muscle segment homeobox

EVOLUTIONARY HOMOLOGS (part 1/3)

msh is homologous to a growing number of divergent homeobox genes from Xenopus and mouse. The vertebrate genes are expressed in mesodermal cells, neural crest cells and neural crest derived tissues, as well as epithelial and mesenchymel cells of the developing eye, tooth and limb (Lord, 1995).

The conservation of developmental functions exerted by Antp-class homeoproteins in protostomes and deuterostomes has suggested that homologs with related functions are present in diploblastic animals, in particular, in Hydra. Phylogenetic analyses show that Antp-class homeodomains belong either to non-Hox or to Hox/paraHox families. See Phylogenetic relationships among 200 Antp-class genes. Among the 13 non-Hox families, 9 reported here have diploblastic homologs: Msx, Emx, Barx, Evx, Tlx, NK-2, and Prh/Hex, Not, and Dlx. Among the Hox/paraHox, poriferan sequences are not found, and the cnidarian sequences form at least five distinct cnox families. Cnox-1 shows some affinity to paralogous group (PG) 1; this group includes Drosophila Labial. Cnox-2 is related to Drosophila Intermediate neuroblast defective. Cnox-3 and 5 show some affinity to PG9-10; this group includes Drosophila AbominalB. Cnox-4 has no counterparts in Drosophila or vertebrates. Intermediate Hox/paraHox genes (PG 3 to 8 and lox) do not have clear cnidarian counterparts. In Hydra, cnox-1, cnox-2, and cnox-3 are not found chromosomally linked within a 150-kb range and display specific expression patterns in the adult head. During regeneration, cnox-1 is expressed as an early gene whatever the polarity, whereas cnox-2 is up-regulated later during head but not foot regeneration. Finally, cnox-3 expression is reestablished in the adult head once the head is fully formed. These results suggest that the Hydra genes related to anterior Hox/paraHox genes are involved at different stages of apical differentiation. However, the positional information defining the oral/aboral axis in Hydra cannot be correlated strictly to that characterizing the anterior-posterior axis in vertebrates or arthropods (Gauchat, 2000)

A novel single-sided specific polymerase chain reaction (PCR) strategy inspired by ligation-mediated PCR has been used to clone fragments of divergent homeobox genes from a flatworm, the planarian Polycelis nigra. Eight homeobox-containing fragments were amplified, belonging to the Hox, msh, NK-1 and NK-2 classes. Together with the results obtained from several genomes of platyhelminths, this screening shows the presence of the same array of homeodomain developmental regulators in planarians, traditionally regarded as primitive metazoans in terms of body plan, as in coelomate organisms. However, the presence of a Ubx/abd-A homolog may indicate that platyhelminths are more closely related to protostomes than to deuterostomes and supports the idea that flatworms have inherited an elaborate HOX cluster (seven or eight genes) from their ancestor. Likely homologs of the fly genes tinman, bagpipe and S59 suggest that the mesoderm might be patterned by the same genes in all bilaterally symmetrical animals. Finally, a msh-like gene, a family known to be involved in inductive mechanisms in vertebrates, has been found. These results support the hypothesis that the tremendous diversity of metazoan body plans is specified by a largely conserved array of homeobox-containing developmental genes (Balavoine, 1996).

