paired


EVOLUTIONARY HOMOLOGS


Table of contents

Domain structure and evolution of paired domain proteins

Drosophila Paired like proteins include Gooseberry-proximal and -distal, and Pox-neuro and -meso (The latter two lack homeoboxes). The mouse pax gene family includes Pax-6, the vertebrate homolog of eyeless (Cai, 1994).

The closest mammalian homolog of Paired is Pax-3. The Drosophila gooseberrys are also part of the same group. Information about Pax-3 and Pax-7 roles in neurogenesis is found in Gooseberry-distal.

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).

A novel Drosophila paired-like homeobox gene, DPHD-1, has been isolated. The homeodomain of DPHD-1 shows 85% amino-acid identity with that of the C. elegans Unc-4 protein. Whole-mount in situ hybridization of embryos and third-instar larvae reveal that the DPHD-1 mRNA is specifically localized in subsets of postmitotic neurons in the central nervous system (CNS) and in the developing epidermis, where it displays a segmentally repeated pattern. Double staining with a posterior compartment marker, an anti-Engrailed antibody, has shown that DPHD-1 expressing neurons in the CNS are present in the posterior compartment, whereas DPHD-1 expression in the epidermis is restricted to the anterior compartment in each segment. This temporal and spatial expression pattern suggests that DPHD-1 may play a role in determining the distinct cell types in each segment (Tabuchi, 1998).

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the N-terminal PAI and the C-terminal RED domains, have been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. The PAI domain is necessary and sufficient for DNA binding in vitro. In fact, the Drosophila Paired protein does not need the RED domain to confer full function to the protein in vivo. There are, however, situations in which the RED domain may play a role. The bipartite organization of the PD allows for the recognition of composite sites by both PAI and RED domain. Furthermore, there is an isoform of the Pax-6 protein (Pax-6 5a) that contains, in the middle of the recognition helix of the PAI domain, an 11-residue insertion that inactivates DNA binding to sequences normally bound by the Pax-6 PD. Pax-6 5a, however, is able to bind to a recently described sequence called 5aCON. Other Pax proteins also contain insertions in the PAI domain that inactivate DNA binding and uncover RED-domain DNA-binding activity. However, to date, there has been no report of a Pax protein containing only a PAI or a RED domain (Jun, 1998 and references).

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).

A Pax protein, originally called Lune, is the product of the Drosophila eyegone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. Most PDs are located in close proximity to the N terminus of Pax proteins: the RED domain of Eyg starts at position 36 of the protein. It exhibits very limited homology with the end of the PAI domain until residue 47 of the full-length PD. At position 48, the homology becomes higher and extends 7 aa to position 54. The sequence is then interrupted by a 6-aa gap, at the position of a splice site that is not found in other PDs. The homology then continues from position 61 in the PD and remains uninterrupted to include the entire RED domain. There is a proline residue inserted after position 65 that is not found in other PDs. Alignment of the Eyg RED domain with that of other complete PDs indicates that it is distinct from other known Pax classes. The linker region between PAI and RED domains is most related to that of Pax-2, Pax-5, and Pax-8 (Drosophila homolog: Sparkling/Shaven), whereas the HTH region of its RED domain is most related to Pax-6 (Drosophila homolog: Eyeless). The most closely related PDs are those of Pax-6 and Pax-8. Eyg also contains a Prd-class HD with a serine at position 50. Overall, the HD is most closely related to that of Pax-3 (Drosophila homolog: Paired) and Pax-7, although it is significantly distinct. The numbers of identical residues between Eyg and other HDs are 40 for Pax-7; 38 for Pax-6, and 36 for Gsb-p. The rest of the Eyg protein has few distinguishing features other than a glutamine-rich region found frequently in Drosophila proteins (Jun, 1998).

