Visceral Arches Research Articles

Zinc deficiency causes apoptosis but not cell cycle alterations in organogenesis-stage rat embryos: effect of varying duration of deficiency.

Zinc deficiency is teratogenic in all species in which it has been examined. Zinc is an essential component of enzymes involved in DNA synthesis and cell proliferation, and may play an as yet undetermined role in apoptosis. To further our understanding of the role of zinc in normal development, we examined cell death and cell cycle parameters in embryos of pregnant rats fed a zinc-deficient diet for 2 to 10 days (0.5 microgram zinc/g diet; zinc-adequate diet was 25 micrograms zinc/g). To elucidate sensitive periods of development and susceptible cell populations, dams were fed the zinc-deficient diet from gestation day 1, 3, 7, or 9 and killed on day 11. Embryos were examined for morphology and developmental stage. From each litter, 2-3 embryos were stained with Nile blue sulfate (NBS) to visualize cell death, 3 embryos were frozen for flow cytometric cell cycle analysis and cell counts, and selected embryos were preserved for histological examination. Dams fed the zinc-deficient diet for more than 3 days reduced their food intake through gestation day 8 but increased food intake on day 9. Maternal plasma zinc dropped to 10-25% of control levels in the zinc-deficient groups. Zinc deficiency from gestation day 1 or 3 resulted in two categories of affected litters on day 11. One category had embryos which were morphologically normal but displayed extensive NBS staining in the visceral arches, neural tube, and somites. The second category had developmentally retarded or maldeveloped embryos which showed little NBS staining. Zinc deficiency from gestation day 7 produced cell death in the posterior dorsal midline in the area of premigratory neural crest cells, which was confirmed by histological examination. Zinc deficiency from gestation day 9 did not affect morphology or NBS staining. Percentages of cells in the G0/G1, S, and G2M phases of the cell cycle on gestation day 11, determined by flow cytometry, were similar to controls in all groups. This study shows that as few as 4 days of maternal zinc deficiency can produce excess embryonal cell death, and that neural crest cells may be particularly sensitive.

Cranial features of dendrobatid larvae (Amphibia: Anura: Dendrobatidae)

The larval neurocranium and visceral arches of seven dendrobatid species representing four genera are described, based on cleared-and-stained and serially sectioned specimens. A variety of characters is shared by all seven species. Larval features do not substantiate the assumption of close ranoid affinities of the Dendrobatidae. Instead dendrobatid larvae share features such as the special quadripartite cartilago suprarostralis, the lack of the larval processus oticus, the presence of three foramina acustica, and the lack of a foramen perilymphaticum accessorius with many bufonoid larvae. The first of these characters is unique to bufonids, hylids, dendrobatids, and some New World leptodactylids; the other characters also occur in pelobatids and are presumably plesiomorphic for the Neobatrachia. The free proximal ends of Ceratobranchialia II and III are an autapomorphy of the Dendrobatidae supporting the monophyly of the family. Some features of the cranium are paedomorphic: low cartilago orbitalis, lack of connection between cartilage orbitalis and otic capsule (most species), and vestigal taeniae tecti. New anatomical terms are introduced.

Cell death in rat and mouse embryos exposed to methanol in whole embryo culture

Methanol induces developmental toxicity in rats and mice producing exencephaly, cleft palate, cervical skeletal defects, reduced body weight, and increased embryo/fetal death. Exposure to methanol in whole embryo culture also induces developmental retardation, dysmorphogenesis, and embryo lethality. In the present study, cultured rat and mouse embryos were exposed to methanol and subsequently observed for morphological effects and increased cell death using modified Feulgen staining which allowed nuclei throughout the embryo to be examined in situ. Growth and developmental scores were reduced by methanol in both rat and mouse embryos and the mouse embryos were affected at lower concentrations when compared to the rat. Methanol increased cell death in specific regions of both rat and mouse embryos, including the forebrain, the visceral arches, otic and optic placodes. These regions form derivatives which manifest morphological abnormalities following exposure in vivo. Methanol did not increase cell death in the neuroepithelium or neural folds and neural tube defects cannot be explained by excess cell death. The results of this study suggest that increased cell death in specific regions of the exposed embryos has a role in producing cranial malformations, abnormalities of the eye and ear, and cleft palate.

