. Using the transposase sequences as an outgroup the early evolution of the Pax proteins was examined. This novel evolutionary scenario features a single homeobox capturing event and an early duplication of Pax genes before the divergence of porifera, indicating a more diverse role of Pax proteins in primitive animals than previously expected (Breitling, 2000).

An attemp has been made to reconstruct the phylogeny and to reliably root the phylogenetic tree of Pax proteins. Since homeodomains, which have been compared for that purpose, are only present in some of the Pax proteins and are conspicuously absent in the PaxA/neuro and Pax1-9 group, the analysis was restricted to the paired box itself. This was facilitated by the introduction of a novel outgroup. Comparison of the X-ray structures of the paired box of Drosophila Paired (1PDN) and human Pax6 (6PAX) within the database of 3D-structures has revealed that the N-terminal subdomain (PAI domain) is closely related to the DNA binding domain of Tc3 transposase of Caenorhabditis elegans (1TC3). A general similarity between transposase DNA binding domains and the paired domain has been reported and their structural relationship has been observed during the analysis of the transposase structure. Initial Blast searches identified a group of transposases from C. elegans whose DNA binding domain seems to be more closely related to the paired box than to most other transposases. The DNA binding domain of these C. elegans transposases (proteins K03H6.3, W04G5.1, F26H9.3, F49C5.8, and C27H2.1; accession numbers T33011, T26169, T21438, T22423, and T19530) shows highly significant similarity only to Bmmar1, a transposase from Bombyx mori [accession number AAB47739, E-score (E)=2e-27 compared to K03H6.3], and to many Pax proteins (e.g. Hydra magnapapillata Pax2/5/8, E=9e-05; Phallusia mammilata Pax6 E=3e-04; or Paracentrotus lividus Pax1/9 E=6e-04). The DNA binding domains of other transposases yield E-scores worse than 1e-03 (e.g. Anopheles albimanus transposase AAB02109, E=9e-03). It is supposed that the transposases of C. elegans and B. mori might represent molecular fossils (proto-Pax) from the time before a homeobox capturing event took place, during which the catalytic domain of the transposase was lost and the DNA-binding domain was fused to a homeobox yielding the first PAX protein. If this is indeed the case, the proto-Pax transposases should also contain the C-terminal subdomain (RED domain) of the paired box. This subdomain is less conserved among Pax proteins than the PAI domain and does not show significant homology in sequence alignments between transposases and Pax proteins. A secondary structure analysis of the proto-Pax transposases was performed using a consensus method (Jpred2), which predicted that they indeed contain two helix-turn-helix motifs, homologous to both the PAI and the RED domain of Pax proteins (Breitling, 2000).

The observation that the DNA binding domain of transposases is in fact closely related to the paired box indicates that it should be possible to use them as an outgroup in the phylogenetic analysis of Pax proteins to determine the most likely evolutionary sequence. The transposase sequence (C. elegans K03H6.3, E = 2e-27) with the highest Blast score was compared to Pax proteins to generate a multiple sequence alignment of Pax-like transposases using the JPred2 server. The JPred2 algorithm was also used to generate a multiple sequence alignment for Pax proteins. Both alignments were combined and realigned by using ClustalW. The resulting data set contains transposases of the Tc1 and mariner families, as well as a wide range of Pax proteins from all known subgroups. The complete alignment was then used for phylogenetic analysis (Breitling, 2000).

Neighbor-joining and parsimony analysis reliably subdivides the Pax proteins into five large groups, which correspond to the classical subfamilies Pax1-9/Pax meso, PaxD/3-7/Gooseberry/Paired, PaxB/2-5-8/Sparkling, Pax4-6/Eyeless and PaxA/Pax neuro. The internal topology of the subfamilies agrees fairly well with the accepted evolutionary relationship of the organisms. One exception is the Pax4-6/Eyeless subfamily which is extremely conserved, so that an unambiguous determination of the internal branching order was not possible. The position of Drosophila Eyegone is also unreliable, because this protein contains only a partial paired domain. In both trees PaxC is significantly associated with the PaxA/Pax neuro subfamily, although PaxC carries a homeobox, and PaxA/Pax neuro proteins do not. Neighbor-joining and parsimony tree reconstruction place the Pax family within the Tc1 family of transposases, while it was not possible to identify a single closest relative of the paired box. The supposed proto-Pax transposases from C. elegans and B. mori, as identified by Blast searches, are not reliably placed as a sister-group of the Pax proteins. This might be due to the general difficulty of reconstructing well-resolved phylogenetic trees of the transposase family (Breitling, 2000).

This focus on the paired box as a descendant of a Tc1- like transposase DNA binding domain allowed for a reevaluation of the early evolution of the paired domain. These results show that the evolutionary scenario proposed by Galliot and Miller (2000) is unlikely to correctly represent the evolution of Pax proteins. This hypothesis was based mainly on the assumption that PaxA, which consists only of a paired box, resembles the probable ancestor of Pax proteins. Contrary to that idea, the scenario developed here is based on the assumption that the paired box is originally derived from a transposase and indicates that PaxA is probably derived by a secondary loss of the homeobox of a PaxC-like protein. These observations also make unlikely the hypothesis that there was more than one homeodomain capturing event. Furthermore, they suggest that the first duplication of Pax proteins occurred before the divergence of the porifera. This consequently implies that sponges, which lack nerve cells and most of the organs patterned by Pax genes in higher animals, already contained (at least) two Pax genes. The function of these early Pax proteins remains a mystery (Breitling, 2000).

Phosphorylation and dephosphorylation of Pax-6

Pax-6 is an evolutionarily conserved transcription factor and acts high up in the regulatory hierarchy controlling eye and brain development in humans, mice, zebrafish, and Drosophila. Previous studies have shown that Pax-6 is a phosphoprotein, and its phosphorylation by ERK, p38, and homeodomain-interacting protein kinase 2 greatly enhances its transactivation activity. However, the protein phosphatases responsible for the dephosphorylation of Pax-6 remain unknown. This study presents both in vitro and in vivo evidence to show that protein serine/threonine phosphatase-1 is a major phosphatase that directly dephosphorylates Pax-6: (1) purified protein phosphatase-1 directly dephosphorylates Pax-6 in vitro; (2) immunoprecipitation-linked Western blot revealed that both protein phosphatase-1α and protein phosphatase-1β interact with Pax-6; (3) overexpression of protein phosphatase-1 in human lens epithelial cells leads to dephosphorylation of Pax-6; (4) inhibition of protein phosphatase-1 activity by calyculin A or knockdown of protein phosphatase-1α and protein phosphatase-1α by RNA interference leads to enhanced phosphorylation of Pax-6. Moreover, these results also demonstrate that dephosphorylation of Pax-6 by protein phosphatase-1 significantly modulates its function in regulating expression of both exogenous and endogenous genes. These results demonstrate that protein phosphatase 1 acts as a major phosphatase to dephosphorylate Pax-6 and modulate its function (Yan, 2007).

