Biomedical subjects

J Heasman

Publications and source records attributed to J Heasman.

At least 19 recordsLinked to original sources

PubMed2001

Vegetal localization of maternal mRNAs is disrupted by VegT depletion.

VegT is an essential maternal regulator of germ layer specification in Xenopus. The localization of VegT mRNA to the vegetal cortex of the oocyte during oogenesis ensures its inheritance by vegetal and not animal cells, and directs the differentiation of vegetal cells into endoderm. Similarly localized mRNAs, Vg1 and Bicaudal-C, are also inherited by vegetal cells, while germ plasm-associated mRNAs, such as Xcat2, become incorporated into vegetally derived primordial germ cells. Although mRNA localization is clearly important for tissue specification, the mechanism of mRNA anchoring to the oocyte vegetal cortex is not understood. Here, we examine the role of VegT in cortical localization. We report that depletion of VegT mRNA caused the release of Vg1 mRNA from the vegetal cortex and a reduction of Vg1 protein, without affecting the total amount of Vg1 transcript. Furthermore, we found that Bicaudal-C and Wnt11 mRNAs were also dispersed, but not degraded, by VegT depletion, while the localization of Xcat2 and Xotx1 mRNAs was unaffected. This effect was specific to the loss of VegT mRNA and not VegT protein, since a morpholino oligo against VegT, that blocked translation without degrading mRNA, did not disperse the vegetally localized mRNAs. Therefore, a subset of localized mRNAs is dependent on VegT mRNA for anchoring to the vegetal cortex, indicating a novel function for maternal VegT mRNA.

Animals↗

PubMed2001

The role of maternal axin in patterning the Xenopus embryo.

Regulation of the stability of beta catenin protein is a critical role of Wnt signaling cascades. In early Xenopus development, dorsal axis specification depends on regulation of beta catenin by both cytoplasmic and nuclear mechanisms. While the cytoplasmic protein axin is known as a key component of the cytoplasmic beta catenin degradation complex, loss-of-function studies are needed to establish whether it is required for dorso-ventral patterning in the embryo, and to test where in the embryo it carries out its function. Here, we show that embryos lacking maternal axin protein have increased levels of soluble beta catenin protein and increased nuclear localization of beta catenin in ventral nuclei at the blastula stage. These embryos gastrulate abnormally and develop with excessive notochord and head structures, and reduced tail and ventral components. They show increased expression of dorsal markers, including siamois, Xnr3, chordin, gsc, Xhex, and Otx2, decreased expression of Xwnt 8 and Xbra, and little alteration of BMP4 and Xvent1 and -2 mRNA levels. The ventral halves of axin-depleted embryos at the gastrula stage have dramatically increased levels of chordin expression, and severely decreased levels of Xwnt 8 mRNA expression, while BMP4 transcript levels are only slightly reduced. This dorso-anterior phenotype is rescued by axin mRNA injected into the vegetal pole of axin-depleted oocytes before fertilization. Interestingly, the phenotype was rescued by ventral but not dorsal injection of axin mRNA, at the 4-cell stage, although dorsal injection into wild-type embryos does cause ventralization. These results show directly that the localized ventral activity of maternal axin is critical for the correct patterning of the early Xenopus embryo.

Animals↗

PubMed2001

Early events in the mammalian germ line.

Germ cells represent the genetic and cellular link between generations, as well as the transmitters of inherited diseases. Despite their central importance, not much is known about the molecular mechanisms whereby a germ cell lineage becomes set aside during development, or how the germ cells, once formed, migrate to the gonads and combine with somatic cells to make a gonad. This article provides a brief review of current knowledge on these issues, with particular focus on the mammalian germ line.

Animals↗

PubMed2001

Timing of endogenous activin-like signals and regional specification of the Xenopus embryo.

