Origin and evolution of the paired domain

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).

A Pax gene from the hydrozoan Podocoryne carnea has been characterized. It is most similar to cnidarian Pax-B genes and encodes a paired domain, a homeodomain and an octapeptide. Expression analysis demonstrates the presence of Pax-B transcripts in eggs, the ectoderm of the planula larva and in a few scattered cells in the apical polyp ectoderm. In developing and mature medusae, Pax-B is localized in particular endodermal cells, oriented toward the outside. Pax-B is not expressed in muscle cells. However, if isolated striated muscle tissue is activated for transdifferentiation, the gene is expressed within 1 h, before new cell types, such as smooth muscle and nerve cells, have formed. The expression data indicate that Pax-B is involved in nerve cell differentiation (Groger, 2000).

The degree of conservation within the paired domain of Pax genes from other phyla is remarkable: more than 80% identity to Pax-2/5/8-like paired domains; more than 70% identity to Pax-6-like paired domains, and about 70% to other paired domains. As in all other Pax proteins, the substitutions are mainly found in the first part of helix 1, in helix 4 and at the beginning of helix 6. Moreover, the positions of phosphate backbone DNA contacts and for contacting major and minor grooves are perfectly conserved in the Podocoryne Pax-B paired domain and suggest DNA binding capacity. The second DNA-binding motif in Podocoryne Pax-B, the homeodomain, is also conserved and the serine at position nine of helix 3 identifies it as a paired-type homeodomain. The Podocoryne Pax-B homeodomain is most similar to its cnidarian counterparts encoded by the Pax-B-type genes (Sparkling in Drosophila). The degree of identity compared to the homeodomain of Drosophila paired (60%), murine Pax-7 (57%) or Pax-6 (53%) is noteworthy, and again, DNA contact positions are completely preserved. A stretch of 57 amino acids separates the octapeptide from the paired domain. The octapeptide most resembles that of mammalian Pax-2. The octapeptide of mammalian Pax-2 and Pax-8 has been shown to act as a transcriptional activator. However, outside of the paired domain, the octapeptide and the homeodomain, no sequence homologies to other Pax genes are identifiable (Groger, 2000).

The Pax gene family consists of tissue-specific transcriptional regulators that always contain a highly conserved DNA-binding domain, including six alpha-helices (paired domain), and, in many cases, a complete or residual homeodomain. Numerous genes of this family have been identified in animals, the largest numbers being found in vertebrates. Evolutionary analyses indicate that the vertebrate Pax gene family consists of four well-defined and statistically supported groups: group I (Pax-1, 9), II (Pax-2, 5, 8), III (Pax-3, 7), and IV (Pax-4, 6). Group I paired domains share a most recent common ancestor with Drosophila Pox meso, group II with Pox neuro, group III with paired and gooseberry, and group IV with the eyeless gene. Two groups containing complete homeodomains (III and IV) are distantly related, and the intergroup relationships are (I,III) and (II,IV). These four major groups arose before the divergence of Drosophila and vertebrates prior to the Cambrian radiation of triploblastic metazoan body plans. Analysis of fixed radical amino acid differences between groups was performed in a phylogenetic context. All four fixed radical amino acid differences between groups I and III are located exclusively in the N-terminal alpha-helices. Similarly, groups II and IV show three fixed radical differences in these alpha-helices but at positions different from those in groups I and III. Implications of such fixed amino acid differences in potentially generating sequence recognition specificities are discussed in the context of some recent experimental findings (Balczarek, 1997).

Domain structure and function of Pax proteins

Pax-5 encodes the transcription factor BSAP which plays an essential role in early B cell development and midbrain patterning. The structural requirements have been examined for transcriptional activation by BSAP. In vitro mutagenesis and transient transfection experiments indicate that the C-terminal serine/threonine/proline-rich region of BSAP contains a potent transactivation domain of 55 amino acids which is active from both the promoter and the enhancer positions. This transactivation domain is found to be inactivated by a naturally occurring frameshift mutation in one PAX-5 allele of the acute lymphoblastic leukemia cell line REH. The function of the transactivation domain is negatively regulated by adjacent sequences from the extreme C-terminus. The activating and inhibitory domains function together as an independent regulatory module in different cell types, as shown by fusion to the GAL4 DNA binding domain. The same arrangement of positively and negatively acting sequences has been conserved in mammalian Pax-2 and Pax-8, and zebrafish Pax-b, as well as sea urchin Pax-258 proteins. These data demonstrate that the transcriptional competence of a subfamily of Pax proteins is determined by a C-terminal regulatory module composed of activating and inhibitory sequences (Dorfler, 1997).