Invertebrate Eyeless homologs

PaxB from Tripedalia cystophora, a cubomedusan jellyfish possessing complex eyes (ocelli), was characterized. PaxB, the only Pax gene found in this cnidarian, is expressed in the larva, retina, lens, and statocyst. PaxB contains a Pax2/5/8-type paired domain and octapeptide, but a Pax6 prd-type homeodomain. Pax2/5/8-like properties of PaxB include a DNA binding specificity of the paired domain, activation and inhibitory domains, and the ability to rescue spapol, a Drosophila Pax2 eye mutant. Like Pax6, PaxB activates jellyfish crystallin and Drosophila rhodopsin rh6 promoters and induces small ectopic eyes in Drosophila. Pax6 has been considered a master control gene for eye development. These data suggest that the ancestor of jellyfish PaxB, a PaxB-like protein, was the primordial Pax protein in eye evolution and that Pax6-like genes evolved in triploblasts after separation from Cnidaria, raising the possibility that cnidarian and sophisticated triploblastic eyes arose independently (Kozmik. 2003).
The present study shows that the structure of the Tripedalia PaxB gene, like that of other cnidarians and of a sponge, corresponds to an ancestral Pax gene, encoding a paired domain, an octapeptide, and a homeodomain. The PaxB protein is a functional hybrid of Pax2/5/8 and Pax6. On the basis of DNA sequence and DNA binding assays, it has been proposed that cnidarian PaxB has maintained the structure of a Pax gene ancestral to modern Pax6 and Pax2/5/8. The sequence and DNA binding specificity of the PaxB paired domain of Tripedalia are characteristic for the Pax2/5/8 subfamily, the DNA binding specificity of which is generally broader than that of Pax6 proteins. In addition, PaxB includes in its C terminus adjacent activation and inhibitory domains, a characteristic of Pax2/5/8. By contrast, Pax6 contains a transactivation domain composed of short regions that act in synergy with each other. Importantly, PaxB can rescue the Drosophila spapol mutant whose eye-specific enhancer of Pax2 is deleted. However, other properties of Tripedalia PaxB are clearly Pax6-like. First, unlike Pax2, PaxB does not induce phosphorylation of Grg4, a Groucho-type transcriptional corepressor that interacts with vertebrate Pax5 via the octapeptide. It seems unlikely that this negative result is caused by interspecies differences because various vertebrate Pax2/5/8 proteins and Drosophila D-Pax2 induce phosphorylation of Drosophila Groucho and mouse Grg4, which argues for an evolutionarily conserved mechanism. Second, like Pax6, PaxB has a prd-type homeodomain with a cognate DNA binding specificity. This was deduced from an even greater activation of the Drosophila rh6 promoter by PaxB than authentic Pax6 in transient transfection assays. Finally, even though PaxB rescued the D-Pax2 spapol mutant, it was also able to induce ectopic eyes in Drosophila, although with lower efficiency than PaxB(IQN). This is a striking difference from zebrafish Pax2, which is unable to generate ectopic eyes in Drosophila (Kozmik. 2003).

Induction of ectopic eyes in Drosophila by PaxB appears intriguing, particularly if one considers that rescue of the Drosophila ey2 mutant requires a protein with a Pax6-type paired domain, quite different from the Pax2-type paired domain of jellyfish PaxB, but not a homeodomain. It is less surprising, however, in view of the finding that D-Pax2 is also able to induce ectopic eyes. Moreover, induction of ectopic eyes depends on a wild-type endogenous ey gene, which is initially activated by Toy and subsequently maintains the genetic program for eye development by positive feedback loops. Accordingly, PaxB needs to bind to the Pax6 binding sites of the ey enhancer to induce ectopic eye development. This requirement is improved by altering the binding specificity of PaxB to that of Pax6 in PaxB(IQN), which is as efficient as Toy in its capacity to induce ectopic eyes. These results, therefore, suggest that Pax6 target sites of the ey enhancer are recognized by PaxB and even D-Pax2, when expressed at high levels, with affinities that suffice to turn on the program of eye development in a few susceptible cells of the leg disc that would normally form tibial structures. Although D-Pax2 can induce ectopic eyes, other Drosophila Pax proteins (Gsb, Prd, Poxm, and Poxn) are unable to do so, which implies that D-Pax2 is more closely related to PaxB than Poxn or any of the Pax proteins of the Pax3/7 and Pax1/9 subfamilies. Conversely, Toy or Ey are both able to rescue the spapol phenotype to some extent, which indicates that these Pax6 proteins are still able to perform some of the Pax2 functions. It follows that the Pax2 and Pax6 proteins have retained the capability to substitute for some of each other's functions. One implication of this is that eyes (ocelli) and future ears (statocysts/mechanoreceptors), which both express PaxB in Tripedalia, are developmentally and evolutionarily linked (Kozmik. 2003).

