Showing papers in "Developmental Dynamics in 1995"

Stages of embryonic development of the zebrafish.

Abstract: We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis--the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts.

Vascularization of the mouse embryo: A study of flk‐1, tek, tie, and vascular endothelial growth factor expression during development

Abstract: We report the detailed developmental expression profiles of three endothelial specific receptor tyrosine kinases (RTKs) flk-1, tek, tie, as well as vascular endothelial growth factor (VEGF), the flk-1 ligand. We also examined the expression of the other VEGF receptor, flt-1, during placental development.flk-1, tek, and tie transcripts were detected sequentially at one-half day intervals starting at E7.0, suggesting that each of these RTKs play a unique role during vascularization of the mouse embryo. All three RTKs were expressed in the extraembryonic and embryonic mesoderm in regions that eventually give rise to the vasculature. Except for the expression of tek and flk-1 in the mesoderm of the amnion, the expression of these RTKs from E8.5 onwards was virtually indistinguishable. An abundant amount of flt-1 transcripts was found in the spongiotrophoblast cells of the developing placenta from E8.0 onwards. This cellular compartment is located between the maternal and labyrinthine layers of the placenta, which both express VEGF. VEGF transcripts were detected as early as E7.0 in the endoderm juxtaposed to the flk-1 positive mesoderm, and later in development VEGF expression displayed an expression profile both contiguous with that of flk-;1, and also in tissues found some distance from the flk-1-expressing endothelium. These results suggest a possible dual role for VEGF which includes a chemotactic and/or a cellular maintenance role for VEGF during vascularization of the mouse embryo. ©1995 Wiley-Liss, Inc.

Expression of Cdx-2 in the mouse embryo and placenta: possible role in patterning of the extra-embryonic membranes.

Abstract: Three mouse homologues of the Drosophila homeotic gene Caudal (Cad) have been described. They are currently designated Cdx-1, Cdx-2, and Cdx-4. Cdx-1 and 2 are both strongly expressed in the adult mid- and hindgut, while Cdx-1 and 4 have been shown to be activated in the embryonic primitive streak. Using a polyclonal antibody against a fusion protein containing the amino terminal 109 amino acids of murine Cdx-2, we here describe the topographical location of the gene product from early cleavage to 12.5 days of embryonic development. Cdx-2 expression begins at 3.5 days and is confined to the trophectoderm, being absent from the inner cell mass. Subsequently, staining is located in the extra-embryonic ectoderm adjacent to the epiblast, but sparing the more superficially placed polar, as well as the mural trophoblastic cells. Continuing expression in the fetal membranes involves the chorion, the allantoic bud, and, at even later stages, the spongiotrophoblast. From 8.5 days, Cdx-2 begins to be expressed in embryonic tissues, principally (unlike Cdx-1) in the posterior part of the gut from its earliest formation, as well as in the tail bud and in the caudal part of the neural tube. Cdx-2 is, therefore, transcribed well before any other membrane of the Cad homologue group and of the related Hox-C group; its expression in the extra-embryonic membranes and in the hindgut reflects the phylogenetic relationship between the cloaca and the chorio-allantois and suggests the possibility that homeobox genes may be involved in placental development and/or patterning. © 1995 wiley-Liss, Inc.

Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz‐F1 disrupted mice

Abstract: Ad4BP (or SF-1) has been iden- tified as a transcription factor which regulates all the steroidogenic P450 genes in the peripheral or- gans, and is encoded by the mammalian homo- logue of Drosophila FTZ-F1 gene. mRNA coding for Ad4BP was detected in the hypothalamus and pituitary of rats by RT-PCR. Immunohistochemi- cal analyses using an antiserum to Ad4BP in the brain and pituitary revealed that the transcrip- tion factor is expressed in nuclei of the dorso- medial part of the ventromedial hypothalamus (dmVMH) and in some subpopulation of the ade- nohypophysial cells. Double immunostaining of the pituitary for Ad4BP and trophic peptide hor- mones, FSH, TSH, and ACTH, indicated a re- stricted localization of Ad4BP to the gonadotroph. Disruption of the mouse Ftz-FI gene was clarified to induce severe defects in the organization of the dmVMH and the function of the pituitary gona- dotroph. However, some of the dm VMH neurons and pituitary gonadotrophs persisted, which pro- vided a sharp contrast to complete agenesis of the peripheral steroidogenic tissues (adrenal and go- nads) in the mutant mouse. Additional abnormal- ities were seen in the ventrolateral part of VMH and dorsomedial hypothalamic nucleus, both of which do not express Ad4BP but have strong reciprocal fiber-connections with the dmVMH. Aromatase P450-containing cells in the medial preoptico-amygdaloid region, which were devoid of Ad4BP, persisted even in the brain of the gene disrupted mice. The present results clearly showed that the hypothalamic and pituitary Ad4BPs are essential to normal development of the functional VMH and gonadotroph through some mechanism distinct from that in the periph-

Coordinate expression of vascular endothelial growth factor receptor‐1 (fit‐1) and its ligand suggests a paracrine regulation of murine vascular development

