Role of TrkA signaling during tadpole tail regeneration and early embryonic development in Xenopus laevis.

Neurotrophic signaling regulates neural cell behaviors in development and physiology, although its role in regeneration has not been fully investigated. Here, we examined the role of neurotrophic signaling in Xenopus laevis tadpole tail regeneration. After the tadpole tails were amputated, the expression of neurotrophin ligand family genes, especially ngf and bdnf, was up-regulated as regeneration proceeded. Moreover, notochordal expression of the NGF receptor gene TrkA, but not that of other neurotrophin receptor genes TrkB and TrkC, became prominent in the regeneration bud, a structure arising from the tail stump after tail amputation. The regenerated tail length was significantly shortened by the pan-Trk inhibitor K252a or the TrkA inhibitor GW-441756, but not by the TrkB inhibitor ANA-12, suggesting that TrkA signaling is involved in elongation of regenerating tails. Furthermore, during Xenopus laevis embryonic development, TrkA expression was detected in the dorsal mesoderm at the gastrula stage and in the notochord at the neurula stage, and its knockdown led to gastrulation defects with subsequent shortening of the body axis length. These results suggest that Xenopus laevis TrkA signaling, which can act in the mesoderm/notochord, plays a key role in body axis elongation during embryogenesis as well as tail elongation during tadpole regeneration.

The atypical mitogen-activated protein kinase ERK3 is essential for establishment of epithelial architecture.

Epithelia contribute to physical barriers that protect internal tissues from the external environment and also support organ structure. Accordingly, establishment and maintenance of epithelial architecture are essential for both embryonic development and adult physiology. Here, using gene knockout and knockdown techniques along with gene profiling, we show that extracellular signal-regulated kinase 3 (ERK3), a poorly characterized atypical mitogen-activated protein kinase (MAPK), regulates the epithelial architecture in vertebrates. We found that in Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight-junction protein distribution, as well as tight-junction barrier function, resulting in epidermal breakdown. Moreover, in human epithelial breast cancer cells, inhibition of ERK3 expression induced thickened epithelia with aberrant adherens and tight junctions. Results from microarray analyses suggested that transcription factor AP-2α (TFAP2A), a transcriptional regulator important for epithelial gene expression, is involved in ERK3-dependent changes in gene expression. Of note, TFAP2A knockdown phenocopied ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 was required for full activation of TFAP2A-dependent transcription. Our findings reveal that ERK3 regulates epithelial architecture, possibly together with TFAP2A.

The E3 ubiquitin ligase Hace1 is required for early embryonic development in Xenopus laevis.

BACKGROUND: HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) regulates a wide variety of cellular processes. It has been shown that one of the targets of HACE1 is the GTP-bound form of the small GTPase Rac1. However, the role of HACE1 in early development remains unknown. RESULTS: In situ hybridization revealed that Xenopus laevis hace1 is specifically expressed in the ectoderm at the blastula and gastrula stages and in the epidermis, branchial arch, kidney, and central nervous system at the tailbud stage. Knockdown of hace1 in Xenopus laevis embryos via antisense morpholino oligonucleotides led to defects in body axis elongation, pigment formation, and eye formation at the tadpole stage. Experiments with Keller sandwich explants showed that hace1 knockdown inhibited convergent extension, a morphogenetic movement known to be crucial for body axis elongation. In addition, time lapse imaging of whole embryos during the neurula stage indicated that hace1 knockdown also delayed neural tube closure. The defects caused by hace1 knockdown were partly rescued by knockdown of rac1. Moreover, embryos expressing a constitutively active form of Rac1 displayed phenotypes similar to those of embryos with hace1 knocked down. CONCLUSIONS: Our results suggest that Xenopus laevis hace1 plays an important role in early embryonic development, possibly via regulation of Rac1 activity.

cnrip1 is a regulator of eye and neural development in Xenopus laevis.