The Pax-6 gene encodes a transcription factor containing both a paired domain and a homeodomain and is highly conserved among Metazoa. In both vertebrates and invertebrates, Pax-6 is required for eye morphogenesis; for development of parts of the central nervous system, and, in some phyla, for the development of olfactory sense organs. Ectopic expression of Pax-6 from insects, mammals, cephalopods (phylum Chordata), and ascidians (phylum Urochordata) induces ectopic eyes in Drosophila, suggesting that Pax-6 may be a universal master control gene for eye morphogenesis. Platyhelminthes are members of an ancient phylum, originating from the base of spiralian protostomes, which bear primitive eyes. These eyes consist of a group of rhabdomeric photoreceptor cells enclosed in a cup of pigment cells. The eye spots of planarians consist of two cell types, a bipolar nerve cell with a rhabdomere as a photoreceptive structure and a cup-shaped structure composed of pigmented cells. Planarian eyes represent one of the most ancestral and simple types of visual systems. The turnover of the eyes is supported by the differentiation of undifferentiated and totipotent cells (neoblasts) present in the adult. In the early stages of regeneration the primitive pigmented cells originate from the blastema (regenerative mesenchymal tissue) close to the primitive visual cells derived from nerve cells from the regenerated cephalic ganglia. This mixture of cells is then rearranged; each cell moves to its respective position, and a normal eye spot is formed. The analysis of Pax-6 and its expression pattern should provide insights into the ancestral function of Pax-6 in eye morphogenesis. The Pax-6 gene of the planarian Dugesia tigrina has been identifed. This gene shares significant sequence identity and conserved genomic organization with Pax-6 proteins from other phyla. Phylogenetic analysis indicates that it clusters with the other Pax-6 genes, but in the most basal position. DtPax-6 is expressed as a single transcript in both regenerating and fully grown eyes, and electron microscopy studies show strong expression in the perykarion of both photoreceptor and pigment cells. DtPax-6 expression in adults could be related to the capacity to regenerate the eyes. The RT-PCR amplification experiments show a distribution of DtPax-6 along the anteroposterior axis, indicating that, as in other organisms, DtPax-6 may be used for developmental processes in structures other than the eye. Because a bona fide Pax-6 homolog has not yet been detected in diploblastic animals, it is speculated that Pax-6 may be typical for triploblasts and that the appearance of additional Pax genes may have coincided with increasingly complex body plans. Attempts to induce ectopic eyes by expressing DtPax-6 in Drosophila have been unsuccessful (Callaerts, 1999).

Two Pax6-related genes, Pax6A and Pax6B, are highly conserved in two planarian species Dugesia japonica and Girardia tigrina (Platyhelminthes, Tricladida). Pax6A is more similar to other Pax6 proteins than Pax6B, which is the most divergent Pax6 described so far. The planarian Pax6 homologs do not show any clear orthology to the Drosophila duplicated Pax6 genes, eyeless and twin of eyeless, which suggests an independent Pax6 duplication in a triclad or platyhelminth ancestor. Pax6A is expressed in the central nervous system of intact planarians, labeling a subset of cells of both cephalic ganglia and nerve cords, and is activated during cephalic regeneration. Pax6B follows a similar pattern, but shows a lower level of expression. Pax6A and Pax6B transcripts are detected in visual cells only at the ultrastructural level, probably because a limited amount of transcripts is present in these cells. Inactivation of both Pax6A and Pax6B by RNA-mediated gene interference (RNAi) inhibits neither eye regeneration nor eye maintenance, suggesting that the genetic network that controls this process is not triggered by Pax6 in planarians (Pineda, 2002).

The C. elegans Pax-6 locus encodes two protein isoforms. One contains a Paired DNA binding domain as well as a homeodomain; the other consists only of the carboxy-terminal portion of the locus encoding the homeodomain. These two isoforms are expressed in a variety of postembryonic cell lineages. In one set of lineages, the sensory ray cells of the male tail, nuclear localization of a homeodomain-only form (MAB-18 isoform) appears to be under temporal and spatial control. MAB-18 is first detected three generations before the birth of ray cells in cells known as ray precursor cells, or Rn cells. Nine bilateral pairs of Rn cells are present in the lateral epidermis of the late L3 larval male. Each Rn cell executes the ray sublineage, giving rise to the two sensory neurons and support cell that comprises a ray. MAB-18 is detected in the ray precursor cells of ray 6, ray 7 and ray 8. Nuclear localization of MAB-18 is correlated with the genetic requirement for mab-18 and with activation of a reporter gene driven by a mab-18 promoter. Reporter gene expression is dependent on mab-18 gene activity. It is hypothesized that a positive feedback loop is activated by regulated nuclear entry. Nuclear entry is delayed for approximately 7 hours while the ray sublineage is in progress. When the ray cells begin to differentiate, lineage-specific or spatial cues presumably trigger nuclear entry and the continued expression of MAB-18 in ray 6, but gene expression ceases and protein is degraded in rays 7 and 8. A similar transition from cytoplasmic staining to nuclear staining is also observed in preanal ganglion neurons derived from P11.a. An alternative explanation for these observations is that there are two forms of the protein, one cytoplasmic and the other nuclear, with expression shifting from one form to the other. The mab-18(bx23) mutation results in the transformation of the morphological identity of ray 6 to that of ray 4 (Zhang, 1998).

The Pax-6 gene of the nematode C. elegans has been identified in genetic studies of male tail morphology. C. elegans Pax-6 encodes at least two independent genetic functions. One, like other Pax-6 genes, contains both paired and homeodomains; this constitutes the genetic locus vab-3. The other, described here, is expressed from an internal promoter and contains only the homeodomain portion; this constitutes the genetic locus mab-18. The mab-18 form of the gene is expressed in a peripheral sense organ and is necessary for specification of sense-organ identity. Its function in this context could be to regulate the expression of cell recognition and adhesion proteins required for sense-organ assembly (Zhang, 1995).

In C. elegans, vab-3 mutants display many defects in head-region development, including aberrant morphogenesis, transformation of hypodermal (epidermal-like) cell fates to those of posterior homologues, and abnormal specification of neurons. vab-3 is a member of the paired-domain-containing Pax-6 gene family and is expressed in head-region cells. This C. elegans Pax-6 locus can also encode proteins lacking the paired domain. These results suggest that a primordial role of the Pax-6 gene family could have been to pattern part of the head region, and that Pax-6 genes subsequently evolved to be more specifically involved in eye development (Chisholm, 1995).

C. elegans has four members of the Six/sine oculis class of homeobox genes: ceh-32, ceh-33, ceh-34, and ceh-35. Proteins encoded by this gene family are transcription factors sharing two conserved domains, the homeodomain and the Six/sine oculis domain, both involved in DNA binding. ceh-32 expression is detected during embryogenesis in hypodermal and neuronal precursor cells and later in descendants of these cells as well as in gonadal sheath cells. RNAi inactivation studies suggest that ceh-32 plays a role in head morphogenesis, like vab-3, the C. elegans Pax-6 ortholog. ceh-32 and vab-3 are coexpressed in head hypodermal cells and ceh-32 mRNA levels are reduced in vab-3 mutants. Moreover, ectopic expression of VAB-3 in transgenic worms is able to induce ceh-32 ectopically. In addition, VAB-3 is able to bind directly to the ceh-32 upstream regulatory region in vitro and to activate reporter gene transcription in a yeast one-hybrid system. These results suggest that VAB-3 acts upstream of ceh-32 during head morphogenesis and directly induces ceh-32. Thus, ceh-32 appears to be the first target gene of VAB-3 identified so far (Dozier, 2001).

