Presumptive Muscle Research Articles

Mitochondria-Specific Monoclonal Antibodies in Eggs and Embryos of the Ascidian Halocynthia roretzi.

ABSTRACTAscidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that the asymmetric segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. However, it is still unclear which signaling pathway is involved in this process. To obtain molecular markers for studying mechanisms involved in the asymmetric distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondriarich cytoplasm in cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like reticular structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. Immunostaining with these antibodies showed that mitochondria are evenly distributed in the animal hemisphere blastomeres at cleavage stages, whereas not in the vegetal hemisphere blastomeres. Mitochondria were transferred to the presumptive muscle and nerve cord lineage cells of the marginal zone in the vegetal hemisphere more than to the presumptive mesenchyme, notochord and endoderm lineage of the central zone. Therefore, it is suggested that these antibodies will be useful markers for studying mechanisms involved in the polarized distribution of mitochondria during ascidian embryogenesis.

First Neuromuscular Contact Correlates with Onset of Primary Myogenesis in Rat and Mouse Limb Muscles.

Skeletal muscle development has been the focus of intensive study for many decades. Recent advances in genetic manipulation of the mouse have increased our understanding of the cell signalling involved in the development of muscle progenitors which give rise to adult skeletal muscles and their stem cell populations. However, the influence of a vital tissue type – the peripheral nerve—has largely been ignored since its earliest descriptions. Here we carefully describe the timing in which myogenic progenitors expressing Pax3 and Pax7 (the earliest markers of myogenic cells) enter the limb buds of rat and mouse embryos, as well as the spatiotemporal relationship between these progenitors and the ingrowing peripheral nerve. We show that progenitors expressing Pax3 enter the limb bud one full day ahead of the first neurites and that Pax7-expressing progenitors (associated with secondary myogenesis in the limb) are first seen in the limb bud at the time of nerve entry and in close proximity to the nerve. The initial entry of the nerve also coincides with the first expression of myosin heavy chain showing that the first contact between nerves and myogenic cells correlates with the onset of myogenic differentiation. Furthermore, as the nerve grows into the limb, Pax3 expression is progressively replaced by Pax7 expression in myogenic progenitors. These findings indicate that the ingrowing nerve enters the limb presumptive muscle masses earlier than what was generally described and raises the possibility that nerve may influence the differentiation of muscle progenitors in rodent limbs.

Cell contact induces multiple types of electrical excitability from ascidian two-cell embryos that are cleavage arrested and contain all cell fate determinants

Ascidian early embryonic cells undergo cell differentiation without cell cleavage, thus enabling mixture of cell fate determinants in single cells, which will not be possible in mammalian systems. Either cell in a two-cell embryo (2C cell) has multiple fates and develops into any cell types in a tadpole. To find the condition for controlled induction of a specific cell type, cleavage-arrested cell triplets were prepared in various combinations. They were 2C cells in contact with a pair of anterior neuroectoderm cells from eight-cell embryos (2C-aa triplet), with a pair of presumptive notochordal neural cells (2C-AA triplet), with a pair of presumptive posterior epidermal cells (2C-bb triplet), and with a pair of presumptive muscle cells (2C-BB triplet). The fate of the 2C cell was electrophysiologically identified. When two-cell embryos had been fertilized 3 h later than eight-cell embryos and triplets were formed, the 2C cells became either anterior-neuronal, posterior-neuronal or muscle cells, depending on the cell type of the contacting cell pair. When two-cell embryos had been fertilized earlier than eight-cell embryos, most 2C cells became epidermal. When two- and eight-cell embryos had been simultaneously fertilized, the 2C cells became any one of three cell types described above or the epidermal cell type. Differentiation of the ascidian 2C cell into major cell types was reproducibly induced by selecting the type of contacting cell pair and the developmental time difference between the contacting cell pair and 2C cell. We discuss similarities between cleavage-arrested 2C cells and vertebrate embryonic stem cells and propose the ascidian 2C cell as a simple model for toti-potent stem cells.

