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Self-organization of vertebrate mesoderm based on simple boundary conditions.

Embryonic development requires cell movements whose coordination is robust and reproducible. A dramatic example is the primary body axis of vertebrates: despite perturbation, cells in prospective axial tissue coordinate their movements to make an elongated body axis. The spatial cues coordinating these movements are not known. We show here that cells deprived of preexisting spatial cues by physical dissociation and reaggregation nonetheless organize themselves into an axis. Activin-induced cells that are reaggregated into a flat disc initially round up into a ball before elongating perpendicular to the disc. Manipulations of the geometry of the disc and immunofluorescence micrography reveal that the edge of the disc provides a circumferential alignment zone. This finding indicates that physical boundaries provide alignment cues and that circumferential "hoop stress" drives the axial extrusion in a manner resembling late-involuting mesoderm of Xenopus and archenteron elongation in other deuterostome species such as sea urchins. Thus, a population of cells finds its own midline based on the form of the population's boundaries using an edge-aligning mechanism. This process provides a remarkably simple organizing principle that contributes to the reliability of embryonic development as a whole.

Activins↗

Cell fate specification along the anterior-posterior axis of the intermediate mesoderm.

The vertebrate intermediate mesoderm (IM) is highly patterned along the anterior-posterior (A-P) axis. In the chick embryo, the kidney tissue, which is a derivative of the IM, is generated only from IM located posterior to the sixth somite axial level, which also marks the border between cranial and trunk segments. The cellular and molecular mechanisms that govern the formation of the anterior border of the kidney morphogenetic field are currently unknown. In this study, we asked whether specific A-P patterning information is conveyed by the movement of cells through the primitive streak (PS) at different time points that consequently affects the expression of kidney genes, or by the environment that these cells encounter during their migration to the IM. In this study, we show that kidney-inductive signals are present along the whole axis, including anterior non-kidney-generating regions. These inductive signals are generated by tissues that are located medial to the anterior IM. We also demonstrate that cells that migrate through the PS of early embryonic stages (Hamburger and Hamilton stage 3-4 and earlier), which will give rise to anterior nonkidney IM, are competent to respond to these inductive factors. This prospective anterior IM tissue loses its competence to respond to kidney inducing signals during its migration from the PS to its final location in the anterior IM. We present here a model in which changes in cell competence determine the formation of the anterior border of kidney gene expression and discuss the possible evolutionary implications of this developmental mechanism.

Animals↗

Establishment of mesodermal gene expression patterns in early Xenopus embryos: the role of repression.

In Xenopus, activin-like signals are able to induce and pattern mesoderm in a concentration-dependent manner. Previous experiments demonstrated that discrete gene expression patterns can be formed in animal cap explants as a response to graded activin signals. We analyzed the spatiotemporal appearance of goosecoid (gsc), chordin (chd), and Xbrachyury (Xbra) mRNAs in whole Xenopus embryos ectopically expressing activin or BVg1. To discriminate between direct transcriptional regulation and indirect, protein synthesis-dependent effects of ectopic signals, we combined overexpression studies and cycloheximide treatment. Our experiments revealed long-range signaling of activin/BVg1, but the expression patterns of gsc, chd, and Xbra in response to activin/BVg1 indicated that repressors are essential to establish the proper expression of these genes. Analysis of endogenous gsc, chd, and Xbra transcript distribution in embryos treated with cycloheximide supported this concept. We, therefore, conclude that inhibition is fundamental during early embryonic patterning.

Animals↗

Zebrafish protocadherin 10 is involved in paraxial mesoderm development and somitogenesis.

Here, we present the first report of the molecular cloning of zebrafish protocadherin 10 (Pcdh10, OL-protocadherin) and describe its functional analyses in the development of segmental plate. Epitope-tagged Pcdh10 expressed in embryos was localized on cell peripheries of adjacent cells. In situ hybridization showed that pcdh10 was expressed in the paraxial mesoderm (PAM) and developing somites, and in the pineal body, the diencephalon, and the vicinity of otocysts. Expression in PAM increased in the last few presumptive somites, reached the maximum level in the latest segmenting somites, and decreased thereafter during somite maturation. These expression patterns suggested that Pcdh10 is involved in development of PAM and somites. This was confirmed by morpholino knockdown and dominant-negative inhibition of Pcdh10 in embryos, which disturbed movements of PAM cells and somite segmentation. Comparative studies showed that pcdh10 expression lasted up to approximately three times longer in maturing somites than that of paraxial protocadherin (pcdh8). They also indicated that the adaxial cells expressed pcdh8 but not pcdh10. We propose that Pcdh10 is involved in the morphogenic movements of PAM cells and somite segmentation and that differential adhesion of Pcdh8 and Pcdh10 plays a role in the morphogenic machinery of somites and adaxial cells.