Signaling by activin-like ligands is important for induction and patterning of mesoderm and endoderm. We have used an antibody that specifically recognizes the phosphorylated and activated form of Smad2, an intracellular transducer of activin-like ligands, to examine how this signaling pathway patterns the early mesendoderm. In contrast to the simple expectation that activin-like signaling should be highest on the dorsal side of the gastrula stage embryo, we have found that while Smad2 phosphorylation is highest dorsally before gastrulation, signaling is attenuated dorsally and is highest on the ventral side by mid-gastrulation. Early dorsal initiation of Smad2 phosphorylation results from cooperation between the vegetally localized maternal transcription factor VegT and dorsally localized beta-catenin. The subsequent ventral appearance of Smad2 phosphorylation is dependent on VegT, but not on signaling from the dorsal side. Dorsal attenuation of Smad2 phosphorylation during gastrulation is mediated by early dorsal expression of feedback inhibitors of activin-like signals. In addition to regulation of Smad2 phosphorylation by the expression of activin-like ligands and their antagonists, the responsiveness of embryonic cells to activin-like ligands is also temporally regulated. Ectopic Vg1, Xnr1 and derrière all fail to activate Smad2 phosphorylation until after the midblastula transition, and the onset of responsiveness to these ligands is independent of transcription. Furthermore, the timing of cellular responsiveness differs for Xnr1 and derrière, and these distinct temporal patterns of responsiveness can be correlated with their distinctive phenotypic effects. These observations suggest that the timing of endogenous activin-like signaling is a determinant of patterning in the early Xenopus embryo.

Activins↗

PubMed2001

Maternal VegT is the initiator of a molecular network specifying endoderm in Xenopus laevis.

During cleavage stages, maternal VegT mRNA and protein are localized to the Xenopus embryo's vegetal region from which the endoderm will arise and where several zygotic gene transcripts will be localized. Previous loss-of-function experiments on this T-box transcription factor suggested a role for VegT in Xenopus endoderm formation. Here, we test whether VegT is required to initiate endoderm formation using a loss of function approach. We find that the endodermal genes, Bix1, Bix3, Bix4, Milk (Bix2), Mix.1, Mix.2, Mixer, Xsox17 alpha, Gata4, Gata5, Gata6 and endodermin, as well as the anterior endodermal genes Xhex and cerberus, and the organizer specific gene, Xlim1, are downstream of maternal VegT. We also find that the TGF beta s, Xnr1, Xnr2, Xnr4 and derrière rescue expression of these genes, supporting the idea that cell interactions are critical for proper endoderm formation. Additionally, inhibitory forms of Xnr2 and Derrière blocked the ability of VegT mRNA injection to rescue VegT-depleted embryos. Furthermore, a subset of endodermal genes was rescued in VegT-depleted vegetal masses by induction from an uninjected vegetal mass. Finally, we begin to establish a gene hierarchy downstream of VegT by testing the ability of Mixer and Gata5 to rescue the expression of other endodermal genes. These results identify VegT as the maternal regulator of endoderm initiation and illustrate the complexity of zygotic pathways activated by VegT in the embryo's vegetal region.

Animals↗

PubMed2000

Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach.

Xenopus embryos develop dorsal/ventral and anterior/posterior axes as a result of the activity of a maternal Xwnt pathway, in which beta-catenin is an essential component, acting as a transactivator of transcription of zygotic genes. However, the questions of where and when beta-catenin is required in early embryogenesis have not been addressed directly, because no loss-of-function method has been available. Here we report the use of a novel antisense approach that allows us to target depletion of protein to individual blastomeres. When a "morpholino" oligo complementary to beta-catenin mRNA is injected into early embryos, it depletes beta-catenin protein effectively through the neurula stage. By targeting the oligo to different cleavage blastomeres, we block beta-catenin activity in different areas and at different times. Dorsal vegetal injection at the 2- and 4-cell stages blocks dorsal axis formation and at the 8-cell stage blocks head formation, while A-tier injection at the 32-cell stage causes abnormal cement gland formation. This approach shows the complex involvement of Xwnt pathways in embryonic patterning and offers a rapid method for the functional analysis of both maternal and early zygotic gene products in Xenopus.

Animals↗

PubMed2000

The onset of germ cell migration in the mouse embryo.

