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Cytoplasmic and cortical determinants interact to specify ectoderm and mesoderm in the leech embryo.

In leech embryos, segmental ectoderm and mesoderm are produced by a pair of sister cells located near the animal and vegetal poles, respectively. We have investigated the mechanism that localizes ectodermal and mesodermal fates along the animal-vegetal axis. The results of cleavage arrest and cell ablation experiments suggest that the full range of normal cell interactions are not required for this process. However, when the animal and vegetal hemispheres are separated by re-orientation of the first cleavage plane from meridional to equatorial, the ectodermal fate co-segregates with the animal hemisphere and the mesodermal fate with the vegetal hemisphere. Two pools of yolk-deficient cytoplasm, called teloplasm, are located at the animal and vegetal poles of the zygote, but separation of the animal and vegetal teloplasms is not sufficient for the segregation of ectodermal and mesodermal fates. Rather, complete segregation of fates requires an equatorial cleavage orientation that separates not only the two teloplasms, but also the animal and vegetal cortical regions. This, in conjunction with previous findings, indicates that ectodermal determinants are localized to the cell cortex in the animal hemisphere of the zygote. We propose that these determinants segregate to the ectodermal precursor and interact with factors in teloplasm to transform the fate of this cell from a mesodermal ground state to ectoderm.

Animals↗

Apical ridge dependent and independent mesodermal domains of GHox-7 and GHox-8 expression in chick limb buds.

The homeobox-containing genes GHox-7 and GHox-8 have been proposed to play fundamental roles in limb development. The expression of GHox-8, by the apical ridge cells, and GHox-7, in the subridge mesoderm, suggests the involvement of these two genes in limb outgrowth and proximo-distal pattern formation. A straightforward way to test this is to remove the apical ridge. Here we report the relationship between the mesodermal expression of GHox-7 and GHox-8 and the apical ectodermal ridge in the chick limb bud. The data from ridge removal experiments indicate that there are at least two domains of GHox-7 expression in the apical limb bud mesoderm. The posterior subridge GHox-7 domain in the progress zone requires the influence of the apical ridge for continued expression, while the anterior GHox-7 domain continues expression after ridge removal. Posterior subridge mesoderm is exquisitely sensitive to the loss of the ridge in that GHox-7 expression by these cells is reduced in only two hours and undetectable by three hours after ridge removal. It would appear that one of the ways progress zone cells respond to the apical ridge signal is by expressing GHox-7. The loss of ridge influence whether by growth at the apex or by ridge removal is followed by an unusually rapid decline in detectable GHox-7 transcripts. Maintenance of GHox-8 expression by the anterior mesoderm appears to be independent of the presence of the apical ridge.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

A receptor protein tyrosine kinase implicated in the segmental patterning of the hindbrain and mesoderm.

Pattern formation in the hindbrain and paraxial mesoderm of vertebrates occurs by the formation of a series of repeated segments. These processes of segmentation appear different at the morphological level, since hindbrain segments, the rhombomeres, form by the subdivision of the neural epithelium into compartments, whereas the mesodermal somites form by the sequential aggregation of mesenchymal cells into epithelial balls. Previous studies have implicated genes encoding transcription factors in the development of hindbrain segments, but nothing is known of genes involved in the formation of somites. Cellular interactions and signal transduction must be an important aspect of hindbrain segmentation, so we have screened for tyrosine kinases expressed in rhombomere-restricted patterns in the developing mouse embryo. We have identified a receptor protein tyrosine kinase, Sek, that has high relative levels of expression in rhombomeres 3 and 5. This alternating pattern is established coincidentally, both spatially and temporally, with the expression of Krox-20, a zinc-finger gene expressed prior to the morphological formation of rhombomeres. In addition, Sek expression occurs in several other developing tissues, including a dynamic regulation in the developing forebrain, spinal cord, early mesoderm and anterior presomitic mesoderm (segmental plate). The latter expression occurs in two stripes that correlate with, and presage, the formation of somites. Sek expression initially occurs throughout the presumptive somite, then becomes restricted anteriorly, and finally is down-regulated as the definitive somite is formed. These data suggest that despite the morphological differences in the segmentation of the hindbrain and mesoderm, Sek is involved in the segmental patterning of both of these tissues.