Abstract: Vascular endothelial growth factor (VEGF) is a candidate regulator of blood vessel growth during embryonic development and in tumors. To evaluate the role of VEGF receptor-1/flt-1 (VEGFR1/flt-1) in the development of the vascular system, we have characterized the murine homolog of the human flt-1 gene and have analyzed its expression pattern during mouse embryogenesis. Receptor binding studies using transfected COS cells revealed that the murine flt-1 gene encodes a high affinity receptor for VEGF. The apparent Kd for VEGF binding, as determined by Scatchard analysis, was 114 pM, demonstrating that VEGFR1/flt-1 has a higher affinity to VEGF than VEGF receptor-2/flk-1 (VEGFR2/flk-1). By in situ hybridization, VEGFR1/flt-1 was detected in the yolk sac mesoderm already at the early stages of vascular development, while the receptor ligand was expressed in the entire endoderm of 7.5-day mouse embryos. A comparison with VEGFR2/flk-1 showed that the two receptors shared a common expression domain in the yolk sac mesoderm, but were expressed at different sites in the ectoplacental cone. The differential expression of the two VEGF receptors persisted in the developing placenta, where VEGFR1/flt-1 mRNA was detected in the spongiotrophoblast layer, whereas VEGFR2/flk-1 transcripts were present in the labyrinthine layer which is the site of VEGF expression. In the embryo proper, VEGFR1/flt-1 mRNA was specifically localized in blood vessels and capillaries of the developing organs, closely resembling the pattern of VEGFR2/flk-1 transcript distribution. In the developing brain, the expression of VEGF receptors in the perineural capillary plexus and in capillary sprouts which have invaded the neuro-ectoderm correlated with endothelial cell proliferation and brain angiogenesis. The data are consistent with the hypothesis that VEGF and its receptors have an important function both in the differentiation of the endothelial lineage and in the neovascularization of developing organs, and act in a paracrine fashion.

Mesenchymal stem cells reside within the connective tissues of many organs

Abstract: Previous studies have noted the presence of mesenchymal stem cells located within the connective tissue matrices of avian skeletal muscle, dermis, and heart. In these studies, clonal analysis coupled with dexamethasone treatment revealed the presence of multiple populations of stem cells composed of both lineage-committed progenitor mesenchymal stem cells and lineage-uncommitted pluripotent mesenchymal stem cells. The present study was undertaken to assess the distribution of these stem cells in the connective tissues throughout various regions of the body. Day 11 chick embryos were divided into 26 separate regions. Heart, limb skeletal muscle, and limb dermis were included as control tissues. Cells were harvested enzymatically and grown using conditions optimal for the isolation, cryopreservation, and propagation of avian mesenchymal stem cells. Cell aliquots were plated, incubated with various concentrations of dexamethasone, and examined for differentiated phenotypes. Four recurring phenotypes appeared in dexamethasone-treated stem cells: skeletal muscle myotubes, fat cells, cartilage nodules, and bone nodules. These results suggest that progenitor mesenchymal stem cells and putative pluripotent mesenchymal stem cells with the potential to form at least four tissues of mesodermal origin have a widespread distribution throughout the body, being located within the connective tissue compartments of many organs and organ systems. © 1995 Wiley-Liss, Inc.

Pattern of keratinocyte growth factor and keratinocyte growth factor receptor expression during mouse fetal development suggests a role in mediating morphogenetic mesenchymal‐epithelial interactions

Abstract: Mesenchymal cells are required for the induction of epithelial development during mammalian organogenesis. Keratinocyte growth factor (KGF) is a mesenchymally derived mitogen with specific activity for epithelial cells, suggesting that it may play a role in mediating these interactions. To further evaluate this hypothesis, in situ hybridization was used to examine the spatial distribution of KGF and KGF receptor (KGFR) transcripts during organogenesis and limb formation in mouse embryos (days 14.5 through 16.5). To facilitate this aim, mouse KGF cDNA clones were isolated. There was extensive identity between the deduced mouse KGF protein sequence and that of its human and rat cognates, indicating that this gene has been highly conserved during mammalian evolution. In addition, mouse KGF protein was purified from fibroblasts and demonstrated to be structurally and functionally similar to human KGF protein. For organs within the integumental, respiratory, gastrointestinal, and urogenital systems, whose development is dependent upon mesenchymal-epithelial interactions, KGF mRNA was detected in mesenchymal cells, while epithelial cells expressed transcripts for the KGFR, KGF and KGFR mRNA was also expressed in certain other tissues such as perichondrium, cartilage of developing bones, developing skeletal muscle, and visceral smooth muscle whose development is not regulated by mesenchymal-epithelial interactions. KGF expression was also detected in tissues isolated from human embryos, suggesting similar functions for KGF in human development. Taken together, our results suggest that KGF plays an important role in mediating mesenchymal-epithelial interactions during organogenesis, but may also have other developmental functions in tissues not governed by such interactions.

Endogenous distribution of retinoids during normal development and teratogenesis in the mouse embryo

Abstract: We have analysed the endogenous retinoids present in whole mouse embryos from day 9 to day 14 of development and in individual components of the embryo at two stages, day 10.5 and day 13, by HPLC. We can only detect two retinoids, all-trans-RA (tRA) and all-trans-retinol (t-retinol), and t-retinol is 5-10-fold in excess over tRA. We cannot detect 9-cis-RA or any didehydroretinoids; thus mammalian embryos seem to differ in their retinoid content from other embryos such as chick, Xenopus, and fish. The levels of tRA do not change significantly over the 6 days of development analysed, whereas t-retinol rises sharply as the liver develops. Within the embryo, tRA is present at high levels in the developing spinal cord and at very low levels in the forebrain; indeed there is a gradient of endogenous tRA from the forebrain to the spinal cord. Other parts of the embryo had intermediate levels of tRA. When a teratogenic dose of RA was administered to day 10.5 embryos, the levels of tRA present in individual tissues of the embryo rose dramatically--from 175-fold to 1,400-fold--and the levels rose in all tissues not in any exclusive areas. We then determined which areas of the embryo were malformed by such a teratogenic dose. The lower jaw, palate, vertebrae, tail, and limbs were consistently abnormal, and since these areas received a dose of tRA no higher than any other it was concluded that cell-specific factors must determine the teratogenic response of these tissues. We then considered whether cellular retinoic acid-binding protein I or II (CRABP I or II) played any role in this response by determining their relative levels in each of the tissues analysed. There was no correlation between the presence of CRABP I and II and the distribution of administered RA. Neither was there a clear correlation in detail between the presence of CRABP I and II and the sites of teratogenesis. We therefore conclude that other factors, for example, nuclear factors, must be responsible for the teratogenic response to RA.