Mouse primordial germ cells (PGCs) are specified between embryonic day 6.5 (E6.5) and E7.5, when they have been visualized as an alkaline phosphatase-positive (AP+) cell population in the developing allantois. By E8.5, they are embedded in the hind-gut epithelium. Previous experiments have suggested different sites for PGCs' origin, and it is unclear how they reach the gut epithelium. We have used transgenic mice expressing GFP under a truncated Oct4 promoter to visualize living PGCs. We find GFP+/AP+ cells in the posterior end of the primitive streak as a dispersed population of cells actively migrating into the allantois, and directly into the adjacent embryonic endoderm. Time-lapse analysis shows these cells to be actively migratory from the time they exit the primitive streak.

Alkaline Phosphatase↗

PubMed2000

The role of cadherins during primordial germ cell migration and early gonad formation in the mouse.

Primordial germ cells (PGCs) are the founder cells of the gametes. In mammals, PGCs migrate from the hindgut to the genital ridges, where they coalesce with each other and with somatic cells to form the primary sex cords. We show here that, in both sexes, PGCs express P- and E-cadherins during and after migration, and N-cadherin at post-migratory stages. E-Cadherin is not expressed by PGCs whilst in the hindgut, but is upregulated as they leave. Blocking antibodies against E-, but not P-cadherin cause defective PGC-PGC coalescence, and in some cases, ectopic PGCs.

Animals↗

PubMed2000

The putative wnt receptor Xenopus frizzled-7 functions upstream of beta-catenin in vertebrate dorsoventral mesoderm patterning.

We have isolated one member of the frizzled family of wnt receptors from Xenopus (Xfz7) to study the role of cell-cell communication in the establishment of the vertebrate axis. We demonstrate that this maternally encoded protein specifically synergizes with wnt proteins in ectopic axis induction. Embryos derived from oocytes depleted of maternal Xfz7 RNA by antisense oligonucleotide injection are deficient in dorsoanterior structures. Xfz7-depleted embryos are deficient in dorsal but not ventral mesoderm due to the reduced expression of the wnt target genes siamois, Xnr3 and goosecoid. These signaling defects can be restored by the addition of beta-catenin but not Xwnt8b. Xfz7 thus functions upstream of the known GSK-3/axin/beta-catenin intracellular signaling complex in vertebrate dorsoventral mesoderm specification.

Amino Acid Sequence↗

PubMed1999

Expression of the Lewis group carbohydrate antigens during Xenopus development.

We have examined the pattern of expression of the Lewis group carbohydrate antigens during the development of African toad Xenopus laevis. One of these antigens, Lewis x (Le(x), also known as SSEA-1), was previously shown to be involved in cell-cell adhesion in early mouse embryos and teratocarcinoma stem cells. Recently another member of these antigens, sialyl-Le(x), was found to be one of the major ligands for the selectin family of cell-cell adhesion molecules. In order to study the role of carbohydrate-mediated cell adhesion during Xenopus development, we first studied the expression pattern of the Le(x). We found that Le(x)was not expressed in early embryos, started to be expressed at the tail bud stage in anterior regions of the body such as the cement gland or head skin, and was gradually showed more posterial expression at later stages. At tadpole stage, it was also expressed on specific cell bodies in brain, and in axon region in brain and neural retina. Antibodies against Le(x)blocked neurite outgrowth in the explant culture of tadpole brain. One of the candidates for Le(x)carrier protein in the tadpole brain is a 200 kDa glycoprotein detected by Western blotting. In adult tissues, it was expressed in brain, testis, and gut, but not in kidney, lung, spleen, ovary, or muscle. We also examined the expression patterns of other Lewis group antigens. Among them, sialyl-Le(x)was expressed on endothelial cells and on leukocytes, suggesting the possibility that it functions as a ligand for selectin in Xenopus.

Animals↗

PubMed1999

Bix4 is activated directly by VegT and mediates endoderm formation in Xenopus development.

