Dale Frank

A Meis family protein caudalizes neural cell fates in Xenopus

A homologue of the Drosophila homothorax (hth) gene, Xenopus Meis3 (XMeis3), was cloned from Xenopus laevis. XMeis3 is expressed in a single stripe of cells in the early neural plate stage. By late neurula, the gene is expressed predominantly in rhombomeres two, three and four, and in the anterior spinal cord. Ectopic expression of RNA encoding XMeis3 protein causes anterior neural truncations with a concomitant expansion of hindbrain and spinal cord. Ectopic XMeis3 expression inhibits anterior neural induction in neuralized animal cap ectoderm explants without perturbing induction of pan-neural markers. In naive animal cap ectoderm, ectopic XMeis3 expression activates transcription of the posteriorly expressed neural markers, but not pan-neural markers. These results suggest that caudalizing proteins, such as XMeis3, can alter A-P patterning in the nervous system in the absence of neural induction. Regionally expressed proteins like XMeis3 could be required to overcome anterior signals and to specify posterior cell fates along the A-P axis.

Xenopus laevis POU91 protein, an Oct3/4 homologue, regulates competence transitions from mesoderm to neural cell fates

Cellular competence is defined as a cells ability to respond to signaling cues as a function of time. In Xenopus laevis, cellular responsiveness to fibroblast growth factor (FGF) changes during development. At blastula stages, FGF induces mesoderm, but at gastrula stages FGF regulates neuroectoderm formation. A Xenopus Oct3/4 homologue gene, XLPOU91, regulates mesoderm to neuroectoderm transitions. Ectopic XLPOU91 expression in Xenopus embryos inhibits FGF induction of Brachyury (Xbra), eliminating mesoderm, whereas neural induction is unaffected. XLPOU91 knockdown induces high levels of Xbra expression, with blastopore closure being delayed to later neurula stages. In morphant ectoderm explants, mesoderm responsiveness to FGF is extended from blastula to gastrula stages. The initial expression of mesoderm and endoderm markers is normal, but neural induction is abolished. Churchill (chch) and Sip1, two genes regulating neural competence, are not expressed in XLPOU91 morphant embryos. Ectopic Sip1 or chch expression rescues the morphant phenotype. Thus, XLPOU91 epistatically lies upstream of chch/Sip1 gene expression, regulating the competence transition that is critical for neural induction. In the absence of XLPOU91 activity, the cues driving proper embryonic cell fates are lost.

Mesodermal Wnt signaling organizes the neural plate via Meis3

In vertebrates, canonical Wnt signaling controls posterior neural cell lineage specification. Although Wnt signaling to the neural plate is sufficient for posterior identity, the source and timing of this activity remain uncertain. Furthermore, crucial molecular targets of this activity have not been defined. Here, we identify the endogenous Wnt activity and its role in controlling an essential downstream transcription factor, Meis3. Wnt3a is expressed in a specialized mesodermal domain, the paraxial dorsolateral mesoderm, which signals to overlying neuroectoderm. Loss of zygotic Wnt3a in this region does not alter mesoderm cell fates, but blocks Meis3 expression in the neuroectoderm, triggering the loss of posterior neural fates. Ectopic Meis3 protein expression is sufficient to rescue this phenotype. Moreover, Wnt3a induction of the posterior nervous system requires functional Meis3 in the neural plate. Using ChIP and promoter analysis, we show that Meis3 is a direct target of Wnt/β-catenin signaling. This suggests a new model for neural anteroposterior patterning, in which Wnt3a from the paraxial mesoderm induces posterior cell fates via direct activation of a crucial transcription factor in the overlying neural plate.

PTK7 modulates Wnt signaling activity via LRP6

Protein tyrosine kinase 7 (PTK7) is a transmembrane protein expressed in the developing Xenopus neural plate. PTK7 regulates vertebrate planar cell polarity (PCP), controlling mesodermal and neural convergent-extension (CE) cell movements, neural crest migration and neural tube closure in vertebrate embryos. Besides CE phenotypes, we now show that PTK7 protein knockdown also inhibits Wnt/β-catenin activity. Canonical Wnt signaling caudalizes the neural plate via direct transcriptional activation of the meis3 TALE-class homeobox gene, which subsequently induces neural CE. PTK7 controls meis3 gene expression to specify posterior tissue and downstream PCP activity. Furthermore, PTK7 morphants phenocopy embryos depleted for Wnt3a, LRP6 and Meis3 proteins. PTK7 protein depletion inhibits embryonic Wnt/β-catenin signaling by strongly reducing LRP6 protein levels. LRP6 protein positively modulates Wnt/β-catenin, but negatively modulates Wnt/PCP activities. The maintenance of high LRP6 protein levels by PTK7 triggers PCP inhibition. PTK7 and LRP6 proteins physically interact, suggesting that PTK7 stabilization of LRP6 protein reciprocally regulates both canonical and noncanonical Wnt activities in the embryo. We suggest a novel role for PTK7 protein as a modulator of LRP6 that negatively regulates Wnt/PCP activity.

Focal Adhesion Kinase protein regulates Wnt3a gene expression to control cell fate specification in the developing neural plate.

FAK is linked to aggressive tumors, but its normal function is not clear. FAK knockdown early in Xenopus development anteriorizes the embryo via a loss of Wnt signaling. Wnt3a expression is FAK dependent in both embryos and human breast cancer cells, suggesting that a FAK–Wnt linkage is highly conserved.