Collagen fibrillogenesis in situ: Fibril segments undergo post‐depositional modifications resulting in linear and lateral growth during matrix development

Abstract: Elucidating how collagen fibril growth is regulated is important in determining how tissues are assembled. Fibrils are deposited as segments. The growth of these segments is an important determinant of tissue architecture, sta- bility, and mechanical attributes. Fibril segments were isolated from developing tendons and their structure characterized. The post-depositional changes leading to linear and lateral growth of fibrils also were examined. Segments extracted from 14-day chicken embryo tendons had a mean length of 29 pm. The segments were asymmetric, having a short and a long tapered end. Most of the segments were centrosymmetric with respect to molecular packing. Segments extracted from 12- to 16-day tendons had the same structure, but mean segment length increased incrementally due to the addition of an increasingly large population of longer segments. At 17 days of development there was a precipitous increase in segment length. The morphological data indicate that the increase in length was the result of lateral associ- ations among adjacent segments. Analysis demon- strated that this fibril growth was associated with a significant decrease in fibril associated decorin. Using immunoelectron microscopy, decorin was seen to decrease significantly at 18 days of devel- opment. When decorin content was biochemically determined, a decrease also was observed. Decorin mRNA also decreased relative to fibrillar collagen mRNA during the same period. These data support the hypothesis that a decrease in fibril-associated decorin is necessary for fibril growth associated with tissue maturation. Growth through post-depositional fusion allows for appo- sitional and intercalary growth and would be es- sential for normal development, growth, and repair. 0 1995 Wiley-Liss, Inc.

Expression of a type II collagen gene in the zebrafish embryonic axis

Abstract: To understand the hierarchy of developmental controls underlying axis specification in vertebrate embryos, it is helpful to identify relationships between regulatory molecules and the genes that given axial cells their differentiated phenotypes. This work reports the cloning and expression pattern of one of these differentiation genes, a type II collagen (col2a1) gene from the zebrafish Danio rerio. Along th8e embryonic axis, col2a1 is expressed dynamically in three rows that are each a single cell wide: the notochord and the rows of cells immediately dorsal and ventral to it—the floor plate of the central nervous system, and the hypochord. In addition, col2a1 is expressed in the pharyngeal arches, the epithelium of the otic capsule, and in the mesenchyme of the neurocranium. Experiments probed the expression pattern of col2a1 relative to that of known or potential regulators of axis development, including axial, sonic hedgehog, twist, and cyclops. The results showed that the spatial and temporal pattern of col2a1 expression in axial mesoderm follows the expression of twist closer than other genes tested. In cyclops embryos, which lack an intact floor plate, col2a1 expression was usually low, but not missing in cells in the ventral spinal cord. Because col2a1 expression reveals abnormalities in the notochord of cyclopsb16 embryos, and less col2a1-expressing mesenchyme accumulates rostral to the notochord in cyclops embryos, the effects of the cyclopsb16 mutation are not confined to the central nervous system. ©1995 Wiley-Liss, Inc.

Novel mouse endothelial cell surface marker is suppressed during differentiation of the blood brain barrier

Abstract: Few markers specific for mouse endothelium exist. We describe here one such marker, MECA-32, a monoclonal antibody which shows high specificity for mouse endothelium in both embryonic and mature tissues. The MECA-32 antigen has a M(r) of 50-55 x 10(3) under reducing conditions and M(r) of 100-120 x 10(3) under nonreducing conditions. It is expressed on most endothelial cells in the embryonic and in the adult mouse, with the exception of the brain, skeletal, and cardiac muscle, where it has a more restricted distribution. In skeletal and cardiac muscle only small arterioles and venules express the MECA-32 antigen, while in the brain its expression is negatively correlated with the differentiation of the vasculature to form the blood brain barrier. Interestingly, during embryonic development the antigen occurs on the brain vasculature up to day 16 of gestation (E16), whereupon it disappears. The embryonic brain is an avascular organ anlage which is vascularized by ingrowth of external blood vessels. Differentiation of the vasculature to form the blood brain barrier occurs at approximately E16 in the mouse. This differentiation correlates with the downregulation of MECA-32 antigen expression. Between E12 and E16 MECA-32 detects most endothelial cell surfaces of the blood vessels in the brain. No MECA-32 antigen is found in the brain at E17 or any later stage of development with the exception of the vasculature of the circumventricular organs. The results suggest that MECA-32 antigen expression is temporally and spatially correlated with the development of the blood brain barrier.