Pax-6 is known to be a key regulator of vertebrate eye development. cDNA for an invertebrate Pax-6 protein has been isolated from sea urchin embryos. Transcripts of this gene first appear during development at the gastrula stage and are later expressed at high levels in the tube foot of the adult sea urchin. The sea urchin Pax-6 protein is highly homologous throughout the whole protein to its vertebrate counterpart, with almost identical paired domains and almost identical homeodomains. Consequently, the DNA-binding and transactivation properties of the sea urchin and mouse Pax-6 proteins are very similar, if not identical. A potent activation domain capable of stimulating transcription from proximal promoter and distal enhancer positions was localized within the C-terminal sequences of both the sea urchin and mouse Pax-6 proteins. The homeodomain of Pax-6 was shown to cooperatively dimerize on DNA sequences consisting of an inverted repeat of the TAAT motif with a preferred spacing of 3 nucleotides. The consensus recognition sequence of the Pax-6 paired domain deviates primarily at only one position from that of BSAP (Pax-5), and yet the two proteins exhibit largely different binding specificities for individual, naturally occurring sites. By creating Pax-6-BSAP fusion proteins, a short amino acid stretch has been identified in the N-terminal part of the paired domain that is responsible for these differences in DNA-binding specificity. The mutation of three Pax-6-specific residues in this region (at positions 42, 44, and 47 of the paired domain) to the corresponding amino acids of BSAP results in a complete switch of the DNA-binding specificity from Pax-6 to BSAP. These three amino acids have been shown to discriminate between the Pax-6- and BSAP-specific nucleotides at the divergent position of the two consensus recognition sequences (Czerny, 1995).

Invertebrate Pax-2/5/8 homologs

Pax proteins form a family of transcription factors sharing 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 either 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 type of 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 the hydra Pax-B paired domain binds 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) than to either Pax-1/9 (Drosophila homolog: Pox meso) or Pax-3/7 (Drosophila homologs: 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, which contains both the homeodomain and the octapeptide (Sun, 1997).

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).

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).

Mutations in the C. elegans gene egl-38 result in a discrete set of defects in developmental pattern formation. In the developing egg-laying system of egl-38 mutant hermaphrodites, the identity of four uterine cells is disrupted and they adopt the fate of their neighbor cells. Likewise, the identity of two rectal epithelial cells in the male tail is disrupted; one of these cells adopts the fate of its neighbor cell. Genetic analysis suggests that egl-38 functions in the tail and the egg-laying system are partially separable, as different egl-38 mutations can preferentially disrupt the different functions. egl-38 is a member of the PAX family of genes, which encodes transcription factors implicated in a variety of developmental patterning events. The predicted EGL-38 protein is most similar to the mammalian class of proteins that includes PAX2, PAX5 and PAX8. The sequence of egl-38 mutant DNA indicates that the tissue-preferential defects of egl-38 mutations result from substitutions in the DNA-binding paired domain of the EGL-38 protein. egl-38 thus provides the first molecular genetic insight into two specific patterning events that occur during C. elegans development and also provides the opportunity to investigate the in vivo functions of this class of PAX proteins with single cell resolution (Chamberlin, 1997).

Three novel Pax genes of the sea urchin were isolated for which no homolog s are yet known in other animal phyla. One of these genes, suPaxB, codes for the previously characterized transcription factor TSAP, which is involved in the developmental regulation of two pairs of late histone genes. Conserved members of the Pax2/5/8 subfamily, which have so far been described only in vertebrates, were isolated not only from the sea urchin, but also from Drosophila and C. elegans. Hence, the Pax2/5/8 transcription factors constitute an ancient subfamily of highly conserved Pax proteins. During Drosophila embryogenesis, the Pax258 gene is shown to be expressed in the precursor cells of the external sensory organs, suggesting a role for Pax258 in the early development of the peripheral nervous system of insects (Czerny, 1997).