C. elegans vab-15, a msh-like homeobox gene

To identify genes regulating the development of the six touch receptor neurons, the F₂ progeny were screened of mutated animals expressing an integrated mec-2::gfp transgene that is expressed mainly in these touch cells. From 2638 mutated haploid genomes, 11 mutations were obtained representing 11 genes that affected the production, migration, or outgrowth of the touch cells. Eight of these mutations were in known genes, and 2 defined new genes (mig-21 and vab-15). The mig-21 mutation is the first known to affect the asymmetry of the migrations of Q neuroblasts, the cells that give rise to two of the six touch cells. vab-15 is a msh-like homeobox gene that appears to be needed for the proper production of touch cell precursors, since vab-15 animals lack the four more posterior touch cells. The remaining touch cells (the ALM cells) are present but mispositioned. lin-32 is a basic helix-loop-helix gene that is most similar to atonal in Drosophila. A lin-32 mutation, u282, produces touch cell defects similar to those produced by vab-15(u781): the AVM, PVM, and PLM cells are missing, and the ALM cells are more anteriorly displaced. To see whether these two genes act together in regulating ALM cell fate, touch cells were examined in a lin-32(u282) vab-15(u781) double mutant by mec-7 immunocytochemistry. The single mutations cause the displacement, but not the loss, of the ALM cells as seen for uIs9 expression and mec-3 expression with u781 and for mec-7 in situ hybridization. In contrast, 49% of the double mutant animals lacked ALM cells and another 38% had only one ALM cell. In addition, when the ALM cells were present, their cell bodies were often anterior to the rear bulb of the pharynx, indicating more severe defects in ALM migration. These additive effects suggest that vab-15 and lin-32 have somewhat redundant roles in activating touch cell fate in ALM cells. Since lin-32 is needed for the generation of the posterior touch cells, by extension, vab-15 may also be needed for their production. In addition to the touch cell abnormalities, vab-15 animals variably exhibit embryonic or larval lethality, cell degenerations, malformation of the posterior body, uncoordinated movement, and defective egg laying (Du, 2001).

Conserved gene regulatory module specifies lateral neural borders across bilaterians

The lateral neural plate border (NPB), the neural part of the vertebrate neural border, is composed of central nervous system (CNS) progenitors and peripheral nervous system (PNS) progenitors. In invertebrates, PNS progenitors are also juxtaposed to the lateral boundary of the CNS. Whether there are conserved molecular mechanisms determining vertebrate and invertebrate lateral neural borders remains unclear. This study presents evidence that orthologs of the NPB specification module specify the invertebrate lateral neural border, which is composed of CNS and PNS progenitors. First, like in vertebrates, the conserved neuroectoderm lateral border specifier Msx/vab-15 (see Drosophila Drop/Msh) specifies lateral neuroblasts in Caenorhabditis elegans. Second, orthologs of the vertebrate NPB specification module [Msx/vab-15, Pax3/7/pax-3 (see Drosophila Paired), and Zic/ref-2 (see Drosophila Odd-paired)] are significantly enriched in worm lateral neuroblasts. Third, Msx/vab-15 was shown to regulate the development of mechanosensory neurons derived from lateral neural progenitors in multiple invertebrate species, including C. elegans, Drosophila, and Ciona. These data suggest a common origin of the molecular mechanism specifying lateral neural borders across bilaterians (Li, 2017).

Molecular architecture of annelid nerve cord supports common origin of nervous system centralization in bilateria

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).

Patterning of brain precursors in ascidian embryos

In terms of their embryonic origins, the anterior and posterior parts of the ascidian central nervous system (CNS) are associated with distinct germ layers. The anterior part of the sensory vesicle, or brain, originates from ectoderm lineages following a neuro-epidermal binary fate decision. In contrast, a large part of the remaining posterior CNS is generated following neuro-mesodermal binary fate decisions. This study addresses the mechanisms that pattern the anterior brain precursors along the medial-lateral axis (future ventral-dorsal) at neural plate stages. Functional studies show that Nodal signals are required for induction of lateral genes, including Delta-like, Snail, Msxb and Trp (see Drosophila Delta, Snail, Msx and Trp). Delta-like/Notch signalling induces intermediate (Gsx; see Drosophila Ind) over medial (Meis; see Drosophila Hth) gene expression in intermediate cells, whereas the combinatorial action of Snail and Msxb prevents the expression of Gsx in lateral cells. It is concluded that despite the distinct embryonic lineage origins within the larval CNS, the mechanisms that pattern neural precursors are remarkably similar (Esposito, 2017).