All chordates possess a dorsal tubular central nervous system, but elaboration of dorsoventral and segmental patterning is far more pronounced in cephalochordates and vertebrates than in the more basal urochordates. Analysis of the urochordates, therefore, should allow deduction of the neural organization and neuronal patterning mechanisms that predated overt dorsoventral and segmental complexity. Functional studies of the ascidian Pax gene (HrPax-37) are reported. The spatiotemporal expression pattern of HrPax-37 suggests involvement in two distinct developmental processes: specification of dorsal cell fates of ectoderm during neurulation, and regional differentiation of the neural tube at later stages. HrPax-37 is descendent from the precursor of the Pax-3 and Pax-7 genes implicated in specification of dorsal fate in the vertebrate neural tube. Injection of HrPax-37 RNA into fertilized eggs causes ectopic expression of the dorsal neural marker tyrosinase gene in neurulae, confirming a regulatory role in dorsal patterning of the neural tube comparable to its vertebrate homologs. These results suggest that dorsal specification in the neural tube by Pax-3/7 subfamily genes was established in the ancestors of extant chordates during emergence of the dorsal tubular nervous system. HrPax-37 is more closely related to vertebrate Pax-3 and Pax7 than to Drosophila gooseberry and paired, suggesting that HrPax-37 is descendent from the precursor of Pax-3 and Pax-7; hence, a gene duplication must have occurred after the divergence of vertebrates and urochordates in the vertebrate lineage. Therefore the duplications that gave rise to paired, gooseberry and gooseberry neural must have occurred in the lineage leading to Drosophila after the divergence of protostomes and deuterostomes. The ascidian Pax-37 gene can be considered representative of the single, ancestral member of the entire Pax-3/7 subfamily (Wada, 1997).

This study describes the isolation and characterization of zebrafish homologs of the mammalian Pax3 and Pax7 genes. The proteins encoded by both zebrafish genes are highly conserved (>83%) relative to the known mammalian sequences. Also the neural expression patterns during embryogenesis are very similar to the murine homologs. Transcripts for pax3 are first detected as bilateral stripes along the lateral margins of the neural plate during early stages of neurulation. In the prospective brain region of 10-11 hour embryos, three paired transverse segment-like stripes of pax3 expression extend towards the midline from the longitudinal stripes. While the two rostralmost stripes appear in regions corresponding to the posterior parts of the diencephalon and midbrain, respectively, the third stripe colocalizes with the area from which the second rhombomere subsequently develops. Expression of pax7 is initiated later than for pax3 and is first detected within a segment-like domain in the dorsal half of the presumptive midbrain. Observed differences in neural crest and mesodermal expression relative to mammals could reflect some functional divergence in the development of these tissues. For zebrafish pax7, no expression was detected in neural crest cells. This finding differs from that observed in the mouse, where defects in cephalic neural crest derivatives are found in Pax7 mutants. There appears to be less expression of zebrafish pax3 and pax7 in mesoderm, when compared with expression of the homologs in mice. For the zebrafish Pax7 protein, the first full-length amino acid sequences in vertebrates is reported and the existence of three additional isoforms are shown which have truncations in the homeodomain and/or the C-terminal region. These novel variants provide evidence for additional isoform diversity of vertebrate Pax proteins (Seo, 1998).

Different cDNA clones encoding a rat homeobox gene and the mouse homolog OG-12 were cloned from adult rat brain and mouse embryo mRNA, respectively. The predicted amino acid sequences of the proteins belong to the paired-related subfamily of homeodomain proteins (Prx homeodomains), which includes Phox2, Prx1/Mhox1 and Prx2. Pax3 and Pax7 are close relatives. Hence, the gene was named Prx3 and the mouse and rat genes are indicated as mPrx3 and rPrx3, respectively. In the mouse as well as in the rat, the predicted Prx3 proteins share the homeodomain but have three different N termini, a 12-aa residue variation in the C terminus, and contain a 14-aa residue motif common to a subset of homeodomain proteins, termed the "aristaless domain." Genetic mapping of Prx3 in the mouse placed this gene on chromosome 3. In situ hybridization on whole mount 12.5-day-old mouse embryos and sections of rat embryos at 14.5 and 16.5 days postcoitum reveals marked neural expression in discrete regions in the lateral and medial geniculate complex, superior and inferior colliculus, the superficial gray layer of the superior colliculus, pontine reticular formation, and inferior olive. In rat and mouse embryos, nonneuronal structures around the oral cavity and in hip and shoulder regions also express the Prx3 gene. In the adult rat brain, Prx3 gene expression is restricted to thalamic, tectal, and brainstem structures that include relay nuclei of the visual and auditory systems as well as other ascending systems conveying somatosensory information. Prx3 may have a role in specifying neural systems involved in processing somatosensory information, as well as in face and body structure formation (van Schaick, 1997).

