Injury-induced cooperation of InhibinßA and JunB is essential for cell proliferation in Xenopus tadpole tail regeneration.

In animal species that have the capability of regenerating tissues and limbs, cell proliferation is enhanced after wound healing and is essential for the reconstruction of injured tissue. Although the ability to induce cell proliferation is a common feature of such species, the molecular mechanisms that regulate the transition from wound healing to regenerative cell proliferation remain unclear. Here, we show that upon injury, InhibinßA and JunB cooperatively function for this transition during Xenopus tadpole tail regeneration. We found that the expression of inhibin subunit beta A (inhba) and junB proto-oncogene (junb) is induced by injury-activated TGF-ß/Smad and MEK/ERK signaling in regenerating tails. Similarly to junb knockout (KO) tadpoles, inhba KO tadpoles show a delay in tail regeneration, and inhba/junb double KO (DKO) tadpoles exhibit severe impairment of tail regeneration compared with either inhba KO or junb KO tadpoles. Importantly, this impairment is associated with a significant reduction of cell proliferation in regenerating tissue. Moreover, JunB regulates tail regeneration via FGF signaling, while InhibinßA likely acts through different mechanisms. These results demonstrate that the cooperation of injury-induced InhibinßA and JunB is critical for regenerative cell proliferation, which is necessary for re-outgrowth of regenerating Xenopus tadpole tails.

Zbtb21 is required for the anterior-posterior patterning of neural tissue in the early Xenopus embryo.

The vertebrate body is organized along the dorsal-ventral (DV) and anterior-posterior (AP) axes by the BMP and Wnt pathways, respectively. We previously reported that Xenopus Zbtb14 promotes dorsalization (neural induction) of ectoderm by inhibiting BMP signaling and also posteriorizes neural tissue by activating Wnt signaling, thereby coordinating the patterning of the DV and AP axes during early development. Although it has been reported that human ZBTB21 binds to ZBTB14 and is involved in gene expression in cultured mammalian cells, the function of Zbtb21 in early embryonic development remains unknown. Here, we show that Xenopus Zbtb21 plays an essential role in AP axis formation in the early Xenopus embryo. zbtb21 and zbtb14 are co-expressed in the dorsal region of embryos during gastrulation. Simultaneous overexpression of Zbtb21 and Zbtb14 in ectodermal explants enhances the neural-inducing activity of Zbtb14. Moreover, knockdown experiments showed that Zbtb21 is required for the formation of posterior neural tissue and the suppression of anterior neural development. Collectively, these results suggest that in cooperation with Zbtb14, Zbtb21 has a crucial function in AP patterning during early Xenopus embryogenesis.

The dual-specificity protein kinase Clk3 is essential for Xenopus neural development.

During vertebrate development, the formation of the central nervous system (CNS) is initiated by neural induction and patterning of the embryonic ectoderm. We previously reported that Cdc2-like kinase 2 (Clk2) promotes neural development in Xenopus embryos by regulating morphogen signaling. However, the functions of other Clk family members and their roles in early embryonic development remain unknown. Here, we show that in addition to Clk2, Clk1 and Clk3 play a role in the formation of neural tissue in Xenopus. clk1 and clk3 are co-expressed in the developing neural tissue during early Xenopus embryogenesis. We found that overexpression of clk1 and clk3 increases the expression of neural marker genes in ectodermal explants. Furthermore, knockdown experiments showed that clk3 is required for the formation of neural tissues. These results suggest that Xenopus Clk3 plays an essential role in promoting neural development during early embryogenesis.

TGF-ß1 signaling is essential for tissue regeneration in the Xenopus tadpole tail.

Amphibians such as Xenopus tropicalis exhibit a remarkable capacity for tissue regeneration after traumatic injury. Although transforming growth factor-ß (TGF-ß) receptor signaling is known to be essential for tissue regeneration in fish and amphibians, the role of TGF-ß ligands in this process is not well understood. Here, we show that inhibition of TGF-ß1 function prevents tail regeneration in Xenopus tropicalis tadpoles. We found that expression of tgfb1 is present before tail amputation and is sustained throughout the regeneration process. CRISPR-mediated knock-out (KO) of tgfb1 retards tail regeneration; the phenotype of tgfb1 KO tadpoles can be rescued by injection of tgfb1 mRNA. Cell proliferation, a critical event for the success of tissue regeneration, is downregulated in tgfb1 KO tadpoles. In addition, tgfb1 KO reduces the expression of phosphorylated Smad2/3 (pSmad2/3) which is important for TGF-ß signal-mediated cell proliferation. Collectively, our results show that TGF-ß1 regulates cell proliferation through the activation of Smad2/3. We therefore propose that TGF-ß1 plays a critical role in TGF-ß receptor-dependent tadpole tail regeneration in Xenopus.

