Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

Expression of XMyoD protein in early Xenopus laevis embryos.

A monoclonal antibody specific for Xenopus MyoD (XMyoD) has been characterized and used to describe the pattern of expression of this myogenic factor in early frog development. The antibody recognizes an epitope close to the N terminus of the products of both XMyoD genes, but does not bind XMyf5 or XMRF4, the other two myogenic factors that have been described in Xenopus. It reacts in embryo extracts only with XMyoD, which is extensively phosphorylated in the embryo. The distribution of XMyoD protein, seen in sections and whole-mounts, and by immunoblotting, closely follows that of XMyoD mRNA. XMyoD protein accumulates in nuclei of the future somitic mesoderm from the middle of gastrulation. In neurulae and tailbud embryos it is expressed specifically in the myotomal cells of the somites. XMyoD is in the nucleus of apparently every cell in the myotomes. It accumulates first in the anterior somitic mesoderm, and its concentration then declines in anterior somites from the tailbud stage onwards.     

Regulation of Hex gene expression and initial stages of avian hepatogenesis by Bmp and Fgf signaling

The vertebrate liver and heart arise from adjacent cell layers in the anterior lateral (AL) endoderm and mesoderm of late gastrula embryos, and the earliest stages of liver and heart development are interrelated through reciprocal tissue interactions. Although classical embryological studies performed several decades ago in chick and quail defined the timing of hepatogenic induction in birds and the important role for cardiogenic mesoderm in this process, almost nothing is known about the molecular aspects of avian liver development. Here we use in vivo and explantation assays to investigate tissue interactions and signaling pathways regulating Hex, a homeobox gene required for liver development, and the earliest stages of hepatogenesis in the chick embryo. We find that explants of late gastrula anterior lateral endoderm plus mesoderm, which have been used extensively for studies relating to heart development, also produce albumin-expressing hepatoblasts. Expression of Hex, the earliest known molecular marker for the hepatogenic endoderm, and albumin, indicative of early committed hepatoblasts, requires both autocrine Bmp signaling and a specific paracrine signal from the cardiogenic (anterior lateral) mesoderm. Endodermal expression of Fox2a, in contrast, requires the mesoderm but is independent of Bmp signaling. In vivo induction assays show that the ability of BMP2 to activate Hex expression in the endoderm is restricted to a region that is only slightly larger than the endogenous domain of Hex expression. Although Fgfs can substitute for the cardiogenic mesoderm to support the expression of Hex and albumin in the endoderm, several Fgf genes are expressed in the anterior lateral endoderm but an Fgf expressed predominantly in the mesoderm was not identified. Studies also showed that Fgf gene expression in the endoderm does not require a signal from the mesoderm. Mechanisms regulating endodermal signaling pathways activated by Fgfs may therefore be more complex than previously appreciated.     

Proteolipid protein 2 mRNA is expressed in the rabbit embryo during gastrulation

The expression pattern of the receptor tyrosine kinase gene EphB3 was examined during the early stages of chick embryogenesis, and is described in this report. In the gastrula, EphB3 is expressed in epiblast cells adjacent to and entering the anterior portion of the primitive streak; expression is extinguished once cells have ingressed. At headfold stages, EphB3 is strongly transcribed in the floor of the foregut and in anterior lateral endoderm, and is expressed in the subjacent cardiogenic mesoderm. EphB3 is transiently expressed in the lateral ectoderm, neural tube, and neural crest during these stages. Later neural expression is localized to the mesencephalon. In the somitic mesoderm, EphB3 is initially expressed in the sclerotome, but later is expressed predominantly in the dermatome. Prominent expression is also detected in the developing heart, liver, posterior ventral limb bud mesenchyme, pharyngeal arches, and head mesenchyme.     

Expression of the cardiac actin gene in axolotl embryos

Axolotis are an important model system for studying heart development. Patterning of the somitic mesoderm occurs in axolotis in a manner that is much more similar to the pattern observed in higher vertebrates than in Xenopus. For these reasons we cloned the axolotl cardiac actin gene, since this gene is expressed during the development of both somitic and cardiac muscle in other vertebrates. In this paper we characterize its expression. Expression of cardiac actin RNA is switched on during gastrula stages and appears in the somitic mesoderm when it is formed; expression is later activated in the embryonic heart. In adults the gene is expressed only in the heart. The results demonstrate that the clone encoding cardiac actin provides a useful marker for studying development of both skeletal and cardiac muscle development in axolotls.     

eFGF regulates Xbra expression during Xenopus gastrulation.