In vitro clonal analysis of cardiac outflow tract mesenchyme.

In vitro clonal analyses were performed to gain insight into the mechanisms that control development of neural crest-derived cardiac outflow tract mesenchyme of quail embryos. The cardiac neural crest originates from the posterior rhombencephalic neural tube. The cells leave the neural tube and migrate through the posterior visceral arches to the outflow tract of the heart, where they form the conotruncal ridges. Mesenchyme cells derived from the neural tube from somitic levels 1-3 contained 5 types of clone-forming cells. One class of clones contained up to 6 phenotypes; smooth muscle cells, connective tissue cells, chondrocytes, sensory neuron precursors, serotonergic (putative enteric) neurons, and pigment cells. Another type of clone was totally pigmented, containing melanocytes only, whereas a third type consisted entirely of smooth muscle cells. The remaining classes of clones contained 3 and 4 phenotypes, respectively. Subsequently, mesenchymal cells obtained from posterior visceral arches were cloned in vitro. The major observations from these experiments are the following. 1) The cells have lost the capacity to form sensory neurons. 2) The capacity to form pigment cells is lost as well. 3) Four types of morphologically distinguishable clones were found. The frequency of one type of clone that contains ectomesenchymal cells only (smooth muscle cells, connective tissue cells, and chondrocytes) increased from zero at Hamburger and Hamilton stage 19+/20 to 67% at stage 24, possibly giving rise to visceral arch-derived structures. The frequency of the other 3 types of clones decreased with progressing embryonic development, indicating that these clone-forming cells either pass through the visceral arches, and/or are being converted to cells with fewer developmental potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Early cranial neural crest migration in the direct-developing frog, Eleutherodactylus coqui.

Direct development is a common reproductive mode in living amphibians characterized by absence of the free-living, aquatic larval stage. In Eleutherodactylus, a species-rich genus of New World frogs, evolution of direct development from the ancestral biphasic ontogeny is correlated with a comprehensive modification in embryonic cranial patterning, including the loss of many larval-specific components and the precocious formation of many adult (postmetamorphic) structures. We use scanning electron microscopy (SEM) to examine the emergence and early migration of cranial neural crest cells in Eleutherodactylus coqui to begin to assess the possible role of the neural crest in mediating these evolutionary changes. As in metamorphosing frogs, cranial crest cells emerge prior to neural fold closure and assemble into three streams: rostral otic, and caudal otic. These streams contribute to the face and first visceral (mandibular) arch, to the second (hyoid) arch, and to posterior (branchial) arches, respectively. Rostrocaudal position, morphology, and/or migration patterns distinguish subpopulations of cells within the rostral stream and caudal otic stream. With the possible exception of the small size of the rostral otic caudal otic streams, evolution of direct development in E. coqui has not altered basic patterns of neural crest emergence or early migration as assessed by SEM. If observed evolutionary changes in embryonic cranial patterning are mediated by the neural crest, then they likely involve later aspects of crest migration or more subtle features related to pattern formation such as cell behavior and commitment, or gene expression.

Localization of PDGF A and PDGFRα mRNA in Xenopus embryos suggests signalling from neural ectoderm and pharyngeal endoderm to neural crest cells

In situ hybridization analysis of Xenopus laevis embryos reveals that mRNA encoding the platelet-derived growth factor α receptor (PDGFRα) is expressed in cephalic neural crest masses prior to migration from the future neural tube and during their migration into the visceral arches. The analysis of fluorescently labeled neural crest tissue transplanted to unlabeled host embryos demonstrates that neural crest cells are the only detectable source of PDGFRα mRNA within visceral arches. Transcripts encoding PDGF A are present in neural ectoderm, otic vesicle and pharyngeal endoderm. Their location suggests that PDGF A provides a signal, first from the neural epithelium and later from the otic vesicle and pharyngeal endoderm, to cephalic neural crest cells during their migration in the arch region.