Intracellular Communication among Morphogen Signaling Pathways during Vertebrate Body Plan Formation.

During embryonic development in vertebrates, morphogens play an important role in cell fate determination and morphogenesis. Bone morphogenetic proteins (BMPs) belonging to the transforming growth factor-ß (TGF-ß) family control the dorsal-ventral (DV) patterning of embryos, whereas other morphogens such as fibroblast growth factor (FGF), Wnt family members, and retinoic acid (RA) regulate the formation of the anterior-posterior (AP) axis. Activation of morphogen signaling results in changes in the expression of target genes including transcription factors that direct cell fate along the body axes. To ensure the correct establishment of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated by a fine-tuning of morphogen signaling. In this review, we focus on the interplay of various intracellular regulatory mechanisms and discuss how communication among morphogen signaling pathways modulates body axis formation in vertebrate embryos.

The AP-1 transcription factor JunB functions in Xenopus tail regeneration by positively regulating cell proliferation.

Xenopus tropicalis tadpoles can regenerate an amputated tail, including spinal cord, muscle and notochord, through cell proliferation and differentiation. However, the molecular mechanisms that regulate cell proliferation during tail regeneration are largely unknown. Here we show that JunB plays an important role in tail regeneration by regulating cell proliferation. The expression of junb is rapidly activated and sustained during tail regeneration. Knockout (KO) of junb causes a delay in tail regeneration and tissue differentiation. In junb KO tadpoles, cell proliferation is prevented before tissue differentiation. Furthermore, TGF-ß signaling, which is activated just after tail amputation, regulates the induction and maintenance of junb expression. These findings demonstrate that JunB, a downstream component of TGF-ß signaling, works as a positive regulator of cell proliferation during Xenopus tail regeneration.

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos.

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.

Coordinated regulation of the dorsal-ventral and anterior-posterior patterning of Xenopus embryos by the BTB/POZ zinc finger protein Zbtb14.

During early vertebrate embryogenesis, bone morphogenetic proteins (BMPs) belonging to the transforming growth factor-ß (TGF-ß) family of growth factors play a central role in dorsal-ventral (DV) patterning of embryos, while other growth factors such as Wnt and fibroblast growth factor (FGF) family members regulate formation of the anterior-posterior (AP) axis. Although the establishment of body plan is thought to require coordinated formation of the DV and AP axes, the mechanistic details underlying this coordination are not well understood. Here, we show that a Xenopus homologue of zbtb14 plays an essential role in the regulation of both DV and AP patterning during early Xenopus development. We show that overexpression of Zbtb14 promotes neural induction and inhibits epidermal differentiation, thereby regulating DV patterning. In addition, Zbtb14 promotes the formation of posterior neural tissue and suppresses anterior neural development. Consistent with this, knock-down experiments show that Zbtb14 is required for neural development, especially for the formation of posterior neural tissues. Mechanistically, Zbtb14 reduces the levels of phosphorylated Smad1/5/8 to suppress BMP signaling and induces an accumulation of ß-Catenin to promote Wnt signaling. Collectively, these results suggest that Zbtb14 plays a crucial role in the formation of DV and AP axes by regulating both the BMP and Wnt signaling pathways during early Xenopus embryogenesis.

Genome organization of the vg1 and nodal3 gene clusters in the allotetraploid frog Xenopus laevis.

Extracellular factors belonging to the TGF-ß family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-ß family genes in response to allotetraploidization.

Genomic organization and modulation of gene expression of the TGF-ß and FGF pathways in the allotetraploid frog Xenopus laevis.

