Activation of Yap-Directed Transcription by Knockdown of Conserved Cellular Functions.

The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription coactivators, YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription coactivator at high cell density. Activation of the YAP transcription coactivator was monitored using a high-content, image-based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known YAP target gene). The effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology, such as components of the nuclear pore, by which YAP cytoplasmic-nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays.

Xwig1, a novel putative endoplasmic reticulum protein expressed during epithelial morphogenesis and in response to embryonic wounding.

In a subtractive differential screening, we identified a novel gene with interesting characteristics, termed Xenopus wounding induced gene 1 (Xwig1). Xwig1 encodes a novel protein of 912 amino acids containing 13 putative transmembrane segments and an evolutionarily conserved carboxy-terminal domain. Protein localization studies revealed that Xwig1 is anchored in cytoplasmic structures, presumably the endoplasmic reticulum. Expression is largely confined to epithelial cells in regions that undergo morphogenetic processes, such as blastopore closure, hindgut closure, dorsal closure and optic vesicle invagination. Interestingly, Xwig1 transcription is activated in response to embryonic epidermal wounding. The wounding-induced transcription occurs downstream of the transient phosphorylation of extracellular signal-regulated protein kinases and is in part mediated by Elk-1, but independent of dissection-induced FGF signalling. Thus, Xwig1 provides a molecular link between epithelial morphogenesis and wound healing.

The Spemann-Mangold organizer: the control of fate specification and morphogenetic rearrangements during gastrulation in Xenopus.

Vertebrate embryonic development is controlled by sequentially operating signalling centres that organize spatial pattern by inductive interactions. The embryonic body plan is established during gastrulation through the action of the Spemann-Mangold or gastrula organizer, a signalling source discovered 75 years ago by Hans Spemann and Hilde Mangold. Transplantation of the organizer to a heterotopic location in a recipient embryo results in the formation of a secondary embryonic body axis, in which several tissue types, most notably somites and the neural tube, are derived from ventral host cells. Because of these non-cell autonomous recruiting or inducing activities the organizer has become a paradigm for studying intercellular communication in the vertebrate embryo. Here, I review some of the recent advances in understanding 1) the initiation of the Spemann-Mangold organizer, 2) its function in pattern formation along the dorsal-ventral and anterior-posterior axes and 3) the integration of cell fate specification events and downstream execution of morphogenetic movements during gastrulation in Xenopus laevis.

Xgravin-like (Xgl), a novel putative a-kinase anchoring protein (AKAP) expressed during embryonic development in Xenopus.

A-kinase anchoring proteins (AKAPs) are a heterogeneous family of scaffolding proteins that regulate the compartmentalization of signaling components, in particular that of the broad specificity kinase PKA. Here we describe the identification of a new member of this gene family, termed Xenopus gravin-like (Xgl), which encodes a highly acidic protein of 268 kDa that shares extensive homology with human Gravin and murine SSeCKS. Xgl is zygotically expressed in a highly dynamic fashion. During gastrulation Xgl is expressed in posterior mesoderm of the dorsal blastopore lip. During neurulation expression is transiently detected in the forebrain, two bilateral neuroectodermal stripes and the notochord. At tailbud stages expression commences in the mandibular neural crest and the roof of the spinal cord from where neural crest cells migrate into the intersomitic region. In addition expression is detected in the heart and the anterior aspect of the chordoneural hinge.

Expression of the RNA recognition motif-containing protein SEB-4 during Xenopus embryonic development.

RNA binding proteins play key roles in the post-transcriptional regulation of gene expression. Here we present the molecular cloning and spatio-temporal expression of Xseb-4, which codes for a putative RNA binding protein containing a single RNA recognition motif (RRM). XSEB-4 shares 60-65% identity with the mammalian SEB-4 proteins. Xseb-4 is strongly expressed maternally. Zygotic transcription is initiated in the early gastrula embryo in paraxial mesoderm that is fated to give rise to somites. During the course of gastrulation and neurulation Xseb-4 expression in somitic paraxial mesoderm is centered within the XmyoD expression domain. As development proceeds Xseb-4 expression is in addition initiated in the cardiac primordium and the lens vesicle. In the heart expression is confined to the myocardium. Thus, the RRM-containing putative RNA binding protein XSEB-4 is differentially expressed during embryonic development in Xenopus.

Signaling specificity by Frizzled receptors in Drosophila.

