Characterization of Danio rerio Nanog and functional comparison to Xenopus Vents.

Nanog is a homeodomain transcription factor associated with the acquisition of pluripotency. Genome analyses of lower and higher vertebrates revealed that the existence of Nanog is restricted to gnathostomata but absent from agnatha and invertebrates. To elucidate the function of Nanog in nonmammalia, we identified the Danio rerio ortholog of Nanog and characterized its role in gain and loss of function experiments. We found Nanog to be crucial for survival of early zebrafish embryos, because depletion of Nanog led to gastrulation defects with subsequent lethality. Mouse Nanog overexpression could rescue these defects. Vice versa, zebrafish Nanog was found to promote proliferation and to inhibit differentiation of mouse embryonic stem cells in the absence of leukemia inhibitory factor. These findings indicate functional conservation of Nanog from teleost fishes to mammals. However, Nanog was lost in the genome of the anurans Xenopus laevis and Xenopus tropicalis. Phylogenetic analysis revealed that deletion probably occurred in a common anuran ancestor along with chromosomal translocations. The closest homologs of Nanog in Xenopus are the Vent proteins. We, therefore, investigated whether the Xvent genes might substitute for Nanog function in Xenopus. Although we found some similarities in phenotypes after overexpression and in the regulation of several marker genes, Xvent1/2 and Nanog cannot substitute each other. Depletion of Nanog in zebrafish cannot be rescued by ectopic expression of Xvent, and Xvent depletion in Xenopus cannot be overcome by ectopic expression of zebrafish Nanog.

Neuron navigator 3a regulates liver organogenesis during zebrafish embryogenesis.

Endodermal organogenesis requires a precise orchestration of cell fate specification and cell movements, collectively coordinating organ size and shape. In Caenorhabditis elegans, uncoordinated-53 (unc-53) encodes a neural guidance molecule that directs axonal growth. One of the vertebrate homologs of unc-53 is neuron navigator 3 (Nav3). Here, we identified a novel vertebrate neuron navigator 3 isoform in zebrafish, nav3a, and we provide genetic evidence in loss- and gain-of-function experiments showing its functional role in endodermal organogenesis during zebrafish embryogenesis. In zebrafish embryos, nav3a expression was initiated at 22 hpf in the gut endoderm and at 40 hpf expanded to the newly formed liver bud. Endodermal nav3a expression was controlled by Wnt2bb signaling and was independent of FGF and BMP signaling. Morpholino-mediated knockdown of nav3a resulted in a significantly reduced liver size, and impaired development of pancreas and swim bladder. In vivo time-lapse imaging of liver development in nav3a morphants revealed a failure of hepatoblast movement out from the gut endoderm during the liver budding stage, with hepatoblasts being retained in the intestinal endoderm. In hepatocytes in vitro, nav3a acts as a positive modulator of actin assembly in lamellipodia and filipodia extensions, allowing cellular movement. Knockdown of nav3a in vitro impeded hepatocyte movement. Endodermal-specific overexpression of nav3a in vivo resulted in additional ectopic endodermal budding beyond the normal liver and pancreatic budding sites. We conclude that nav3a is required for directing endodermal organogenesis involving coordination of endodermal cell behavior.

FoxO genes are dispensable during gastrulation but required for late embryogenesis in Xenopus laevis.

Forkhead box (Fox) transcription factors of subclass O are involved in cell survival, proliferation, apoptosis, cell metabolism and prevention of oxidative stress. FoxO genes are highly conserved throughout evolution and their functions were analyzed in several vertebrate and invertebrate organisms. We here report on the identification of FoxO4 and FoxO6 genes in Xenopus laevis and analyze their expression patterns in comparison with the previously described FoxO1 and FoxO3 genes. We demonstrate significant differences in their temporal and spatial expression during embryogenesis and in their relative expression within adult tissues. Overexpression of FoxO1, FoxO4 or FoxO6 results in severe gastrulation defects, while overexpression of FoxO3 reveals this defect only in a constitutively active form containing mutations of Akt-1 target sites. Injections of FoxO antisense morpholino oligonucleotides (MO) did not influence gastrulation, but, later onwards, the embryos showed a delay of development, severe body axis reduction and, finally, a high rate of lethality. Injection of FoxO4MO leads to specific defects in eye formation, neural crest migration and heart development, the latter being accompanied by loss of myocardin expression. Our observations suggest that FoxO genes in X. laevis are dispensable until blastopore closure but are required for tissue differentiation and organogenesis.

Functional dissection of XDppa2/4 structural domains in Xenopus development.

