The mouse KRAB zinc-finger protein CHATO is required in embryonic-derived tissues to control yolk sac and placenta morphogenesis

Yolk sac and placenta are required to sustain embryonic development in mammals, yet our understanding of the genes and processes that control morphogenesis of these extraembryonic tissues is still limited. The chato mutation disrupts ZFP568, a Krüppel-Associated-Box (KRAB) domain Zinc finger protein, and causes a unique set of extraembryonic malformations, including ruffling of the yolk sac membrane, defective extraembryonic mesoderm morphogenesis and vasculogenesis, failure to close the ectoplacental cavity, and incomplete placental development. Phenotypic analysis of chato embryos indicated that ZFP568 does not control proliferation or differentiation of extraembryonic lineages but rather regulates the morphogenetic events that shape extraembryonic tissues. Analysis of chimeric embryos showed that Zfp568 function is required in embryonic-derived lineages, including the extraembryonic mesoderm. Depleting Zfp568 affects the ability of extraembryonic mesoderm cells to migrate. However, explanted Zfp568 mutant cells could migrate properly when plated on appropriate extracellular matrix conditions. We show that expression of Fibronectin and Indian Hedgehog are reduced in chato mutant yolk sacs. These data suggest that ZFP568 controls the production of secreted factors required to promote morphogenesis of extraembryonic tissues. Our results support previously undescribed roles of the extraembryonic mesoderm in yolk sac morphogenesis and in the closure of the ectoplacental cavity and identify a novel role of ZFP568 in the development of extraembryonic tissues.     

Bmp activity gradient regulates convergent extension during zebrafish gastrulation

During vertebrate gastrulation, a ventral to dorsal gradient of bone morphogenetic protein (Bmp) activity establishes cell fates. Concomitantly, convergent extension movements narrow germ layers mediolaterally while lengthening them anteroposteriorly. Here, by measuring movements of cell populations in vivo, we reveal the presence of three domains of convergent extension movements in zebrafish gastrula. Ventrally, convergence and extension movements are absent. Lateral cell populations converge and extend at increasing speed until they reach the dorsal domain where convergence speed slows but extension remains strong. Using dorsalized and ventralized mutants, we demonstrate that these domains are specified by the Bmp activity gradient. In vivo cell morphology and behavior analyses indicated that low levels of Bmp activity might promote extension with little convergence by allowing mediolateral cell elongation and dorsally biased intercalation. Further, single cell movement analyses revealed that the high ventral levels of Bmp activity promote epibolic migration of cells into the tailbud, increasing tail formation at the expense of head and trunk. We show that high Bmp activity limits convergence and extension by negatively regulating expression of the wnt11 (silberblick) and wnt5a (pipetail) genes, which are required for convergent extension but not cell fate specification. Therefore, during vertebrate gastrulation, a single gradient of Bmp activity, which specifies cell fates, also regulates the morphogenetic process of convergent extension.     

A novel role for a nodal-related protein; Xnr3 regulates convergent extension movements via the FGF receptor

Convergent extension behaviour is critical for the formation of the vertebrate body axis. In Xenopus, components of the Wnt signaling pathway have been shown to be required for convergent extension movements but the relationship between cell fate and morphogenesis is little understood. We show by loss of function analysis that Xnr3 activates Xbra expression through FGFR1. We show that eFGF activity is not essential in the pathway, and that dishevelled acts downstream of Xnr3 and not in a parallel pathway. We provide evidence for the involvement of the EGF-CFC protein FRL1, and suggest that the pro-domain of Xnr3 may be required for its activity. Since Xnr3 is a direct target of the maternal betacatenin/XTcf3 signaling pathway, it provides the link between the initial, maternally controlled, allocation of cell fate, and the morphogenetic movements of cells derived from the organizer.     

Genetic evidence for posterior specification by convergent extension in the Xenopus embryo

Genetic studies substantiate that mesodermal convergent extension expressed behind the anteroposterior borderline, in the form of a gradient with the posterior apex after gastrulation, regulates morphogenesis of the posterior zone at the dorsal and dorso-lateral levels which is in full agreement with the model of dorsalization–caudalization. In contrast, how anteroposterior specification of mesodermal tissues occurs at the ventral and latero-ventral levels is not yet understood.     

The planar polarity gene strabismus regulates convergent extension movements in Xenopus

The signaling mechanisms that specify, guide and coordinate cell behavior during embryonic morphogenesis are poorly understood. We report that a Xenopus homolog of the Drosophila planar cell polarity gene strabismus (stbm) participates in the regulation of convergent extension, a critical morphogenetic process required for the elongation of dorsal structures in vertebrate embryos. Overexpression of Xstbm, which is expressed broadly in early development and subsequently in the nervous system, causes severely shortened trunk structures; a similar phenotype results from inhibiting Xstbm translation using a morpholino antisense oligo. Experiments with Keller explants further demonstrate that Xstbm can regulate convergent extension in both dorsal mesoderm and neural tissue. The specification of dorsal tissues is not affected. The Xstbm phenotype resembles those obtained with several other molecules with roles in planar polarity signaling, including Dishevelled and Frizzled-7 and -8. Unlike these proteins, however, Stbm has little effect on conventional Wnt/beta-catenin signaling in either frog or fly assays. Thus our results strongly support the emerging hypothesis that a vertebrate analog of the planar polarity pathway governs convergent extension movements.     

