Restricted expression of the zebrafish hsp90alpha gene in slow and fast muscle fiber lineages

Members of the heat shock protein 90 (Hsp90) family of molecular chaperones play important roles in allowing a select group of intracellular signaling molecules reach and maintain functionally active conformations. We have previously shown that hsp90alpha gene expression in early zebrafish embryos is restricted to a subgroup of paraxial-mesoderm derived somitic cells prior to muscle formation and that the gene is downregulated in mature trunk and tail muscle fibers. Here we have compared the expression of the hsp90alpha gene to muscle regulatory genes during development of slow and fast muscle fibers in normal embryos and in embryos carrying mutations which affect somitic muscle formation. We show that hsp90alpha is first expressed early during the development of slow somitic muscle progenitors shortly following myoD activation and at a point prior to or co-incident with the expression of other known muscle regulatory genes. Expression of hsp90alpha is also activated in the midline of flh mutants when these cells switch from a notochord to a muscle fate. Conversely, expression is not detectable in cells of the paraxial mesoderm lineage which fail to converge in spt mutants and which do not activate expression of other muscle specific marker genes. Finally, expression of hsp90alpha is downregulated in slow muscle fibers by 24 h of age but becomes detectable in the later developing fast fibers at this time. Thus, hsp90alpha is expressed in developing muscle progenitors during short temporal and spatial windows of both slow and fast fiber lineages in the zebrafish somite.     

Lamprey contractile protein genes mark different populations of skeletal muscles during development

Agnathan lampreys retain ancestral characteristics of vertebrates in the morphology of skeletal muscles derived from two mesodermal regions: trunk myotomes and unsegmented head mesoderm. During lamprey development, some populations of myoblasts migrate via pathways that differ from those of gnathostomes. To investigate the evolution of skeletal muscle differentiation in vertebrates, we characterize multiple contractile protein genes expressed in the muscle cells of the Japanese lamprey, Lethenteron japonicum. Lamprey actin gene LjMA2, and myosin heavy chain (MyHC) genes LjMyHC1 and LjMyHC2 are all expressed in the developing skeletal muscle cells of early embryos. However, LjMyHC1 and LjMyHC2 are expressed only in cells originating from myotomes, while LjMA2 is expressed in both myotomal and head musculature. Thus, in lampreys, myotomes and head mesoderm differ in the use of genes encoding contractile protein isoforms. Phylogenetic tree analyses including lamprey MyHCs suggest that the variety of muscle MyHC isoforms in different skeletal muscles may correspond to the morphological complexity of skeletal muscles of different vertebrate species. Another lamprey actin gene LjMA1 is likely to be the first smooth muscle actin gene isolated from non-tetrapods. We conclude that, in vertebrate evolution, the different regulatory systems for striated and smooth muscle-specific genes may have been established before the agnathan/gnathostome divergence.     

GATA-6 maintains BMP-4 and Nkx2 expression during cardiomyocyte precursor maturation

GATA-6 is expressed in presumptive cardiac mesoderm before gastrulation, but its role in heart development has been unclear. Here we show that Xenopus and zebrafish embryos, injected with antisense morpholino oligonucleotides designed specifically to knock-down translation of GATA-6 protein, are severely compromised for heart development. Injected embryos express greatly reduced levels of contractile machinery genes and, at the same stage, of regulatory genes such as bone morphogenetic protein-4 (BMP-4) and the Nkx2 family. In contrast, initial BMP and Nkx2 expression is normal, suggesting a maintenance role for GATA-6. Endoderm is critical for heart formation in several vertebrates including Xenopus, and separate perturbation of GATA-6 expression in the deep anterior endoderm and in the overlying heart mesoderm shows that GATA-6 is required in both for cardiogenesis. The GATA-6 requirement in cardiac mesoderm was confirmed in zebrafish, an organism in which endoderm is thought not to be necessary for heart formation. We therefore conclude that proper maturation of cardiac mesoderm requires GATA-6, which functions to maintain BMP-4 and Nkx2 expression.     