Developmental expression of two members of a new class of transcription factors: I. Expression of aryl hydrocarbon receptor in the C57BL/6N mouse embryo

Abstract: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with a basic region/helix-loop-helix (bHLH) motif. AhR has been sequenced and the functional domains defined and there is information on the formation of complexes with other peptides and interactions with DNA, although these areas continue to be investigated. AhR mediates many biological effects such as developmental toxicity, including induction of cleft palate and hydronephrosis. This regulatory protein is expressed in embryonic liver and has been immunohistochemically localized in cells of human and mouse secondary palate. The expression of AhR in embryonic tissues and its ability to disrupt development suggests a significant role for this protein in development. The present study examines the pattern of AhR expression in the C57BL/6N mouse embryo from gestation days (GD) 10–16, using in situ hybridization and immunohistochemical analysis. AhR mRNA was localized with 35S-RNA antisense riboprobe (cAh1 probe, 1.8 Kb amino terminal DNA). AhR protein was localized with purified monoclonal antibody (RPT-9) raised against the N-terminal peptide sequence. AhR mRNA and protein were expressed in GD 10–13 neuroepithelium, and as development progressed the levels in brain decreased. GD 10–12 embryos also showed AhR in branchial arches, heart, somites, and liver. AhR protein and mRNA in heart were highest at GD 10–11 and decreased with age. In liver, AhR mRNA and protein levels increased and nuclear localization became more pronounced with gestational age. In GD 14–16 embryos levels in liver and adrenal were highest, but AhR was present in ectoderm, bone, and muscle. AhR expression was specific for both cell type, organ/tissue, and developmental stage, suggesting that this novel ligand-activated transcriptional regulator may be important in normal embryonic development. © 1995 wiley-Liss, Inc.1

Expression of M-twist during postimplantation development of the mouse.

Abstract: The murine homologue of the Drosophila twist gene has been shown to be essential for head mesenchyme formation and to act as an inhibitor of muscle differentiation. This paper presents a detailed analysis of M-twist expression patterns from day 7 post coitum (p.c.) to day 18 p.c., indicating a more general function of the M-twist gene. At day 7 p.c., M-twist is expressed in the mesoderm outside the primitive streak. Later M-twist message is predominantly found in the somites, the head mesenchyme, the branchial arches, the limbs, and in the mesenchyme underneath the epidermis. Beginning at day 8 p.c., M-twist is mainly expressed in undifferentiated cells committed to muscle and cartilage development: this expression is consistent with a suggested role of M-twist in inhibiting overt muscle and cartilage differentiation. However, during organogenesis, M-twist is expressed in several areas of mesenchyme-epithelia interactions, suggesting additional tissue specific functions.

Homozygosity for the Min Allele of Apc Results in Disruption of Mouse Development Prior to Gastrulation

Abstract: Mutation of the APC (adenomatous polyposis coli) gene is an early event in colon tumor development in humans. Mice carrying Min (multiple intestinal neoplasia), a mutant allele of Apc, develop intestinal and mammary tumors as adults. To study the role of the Apc gene in development, we have investigated the phenotype of embryos homozygous for ApcMin (Min). Development of the primitive ectoderm fails prior to gastrulation in homozygous Min embryos. By midgestation, the presumed homozygotes consist of a mass of trophoblast giant cells with an additional cluster of much smaller embryonic cells. These results indicate that functional Apc is required for normal growth of inner cell mass derivatives.

Developmental regulation of connexin 40 gene expression in mouse heart correlates with the differentiation of the conduction system

Abstract: In adult mouse heart, CX40 is expressed in the atria and the proximal part of the ventricular conduction system (the His bundle and the upper parts of the bundle branches). This cardiac tissue is specialized in the conduction of the electrical impulse. CX40 is the only mouse connexin known to be expressed in these parts of the adult conductive tissue and is thus considered as a marker of the conduction system. In the present report, we investigated CX40 expression and distribution during mouse heart development. We first demonstrate that CX40 mRNA is regulated throughout development, as are other heart connexin transcripts, i.e., CX37, CX43, and CX45, with a decreasing abundance as development proceeds. We also show that the CX40 transcript and protein are similarly regulated, CX40 being expressed as two different phosphorylated and un-phosphorylated forms of 41 and 40 kDa, respectively. Surprisingly, distribution studies demonstrated that CX40 is widely expressed in 11 days post-coitum (dpc) embryonic heart, where it is detected in both the atria and ventricle primordia. As development proceeds, the CX40 distribution pattern in the atria is maintained, whereas a more dynamic pattern is observed in the ventricles. From 14 dpc onwards, as the adult ventricular conduction system differentiates, CX40 decreases in the trabecular network and it is preferentially distributed in the ventricular conduction system. CX40 is thus the marker of the early differentiating conduction system. It is hypothesized that the conduction system is present in unorganized “embryonic” form at 11 dpc and trans-differentiates by 14 dpc into the adult conduction system. © 1995 wiley-Liss, Inc.

Angiogenic Potential of the Avian Somite

Abstract: We have studied the angiogenic potential of the unsegmented paraxial mesoderm and epithelial somites of the trunk with homotopical grafts between quail and chick embryos. Quail endothelial cells of the grafts were stained with the QH-1 antibody after 1-6 days of reincubation. The unsegmented paraxial mesoderm and all parts of the epithelial somite were found to contain angioblasts which develop into QH-1 positive endothelial cells. These cells are incorporated into the lining of the host's blood vessels such as the perineural vascular plexus and the dorsal branches of the aorta. There is a certain preference as concerns the location of endothelial cells derived from different parts of the somites. Angioblasts from ventral somite halves are mainly found in ventrolateral blood vessels. Those from dorsomedial quadrants form vessels in the dermis of the back, and those from dorsolateral quadrants can be found in the ventrolateral body wall and the wing. With the exception of the dorsal perineural vascular plexus, angioblasts do not cross the median plane of the body. This shows that, although angioblasts migrate extensively, there is bilaterality of the vascular system in the trunk. It remains to be studied whether the notochord plays a role in the establishment of this bilaterality.

