Anteroposterior neural tissue specification by activin-induced mesoderm

The transforming growth factor β superfamily member, activin, is able to induce mesodermal tissues in animal cap explants from Xenopus laevis blastula stage embryos. Activin can act like a morphogen of the dorsoventral axis in that lower doses induce more ventral, and higher doses more dorsal, tissue types. Activin has also previously been reported to induce neural tissues in animal caps. From cell mixing experiments it was inferred that this might be an indirect effect of induced mesoderm signaling to uninduced ectoderm. Here we demonstrate directly that neural tissues do indeed arise by the action of induced mesoderm on uninduced ectoderm. Dorsal mesoderm is itself subdivided into posterior and anterior domains in vivo, but this had not been demonstrated for induced mesoderm. We therefore tested whether different concentrations of activin recreate these different anteroposterior properties as well. We show that the anteroposterior positional value of induced mesoderm, including its neuroinductive properties, depends on the dose of activin applied to the mesoderm, with lower doses inducing more posterior and higher doses giving more anterior markers. We discuss the implications of these results for patterning signals and the relationship between anteroposterior and dorsoventral axes.

Identification of novel genes in Drosophila reveals the complex regulation of early gene activity in the mesoderm.

Two zygotic genes, twist and snail, are indispensable for the correct establishment of the mesoderm primordium in the early Drosophila embryo. They are also needed for morphogenesis and differentiation of the mesoderm. Both genes code for transcription factors with different, albeit complementary, functions. Therefore, to understand the early development of the mesoderm, it will be necessary to identify and study the genes regulated by twist and snail. We have searched for downstream genes using a subtractive cDNA library enriched in sequences expressed in the mesoderm. We have isolated sequences that correspond to 13 novel early mesoderm genes. These novel genes show a variety of expression patterns and also differ in their dependence on twist and snail functions. This indicates that the regulation of early gene activity in the mesoderm is more complex than previously thought.

Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase.

Abnormal mesoderm movement, leading to defects in axial organization, is observed in mouse and Xenopus laevis embryos deprived of platelet-derived growth factor (PDGF) AA signaling. However, neither the cellular response to PDGF nor the signaling pathways involved are understood. Herein we describe an in vitro assay to examine the direct effect of PDGF AA on aggregates of Xenopus embryonic mesoderm cells. We find that PDGF AA stimulates aggregates to spread on fibronectin. This behavior is similar to that of migrating mesoderm cells in vivo that spread and form lamellipodia and filipodia on contact with fibronectin-rich extracellular matrix. We go on to show two lines of evidence that implicate phosphatidylinositol 3-kinase (PI3K) as an important component of PDGF-induced mesoderm cell spreading. (i) The fungal metabolite wortmannin, which inhibits signaling by PI3K, blocks mesoderm spreading in response to PDGF AA. (ii) Activation of a series of receptors with specific tyrosine-to-phenylalanine mutations revealed PDGF-induced spreading of mesoderm cells depends on PI3K but not on other signaling molecules that interact with PDGF receptors including phospholipase C gamma, Ras GTPase-activating protein, and phosphotyrosine phosphatase SHPTP2. These results indicate that a PDGF signal, medicated by PI3K, can facilitate embryonic mesoderm cell spreading on fibronectin. We propose that PDGF, produced by the ectoderm, influences the adhesive properties of the adjacent mesoderm cells during gastrulation.

A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos.

The formation of ventral mesoderm has been traditionally viewed as a result of a lack of dorsal signaling and therefore assumed to be a default state of mesodermal development. The discovery that bone morphogenetic protein 4 (BMP4) can induce ventral mesoderm led to the suggestion that the induction of the ventral mesoderm requires a different signaling pathway than the induction of the dorsal mesoderm. However, the individual components of this pathway remained largely unknown. Here we report the identification of a novel Xenopus homeobox gene PV.1 (posterior-ventral 1) that is capable of mediating induction of ventral mesoderm. This gene is activated in blastula stage Xenopus embryos, its expression peaks during gastrulation and declines rapidly after neurulation is complete. PV.1 is expressed in the ventral marginal zone of blastulae and later in the posterior ventral area of gastrulae and neurulae. PV.1 is inducible in uncommited ectoderm by the ventralizing growth factor BMP4 and counteracts the dorsalizing effects of the dominant negative BMP4 receptor. Overexpression of PV.1 yields ventralized tadpoles and rescues embryos partially dorsalized by LiCl treatment. In animal caps, PV.1 ventralizes induction by activin and inhibits expression of dorsal specific genes. All of these effects mimic those previously reported for BMP4. These observations suggest that PV.1 is a critical component in the formation of ventral mesoderm and possibly mediates the effects of BMP4.