Wnt-Frizzled (Fz) signaling pathways play recurring important roles during the development and homeostasis of vertebrates and invertebrates. Fz receptors can signal through beta-catenin-dependent and -independent pathways. In Drosophila, Fz and Fz2 are redundant receptors for Wg. In addition, Fz conveys signals through a distinct pathway to organize planar polarization of epithelial structures. We demonstrate that the cytoplasmic sequences of Fz2 and Fz preferentially activate the beta-catenin and planar polarity cascade, respectively. Both receptors activate either pathway, but with different efficiencies. Intrinsic differences in signaling efficiency in closely related receptors might be a general mechanism for generating signaling specificity in vivo.

Neuroectodermal specification and regionalization of the Spemann organizer in Xenopus.

During gastrulation in Xenopus convergence and extension movements, mediated by mediolateral intercalations, are the driving force for early neural plate morphogenesis. Here we show that the winged helix transcriptional regulator, Xfd-12' is dynamically expressed in medial neural plate precursors that undergo convergence and extension movements. These medial neuraxial progenitors are specified in and beyond the Spemann organizer prior to specification of the basal anlage of the neural plate. The initiation of Xfd-12' expression coincides with the induction of mesendoderm by Nodal-related growth factors at the late blastula stage. Comparative expression analysis suggests that cellular rearrangements at the pre-gastrulation stage account for regionalization of the Spemann organizer into head and trunk organizer compartments, the latter in which medial neural plate progenitors reside. While the maintenance of Xfd-12' expression in the dorsal non-involuting marginal zone requires FGF signalling, its subsequent positioning along the medial aspect of the neuraxis depends on signalling by Wnt and Nodal-related family members. Based on these findings we propose that XFD-12' is a trunk organizer component that might control convergence and extension movements of medial neural plate precursors during gastrulation.

Essential role of Bmp7 (snailhouse) and its prodomain in dorsoventral patterning of the zebrafish embryo.

Bone morphogenetic proteins (Bmps) are signaling molecules that have been implicated in a variety of inductive processes. We report here that zebrafish Bmp7 is disrupted in snailhouse (snh) mutants. The allele snh(st1) is a translocation deleting the bmp7 gene, while snh(ty68) displays a Val->Gly exhange in a conserved motif of the Bmp7 prodomain. The snh(ty68) mutation is temperature-sensitive, leading to severalfold reduced activity of mutant Bmp7 at 28 degrees C and non-detectable activity at 33 degrees C. This prodomain lesion affects secretion and/or stability of secreted mature Bmp7 after processing has occurred. Both snh(st1) and snh(ty68) mutant zebrafish embryos are strongly dorsalized, indicating that bmp7 is required for the specification of ventral cell fates during early dorsoventral patterning. At higher temperature, the phenotype of snh(ty68) mutant embryos is identical to that caused by the amorphic bmp2b mutation swirl swr(ta72) and similar to that caused by the smad5 mutation somitabun sbn(dtc24). mRNA injection studies and double mutant analyses indicate that Bmp2b and Bmp7 closely cooperate and that Bmp2b/Bmp7 signaling is transduced by Smad5 and antagonized by Chordino.

The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo.

Signaling by members of the TGFbeta superfamily is thought to be transduced by Smad proteins. Here, we describe a zebrafish mutant in smad5, designated somitabun (sbn). The dominant maternal and zygotic effect of the sbntc24 mutation is caused by a change in a single amino acid in the L3 loop of Smad5 protein which transforms Smad5 into an antimorphic version, inhibiting wild-type Smad5 and related Smad proteins. sbn mutant embryos are strongly dorsalized, similarly to mutants in Bmp2b, its putative upstream signal. Double mutant analyses and RNA injection experiments show that sbn and bmp2b interact and that sbn acts downstream of Bmp2b signaling to mediate Bmp2b autoregulation during early dorsoventral (D-V) pattern formation. Comparison of early marker gene expression patterns, chimera analyses and rescue experiments involving temporally controlled misexpression of bmp or smad in mutant embryos reveal three phases of D-V patterning: an early sbn- and bmp2b-independent phase when a coarse initial D-V pattern is set up, an intermediate sbn- and bmp2b-dependent phase during which the putative morphogenetic Bmp2/4 gradient is established, and a later sbn-independent phase during gastrulation when the Bmp2/4 gradient is interpreted and cell fates are specified.

The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals.

Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk-tail development. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures. Here we show that the cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signalling by BMP and Wnt. In order for the head territory to form, we propose that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions.

Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signaling activity.