Three-dimensional gradients of voltage during development of the nervous system as invisible coordinates for the establishment of embryonic pattern.

Abstract: We are interested in the generation of endogenous electric fields associated with ionic currents driven through the vertebrate embryo by the transepithelial potential of its surface ectoderm. Using a non-invasive vibrating electrode for the measurement of ionic current, we have provided measurements of currents traversing amphibian embryos, and a preliminary report of the internal, extracellular voltage gradient under the neural plate which polarizes the embryo in the rostral/caudal axis (Metcalf et al. [1994] J. Exp. Zool. 268:307-322). Here we complete a description of this gradient in electrical potential (ca. 10 mV/mm, caudally negative), describe a simultaneous gradient organized in the medial/lateral axis (ca. 5-18 mV/mm, negative at the margins of the neural folds), and describe their appearance and disappearance during ontogeny of the axolotl embryo. Both voltage gradients are not expressed until neurulation, and disappear at its climax. This appearance and disappearance correlates with the shunting of current out of the lateral margins of the neural folds in rostral regions of the embryo beginning at stage 15, and is not associated with a more substantial current leak from the blastopore which appears at gastrulation. A steady blastopore current is still present after neural tube formation when intra-embryonic electric fields have been extinguished. We discuss the direct experimental tests supporting the hypothesis that these extracellular electric fields both polarize the early vertebrate embryo and serve as cues for morphogenesis and pattern.

Expression pattern of the murine lim class homeobox gene lhx3 in subsets of neural and neuroendocrine tissues

Abstract: Murine Lhx3 cDNA isolated from the mouse pituitary cDNA library encodes a LIM-type homeodomain protein that contains two tandemly repeated LIM domains and the homeodomain. The identities of predicted amino acid sequences between the mouse of Lhx3 and Xenopus Xlim-3 genes are 80, 95, and 97% in the LIM domains 1 and 2, and the homeodomain, respectively, and 84% in the entire protein. 5'-RACE procedures and genomic cloning revealed that two distinct N-terminal sequences arise from two different exons 1a and 1b. Exon 1a encodes a sequence similar to that of Xlim-3, whereas exon 1b encodes a different N-terminus. It is likely that there are two transcription initiation sites in the Lhx3 gene. The Lhx3 transcripts were detected by whole mount in situ hybridization as early as day E9.5 post coitum in Rathke's pouch and the closing neural tube. During subsequent development, Lhx3 expression was observed in the anterior and intermediate but not in the posterior lobes of the pituitary, and in the ventral hindbrain and spinal cord. Northern blot analysis of adult tissues showed that Lhx3 mRNA persists in the pituitary. The expression pattern of Lhx3 is well conserved between Xenopus and mouse, underscoring the functional importance of this gene as a regulator of development. A number of established cell lines of pituitary origin express Lhx3 and therefore constitute a useful tool for further study of Lhx3 gene function.

MyoD protein accumulates in satellite cells and is neurally regulated in regenerating myotubes and skeletal muscle fibers

Abstract: MyoD belongs to a family of helix-loop-helix proteins that control myogenic differentiation. Transfection of various non-myogenic cell lines with MyoD transforms them into myogenic cells. In normal embryonic development MyoD is upregulated at the time when the hypaxial musculature begins to form, but its role in the function of adult muscle remains to be elucidated. In this study we examined the cellular locations of MyoD protein in normal and abnormal muscles to see whether the presence of MyoD protein is correlated with a particular cellular behaviour and to assess the usefulness of MyoD as a marker for satellite cells. Adult rats were anaesthetised and their tibialis anterior or soleus muscles either denervated, tenotomised, freeze lesioned, lesioned and denervated, or lesioned and tenotomised. At various intervals after the operations the rats were killed and their muscles removed, snap frozen, and sectioned with a cryostat along with muscles from unoperated neonatal and adult rats. The sections were processed for immunohistochemistry using a rabbit affinity-purified antibody to recombinant MyoD. MyoD proved to be an excellent marker for active satellite cells; satellite cells in neonatal and regenerating muscles contained high levels of MyoD protein. MyoD positive cells were not observed in the muscles of old adults, in which the satellite cells are fully quiescent. MyoD immunoreactivity was rapidly lost from satellite cell nuclei after they fused into myotubes and was not detected in either sub-synaptic or non-synaptic nuclei of mature fibers. Denervation, and to a lesser extent tenotomy, of lesioned muscles induced expression of MyoD in myotubal nuclei. Denervation of normal muscles also upregulated MyoD in muscle fiber nuclei, an effect which was maximal after 3 days. We conclude that MyoD protein is neurally regulated in both myotubes and muscle fibers.

