Sarah Elias

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.

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.

A POU protein regulates mesodermal competence to FGF in Xenopus

XLPOU91, a POU-homeobox gene is expressed in a narrow window during early Xenopus development. We show that ectopic expression of XLPOU91 RNA causes severe posterior truncations in embryos without inhibiting the formation of Spemanns organizer. Ectopic XLPOU91 expression also inhibits mesoderm induction by fibroblast growth factor (FGF) and activin in animal cap explants. Using antisense RNA, we depleted endogenous XLPOU91 protein in animal caps. Gastrula-stage animal caps expressing XLPOU91 antisense RNA do not lose competence to FGF, unlike controls, these animal caps express XBra after FGF treatment. Endogenous XLPOU91 levels are peaking when FGF mesoderm-inducing competence is lost in animal caps. Thus XLPOU91 protein may act as a competence switch during early development, as XLPOU91 levels increase in the embryo, the mesoderm response to FGF is lost.

Aggregation of maternal pigment granules is induced by the cytosolic discoidin domain of the Xenopus Del1 protein.

Xenopus oocytes generate pigment granules (melanosomes) that predominantly localize to the animal hemisphere cortex. During embryonic development, these granules are located near the membranes of outer layer ectoderm cells. We report a novel phenotype found during an expression cloning screen in Xenopus laevis embryos. The phenotype is characterized by dissociation of pigment granules from the cell membrane to form large central aggregates. This phenomenon was induced by a truncated form of the Xenopus Del1 (XDel1) protein that contains only the C‐terminal discoidin (D2) domain. This truncated form of XDel1 localized to membranes as shown by a chimeric enhanced green fluorescent protein construct. Although a similar localization occurred in immature oocytes, dissociation of pigment granules was limited to the oocyte vegetal hemisphere. The full‐length XDel1 cDNA was cloned, and XDel1 mRNA expression was found to be ubiquitous and continuous from early oocyte to tail bud stages, with a transient enrichment in the cement gland. Ectopic expression of various deletion or full‐length constructs or antisense morpholino oligonucleotides did not induce any significant developmental phenotypes. Developmental Dynamics 233:224–232, 2005.

BMP regulates vegetal pole induction centres in early xenopus development.

Bone morphogenetic protein (BMP) plays an important role in mesoderm patterning in Xenopus. The ectopic expression of BMP‐4 protein hyperventralizes embryos, whereas embryos expressing a BMP‐2/4 dominant‐negative receptor (DNR) are hyperdorsalized. Mesoderm is initially induced in the marginal zone by cells in the underlying vegetal pole. While much is known about BMPs expression and role in patterning the marginal zone, little is known about its early role in regulating vegetal mesoderm induction centre formation.