The Pax gene egl-38 plays an important role in the development of several organs in C. elegans. egl-38 encodes a Pax transcription factor that is most similar to the mammalian Pax2/5/8 subclass of factors. To understand how a Pax transcription factor influences distinct developmental choices in different cells and tissue types, a second gene, lin-48, has been characterized. lin-48 functions with egl-38 in the development of one structure, the hindgut, but not in other tissues such as the egg-laying system. lin-48 encodes a C2H2 zinc-finger protein that is similar to the product of the Drosophila gene ovo and is expressed in the hindgut cells that develop abnormally in lin-48 mutants. Evidence is presented that lin-48 is a target for EGL-38 in hindgut cells. lin-48 requires egl-38 for its expression in the hindgut. Using deletion analysis, two elements in the lin-48 promoter have been identified that are necessary for lin-48 expression. EGL-38 binds with high affinity to one of these elements. In addition, genetic interactions have been observed between mutations in the lin-48 promoter and specific alleles of egl-38. These experiments demonstrate a functional link between Pax and Ovo transcription factors, and provide a model for how Pax transcription factors can regulate different target genes in different cells (Johnson, 2001).

Work with ovo genes in Drosophila and mouse has focused on their roles in fertility and epidermal development. Although lin-48 plays no apparent role in fertility or development of epidermis, ovo genes in mouse, Drosophila and C. elegans exhibit parallels in that they all play a role in the differentiation and maintenance of specific cell types. In addition, C. elegans and mouse ovo genes are similar in that they play a role in urogenital development. Mouse Ovo1 is important in development of the genital tract and kidney, and lin-48 plays a role in development of the hindgut (which develops into the C. elegans adult male cloaca) and potentially the excretory system. The experiments reported here indicate lin-48 is a direct target for EGL-38. A direct link between Pax factors and ovo genes has not been previously reported. However, genetic parallels in mammals indicate the potential for a conserved functional relationship between these classes of genes. In vertebrates, the Pax2 gene is essential for development of kidney, brain and ear, and the Pax8 gene plays a role in thyroid and kidney development. Mouse Ovo1 is expressed abundantly in the kidney, and is required for its normal differentiation. Thus, as in C. elegans, Ovo1 acts in a subset of the cells that require Pax2/5/8 factors. Future experiments will be required to test whether Ovo1 is a target for Pax2 or Pax8 during kidney development. Since all of the functions of the Drosophila Pax2/5/8 gene sparkling (shaven) have not been characterized, it is not known whether there are developmental functions shared by ovo and sparkling (Johnson, 2001).

Ascidians (phylum Urochordata) and vertebrates both have dorsal tubular central nervous systems. The structure of the ascidian neural tube is extremely simple, containing less than 400 cells, among which less than 100 cells are neurons. Recent studies suggest that despite its simple organization, the mechanisms patterning the ascidian neural tube are similar to those of the more complex vertebrate brain. However, identification of homologous regions between vertebrate and ascidian nervous systems remains to be resolved. This study describes the expression of the HrPax-258 gene, an ascidian homolog of vertebrate Pax-2, Pax-5 and Pax-8 genes. Molecular phylogenetic analyses indicate that HrPax-258 is descendant from a single precursor gene that gave rise to the three vertebrate genes. The expression pattern of HrPax-258 suggests that this subfamily of Pax genes has conserved roles in regional specification of the brain. Comparison with expression of ascidian Otx (Hroth) and a Hox gene (HrHox1) by double-staining in situ hybridizations indicates that the ascidian brain region can be subdivided into three parts: an anterior region marked by Hroth, and probably homologous to the vertebrate forebrain and midbrain; a middle region, marked by HrPax-258 and probably homologous to the vertebrate anterior hindbrain (and maybe also the midbrain), and a posterior region marked by Hox genes, which is homologous to the vertebrate hindbrain and spinal cord. Later expression of HrPax-258 in atrial primordia implies that basal chordates such as ascidians have already acquired a sensory organ that develops from epidermal thickenings (placodes) and expresses HrPax-258; this organ may be homologous to the vertebrate ear. The atrial primordia develop as a pair of ectodermal invaginations that fuse to form one atrial siphon. This mode of formation is strikingly similar to that of vertebrate placodes. The atrium of adult ascidians develops ciliated sensory cells in cupular organs resembling those of the vertebrate acoustico-lateralis system. Therefore, placodes are not likely to be a newly acquired feature in vertebrates, but may have already been possessed by the earliest chordates. It is suggested that the atrial primordia of ascidian larvae are homologous to the vertebrate inner ear (otic system). HrPax-258 is also expressed in the primordial pharynx. This organ develops into the oral siphon of adult ascidians. The oral siphon of adult ascidians can be regarded as a mouth. The similarity of cell lineage strongly supports the homology between the ascidian and vertebrate mouth (Wada, 1998).