Pattern of muscle fiber type formation in the pig

Abstract: The aim of this study was to analyze the temporal sequence of expression of the myosin isoforms in the populations of muscle fibers in the pig and to bring more information on the origin of the strikingly different pattern of fiber composition and distribution between the deep medial red (oxido-glycolytic) and superficial white (glycolytic) portions of semitendinosus (ST) muscle. Muscle samples were taken from 49-, 55-, 75-, 90-, 103-, and 113- (birth) day-old fetuses, from 6-, 11-, 21-, 35-, 50-, and 80-day-old piglets, and from a 3-year-old pig. Our results confirm the sequential formation of primary and secondary generation fibers. The use of immunohistochemistry and heterologous monoclonal antibodies (mAb) directed against specific myosin heavy chain (MHC) isoforms revealed a different pattern of gene expression between the two portions of the ST muscle for both generations of fibers. By 75 days of gestation (dg), primary myotubes from the deep medial portion stained positively for the anti-slow MHC mAb and negatively for the adult anti-fast MHC, whereas the opposite was observed in the superficial portion. Secondary fibers never expressed slow MHC until late gestation. Instead, they expressed an adult fast MHC isoform as soon as they formed in the deep medial portion and later on in the superficial portion. From late gestation to the first 3 postnatal weeks, slow MHC began to be expressed in a subpopulation of secondary fibers. These fibers were in the direct vicinity of primary myotubes in the deep medial portion, whereas their location could not be established in the superficial portion. The remaining secondary fibers matured to type IIA in the direct vicinity of these type I fibers and to type IIB at the periphery of the islets. In both portions of the muscle, a subpopulation of secondary fibers, the first ones to express slow MHC, also transitorily expressed a MHC that was identical or closely related to the alpha-cardiac MHC during the early postnatal period. A third generation of small diameter fibers was observed shortly after birth and reacted with the anti-fetal MHC mAb; their destiny remains to be established.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular analysis of smooth muscle development in the mouse

Abstract: Little is currently known regarding the ontogeny of smooth muscle tissues during normal mammalian development. The alpha-smooth muscle and gamma-smooth muscle isoactins have been shown to be excellent molecular markers of smooth muscle cell phenotype. This study characterizes both the temporal and spatial patterns of alpha-smooth muscle and gamma-smooth muscle isoactin expression in the developing mouse. In situ analysis was performed on serial sections of whole mouse embryos on embryonic day 9, 11, 13, 15, and 17 using alpha-smooth muscle and gamma-smooth muscle isoactin-specific riboprobes. Distinct temporal and spatial patterns of alpha-smooth muscle and gamma-smooth muscle isoactin gene expression were observed in the developing gastrointestinal tract, urogenital tract, respiratory tract, and vascular system. Independent expression of the alpha-smooth muscle isoactin was observed during the early stages of skeletal, cardiac, and smooth muscle myogenesis as well as in a novel subset of distinct organs including the postnatal component of the hindgut, allantois, and primitive placenta. The results of this study indicate that distinct cellular phenotypes are involved in smooth muscle myogenesis and suggest that organ-specific mechanisms might exist for the initiation of smooth muscle development in vivo. In addition, the pattern of independent alpha-smooth muscle isoactin expression observed in this study provides novel information regarding the early stages of hindgut and placental development, and suggests that a common functional phenotype may be associated with the early stages of skeletal, cardiac, and smooth muscle myogenesis.

Chondrocyte apoptosis in endochondral ossification of chick sterna.

Abstract: In the process of endochondral ossification, chondrocytes progress through a series of maturational changes, including division and hypertrophy, that culminate in chondrocyte loss and cartilage resorption. From an investigation of morphology, DNA fragmentation, and collagen synthesis in the developing chick sterna we have characterized chondrocytes death in this process. Light microscopy of resorbing sterna demonstrated chondrocyte condensation at the interface with the invading vasculature and electron microscopy demonstrated a range of chondrocyte morphologies, including retraction from the pericellular matrix, cytoplasmic and nuclear condensation, and vesiculation suggestive of sequential changes characteristic of apoptosis. Isolation and end-labeling of DNA from chick primary ossification centers demonstrated fragmentation to nucleosome sized units, only in primary ossification centers exhibiting active resorption, and in situ detection of DNA fragmentation showed a restriction to chondrocytes at the interface with invading blood. We conclude that terminal differentiation of chondrocytes results in death by an apoptotic process prior to resorption of the tissue and invasion by blood vessels. The extent of DNA fragmentation correlated closely with the proportion of cells displaying a condensed phenotype in contralateral primary ossification centers and peaked at an early stage of resorption, suggesting that chondrocyte apoptosis may be an initiating event in tissue resorption and vascular invasion. Comparison of DNA fragmentation with expression of the hypertrophic chondrocyte phenotype, as indicated by type X collagen synthesis, suggested that DNA fragmentation was a late event in the process of chondrocyte hypertrophy and probably corresponded with chondrocyte condensation. ©1995 Wiley-Liss, Inc.

Cell and matrix specialisations of rhombomere boundaries

Abstract: Hindbrain segments, rhombomeres, define distinct cellular and molecular domains which furnish the ground plan for important aspects of neural and cranial development. In this study, further evidence is presented that the interfaces between rhombomeres, rhombomere boundaries, contain both cells and extracellular matrix with specialised characteristics. Cells at rhombomere boundaries show temporally and spatially distinct expression patterns of developmentally important genes. Towards the end of the developmental period when rhombomeres are present, a fan-shaped array of cells at rhombomere boundaries, that constitute the ventricular ridge, shows decreased expression of two genes (Hoxb-1 and Krox-20), which earlier in development were expressed in all cells of specific rhombomeres. In contrast, these boundary cells show increased expression of another gene, Pax-6, which earlier in development has a rhombomere-specific expression pattern. A specialised identity for boundary cells is further suggested by increased labelling with an anti-vimentin antibody at rhombomere boundaries, indicating that at least some boundary cells are radial glia or glial precursors. In addition to distinct cellular properties, the extracellular domain at rhombomere boundaries is also specialised. Chondroitin sulphate proteoglycan (CSPG) immunoreactivity is increased and, as revealed by immuno-electron microscopy, localised to extracellular spaces. CSPG is also enriched in boundaries regenerated after ablation, or boundaries generated ectopically by rhombomere transplantation. We propose that rhombomere boundaries form their characteristic morphology at the interface between groups of cells with differing molecular characteristics, representing different cell states. A specialised band of cells then develops at the interface. Both the boundary cells and extracellur matrix have characteristics which could be important in later events of neural development such as axon guidance and cell migration.