Animals↗

The 412 retrotransposon and the development of gonadal mesoderm in Drosophila.

We have shown that the expression of the 412 retrotransposon provides a useful early marker for the development of the gonadal mesoderm in Drosophila embryos. 412 is initially expressed in a set of parasegmentally repeated stripes from parasegments (PS) 2-14 in the mesoderm at the extended germ band stage. During germ band retraction the bulk of 412 expression declines except in dorsolateral clusters of cells in PS10, 11 and 12, where high levels of 412 expression remain. These mesodermal cell clusters are associated with germ cells and subsequently they coalesce, rounding up to form the gonads. The gonadal mesoderm thus appears to originate specifically from three abdominal parasegments, PS10, 11 and 12. We show that the maintenance of high levels of 412 expression in gonadal mesoderm is not induced by contact with germ cells, but rather depends on genetic control by the homeotic genes abdominal-A and Abdominal-B.

Animals↗

Patterns of cell motility in the organizer and dorsal mesoderm of Xenopus laevis.

In a companion paper (Shih, J. and Keller, R. (1992) Development 116, 901-914), we described a sequence of cell behaviors, called mediolateral intercalation behavior (MIB), that produces mediolateral cell intercalation, the process that drives convergence and extension of the axial and paraxial mesoderm of Xenopus. In this paper, we describe the pattern of expression of MIB in the mesoderm during gastrulation, using video image processing and recording of cell behavior in 'shaved', open-faced explants of the marginal zone. At midgastrula stage (10.5), MIB begins at two dorsolateral sites in the prospective anterior mesoderm and progresses medially along two arcs that lengthen toward and meet at the midline to form a single arc of cells expressing MIB, called the vegetal alignment zone (VgAZ). The notochordal-somitic mesodermal boundary forms within the VgAZ at stage 11, and then progresses animally and laterally, along the prospective anterior-posterior axis, eventually bounding a trapezoidal area the shape of the fate-mapped notochord. Meanwhile, from its origin in the VgAZ, MIB spreads in the prospective posterior direction along the lateral boundaries of both the notochordal and somitic mesoderm. From there it spreads medially in both tissues. Subsequently, vacuolation of notochord cells, and segmentation and expression of a somite-specific marker repeat the progression of mediolateral intercalation behavior. Thus cells in the posterior, medial regions of the notochordal and the somitic territories are the last to express mediolateral intercalation behavior and subsequent tissue differentiations. In explants that do not converge, these cells neither express mediolateral intercalation behavior nor differentiate. These facts suggest that progressions of MIB in the anterior-posterior and lateral-medial directions may be organized by signals emanating from the lateral somitic and notochordal boundaries. These signals may have limited range and may be dependent on convergence, driven by mediolateral cell intercalation, to bring cells within their range. In the embryo, the posterior progression of MIB results in arcs of convergence, anchored in the vegetal endoderm at each end, acting on the inside of the blastoporal lip to produce involution of the IMZ.

Animals↗

Synergistic principles of development: overlapping patterning systems in Xenopus mesoderm induction.

The first inductive event in Xenopus development establishes the mesoderm at the equator of the developing embryo. As part of this process, the dorsal-ventral and anterior-posterior axes of the embryo are initially established. A number of signalling molecules which may play a role in mesodermal induction and patterning have been identified in the last several years, including members of the FGF, TGF-beta and Wnt gene families. A variety of experiments, using either purified factors or injection of RNA encoding these factors, have added to the wealth of classical embryological experimental data collected over the last century. We have synthesized some recent results with the classical data to provide a framework for examining the process of mesoderm induction, and to formulate putative roles for some of the different factors. We incorporate these ideas into a working model of mesoderm induction that provides a basis for future experimental directions. Finally, we suggest that mesoderm induction may not be a discrete set of well separated events, but instead may be a process involving partially overlapping signals that produce the same pattern.