Phylogenetic Trends in the Baroreceptor Control of Arterial Blood Pressure

This article attempts to draw out current themes in the comparative physiology of baroreceptors in order to point out potential areas of future research. The first theme concerns the distribution of baroreceptive zones in the central cardiovascular system. Baroreceptor populations are widely distributed among jawed vertebrates. Multiple baroreceptive zones commonly occur within segments of the central cardiovascular system derived from the embryonic visceral arch arteries and are innervated by the corresponding visceral arch nerves. Aortic (fourth arch) and pulmonary (sixth arch), but not carotid, baroreceptors are the most conspicuous populations, at least in terms of the consistency with which they have been functionally identified. A second theme that links baroreceptor structure to function has emerged recently. Unmyelinated baroreceptors have been identiied in amphibians, reptiles, and mammals. Such receptors have thresholds ranging from near to well above normal arterial pressures and act predominantly to mediate reflex responses to increases in arterial blood pressure above the normal range. In contrast, myelinated baroreceptors with thresholds well below normal arterial pressures have so far only been positively identiied in mammals. Quantitative data suggest that the reflex regulation of arterial pressure in nonmammalian vertebrates is of physiological importance. In the toad, open-loop analysis of the pulmocutaneous baroreflex suggests that the peak capacity of this reflex to regulate arterial blood pressure is of the same order of magnitude as that of the carotid sinus baroreflex in the dog. However, as only unmyelinated baroreceptors are present in the toad, expression of this reflex is largely restricted to arterial pressures well above the normal range. Finally, it is appealing to speculate that differences in the structure and function of baroreceptors between vertebrate classes are linked to corresponding differences in vascular anatomy and metabolic rate. Unmyelinated baroreceptors may be widely distributed in vertebrates, which would reflect a widespread need to protect the circulation from damaging increases in arterial pressure. In vertebrates with incompletely divided hearts, baroreflexes may be of particular importance in protecting the circulation of the gas exchanger from excessive pressures. Low-threshold myelinated baroreceptors may represent a relatively specialized addition topressure-regulating systems in endothermic vertebrates that have poor tolerance to even transient hypotension.

Pagliaccio, a member of the Eph family of receptor tyrosine kinase genes, has localized expression in a subset of neural crest and neural tissues in Xenopus laevis embryos

Cranial neural crest cells arise from neural folds in the embryonic head and differentiate to produce most of the cartilages and bones of the skull and the somatosensory ganglia of several cranial nerves, among other tissues. Since the molecular basis of the determination of these cells is poorly understood, we have begun a search for molecules involved in signal transduction in cranial neural crest. From a Xenopus laevis cranial neural crest cDNA bank, we have cloned a cDNA encoding a putative receptor tyrosine kinase, which we call Pagliaccio ( Pag). Pag RNA is present transiently in visceral arch 3, probably representing neural crest cells in this tissue. Pag is also expressed in the forebrain, rhombomeres r3 and r5 of the hindbrain and in the pronephros. Based on this localized expression, we propose that Pag may play a role in the differentiation of cranial neural crest and other tissues.

Combinatorial expression of three zebrafish genes related to distal- less: part of a homeobox gene code for the head

We describe analysis of zebrafish distal-less-related homeobox genes that may serve as specifiers of positional information in anterior regions of the CNS and in peripheral structures. We isolated three zebrafish genes, dlx2, dlx3, and dlx4, by screening embryonic cDNA libraries. Comparisons of the predicted sequences of the Dlx2, Dlx3, and Dlx4 proteins with distal-less proteins from other species suggest that vertebrate distal-less genes can be divided into four orthologous groups. We observed similarities but also unique features of the expression patterns of the zebrafish dlx genes. Among the three genes, dlx3 alone is expressed during gastrulation. Shortly after gastrulation, cells in the ventral forebrain rudiment express dlx2 and dlx4, but not dlx3, and hindbrain neural crest cells express only dlx2. Presumptive precursor cells of the olfactory placodes express dlx3 and dlx4 but not dlx2. Transcripts of dlx3 and dlx4 are present in overlapping subsets of cells in the auditory vesicle and in cells of the median fin fold, whereas dlx2 is never expressed in the auditory vesicle and only at low levels in localized regions of the median fin fold. Cells of the visceral arches and their primordia express all three dlx genes, but with different developmental time courses. We suggest that combinatorial expression of the dlx genes is part of a homeobox gene code specifying pattern formation or cell fate determination in the forebrain, in peripheral structures of the head, and in the fins.