The maintenance of pluripotency in mammalian embryonic stem cells depends upon the expression of regulatory genes like Oct3/4 and Sox2. While homologues of these genes are also characterized in non-mammalian vertebrates, like birds, amphibians and fish, existence and function of developmental pluripotency associated genes (Dppa) in lower vertebrates have not yet been reported. Here we describe a Dppa2/4-like gene, XDppa2/4, in Xenopus. The protein contains a SAP domain and a conserved C-terminal region. Overexpression of XDppa2/4, murine Dppa2 or Dppa4 produces similar phenotypes (defects in blastopore closure), while injection of XDppa2/4 morpholino generates a loss of blastopore closure and neural fold formation. Embryos die up to tailbud stage. mDppa2 (but not mDppa4) rescues blastopore closure and neurulation defects caused by XDppaMO, but does not prevent subsequent death of embryos. Although XDppa2/4 exhibits a Dppa-like expression pattern and is indispensable for embryogenesis, analyses of various marker genes make its role as a pluripotency factor rather unlikely. Both the gain and loss of function effects until the end of neurulation are caused by the conserved C-terminal region but not by the SAP domain. The SAP domain is required for association of XDppa2/4 to chromatin and for embryonic survival at later stages of development suggesting epigenetic programming events.

Oct25 represses transcription of nodal/activin target genes by interaction with signal transducers during Xenopus gastrulation.

The balance between differentiation signals and signals maintaining the undifferentiated state of embryonic cells ensures proper formation of germ layers. The nodal/activin pathway represents one of the major signaling chains responsible for the differentiation of embryonic cells into mesodermal and endodermal germ layers, while Oct4 is one of the major players in the maintenance of an undifferentiated state. Here we show that Oct25, an Oct4 homologue in Xenopus, antagonizes the activity of nodal/activin signaling by inhibiting the transcription of its target genes, Gsc and Mix2. The inhibitory effect is achieved by forming repression complexes on the promoters of Gsc and Mix2 between Oct25 and the signal transducers of the nodal/activin pathway, WBSCR11, FAST1, and Smad2. We have analyzed the significance of the Oct binding site for its inhibitory effect within the Gsc promoter. Albeit VP16-Oct25 fusion protein demonstrated a stimulating effect and EVE-Oct25 revealed a repression effect on an artificial reporter that is composed of eight repeats of Oct binding motifs, both fusions, like wild-type Oct25, inhibited mesendoderm formation and the activity of Gsc and Mix2 promoters. These results suggest that the regulatory effect of Oct25 on the expression of Gsc and Mix2 is mediated by specific protein/protein interactions. Furthermore, we demonstrate that histone deacetylase activities are not required for the inhibitory effect of Oct25. Our results provide a novel view in that Oct25 controls the nodal/activin pathway and thus maintains the undifferentiated state of embryonic cells in preventing them from premature differentiation.

POU-V factors antagonize maternal VegT activity and beta-Catenin signaling in Xenopus embryos.

VegT and beta-Catenin are key players in the hierarchy of factors that are required for induction and patterning of mesendoderm in Xenopus embryogenesis. By descending the genetic cascades, cells lose their pluripotent status and are determined to differentiate into distinct tissues. Mammalian Oct-3/4, a POU factor of subclass V (POU-V), is required for the maintenance of pluripotency of embryonic stem cells. However, its molecular function within the early embryo is yet poorly understood. We here show that the two maternal Xenopus POU-V factors, Oct-60 and Oct-25, inhibit transcription of genes activated by VegT and beta-Catenin. Maternal POU-V factors and maternal VegT show an opposite distribution along the animal/vegetal axis. Oct-25, VegT and Tcf3 interact with each other and form repression complexes on promoters of VegT and beta-Catenin target genes. We suggest that POU-V factors antagonize primary inducers to allow germ layer specification in a temporally and spatially coordinated manner.

Xenopus POU factors of subclass V inhibit activin/nodal signaling during gastrulation.

Three POU factors of subclass V, Oct-25, Oct-60 and Oct-91 are expressed in Xenopus oocytes and early embryos. We here demonstrate that vegetal overexpression of Oct-25, Oct-60, Oct-91 or mammalian Oct-3/4 suppresses mesendoderm formation in Xenopus embryos. Oct-25 and Oct-60 are shown to inhibit activin/nodal and FGF signaling pathways. Loss of Oct-25 and Oct-60 function results in elevated transcription of mesendodermal marker genes and ectopic formation of endoderm in the equatorial region of gastrula stage embryos. Within the ectoderm, Oct-25 promotes neural fate by upregulating neuroectodermal genes, such as Xsox2, which prevent differentiation of neural progenitors into neurons. We also show that mouse Oct-3/4 and Xenopus Oct-25 or Oct-60 behave as functional homologues. We conclude that Xenopus Oct proteins are required to control the levels of embryonic signaling pathways, thereby ensuring the correct specification of germ layers.