Animals↗

FGF signalling in the early specification of mesoderm in Xenopus.

We have examined the role of FGF signalling in the development of muscle and notochord and in the expression of early mesodermal markers in Xenopus embryos. Disruption of the FGF signalling pathway by expression of a dominant negative construct of the FGF receptor (XFD) generally results in gastrulation defects that are later evident in the formation of the trunk and tail, though head structures are formed nearly normally. These defects are reflected in the loss of notochord and muscle. Even in embryos that show mild defects and gastrulate properly, muscle formation is impaired, suggesting that morphogenesis and tissue differentiation each depend on FGF. The XFD protein inhibits the expression of the immediate early gene brachyury throughout the marginal zone, including the dorsal side; it does not, however, inhibit the dorsal lip marker goosecoid, which is expressed in the first involuting mesoderm at the dorsal side that will underlie the head. The XFD protein also inhibits Xpo expression, an immediate early marker of ventral and lateral mesoderm. These results suggest that FGF is involved in the earliest events of most mesoderm induction that occur before gastrulation and that the early dorsal mesoderm is already composed of two cell populations that differ in their requirements for FGF.

Animals↗

Distinct elements of the xsna promoter are required for mesodermal and ectodermal expression.

Xsna, the Xenopus homologue of Drosophila snail, is expressed in both mesoderm and ectoderm. Expression occurs in all mesoderm initially but is down regulated in a tissue-specific fashion at the end of gastrulation in a way that reveals the subdivision of the mesoderm before its derivatives are overtly differentiated. Xsna is also expressed in the ectoderm of the prospective neural fold from stage 11, in a distinct band of cells surrounding the prospective neural plate, which we designate the neural plate border. The deep and superficial ectoderm compartments labelled by Xsna represent the prospective neural crest and the prospective roof of the neural tube, respectively. Xsna expression persists in neural crest cells during their subsequent migration. The role of the Xsna promoter in creating this pattern of expression has been investigated by injecting fertilised eggs with constructs containing the 5' upstream sequence of the gene fused to a reporter. An element of 115 base pairs (-160 to -45 relative to the transcriptional start) is sufficient to drive appropriate reporter gene expression. The promoter does not contain a TATA or CAAT box and does not have a high GC content, but RNA synthesis starts precisely at 33 bases upstream to the translational start. The start sequence can be deleted so that transcription is initiated elsewhere without affecting the expression pattern. The distribution of Xsna promoter activity within the embryo, examined using beta-galactosidase (beta-gal) fusions, is similar to that of the endogenous mRNA seen by in situ hybridisation. The contribution of elements within the 5' sequence have been assessed by comparing the expression patterns of constructs that have deletions in this region. Sequences from -112 to -97 are required for mesodermal expression and sequences from -96 to -44 are required for ectodermal expression. The behaviour of the injected promoter constructs differ in one important respect from the endogenous gene in that expression in an animal cap assay is not inducible by mesoderm-inducing factors but is inducible by cells of the vegetal pole.

Animals↗

Morphological differences in Xenopus embryonic mesodermal cells are specified as an early response to distinct threshold concentrations of activin.

The involution of presumptive mesoderm that occurs during amphibian gastrulation is a complex process requiring the coordinated action of a diverse range of cells. We show that cells with distinct morphologies, resembling each of those normally found in the involuting tissue of the Xenopus embryo, are induced in dispersed animal pole cells by different doses of the potent mesoderm-inducing factor activin. Each cell type is induced within a restricted dose range of activin concentrations, the boundaries of which are well demarcated shortly after activin treatment. In contrast, Brachyury and goosecoid, two genes thought to pattern the presumptive mesoderm, and the gene encoding platelet-derived growth factor receptor alpha, which is expressed in the mesoderm of gastrula stage embryos, are induced by broad, overlapping ranges of high activin concentrations at such early times. Similarly, the response of the gene encoding platelet-derived growth factor A, which is expressed normally in ectoderm of gastrula stage embryos, diminishes gradually as the activin concentration increases. Dose windows for the expression of these four genes narrow and become distinct from one another in cell aggregates after several hours in culture, suggesting that activin prompts a dynamic program of gene expression in induced mesoderm.