Cannabinoid receptor interacting protein 1 (CNRIP1), which has been originally identified as the binding partner of cannabinoid receptor 1 (CNR1), is evolutionarily conserved throughout vertebrates, but its physiological function has been unknown. Here, we identify a developmental role of CNRIP1 using Xenopus laevis embryos. During early embryogenesis, expression of Xenopus laevis cnrip1 is highly restricted to the animal region of gastrulae where neural and eye induction occur, and afterward it is seen in neural and other tissues with a temporally and spatially regulated pattern. Morpholino-mediated knockdown experiments indicate that cnrip1 has an essential role in early eye and neural development by regulating the onset of expression of key transcription factor genes, sox2, otx2, pax6 and rax. Also, over-expression experiments suggest that cnrip1 has a potential to expand sox2, otx2, pax6 and rax expression. These results suggest an instructive role of Xenopus laevis cnrip1 in early eye and neural development. Furthermore, Xenopus laevis cnr1 knockdown leads to eye defects, which are partly similar to, but milder than, those caused by cnrip1 knockdown, suggesting a possible functional similarity between CNRIP1 and CNR1. This study is the first characterization of an in vivo role of CNRIP1 in the context of whole organisms.

The protein kinase MLTK regulates chondrogenesis by inducing the transcription factor Sox6.

Sox9 acts together with Sox5 or Sox6 as a master regulator for chondrogenesis; however, the inter-relationship among these transcription factors remains unclear. Here, we show that the protein kinase MLTK plays an essential role in the onset of chondrogenesis through triggering the induction of Sox6 expression by Sox9. We find that knockdown of MLTK in Xenopus embryos results in drastic loss of craniofacial cartilages without defects in neural crest development. We also find that Sox6 is specifically induced during the onset of chondrogenesis, and that the Sox6 induction is inhibited by MLTK knockdown. Remarkably, Sox6 knockdown phenocopies MLTK knockdown. Moreover, we find that ectopic expression of MLTK induces Sox6 expression in a Sox9-dependent manner. Our data suggest that p38 and JNK pathways function downstream of MLTK during chondrogenesis. These results identify MLTK as a novel key regulator of chondrogenesis, and reveal its action mechanism in chondrocyte differentiation during embryonic development.

A transmembrane protein EIG121L is required for epidermal differentiation during early embryonic development.

Epidermal differentiation in the ventral ectoderm of Xenopus embryos is regulated by the bone morphogenetic protein (BMP) pathway. However, it remains unclear how the BMP pathway is activated and induces the epidermal fate in the ventral ectoderm. Here, we identify a novel player in the BMP pathway that is required for epidermal differentiation during Xenopus early embryonic development. We show that Xenopus EIG121L (xEIG121L) protein, an evolutionarily conserved transmembrane protein, is expressed in the ventral ectoderm at the gastrula and neurula stages. Almost complete knockdown of xEIG121L protein with antisense morpholino oligonucleotides in early Xenopus embryos results in severe developmental defects, including the inhibition of epidermal differentiation and the induction of neural genes. Remarkably, our analysis shows that BMP/Smad1 signaling is severely suppressed in the xEIG121L knockdown ectoderm. Moreover, immunoprecipitation and immunostaining experiments suggest that xEIG121L protein physically interacts, and co-localizes, with BMP receptors. Thus, our results identify a novel regulator of the BMP pathway that has a positive role in BMP signaling and plays an essential role in epidermal differentiation during early embryonic development.

XGAP, an ArfGAP, is required for polarized localization of PAR proteins and cell polarity in Xenopus gastrulation.

To dissect the molecular mechanisms underlying convergent extension (CE), a prominent set of cell movements during Xenopus gastrulation, we performed a functional expression screen and identified a GTPase-activating protein for ADP ribosylation factors (ArfGAP), which we termed XGAP. We demonstrated that XGAP is required to confine or restrict the cellular protrusive activity to the mediolateral ends of cells, where XGAP is normally localized, and therefore for the proper intercalation of cells participating in CE. We also demonstrated that a C-terminal conserved domain of XGAP, but not its GAP activity, is required and sufficient for this intracellular localization and function. We further showed that XGAP physically interacts with the known polarity proteins 14-3-3epsilon, aPKC, and PAR-6 and directs them to the mediolateral ends of dorsal mesoderm cells during gastrulation. We propose that XGAP controls CE through the restriction and maintenance of partitioning-defective (PAR) proteins in the regions that harbor protrusive activity.