On the basis of developmental gene expression, the vertebrate central nervous system comprises a forebrain plus anterior midbrain; a midbrain-hindbrain boundary region (MHB) having organizer properties, and a rhombospinal domain. The vertebrate MHB is characterized by position, by organizer properties and by being the early site of action of Wnt1 and engrailed genes, and of genes of the Pax2/5/8 subfamily. Ascidian tunicates have a vertebrate-like MHB on the basis of ascidian Pax258 expression there. In another invertebrate chordate, amphioxus, comparable gene expression evidence for a vertebrate-like MHB is lacking. AmphiPax2/5/8, the sole member of this subfamily was therefore isolated and characterized in amphioxus. AmphiPax2/5/8 is initially expressed well back in the rhombospinal domain and not where a MHB would be expected. In contrast, most of the other expression domains of AmphiPax2/5/8 correspond to expression domains of vertebrate Pax2, Pax5 and Pax8 in structures that are probably homologous: support cells of the eye, nephridium, thyroid-like structures and pharyngeal gill slits (although AmphiPax2/5/8 is not transcribed in any structures that could be interpreted as homologs of vertebrate otic placodes or otic vesicles). In sum, the developmental expression of AmphiPax2/5/8 indicates that the amphioxus central nervous system lacks a MHB resembling the vertebrate isthmic region. Additional gene expression data for the developing ascidian and amphioxus nervous systems would help determine whether a MHB is a basal chordate character secondarily lost in amphioxus. The alternative is that the MHB is a vertebrate innovation (Kozmik, 1999).

Pax8 is expressed in the developing thyroid of most vertebrates. These Pax genes are expressed in the thyroid rudiment during its differentiation from the floor of the pharynx. The thyroid of adult vertebrates also expresses Pax8, evidently to maintain the differentiated state by modulating cell proliferation and the expression of two thyrocyte-specific genes. In amphioxus embryos, Amphipax2/5/8 is expressed in the thickened endoderm in the right anterior region of the pharynx. At the end of the larval stage of amphioxus, this thickening, called the endostyle, migrates ventrally and then to the posterior, finally taking the form of a groove along the pharyngeal floor of the juveniles and adults. It was originally proposed that the amphioxus endostyle is the homolog of the vertebrate thyroid gland, because larval lampreys have an endostyle-like structure that becomes converted into a thyroid gland later in development. The amphioxus endostyle and the vertebrate thyroid gland have since been found to synthesize iodothyronines, thyroglobulins and thyroperoxidases. The homology between the vertebrate thyroid and the amphioxus endostyle has been questioned, since the latter has no known endocrine function (it makes food-trapping extracellular secretions). Most recently, the expression of AmphiNk2-1 (amphioxus TTF-1) and Amphipax2/5/8 in the endostyle lends additional support to its proposed homology to the vertebrate thyroid gland (Kozmik, 1999 and references).