PAX-6 proteins are involved in eye and brain development in many animals. In the nematode Caenorhabditis elegans the pax-6 locus encodes multiple PAX-6 isoforms both with and without a paired domain. Mutations in the C. elegans pax-6 locus can be grouped into three classes. Mutations that affect paired domain-containing isoforms cause defects in epidermal morphogenesis, epidermal cell fates, and gonad cell migration and define the class I (vab-3) complementation group. The class II mutation mab-18(bx23) affects nonpaired domain-containing isoforms and transforms the fate of a sensory organ in the male tail. Class III mutations affect both paired domain and nonpaired domain isoforms; the most severe class III mutations are candidate null mutations in pax-6. Class III mutant phenotypes do not resemble a simple sum of class I and class II phenotypes. A comparison of class I and class III phenotypes indicates that PAX-6 isoforms can interact additively, synergistically, or antagonistically, depending on the cellular context (Cindar, 2004).

The C. elegans pax-6 locus expresses multiple transcripts that encode both typical PAX6-like proteins and smaller isoforms lacking the paired domain. Both previous work and this analysis of the pax-6 allelic series are consistent with these products having both independent and redundant functions in development. PD-containing isoforms have unique functions in gonadal distal tip cell migration, head epidermal morphogenesis, specification of the H0 ectodermal cell fate, and specification of B.a and Y.p ectodermal blast cell fates. Nonpaired domain-containing isoforms have unique functions in specification of the ray 6 fate. Both PD and non-PD isoforms may function redundantly to specify the H1 cell fate. This comparison of the phenotypic strength of class I and class III mutants suggests that non-PD-containing isoforms may synergize with PD-containing isoforms in head epidermal morphogenesis. Non-PD-containing isoforms may also weakly synergize with PD-containing isoforms in B division asymmetry. Finally, PD-containing isoforms appear to antagonize the role of non-PD-containing isoforms in ray 6 fate specification. pax-6 has also been shown to function in cell fate specification in nondividing head epidermal cells; both PD-containing and non-PD-containing isoforms appear to contribute to regulation of transcription of the sine oculis class homeobox gene ceh-32. An important avenue for future work will be to define additional targets of pax-6 regulation in the many tissues affected in pax-6 mutants (Cindar, 2004).

Integrin receptors for extracellular matrix are critical for cell motility, but the signals that determine when to stop are not known. Analysis of distal tip cell (DTC) migration during gonadogenesis in C. elegans has revealed the importance of transcription factor vab-3/Pax6 in regulating the alpha integrin genes, ina-1 and pat-2. Utilizing vab-3 mutants, it was shown that the down-regulation of ina-1 is necessary for DTC migration cessation and the up-regulation of pat-2 is required for directionality. These results demonstrate concomitant, but distinct roles in migration for each integrin. Notably, transcriptional control of migration termination provides a new mechanism for regulation of morphogenesis and organ size (Meighan, 2007).

The Pax-6 gene encodes a transcription factor essential for the development of eyes and other sensory organs in species ranging from planaria to mice. Because Pax-6 activity can be both necessary and sufficient for eye organogenesis, much work has focused on PAX-6 function and regulation of target genes. However, less is known about the genetic mechanisms that establish the Pax-6 expression pattern. C. elegans was used as a relatively simple model system to characterize the regulation of Pax-6 transcription in sensory organ precursors. In C. elegans males, two sensory mating structures, the copulatory spicules and the post-cloacal sensilla, are formed from stereotyped divisions of the two post-embryonic blast cells, B.a and Y.p, respectively. A C. elegans pax-6 transcript, vab-3, is necessary for the development of these sensory structures. Using a green fluorescent protein (GFP)-based vab-3 transcriptional reporter, it was shown that expression is restricted to the sensory organ lineages of B.a and Y.p. Transcription of vab-3 in the tail region of the worm requires the Abdominal B homeobox gene, egl-5. Opposing this activation, a transcription factor cascade and a Wnt signaling pathway each act to restrict vab-3 expression to the appropriate cell lineages. Thus multiple genetic pathways have been identified that act to restrict pax-6/vab-3 gene expression to the sensory organ precursor cells (Johnson, 2008).

Vertebrate Pax-6 and its Drosophila homolog eyeless play central roles in eye specification, although it is not clear if this represents the ancestral role of this gene class. As the most 'primitive' animals with true nervous systems, the Cnidaria may be informative in terms of the evolution of the Pax gene family. For this reason the Pax gene complement of a representative of the basal cnidarian class (the Anthozoa), the coral Acropora millepora, was surveyed. cDNAs encoding two coral Pax proteins were isolated. Pax-Aam encodes a protein containing only a paired domain, whereas Pax-Cam also contained a homeodomain clearly related to those in the Pax-6 family. The paired domains in both proteins most resemble the vertebrate Pax-2/5/8 class, but share several distinctive substitutions. As in most Pax-6 homologs and orthologs, an intron is present in the Pax-Cam locus at a position corresponding to residues 46/47 in the homeodomain. A model is proposed for evolution of the Pax family, in which the ancestor of all of the vertebrate Pax genes most resembled Pax-6, and arose via fusion of a Pax-Aam-like gene (encoding only a paired domain) with an anteriorly-expressed homeobox gene resembling the paired-like class (Catmull, 1998).

Pax genes encode a family of transcription factors, many of which play key roles in animal embryonic development, however, their evolutionary relationships and ancestral functions are unclear. To address these issues, the Pax gene complement of the coral Acropora millepora, an anthozoan cnidarian, has been characterized. As the simplest animals at the tissue level of organization, cnidarians occupy a key position in animal evolution; the Anthozoa are the basal class within this diverse phylum. Four Pax genes have been identifed in Acropora: two (Pax-Aam and Pax-Bam) are orthologs of genes identified in other cnidarians; the others (Pax-Cam and Pax-Dam) are unique to Acropora. Pax-Aam may be orthologous with Drosophila Pox neuro, and Pax-Bam clearly belongs to the Pax-2/5/8 class. The Pax-Bam Paired domain binds specifically and preferentially to Pax-2/5/8 binding sites. The recently identified Acropora gene Pax-Dam belongs to the Pax-3/7 class. Clearly, substantial diversification of the Pax family occurred before the Cnidaria/higher Metazoa split. The fourth Acropora Pax gene, Pax-Cam, may correspond to the ancestral vertebrate Pax gene and most closely resembles Pax-6. The expression pattern of Pax-Cam, in putative neurons, is consistent with an ancestral role of the Pax family in neural differentiation and patterning. The genomic structure of each Acropora Pax gene has been determined and some splice sites are shown to be shared both between the coral genes and between these and Pax genes in triploblastic metazoans. Together, these data support the monophyly of the Pax family and indicate ancient origins of several introns (Miller, 2000).