The core enhancer is essential for proper timing of MyoD activation in limb buds and branchial arches

Transgenic analyses have defined two transcriptional enhancers that regulate MyoD expression in mammals, the core enhancer and distal regulatory region; these enhancers exhibit complementary activities and together are sufficient to recapitulate MyoD expression in developing and mature skeletal muscle. The core enhancer is activated in presumptive muscle cells and determined myoblasts, suggesting an important role in initiating MyoD expression. Here, targeted mutagenesis in the mouse is used to identify necessary and redundant core enhancer functions. The core enhancer is essential for the timely initiation of MyoD expression in limb buds and branchial arches, as enhancer deletion delayed MyoD activation by 1 to 2 days in these muscle lineages. Functionally, this delay in MyoD transcription delayed the onset of muscle differentiation, as assayed by expression of the gene encoding for the early differentiation marker, Myogenin. In addition to these lineage-specific defects, a generalized, modest reduction in MyoD expression was observed in all muscle lineages and at all embryonic stages examined. Interestingly, however, a specific defect was not observed in the nascent myocytes at the medial and lateral aspects of the myotome, suggesting the existence of at least one other enhancer with this specificity. The core enhancer was also dispensable for Myf-5- and Pax-3-dependent regulation of MyoD transcription. These data demonstrate a differential requirement for core enhancer activity in muscle lineages derived from migratory precursors and suggest redundancy in cis regulatory mechanisms controlling myotomal MyoD expression.

Migrating Mesoderm Establish a Uniform Distribution of Laminin in the Developing Grasshopper Embryo

The basal lamina is composed of molecules which physically interact to form a network that serves as a migrational scaffold for many cell types. In the developing peripheral nervous system of the grasshopper, neuronal growth cones are intimately associated with the basal lamina as they migrate. Laminin is a major component of the basal lamina and is a potent promoter of neurite outgrowth in vitro. However, it is unclear what the source of laminin is or how the distribution of laminin within the basal lamina is established. To address this question, grasshopper laminin subunit genes were cloned. As expected, laminin was found within the basal lamina throughout the embryo, in particular in the limb bud, where its expression is coincident with the outgrowth and guidance of the Tibial (Til) pioneer neurons. Surprisingly, the synthesis of β and γ chains of laminin was restricted to migratory mesodermal cells, while in other nonmigratory tissues, such as epithelium and presumptive muscle, β and γ chains of laminin were not detected. In spite of this, laminin immunoreactivity in the basal lamina appears uniform and is available as a substrate for axonal outgrowth.

Expression and regulation of the retinoic acid synthetic enzyme RALDH-2 in the embryonic chicken wing.

Retinaldehyde dehydrogenase type 2 (RALDH-2) is a major retinoic acid (RA) generating enzyme in the embryo. Here, we report immunolocalization of this enzyme (RALDH-2-IR) in the developing wings of stage 17-30 chicken embryos. RALDH-2-IR is located in the area of the presumptive muscle masses, although it is not colocalized with developing muscle cells. RALDH-2-IR is located in tendon precursor cells and may be present in muscular connective tissue. We show that motor neurons and blood vessels, tissues showing RALDH-2-IR as they enter the limb, are capable of synthesizing and releasing RA in culture. RALDH-2-IR in the limb mesenchyme is under the control of both the vasculature and the motor innervation; it is decreased with denervation and increased with hypervascularization. RALDH-2-IR is present in the motor neuron pool of the brachial spinal cord, but this expression pattern is apparently not under the control of limb target tissues, RA in the periphery, or somitic factors. RA is known to be a potent inducer of cellular differentiation; we propose that locally synthesized RA may be involved in aspects of wing tissue specification, including cartilage condensation and outgrowth, skeletal muscle differentiation, and recruitment of smooth muscle cells to the vasculature.