92-kDa Type IV Collagenase and TIMP-3, But Not 72-kDa Type IV Collagenase or TIMP-1 or TIMP-2, Are Highly Expressed During Mouse Embryo Implantation

Abstract: Expression of 72 kDa and 92 kDa type IV collagenases and the metalloproteinase inhibitors TIMPs 1, 2, and 3 was studied by in situ hybridization in implanting mouse embryos of days 5.5 to 7.5. The 92 kDa type IV collagenase was strongly expressed in invading trophoblasts, signals above background not being observed in the embryonic proper or placental tissue. In contrast, signals above background were not seen for the 72 kDa enzyme in any cells of the implantation region, including trophoblasts and stromal cells of the decidual tissue. Only cells in the mucosal stroma outside the decidual region displayed some expression. TIMP-3 was intensily expressed in maternal cells in the area surrounding the invading embryonic tissue. No expression was observed for TIMP-1 or TIMP-2 in the embryo proper, trophoblasts, or the area of the uterine decidual reaction. Weak signals appeared for TIMP-1 only in the circular layer of myometrial smooth muscle and in some uterine stroma cells distant from the site of embryo implantation. The results suggest a central role for 92 kDa type IV collagenase and TIMP-3 in the extracellular proteolysis associated with implantation of the early embryo.

Synthesis and secretion of matrix-degrading metalloproteases by human skeletal muscle satellite cells.

Abstract: The expression of matrix-degrading metalloproteases (MMPs) by human skeletal muscle satellite cells was investigated by zymography of cell culture media and by Northern blot analysis of mRNA prepared from satellite cells. Zymography in gelatin substrate gels revealed that satellite cells constitutively synthesize and secrete 72 kDa gelatinase (MMP-2). In addition, treatment of satellite cell cultures with phorbol ester resulted in an induction of 92 kDa gelatinase (MMP-9) activity. On casein substrate gels, little or no proteolytic activity was detectable in control or phorbol ester treated satellite cell cultures, suggesting that compared to fibroblasts, satellite cells secrete little or no interstitial collagenase (MMP-1) or stromelysin (MMP-3) activity. Northern blotting, however, revealed that there is detectable expression of mRNA transcripts encoding MMP-1 in satellite cell cultures, and that increased accumulation of MMP-1 mRNA transcripts occurs upon treatment of these cells with phorbol ester. In contrast, no constitutive, or induced expression of transcripts encoding MMP-3 was detectable in satellite cells. These findings show that satellite cells can synthesize and secrete selected members of the MMP family and suggest that skeletal muscle cells may participate directly in remodelling of the extracellular matrix during myogenesis and the regeneration of skeletal muscle.

Differential expression of myogenic regulatory genes and Msx‐1 during dedifferentiation and redifferentiation of regenerating amphibian limbs

Abstract: An amputated limb of an adult urodele amphibian is capable of undergoing regeneration. The new structures form from an undifferentiated mass of cells called the regenerative blastema. The cells of the blastema are believed to derive from differentiated tissues of the adult limb. However, the exact source of these cells and the process by which they undergo dedifferentiation are poorly understood. In order to elucidate the molecular and cellular basis for dedifferentiation we isolated a number of genes which are potential regulators of the process. These include Msx-1, which is believed to support the undifferentiated and proliferative state of cells in the embryonic limb bud; and two members of the myogenic regulatory gene family, MRF-4 and Myf-5, which are expressed in differentiated muscle and regulate muscle-specific gene activity. As anticipated, we find that Msx-1 is strongly up-regulated during the initiation of regeneration. It remains expressed throughout regeneration but is not found in the fully regenerated limb. The myogenic gene MRF-4 has the reverse expression pattern. It is expressed in adult limb muscle, is rapidly shut off in early regenerative blastemas, and is only reexpressed at the completion of regeneration. These kinetics are paralleled by those of a muscle-specific Myosin gene. In contrast Myf-5, a second member of the myogenic gene family, continues to be expressed throughout the regenerative process. Thus, MRF-4 and Myf-5 are likely to play distinct roles during regeneration. MRF-4 may directly regulate muscle phenotype and as such its repression may be a key event in dedifferentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of genes encoding bone morphogenetic proteins and sonic hedgehog in talpid (ta3) limb buds: Their relationships in the signalling cascade involved in limb patterning