Sef is a spatial regulator for Ras/MAP kinase signaling.

Spatiotemporal control of the Ras/ERK MAP kinase signaling pathway is among the key mechanisms for regulating a wide variety of cellular processes. In this study, we report that human Sef (hSef), a recently identified inhibitor whose action mechanism has not been fully defined, acts as a molecular switch for ERK signaling by specifically blocking ERK nuclear translocation without inhibiting its activity in the cytoplasm. Thus, hSef binds to activated forms of MEK, inhibits the dissociation of the MEK-ERK complex, and blocks nuclear translocation of activated ERK. Consequently, hSef inhibits phosphorylation and activation of the nuclear ERK substrate Elk-1, while it does not affect phosphorylation of the cytoplasmic ERK substrate RSK2. Downregulation of endogenous hSef by hSef siRNA enhances the stimulus-induced ERK nuclear translocation and the activity of Elk-1. These results thus demonstrate that hSef acts as a spatial regulator for ERK signaling by targeting ERK to the cytoplasm.

ERK7 regulates ciliogenesis by phosphorylating the actin regulator CapZIP in cooperation with Dishevelled.

Cilia are essential for embryogenesis and maintenance of homeostasis, but little is known about the signalling pathways that regulate ciliogenesis. Here, we identify ERK7, an atypical mitogen-activated protein kinase, as a key regulator of ciliogenesis. ERK7 is strongly expressed in ciliated tissues of Xenopus embryos. ERK7 knockdown markedly diminishes both the number and the length of cilia in multiciliated cells, and it inhibits the apical migration of basal bodies. Moreover, ERK7 knockdown results in a loss of the apical actin meshwork, which is required for the proper migration of basal bodies. We find that the actin regulator CapZIP, which has been shown to regulate ciliogenesis in a phosphorylation-dependent manner, is an ERK7 substrate, and that Dishevelled, which has also been shown to regulate ciliogenesis, facilitates ERK7 phosphorylation of CapZIP through binding to both ERK7 and CapZIP. Collectively, these results identify an ERK7/Dishevelled/CapZIP axis that regulates ciliogenesis.

mab21-l3 regulates cell fate specification of multiciliate cells and ionocytes.

Cell fate specifications of multiciliate cells (MCCs) and ionocytes are commonly suppressed by the Notch pathway in developing epithelia, but are governed by different master regulators, suggesting the existence of a common regulator linking the Notch pathway to both MCC and ionocyte specifications. Here we show that a mab21 family gene, mab21-l3, represents the missing link. In Xenopus embryonic epidermis, mab21-l3 expression is specifically found in MCCs and ionocytes and is downregulated by the Notch pathway. Knockdown of mab21-l3 in Xenopus downregulates both MCC-specific and ionocyte-specific master genes, resulting in drastic loss of MCCs and ionocytes. In mouse tracheal epithelial cells, mab21-l3 expression is also downregulated by the Notch pathway and is required for MCC differentiation. Moreover, conditional gain of function of mab21-l3 rescues Notch-induced loss of MCCs and ionocytes in Xenopus. These results indicate that mab21-l3 acts downstream of the Notch pathway in cell fate specifications of MCCs and ionocytes.

Identification and characterization of Xenopus kctd15, an ectodermal gene repressed by the FGF pathway.