Activins↗

Cell-autonomous shift from axial to paraxial mesodermal development in zebrafish floating head mutants.

Zebrafish floating head mutant embryos lack notochord and develop somitic muscle in its place. This may result from incorrect specification of the notochord domain at gastrulation, or from respecification of notochord progenitors to form muscle. In genetic mosaics, floating head acts cell autonomously. Transplanted wild-type cells differentiate into notochord in mutant hosts; however, cells from floating head mutant donors produce muscle rather than notochord in wild-type hosts. Consistent with respecification, markers of axial mesoderm are initially expressed in floating head mutant gastrulas, but expression does not persist. Axial cells also inappropriately express markers of paraxial mesoderm. Thus, single cells in the mutant midline transiently co-express genes that are normally specific to either axial or paraxial mesoderm. Since floating head mutants produce some floor plate in the ventral neural tube, midline mesoderm may also retain early signaling capabilities. Our results suggest that wild-type floating head provides an essential step in maintaining, rather than initiating, development of notochord-forming axial mesoderm.

Animals↗

The eed mutation disrupts anterior mesoderm production in mice.

Mouse embryos homozygous for the mutation embryonic ectoderm development (eed) exhibit a growth defect and fail to gastrulate normally. While extraembryonic mesoderm is produced extensively, very little embryonic mesoderm is detected in eed mutant embryos, and there is no subsequent organization of mesoderm into node, notochord, or somites. The phenotype is consistent with a defect in the distal primitive streak. Here we report additional phenotypic analyses that include mRNA in situ hybridization of genes whose expression reflects the function of different regions of the primitive streak and their derivatives. These studies have confirmed that mesoderm derived from the proximal primitive streak is specified appropriately. Despite the absence of a morphologically distinct node, sparse axial mesoderm cells in eed mutant embryos are specified, as reflected by expression of Brachyury (T), Sonic hedgehog, and Tcf3b/HNF-3 beta, and definitive endoderm is produced. Specification of these cell types is also independent of correct expression of nodal, Fgf4, and gsc. Finally, T and Evx1 display ectopic expression in cells not normally fated to ingress through the primitive streak. The data presented are discussed in terms of mechanisms for establishment of the eed phenotype, and are consistent with the eed gene product playing an early role in primitive streak formation and/or organization.

Animals↗

The somatic-visceral subdivision of the embryonic mesoderm is initiated by dorsal gradient thresholds in Drosophila.

The maternal dorsal regulatory gradient initiates the differentiation of the mesoderm, neuroectoderm and dorsal ectoderm in the early Drosophila embryo. Two primary dorsal target genes, snail (sna) and decapentaplegic (dpp), define the limits of the presumptive mesoderm and dorsal ectoderm, respectively. Normally, the sna expression pattern encompasses 18-20 cells in ventral and ventrolateral regions. Here we show that narrowing the sna pattern results in fewer invaginated cells. As a result, the mesoderm fails to extend into lateral regions so that fewer cells come into contact with dpp-expressing regions of the dorsal ectoderm. This leads to a substantial reduction in visceral and cardiac tissues, consistent with recent studies suggesting that dpp induces lateral mesoderm. These results also suggest that the dorsal regulatory gradient defines the limits of inductive interactions between germ layers after gastrulation. We discuss the parallels between the subdivision of the mesoderm and dorsal ectoderm.

Animals↗

Cranial paraxial mesoderm and neural crest cells of the mouse embryo: co-distribution in the craniofacial mesenchyme but distinct segregation in branchial arches.