Abstract: The chicken mutant talpid3 (ta3) has polydactylous limbs with up to 7-8 morphologically similar digits. This lack of antero-posterior polarity in digit pattern is correlated with symmetrical expression of genes of the HoxD complex. We determined the distribution of polarizing activity in limb buds of the chick mutant ta3 by assessing the ability of mesenchyme from various positions along the antero-posterior axis to induce digit duplications when grafted anteriorly into a normal limb. Cells with highest polarizing activity were found at the posterior margin of the wing as in the polarizing region of normal limb buds. However, in contrast to normal limb buds, ta3 anterior mesenchyme also had low polarizing activity. Application of retinoic acid or a polarizing region graft to the anterior of ta3 limb buds changed digit morphology but did not induce digit duplications or digits with any characteristic a-p pattern. To determine which genes are associated with polarizing activity and which are associated with patterning of the digits, we examined expression of the genes Sonic hedgehog (shh), Bmp-2, and Bmp-7, whose expression is normally confined to the posterior margin of the early wing bud and is associated with the polarizing region. In addition, we determined the distribution of Fgf-4 transcripts which in normal limb buds are restricted to the posterior part of the apical ectodermal ridge. In ta3 limb buds, shh expression is restricted to the posterior limb mesenchyme, which has high polarizing activity, but is not expressed in regions which have low polarizing activity. In contrast, Bmp-2 and Bmp-7 are expressed uniformly along the a-p axis. Fgf-4 transcripts are present throughout the apical ectodermal ridge in ta3 limb buds. In the ta3 mutant, there is both an abnormal distribution of signalling activity and response to polarizing signals. In addition, the dissociation between the expression of shh and Bmps suggests distinct roles for the encoded molecules in signalling and response in a-p patterning of limb buds.

Gsh-1: a novel murine homeobox gene expressed in the central nervous system

Abstract: We report the characterization of Gsh-1, a novel murine homeobox gene. Northern blot analysis revealed a transcript of approximately 2 kb in size present at embryonic days 10.5, 11.5, and 12.5 of development. The cDNA sequence encoded a proline rich motif, a polyalanine tract, and a homeodomain with strong homology to those encoded by the clustered Hox genes. The Gsh-1 expression pattern was determined for days E8.5 to E13.5 by whole mount and serial section in situ hybridizations. Gsh-1 transcription was restricted to the central nervous system. Expression is present in the neural tube and hindbrain as two continuous, bilaterally symmetrical stripes within neural epithelial tissue. In the mesencephalon, expression is seen as a band across the most anterior portion. There is also diencephalon expression in the anlagen of the thalamus and the hypothalamus as well as in the optic stalk, optic recess, and the ganglionic eminence. Moreover, through the use of fusion proteins containing the Gsh-1 homeodomain, we have determined the consensus DNA ninding site of the Gsh-1 homeoprotein to be GCT/CA/CATTAG/A. ©1995 Wiley-Liss, Inc.

Number of adrenergic and islet-1 immunoreactive cells is increased in avian trunk neural crest cultures in the presence of human recombinant osteogenic protein-1.

Abstract: OP-1, also known as BMP-7, is a member of the TGF-β superfamily of proteins and was originally identified on the basis of its ability to induce new bone formation in vivo. OP-1 mRNA is found in the developing kidney and adrenal gland as well as in some brain regions (Ozkaynak et al. [1991] Biochem. Biophys. Res. Commun. 179:116–123). We have tested the effect of recombinant human OP-1 on quail trunk neural crest cultures. The number of catechol-amine-positive cells which developed after 7 days in vitro in the presence of OP-1 was increased in a dose-dependent manner, with a greater than 100-fold maximal stimulation observed. The increase in the number of catecholamine-positive cells in the presence of OP-1 was paralleled by an increase in the number of tyrosine hydroxylase (TH)-positive cells. In contrast, total and melanocyte cell number were unaffected by the presence of OP-1. The number of Islet-1-immunoreactive cells was also increased by OP-1, but to only about half the value seen for TH. Double label experiments revealed these Islet-1-positive cells were a subset of the TH-positive cells. Inhibitors of DNA synthesis prevented the OP-1-mediated increase in adrenergic cell number, indicating that OP-1 does not act on a postmitotic cell population. However, labeling studies with bromodeoxyuridine indicated that OP-1 did not increase the proportion of the cell population engaged in DNA synthesis. Thus, the OP-1-mediated increase in adrenergic cell number most likely occurs as a result of the enhanced survival of a subpopulation of adrenergic precursors or an increase in their probability of adrenergic differentiation, but not by increasing the mitotic rate of adrenergic precursors or adrenergic cells themselves. In contrast to OP-1, TGF-β1 decreased adrenergic cell number. When OP-1 and TGF-β1 were added simultaneously, TGF-β1 antagonized the OP-1-mediated increase in adrenergic cell number in a dose-dependent manner. �1995 Wiley-Liss, Inc.

Cell cycle of globose basal cells in rat olfactory epithelium

Abstract: The olfactory epithelium of adult mammals has the unique property of generating olfactory sensory neurons throughout life. Cells of the basal compartment, which include horizontal and globose basal cells, are responsible for the ongoing process of neurogenesis in this system. We report here that the globose basal cells in olfactory epithelium of rats, as in mice, are the predominant type of proliferating cell, and account for 97.6% of the actively dividing cells in the basal compartment of the normal epithelium. Globose basal cells have not been fully characterized in terms of their proliferative properties, and the dynamic aspects of neurogenesis are not well understood. As a consequence, it is uncertain whether cell kinetic properties are under any regulation that could affect the rate of neurogenesis. To address this gap in our knowledge, we have determined the duration of both the synthesis phase (S-phase) and the full cell cycle of globose basal cells in adult rats. The duration of the S-phase was found to be 9 hr in experiments utilizing sequential injections of either IdU followed by BrdU or 3H-thy followed by BrdU. The duration of the cell cycle was determined by varying the time interval between the injections of 3H-thy and BrdU and tracking the set of cells that exit S shortly after the first injection. With this paradigm, the interval required for these cells to traverse G2, M, G1, and a second S-phase, is equivalent to the duration of one mitotic cycle and equals 17 hr. These observations serve as the foundation to assess whether the cell cycle duration is subject to regulation in response to experimental injury, and whether such regulation is partly responsible for changes in the rate of neurogenesis in such settings.