Pax-3 group III genes in grasshoppers

Pair-rule genes were identified and named for their role in segmentation in embryos of the long germ insect Drosophila. Among short germ insects these genes exhibit variable expression patterns during segmentation and thus are likely to play divergent roles in this process. Understanding the details of this variation should shed light on the evolution of the genetic hierarchy responsible for segmentation in Drosophila and other insects. The expression of homologs of the Drosophila Pax group III genes paired, gooseberry and gooseberry-neuro have been examined in short germ flour beetles and grasshoppers. During Drosophila embryogenesis, paired acts as one of several pair-rule genes that define the boundaries of future parasegments and segments, via the regulation of segment polarity genes such as gooseberry, which in turn regulates gooseberry-neuro, a gene expressed later in the developing nervous system. Using a crossreactive antibody, it has been shown that the embryonic expression of Pax group III genes in both the flour beetle Tribolium and the grasshopper Schistocerca is remarkably similar to the pattern in Drosophila. Two Pax group III genes, pairberry1 and pairberry2, are responsible for the observed protein pattern in grasshopper embryos. Both pairberry1 and pairberry2 are expressed in coincident stripes of a one-segment periodicity, in a manner reminiscent of Drosophila gooseberry and gooseberry-neuro. pairberry1, however, is also expressed in stripes of a two-segment periodicity before maturing into its segmental pattern. This early expression of pairberry1 is reminiscent of Drosophila paired and represents the first evidence for pair-rule patterning in short germ grasshoppers or any hemimetabolous insect (Davis, 2001).

With the exception of possible nematode homologs, protostome Pax group III (PgIII) genes have thus far not been reported outside Drosophila. Two PgIII genes from Schistocerca have been named pairberry1 (pby1) and pairberry2 (pby2). Each gene possesses both a paired box and an extended S50 paired-like homeobox. Phylogenetic analysis and high sequence similarity to Drosophila prd, gsb and gsbn supports the inclusion of pby1 and pby2 within PgIII. Additionally, pby1 and pby2 appear to be more closely related to each other than either is to prd, gsb or gsbn, suggesting they may be the result of an independent duplication. This conclusion is tempered, however, by the possibility of homogenization of pby1 and pby2 via gene conversion (Davis, 2001).

Although the two grasshopper genes may be closely related, their relationship to the fly genes could not be unequivocally resolved. Although it is possible that pby1 and pby2 result from the duplication of the ancestral gsb/gsbn gene along the lineage leading to Schistocerca after its split with Drosophila, this scenario implies that a grasshopper prd ortholog either exists and has not been found, or was subsequently lost. Since the expression patterns of both pby1 and pby2 include elements similar to the expression of each of the three Drosophila genes, it is thought more likely that pby1 and pby2 result from an independent duplication of a single ancestral insect PgIII (prd/gsb/gsbn) gene (Davis, 2001).

The early transcript and protein expression patterns of pby1 provide, for the first time, evidence of pair-rule patterning in the grasshopper Schistocerca. Indirect evidence is provided by the order of appearance of the gnathal and thoracic Pby1 stripes. In particular, the onset of the Mx and T1 stripes is delayed relative to their adjacent stripes. Thus, like many segment polarity genes in Drosophila, the order of appearance of these segmental stripes follows a two-segment periodicity. This may reflect, as it does in Drosophila, regulation by an underlying pair-rule patterning mechanism (Davis, 2001).

Stronger evidence for pair-rule patterning lies with the early domains of pby1 expression from T2 to A10. Stripes of these segments originate as broad domains of a two-segment periodicity at the extending posterior tip, each of which subsequently splits into a pair of adjacent segmental stripes. Thus, adjacent stripes arise by subtly different means. The segmental stripes of T2, A1, A3, A5, A7 and A9 resolve from the anterior edge of sequentially appearing broad domains. By contrast, the segmental stripes of T3, A2, A4, A6, A8 and A10 resolve from within the posterior portions of the same respective broad domains. This resolution of broad domains into adjacent pairs of segmental stripes is analogous to the process by which Drosophila prd acquires its segmental pattern from initial stripes of a two-segment periodicity (Davis, 2001).

Although similar to Drosophila and flour beetles, the broad domains in grasshopper exhibit at least one notable difference. When compared with either Drosophila or flour beetles, the pairing of stripes in grasshoppers is shifted by one segment. For example, in grasshoppers, the Pby1 stripes of A1 and A2 derive from a single A1/A2 broad domain, while in flies and flour beetles the segmental Pby stripes of A1 and A2 derive from the T3/A1 and A2/A3 broad domains (primary Prd stripes 4 and 5 in Drosophila). The shift in phasing of stripe pairs in Schistocerca when compared with Tribolium and Drosophila is reflected in the fact that the grasshopper A11 Pby1 stripe, which appears relatively late, arises without a sister stripe. Such variation in phasing is likely to reflect a spatial shift in the expression of upstream components of the segmentation hierarchy (Davis, 2001).