Animals↗

Dact1 presomitic mesoderm expression oscillates in phase with Axin2 in the somitogenesis clock of mice.

During segmentation (somitogenesis) in vertebrate embryos, somites form in a rostral-to-caudal sequence according to a species-specific rhythm called the somitogenesis clock. The expression of genes participating in somitogenesis oscillates in the presomitic mesoderm (PSM) in time with this clock. We previously reported that the Dact1 gene (aka Dpr1/Frd1/ThyEx3), which encodes a Dishevelled-binding intracellular regulator of Wnt signaling, is prominently expressed in the PSM as well as in a caudal-rostral gradient across the somites of mouse embryos. This observation led us to examine whether Dact1 expression oscillates in the PSM. We have found that Dact1 PSM expression does indeed oscillate in time with the somitogenesis clock. Consistent with its known signaling functions and with the "clock and wavefront" model of signal regulation during somitogenesis, the oscillation of Dact1 occurs in phase with the Wnt signaling component Axin2, and out of phase with the Notch signaling component Lfng.

Animals↗

Chordin affects pronephros development in Xenopus embryos by anteriorizing presomitic mesoderm.

Spemann's organizer emits signals that pattern the mesodermal germ layer during Xenopus embryogenesis. In a previous study, we demonstrated that FGFR1 activity within the organizer is required for the production of both the somitic muscle- and pronephros-patterning signals by the organizer and the expression of chordin, an organizer-specific secreted protein (Mitchell and Sheets [2001] Dev. Biol. 237:295-305). Studies from others in both chicken and Xenopus embryos provide compelling evidence that pronephros forms by means of secondary induction signals emitted from anterior somites (Seufert et al. [1999] Dev. Biol. 215:233-242; Mauch et al. [2000] Dev. Biol. 220:62-75). Here we provide several lines of evidence in support of the hypothesis that chordin influences pronephros development by directing the formation of anterior somites. Chordin mRNA was absent in ultraviolet (UV) -irradiated embryos lacking pronepheros (average DAI<2) but was always found in UV-irradiated embryos that retain pronepheros (average DAI>2). Furthermore, ectopic expression of chordin in embryos and in tissue explants leads to the formation of anterior somites and pronephros. In these experiments, pronephros was only observed in association with muscle. Chordin diverted somatic muscle cells to more anterior positions within the somite file in chordin-induced secondary trunks and induced the expression of the anterior myogenic gene myf5. Finally, depletion of chordin mRNA with DEED antisense oligonucleotides substantially reduced somitic muscle and pronephric tubule and duct formation in whole embryos. These data and previous studies on ectoderm and endoderm (Sasai et al. [1995] Nature 377:757) support the idea that chordin functions as an anteriorizing signal in patterning the germ layers during vertebrate embryogenesis. Our data support the hypothesis that chordin directs the formation of anterior somites that in turn are necessary for pronephros development.

Animals↗

Synergistic growth stimulation of corneal fibroblasts by components of mesodermal growth factor from murine submaxillary glands.

Recycled mesodermal growth factor (R-MGF) is a pool of proteins of 26,000 molecular weight obtained by recycling gel chromatography of male murine submaxillary gland extracts. R-MGF strikingly accelerates corneal stromal wound healing in vivo, fibroblast growth and migration in cultured corneal buttons and is showing here to stimulate stromal fibroblast growth and division in tissue culture. Chromatographic fractionation of R-MGF has yielded several components, none of which has a greater biological potency than the parent R-MGF. In contrast, two components, MGF-I and -II, when recombined synergistically stimulate fibroblast response in tissue culture and organ culture in excess of those obtained with the parent R-MGF. Three MGF components (I, III, and IV) have been purified and are inactive at 10-15 micrograms/ml in organ culture but potently stimulate fibroblast responses when combined in pairs containing 7.5 micrograms of each component. The striking synergism in organ culture suggests that the stimulation of wound healing by R-MGF in vivo may also reflect synergistic action of more than one R-MGF component. Procedures for isolating gram quantities of R-MGF and for the purification of different R-MGF components by ion exchange chromatography are detailed.