Alternatively spliced RNAs derived from a single Pax-6 gene in the squid (Loligo opalescens) are expressed in the embryonic eye, olfactory organ, brain, and arms. Despite significant sequence differences between squid Pax-6 and Drosophila Eyeless in the region outside the paired domains and homeodomains, squid Pax-6 is able to induce the formation of ectopic eyes in Drosophila. These results support the idea that Pax-6 related genes are necessary for eye and olfactory system formation throughout the animal kingdom (Tomarev, 1997).

The cloning of a Pax6 orthologue from the sepiolid squid Euprymna scolopes and its developmental expression pattern are described. The data are consistent with the presence of a single gene encoding a protein with highly conserved DNA-binding paired and homeodomains. A detailed expression analysis by in situ hybridization and immunodetection reveals Pax6 mRNA and protein with predominantly nuclear localization in the developing eye, olfactory organ, brain lobes (optic lobe, olfactory lobe, peduncle lobe, superior frontal lobe and dorsal basal lobe), arms and mantle, suggestive of a role in eye, brain, and sensory organ development (Hartmann, 2002).

Pax-6 genes have been identified from a broad range of invertebrate and vertebrate animals and shown to be always involved in early eye development. Therefore, it has been proposed that the various types of eyes evolved from a single eye prototype, including conserved Pax-6-dependent functions. The characterization of a cephalochordate Pax-6 gene is described. The single amphioxus Pax-6 gene (AmphiPax-6) can produce several alternatively spliced transcripts, resulting in proteins with markedly different amino and carboxy termini. The amphioxus Pax-6 proteins are 92% identical to mammalian Pax-6 proteins in the paired domain and 100% identical in the homeodomain. Expression of AmphiPax-6 in the anterior epidermis of embryos may be related to development of an olfactory epithelium. Expression is also detectable in Hatschek's left diverticulum during the time when it forms the preoral ciliated pit; part of this tissue gives rise to the homolog of the vertebrate anterior pituitary. A zone of expression in the anterior neural plate of early embryos is carried into the cerebral vesicle (a probable diencephalic homolog) during neurulation. This zone includes cells that will differentiate into the lamellar body, a presumed homolog of the vertebrate pineal eye. In neurulae, AmphiPax-6 is also expressed in ventral cells at the anterior tip of the nerve cord; these cells are precursors of the photoreceptive neurons of the frontal eye, the presumed homolog of the vertebrate paired eyes. However, AmphiPax-6 expression has not been detected in two additional types of photoreceptors, the Joseph cells or the organs of Hesse, which are evidently relatively recent adaptations (ganglionic photoreceptors) and appear to be rare exceptions to the general rule that animal photoreceptors develop from a genetic program triggered by Pax-6 (Glardon, 1998).

A Pax-6 homologous gene has been isolated from the ascidian Phallusia mammillata. Ascidians embryos exhibit a bilateral cleavage and a constant cell lineage. Ascidians occupy an important position in early chordate evolution; the Phallusia larva has a simple photosensitive ocellus. Two sensory organs, the ocellus and otolith (a gravity sense organ) arise from two precursor cells that are located symmetrically in the anterior half of the gastula and form an equivalence group. Either one of the precursor cells can differentiate into the ocellus or the otolith, depending on their final position in the brain vesicle. Phallusia Pax-6 shares extensive sequence identity and conserved genomic organization with the known Pax-6 genes of vertebrates and invertebrates. Expression of Phallusia Pax-6 is first detected at late gastrula stages in distinct regions of the developing neural plate. At the tailbud stage, it is expressed in the spinal cord, along the whole anteroposterior axis, and the entire prosencephalon (sensory vesicle), where the sensory organs (ocellus and otolith) form. Ectopic expression of the ascidian Pax-6 gene in Drosophila leads to the induction of supernumerary eyes, indicating a highly conserved gene regulatory function for Pax-6 genes (Glardon, 1997).

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Twenty-two full-length coding sequences of orthologs associated with neural patterning in chordates were isolated. These genes are probably present as single copies in S. kowalevskii because orthologs of most of them are present as single copies in lower chordates and echinoderms, and many of the genes were recovered multiple times in the EST analysis without finding any closely related sequences (Lowe, 2003).

Using full-length probes for in situ hybridization, all 22 genes were found to be expressed strongly in the ectoderm as single or multiple bands around the animal, in most cases without dorsal or ventral differences (rx, hox4, nkx2-1, en, barH, lim1/5, and otx are exceptions). Circumferential expression is consistent with diffuse neurogenesis in the ectoderm. The domains resemble the circumferential expression of orthologs in Drosophila embryos. In chordates, by contrast, most of these neural patterning genes are expressed in stripes or patches only within the dorsal neurectoderm and not in the epidermal ectoderm. Also, in chordates, the domains are often broader medially or laterally within the neurectoderm, and there are usually additional expression domains in the mesoderm and endoderm. In most of the 22 cases in S. kowalevskii, the ectodermal domain is the only expression domain (six3, otx, gbx, otp, nkx2-1, dbx, hox11/13, and irx are exceptions) (Lowe, 2003).

Although each of the 22 genes has a distinct expression domain along the anteroposterior dimension of the chordate body, attempts were made to divide them into three broad groups to facilitate the comparison with hemichordates: anterior, midlevel, and posterior genes. Anterior genes are those which in chordates are expressed either throughout or within a subdomain of the forebrain. Midlevel genes are those expressed at least in the chordate midbrain, having anterior boundaries of expression in the forebrain or midbrain, and posterior boundaries in the midbrain or anterior hindbrain. Posterior genes are those expressed entirely within the hindbrain and spinal cord of chordates. Many of the chordate genes have additional domains of expression elsewhere in the nervous system and in other germ layers, but comparisons were restricted to domains involved in specifying the neuraxis in the anteroposterior dimension. Taking these groups of genes one at a time, it was asked where the orthologous genes are expressed in S. kowalevskii. In all comparisons, no morphological homology is implied between the subregions of the chordate and hemichordate nervous systems (Lowe, 2003).