Expression of estrogen-receptor related receptors in amphioxus and zebrafish: implications for the evolution of posterior brain segmentation at the invertebrate-to-vertebrate transition

Summary The evolutionary origin of vertebrate hindbrain segmentation is unclear since the amphioxus, the closest living invertebrate relative to the vertebrates, possesses a hindbrain homolog that displays no gross morphological segmentation. Three of the estrogen-receptor related (ERR) receptors are segmentally expressed in the zebrafish hindbrain, suggesting that their common ancestor was expressed in a similar, reiterated manner. We have also cloned and determined the developmental expression of the single homolog of the vertebrate ERR genes in the amphioxus (AmphiERR). This gene is also expressed in a segmented manner in a region considered homologous to the vertebrate hindbrain. In contrast to the expression of amphioxus islet (a LIM-homeobox gene that also labels motoneurons), AmphiERR expression persists longer in the hindbrain homolog and does not later extend to additional posterior cells. In addition, AmphiERR and one of its vertebrate homologs (ERRalpha) are expressed in the developing somitic musculature of amphioxus and zebrafish, respectively. Altogether, our results are consistent with fine structural evidence suggesting that the amphioxus hindbrain is segmented, and indicate that chordate ERR gene expression is a marker for both hindbrain and muscle segmentation. Furthermore, our data support an evolution model of chordate brain segmentation: originally, the program for anterior segmentation in the protochordate ancestors of the vertebrates resided in the developing axial mesoderm which imposed reiterated patterning on the adjacent neural tube; during early vertebrate evolution, this segmentation program was transferred to and controlled by the neural tube.     

Pax3 functions in cell survival and in pax7 regulation

In developing vertebrate embryos, Pax3 is expressed in the neural tube and in the paraxial mesoderm that gives rise to skeletal muscles. Pax3 mutants develop muscular and neural tube defects; furthermore, Pax3 is essential for the proper activation of the myogenic determination factor gene, MyoD, during early muscle development and PAX3 chromosomal translocations result in muscle tumors, providing evidence that Pax3 has diverse functions in myogenesis. To investigate the specific functions of Pax3 in development, we have examined cell survival and gene expression in presomitic mesoderm, somites and neural tube of developing wild-type and Pax3 mutant (Splotch) mouse embryos. Disruption of Pax3 expression by antisense oligonucleotides significantly impairs MyoD activation by signals from neural tube/notochord and surface ectoderm in cultured presomitic mesoderm (PSM), and is accompanied by a marked increase in programmed cell death. In Pax3 mutant (Splotch) embryos, MyoD is activated normally in the hypaxial somite, but MyoD-expressing cells are disorganized and apoptosis is prevalent in newly formed somites, but not in the neural tube or mature somites. In neural tube and somite regions where cell survival is maintained, the closely related Pax7 gene is upregulated, and its expression becomes expanded into the dorsal neural tube and somites, where Pax3 would normally be expressed. These results establish that Pax3 has complementary functions in MyoD activation and inhibition of apoptosis in the somitic mesoderm and in repression of Pax7 during neural tube and somite development.     

Inductive signals from the somatopleure mediated by bone morphogenetic proteins are essential for the formation of the sternal component of avian ribs

The posterior five pairs of avian ribs are composed of vertebral and sternal components, both derived from the somitic mesoderm. For the patterning of the rib cartilage, inductive signals from neighboring tissues on the somitic mesoderm have been suggested to play critical roles. The notochord and surface ectoderm overlying the somitic mesoderm are essentially required for the development of proximal and distal regions of the ribs, respectively. Involvement of the somatopleure in rib development has already been suggested but is less understood than those of the notochord and surface ectoderm. In this study, we reinvestigated the role of the somatopleure during rib development. We first identified the chicken homologue of the mouse Mesenchymal forkhead-1 (cMfh-1) gene based on sequence similarities. cMfh-1 was observed to be expressed in the nonaxial mesoderm, including the somitic mesoderm, and, subsequently, in cartilage forming the ribs, vertebrae, and appendicular skeletal system. In the interlimb region, corresponding to somites 21-25 (or 26), cMfh-1-positive somitic mesoderm was seen penetrating the somatopleure of E4 embryos, and cMfh-1 was used as a molecular marker demarcating prospective rib cartilage. A series of experiments affecting the penetration of the somitic mesoderm into the somatopleure was performed in the present study, resulting in defects in sternal rib formation. The inductive signals emanating from the somatopleure mediated by BMP family proteins were observed to be essentially involved in the ingrowth of the somitic mesoderm. BMP4 alone, however, could not completely replace inductive signals from the somatopleure, suggesting the involvement of additional signals for rib formation.     