A type 1 serine/threonine kinase receptor that can dorsalize mesoderm in Xenopus.

We have cloned a type I serine/threonine kinase receptor, XTrR-I, from Xenopus. XTrR-I (Xenopus transforming growth factor beta-related receptor type I) is expressed in all regions of embryos throughout early development. Overexpression of this receptor does not affect ectoderm or endoderm but dorsalizes the mesoderm such that muscle appears in ventral mesoderm and notochord appears in lateral mesoderm normally fated to become muscle. In addition, overexpression of XTrR-I in UV-treated embryos is able to cause formation of a partial dorsal axis. These results suggest that XTrR-I encodes a receptor which responds in normal development to a transforming growth factor beta-like ligand so as to promote dorsalization. Its function would therefore be to direct mesodermalized tissue into muscle or notochord.

Meso1, a basic-helix-loop-helix protein involved in mammalian presomitic mesoderm development.

To identify genes involved in the regulation of early mammalian development, we have developed a dominant-negative mutant basic-helix-loop-helix (bHLH) protein probe for interaction cloning and have isolated a member of the bHLH family of transcription factors, Meso1. Meso1-E2A heterodimers are capable of binding to oligonucleotide probes that contain a bHLH DNA recognition motif. In mouse embryos, Meso1 is expressed prior to MyoD1 family members. Meso1 expression is first detected at the neural plate stage of development in the paraxial mesoderm of the head and in presomitic mesodermal cells prior to their condensation into somites. Our findings suggest that Meso1 may be a key regulatory gene involved in the early events of vertebrate mesoderm differentiation.

Morphogenetic movements at gastrulation require the SH2 tyrosine phosphatase Shp2

The SH2 domain-containing tyrosine phosphatase Shp2 plays a pivotal role during the gastrulation of vertebrate embryos. However, because of the complex phenotype observed in mouse mutant embryos, the precise role of Shp2 during development is unclear. To define the specific functions of this phosphatase, Shp2 homozygous mutant embryonic stem cells bearing the Rosa-26 LacZ transgene were isolated and used to perform a chimeric analysis. Here, we show that Shp2 mutant cells amass in the tail bud of embryonic day 10.5 chimeric mouse embryos and that this accumulation begins at the onset of gastrulation. At this early stage, Shp2 mutant cells collect in the primitive streak of the epiblast and thus show deficiencies in their contribution to the mesoderm lineage. In high-contribution chimeras, we show that overaccumulation of Shp2 mutant cells at the posterior end of the embryo results in two abnormal phenotypes: spina bifida and secondary neural tubes. Consistent with a failure to undergo morphogenic movements at gastrulation, Shp2 is required for embryo fibroblast cells to mount a positive chemotactic response to acidic fibroblast growth factor in vitro. Our results demonstrate that Shp2 is required at the initial steps of gastrulation, as nascent mesodermal cells form and migrate away from the primitive streak. The aberrant behavior of Shp2 mutant cells at gastrulation may result from their inability to properly respond to signals initiated by fibroblast growth factors.

Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Cα

Protein phosphatase 2A (PP2A) is a multimeric enzyme, containing a catalytic subunit complexed with two regulatory subunits. The catalytic subunit PP2A C is encoded by two distinct and unlinked genes, termed Cα and Cβ. The specific function of these two catalytic subunits is unknown. To address the possible redundancy between PP2A and related phosphatases as well as between Cα and Cβ, the Cα subunit gene was deleted by homologous recombination. Homozygous null mutant mice are embryonically lethal, demonstrating that the Cα subunit gene is an essential gene. As PP2A exerts a range of cellular functions including cell cycle regulation and cell fate determination, we were surprised to find that these embryos develop normally until postimplantation, around embryonic day 5.5/6.0. While no Cα protein is expressed, we find comparable expression levels of PP2A C at a time when the embryo is degenerating. Despite a 97% amino acid identity, Cβ cannot completely compensate for the absence of Cα. Degenerated embryos can be recovered even at embryonic day 13.5, indicating that although embryonic tissue is still capable of proliferating, normal differentiation is significantly impaired. While the primary germ layers ectoderm and endoderm are formed, mesoderm is not formed in degenerating embryos.

Postgastrulation Smad2-deficient embryos show defects in embryo turning and anterior morphogenesis

SMAD2 is a member of the transforming growth factor β and activin-signaling pathway. To examine the role of Smad2 in postgastrulation development, we independently generated mice with a null mutation in this gene. Smad2-deficient embryos die around day 7.5 of gestation because of failure of gastrulation and failure to establish an anterior–posterior (A-P) axis. Expression of the homeobox gene Hex (the earliest known marker of the A-P polarity and the prospective head organizer) was found to be missing in Smad2-deficient embryos. Homozygous mutant embryos and embryonic stem cells formed mesoderm derivatives revealing that mesoderm induction is SMAD2 independent. In the presence of wild-type extraembryonic tissues, Smad2-deficient embryos developed beyond 7.5 and up to 10.5 days postcoitum, demonstrating a requirement for SMAD2 in extraembryonic tissues for the generation of an A-P axis and gastrulation. The rescued postgastrulation embryos showed malformation of head structures, abnormal embryo turning, and cyclopia. Our results show that Smad2 expression is required at several stages during embryogenesis.

Transcriptional regulation of the Xlim-1 gene by activin is mediated by an element in intron I

The Xlim-1 gene is activated in the late blastula stage of Xenopus embryogenesis in the mesoderm, and its RNA product becomes concentrated in the Spemann organizer at early gastrula stage. A major regulator of early expression of Xlim-1 is activin or an activin-like signal. We report experiments aiming to identify the activin response element in the Xlim-1 gene. The 5′ flanking region of the gene contains a constitutive promoter that is not activin responsive, whereas sequences in the first intron mediate repression of basal promoter activity and stimulation by activin. An intron-derived fragment of 212 nt is the smallest element that could mediate activin responsiveness. Nodal and act-Vg1, factors with signaling properties similar to activin, also stimulated Xlim-1 reporter constructs, whereas BMP-4 did not stimulate or repress the constructs. The mechanism of activin regulation of Xlim-1 and the sequence of the response element are distinct from activin response elements of other genes studied so far.

Mouse Eya genes are expressed during limb tendon development and encode a transcriptional activation function

Vertebrate limb tendons are derived from connective cells of the lateral plate mesoderm. Some of the developmental steps leading to the formation of vertebrate limb tendons have been previously identified; however, the molecular mechanisms responsible for tendinous patterning and maintenance during embryogenesis are largely unknown. The eyes absent (eya) gene of Drosophila encodes a novel nuclear protein of unknown molecular function. Here we show that Eya1 and Eya2, two mouse homologues of Drosophila eya, are expressed initially during limb development in connective tissue precursor cells. Later in limb development, Eya1 and Eya2 expression is associated with cell condensations that form different sets of limb tendons. Eya1 expression is largely restricted to flexor tendons, while Eya2 is expressed in the extensor tendons and ligaments of the phalangeal elements of the limb. These data suggest that Eya genes participate in the patterning of the distal tendons of the limb. To investigate the molecular functions of the Eya gene products, we have analyzed whether the highly divergent PST (proline-serine-threonine)-rich N-terminal regions of Eya1–3 function as transactivation domains. Our results demonstrate that Eya gene products can act as transcriptional activators, and they support a role for this molecular function in connective tissue patterning.