Ten genes expressed in midlevel neural domains were examined, namely tailless (tll), paired box homeobox 6 (pax6), emptyspiracles-like (emx), barH, orthopedia (otp), developing brain homeobox (dbx), lim domain homeobox 1/5 (lim1/5), iroquois (irx), orthodenticle-like (otx), and engrailed (en). These genes are all expressed in chordates at least in the midbrain of the central nervous system, and thus, as a group, their domains are more posteriorly located than the anterior set. Some have the anterior border of the domain in the forebrain (tll, pax6, emx, lim1/5, and otx), and some have their anterior border in the midbrain (otp, barH, dbx, irx, and en). Most have posterior borders in the midbrain, but two (en and irx) have posterior borders in the anterior hindbrain. Thus, while all are expressed in the midbrain, each differs in its anterior and posterior extent. Several of the chordate genes (pax6, dbx, en, and irx) have separate posterior expression domains running the length of the chordate hindbrain and spinal cord at different dorsoventral levels of the neural tube (Lowe, 2003).

In S. kowalevskii, these ten orthologs are expressed in circumferential bands in the ectoderm at least of the mesosome (collar) or anterior metasome, that is, more posteriorly than the anterior group. Each gene differs in the exact anteroposterior extent of its domain -- some are expressed in part or all of the prosome. The most broadly expressed orthologs of this group are pax6, otp, lim1/5, irx, and otx. All are expressed in the prosome (relatively weakly for otx), mesosome (weakly in the case of otp and lim1/5), and anterior metasome, all ceasing by the level of the first gill slit. pax6 is strongest at the base of the proboscis, and lim1/5 is expressed most strongly in a dorsal patch at the base of the proboscis. The most narrowly expressed orthologs are barH, tll, emx, and en. tll is detected in early stages in the anterior prosome, posterior prosome, and anterior mesosome and in later stages restricted to the anterior mesosome. The emx domain is a single ring in the anterior mesosome plus an additional domain in the ciliated band in the posterior metasome, the only gene of the 25 to be expressed in the band cells. barH and en are both expressed in narrow ectodermal bands; barH in the anterior mesosome and en in the anterior metasome. A dorsal view of both en and barH reveals a dorsal narrow gap in expression in the midline. Ventrally, no such gap is observed. Two additional spots of en expression are detected in the ectoderm on either side of the dorsal midline in the proboscis. In the most posterior ring of otx expression in the metasome, a similar gap in expression is observed. otp is expressed predominantly in a punctate pattern in the apical layer of prosome ectoderm and in a diffuse pattern in the basal layer of prosome ectoderm, similar to dlx. It is also expressed in a circumferential ring of intermittant ectodermal cells in the posterior mesosome and then in two parallel lines of cells bilateral to the dorsal axon tract of the anterior metasome. Early dbx expression is most strongly detected in an ectodermal ring in the developing mesosome overlapping the posterior domain of tll. dbx is also expressed in the prosome at low levels throughout the ectoderm and at high levels in scattered individual cells or groups of cells. Later expression is restricted to two ectodermal bands marking the anterior and posterior limits of the mesosome. An additional endodermal domain of expression is observed predominantly in the ventral anterior pharyngeal endoderm (Lowe, 2003).

otx, en, and irx deserve description in more detail because in chordates, especially vertebrates, the products of these regionally expressed genes are thought to interact in setting up the midbrain-hindbrain boundary and the isthmic organizer. Furthermore, the otx domain at the midbrain level is the site from which neural crest cells migrate ventrally to the first branchial arch. In S. kowalevskii, otx is expressed at low but readily detectable levels in the prosome ectoderm and at high levels in four closely spaced ectodermal rings: one at the base of the prosome, two in the mesosome, and one in the anterior metasome. This fourth stripe of otx expression crosses the site where the first gill slit perforates the ectoderm. As evidence, beyond morphology, that the hemichordate gill slit is homologous to the chordate gill slit/branchial arch, the pax1/9 ortholog, known to be expressed in chordate gill slits, is expressed in the endoderm of the developing S. kowalevskii gill slit. Gill slit expression of pax1/9 is observed in the adult of P. flava. Thus, chordates and hemichordates have in common the association of the posterior limit of the otx domain with the position of the first gill slit or branchial arch (Lowe, 2003).

In hemichordates, the en domain overlaps the posterior part of the otx domain, and the irx domain runs through both of these, as is also the case in chordates. However, otx expression in S. kowalevskii extends slightly more posteriorly than does en, whereas in chordates the en domain extends slightly more posteriorly (Lowe, 2003).

In summary, for this midlevel group of genes, the S. kowalevskii orthologs are expressed in the mesosome and anterior metasome (with some domains extending anteriorly into the prosome), that is, more posteriorly than those genes of the anterior group. In general, expression domains that end posteriorly near the midbrain-hindbrain boundary in chordates, end in the anterior metasome in hemichordates. Although the anterior metasome is not the site of an obvious morphological boundary, it is the site of the first gill slit. The first gill slit/branchial arch in chordates is at the same body level as the midbrain-hindbrain boundary (Lowe, 2003).

The 22 expression domains of orthologs of chordate neural patterning genes of S. kowalevskii correspond strikingly to those in chordates. There are differences such as the extent of overlap of edges of domains of otx, en, and gbx and other midlevel genes that are critical for forming boundaries within the chordate brain, but the relative domain locations are nonetheless very similar. This similar topography of domains is most parsimoniously explained by conservation in both lineages of a domain arrangement (a map) already present in the common ancestor, the ancestor of deuterostomes (Lowe, 2003).