We show that, in addition to a role in mesoderm induction during blastula stages, FGF signalling plays an important role in maintaining the properties of the mesoderm in the gastrula of Xenopus laevis. eFGF is a maternally expressed secreted Xenopus FGF with potent mesoderm-inducing activity. However, it is most highly expressed in the mesoderm during gastrulation, suggesting a role after the period of mesoderm induction. eFGF is inhibited by the dominant negative FGF receptor. Embryos overexpressing the dominant negative receptor show a change of behaviour of the dorsal mesoderm such that it moves around the blastopore lip instead of elongating in an antero-posterior direction. In such embryos there is a reduction in Xbra expression during gastrulation. We show that during blastula stages eFGF and Xbra are able to activate the expression of each other, suggesting that they are components of an autocatalytic regulatory loop. Moreover, we show that Xbra expression in isolated gastrula mesoderm cells is maintained by eFGF, suggesting that eFGF continues to regulate the expression of Xbra in the blastopore region. In addition, overexpression of eFGF after the mid-blastula transition results in the up-regulation of Xbra expression during gastrula stages and causes suppression of the head and enlargement of the proctodeum, which is the converse of the posterior reductions of the FGF dominant negative receptor phenotype. These data suggest an important role for eFGF in regulating the expression of Xbra and for the eFGF-Xbra regulatory pathway in the control of mesodermal cell behaviour during gastrula stages.     

Gastrulation in the mouse: the role of the homeobox gene goosecoid.

Mouse goosecoid is a homeobox gene expressed briefly during early gastrulation. Its mRNA accumulates as a patch on the side of the epiblast at the site where the primitive streak is first formed. goosecoid-expressing cells are then found at the anterior end of the developing primitive streak, and finally in the anteriormost mesoderm at the tip of the early mouse gastrula, a region that gives rise to the head process. Treatment of early mouse embryos with activin results in goosecoid mRNA accumulation in the entire epiblast, suggesting that a localized signal induces goosecoid expression during development. Transplantation experiments indicate that the tip of the murine early gastrula is the equivalent of the organizer of the amphibian gastrula.     

Delta-Notch signaling induces hypochord development in zebrafish

Different cell types that occupy the midline of vertebrate embryos originate within the Spemann-Mangold or gastrula organizer. One such cell type is hypochord, which lies ventral to notochord in anamniote embryos. We show that hypochord precursors arise from the lateral edges of the organizer in zebrafish. During gastrulation, hypochord precursors are closely associated with no tail-expressing midline precursors and paraxial mesoderm, which expresses deltaC and deltaD. Loss-of-function experiments revealed that deltaC and deltaD were required for her4 expression in presumptive hypochord precursors and for hypochord development. Conversely, ectopic, unregulated Notch activity blocked no tail expression and promoted her4 expression. We propose that Delta signaling from paraxial mesoderm diversifies midline cell fate by inducing a subset of neighboring midline precursors to develop as hypochord, rather than as notochord.     

Zic3 is involved in the left-right specification of the Xenopus embryo

Establishment of left-right (L-R) asymmetry is fundamental to vertebrate development. Several genes involved in L-R asymmetry have been described. In the Xenopus embryo, Vg1/activin signals are implicated upstream of asymmetric nodal related 1 (Xnr1) and Pitx2 expression in L-R patterning. We report here that Zic3 carries the left-sided signal from the initial activin-like signal to determinative factors such as Pitx2. Overexpression of Zic3 on the right side of the embryo altered the orientation of heart and gut looping, concomitant with disturbed laterality of expression of Xnr1 and Pitx2, both of which are normally expressed in the left lateral plate mesoderm. The results indicate that Zic3 participates in the left-sided signaling upstream of Xnr1 and Pitx2. At early gastrula, Zic3 was expressed not only in presumptive neuroectoderm but also in mesoderm. Correspondingly, overexpression of Zic3 was effective in the L-R specification at the early gastrula stage, as revealed by a hormone-inducible Zic3 construct. The Zic3 expression in the mesoderm is induced by activin (beta) or Vg1, which are also involved in the left-sided signal in L-R specification. These findings suggest that an activin-like signal is a potent upstream activator of Zic3 that establishes the L-R axis. Furthermore, overexpression of the zinc-finger domain of Zic3 on the right side is sufficient to disturb the L-R axis, while overexpression of the N-terminal domain on the left side affects the laterality. These results suggest that Zic3 has at least two functionally important domains that play different roles and provide a molecular basis for human heterotaxy, which is an L-R pattern anomaly caused by a mutation in human ZIC3.     