An additional similarity of early pby1 expression to Drosophila prd is its timing relative to segment polarity genes. In Drosophila, prd is expressed before en and wg. In Schistocerca, pby1 is expressed before En protein by approximately four to five stripes from ~20%-27% of development. Hence, pby1 is also likely to be expressed ahead of wg. As in Schistocerca gregaria, wg transcript appears only two to three stripes ahead of En protein. Another feature shared by the early pby1 pattern and that of Drosophila prd is the gnathal arc. This early domain comprises the future Pby1 stripes of the Mn, Mx and La segments. In Drosophila, prd is also expressed as a single broad stripe before splitting into primary Prd stripes 1 and 2 at the onset of cellularization, just as stripes 3-7 begin to appear. Primary stripes 1 and 2 in turn give rise to the future Mn, Mx and La secondary stripes of Prd. This early Prd domain in flies is thus remarkably similar to the Pby1 gnathal arc in grasshoppers. A similar pattern in flour beetle embryos could not be detected, since the Mn Pby stripe appears de novo (Davis, 2001).

The position of Pby1 stripes just anterior to En with an overlap of ~one cell row, along with their subsequent restriction to the neuroectoderm, is reminiscent of gsb expression in Drosophila. Similarly, the delayed appearance of Pby2 stripes, their restricted form, and their coincident expression with Pby1 anterior to En is reminiscent of late gsb expression. Additionally, the striped neural expression of both pby1 and pby2 as late as 40% of development is reminiscent of gsbn expression. Thus, only one of two PgIII genes identified in Schistocerca, pby1, is potentially functioning in the capacity of all three PgIII genes in Drosophila (prd, gsb and gsbn), while pby2 is potentially functioning in the capacity of one, or perhaps two, of the Drosophila genes (gsb and gsb-n). Finally, although the behavior pby2 is most similar to Drosophila gsb and gsbn, the late expression of both pby1 and pby2 at the base of the developing gnathal appendages is reminiscent of the late expression of prd at the base of the gnathal protuberances in Drosophila embryos (Davis, 2001).

During Drosophila embryogenesis, the pair-rule gene prd activates the segment polarity gene gsb, which, in turn, activates gsbn. Additionally, the products of these three genes are for the most part functionally interchangeable. Given both their similarity to the three fly genes and their coincident expression, pby1 may be required for the activation of pby2 (Davis, 2001).

In Drosophila, prd is also required for the activation of odd-numbered wg stripes. Thus, Pby1 may be required for activation of wg in Schistocerca americana. The temporal dynamics of wg mRNA in the closely related grasshopper Schistocerca gregaria are consistent with this suggestion. In Drosophila, prd is also responsible for activating and defining the posterior border of odd-numbered En stripes. This is suggested by the absence of odd-numbered En stripes in prd-negative embryos, as well as their posterior expansion in heat shocked prd embryos. Consistent with this role, the posterior borders of secondary Prd stripes in Drosophila are coincident with the posterior borders of En stripes. In Schistocerca, however, Pby1 does not simultaneously share a posterior border with En. Instead, nascent segmental stripes spanning four cell rows narrow to two cell rows just before the appearance of an adjacent En stripe, which overlaps by only a single row of cells. This lack of temporally coincident expression does not, however, rule out a possible role in activating en, for it is conceivable that the four-cell row domain of Pby1 may activate en before narrowing, with the result that En appears specifically in cells that were previously expressing pby1. A similar situation may hold true for Drosophila, as it has been proposed that, despite the coincident expression of secondary Prd stripes and En, it is instead the earlier primary stripes of Prd that are responsible for the activation of en. Finally, it is important to note that a fully functioning pair-rule mechanism in grasshoppers may well require genes in addition to pby1 that exhibit pair-rule like expression patterns (Davis, 2001).

Based on widespread conservation of expression patterns, it seems likely that the Drosophila segment polarity genes functioned as such in the context of the ancestral insect segmentation system. The picture is less clear for pair-rule genes. In light of the more basal phylogenetic position of Schistocerca, it is tempting to view the posterior expression domains of eve and ftz as ancestral for insects, existing before the evolutionary recruitment of these genes to play a role in segmentation. In support of this conjecture, vertebrate orthologs of eve are linked to the Hox clusters and expressed in broad Hox-like domains, while the C. elegans eve ortholog, vab-7, is both expressed in a broad posterior domain and required for posterior cell fates. ftz, a gene closely related to the Antp-class Hox genes, is likewise expressed in a broad Hox-like domain in mites (Davis, 2001).