Inductive interactions mediated by the TGF-ß and FGF-MAPK pathways are essential for specification of the germ layers and embryonic body axes during early vertebrate embryogenesis. TGF-ß and FGF ligands signal through receptor Ser/Thr and Tyr kinases, respectively, and these signaling pathways cross-talk to regulate transcription and cell behavior. The allotetraploid Xenopus laevis and its ancestral diploid Xenopus tropicalis are versatile model organisms with which to study the inductive interactions and mechanisms of these signal transduction pathways. Here we have analyzed the draft genome of X. laevis with respect to the genomic organization and differential expression of genes in the TGF-ß and FGF pathways. Genomic structure and gene expression analyses of pathway components in X. laevis revealed that genetic modulations, including deletions resulting in singletons and differential expression of homeologs, have occurred frequently among extracellular regulatory factors of the TGF-ß pathway after allotetraploidization. Moreover, differential gene expression was found for factors regulating various cellular responses including co-receptors, decoy receptors, and intracellular negative regulators in both the TGF-ß and FGF-MAPK pathways. We summarize the patterns of genetic alterations in the allotetraploid frog X. laevis and discuss the importance of these changes with regard to developmental processes.

Involvement of JunB Proto-Oncogene in Tail Formation During Early Xenopus Embryogenesis.

Integration of signaling pathways is important for the establishment of the body plan during embryogenesis. However, little is known about how the multiple signals interact to regulate morphogenesis. Here, we show that junb is expressed in the posterior neural plate and the caudal fin during Xenopus embryogenesis and that overexpression of wild-type JunB induces small head phenotypes and ectopic tail-like structures. A mutant form of JunB that lacked GSK3 and MAPK phosphorylation sites showed stronger tail-like structure-inducing activity than wild-type JunB. Moreover, the mutant JunB induced expression of tailbud and neural marker genes, but not somite and chordoneural hinge (CNH) marker genes in ectopic tail-like structures. In ectodermal explants of Xenopus embryos, overexpression of JunB increased the expression of tailbud and posterior marker genes including fgf3, xbra (t) and wnt8. These results indicate that JunB is capable of inducing the ectopic formation of tissues similar to the tailbud, and that the tailbud-inducing activity of JunB is likely to be regulated by FGF and Wnt pathways. Overall, our results suggest that JunB is a regulator of tail organization possibly through integration of several morphogen signaling pathways.

The forkhead transcription factor FoxB1 regulates the dorsal-ventral and anterior-posterior patterning of the ectoderm during early Xenopus embryogenesis.

The formation of the dorsal-ventral (DV) and anterior-posterior (AP) axes, fundamental to the body plan of animals, is regulated by several groups of polypeptide growth factors including the TGF-ß, FGF, and Wnt families. In order to ensure the establishment of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated. However, the molecular mechanisms responsible for these interactions remain unclear. Here, we demonstrate that the forkhead box transcription factor FoxB1, which is upregulated by the neuralizing factor Oct-25, plays an important role in the formation of the DV and AP axes. Overexpression of FoxB1 promoted neural induction and inhibited BMP-dependent epidermal differentiation in ectodermal explants, thereby regulating the DV patterning of the ectoderm. In addition, FoxB1 was also found to promote the formation of posterior neural tissue in both ectodermal explants and whole embryos, suggesting its involvement in embryonic AP patterning. Using knockdown analysis, we found that FoxB1 is required for the formation of posterior neural tissues, acting in concert with the Wnt and FGF pathways. Consistent with this, FoxB1 suppressed the formation of anterior structures via a process requiring the function of XWnt-8 and eFGF. Interestingly, while downregulation of FoxB1 had little effect on neural induction, we found that it functionally interacted with its upstream factor Oct-25 and plays a supportive role in the induction and/or maintenance of neural tissue. Our results suggest that FoxB1 is part of a mechanism that fine-tunes, and leads to the coordinated formation of, the DV and AP axes during early development.

The Xenopus POU class V transcription factor XOct-25 inhibits ectodermal competence to respond to bone morphogenetic protein-mediated embryonic induction.

Bone morphogenetic proteins (BMPs) have been shown to play a key role in controlling ectodermal cell fates by inducing epidermis at the expense of neural tissue during gastrulation. Here, we present evidence that the Xenopus POU class V transcription factor XOct-25 regulates ectodermal cell fate decisions by inhibiting the competence of ectodermal cells to respond to BMP during Xenopus embryogenesis. When overexpressed in the ectoderm after the blastula stage, XOct-25 suppressed early BMP responses of ectodermal cells downstream of BMP receptor activation and promoted neural induction while suppressing epidermal differentiation. In contrast, inhibition of XOct-25 function in the prospective neuroectoderm resulted in expansion of epidermal ectoderm at the expense of neuroectoderm. The reduction of neural tissue by inhibition of XOct-25 function could be rescued by decreasing endogenous BMP signaling, suggesting that XOct-25 plays a role in the formation of neural tissue at least in part by inhibiting BMP-mediated epidermal induction (neural inhibition). This hypothesis is supported by the observation that ectodermal cells from XOct-25 morphants were more sensitive to BMP signaling than cells from controls in inducing both immediate early BMP target genes and epidermis at the expense of neural tissue, while cells overexpressing XOct-25 are less competent to respond to BMP-mediated induction. These results document an essential role for XOct-25 in commitment to neural or epidermal cell fates in the ectoderm and highlight the importance of a regulatory mechanism that limits competence to respond to BMP-mediated embryonic induction.