Effects of the biguanide class of oral hypoglycemic agents on mouse embryogenesis.

The incidence of birth defects among offspring of mothers with non-insulin dependent diabetes mellitus (NIDDM) is 2-3-fold higher than among infants of non diabetics. Since mothers with NIDDM are frequently given oral hypoglycemic agents, including sulphonylureas and biguanides, to control their disease and since these agents have been associated with the occurrence of congenital malformations in humans and animals, the embryotoxic effects of the most commonly employed biguanides, metformin and phenformin, were evaluated in whole embryo culture. Neurulating mouse embryos were exposed to therapeutic concentrations (metformin 500-2,550 mg per day; phenformin 50-400 mg per day, respectively) of the compounds for 24-48 h. Concentrations of metformin in culture ranged from 0.15 to 1.8 mg/ml and phenformin from 2.5 x 10(-5) to 0.4 mg/ml. Cultures were terminated and scored for gross morphological alterations and total protein content. Metformin produced no alterations in embryonic growth and no major malformations. Approximately 10% of all embryos exposed to metformin regardless of dose, exhibited open cranial neuropores after 24 h of culture. However, this anomaly appeared to represent a delay in closure as opposed to an overt defect, since no embryos exposed to the highest concentration of the drug and cultured for 48 h showed open neural tubes. In contrast, phenformin produced dose dependent changes in incidence of malformations, protein content, and embryolethality. Malformations included neural tube closure defects, craniofacial hypoplasia, and reduction in size of the first and second visceral arches. Doses above 0.1 mg/ml produced embryolethality and all embryos were killed at the 0.4 mg/ml concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

A Chicken Homeobox Gene Related to Drosophila paired Is Predominantly Expressed in the Developing Limb

We identified a homeobox-containing gone, Prx-1, isolated from the chick limb bud cDNA library. The homeodomain sequence is related to Drosophila paired and gooseberry and mouse Pax-3, Pax-6, and Pax-7. The deduced amino acid sequence of the Prx-1 gene product reveals the absence of a paired-box sequence and extensive similarity to mouse S8 and MHox homeodomain proteins, thus constituting a new class of homeobox gene. Using an in situ hybridization method, the Prx-1 gene is shown to be expressed predominantly in the limb bud and visceral arches. At early stages of limb development, distal mesodermal cells express the homeobox gene with an apparent gradient along the proximal-distal axis. The signal is absent in the apical and nonridge ectoderm. Removal of the apical ectodermal ridge had no apparent effect on the subsequent expression of Prx-1 in the limb mesenchyme. The Prx-1-expressing cells are later confined to the interdigital and perichondrial regions. The Prx-1 transcripts are also detectable in the mesenchyme of the visceral arches and facial primordia subjacent to the ectoderm. The Prx-1 gene is weakly expressed in somites and condensing vertebrae. No signal is detectable in neural tube and ectodermal epithelium. These results suggest that the Prx-1 homeodomain protein is involved in the differentiation of bone, muscle, and other tissues of mesodermal origin during limb development.

Isolated sequences from the linked Myf-5 and MRF4 genes drive distinct patterns of muscle-specific expression in transgenic mice.