A Twist in fate: evolutionary comparison of Twist structure and function

The general requirement to induce mesoderm and allocate cells into different mesodermal tissues such as body muscle or heart is common in many animal embryos. Since the discovery of the twist gene, there has been great progress toward unraveling the molecular mechanisms that control mesoderm specification and differentiation. Twist was first identified in Drosophila as a gene crucial for proper gastrulation and mesoderm formation. In the fly embryo, Twist continues to play additional roles, allocating mesodermal cells into the body wall muscle fate and patterning a subset of these muscles. Twist is also required for proper differentiation of the adult musculature. Twist homologues have been identified in a great variety of organisms, which span the phylogenetic tree. These organisms include other invertebrates such as jellyfish, nematode, leech and lancelet as well as vertebrates such as frog, chick, fish, mouse and human. The Twist family shares both homology in structure across the basic helix-loop-helix domain and in expression during mesoderm and muscle development in most species. Here we review the current state of knowledge of the Twist family and consider how Twist functions during development. Moreover, we highlight experimental evidence that shows common themes that Twist employs during specification and patterning of the mesoderm among evolutionarily distant organisms. Conserved principles and the molecular mechanisms underlying them are discussed.     

Expression of the Dan gene during chicken embryonic development.

The Dan gene was first identified as the putative rat tumor suppressor gene and encodes a protein structurally related to Cerberus and Gremlin in vertebrates. Xenopus DAN, as with Cerberus and Gremlin, was demonstrated to block bone morphogenetic protein (BMP) signaling by binding BMPs, and to be capable of inducing additional anterior structures by ectopic overexpression in Xenopus embryos. DAN, thus, is suggested to play pivotal roles in early patterning and subsequent organ development, as in the case of other BMP antagonists. In this report, we isolated the chicken counterpart of Dan. Chicken Dan is mainly expressed in the cephalic and somitic mesoderm and several placodes during organ development.     

Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.     

Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during early mesodermal patterning in mouse embryos.

We have isolated two mouse genes, Mox-1 and Mox-2 that, by sequence, genomic structure and expression pattern, define a novel homeobox gene family probably involved in mesodermal regionalization and somitic differentiation. Mox-1 is genetically linked to the keratin and Hox-2 genes of chromosome 11, while Mox-2 maps to chromosome 12. At primitive streak stages (approximately 7.0 days post coitum), Mox-1 is expressed in mesoderm lying posterior of the future primordial head and heart. It is not expressed in neural tissue, ectoderm, or endoderm. Mox-1 expression may therefore define an extensive 'posterior' domain of embryonic mesoderm before, or at the earliest stages of, patterning of the mesoderm and neuroectoderm by the Hox cluster genes. Between 7.5 and 9.5 days post coitum, Mox-1 is expressed in presomitic mesoderm, epithelial and differentiating somites (dermatome, myotome and sclerotome) and in lateral plate mesoderm. In the body of midgestation embryos, Mox-1 signal is restricted to loose undifferentiated mesenchyme. Mox-1 signal is also prominent over the mesenchyme of the heart cushions and truncus arteriosus, which arises from epithelial-mesenchymal transformation and over a limited number of craniofacial foci of neural crest-derived mesenchyme that are associated with muscle attachment sites. The expression profile of Mox-2 is similar to, but different from, that of Mox-1. For example, Mox-2 is apparently not expressed before somites form, is then expressed over the entire epithelial somite, but during somitic differentiation, Mox-2 signal rapidly becomes restricted to sclerotomal derivatives. The expression patterns of these genes suggest regulatory roles for Mox-1 and Mox-2 in the initial anterior-posterior regionalization of vertebrate embryonic mesoderm and, in addition, in somite specification and differentiation.     

Proteolipid protein 2 mRNA is expressed in the rabbit embryo during gastrulation

The expression pattern of the receptor tyrosine kinase gene EphB3 was examined during the early stages of chick embryogenesis, and is described in this report. In the gastrula, EphB3 is expressed in epiblast cells adjacent to and entering the anterior portion of the primitive streak; expression is extinguished once cells have ingressed. At headfold stages, EphB3 is strongly transcribed in the floor of the foregut and in anterior lateral endoderm, and is expressed in the subjacent cardiogenic mesoderm. EphB3 is transiently expressed in the lateral ectoderm, neural tube, and neural crest during these stages. Later neural expression is localized to the mesencephalon. In the somitic mesoderm, EphB3 is initially expressed in the sclerotome, but later is expressed predominantly in the dermatome. Prominent expression is also detected in the developing heart, liver, posterior ventral limb bud mesenchyme, pharyngeal arches, and head mesenchyme.     

Distinct enhancers regulate skeletal and cardiac muscle-specific expression programs of the cardiac alpha-actin gene in Xenopus embryos

During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the alpha-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac alpha-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box 1 faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC(A/T)(6)GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac alpha-actin promoter, to confer appropriate cardiac alpha-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac alpha-actin gene promoter is to act solely as a high-affinity SRF binding site.