Morioh Kusakabe

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.

Identification of Two Smad4 Proteins in Xenopus THEIR COMMON AND DISTINCT PROPERTIES

Smad family proteins have been identified as mediators of intracellular signal transduction by the transforming growth factor-β (TGF-β) superfamily. Each member of the pathway-restricted, receptor-activated Smad family cooperates and synergizes with Smad4, called co-Smad, to transduce the signals. Only Smad4 has been shown able to function as a common partner of the various pathway-restricted Smads in mammals. Here we have identified a novel Smad4-like molecule in Xenopus (XSmad4β) as well as a Xenopus homolog of a well established Smad4 (XSmad4α). XSmad4β is 70% identical to XSmad4α in amino acid sequence. Both of the Xenopus Smad4s can cooperate with Smad1 and Smad2, the pathway-restricted Smads specific for bone morphogenetic protein and TGF-β, respectively. However, they show distinct properties in terms of their developmental expression patterns, subcellular localizations, and phosphorylation states. Moreover, XSmad4β, but not XSmad4α, has the potent ability to induce ventralization when microinjected into the dorsal marginal region of the 4-cell stage of the embryos. These results suggest that the two XenopusSmad4s have overlapping but distinct functions.

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.

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 FRS2 is involved in early embryogenesis in cooperation with the Src family kinase Laloo

FRS2 has been identified in mammalian cells as a protein that is tyrosine phosphorylated and binds to Grb2 and Shp2 in response to fibroblast growth factor (FGF) or nerve growth factor (NGF) stimulation. But neither its existence in other vertebrate classes or invertebrates nor its function during embryonic development has been defined. Here we have identified and characterized a Xenopus homolog of FRS2 (xFRS2). xFRS2 is tyrosine phosphorylated in early embryos, and overexpression of an unphosphorylatable form of xFRS2 interferes with FGF‐dependent mesoderm formation. The Src family Kinase Laloo, which was shown to function in FGF signaling during early Xenopus development, binds to xFRS2 and promotes tyrosine phosphorylation of xFRS2. Moreover, xFRS2 and Laloo are shown to bind to Xenopus FGF receptor 1. These results suggest that xFRS2 plays an important role in FGF signaling in cooperation with Laloo during embryonic development.

The TGF‐β family member derrière is involved in regulation of the establishment of left–right asymmetry

Although a number of genes that are involved in the establishment of left–right asymmetry have been identified, earlier events in the molecular pathway developing left–right asymmetry remain to be elucidated. Here we present evidence suggesting that the transforming growth factor‐β family member derrière is involved in the development of left–right asymmetry in Xenopus embryos. Ectopic expression of derrière on the right side can fully invert cardiac and visceral left–right orientation and nodal expression, and expression of a dominant‐negative form of derrière on the left side can partially randomize the left–right orientation and nodal expression. Moreover, while expression of the dominant‐negative derrière does not inhibit the activity of Vg1 directly, it can rescue the altered left–right orientation induced by Vg1. Vg1 can induce derrière in animal cap explants. These results suggest that derrière is involved in earlier molecular pathways developing the left–right asymmetry.

The Kinase SGK1 in the Endoderm and Mesoderm Promotes Ectodermal Survival by Down-Regulating Components of the Death-Inducing Signaling Complex

The kinase SGK1 participates in an intertissue survival pathway during the early stages of development. Survival Signal from Afar All of the tissues and organs in vertebrates develop from just three germ layers—ectoderm, mesoderm, and endoderm. Although programmed cell death plays key roles in proper development, inappropriate apoptosis must be prevented. Endo et al. report an intertissue signaling pathway by which the endodermal and mesodermal layers promote the survival of the ectodermal layer. Depletion of serum- and glucocorticoid-inducible kinase 1 (SGK1) in Xenopus embryos caused apoptosis in the ectoderm in vivo but not in ectodermal explants. SGK1 participated in an endodermal and mesodermal signaling pathway that resulted in nuclear factor κB (NF-κB)–mediated transcription and release of bone morphogenetic protein 7, which suppressed the transcription of genes encoding components of death-inducing signaling complex (DISC) in the ectoderm. Thus, the embryonic ectoderm receives apoptotic signals, but an SGK1 signaling pathway in the other two germ layers opposes these cues to promote survival. 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.

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.

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.

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.