The spatial distribution of the cranial paraxial mesoderm and the neural crest cells during craniofacial morphogenesis of the mouse embryo was studied by micromanipulative cell grafting and cell labelling. Results of this study show that the paraxial mesoderm and neural crest cells arising at the same segmental position share common destinations. Mesodermal cells from somitomeres I, III, IV and VI were distributed to the same craniofacial tissues as neural crest cells of the forebrain, the caudal midbrain, and the rostral, middle and caudal hindbrains found respectively next to these mesodermal segments. This finding suggests that a basic meristic pattern is established globally in the neural plate ectoderm and paraxial mesoderm during early mouse development. Cells from these two sources mixed extensively in the peri-ocular, facial, periotic and cervical mesenchyme. However, within the branchial arches a distinct segregation of these two cell populations was discovered. Neural crest cells colonised the periphery of the branchial arches and enveloped the somitomere-derived core tissues on the rostral, lateral and caudal sides of the arch. Such segregation of cell populations in the first three branchial arches is apparent at least until the 10.5-day hindlimb bud stage and could be important for the patterning of the skeletal and myogenic derivatives of the arches.

Animals↗

MesP1: a novel basic helix-loop-helix protein expressed in the nascent mesodermal cells during mouse gastrulation.

A subtractive hybridization strategy was used to isolate putative genes involved in the development of mouse primordial germ cells (PGC). Complimentary DNA was amplified on RNA isolated from the base of the allantois where PGC are located in the 7.5 days post coitum (dpc) mouse embryo. It was then subtracted by hybridization with cDNA amplified on RNA of the anterior region where PGC are absent. A novel gene thus isolated is designated as Mesp1 and encodes a possible transcription factor MesP1 containing a basic helix-loop-helix motif. Its earliest expression was observed at the onset of gastrulation, as early as 6.5 dpc, in the nascent mesodermal cells that first ingressed at the end of the primitive streak. These expressing cells in the lateral and extraembryonic mesoderm showed a wing-shaped distribution. Its initial expression was soon down-regulated at 7.5 dpc before the completion of gastrulation, except at the proximal end of the primitive streak which included the extraembryonic mesoderm and the base of allantois. At 8 dpc, the expression at the base of the allantois moved laterally. This distribution between 7.0 and 8.0 dpc was similar to that of PGC detected by the alkaline phosphatase activity. However, the expression of Mesp1 was down-regulated thereafter, when PGC entered in the migration stage. After birth, Mesp1 expression was detected only in mature testes, but in a different isoform from that expressed in the embryo. Mesp1 was mapped to the mid region of chromosome 7, near the mesodermal deficiency gene (mesd). However, a Southern hybridization study clearly showed that Mesp1 was distinctly different from mesd. The amino acid sequence and its expression pattern suggest that MesP1 plays an important role in the development of the nascent mesoderm including PGC.

Amino Acid Sequence↗

Requirements of DFR1/Heartless, a mesoderm-specific Drosophila FGF-receptor, for the formation of heart, visceral and somatic muscles, and ensheathing of longitudinal axon tracts in CNS.

DFR1 encodes a mesoderm-specific fibroblast growth factor receptor in Drosophila. Here, we identified and characterized a protein-null mutant of DFR1 and examined DFR1 expression in embryos using anti-DFR1 antibody. Mutant phenotypes were completely rescued by a genomic fragment from the DFR1 locus. After invagination, mesodermal cells expressing DFR1 undergo proliferation and spread out dorsally to form a monolayer beneath the ectoderm. In mutant embryos, however, the mesoderm is not capable of extending to the normal dorsal limit and consequently mesodermal cells fail to receive ectodermal signals and thus rendered incapable of differentiating into primordia for the heart, visceral and somatic muscles. DFR1 is also required for normal development of the central nervous system. The absence of DFR1 resulted in the failure of longitudinal glia to enwrap longitudinal axon tracts. DFR1 mutant phenotypes were partially mimicked by the targeted expression of activated Yan, thus demonstrating the MAP kinase pathway to be involved in differentiation of mesoderm.

Abdomen↗

Bmp-4 acts as a morphogen in dorsoventral mesoderm patterning in Xenopus.