At least 14 of the 22 conserved domains have similar locations in one or more protostome groups. Such similarities are most parsimoniously explained as a conservation of domains from the ancestral bilaterian. In the case of the hox genes, otx, emx, pax6, six3, gbx, and tll, there is strong evidence for such conservation, but less so for the others (barH and rx). At least four of the chordate-hemichordate conserved domains may not be shared by protostomes. Namely, three of these genes (dbx, vax, and hox11/13) are absent from the Drosophila genome and have not been cloned from other protostome groups. Also, one gene, engrailed, has no clear corresponding domain of expression known in protostomes. In Drosophila, en is expressed in the posterior compartments of 14 body segments and at three or more sites in the head that probably derive from ancient preoral segments. This pattern for en appears very different from the single ectodermal band in deuterostomes (Lowe, 2003).

The nerve net of hemichordates could represent the basal condition of the deuterostome ancestor, or it could represent the secondary loss of a central nervous system from an ancestor. Was the complex map of the ancestor associated with a complex diffuse nerve net or a central nervous system in the ancestor? It is suggested that the deuterostome ancestor may have had a diffuse basiepithelial nervous system with a complex map of expression domains, though not necessarily a diffuse net exactly like that of extant hemichordates. Hemichordates would then have retained a diffuse system in their lineage and early in the chordate lineage, centralization would have taken place. In this proposal, the domain map predates centralization and is carried into the nervous system. In this respect, the core questions of nervous system evolution would concern the modes of centralization utilized by the ancestor's various descendents rather than a dorsoventral inversion, per se. Thus, it is proposed that in chordates, especially vertebrates, the major innovation may have been the formation of a large contiguous nonneural (epidermogenic) region (Lowe, 2003).

To elucidate the evolutionary origin of nervous system centralization, the molecular architecture of the trunk nervous system was investigated in the annelid Platynereis dumerilii. Annelids belong to Bilateria, an evolutionary lineage of bilateral animals that also includes vertebrates and insects. Comparing nervous system development in annelids to that of other bilaterians could provide valuable information about the common ancestor of all Bilateria. The Platynereis neuroectoderm is subdivided into longitudinal progenitor domains by partially overlapping expression regions of nk and pax genes. These domains match corresponding domains in the vertebrate neural tube and give rise to conserved neural cell types. As in vertebrates, neural patterning genes are sensitive to Bmp signaling. These data indicate that this mediolateral architecture was present in the last common bilaterian ancestor and thus support a common origin of nervous system centralization in Bilateria (Denes, 2007).

Given the obvious paucity of information from the fossil record, the main strategy to elucidate CNS evolution is to compare nervous system development in extant forms. This comparative study of mediolateral neural patterning and neuron-type distribution in the developing trunk CNS of the annelid Platynereis revealed an unexpected degree of similarity to the mediolateral architecture of the developing vertebrate neural tube (Denes, 2007).

Three similarities are described. (1) The Platynereis and vertebrate neuroepithelium are similarly subdivided (from medial to lateral) into a sim+ midline and four longitudinal CNS progenitor domains (nk2.2+/nk6+, pax6+/nk6+, pax6+/pax3/7+, and msx+/pax3/7+), laterally bounded by an msx+, dlx+ territory. This strongly indicates a common evolutionary origin from an equally complex ancestral pattern. It is highly unlikely that precisely this mediolateral order and overlap in expression of orthologous genes in the CNS neuroectoderm should evolve twice independently. One can also discount the possibility that these genes are necessarily linked and thus co-opted as a package because they also act independently of each other in other developmental contexts (nk2.2 in endoderm development; pax6 in eye development, pax3/7 in segmentation, and msx in muscle development). Following similar reasoning, the complex conserved topography of gene expression along the anteroposterior axis in the enteropneust and vertebrate head is considered homologous (Denes, 2007).

(2) Evidence was found for conserved neuron types emerging from corresponding domains in Platynereis and in vertebrates. Serotonergic neurons involved in locomotor control form from the medial nk2.2+/nk6+ domain. A conserved population of hb9+ cholinergic somatic motoneurons emerges from the adjacent pax6+/nk6+ domain. Neurons expressing interneuron markers are found at the same level and more laterally, and single cells positive for sensory marker genes populate the lateral dlx+ domain. Notably, characterization of neuron types in the developing Platynereis nervous system is yet incomplete so that the full extent of conservation in neuron type distribution remains to be determined (Denes, 2007).

(3) Bmp signaling is similarly involved in the dose-dependent control of the neural genes. The finding that exogenous Bmp4 protein differentially regulates neural patterning genes in Platynereis nervous system development corroborates recent evidence that Bmps play an ancestral role in the mediolateral patterning of the bilaterian CNS neuroectoderm. Also, the strong upregulation of Pdu-atonal in the larval ectoderm goes in concert with Drosophila data that indicate that Dpp signaling positively regulates atonal expression in the lateral PNS anlage, and it supports the view that Bmp signaling also plays a conserved role in the specification of peripheral sensory neurons. Conservation of the molecular mediolateral CNS architecture concomitant with its sensitivity to Bmp signaling indicates that the developmental link between Bmp signaling and nervous system centralization predates Bilateria (Denes, 2007).

Taken together, these data make a very strong case that the complex molecular mediolateral architecture of the developing trunk CNS, as shared between Platynereis and vertebrates, was already present in their last common ancestor, Urbilateria. The concept of bilaterian nervous system centralization implies that neuron types concentrate on one side of the trunk, as is the case in vertebrates and many invertebrates including Platynereis, where they segregate and become spatially organized (as opposed to a diffuse nerve net). The data reveal that a large part of the spatial organization of the annelid and vertebrate CNS was already present in their last common ancestor, which implies that Urbilateria had already possessed a CNS (Denes, 2007).

Evolutionary conservation of the molecular mediolateral architecture as shared between Platynereis and vertebrates would imply that it was initially present also in the evolutionary lines leading to Drosophila, the nematode Caenorhabditis, and the enteropneust Saccoglossus. Yet it is clear from the available data that these animals are missing or have modified at least part of this pattern, although the extent of conservation may actually be larger than is currently apparent. For example, nk2.2/vnd and pax6 expression were costained in the fly, and a complementary pattern was found at germ-band-extended stage, reminiscent of the Platynereis and vertebrate situation. Strikingly, however, there is no trace so far of the conserved mediolateral architecture in the nematode Caenorhabditis and hardly any in the enteropneust Saccoglossus. How did this come about? Fly and nematode exhibit very fast development, making it plausible that they have (partially) omitted the transitory formation of longitudinal progenitor domains to speed up neurodevelopment. For the enteropneust, however, the situation is less clear. Why is the pattern absent in an animal that otherwise shows strong evolutionary conservation? One possible explanation is that the enteropneust trunk has lost part of its neuroarchitecture due to an evolutionary change in locomotion. While annelids and vertebrates propel themselves through trunk musculature (and associated trunk CNS), the enteropneust body is mainly drawn forward by means of the contraction of the longitudinal muscles in their anterior proboscis and collar. Possibly, enteropneusts have partially reduced their locomotor trunk musculature concomitant with motor parts of the CNS (while the peripheral sensory neurons prevailed in 'diffuse' arrangement). In line with this, expression of the conserved somatic motoneuron marker hb9/mnx is mostly absent from the Saccoglossus trunk ectoderm except for few patches. A more detailed understanding of enteropneust nervous system organization, neuron type distribution, and locomotion will help with resolving this issue (Denes, 2007).