Sea urchin, amphioxus, amphibians and fish MSH genes

Msx- class homeobox genes, characterized by a distinct and highly conserved homeodomain, have been identified in a wide variety of metazoans from vertebrates to coelenterates. Although there is evidence that they participate in inductive tissue interactions that underlie vertebrate organogenesis, including those that pattern the neural crest, there is little information about their function in simple deuterostomes. Both to learn more about the ancient function of Msx genes, and to shed light on the evolution of developmental mechanisms within the lineage that gave rise to vertebrates, Msx genes have been isolated and characterized from ascidians (Urochordates) and echinoderms (Echinoderms). The sequence and expression of a sea urchin (Strongylocentrotus purpouratus) Msx gene whose homeodomain is very similar to that of vertebrate Msx2 is described. This gene, designated SpMsx, is first expressed in blastula stage embryos, apparently in a non-localized manner. Subsequently, during the early phases of gastrulation, SpMsx transcripts are expressed intensely in the invaginating archenteron and secondary mesenchyme, and at reduced levels in the ectoderm. In the latter part of gastrulation, SpMsx transcripts are concentrated in the oral ectoderm and gut, and continue to be expressed at those sites through the remainder of embryonic development. The fact that vertebrate Msx genes are regulated by inductive tissue interactions and growth factors suggests that the restriction of SpMsx gene expression to the oral ectoderm and derivatives of the vegetal plate might similarly be regulated by the series of signaling events that pattern these embryonic territories. As a first test of this hypothesis, the influence of exogastrulation and cell-dissociation were examined on SpMsx gene expression. In experimentally-induced exogastrulae, SpMsx transcripts are distributed normally in the oral ectoderm, evaginated gut, and secondary mesenchyme. However, when embryos are dissociated into their component cells, SpMsx transcripts failed to accumulate. These data show that the localization of SpMsx transcripts in gastrulae does not depend on interactions between germ layers, yet the activation and maintenance of SpMsx expression does require either cell-cell or cell-matrix interactions. Given that echinoderms are likely to be closely related to stem organsims of the deuterstome group, common feature of Msx gene expression uniting echinoderms and chordates can be described. Both groups exhibit endodermal and/or mesodermal expression in cells undergoing morphogentic movements during gastrulation, and neural and/or ectodermal expression in cells in the anterior portion of the embryo. It is concluded that the restriction of SpMsx to the oral ectoderm and archenteron is not a response to an interaction between germ layers (Dobias, 1997).

Genomic and cDNA clones of an Msx class homeobox gene were isolated from amphioxus (Branchiostoma floridae). The gene, AmphiMsx, is expressed in the neural plate from late gastrulation; in later embryos it is expressed in dorsal cells of the neural tube, excluding anterior and posterior regions, in an irregular reiterated pattern. There is transient expression in dorsal cells within somites, reminiscent of migrating neural crest cells of vertebrates. In larvae, mRNA is detected in two patches of anterior ectoderm proposed to be placodes. Evolutionary analyses show there is little phylogenetic information in Msx protein sequences; however, it is likely that duplication of Msx genes occurred in the vertebrate lineage (Sharman, 1999).

Differential induction of four zebrafish msx homeobox genes during fin development and regeneration suggest distinct roles for each gene. During development, the median fin fold, which gives rise to the unpaired fins, express the four msx genes. Transcripts of the genes are also present in cells of the presumptive pectoral fin buds. The most distal cells, the apical ectodermal ridge of the paired fins, and the cleft and ectodermal ridge of the paired fins and cleft and flanking cells of the medial fin fold express all the msx genes with the exception of msxC. Mesenchymal cells underlying the most distal cells express all four genes. Expression of the msx genes is transient, and expression is undetectable by three days after fertilization. Induction of msx gene expression in regenerating caudal fins reach a maximum between the third and fifth days postamputation. In the regenerating fin, the blastema cells that develop at the tip of each fin ray express msxB and msxC. Cells of the overlying epithelium express msxA and msxD, but do not express msxB or msxC. Rapidly prolifrating cells of the proximal blastema express higher levels than cells of the less rapidly proliferating distal blastema (Akimenko, 1995).