Pax2 and Pax8 are transcribed in the developing vertebrate kidney. In amniotes, there is a successive, anterior-to-posterior formation of a pronephros, mesonephros and metanephros, with only the last functioning as the adult kidney. In fishes and amphibians, the metanephros does not form and the mesonephros becomes the adult kidney; in agnathans, the pronephros is the only kidney to develop. The vertebrate pronephros typically develops bilaterally in a few anterior metameres from a thickening of the somatic mesothelium of the intermediate mesoderm. On either side of the body, these mesothelial cells become rearranged into a compact bud that acquires a small lumen, the nephrocoel, and produces the pronephric tubules and their common nephric duct. The pronephric tubules and duct are subsequently completed without any contribution from the surrounding mesenchyme cells. In contrast, the mesonephros and metanephros have a dual origin: from side branches of the nephric duct as well as from nearby mesenchyme cells that form kidney tubules by a mesenchyme-to-epithelium transition. In amphioxus, the excretory system develops as a segmentally arranged series of tubules without any contribution from condensing mesenchyme. These tubules are widely thought to be homologous to the mesodermal pronephric tubules of vertebrates. Amphipax2/5/8 is expressed in the rudiment of the earliest excretory tubule (often called Hatschek’s nephridium) to appear during the embryonic development, thus strengthening the homology between amphioxus excretory organs and the vertebrate pronephros, which develops under the influence of homologous Pax genes. This is additional evidence against the alternative idea that the excretory tubules of amphioxus are ectodermally derived and thus not homologous to the vertebrate kidney tubules (Kozmik, 1999 and references).

In Xenopus embryos, Pax2 is expressed in the ectodermal furrows of the visceral arches, which suggests that the gene might play a role in perforation of the gill slits. For amphioxus, formation of the gill slits and mouth (which is usually considered to be a modified gill slit) may well be under similar genetic control, because the pharyngeal endoderm and the ectoderm express Amphipax2/5/8 in regions where the two epithelial layers will later make contact and fuse. It is possible that Amphipax2/5/8 may be interacting with downstream genes that control mechanochemical properties of the extracellular matrix to facilitate fusion of the adjacent endodermal and ectodermal epithelia. It has been suggested that Pax genes may interact with genes encoding extracellular materials and thereby influence tissue-level morphogenesis; e.g. mouse Pax8 binds to and activates the N-CAM gene. One kind of this morphoregulation would be the local dissolution of basal laminae to permit fusion of two adjacent epithelia that have made intimate contact. Further examples may be the regulation of the closure of the neural tube or optic fissure by mouse Pax2 and regulation of processes connecting the internal and external epithelia by egl-38, a somewhat aberrant member of the Pax2/5/8 subfamily in Caenorhabditis. Vertebrate Pax2 and Pax8 are expressed in the otic placode and in the otic vesicle formed by its invagination, and Pax2 plays a key role in the development of the cochlea and spiral ganglion of the inner ear. In zebrafish, but evidently not in higher vertebrates, Pax5 is expressed in addition to Pax8 during the differentiation of the otic vesicle. There are no previous reports that amphioxus embryos or larvae have otic placodes or otic vesicles and no patterns of Amphipax2/5/8 expression were found suggesting that such structures were overlooked in earlier studies. In contrast, in the larvae of another group of invertebrate chordates (namely the ascidian tunicates), HrPax258 is expressed in two patches of epidermis that are destined to invaginate to form the atria. This pattern of gene expression, together with the presence of cupula-like structures in ascidian atria has prompted a suggestion that the epidermal regions giving rise to the atria might be homologs of vertebrate otic placodes and that the atria themselves might be homologs of vertebrate otic vesicles. If these homologies are valid, it implies that otic placodes/vesicles are a fundamental chordate character that must have been lost in the evolutionary line leading to amphioxus. Alternatively, it is possible that ascidian atria represent not otic vesicles, but the outer part of gill slits; in this case, the epidermal expression of HrPax258 would represent not otic placodes, but regions of epidermis destined to invaginate and then form the outer part of the gill slits by fusion with the endoderm. More light might be shed on the atrial homologies of tunicates by a study of Pax2/5/8 expression during gill slit development in appendicularians, which represent the most basal group of tunicates and have only one pair of simple gill slits. Amphioxus, like ascidians, develops a peribranchial space called the atrium. Unlike ascidian atria, the one in amphioxus develops relatively late in development (in one-month-old larvae undergoing metamorphosis) as a single, not a paired, structure. Moreover, the amphioxus atrium forms not by epidermal invagination, as in ascidians, but by the outgrowth of bilateral flanges of body wall that meet midventrally except in a region that becomes the atriopore. In spite of these differences, it has been claimed that the atria of ascidians and amphioxus are homologous. In 1- month-old metamorphic amphioxus larvae, no Amphipax2/5/8 expression was found in structures associated with atrium formation, although expression was detected of the amphioxus actin gene BfMA1 in the muscles. It is thought that the amphioxus atrium may be homologous with the efferent gill chambers of myxinoid hagfishes (Kozmik, 1999 and references).