FGF5 stimulates expansion of connective tissue fibroblasts and inhibits skeletal muscle development in the limb

FGF5 is expressed in the mesenchyme and skeletal muscle of developing and adult mouse limbs. However, the function of FGF5 during development of the limb and limb musculature is unknown. To elucidate the inherent participation of FGF5 during limb organogenesis, a retroviral delivery system (RCAS) was used to overexpress human FGF5 throughout developing hind limb of chicken embryos. Misexpression of the soluble growth factor severely inhibited the formation of mature myocytes. Limbs infected with RCAS-FGF5 contained smaller presumptive muscle masses as evidenced by a decrease in MyoD and myosin heavy chain expressing cells. In contrast, ectopic expression of FGF5 significantly stimulated proliferation and expansion of the tenascin-expressing, connective-tissue fibroblast lineage throughout the developing limb. Histological analysis demonstrated that the increase in tenascin immunostaining surrounding the femur, ileum, and pubis in the FGF5 infected limbs corresponded to the fibroblasts forming the stacked-cell perichondrium. Furthermore, pulse labeling experiments with the thymidine analog, BrdU, revealed that the increased size of the perichondrium was attributable to enhanced cell proliferation. These results support a model whereby FGF5 acts as a mitogen to stimulate the proliferation of mesenchymal fibroblasts that contribute to the formation of connective tissues such as the perichondrium, and inhibits the development of differentiated skeletal muscle. These results also contend that FGF5 is a candidate mediator of the exclusive spatial patterning of the hind limb connective tissue and skeletal muscle.

Myogenic Determination Occurs Independently in Somites and Limb Buds

Gene targeting has indicated that the bHLH transcription factorsMyf-5andMyoDare required for myogenic determination because skeletal myoblasts and myofibers are entirely ablated in mouse embryos lacking bothMyf-5andMyoD. Entrance into the skeletal myogenic program during development occurs following the independent transcriptional induction of eitherMyf-5orMyoD. To identify sequences required for thede novoinduction ofMyoDtranscription during development, we investigated the expression patterns ofMyoD-lacZtransgenes in embryos deficient in bothMyf-5andMyoD. We observed that a 258-bp fragment containing the core of the −20-kbMyoDenhancer activated expression in newly formed somites and limb buds in compound mutant embryos lacking bothMyf-5andMyoD. Importantly,Myf-5-andMyoD-deficient presumptive muscle precursor cells expressing β-galactosidase were observed to assume nonmuscle fates primarily as precartilage primordia in the trunk and the limbs, suggesting that these cells were multipotential. Therefore, cells are recruited into theMyoD-dependent myogenic lineage through activation of the −20-kbMyoDenhancer and this occurs independently in somites and limb buds.

Drosophila mef2Expression during Mesoderm Development Is Controlled by a Complex Array ofcis-Acting Regulatory Modules

The function of theDrosophila mef2gene, a member of the MADS box supergene family of transcription factors, is critical for terminal differentiation of the three major muscle cell types, namely somatic, visceral, and cardiac. During embryogenesis,mef2undergoes multiple phases of expression, which are characterized by initial broad mesodermal expression, followed by restricted expression in the dorsal mesoderm, specific expression in muscle progenitors, and sustained expression in the differentiated musculatures. In this study, evidence is presented that temporally and spatially specificmef2expression is controlled by a complex array ofcis-acting regulatory modules that are responsive to different genetic signals. Functional testing of ∼12 kb of 5′ flanking region of themef2gene showed that the initial widespread mesodermal expression is achieved through a 280-bptwist-dependent enhancer. The subsequent dorsal mesoderm-restrictedmef2expression is mediated through a 460-bpdpp-responsive regulatory module, which involves the function of the Smad4 homologMedeaand contains several binding sites for Medea and Mad. The analysis also showed that regulatedmef2expression in the caudal and trunk visceral mesoderm, which give rise to longitudinal and circular gut musculatures, respectively, is under the control of distinct enhancer elements. In addition,mef2expression in the cardioblasts of the heart is dependent upon at least two distinct enhancers, which are active at different periods during embryogenesis. Moreover, multiple regulatory elements are differentially activated for specific expression in presumptive muscle founders, prefusion myoblasts, and differentiated muscle fibers. Taken together, the presented data suggest that specific expression of themef2gene in myogenic lineages in theDrosophilaembryo is the result of multiple genetic inputs that act in an additive manner upon distinct enhancers in the 5′ flanking region.