The maternal T-box gene VegT, whose transcripts are restricted to the vegetal hemisphere of the Xenopus embryo, plays an essential role in early development. Depletion of maternal VegT transcripts causes embryos to develop with no endoderm, while vegetal blastomeres lose the ability to induce mesoderm (Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C. and Heasman, J. (1998) Cell 94, 515-524). The targets of VegT, a transcription activator, must therefore include genes involved both in the specification of endoderm and in the production of mesoderm-inducing signals. We recently reported that the upstream regulatory region of the homeobox-containing gene Bix4 contains T-box binding sites. Here we show that expression of Bix4 requires maternal VegT and that two T-box binding sites are necessary and sufficient for mesodermal and endodermal expression of reporter genes driven by the Bix4 promoter in transgenic Xenopus embryos. Remarkably, a single T-box binding site is able to act as a mesoderm-specific enhancer when placed upstream of a minimal promoter. Finally, we show that Bix4 rescues the formation of endodermal markers in embryos in which VegT transcripts have been ablated but does not restore the ability of vegetal pole blastomeres to induce mesoderm. These results demonstrate that Bix4 acts directly downstream of VegT to specify endodermal differentiation in Xenopus embryos.

Animals↗

PubMed1999

Mesoderm induction in Xenopus is a zygotic event regulated by maternal VegT via TGFbeta growth factors.

The maternal transcription factor VegT is important for establishing the primary germ layers in Xenopus. In previous work, we showed that the vegetal masses of embryos lacking maternal VegT do not produce mesoderm-inducing signals and that mesoderm formation in these embryos occurred ectopically, from the vegetal area rather than the equatorial zone of the blastula. Here we have increased the efficiency of the depletion of maternal VegT mRNA and have studied the effects on mesoderm formation. We find that maternal VegT is required for the formation of 90% of mesodermal tissue, as measured by the expression of mesodermal markers MyoD, cardiac actin, Xbra, Xwnt8 and alphaT4 globin. Furthermore, the transcription of FGFs and TGFbetas, Xnr1, Xnr2, Xnr4 and derrière does not occur in VegT-depleted embryos. We test whether these growth factors may be endogenous factors in mesoderm induction, by studying their ability to rescue the phenotype of VegT-depleted embryos, when their expression is restricted to the vegetal mass. We find that Xnr1, Xnr2, Xnr4 and derrière mRNA all rescue mesoderm formation, as well as the formation of blastopores and the wild-type body axis. Derrière rescues trunk and tail while nr1, nr2 and nr4 rescue head, trunk and tail. We conclude that mesoderm induction in Xenopus depends on a maternal transcription factor regulating these zygotic growth factors.

Animals↗

PubMed1999

Mouse primordial germ cells lacking beta1 integrins enter the germline but fail to migrate normally to the gonads.

Primordial germ cells are the founder cells of the gametes. They are set aside at the initial stages of gastrulation in mammals, become embedded in the hind-gut endoderm, then actively migrate to the sites of gonad formation. The molecular basis of this migration is poorly understood. Here we sought to determine if members of the integrin family of cell surface receptors are required for primordial germ cell migration, as integrins have been implicated in the migration of several other motile cell types. We have established a line of mice which express green fluorescent protein in germline cells that has enabled us to efficiently purify primordial germ cells at different stages by flow cytometry. We have catalogued the spectrum of integrin subunit expression by primordial germ cells during and after migration, using flow cytometry, immunocytochemistry and RT-PCR. Through analysis of integrin beta1(-/-)-->wild-type chimeras, we show that embryonic cells lacking beta1 integrins can enter the germline. However, integrin beta1(-/-) primordial germ cells do not colonize the gonad efficiently. Embryos with targeted deletion of integrin subunit alpha3, alpha6, or alphaV show no major defects in primordial germ cell migration. These results demonstrate a role for beta1-containing integrins in the development of the germline, although an equivalent role for * integrin subunit(s) has yet to be established.

Animals↗

PubMed1999

Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line.

During normal embryonic development, mammalian germ cells use both cell migration and aggregation to form the primitive sex cords. Germ cells must be able to interact with their environment and each other to accomplish this; however, the molecular basis of early germ cell adhesion is not well characterized. Differential adhesion is also thought to occur in the adult seminiferous tubules, since germ cells move from the periphery to the lumen as they differentiate. In a screen for additional adhesion molecules expressed by the germ line, expression of the homophilic adhesion molecule, Ep-CAM, was identified in embryonic, neonatal and adult germ cells using immunocytochemistry and flow cytometry with an Ep-CAM-specific monoclonal antibody. At embryonic stages, germ cells were found to express Ep-CAM during migration at embryonic day 10.5 and early gonad assembly at embryonic day 12.5. Expression of Ep-CAM was also found on neonatal male and female germ cells. In the adult testis, Ep-CAM was detected only on spermatogonia, and was absent from more differentiated cells. Finally, embryonic stem cells were shown to express this receptor. It is proposed that Ep-CAM plays a role in the development of the germ line and the behaviour of totipotent cells.