Neural expression of the Xenopus homeobox gene Xhox3: evidence for a patterning neural signal that spreads through the ectoderm

The Xenopus laevis homeobox gene Xhox3 is expressed in the axial mesoderm of gastrula and neurula stage embryos. By the late neurula-early tailbud stage, mesodermal expression is no longer detectable and expression appears in the growing tailbud and in neural tissue. In situ hybridization analysis of the expression of Xhox3 in neural tissue shows that it is restricted within the neural tube and the cranial neural crest during the tailbud-early tadpole stages. In late tadpole stages, Xhox3 is only expressed in the mid/hindbrain area and can therefore be considered a marker of anterior neural development. To investigate the mechanism responsible for the anterior-posterior (A-P) regionalization of the neural tissue, the expression of Xhox3 has been analysed in total exogastrula. In situ hybridization analyses of exogastrulated embryos show that Xhox3 is expressed in the apical ectoderm of total exogastrulae, a region that develops in the absence of anterior axial mesoderm. The results provide further support for the existence of a neuralizing signal, which originates from the organizer region and spreads through the ectoderm. Moreover, the data suggest that this neural signal also has a role in A-P patterning the neural ectoderm.     

Antagonistic role of XESR1 and XESR5 in mesoderm formation in Xenopus laevis

The notch signaling pathway is widely conserved from vertebrates to invertebrates and mediates the specification of numerous cell fates during developmental processes. In the Xenopus gastrula embryo, Xdelta1, one of the Notch ligands, is expressed in the prospective mesoderm prior to Xbra expression. Here, we examined the role of Notch signaling in mesoderm formation. Embryos injected with Xdelta1 morpholino oligo DNA showed a severe gastrulation defect and suppression of Xbra expression, which were completely rescued by co-injection with the active form of Notch. In order to fully understand the role of Notch signaling, we examined the expression of the Notch target genes XESR1 and XESR5. RT-PCR and whole-mount in situ hybridization analyses showed that XESR5 was highly expressed in the marginal zone of the early gastrula embryo, whereas expression of XESR1 was not detected. Animal cap assays indicated that expression of XESR5 was not induced by Notch signaling but by nodal signaling. To clarify the role of XESR5 in the gastrula embryo, a dominant negative form of XESR5 was injected into the prospective mesoderm. The truncated form of XESR5 induced the ectopic expression of XESR1, which caused a decrease in Xbra expression and defective gastrulation. In contrast, the truncated form of XESR1 caused an upregulation of XESR5 resulting in an increase in Xbra expression. The antagonistic effect of XESR1 and XESR5 suggests a dual regulation in which XESR5 produces a competent area for mesoderm formation by suppressing the gene expression of XESR1, while XESR1 sharpens the boundary of Xbra expression.     

Pintallavis, a gene expressed in the organizer and midline cells of frog embryos: involvement in the development of the neural axis.

We have identified a novel frog gene, Pintallavis (the Catalan for lipstick), that is related to the fly fork head and rat HNF-3 genes. Pintallavis is expressed in the organizer region of gastrula embryos as a direct zygotic response to dorsal mesodermal induction. Subsequently, Pintallavis is expressed in axial midline cells of all three germ layers. In axial mesoderm expression is graded with highest levels posteriorly. Midline neural plate cells that give rise to the floor plate transiently express Pintallavis, apparently in response to induction by the notochord. Overexpression of Pintallavis perturbs the development of the neural axis, suppressing the differentiation of anterior and dorsal neural cell types but causing an expansion of the posterior neural tube. Our results suggest that Pintallavis functions in the induction and patterning of the neural axis.     

Expression of zebrafish btg-b, an anti-proliferative cofactor, during early embryogenesis

BTG/tob family proteins are thought to be a potential tumor suppressor due to their anti-proliferative activity. We cloned zebrafish btg-b, a member of the BTG1/2 subfamily, using in situ hybridization screening. The tissue-specific expression of btg-b is first observed in the organizer region at the early gastrula stage. Later in development, the forebrain, the hindbrain, the polster and the paraxial mesoderm transiently express btg-b. Recently, mouse Btg1 and Btg2 have been shown to be a cofactor for Hoxb9. Double in situ hybridization with zebrafish btg-b and hoxb9a indicates that the expression domains of these two genes overlap in the posterior paraxial mesoderm.