Xenopus Meis3 protein lies at a nexus downstream to Zic1 and Pax3 proteins, regulating multiple cell-fates during early nervous system development.

In Xenopus embryos, XMeis3 protein activity is required for normal hindbrain formation. Our results show that XMeis3 protein knock down also causes a loss of primary neuron and neural crest cell lineages, without altering expression of Zic, Sox or Pax3 genes. Knock down or inhibition of the Pax3, Zic1 or Zic5 protein activities extinguishes embryonic expression of the XMeis3 gene, as well as triggering the loss of hindbrain, neural crest and primary neuron cell fates. Ectopic XMeis3 expression can rescue the Zic knock down phenotype. HoxD1 is an XMeis3 direct-target gene, and ectopic HoxD1 expression rescues cell fate losses in either XMeis3 or Zic protein knock down embryos. FGF3 and FGF8 are direct target genes of XMeis3 protein and their expression is lost in XMeis3 morphant embryos. In the genetic cascade controlling embryonic neural cell specification, XMeis3 lies below general-neuralizing, but upstream of FGF and regional-specific genes. Thus, XMeis3 protein is positioned at a key regulatory point, simultaneously regulating multiple neural cell fates during early vertebrate nervous system development.

Molecular insights into the origin of the Hox-TALE patterning system

Despite tremendous body form diversity in nature, bilaterian animals share common sets of developmental genes that display conserved expression patterns in the embryo. Among them are the Hox genes, which define different identities along the anterior–posterior axis. Hox proteins exert their function by interaction with TALE transcription factors. Hox and TALE members are also present in some but not all non-bilaterian phyla, raising the question of how Hox–TALE interactions evolved to provide positional information. By using proteins from unicellular and multicellular lineages, we showed that these networks emerged from an ancestral generic motif present in Hox and other related protein families. Interestingly, Hox-TALE networks experienced additional and extensive molecular innovations that were likely crucial for differentiating Hox functions along body plans. Together our results highlight how homeobox gene families evolved during eukaryote evolution to eventually constitute a major patterning system in Eumetazoans. DOI: http://dx.doi.org/10.7554/eLife.01939.001

Xenopus Meis3 protein forms a hindbrain-inducing center by activating FGF/MAP kinase and PCP pathways

Knockdown studies in Xenopus demonstrated that the XMeis3 gene is required for proper hindbrain formation. An explant assay was developed to distinguish between autonomous and inductive activities of XMeis3 protein. Animal cap explants caudalized by XMeis3 were recombined with explants neuralized by the BMP dominant-negative receptor protein. XMeis3-expressing cells induced convergent extension cell elongations in juxtaposed neuralized explants. Elongated explants expressed hindbrain and primary neuron markers, and anterior neural marker expression was extinguished. Cell elongation was dependent on FGF/MAP-kinase and Wnt-PCP activities. XMeis3 activates FGF/MAP-kinase signaling, which then modulates the PCP pathway. In this manner, XMeis3 protein establishes a hindbrain-inducing center that determines anteroposterior patterning in the brain.

The POU-er of gene nomenclature

The pluripotency factor POU5F1 (OCT4) is well known as a key regulator of stem cell fate. Homologues of POU5F1 exist throughout vertebrates, but the evolutionary and functional relationships between the various family members have been unclear. The level to which function has been conserved within this family provides insight into the evolution of early embryonic potency. Here, we seek to clarify the relationship between POU5F1 homologues in the vertebrate lineage, both phylogenetically and functionally. We resolve the confusion over the identity of the zebrafish gene, which was originally named pou2, then changed to pou5f1 and again, more recently, to pou5f3. We argue that the use of correct nomenclature is crucial when discussing the degree to which the networks regulating early embryonic differentiation are conserved.

FAK and WNT signaling: the meeting of two pathways in cancer and development.

Recent studies connect the FAK and Wnt/β-catenin signaling pathways, both which promote cancer when aberrantly activated in mammalian cells. Over-stimulation of either Wnt/β-catenin or FAK activities was independently shown to promote numerous types of human cancers, including colon, breast, prostate and ovary. Observations in different model systems suggest a complex and dynamic cross-talk between these two pathways. During early vertebrate development, FAK protein is required for the proper regulation of Wnt/β- catenin signaling that controls pattern formation in the developing nervous system. In Xenopus laevis embryos, FAK protein depletion eliminated Wnt3a gene expression in the neural plate. In mouse osteoclast cells, mechanical stimulation through FAK activation stabilized β-catenin protein to promote its nuclear translocation. In contrast, in the mouse intestine, FAK activity was induced downstream of Wnt to promote intestinal regeneration and was also essential for tumorigenesis in an APC deletion model of colorectal cancer. Adding to this complexity, in human cell lines, FAK induced a context-dependent modulation of Wnt signaling to activate target-gene expression. Other diseases are also associated with FAK and Wnt pathway over-activation. Increased FAK and Wnt pathway activities were independently implicated in idiopathic pulmonary fibrosis (IPF), a lung disease of unknown etiology. Revealing the FAK-Wnt connection in IPF could provide a better understanding of disease pathology. There appear to be multiple interactions between the Wnt/β-catenin and FAK signaling pathways in different cell types and organisms. Mutual FAK-Wnt pathway regulation could be a general phenomenon, having many still undetermined roles in either normal physiological or disease processes.