However, grasshoppers in some respects are likely to represent a derived state for insects. This is probably the case for eve, since this gene is expressed in stripes in spiders. Thus, it is possible that eve was primitively expressed in both stripes and a posterior domain, but somewhere along the lineage leading to Schistocerca, the gene lost its striped expression. The observation that a PgIII gene is expressed in stripes of a two-segment periodicity in grasshoppers suggests that pair-rule patterning is part of the ancestral insect segmentation system. However, confirmation of this claim will require closer examination of the striped expression of pair-rule orthologs in primitive insects and non-insect arthropods (Davis, 2001).

An additional consequence of the molecular data presented here is that Tribolium and Schistocerca appear more similar in their embryology than previously appreciated. Before this study, the non-striped expression of eve and ftz did not allow comparison with the striped expression of pair-rule genes in other insects. The Pby pattern, however, allows such a comparison. In the case of Tribolium, only one Pby stripe, that of the mandibular segment, has formed before the onset of gastrulation; eve and ftz stripes at this stage have likewise not formed posterior to the gnathal region. In Schistocerca, no pby1 expression has been detected before the onset of gastrulation (~36 hours AEL), and the first stripe associated with segmentation (the gnathal arc) does not appear until 10% of develoment (~50 hours AEL), well after gastrulation has begun. Thus, neither Tribolium nor Schistocerca has specified segmental or parasegmental boundaries posterior to the head at the start of gastrulation, conforming to the classical idea of short (as opposed to intermediate) germ embryogenesis (Davis, 2001).

Drosophila prd is at the bottom of the genetic hierarchy of pair-rule genes and this fact, coupled with its later segmental expression, have led some to suggest that in flies prd acts as a bridge between the pair-rule and segment polarity levels of the segmentation hierarchy. If pair-rule patterning is an evolutionarily recent specialization of prd, then the segmental secondary Prd stripes of Drosophila are best seen as the remnants of an ancestral dual function as a pair-rule and segment polarity gene. It is perhaps not surprising then, that pby1 -- a PgIII gene from a more phylogenetically primitive insect -- is expressed in both a pair-rule and segment polarity fashion. As one of only two PgIII genes in Schistocerca, pby1 is expressed in a manner reminiscent of the combined pattern of all three PgIII genes in Drosophila. In lacking the specialized expression of the Drosophila genes, pby1 may be the closest approximation of the ancestral insect PgIII gene (Davis, 2001).

Paired in Tribolium

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, Tc-prd and Tc-slp were analyzed to determine the extent to which this paradigm is conserved in Tribolium. It was found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning (Choe, 2007; full text of article).

Pax3/7 expressing cells and limb regeneration in crustaceans: A common cellular basis for muscle regeneration in arthropods and vertebrates

Many animals are able to regenerate amputated or damaged body parts, but it is unclear whether different taxa rely on similar strategies. Planarians and vertebrates use different strategies, based on pluripotent versus committed progenitor cells, respectively, to replace missing tissues. In most animals, however, experimental tools needed to determine the origin of regenerated tissues are lacking. This study presents a genetically tractable model for limb regeneration, the crustacean Parhyale hawaiensis. Regeneration in Parhyale involves lineage-committed progenitors, as in vertebrates. Pax3/7-expressing muscle satellite cells, previously identified only in chordates, are shown to be a source of regenerating muscle in Parhyale. These similarities point to a common cellular basis of regeneration, dating back to the common ancestors of bilaterians (Konstantinides, 2014).

DNA binding by the paired domain of Pax-3

Pax-3, a transcription factor that is required for development of the embryonic neural tube, neural crest, and somitic derivatives, contains two DNA-binding domains, a paired domain, and a paired-type homeodomain. Although Pax-3 binds to sequences related to the e5 element of the Drosophila even-skipped gene, the sequence requirements of an optimal Pax-3 response element have not been well characterized. Using both DNA-binding domains and a pool of random oligonucleotides, a new paired box consensus motif, GTTAT, was identified which was located 1, 4, 5, 8, or 13 base pairs downstream of the homeobox binding motif, ATTA. Binding analysis of these sequences demonstrate that the distance between recognition elements for the homeodomain and the paired domain affects affinity. Specifically, spacing elements 1 or 13 base pairs apart from each other confers low affinity Pax-3 binding, whereas intermediate spacing (5 or 8 base pairs) confers high affinity binding. Contrary to previous reports, oligonucleotides deleted for either the ATTA or the GTTAT can also be bound by Pax-3, although both sites are necessary for maximal affinity. Finally, transient transfections demonstrate that Pax-3 trans-activation correlates with binding affinity. Because the Pax-3-responsive genes identified to date contain almost exclusively low affinity binding sequences, this analysis indicates that they may be responsive to Pax-3 only when cellular levels are high (Phelan, 1998).