Mesodermal cell fate decisions in Drosophila are under the control of the lineage genes numb, Notch, and sanpodo

In Drosophila, much has been learned about the specification of neuronal cell fates but little is known about the lineage of mesodermal cells with different developmental fates. Initially in development, individual mesodermal precursor cells are singled out to become the founder cells for specific muscles. The selection of muscle founder cells is thought to employ a Notch-mediated process of lateral inhibition, similar to what is observed for the specification of neural precursors. These muscle founder cells then seem to fuse with the surrounding, uncommitted myocytes inducing the formation of muscle fiber syncytia. In contrast, the differentiated progeny of neural precursor cells are usually the result of a fixed pattern of asymmetric cell divisions which are directed, in part, by interactions between Numb, a localized intracellular-receptor protein, Sanpodo ( Spdo), a potential tropomodulin homolog, and Notch, a transmembrane receptor protein. Here, we have investigated the role of these neural lineage genes in the cell fate specification of muscle and heart precursors. In particular, we have focused on a progenitor cell that is likely to produce a mixed lineage, generating both a pericardial heart cell and a somatic muscle founder cell. We show that the asymmetric segregation of Numb into one of these daughter cells antagonizes the function of Notch and spdo by preventing the presumptive muscle founder from assuming the same fate as its cardiac sibling. Our results suggest that asymmetric cell divisions, in addition to the previously-documented inductive mechanisms, play a major role in cardiac and somatic muscle patterning and that additionally the cytoskeleton may have a role in the asymmetrical localization of cell fate determinants.

Functional analysis of an ascidian homologue of vertebrate Bmp-2/Bmp-4 suggests its role in the inhibition of neural fate specification.

The ascidian tadpole larva is thought to be close to a prototype of the ancestral chordate. The vertebrate body plan is established by a series of inductive cellular interactions, whereas ascidians show a highly determinate mode of development. Recent studies however, suggest some roles of cell-cell interaction during ascidian embryogenesis. To elucidate the signaling molecules responsible for the cellular interaction, we isolated HrBMPb, an ascidian homologue of the vertebrate bone morphogenetic protein (BMP) gene, from Halocynthia roretzi. The amino acid sequence of HrBMPb closely resembled those of vertebrate BMP-2 and BMP-4 and of Drosophila Decapentaplegic (DPP). In addition to the sequence similarity, HrBMPb overexpression induced the ventralization of Xenopus embryos, suggesting functional conservation. The zygotic expression of HrBMPb was first detected around gastrulation. HrBMPb expression was maintained in some cells at the lateral edges of the neural plate through gastrulation to neurulation, although that in the presumptive muscle cells was downregulated. HrBMPb was not expressed in the presumptive epidermis during gastrulation. When HrBMPb mRNA was injected into fertilized Halocynthia eggs, cells that normally give rise to the neural tissue differentiated into epidermis, causing a loss of anterior neural tissue in the larva. In addition, HrBMPb might function synergistically with HrBMPa, an ascidian homologue of BMPs-5 to 8. However, HrBMPb overexpression did not affect differentiation of the notochord and muscle cells. These results suggest that HrBMPb functions as a neural inhibitor and as an epidermal inducer but not as a ventralizing agent in ascidian development.

A forkhead gene related to HNF-3beta is required for gastrulation and axis formation in the ascidian embryo.

We have isolated a member of the HNF-3/forkhead gene family in ascidians as a means to determine the role of winged-helix genes in chordate development. The MocuFH1 gene, isolated from a Molgula oculata cDNA library, exhibits a forkhead DNA-binding domain most similar to zebrafish axial and rodent HNF-3beta. MocuFH1 is a single copy gene but there is at least one other related forkhead gene in the M. oculata genome. The MocuFH1 gene is expressed in the presumptive endoderm, mesenchyme and notochord cells beginning during the late cleavage stages. During gastrulation, MocuFH1 expression occurs in the prospective endoderm cells, which invaginate at the vegetal pole, and in the presumptive notochord and mesenchyme cells, which involute over the anterior and lateral lips of the blastopore, respectively. However, this gene is not expressed in the presumptive muscle cells, which involute over the posterior lip of the blastopore. MocuFH1 expression continues in the same cell lineages during neurulation and axis formation, however, during the tailbud stage, MocuFH1 is also expressed in ventral cells of the brain and spinal cord. The functional role of the MocuFH1 gene was studied using antisense oligodeoxynucleotides (ODNs), which transiently reduce MocuFH1 transcript levels during gastrulation. Embryos treated with antisense ODNs cleave normally and initiate gastrulation. However, gastrulation is incomplete, some of the endoderm and notochord cells do not enter the embryo and undergo subsequent movements, and axis formation is abnormal. In contrast, the prospective muscle cells, which do not express MocuFH1, undergo involution and later express muscle actin and acetylcholinesterase, markers of muscle cell differentiation. The results suggest that MocuFH1 is required for morphogenetic movements of the endoderm and notochord precursor cells during gastrulation and axis formation. The effects of inhibiting MocuFH1 expression on embryonic axis formation in ascidians are similar to those reported for knockout mutations of HNF-3beta in the mouse, suggesting that HNF-3/forkhead genes have an ancient and fundamental role in organizing the body plan in chordates.