The FGF pathway regulates a variety of developmental processes in animals through activation and/or repression of numerous target genes. Here we have identified a Xenopus homolog of potassium channel tetramerization domain containing 15 (KCTD15) as an FGF-repressed gene. Kctd15 expression is first detected at the gastrula stage and gradually increases until the tadpole stage. Whole-mount in situ hybridization reveals that the spatial expression of kctd15 is tightly regulated during early embryogenesis. While kctd15 is uniformly expressed throughout the presumptive ectoderm at the early gastrula stage, its expression becomes restricted to the non-neural ectoderm and is excluded from the neural plate at the early neurula stage. At the mid-neurula stage, kctd15 shows a more restricted distribution pattern in regions that are located at the anterior, lateral or medial edge of the neural fold, including the preplacodal ectoderm, the craniofacial neural crest and the prospective roof plate. At the tailbud stage, kctd15 expression is mainly detected in neural crest- or placode-derived tissues that are located around the eye, including the mandibular arch, trigeminal ganglia and the olfactory placode. FGF represses kctd15 expression in ectodermal explants, and the inhibition of FGF receptor with a chemical compound dramatically expands the region expressing kctd15 in whole embryos. Dorsal depletion of kctd15 in Xenopus embryos leads to bent axes with reduced head structures, defective eyes and abnormal somites, while ventral depletion causes defects in ventral and caudal morphologies. These results suggest that kctd15 is an FGF-repressed ectodermal gene required for both dorsal and ventral development.

The kinase SGK1 in the endoderm and mesoderm promotes ectodermal survival by down-regulating components of the death-inducing signaling complex.

A balance between cell survival and apoptosis is essential for animal development. Although proper development involves multiple interactions between germ layers, little is known about the intercellular and intertissue signaling pathways that promote cell survival in neighboring or distant germ layers. We found that serum- and glucocorticoid-inducible kinase 1 (SGK1) promoted ectodermal cell survival during early Xenopus embryogenesis through a non-cell-autonomous mechanism. Dorsal depletion of SGK1 in Xenopus embryos resulted in shortened axes and reduced head structures with defective eyes, and ventral depletion led to defective tail morphologies. Although the gene encoding SGK1 was mainly expressed in the endoderm and dorsal mesoderm, knockdown of SGK1 caused excessive apoptosis in the ectoderm. SGK1-depleted ectodermal explants showed little or no apoptosis, suggesting non-cell-autonomous effects of SGK1 on ectodermal cells. Microarray analysis revealed that SGK1 knockdown increased the expression of genes encoding FADD (Fas-associated death domain protein) and caspase-10, components of the death-inducing signaling complex (DISC). Inhibition of DISC function suppressed excessive apoptosis in SGK1-knockdown embryos. SGK1 acted through the transcription factor nuclear factor κB (NF-κB) to stimulate production of bone morphogenetic protein 7 (BMP7), and overexpression of BMP7 in SGK1-knockdown embryos reduced the abundance of DISC components. We show that phosphoinositide 3-kinase (PI3K) functioned upstream of SGK1, thus revealing an endodermal and mesodermal pathway from PI3K to SGK1 to NF-κB that produces BMP7, which promotes ectodermal survival by decreasing DISC function.

Expression of estrogen induced gene 121-like (EIG121L) during early Xenopus development.

Estrogen induced gene 121 (EIG121) and EIG121-like (EIG121L) are evolutionarily conserved genes. But, their function is still unknown. Here, we report the expression pattern of Xenopus EIG121-like (xEIG121L) during early development. Its expression was first detected at stage 9 after mid-blastula transition, attained its maximal level at the gastrula stage, and remained constant until the tadpole stage. Whole-mount in situ hybridization revealed that xEIG121L was expressed strongly in the ventral ectoderm at the gastrula stage, and in the anterior ectoderm surrounding the neural plate at the neurula stage. xEIG121L expression was especially high in the presumptive hatching gland and cement gland regions in the neurula. At the tailbud stage, xEIG121L expression was limited to the hatching gland; an inverted Y type staining, characteristic of the hatching gland, was observed. However, at the tadpole stage, xEIG121L was expressed broadly in the head, heart and fin.