Cells expressing Amphipax2/5/8 near the anterior end of the amphioxus cerebral vesicle are the presumed precursors of the frontal eye pigment cells. Amphipax2/5/8 expression in these neuronal support cells appears to be comparable to transcription of Drosophila Pax2/5/8 (sparkling) in the developing pigment cells of the compound eye and to transcription of vertebrate Pax2 in glial cells lining the choroid fissure and optic stalk/nerve of the paired eyes. Transcription of amphioxus Amphipax2/5/8 in developing pigment cells of the frontal eye is consistent with a proposal that an ancestral Pax2 gene played a role in neuronal support of glial cells during photoreceptor development in the common precursor of the protostomes and deuterostomes. Even so, the absence of ascidian HrPax258 expression in the sensory vesicle cells near the larval ocellus does not fit with the idea and points to the need for genetic studies of photoreceptors in a wider range of animal groups (Kozmik, 1999 and references).

Early neural expression of vertebrate Pax2 appears not only in the MHB region, but also in the rhombospinal domain, where the gene is transcribed in differentiating interneurons located laterally on either side of the neural tube. In the rhombospinal region of amphioxus, the earliest detectable expression of Amphipax2/5/8 begins roughly in the middle of the hindbrain and spreads posteriorly and anteriorly from there. Eventually, the anterior limit of the hindbrain expression domain of Amphipax2/5/8 approaches a region of the neural tube where a MHB might be expected to occur if one existed; however, this spatiotemporal pattern is quite different from the early appearance of vertebrate Pax2, Pax5 and Pax8 in the vertebrate MHB region. In the rhombospinal region of amphioxus, Amphipax2/5/8 can be expressed dorsoventrally anywhere except in the floor plate cells. This expression pattern may reflect a wide-spread distribution of interneurons in the dorsoventral axis of the amphioxus nerve cord; unfortunately, knowledge of the neuroanatomy of the amphioxus rhombospinal domain is still very incomplete and needs to be reinvestigated. HrPax258 transcription has been studied in larval ascidians and its neural expression is limited to the neck region of the central nervous system. Most of the rhombospinal region of the ascidian larval nervous system is composed solely of ependymal cell bodies and a few descending motor axons. Thus the absence of HrPax258 expression in this posterior region of the ascidian nervous system might be related to the lack of interneurons there (Kozmik, 1999 and references).

Genes encoding a novel group of homeodomain transcription factors, ONECUT class homeodomain proteins, have been isolated from vertebrates and insects. Among them, vertebrate HNF-6 is expressed in hepatocytes and the central nervous system during embryogenesis. Although the functions of HNF-6 in hepatocytes have been well studied, the functions of HNF-6 in the central nervous system have remained unknown. In this study, HrHNF-6, which encodes a new ONECUT class homeodomain protein, has been isolated from an ascidian, Halocynthia roretzi. HrHNF-6 mRNA was expressed exclusively in neural cells, just posterior to the expression of Hroth (the ascidian homolog of vertebrate Otx) during embryogenesis. One of the functions of HrHNF-6 in neural cells is the activation of the expression of HrTBB2, the ascidian beta-tubulin gene. Another is the restriction of the expression of HrPax-258 (which is expressed in the neural tube), suggesting that HrHNF-6 functions in the specification of the neural tube. These results indicate that HrHNF-6 functions in the differentiation and regional specification of neural cells during ascidian embryogenesis (Sasakura, 2001).