In developing mouse embryos, MyoD family regulatory genes are expressed specifically in muscle precursors and mature myofibers. This pattern, taken together with the well-established ability of MyoD family members to convert a variety of cell types to skeletal muscle, suggests a significant role for these genes in regulating skeletal myogenesis. The possibility that expression of these genes may be causally associated with segregation of the myogenic lineage from other mesodermal derivatives, or with the subsequent maintenance of muscle phenotypes at later times, raises the issue of how MyoD family genes are themselves regulated during development. In this work, we have initiated studies to identify DNA sequences that govern Myf-5 and MRF4 (herculin, myf-6) transcription. Myf-5 is the first of the MyoD family to be expressed in the developing mouse embryo, while MRF4 is the most abundantly expressed myogenic factor in postnatal animals. In spite of their strikingly divergent patterns of expression, Myf-5 and MRF4 are tightly linked in the mouse genome; their translational start codons are only 8.5 kilobases apart. Here, the 5' flanking regions of the mouse Myf-5 and MRF4 genes were separately linked to a bacterial beta-galactosidase (lacZ) gene, and these constructs were each used to produce several lines of transgenic mice. Transgene expression was monitored by X-gal staining of whole embryos and by in situ hybridization of embryo sections. For the Myf-5/lacZ lines, the most intense transgene expression was in the visceral arches and their craniofacial muscle derivatives, beginning at day 8.75 post coitum (p.c.). This correlates with endogenous Myf-5 expression in visceral arches.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a Xenopus Distal-less homeobox gene involved in forebrain and cranio-facial development

Homeobox-containing genes are thought to perform essential functions in the process of pattern formation in vertebrates and invertebrates. They provide cells with positional information critical for normal embryonic development. Since most of the identified homeobox genes in Xenopus seem to provide positional information for the development of the trunk, we have concentrated on genes that may be specifically involved in the formation of the head region. Using a polymerase chain reaction strategy we have searched for Xenopus homeobox-containing genes that might provide positional cues for correct development of the brain. In this paper we report the identification and cloning of a novel gene that by homology appears to be a member of the Distal-less homeobox gene family. We show that its temporal expression patterns in the cement gland, neural crest derived visceral arches, retina and forebrain, while quite diverse, does suggest shared developmental features which may be required for correct craniofacial development and the regionalization of the Xenopus brain. Furthermore, expression of this gene at later stages is primarily restricted to the tadpole forebrain suggesting that the Distal-less gene product continues to play a role after the initial brain patterning is complete.

Upstream Region of the Myogenin Gene Confers Transcriptional Activation in Muscle Cell Lineages During Mouse Embryogenesis

Myogenin, one of the MyoD-related factors containing basic-helix-loop-helix motifs, is transcriptionally activated in skeletal muscle lineages in somites and limb buds during embryogenesis. In an attempt to understand regulatory mechanisms which govern transcriptional activation of the myogenin gene, transgenic mice bearing the lacZ gene driven by the upstream region of the myogenin gene were generated. Stereoscopic visualization of LacZ-positive cells of these transgenic mouse embryos revealed that the upstream region of the myogenin gene conferred its transcriptional activation in cells of the skeletal muscle lineages in somites, limb buds, and visceral arches. Moreover, transient LacZ expression in newly formed somites in addition to its strong activation in myotomal regions of mature somites with a rostro-caudal gradient raised the possibility that myogenin is transcriptionally activated in immature somites before myotome formation.

Pluripotent and Developmentally Restricted Neural-Crest-Derived Cells in Posterior Visceral Arches