An intrinsic genetic program for autonomous differentiation of muscle cells in the ascidian embryo.

The B-line presumptive muscle cells of ascidian embryos have extensive potential for self-differentiation dependent on determinants prelocalized in the myoplasm of fertilized eggs. Ascidian larval muscle cells therefore provide an experimental system with which to explore an intrinsic genetic program for autonomous specification of embryonic cells. Experiments with egg fragments suggested that maternal mRNAs are one of the components of muscle determinants. Expression of larval muscle actin genes begins as early as the 32-cell stage, prior to the developmental fate restriction of the cells. The timing of initiation of the actin gene expression proceeds the expression of an ascidian homologue of vertebrate MyoD by a few hours. Mutations in the proximal E-box of the 5' flanking region of the actin genes did not alter the promoter activity for muscle-specific expression of reporter gene. These results, together with results of deletion constructs of fusion genes, suggest that muscle determinants regulate directly, or indirectly via regulatory factors other than MyoD, the transcription of muscle-specific structural genes leading to the terminal differentiation.

Cis -Regulatory elements conserved in the proximal promoter region of an ascidian embryonic muscle myosin heavy-chain gene

The B-line muscle cells of the ascidian embryo are specified autonomously depending on determinants prelocalized in the myoplasm of unfertilized eggs. Expression of muscle-specific actin and myosin heavy-chain genes commences in the B-line presumptive muscle cells as early as the 32-cell stage. To explore the intrinsic genetic program for this differentiation, we analysed cis-regulatory elements of the Halocynthia roretzi muscle myosin heavy-chain gene (HrMHC1). Comparison of the entire amino acid sequence of HrMHC1 with those of other invertebrates and vertebrates indicated that HrMHC1 resembles myosin heavy-chain of vertebrate skeletal and cardiac muscles. A fusion gene was constructed consisting of 132 bp upstream the 5'-end of HrMHC1 gene fused to a bacterial lacZ reporter. When the fusion gene was microinjected into fertilized eggs, the reporter gene was eventually expressed only in muscle cells of tailbud embryos. It has been reported that 103 bp of sequence 5' of the transcription start site of the ascidian embryonic muscle actin gene (HrMA4) contains information sufficient for muscle-specific expression (Hikosaka et al. 1994). Comparison of the 132 bp of sequence 5' of the HrMHC1 gene with the 103 bp of sequence 5' of the HrMA4 gene revealed several common motifs shared by the two genes (E-box, GATA box and Boxes A, B, T1 and T2). Point mutations inserted into these motifs suggested that the Box T1/T2 (TTTTTTCTTTCA) is critical for the promoter activity of the HrMHC1 gene.

Bone Morphogenetic Protein-2 (BMP-2) Inhibits Muscle Development and Promotes Cartilage Formation in Chick Limb Bud Cultures

Bone morphogenetic proteins (BMPs) induce ectopic cartilage and bone when implanted intramuscularly in adult rats. Expression data suggest that BMPs signal skeletal development in embryos. An important question is which cells are targets of BMP signaling in adult and embryonic tissues. Here, we examined the effect of BMP-2 on micromass cultures of chick limb bud mesenchyme. We report that BMP-2 promotes formation of cartilage and simultaneously inhibits development of muscle cells. To follow the fate of presumptive muscle cells, we replaced chick somites with quail somites at the wing level and made micromass cultures from the chimeric wings. In untreated cultures, quail cells formed muscle but not cartilage. In BMP-2-treated cultures, quail cells disappeared altogether. This suggests that BMP-2 may simultaneously promote cartilage differentiation and reduce the presumptive myogenic cell populations in regions of skeletal development.