Detection of targeted GFP-Hox gene fusions during mouse embryogenesis

The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13GFPneo embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13lacZneo embryos. Similarly, Hoxa1GFPneo expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1GFPneo embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.

Smad8 mediates the signaling of the receptor serine kinase

Smad proteins are critical intracellular mediators of signaling by growth and differentiation factors of the transforming growth factor β superfamily. We have isolated a member of the Smad family, Smad8, from a rat brain cDNA library and biochemically and functionally characterized its ability to transduce signals from serine kinase receptors. In Xenopus embryo, Smad8 is able to transcriptionally activate a subset of mesoderm target genes similar to those induced by the receptor serine kinase, activin receptor-like kinase (ALK)-2. Smad8 can be specifically phosphorylated by a constitutively active ALK-2 but not the related receptor serine kinase, ALK-4. In response to signaling from ALK-2, Smad8 associates with a common regulatory molecule, Smad4, and this association leads to a synergistic effect on gene transcription. Furthermore, Smad8 is able to rescue the expression of mesoderm genes blocked by truncated ALK-2 in the embryo. These results indicate that Smad8 can function as a downstream signaling mediator of ALK-2.

Mitogen-activated protein kinase and neural specification in Xenopus

We have investigated the activity and function of mitogen-activated protein kinase (MAPK) during neural specification in Xenopus. Ectodermal MAPK activity increased between late blastula and midgastrula stages. At midgastrula, MAPK activity in both newly induced neural ectoderm and ectoderm overexpressing the anterior neural inducer noggin was 5-fold higher than in uninduced ectoderm. Overexpression of MAPK phosphatase-1 (MKP-1) in ectoderm inhibited MAPK activity and prevented neurectoderm-specific gene expression when the ectoderm was recombined with dorsal mesoderm or treated with fibroblast growth factor (FGF). Neurectoderm-specific gene expression was observed, however, in ectoderm overexpressing both noggin and MKP-1. To evaluate the role of MAPK in posterior regionalization, ectodermal isolates were treated with increasing concentrations of FGF and assayed for MAPK activity and neurectoderm-specific gene expression. Although induction of posterior neural ectoderm by FGF was accompanied by an elevation of MAPK activity, relative MAPK activity associated with posterior neural fate was no higher than that of ectoderm specified to adopt an anterior neural fate. Thus, increasingly posterior neural fates are not correlated with quantitative increases in MAPK activity. Because MAPK has been shown to down-regulate Smad1, MAPK may disrupt bone morphogenetic protein 4 (BMP-4) signaling during neural specification. Our results suggest that MAPK plays an essential role in the establishment of neural fate in vivo.

Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2

The existence of a common precursor for endothelial and hemopoietic cells, termed the hemangioblast, has been postulated since the beginning of the century. Recently, deletion of the endothelial-specific vascular endothelial growth factor receptor 2 (VEGFR2) by gene targeting has shown that both endothelial and hemopoietic cells are absent in homozygous null mice. This observation suggested that VEGFR2 could be expressed by the hemangioblast and essential for its further differentiation along both lineages. However, it was not possible to exclude the hypothesis that hemopoietic failure was a secondary effect resulting from the absence of an endothelial cell microenvironment. To distinguish between these two hypotheses, we have produced a mAb directed against the extracellular domain of avian VEGFR2 and isolated VEGFR2+ cells from the mesoderm of chicken embryos at the gastrulation stage. We have found that in clonal cultures, a VEGFR2+ cell gives rise to either a hemopoietic or an endothelial cell colony. The developmental decision appears to be regulated by the binding of two different VEGFR2 ligands. Thus, endothelial differentiation requires VEGF, whereas hemopoietic differentiation occurs in the absence of VEGF and is significantly reduced by soluble VEGFR2, showing that this process could be mediated by a second, yet unidentified, VEGFR2 ligand. These observations thus suggest strongly that in the absence of the VEGFR2 gene product, the precursors of both hemopoietic and vascular endothelial lineages cannot survive. These cells therefore might be the initial targets of the VEGFR2 null mutation.