The early migratory cells of the posterior rhombencephalic neural crest consist of a heterogeneous population of pluripotent and developmentally restricted neural crest cells (Ito and Sieber-Blum, Dev. Biol. 148, 95-106, 1991). To determine if changes in developmental capacities occur during migration, the developmental potentials of posterior visceral arch mesenchymal cells were investigated by in vitro clonal analysis. Most of these cells consisted of the post-migratory cells of the posterior rhombencephalic neural crest. Four morphologically distinct types of clones were observed, and the cells within these clones expressed characteristic phenotypes as shown by their binding of antibodies against cell type-specific markers: (1) "DP" clones consisted of densely packed polygonal cells, with flattened large cells located predominantly at the periphery of these clones. Immunocytochemical analyses showed that DP clones contained smooth muscle cells, connective tissue cells, chondrocytes, and serotonin (5-HT)-positive neurons, and over 90% of the cells per clone were HNK-1 positive. This suggests that DP clone-forming cells are pluripotent neural-crest-derived cells with the capacity to develop into ectomesenchymal derivatives and serotonergic neurons. (2) "DS" clones consisted of densely packed spindle-shaped cells. These clones included smooth muscle cells, connective tissue cells, and chondrocytes. By contrast, neuronal phenotypes were not present. An average of 0.4% of the cells per clone were HNK-1 positive. DS clones appear to be formed by neural-crest-derived cells that are partially restricted, expressing ectomesenchymal phenotypes only. (3) "DF" clones consisted of densely packed small cells and flattened large cells. Although no HNK-1-positive cells were found in DF clones, these clones contained connective tissue cells and/or smooth muscle cells. DF clones appear to be derived from bipotent cells with the ability to differentiate into connective tissue cells and smooth muscle cells, or cells committed to the connective tissue cell lineage. (4) "LF" clones consisted of loosely arranged, flattened large cells. These clones did not contain HNK-1-positive cells. The clones consisted entirely of smooth muscle cells. Therefore, LF clones are most likely formed by precursors that are committed to the smooth muscle cell lineage. These results indicate the presence of pluripotent neural-crest-derived cells, cells with a restricted developmental potential, and apparently committed cells in the posterior visceral arch. Pluripotent cells can generate up to four neuronal and nonneuronal phenotypes. Other cells are restricted to ectomesenchymal cell types, and the proportion of these cells in the posterior visceral arch changes with progressing embryonic development. The present data in combination with our previous study (Ito and Sieber-Blum, Dev. Biol. 148, 95-106, 1991) suggest a characteristic pattern of lineage segregation during posterior rhombencephalic neural crest development, indicating that specification of some cell types can occur at the final sites of the neural crest cell localization.

The structure and expression of the Xenopus Krox-20 gene: conserved and divergent patterns of expression in rhombomeres and neural crest

Recent studies in the chick have indicated that rhombomeres (r) are segments that underlie the patterning of hindbrain nerves. These segments may also be important for the specification of branchial arch structures since alternating rhombomeres, r2, r4 and r6, each contribute crest to a specific arch. Krox-20 has been implicated in the segmental patterning of the hindbrain in the mouse by its expression prior to segment formation in alternating domains, which later correspond to r3 and r5. Here, we describe the sequence and developmental expression of the Xenopus Krox-20 gene, XKrox-20. Alternating domains of XKrox-20 expression appear in the early neurula, later correspond to r3 and r5, and persist until late tadpole stages. In contrast to this conserved spatial expression in rhombomeres, we find a pattern in the neural crest of Xenopus that appears different from that found in the mouse: expression occurs in crest that migrates from r5 into the third visceral arch. We speculate that this may reflect a distinct route of neural crest migration due to anatomical differences between these systems, rather than a difference in the site of origin of Krox-20-expressing crest.

Antisense attenuation of Wnt‐1 and Wnt‐3a expression in whole embryo culture reveals roles for these genes in craniofacial, spinal cord, and cardiac morphogenesis

Wnt-1 and Wnt-3a proto-oncogenes have been implicated in the development of midbrain and hindbrain structures. Evidence for such a role has been derived from in situ hybridization studies showing Wnt-1 and -3a expression in developing cranial and spinal cord regions and from studies of mutant mice whose Wnt-1 genes have undergone targeted disruption by homologous recombination. Wnt-1 null mutants exhibit cranial defects but no spinal cord abnormalities, despite expression of the gene in these regions. The absence of spinal cord abnormalities is thought to be due to a functional compensation of the Wnt-1 deficiency by related genes, a problem that has complicated the analysis of null mutants of other developmental genes as well. Herein, we describe the attenuation of Wnt-1 expression using antisense oligonucleotide inhibition in mouse embryos grown in culture. We induce similar mid- and hindbrain abnormalities as those seen in the Wnt-1 null mutant mice. Attenuation of Wnt-1 expression was also associated with cardiomegaly resulting in hemostasis. These findings are consistent with the possibility that a subset of Wnt-1 expressing cells include neural crest cells known to contribute to septation of the truncus arteriosus and to formation of the visceral arches. Antisense knockout of Wnt-3a, a gene structurally related to Wnt-1, targeted the forebrain and midbrain region, which were hypoplastic and failed to expand, and the spinal cord, which exhibited lateral outpocketings at the level of the forelimb buds. Dual antisense knockouts of Wnt-1 and Wnt-3a targeted all brain regions leading to incomplete closure of the cranial neural folds, and an increase in the number and severity of outpocketings along the spinal cord, suggesting that these genes complement one another to produce normal patterning of the spinal cord. The short time required to assess the mutant phenotype (2 days) and the need for limited sequence information of the target gene (20-25 nucleotides) make this antisense oligonucleotide/whole embryo culture system ideal for testing the importance of specific genes and their interactions in murine embryonic development.