Requirement of the MEK5-ERK5 pathway for neural differentiation in Xenopus embryonic development.

Although previous studies have identified several key transcription factors in the generation process of the vertebrate nervous system, the intracellular signalling pathways that function in this process have remained unclear. Here we identify the evolutionarily conserved mitogen-activated protein kinase kinase 5 (MEK5)-extracellular signal-regulated kinase 5 (ERK5) pathway as an essential regulator in neural differentiation. Knockdown of Xenopus ERK5 or Xenopus MEK5 with antisense morpholino oligonucleotides results in the reduced head structure and inhibition of neural differentiation. Moreover, forced activation of the MEK5-ERK5 module on its own induces neural differentiation. In addition, we show that the MEK5-ERK5 pathway is necessary for the neuralizing activity of SoxD, a regulator of neural differentiation, and is sufficient for the expression of Xngnr1, a proneural gene. These results show that the MEK5-ERK5 pathway has an essential role in the regulation of neural differentiation downstream of SoxD and upstream of Xngnr1.

Xenopus ILK (integrin-linked kinase) is required for morphogenetic movements during gastrulation.

It has been suggested that ILK (integrin-linked kinase) participates in integrin- and growth factor-mediated signaling pathways and also functions as a scaffold protein at cell-extracellular matrix (ECM) adhesion sites. As the recently reported ILK knockout mice were found to die at the peri-implantation stage, the stage specific to mammals, little is known about the function of ILK in early developmental processes common to every vertebrate. To address this, we isolated a Xenopus ortholog of ILK (XeILK) and characterized its role in early Xenopus embryogenesis. XeILK was expressed constitutively and ubiquitously throughout the early embryogenesis. Depletion of XeILK with morpholino oligonucleotides (XeILK MO) caused severe defects in blastopore closure and axis elongation without affecting the mesodermal specification. Furthermore, XeILK MO was found to interfere with cell-cell and cell-ECM adhesions in dorsal marginal zone explants and to result in a significant loss of cell-ECM adhesions in activin-treated dissociated animal cap cells. These results thus indicate that XeILK plays an essential role in morphogenetic movements during gastrulation.

The polarity-inducing kinase Par-1 controls Xenopus gastrulation in cooperation with 14-3-3 and aPKC.

Par (partitioning-defective) genes were originally identified in Caenorhabditis elegans as determinants of anterior/posterior polarity. However, neither their function in vertebrate development nor their action mechanism has been fully addressed. Here we show that two members of Par proteins, 14-3-3 (Par-5) and atypical PKC (aPKC), regulate the serine/threonine kinase Par-1 to control Xenopus gastrulation. We find first that Xenopus Par-1 (xPar-1) is essential for gastrulation but not for cell fate specification during early embryonic development. We then find that xPar-1 binds to 14-3-3 in an aPKC-dependent manner. Our analyses identify two aPKC phosphorylation sites in xPar-1, which are essential for 14-3-3 binding and for proper gastrulation movements. The aPKC phosphorylation-dependent binding of xPar-1 to 14-3-3 does not markedly affect the kinase activity of xPar-1, but induces relocation of xPar-1 from the plasma membranes to the cytoplasm. Finally, we show that Xenopus aPKC and its binding partner Xenopus Par-6 are also essential for gastrulation. Thus, our results identify a requirement of Par proteins for Xenopus gastrulation and reveal a novel interrelationship within Par proteins that may provide a general mechanism for spatial control of Par-1.

JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates.

Recent genetic studies in Drosophila identified a novel non-canonical Wnt pathway, the planar cell polarity (PCP) pathway, that signals via JNK to control epithelial cell polarity in Drosophila. Most recently, a pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the PCP pathway. However, it is not known whether the JNK pathway functions in this non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. Here we show that Wnt5a is capable of activating JNK in cultured cells, and present evidence that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. Our results thus demonstrate that the non-canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and define that JNK has an activity to regulate morphogenetic cell movements.