Animals↗

Gastrulation and mesoderm morphogenesis in the white sturgeon.

This study presents a detailed description of gastrulation in the white sturgeon, Acipenser transmontanus, using scanning electron microscopy, histology, and time-lapse filming and video microscopy. This morphological analysis describes the similarity of gastrula structure in the sturgeon and the amphibian Xenopus laevis, and suggests that the species share many developmental mechanisms. It also identifies important differences, such as the equatorial dorsal lip in sturgeon, and provides a basis for interpreting experiments that test the effect of these differences on gastrulation. The onset of gastrulation in the sturgeon is marked by the appearance of a blastoporal equatorial pigment line that forms as the apices of bottle cells contract and concentrate surface pigment granules. Bottle cell formation at the blastopore lip and involution of surface material through the blastopore are strikingly similar to the equivalent processes in amphibian embryos. As gastrulation continues, a distinct cleft of Brachet forms between pre-involution and post-involution material. Following involution, the prospective axial mesoderm located on the dorsal surface of the late blastula (Ballard and Ginsburg: J. Exp. Zool., 213:69-103, 1980) ingresses from a central zone in the posterior archenteron roof surface in a process that is unlike any in Xenopus, but resembles events in other amphibians (Purcell, 1992; Smith: Dev. Biol., 98:250-254, 1983; King: Biol. Bull., 4:287-300, 1903). The detailed comparison of similarities and differences in gastrulation in different vertebrate lineages yields insights into the function and versatility of common developmental mechanisms.

Animals↗

Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis.

Bone marrow stromal stem cells (MSCs) normally differentiate into mesenchymal derivatives but recently have also been converted into neurons, classical ectodermal cells. To begin defining underlying mechanisms, we extended our characterization of MSCs and the differentiated neurons. In addition to expected mesodermal mRNAs, populations and clonal lines of MSCs expressed germinal, endodermal, and ectodermal genes. Thus, the MSCs are apparently "multidifferentiated" in addition to being multipotent. Conversely, the differentiating neurons derived from populations and clonal lines of MSCs expressed the specific markers beta-III tubulin, tau, neurofilament-M, TOAD-64, and synaptophysin de novo. The transmitter enzymes tyrosine hydroxylase and choline acetyltransferase were localized to neuronal subpopulations. Our observations suggest that MSCs are already multidifferentiated and that neural differentiation comprises quantitative modulation of gene expression rather than simple on-off switching of neural-specific genes.

Age Factors↗

XLPOU-60, a Xenopus POU-domain mRNA, is oocyte-specific from very early stages of oogenesis, and localised to presumptive mesoderm and ectoderm in the blastula.

POU-domain proteins are a large family of transcriptional regulatory proteins, related to the homeodomain proteins, many of which are implicated in the control of gene expression during early development. We describe here the isolation of a cDNA encoding a Xenopus POU-domain protein, XLPOU-60. The predicted protein sequence of this cDNA is most closely related to the mouse germ line-specific transcription factor Oct-3/4. The XLPOU-60 gene is specifically expressed in oocytes of newly metamorphosed frogs, from the earliest stages at which transcription is known to occur. The mRNA is concentrated in the animal half of fully grown oocytes and is inherited maternally by the embryo, where it remains localised to animal cap and marginal zone cells of the blastula. Transcripts decline abruptly to a low level during gastrulation, but remain detectable throughout larval stages. However, unlike Oct-3/4, the transcript is not detectable in primordial germ cells, and XLPOU-60 is therefore probably not the functional homologue of the murine gene. We suggest that XLPOU-60 is one of the earliest genes to be transcribed in oocyte development, and that the XLPOU-60 protein may therefore be involved in initiating oocyte-specific patterns of transcription. Localisation of the transcript in the embryo may indicate that XLPOU-60 is also required for the initiation of mesoderm- and ectoderm-specific patterns of transcription in the embryo.

Amino Acid Sequence↗

Inhibition of precardiac mesoderm cell proliferation by antisense oligodeoxynucleotide complementary to fibroblast growth factor-2 (FGF-2).