Multiple sites of Hox-7 expression during mouse embryogenesis: comparison with retinoic acid receptor mRNA localization.

We report results from a study of Hox-7 expression during mouse embryonic and fetal development and compare the localization of Hox-7 transcripts with those of the retinoic acid receptors. Transcripts were detected by in situ hybridization. Hox-7 expression occurs in (1) cephalic neural crest and its derivatives, (2) sites of ectomesodermal interaction, (3) extraembryonic tissues, and (4) endocardial cells. Hox-7 does not seem to be involved in defining rostrocaudal boundaries, but instead appears to be expressed along the proximodistal axes at these sites. We further investigated the active sites of morphogenesis, which involve an ectomesodermal interaction (e.g., limb buds, visceral arches), including genital tubercle and tail ridge. These are regions highly positive for Hox-7 transcripts, and many are known to be sites for the expression of gamma-retinoic acid receptors (RARs) and cellular retinoic acid binding proteins. Most regions that express Hox-7 are subregions of gamma-RAR expression. In the developing limb bud, expression of Hox-7 takes place in the interdigital region, where it overlaps areas of beta-RAR expression.

MHox: a mesodermally restricted homeodomain protein that binds an essential site in the muscle creatine kinase enhancer.

Myogenic helix-loop-helix (HLH) proteins, such as myogenin and MyoD, can activate muscle-specific transcription when introduced into a variety of nonmuscle cell types. Whereas cells of mesodermal origin are especially permissive to the actions of these myogenic regulators, many other cell types are refractory to myogenic conversion by them. Here we describe a novel homeodomain protein, MHox, that binds an A+T-rich element in the muscle creatine kinase (MCK) enhancer that is essential for muscle-specific transcription and trans-activation by myogenic HLH proteins. MHox is completely restricted to mesodermally derived cell types during embryogenesis and to established cell lines of mesodermal origin. In contrast to most other homeobox genes, MHox expression is excluded from the nervous system, with the highest levels observed in limb bud and visceral arches. In adult mice, MHox is expressed at high levels in skeletal muscle, heart and uterus. The DNA-binding properties and pattern of MHox expression are unique among homeobox genes and suggest a role for MHox as a transcriptional regulator that participates in the establishment of diverse mesodermal cell types.

Tropic effects of otic epithelium on cochleo‐vestibular ganglion fiber growth in vitro

Sensory nerve fibers of the cochleo-vestibular ganglion (CVG) innervate the otic epithelium in the early chick embryo by directed growth. To see if the target tissue could exert a tropic influence, we co-cultured CVGs from chick embryos (Hamburger-Hamilton stages 16-30) in a 3D collagen matrix with their normal target epithelium or with other epithelial tissues taken from the same or different stages of development. The pattern of neurite outgrowth and the viability of the CVG after five days in vitro were assessed histologically with a silver method. On the basis of the patterns of neurite outgrowth directed toward the epithelium, the cultures were classified as having slightly, mostly, exclusively, or no directed outgrowth. Of 49 cultures containing otic epithelium, 33 had mostly or exclusively directed growth patterns. This effect did not depend on any particular stage difference between co-cultures or on their viability in vitro. Cultures of non-sensory otic epithelium (endolymphatic duct) also presented directed growth patterns. Co-cultures with ectoderm from forelimb or visceral arch had little, if any, directed growth. The directed growth could not be explained simply as a result of guidance by non-neuronal cells or of the viability of the explants. The results are consistent with the hypothesis that the otic epithelium provides a tropic factor that attracts growing CVG fibers.