Following amputation of a urodele limb or teleost fin, the formation of a blastema is a crucial step in facilitating subsequent regeneration. Early caudal fin regenerative events can be separated into four stages. (1) During the first 12 h, epidermal cells migrate to cover the stump. (2) Within the next 12 h, the wound epidermis thickens, while fibroblasts and scleroblasts located within one or two bone segments proximal to the amputation plane lose their dense organization and show signs of distal migration. (3) Next, these mesenchymal cells organize and proliferate to form a blastema, a mass of undifferentiated tissue, just distal to the ray stumps. (4) During the outgrowth stage, proximal cells of the regeneration blastema differentiate to participate in bone deposition, while distal cells divide and maintain outgrowth. Using the zebrafish caudal fin regeneration model, the hypothesis that fibroblast growth factors initiate blastema formation from fin mesenchyme was examined. fibroblast growth factor receptor 1 (fgfr1) is expressed in mesenchymal cells underlying the wound epidermis during blastema formation and in distal blastemal tissue during regenerative outgrowth. fgfr1 transcripts colocalize with those of msxb and msxc, putative markers for undifferentiated, proliferating cells. A zebrafish Fgf member, designated wfgf, is expressed in the regeneration epidermis during outgrowth. Furthermore, a specific inhibitor of Fgfr1, applied immediately following fin amputation, blocks blastema formation without obvious effects on wound healing. This inhibitor blocks the proliferation of blastemal cells and the onset of msx gene transcription. Inhibition of Fgf signaling during ongoing fin regeneration prevents further outgrowth while downregulating the established expression of blastemal msx genes and epidermal sonic hedgehog. These findings indicate that zebrafish fin blastema formation and regenerative outgrowth require Fgf signaling (Poss, 2000).

It is proposed that following amputation and wound healing, mesenchymal cells disorganize and begin to migrate toward the amputation plane. At the epidermal-mesenchymal junction, Fgf molecules synthesized in the wound epidermis bind to mesenchymal Fgfr1. Signaling by Fgfr1 triggers proliferation and the induction or maintenance of msxb and msxc expression in these cells, and a blastema forms. During later stages, Wfgf and/or other Fgfs are released from the distalmost epidermal cells and signal through blastemal Fgfr1 to maintain msxb/c expression and cell division, which promotes outgrowth. Meanwhile, Fgfs activate Fgfrs in basal layer epidermal cells to maintain shh transcription during outgrowth. Shh released from these cells is thought to help direct new bone deposition by scleroblasts (Poss, 2000).

cDNAs encoding a novel Xenopus homeodomain-containing protein homologous to the mouse Hox-7.1 and the Drosophila muscle segment homebox (msh) have been isolated. Northern blot and RNAase protection experiments establish that transcripts of the frog gene, termed Xhox-7.1, first appear at about the beginning of gastrulation. After a rapid increase, mRNA levels plateau between the neurula and middle-tailbud stages, and decrease steadily thereafter. In situ hybridization localizes the Xhox-7.1 message to the dorsal mesodermal mantle of gastrula stage embryos. Comparison of the hybridization patterns of progressively more anterior cross-section of tailbud stage embryos localizes the signal to the dorsal neural tube and neural crest, to specific regions of the lateral plate mesoderm, and to the cardiogenic region. By the tadpole stage, the Xhox-7.1 message appears only at specific sites in the central nervous system, such as in the dorsal hindbrain. Thus, during embryonic development, levels of Xhox-7.1 expression decrease as the transcript becomes more progressively localized. Evidence is presented of a distinct msh-like transcript (provisionally termed Xhox-7.1') which begins to accumulate at early-gastrula stage, as well (Su, 1991).