The marginal zone is a ring of tissue that gives rise to a characteristic dorsoventral pattern of mesoderm in amphibian embryos. Bmp-4 is thought to play an important role in specifying ventral mesodermal fate. Here we show (1) that different doses of Bmp-4 are sufficient to pattern four distinct mesodermal cell types and to pattern gene expression in the early gastrula marginal zone into three domains, (2) that there is a graded requirement for a Bmp signal in mesodermal patterning, and (3) that Bmp-4 has long-range activity which can become graded in the marginal zone by the antagonizing action of noggin. The results argue that Bmp-4 acts as a morphogen in dorsoventral patterning of mesoderm.

Animals↗

The ALK-2 and ALK-4 activin receptors transduce distinct mesoderm-inducing signals during early Xenopus development but do not co-operate to establish thresholds.

The TGFbeta family member activin induces different mesodermal cell types in a dose-dependent fashion in the Xenopus animal cap assay. High concentrations of activin induce dorsal and anterior cell types such as notochord and muscle, while low concentrations induce ventral and posterior tissues such as mesenchyme and mesothelium. In this paper we investigate whether this threshold phenomenon involves the differential effects of the two type I activin receptors ALK-2 and ALK-4. Injection of RNA encoding constitutively active forms of the receptors (here designated ALK-2* and ALK-4*) reveals that ALK-4* strongly induces the more posterior mesodermal marker Xbra and the dorsoanterior marker goosecoid in animal cap explants. Maximal levels of Xbra expression are attained using lower concentrations of RNA than are required for the strongest activation of goosecoid, and at the highest doses of ALK-4*, levels of Xbra transcription decrease, as is seen with high concentrations of activin. By contrast, the ALK-2* receptor activates Xbra but fails to induce goosecoid to significant levels. Analysis at later stages reveals that ALK-4* signalling induces the formation of a variety of mesodermal derivatives, including dorsal cell types, in a dose-dependent fashion, and that high levels also induce endoderm. By contrast, the ALK-2* receptor induces only ventral mesodermal markers. Consistent with these observations, ALK-4* is capable of inducing a secondary axis when injected into the ventral side of 32-cell stage embryos whilst ALK-2* cannot. Co-injection of RNAs encoding constitutively active forms of both receptors reveals that ventralising signals from ALK-2* antagonise the dorsal mesoderm-inducing signal derived from ALK-4*, suggesting that the two receptors use distinct and interfering signalling pathways. Together, these results show that although ALK-2* and ALK-4* transduce distinct signals, the threshold responses characteristic of activin cannot be due to interactions between these two pathways; rather, thresholds can be established by ALK-4* alone. Furthermore, the effects of ALK-2* signalling are at odds with it behaving as an activin receptor in the early Xenopus embryo.

Activin Receptors, Type I↗

Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm formation by T-box genes.

Inhibition of fibroblast growth factor (FGF) signaling prevents trunk and tail formation in Xenopus and zebrafish embryos. While the T-box transcription factor Brachyury (called No Tail in zebrafish) is a key mediator of FGF signaling in the notochord and tail, the pathways activated by FGF in non-notochordal trunk mesoderm have been uncertain. Previous studies have shown that the spadetail gene is required for non-notochordal trunk mesoderm formation; spadetail mutant embryos have major trunk mesoderm deficiencies, but relatively normal tail and notochord development. We demonstrate here that spadetail encodes a T-box transcription factor with homologues in Xenopus and chick. Spadetail is likely to be a key mediator of FGF signaling in trunk non-notochordal mesoderm, since spadetail expression is regulated by FGF signaling. Trunk and tail development are therefore dependent upon the complementary actions of two T-box genes, spadetail and no tail. We show that the regulatory hierarchy among spadetail, no tail and a third T-box gene, tbx6, are substantially different during trunk and tail mesoderm formation, and propose a genetic model that accounts for the regional phenotypes of spadetail and no tail mutants.

Amino Acid Sequence↗