Double-staining in situ hybridization had revealed that HrPax-258 is expressed just next to one of the HrHNF-6-positive zones. Moreover, ectopic expression of HrHNF-6 markedly reduces the expression of HrPax-258 in the neural plate. These results suggest that HrHNF-6 func tions in the specification of the neural tube by restricting the HrPax-258 expression. Because HrHNF-6-EnR is associated with strong activity to repress the HrPax-258 expression in the neural plate and epidermis, HrHNF-6 itself likely represses the transcription of HrPax-258. Since HNF-6 has been reported to be a transcriptional activator, the activity of HrHNF-6 as a repressor is surprising. It is possible that HrHNF-6 uses an unknown cofactor, and the Engrailed repressor domain mimics its function. HrPax-258 was expressed as early as the neural plate stage, in one bilateral pair of cells in the neural plate. The timing of this expression is very close to that of HrHNF-6. HrHNF-6 may regulate HrPax-258 from the beginning of its expression. HrHNF-6 has only a very weak effect on the expression of HrPax-258 in the epidermal lineage cells. This suggests that the expression of HrPax-258 is regulated differently in the neural plate and epidermis, through different cis elements and trans acting factors that bind to the cis elements. HrHNF-6 regulates primarily the expression from the cis element corresponding to the neural plate expression. When the amount of HrHNF-6 mRNA injected is increased or when HrHNF-6-EnR mRNA is injected, HrPax-258 expression in the epidermis is reduced, suggesting that HrHNF-6 also regulates the expression of HrPax-258 in the epidermis in a weak fashion (Sasakura, 2001).

HrPax-258 is thought to resemble the ancestral gene of mammal Pax-2, Pax-5 and Pax-8. Mouse Pax-2 and Pax-5 are expressed at the midbrain-hindbrain boundary (MHB), and function in midbrain formation. Mutation at the Pax-2 locus in mice results in a defect of MHB and subsequent defects in midbrain and cerebellum. In chick, the ectopic expression of Pax-2 or Pax-5 at the mesencephalon induces the ectopic midbrain to form at that position. In zebrafish, a mutation in the pax-b gene affects the formation of MHB. The dominance of Pax-2 in brain regionalization implies that there must be mechanisms that restrict the expression of Pax-2 at the MHB. There have been two reports that mammalian HNF-6 is expressed in the brain. Whether HNF-6 functions in the regulation of Pax-2, -5, -8 transcription in vertebrate brain of interest and should be investigated (Sasakura, 2001).

The tripartite organization of the central nervous system (CNS) may be an ancient character of the bilaterians. However, the elaboration of the more complex vertebrate brain depends on the midbrain-hindbrain boundary (MHB) organizer, which is absent in invertebrates such as Drosophila. The Fgf8 signaling molecule expressed in the MHB organizer plays a key role in delineating separate mesencephalon and metencephalon compartments in the vertebrate CNS. This study presents evidence that an Fgf8 ortholog establishes sequential patterns of regulatory gene expression in the developing posterior sensory vesicle, and the interleaved 'neck' region located between the sensory vesicle and visceral ganglion of the simple chordate Ciona intestinalis. The detailed characterization of gene networks in the developing CNS led to new insights into the mechanisms by which Fgf8/17/18 patterns the chordate brain. The precise positioning of this Fgf signaling activity depends on an unusual AND/OR network motif that regulates Snail, which encodes a threshold repressor of Fgf8 expression. Nodal is sufficient to activate low levels of the Snail repressor within the neural plate, while the combination of Nodal and Neurogenin produces high levels of Snail in neighboring domains of the CNS. The loss of Fgf8 patterning activity results in the transformation of hindbrain structures into an expanded mesencephalon in both ascidians and vertebrates, suggesting that the primitive MHB-like activity predates the vertebrate CNS (Imai, 2009).

This study provides a number of key insights into the compartmentalization of the chordate CNS. First, a localized Fgf8 signaling center was probably used by the last shared ancestor of ascidians and vertebrates to delineate two regions of the chordate brain (mesencephalon and metencephalon). Second, Fgf8 signaling in Ciona leads to restricted expression of Otx and FoxB in the PSV, as well as restricted expression of Pax2/5/8-A in the neck. Otx and FoxB might inhibit Hox1 expression in the forebrain via Cyp26, whereas Pax2/5/8-A might coordinate the expression of the regulatory genes required for the differentiation of metencephalon motoneurons, such as Phox2a/Arix. Finally, although the regulatory genes responsible for the compartmentalization of the vertebrate CNS (e.g. Otx, Pax2, Neurogenin, etc.) exhibit comparable patterns of expression in the Ciona CNS, there are both conserved and distinctive features of the underlying mechanism. Localized Fgf8 signaling is used to deploy these expression patterns in both systems, even though different regulatory mechanisms are used to restrict Fgf8 (Imai, 2009).

Vertebrate Pax2 cloning, expression and regulation

Evolutionary homologs continued: part 2/5 | part 3/5 | part 4/5 | part 5/5 |

sparkling: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.