An overall conservation of mediolateral CNS neuroarchitecture as proposed in this study does not imply that everything is similar. It is clear that the lines of evolution leading to annelids and vertebrates diverged for more than 600 million years, and numerous smaller or larger modifications of the ancestral pattern must have accumulated in both lines. The common-ground pattern as elucidated in this study helps in identifying these changes. For example, annelid and vertebrate differ in the deployment of gsx and dbx orthologs. While mouse gsh and dbx genes act early to specify interneuron progenitor domains in the dorsal neural tube, it was found the Platynereis gsx and dbx genes expressed at differentiation stages only. Adding to this, Pdu-gsx is expressed at a different mediolateral position in the nk2.2+ domain, and Pdu-dbx expression is much more restricted than that of its vertebrate counterparts (though the overall mediolateral coordinates correspond). It is hypothesized that these differences relate to the emergence of new interneuron domains (gsx+; dbx+) inside of the ancestral pax6+/pax3/7+ domain in the dorsal vertebrate neural tube. For this, it is conceivable that genes were recruited that had been active already in the differentiation of the diversifying interneuron populations. It is worth mentioning that the role of gsx in neuronal development also varies among vertebrates (Denes, 2007).

Homology of the vertebrate and Platynereis mediolateral molecular architecture is inevitably linked to the notion of dorsoventral axis inversion during early chordate evolution. In his 1875 essay on the origin of vertebrates Anton Dohrn discusses the resemblances between vertebrates and annelids and states that 'what stands most in the way of such a comparison has been the viewpoint that the nervous system of [annelids] is located in the venter, but that of vertebrates in the dorsum. Hence the one is called the ventral nerve cord, the other the dorsal nerve cord. Had we not possessed the terms dorsal and ventral, then the comparison would have been much easier.' How did the relocation of the trunk CNS from ventral to dorsal come about? Anton Dohrn proposed that vertebrate ancestors inverted their entire body dorsoventrally so that the former belly became the new back. This would not necessarily involve a sudden major shift in the lifestyle of an ancestor, as argued by critics of DV axis inversion. One can also imagine that an inversion involved transitional forms, with hemisessile or burrowing lifestyle and changing orientation toward the substrate. These animals had gill slits and lived as filter feeders. Only when early vertebrates left the substrate and acquired a free-swimming lifestyle would their new belly-up orientation have been fixed such that their CNS was then dorsal. Dohrn believed that the foremost gill slits then formed a new mouth on the new ventral body side. More than 130 years later, the molecular data on annelid neurodevelopment corroborate the key aspect of Dohrn's annelid theory, which is the homology of the annelid and vertebrate trunk CNS (Denes, 2007).

The Pax6 gene has attracted intense research interest due to its apparently important role in the development of eyes and the central nervous system (CNS) in many animal groups. Pax6 is also of interest for comparative genomics since it has not been duplicated in tetrapods, making for a direct orthology between the Ciona intestinalis gene CiPax6 and Pax6 in mammals. CiPax6 has been shown to be expressed in the anterior brain, caudal nerve cord, and in parts of the brain associated with the photoreceptive ocellus. This information has been extended in this study using in-situ hybridization. CiPax6 transcripts mark the lateral regions of the nerve cord, remarkably similar to Pax6 expression in the mouse. As a means of dissecting the cis-regulation of CiPax6 8 kb of sequence was tested using transient reporter transgene assays. Three separate regions were found that work together to drive the overall CiPax6 expression pattern. A 211 bp sequence 2 kb upstream of the first exon was found to be a major enhancer driving expression in the sensory vesicle (the anterior portion of the ascidian brain). Other upstream sequences were shown to work with the sensory vesicle enhancer to drive expression in the remainder of the CNS. An 'eye enhancer' was localized to the first intron. It controls specific expression in the central portion of the sensory vesicle, including photoreceptor cells. The fourth intron was found to repress ectopic expression of the reporter gene in middle portions of the embryonic brain. Aspects of this overall regulatory organization are similar to the organization of the Pax6 homologs in mice and Drosophila, particularly the presence of intronic elements driving expression in the eye, brain and nerve cord (Irvine, 2008).

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

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

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

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

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

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

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

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

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

The Pax6 genes eyeless and twin of eyeless are required for global patterning of the ocular segment in the Tribolium embryo

The transcription factor gene Pax6 is widely considered a master regulator of eye development in bilaterian animals. However, the existence of visual organs that develop without Pax6 input and the considerable pleiotropy of Pax6 outside the visual system dictate further studies into defining ancestral functions of this important regulator. Previous work has shown that the combinatorial knockdown of the insect Pax6 orthologs eyeless (ey) and twin of eyeless (toy) perturbs the development of the visual system but also other areas of the larval head in the red flour beetle Tribolium castaneum. To elucidate the role of Pax6 during Tribolium head development in more detail, head cuticle morphology, brain anatomy, embryonic head morphogenesis and developmental marker gene expression were studied in combinatorial ey and toy knockdown animals. The experiments reveal that Pax6 is broadly required for patterning the anterior embryonic head. One of the earliest detectable roles is the formation of the embryonic head lobes, which originate from within the ocular segment and give rise to large parts of the supraesophageal brain including the mushroom body, a part of the posterior head capsule cuticle, and the visual system. Evidence is presented that toy continues to be required for the development of the larval eyes after formation of the embryonic head lobes in cooperation with the eye developmental transcription factor dachshund (dac). The sum of these findings suggests that Pax6 functions as a competence factor throughout the development of the insect ocular segment. Comparative evidence identifies this function as an ancestral aspect of bilaterian head development (Luan, 2014).


Table of contents


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

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