Immunolocalization of α-integrin subunits and extracellular matrix components during human colonic organogenesis

BACKGROUND & AIMS: The role of cell adhesion molecules in colonic organogenesis remains poorly understood. This study examined the expression of alpha-integrin subunits and extracellular matrix ligands during human colonic development. METHODS: Standard immunohistochemistry was used to characterize extracellular matrix and alpha-integrin subunit expression during development. RESULTS: At 9 weeks, type-IV collagen and laminin were present underlying epithelium, around vascular structures, and surrounding inner circular muscle layer fibers. Fibronectin was uniformly expressed in the mesenchyme. Tenascin distribution was restricted to the presumptive muscle layer and, later, to the villus core and muscularis mucosae. The 9-week epithelium expressed alpha 2, alpha 3, alpha 5, and alpha 8, and, by 11 weeks, alpha 9. alpha 3, alpha 6, and alpha 8 expression was accentuated at the basal membrane. During transition from pseudostratified to simple columnar epithelium, a vertical alpha 2 gradient formed. Mesenchymal cells expressed alpha 5 and alpha 8 by 9 weeks. The developing muscularis (propria and mucosae) showed accentuated alpha 5 expression. By 16 weeks, alpha 8 expression localized to the muscularis mucosae and villus core. Mesenchymal vascular elements stained strongly with anti- alpha 2 and alpha 6 by 9 weeks. CONCLUSIONS: These observations show the complexity and overlap of adhesive receptor expression and ligands during development and reveal early cell commitment to the formation of specific structures. (Gastroenterology 1996 Jan;110(1):58-71)

Nitric oxide synthase and chemical coding in cat sympathetic postganglionic neurons.

Nitric oxide synthase-like immunoreactivity was found in a subpopulation of sympathetic postganglionic neurons in the cat stellate and lower lumbar ganglia. In the ganglia of other segments such cells were rare. Double staining for tyrosine hydroxylase-like immunoreactivity and nitric oxide synthase-like immunoreactivity or the reduced nicotinamide adenine dinucleotide phosphate diaphorase reaction indicated that nitric oxide synthase-like immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase reactivity was always co-localized and was confined to tyrosine hydroxylase-negative (presumably cholinergic) ganglion cells, and was present in most of them. The occurrence of nitric oxide synthase in two subpopulations of cholinergic postganglionic neurons was investigated in triple staining experiments. Presumptive sudomotor neurons have been previously defined as scattered cells containing calcitonin gene-related peptide-like immunoreactivity, usually accompanied by vasoactive intestinal peptide-like immunoreactivity: 99% of these contained nitric oxide synthase. Presumptive muscle vasodilator neurons have been previously identified as clumped cells with strong vasoactive intestinal peptide-like immunoreactivity but no calcitonin gene-related peptide-like immunoreactivity: 70% of these contained nitric oxide synthase. Sweat glands were found in the paw pad skin surrounded by varicose fibres showing calcitonin gene-related peptide-like immunoreactivity and vasoactive intestinal peptide-like immunoreactivity, confirming previous work. Such fibres also stained for nitric oxide synthase-like immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase reactivity, although their staining was relatively weaker than in the corresponding cell bodies. Varicose fibres with the same chemical coding were also found around all large and most medium and small arteries in the paw skin as well as around arteriovenous anastomoses. Fibres with the muscle vasodilator coding (vasoactive intestinal peptide-like immunoreactivity without calcitonin gene-related peptide-like immunoreactivity) were not seen in paw skin. These results suggest that nitric oxide may act as a co-transmitter (with acetylcholine, substance P, vasoactive intestinal peptide and calcitonin gene-related peptide) in sudomotor neurons and (with acetylcholine and vasoactive intestinal peptide) in vasodilator neurons. Collateral branches of sudomotor neurons may innervate skin vessels, and release vasodilator transmitters including nitric oxide to cause the vasodilatation which provides the fluid supply for sweat formation. Alternatively, separate vasodilator neurons to skin may share the same chemical code as sudomotor neurons.