Signaling by Notch family receptors is involved in many cell-fate decisions during development. Several modifiers of Notch activity have been identified, suggesting that regulation of Notch signaling is complex. In a genetic screen for modifiers of Notch activity, we identified a gene encoding a novel WD40-repeat protein. The gene is called Notchless, because loss-of-function mutant alleles dominantly suppress the wing notching caused by certain Notch alleles. Reducing Notchless activity increases Notch activity. Overexpression of Notchless in Xenopus or Drosophila appears to have a dominant-negative effect in that it also increases Notch activity. Biochemical studies show that Notchless binds to the cytoplasmic domain of Notch, suggesting that it serves as a direct regulator of Notch signaling activity.

The role of paraxial protocadherin in selective adhesion and cell movements of the mesoderm during Xenopus gastrulation.

Paraxial Protocadherin (PAPC) encodes a transmembrane protein expressed initially in Spemann's organizer and then in paraxial mesoderm. Together with another member of the protocadherin family, Axial Protocadherin (AXPC), it subdivides gastrulating mesoderm into paraxial and axial domains. PAPC has potent homotypic cell adhesion activity in cell dissociation and reaggregation assays. Gain- and loss-of-function microinjection studies indicate that PAPC plays an important role in the convergence and extension movements that drive Xenopus gastrulation. Thus, PAPC is not only an adhesion molecule but also a component of the machinery that drives gastrulation movements in Xenopus. PAPC may provide a link between regulatory genes in Spemann's organizer and the execution of cell behaviors during morphogenesis.

Vertebrate head induction by anterior primitive endoderm.

In vertebrates the antero-posterior organization of the embryonic body axis is thought to result from the activity of two separate centers, the head organizer and the trunk organizer, as operationally defined by Spemann in the 1920s. Current molecular studies have supported the existence of a trunk organizer activity while the presence of a distinct head inducing center has remained elusive. Mainly based on analyses of headless mutants in mice, it has been proposed that the anterior axial mesoderm plays a determining role in head induction. Recent gain- and loss-of-function studies in various organisms, however, provide compelling evidence that a largely ignored region, the anterior primitive endoderm, specifies rostral identity. In this review we discuss the emerging concept that the anterior primitive endoderm, rather than the prechordal plate mesoderm, induces head development in the vertebrate embryo.

Transcription regulation and alternative splicing of an early zygotic gene encoding two structurally distinct zinc finger proteins in Xenopus laevis.

We describe the structural organization of a gene, termed XFDL 141/156, that is transiently activated during early Xenopus development. XFDL 141/156 is first transcribed at the midblastula transition (MBT) and during early gastrulation events. A roughly 200 nucleotide fragment immediately 5' to the transcription start site is sufficient for transient, early zygotic activation of gene expression. The primary transcript is subject to alternative splicing. Corresponding cDNAs encode two structurally related but completely distinct C2H2-type zinc finger proteins of unknown biological function.

Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer.

Frzb-1 is a secreted protein containing a domain similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, it is expressed and regulated as a typical Spemann organizer component. Injection of frzb-1 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Frzb-1 antagonizes the effects of Xwnt-8 ectopic expression in a non-cell-autonomous manner. Cultured cells transfected with a membrane-tethered form of Wnt-1 bind epitope-tagged Frzb-1 in the 10(-10) M range. The results strengthen the view that the Spemann organizer is a source of secreted inhibitory factors.

Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula.

We report the isolation of mouse cerberus-like (cer-l), a gene encoding a novel secreted protein that is specifically expressed in the anterior visceral endoderm during early gastrulation. Expression in the primitive endoderm starts before the appearance of the primitive streak and lasts until the head-fold stage. In later stages, a second region of expression is found in newly formed somites. Mouse cer-l shares some sequence similarity with Xenopus cerberus (Xcer). In Xenopus assays cer-l, like Xcer, mRNA acts as a potent neuralizing factor that induces forebrain markers and endoderm, but is unable to induce ectopic head-like structures as Xcer does. In addition to cer-l, anterior visceral endoderm was found to express the transcription factors Lim1, goosecoid and HNF-3beta that are also present in trunk organizer cells. A model of how head and trunk development might be regulated is discussed. Given its neuralizing activity, the secreted protein Cer-l is a candidate for mediating inductive activities of anterior visceral endoderm.

Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer.

An abundant cDNA enriched in Spemann's organizer, cerberus, was isolated by differential screening. It encodes a secreted protein that is expressed in the anterior endomesoderm. Microinjection of cerberus mRNA into Xenopus embryos induces ectopic heads, and duplicated hearts and livers. The results suggest a role for a molecule expressed in the anterior endoderm in the induction of head structures in the vertebrate embryo.