Induction and patterning of the Purkinje fibre network.

Impulse-conducting Purkinje cells differentiate from myocytes during embryogenesis. In the embryonic chicken heart, this conversion of contractile myocytes into conduction cells occurs subendocardially and periarterially. The unique sites of Purkinje fibre differentiation suggest that a shear stress-induced paracrine signal from the endocardium and arterial beds may induce adjacent myocytes to differentiate into conduction cells. Consistent with this model, Purkinje fibre marker genes can be induced in cultured embryonic myocytes by endothelin (ET), an endothelial cell-derived signalling peptide. This inductive response is, however, gradually lost from myocytes as embryos develop, and mature myocytes express only genes characteristic of hypertrophy in response to ET. In vivo, active ET is produced, through proteolytic processing, from its precursor by ET-converting enzyme 1 (ECE1) and triggers signalling by binding to its receptors, ETA and ETB. In the embryonic heart, the expression of these ET signalling components changes dynamically, defining the site and timing of Purkinje fibre differentiation within the ventricular myocardium during chick embryogenesis.

Interplay between the tumor suppressor p53 and TGF beta signaling shapes embryonic body axes in Xenopus.

The transcription factor p53 has been shown to mediate cellular responses to diverse stresses such as DNA damage. However, the function of p53 in cellular differentiation in response to growth factor stimulations has remained obscure. We present evidence that p53 regulates cellular differentiation by modulating signaling of the TGF beta family of growth factors during early Xenopus embryogenesis. We show that p53 functionally and physically interacts with the activin and bone morphogenetic protein pathways to directly induce the expression of the homeobox genes Xhox3 and Mix.1/2. Furthermore, functional knockdown of p53 in embryos by an antisense morpholino oligonucleotide reveals that p53 is required for the development of dorsal and ventral mesoderm. Our data illustrate a pivotal role of interplay between the p53 and TGF beta pathways in cell fate determination during early vertebrate embryogenesis.

Competency of embryonic cardiomyocytes to undergo Purkinje fiber differentiation is regulated by endothelin receptor expression.

Purkinje fibers of the cardiac conduction system differentiate from heart muscle cells during embryogenesis. In the avian heart, Purkinje fiber differentiation takes place along the endocardium and coronary arteries. To date, only the vascular cytokine endothelin (ET) has been demonstrated to induce embryonic cardiomyocytes to differentiate into Purkinje fibers. This ET-induced Purkinje fiber differentiation is mediated by binding of ET to its transmembrane receptors that are expressed by myocytes. Expression of ET converting enzyme 1, which produces a biologically active ET ligand, begins in cardiac endothelia, both arterial and endocardial, at initiation of conduction cell differentiation and continues throughout heart development. Yet, the ability of cardiomyocytes to convert their phenotype in response to ET declines as embryos mature. Therefore, the loss of responsiveness to the inductive signal appears not to be associated with the level of ET ligand in the heart. This study examines the role of ET receptors in this age-dependent loss of inductive responsiveness and the expression profiles of three different types of ET receptors, ET(A), ET(B) and ET(B2), in the embryonic chick heart. Whole-mount in situ hybridization analyses revealed that ET(A) was ubiquitously expressed in both ventricular and atrial myocardium during heart development, while ET(B) was predominantly expressed in the atrium and the left ventricle. ET(B2) expression was detected in valve leaflets but not in the myocardium. RNase protection assays showed that ventricular expression of ET(A) and ET(B) increased until Purkinje fiber differentiation began. Importantly, the levels of both receptor isotypes decreased after this time. Retrovirus-mediated overexpression of ET(A) in ventricular myocytes in which endogenous ET receptors had been downregulated, enhanced their responsiveness to ET, allowing them to differentiate into conduction cells. These results suggest that the developmentally regulated expression of ET receptors plays a crucial role in determining the competency of ventricular myocytes to respond to inductive ET signaling in the chick embryo.