The Mouse 14-3-3 ϵ Isoform, a Kinase Regulator Whose Expression Pattern Is Modulated in Mesenchyme and Neuronal Differentiation

Kidney development is a complex, little understood process based on inductive interactions and intricate epithelial and mesenchymal morphogenesis. Here, we report the use of subtractive hybridization to clone cDNAs expressed in early nephrogenesis. cDNA made from E14.5 mouse kidney was hybridized with adult mouse liver mRNA employing a technique based on labeling the driver mRNA with photo-activatable biotin and using streptavidin to remove RNA: cDNA complexes. An aliquot of the unhybridized cDNA identified several clones including three isolates that proved to be the ϵ isoform of the 14-3-3 gene family that is, among other functions, implicated in protein kinase C regulation. Northern blot analysis showed a 2.0-kb transcript widely present in mouse embryos from E7.5 onward, but, as expected from the subtractive strategy, absent in adult liver. In situ hybridization was carried out on mouse embryos aged E8.5 to E15.5. These showed that, in the E8.5 embryo, the 14-3-3 ϵ gene was expressed throughout the embryo, but that, within a day, expression was more marked in mesenchyme than elsewhere (e.g., epithelial tissue, where it was generally low), although levels in neural tissue rose again by about E12.5. This difference was maintained until E15.5 when expression levels started to drop in most tissues, with those of the nervous system, tooth, and kidney being exceptions. Perhaps the most intriguing feature of the expression pattern, however, was that, while the gene was strongly expressed in early mesenchyme, the level of expression decreased as the mesenchyme differentiated. This change was particularly noted in mesenchymal condensations that would become cartilage, bone, and myotome-derived muscle, in the presumptive muscle layer of the gut, and in the kidney. In this last case, the gene was strongly expressed in stem cells and mesenchyme, but expression levels dropped markedly as early nephrogenic condensates epithelialized. The results as a whole thus argue for the 14-3-3 ϵ isoform playing roles in neural development and in early mesenchyme, with this latter function being lost or replaced as the tissue differentiates.

The larval development of lateral musculature in gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax

Fibre-type differentiation of the lateral musculature has been studied in Sparus aurata (L.) and Dicentrarchus labrax (L.) during larval development. Histochemical and ultrastructural techniques show two presumptive muscle layers and two germinative zones of presumptive myoblasts. At hatching, myotomal muscle consists of a monolayer of thin undifferentiated cells near the skin (first germinative zone) overlying another mono-layer of small diameter fibres extending hypaxially and epaxially away from the transverse septum. Below this, there is a much thicker, deep layer of fibres, generally large in diameter and polygonal in shape. The presumptive myoblasts are located between these two layers of fibres in the second germinative zone. Initially, the superficial and deep muscle fibres show high and low myosin ATPase activity, respectively. Both layers grow by generating new fibres from the two mentioned germinative zones. At the end of larval life, the superficial layer changes its histochemical profile from high to low myosin ATPase activity and, at the same time, intermediate or pink muscle fibres can be observed by oxidative activity (the NADH-TR reaction). Morphometric analysis shows a significant increase in mean fibre diameter during successive ages, as shown by the Student's t-test (hypertrophic growth). Skewness and kurtosis values of fibre diameters point to the generation of a new fibre population from the germinative zones (hyperplastic growth).

Mammalian myoblasts become fast or slow myocytes within the somitic myotome.

A monoclonal antibody has been prepared to slow skeletal muscle type myosin heavy chain which in the rat distinguishes fast and slow myocytes within the somitic myotome at 11.5 days in utero. The distribution of slow myotubes identified by this antibody in developing limb buds is also restricted to presumptive slow muscle cell type regions only. No slow myoblasts in hindlimb buds, however, were detected at 14.5 day in utero when a small number of fast muscle cells were already present. The presence of slow muscle cell population detected by this specific antibody became apparent a day later. This study thus demonstrated the diversification into different muscle cell types during both early embryonic and foetal development.