This laboratory recently reported the occurrence of concentrated deposits of fibroblast growth factor-2 (FGF-2; basic FGF)-like proteins in the sarcoplasm of embryonic cardiac myocytes from the earliest stage of avian heart development (M. H. Parlow, D. L. Bolender, N. P. Kokan-Moore, and J. Lough, Dev. Biol. 146, 139, 1991). To determine the role, if any, of FGF-2 during embryonic cardiogenesis, the proliferative and functional effects of treating cultured anterior lateral plate mesoderm from Hamburger-Hamilton stage 6 embryos with antisense oligodeoxynucleotide (ODN) complementary to FGF-2 mRNA were determined. Within 2 days of culture in defined medium, the explanted monolayer normally differentiates into a multilayer of proliferative cells that express sarcomeric alpha-actin and exhibit rhythmic contractions. The inclusion of 25 microM ODN that is complementary to the second exon of chicken FGF-2 mRNA caused a 50% inhibition in these cells' proliferative ability as judged by incorporation of 5'-bromodeoxyuridine; contractility was similarly inhibited. These effects were prevented by including recombinant human FGF-2 protein in the medium. Treatment with sense ODN did not cause inhibition. Inhibition of FGF-2 protein synthesis in the explanted tissue by antisense ODN was verified by immunoprecipitation analysis. These results point to a critical role for FGF-2 in the autocrine regulation of proliferation, and perhaps differentiative function, of embryonic cardiac myocytes.

Amino Acid Sequence↗

Cloning and expression analysis of a novel mesodermally expressed cadherin.

Cadherins are calcium-dependent cell adhesion molecules which show developmental and tissue-specific expression. Here we report the cloning of a mouse cadherin which is predominantly expressed in tissues of mesodermal origin. In contrast to other cadherins, cadherin-11 expression is largely restricted to mesenchymal tissues surrounding various organs but is not found in epithelia. Sequence analysis suggests that this cadherin is the mouse homologue of the previously reported human cadherin-11 and is a member of a cadherin subfamily, which is evolutionarily distinct from other cadherin subfamilies identified so far.

Amino Acid Sequence↗

Essential and neural transcripts from the Drosophila shaking-B locus are differentially expressed in the embryonic mesoderm and pupal nervous system.

The shaking-B gene of Drosophila encodes two functions: one specifically neural and the other required for viability. Flies carrying neural mutations show a range of defects, the best characterized of which is a disruption of some synapses in the giant fibre system, while mutations in the essential function cause animals to die as first instar larvae. We have characterised an essential transcript from this locus and show that mutant lesions underlying two lethal shaking-B alleles map to its coding sequence. We also propose a new model for the topologies of Shaking-B proteins and their relatives. Essential shaking-B transcripts are found in embryonic mesodermal derivatives, while during metamorphosis both essential and neural transcripts are dynamically expressed in the pupal nervous system. Although the expression patterns of these transcripts overlap in many cells, only the neural form is expressed in the giant fibre cell bodies and the lamina and medulla of the optic lobes. This observation correlates with the phenotypes of mutations which disrupt the coding region of this neural transcript. On the basis of the expression patterns of shaking-B transcripts and the phenotypes conferred by mutations of shaking-B and homologous genes, we suggest that Shaking-B proteins and their homologues may be involved in the organisation of cellular membranes.

Alleles↗

Virally mediated misexpression of Hoxc-6 in the cervical mesoderm results in spinal nerve truncations.

Members of the Hox gene family appear to regulate anterior-posterior (A-P) regionalization in embryos. Genetic manipulation of numerous Hox genes in the developing trunk region of vertebrates results in changes in the morphology of individual vertebrae. We have used virally mediated, targeted misexpression to ectopically express the Hoxc-6 protein in chick embryos. Hoxc-6 has an anterior border of expression at the cervical-thoracic transition in tetrapods. Misexpression of this gene in the cervical mesoderm of chick embryos results in dramatic truncations of the ventral rami of cervical spinal nerves in the infected region. These data point to a role for Hoxc-6 in axon guidance, and suggest that in addition to regulating proliferative rates, Hox gene expression provides positional information utilized in producing domain-specific extracellular signals.

Animals↗

The secretory protein Sec8 is required for paraxial mesoderm formation in the mouse.