Animals↗

PubMed1998

The role of maternal VegT in establishing the primary germ layers in Xenopus embryos.

VegT is a T-box transcription factor whose mRNA is synthesized during oogenesis and localized in the vegetal hemisphere of the egg and early embryo. We show that maternally expressed VegT controls the pattern of primary germ layer specification in Xenopus embryos. Reduction of the maternal store completely alters the fates of different regions of the blastula so that animal cell fate is changed from epidermis and nervous system to epidermis only, equatorial cell fate is changed from mesoderm to ectoderm, and vegetal cell fate is changed from endoderm to mesoderm and ectoderm. Vegetal cells lose their capacity both to form endoderm and to release mesoderm-inducing signals. These results show that a single maternally expressed gene controls the patterning of the Xenopus blastula.

Animals↗

PubMed1998

Evidence that dorsal-ventral differences in gap junctional communication in the early Xenopus embryo are generated by beta-catenin independent of cell adhesion effects.

Gap junctional communication (GJC) is regulated in the early Xenopus embryo and quantitative differences in junctional communication correlate with the specification of the dorsal-ventral axis. To address the mechanism that is responsible for regulating this differential communication, we investigated the function of beta-catenin during the formation of the dorsal-ventral axis in Xenopus embryos by blocking its synthesis with antisense oligodeoxynucleotides. This method has previously been shown to reduce the level of beta-catenin in the early embryo, prior to zygotic transcription, and to inhibit the formation of the dorsal axis (Heasman et al., 1994, Cell 79, 791-803). We show here that antisense inhibition of beta-catenin synthesis also reduces GJC among cells in the dorsal hemisphere of 32-cell embryos to levels similar to those observed among ventral cells. Full-length beta-catenin mRNA can restore elevated levels of dorsal GJC when injected into beta-catenin-deficient oocytes, demonstrating the specificity of the beta-catenin depletion with the antisense oligonucleotides. Thus, endogenous beta-catenin is required for the observed differential GJC. This regulation of GJC is the earliest known action of the dorsal regulator, beta-catenin, in Xenopus development. Two lines of evidence, presented here, indicate that beta-catenin acts within the cytoplasm to regulate GJC, rather than through an effect on cell adhesion. First, when EP-cadherin is overexpressed and increased adhesion is observed, embryos display both a ventralized phenotype and reduced dye transfer. Second, a truncated form of beta-catenin (i.e., the ARM region), that lacks the cadherin-binding domain, restores dorsal GJC to beta-catenin-depleted embryos. Thus, beta-catenin appears to regulate GJC independent of its role in cell-cell adhesion, by acting within the cytoplasm through a signaling mechanism.

Animals↗

PubMed1998

The origin and migration of primordial germ cells in the mouse.

Primordial germ cells (PGCs) are the founders of the gametes. They migrate from their origin to combine with the somatic component of the gonad. Progenitors in mice have been recognized in the proximal epiblast. When and how these cells are allocated to the germ line has not been elucidated fully. Numerous growth factors have been described which affect PGC survival, proliferation and directional control during their migration. The extracellular matrix also may play a role in PGC migration, particularly fibronectin and laminin. Once they have arrived at the gonad, PGCs aggregate with one another. The molecules governing this PGC-PGC interaction have yet to be identified.

Animals↗

PubMed1998

Early stages in male germ cell differentiation in the mouse. Review article.

Primordial germ cells arise during gastrulation and migrate from the hindgut into the gonad primordium during early organogenesis. In this article, we discuss factors that control migration, proliferation and targeting of the PGCs. In particular we discuss how changes in adhesiveness control germ cell positioning in the gonad, and the molecules involved.

Animals↗