As a further investigation of vertebrate head morphogenesis, expression patterns of several homeobox-containing genes were examined using whole-mount in situ hybridization in a sensory system considered to be primitive for the vertebrate subphylum: the axolotl (class: Amphibia, order: Urodela) lateral lines and the placodes from which they develop. The lateral line system develops from the ectodermal placodes. The lateral line placodes develop in a dorsolateral series parallel to the main body axis; it has been hypothesized that the dorsolateral and ventrolateral placode series may be patterned by a mechanism similar to the Hox code described for the head and branchial regions of amniote embryos. Axolotl Msx-2 and Dlx-3 are expressed in all of the lateral line placodes. Both genes are expressed throughout development of the lateral line system and their expression continues in the fully developed neuromasts. Expression within support cells is highly polarized. In contrast to most other observations of Msx genes in vertebrate organogenesis, expression of Msx-2 in developing lateral line organs is exclusively epithelial and is not associated with epithelial-mesenchymal interactions. A Hox-complex gene, Hoxb-3, is shown to be expressed in the embryonic hindbrain and in a lateral line placode at the same rostrocaudal level, but not in other placodes nor in mature lateral line organs. A Hox gene of a separate paralog group, Hoxa-4, is expressed in a more posterior hindbrain domain in the embryo, but is not expressed in the lateral line placode at that rostrocaudal level. These data provide the first test of the hypothesis that the neurogenic placodes develop in two rostrocaudal series aligned with the rhombomeric segments and are patterned by combinations of Hox genes in parallel with the central nervous system (Metscher, 1997).

This study analyzes the expression and the embryonic function of Xenopus msx-1 (Xmsx-1) in relation to the ventralizing activity of bone morphogenetic protein-4 (Drosophila homolog: Decapentaplegic). Expression of Xmsx-1 is increased in UV-treated ventralized embryos and decreased in LiCl-treated dorsalized embryos at the neurula stage (stage 14). Xmsx-1 is expressed in the marginal zone and animal pole areas, laterally and ventrally, but not dorsally, at mid-gastrula (stage 11) and late-gastrula (stage 13). Injection of BMP-4 messenger RNA, but not activin mRNA, induces Xmsx-1 expression in the dorsal marginal zone at the early gastrula stage (stage 10+). Introduction of a dominant negative form of BMP-4 receptor RNA suppresses Xmsx-1 expression in animal cap and ventral marginal zone explants at stage 14. Thus, Xmsx-1 is a target gene specifically regulated by BMP-4 signaling. Embryos injected with Xmsx-1 mRNA in dorsal blastomeres at the 4-cell stage exhibit a ventralized phenotype, with microcephaly and swollen abdomen. Histological observation and immunostaining reveal that these embryos have a large block of muscle tissue in the dorsal mesodermal area in place of what should be the notochord. On the basis of molecular marker analysis, however, the injection of Xmsx-1 RNA does not induce the expression of alpha-globin, nor reduce cardiac alpha-actin in dorsal marginal zone explants. This contrasts with BMP-4, which reduces cardiac alpha-actin and induces alpha-globin. In other words, BMP-4 is ventralizing while Xmsx-1 induces dorsolateral tissue. A significant amount of alpha-actin is induced and alpha-globin is turned off in the ventral marginal zone explants injected with Xmsx-1. These results indicate that Xmsx-1 is a target gene of BMP-4 signaling, but it carries out a distinct function in relation to dorsal-ventral patterning of mesodermal tissues (Maeda, 1997).

Epidermal fate in Xenopus ectoderm has been shown to be induced by a secreted growth factor, bone morphogenetic protein 4 (BMP4). However, the molecular mechanism mediating this response is poorly understood. The expression of the homeobox gene msx1 is an immediate early response to BMP4 in Xenopus embryos. The timing of expression and embryonic distribution of msx1 parallels the timing and distribution described for BMP4. Overexpression of msx1 in early Xenopus embryos leads to their ventralization, as described for BMP4. Consistent with mediating a BMP type of signaling, overexpression of msx1 is sufficient to induce epidermis in dissociated ectoderm cells, which would otherwise form neural tissue. msx1 can also rescue neuralization imposed by a dominant negative BMP receptor (tBR) in ectodermal explants. It is proposed that Xenopus msx1 acts as a mediator of BMP signaling in epidermal induction and inhibition of neural differentiation (Suzuki, 1997).