The chimeric transcription factor Pax3-FKHR, produced by the t(2;13)(q35;q14) chromosomal translocation in alveolar rhabdomyosarcoma, consists of the two Pax3 DNA binding domains (paired box and homeodomain) fused to the C-terminal forkhead (FKHR) sequences that contain a potent transcriptional activation domain. To determine which of these domains are required for cellular transformation, Pax3, Pax3-FKHR, and selected mutants were retrovirally expressed in NIH 3T3 cells and evaluated for their capacity to promote anchorage-independent cell growth. Mutational analysis has revealed that both the third alpha-helix of the homeodomain and a small region of the FKHR transactivation domain are absolutely required for efficient transformation by the Pax3-FKHR fusion protein. Surprisingly, point mutations in the paired domain that abrogate sequence-specific DNA binding retain a transformation potential equivalent to that of the wild-type protein. This finding suggests that DNA binding mediated through the Pax3 paired box is not required for transformation. These results demonstrate that the integrity of the Pax3 homeodomain recognition helix and the FKHR transactivation domain is necessary for efficient cellular transformation by the Pax3-FKHR fusion protein (Lam, 1999).

pax-3 and vulva formation in Pristionchus pacificus

The Hox gene lin-39 plays a crucial role in the establishment of the nematode vulva equivalence group. Mutations in lin-39 in Caenorhabditis elegans and Pristionchus pacificus result in a vulvaless phenotype because presumptive vulva precursor cells adopt non-vulval fates. Interestingly, the non-vulval fate of anterior and posterior epidermal cells differs between Caenorhabditis and Pristionchus; in C. elegans, non-vulval cells fuse with the hypodermis, whereas, in P. pacificus, they die as a result of programmed cell death. C. elegans lin-39 (Cel-lin-39) indirectly controls the cell fusion gene eff-1 by regulating the GATA transcription factors egl-18 and elt-6. In P. pacificus, the genetic context of its lin-39 (Ppa-lin-39) function was unknown. This study describes the isolation and characterization of gev-2, a second generation-vulvaless mutant in P. pacificus. gev-2 is the Ppa-pax-3 gene, and it has distinct functions in the cell fate specification of epidermal cells. Whereas Ppa-pax-3 regulates cell survival of the presumptive vulval precursor cells, it controls cell death of posterior epidermal cells. Molecular studies indicate that Ppa-pax-3 is a direct target of Ppa-LIN-39. Thus, this study describes the first specific developmental defect of a nematode pax-3 gene and the data reveal different regulatory networks for the specification of the vulva equivalence group (Yi, 2007).

Specification of muscle neurotransmitter sensitivity by a Paired-like homeodomain protein in Caenorhabditis elegans

The effects of neurotransmitters depend on the receptors expressed on the target cells. In Caenorhabditis elegans, there are two types of GABA receptors that elicit opposite effects: excitatory receptors that open cation-selective channels, and inhibitory receptors that open anion-selective channels. The four non-striated enteric muscle cells required for the expulsion step of the defecation behavior are all sensitive to GABA: the sphincter muscle expresses a classical GABA-sensitive chloride channel (UNC-49) and probably relaxes in response to GABA, while the other three cells express a cation-selective channel (EXP-1) and contract. The expression of the exp-1 gene is under the control of dsc-1, which encodes a Paired-like homeodomain protein, a class of transcription factors previously associated with the terminal differentiation of neurons in C. elegans. dsc-1 mutants have anatomically normal enteric muscles but are expulsion defective. This defect is due to the lack of expression of exp-1 in the three cells that contract in response to GABA. In addition, dsc-1, but not exp-1, affects the periodicity of the behavior, revealing an unanticipated role for the enteric muscles in regulating this ultradian rhythm (Branicky, 2005).

Pax-3 in Annelida

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).


Table of contents


paired continued: Biological Overview | Regulation | 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.