The sec8 gene, isolated in a gene trap screen in embryonic stem cells, is required for paraxial mesoderm formation in the mouse. Homozygous sec8 mutant embryos initiate gastrulation but are unable to progress beyond the primitive streak stage and die shortly afterward. The genomic locus and cDNA of the sec8 gene have been cloned. An open reading frame in the cDNA encodes a 971-amino-acid leucine-rich protein, similar to rat rSec8. A description of the mutant phenotype and the cloning of the gene is presented here and the results are considered in light of the possibility that the Sec8 protein is involved in secretion.

Amino Acid Sequence↗

Developmental fates of larval tissues after metamorphosis in ascidian Halocynthia roretzi. I. Origin of mesodermal tissues of the juvenile.

Cell lineages during ascidian embryogenesis are invariant. In a previous study, the developmental fate of each blastomere during embryogenesis of the ascidian Halocynthia roretzi was analyzed in detail by intracellular injection of a tracer molecule, horseradish peroxidase (Nishida, H., Dev. Biol. 121, 526-541, 1987). In the present study, the developmental fates of various larval mesodermal tissues after metamorphosis in H. roretzi were traced by labeling each precursor blastomere of larval tissues. Mesenchyme cells of larvae gave rise to tonic cells of juveniles. Trunk ventral cells gave rise to body-wall (atrial siphon and latitudinal mantle) muscle, heart, and pericardium of juvenile. Trunk lateral cells gave rise to blood (coelomic) cells as well as body-wall (oral siphon and longitudinal mantle) muscle. Larval muscle and notochord cells did not contribute to juvenile tissues. It was revealed that it is predictable to which juvenile tissue a blastomere of the 110-cell embryo will give rise. Therefore, in ascidian development, developmental fates after metamorphosis were almost invariant with regard to cell type of descendants.

Animals↗

Lineage-specific regulation of the Ciona snail gene in the embryonic mesoderm and neuroectoderm.

The snail gene encodes a highly conserved, zinc-finger transcription factor that has been implicated in the specification of mesodermal and neuronal tissues in a variety of organisms. In the ascidian, Ciona intestinalis, snail (Ci-sna) is expressed at the 32-cell stage in derivatives of the B4.1 blastomere, including B6.2, B6.4, and B7.5, which give rise to the primary-lineage tail muscles of the tadpole. At later stages, Ci-sna is expressed in the lineages that will form the secondary tail muscle, the lateral ependymal cells of the spinal cord, and the dorsal cells of the cerebral vesicle. A minimal, 504-bp cis-regulatory sequence from the Ci-sna promoter region, the B4.1 enhancer, is shown to direct the expression of heterologous promoters in primary-lineage muscles. Furthermore, evidence is presented that cis-regulatory elements necessary for expression in both the secondary muscle and neuronal lineages are separate from the B4.1 enhancer. We discuss the possibility that the classical muscle determinant present in ascidian eggs may correspond to bHLH activators, which bind to specific E-box sequences contained in the B4.1 enhancer.

Animals↗

BMP1-related metalloproteinases promote the development of ventral mesoderm in early Xenopus embryos.

Bone morphogenetic protein 1 (BMP1) is a metalloproteinase closely related to Drosophila Tolloid (Tld). Tld regulates dorsoventral patterning in early Drosophila embryos by enhancing the activity of Dpp, a member of the TGF-beta family most closely related to BMP2 and BMP4. In Xenopus BMP4 appears to play an essential role in dorsoventral patterning, promoting the development of ventral fates during gastrula stages. To determine if BMP1 has a role in regulating the activity of BMP4, we have isolated cDNAs for Xenopus BMP1 and a novel closely related gene that we have called xolloid (xld). Whereas xbmp1 is uniformly expressed at all stages tested, the initial uniform expression of xld becomes localized to two posterior ectodermal patches flanking the neural plate and later to the inner ectoderm of the developing tailbud. xld is also expressed in dorsal regions of the brain during tailbud stages and is especially abundant in the ventricular layer of the dorsal hindbrain caudal to the otic vesicle. Overexpression of either gene inhibits the development of dorsoanterior structures in whole embryos and ventralizes activin-induced dorsal mesoderm in animal caps. Since ventralization of activin-induced animal caps can be blocked by coinjecting a dominant-inhibitory receptor for BMP2 and BMP4, we suggest a role for BMP1 and Xld in regulating the ventralizing activity of these molecules.

Activins↗