During early patterning of the vertebrate neuraxis, the expression of the paired-domain transcription factor Pax-3 is induced in the lateral portions of the posterior neural plate via posteriorizing signals emanating from the late organizer and posterior nonaxial mesoderm. Using a dominant-negative approach, in explant assays it has been shown that Pax-3 inductive activities from the organizer do not depend on FGF, retinoic acid, or XWnt-8, either alone or in combination, suggesting that the organizer may produce an unknown posteriorizing factor. However, Pax-3 inductive signals from posterior nonaxial mesoderm are Wnt-dependent. Pax-3 expression in the lateral neural plate expands in XWnt-8-injected embryos and is blocked by dominant-negative XWnt-8. Similarly, the homeodomain transcription factor Msx-1, which like Pax-3 is an early marker of the lateral neural plate, is induced by posterior nonaxial mesoderm and blocked by dominant-negative XWnt-8. Rohon-Beard primary neurons, a cell type that develops within the lateral neural plate, are also blocked in vivo by dominant-negative Xwnt-8. Together these data support a model in which patterning of the lateral neural plate by Wnt-mediated signals is an early event that establishes a posteriolateral domain, marked by Pax-3 and Msx-1 expression, from which Rohon-Beard cells and neural crest will subsequently arise (Bang, 1999).

FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, the role of the transcription factors Msx1 and Pax3 was analyzed. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, it has been show that Msx1 and Pax3 are both required for neural crest formation, display overlapping but nonidentical activities, and that Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction (Monsoro-Burq, 2004).

Chicken MSH homologs

Chicken Nkx-2.8 (cNkx-2.8) has been found to be 68% homologous to Ventral nervous system defective and Msh-2 of Drosophila, and more closely related to vertebrate Nkx-2.5 proteins. cNkx-2.8 transcripts are first detectable at HH stage 7 in the splanchnopleura. At stage 10(+), the cNkx-2.8 gene is expressed in the linear heart tube and the dorsal half of the vitelline vein. However, after looping at HH stage 13, cNkx-2.8 is no longer expressed in the looped heart tube, but is expressed in the ventral pharyngeal endoderm. At stage 15, in addition to the pharyngeal expression pattern, cNkx-2.8 is expressed in the ectoderm of the pharyngeal arches and the aortic sac. By HH Stage 17, cNkx-2.8 expression is detectable in the lateral endoderm of the second and third pharyngeal pouches, the posterior portion of the aortic sac, and the sinus venosus. cNkx-2.8 binds to previously characterized Nkx2-1 and Nkx2-5 DNA-binding sites. Overexpression of cNkx-2.8 transactivates a minimal promoter, which contains multimerized Nkx-2 DNA-binding sites. cNkx-2.8 and serum response factor can coactivate a minimal cardiac alpha-actin promoter. These data are consistent with a model in which cNkx-2.8 performs a unique temporally and spatially restricted function in the developing embryonic heart and pharyngeal region. The coexpression of cNkx-2.5 and -2.8 raises the possibility that cNkx-2.8 may have a redundant role with respect to cNkx-2.5 in the coalescing heart tube and may play an important role in the transcriptional program(s) that underlies thymus formation. It is possible that the position and identities of various organ rudiments are determined by the combinatorial expression of Nkx genes (Reecy, 1997)

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development, as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme, which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. Therefore, it has been suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study, ectopic expression of Msx-2 via a retroviral expression vector in the posterior mesoderm of the progress zone severely impairs limb morphogenesis from the time of the initial formation of the limb bud. Msx-2-infected limbs are typically very narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of ectopic Msx-2 expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. Msx-2 also impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signaling molecule that is coexpressed with Msx-2 during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates that Msx-2 regulates BMP-4 expression and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development, Msx-2 is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. Rather large numbers of apoptotic cells, as well as proliferating cells, were found to be present throughout the AER during all stages of normal limb development examined, indicating that many of the cells of the AER are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death may play a very important role in maintaining the activity of the AER (Ferrari, 1998).

Murine MSH protein expression

Continue: Muscle segment homeobox Evolutionary homologs part 2/3 | part 3/3

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