MZF6D, a novel KRAB zinc-finger gene expressed exclusively in meiotic male germ cells

Spermatogenesis takes place in the seminiferous tubule in the testes and culminates in the production of spermatozoa (male gametes). Here we report the identification of a novel mouse zinc-finger gene, MZF6D, which is selectively expressed in meiotic spermatocytes. The MZF6D protein contains an N-terminally located repressor domain, a KRAB domain, followed by at least seven successive Krüppel zinc-finger motifs. The KRAB domain of MZF6D, which consists of a KRAB A box and the newly identified KRAB C box, has previously been shown to interact with TIF1beta, which is the common corepressor of all KRAB zinc-finger proteins. Northern blot analysis shows that the expression of MZF6D is restricted to testes. This was confirmed by RT-PCR analysis of a panel of mouse tissues. In situ hybridization of sections from adult mouse testes localizes the expression to meiotic spermatocytes, suggesting a specific role for MZF6D in the regulation of spermatogenesis.     

RhoA regulates initiation of invagination, but not convergent extension, during sea urchin gastrulation

During gastrulation, the archenteron is formed using cell shape changes, cell rearrangements, filopodial extensions, and convergent extension movements to elongate and shape the nascent gut tube. How these events are coordinated remains unknown, although much has been learned from careful morphological examinations and molecular perturbations. This study reports that RhoA is necessary to trigger archenteron invagination in the sea urchin embryo. Inhibition of RhoA results in a failure to initiate invagination movements, while constitutively active RhoA induces precocious invagination of the archenteron, complete with the actin rearrangements and extracellular matrix secretions that normally accompany the onset of invagination. Although RhoA activity has been reported to control convergent extension movements in vertebrate embryos, experiments herein show that RhoA activity does not regulate convergent extension movements during sea urchin gastrulation. Instead, the results support the hypothesis that RhoA serves as a trigger to initiate invagination, and once initiation occurs, RhoA activity is no longer involved in subsequent gastrulation movements.     

OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice

The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA₃) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA₁ and real-time PCR quantitative assay analyses confirmed that the decrease of GA₁ in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice.     

Expression pattern of Zac1 mouse gene, a new zinc-finger protein that regulates apoptosis and cellular cycle arrest, in both adult brain and along development

Using in situ hybridization, we analyzed the expression pattern of the Zac1 gene in mouse brain during the embryonic and postnatal development. Zac1 is a new gene that regulates extensive apoptosis and cell cycle arrest through unrelated pathways. At embryonic stages, strong expression was observed in brain areas with active proliferation (ventricular zone and numerous neuroepithelius) and in nervous system (neural retina and neural tube). In addition, some areas with differentiation activity were noticeably labeled such as arcuate nucleus and amygdaloid region of the brain together with other embryonic sites (hindlimb, forelimb and somites). From P0 onwards, the expression appeared in some proliferative areas, such as subventricular zone and cerebellum (external granular layer and Purkinje cells) and in some synaptic plasticity areas, such as the dorso and ventromedial hypothalamic nuclei, arcuate nucleus, ventral thalamic nucleus.     

Geranylgeranyl pyrophosphate-mediated protein geranylgeranylation regulates endothelial cell proliferation and apoptosis during vasculogenesis in mouse embryo

Vascular development is essential for the establishment of the circulatory system during embryonic development and requires the proliferation of endothelial cells. However, the underpinning regulatory mechanisms are not well understood. Here, we report that geranylgeranyl pyrophosphate(GGPP), a metabolite involved in protein geranylgeranylation, plays an indispensable role in embryonic vascular development. GGPP is synthesized by geranylgeranyl pyrophosphate synthase(GGPPS) in the mevalonate pathway. The selective knockout of Ggpps in endothelial cells led to aberrant vascular development and embryonic lethality, resulting from the decreased proliferation and enhanced apoptosis of endothelial cells during vasculogenesis. The defect in protein geranylgeranylation induced by GGPP depletion inhibited the membrane localization of Rho A and enhanced yes-associated protein(YAP) phosphorylation, thereby prohibiting the entry of YAP into the nucleus and the expression of YAP target genes related to cell proliferation and the antiapoptosis process. Moreover, inhibition of the mevalonate pathway by simvastatin induced endothelial cell proliferation defects and apoptosis, which were ameliorated by GGPP. Geranylgeraniol(GGOH), a precursor of GGPP, ameliorated the harmful effects of simvastatin on vascular development of developing fetuses in pregnant mice. These results indicate that GGPP-mediated protein geranylgeranylation is essential for endothelial cell proliferation and the antiapoptosis process during embryonic vascular development.     

The zinc-finger protein CNBP is required for forebrain formation in the mouse

Mouse mutants have allowed us to gain significant insight into axis development. However, much remains to be learned about the cellular and molecular basis of early forebrain patterning. We describe a lethal mutation mouse strain generated using promoter-trap mutagenesis. The mutants exhibit severe forebrain truncation in homozygous mouse embryos and various craniofacial defects in heterozygotes. We show that the defects are caused by disruption of the gene encoding cellular nucleic acid binding protein (CNBP); Cnbp transgenic mice were able to rescue fully the mutant phenotype. Cnbp is first expressed in the anterior visceral endoderm (AVE) and, subsequently, in the anterior definitive endoderm (ADE), anterior neuroectoderm (ANE), anterior mesendoderm (AME), headfolds and forebrain. In Cnbp(-/-) embryos, the visceral endoderm remains in the distal tip of the conceptus and the ADE fails to form, whereas the node and notochord form normally. A substantial reduction in cell proliferation was observed in the anterior regions of Cnbp(-/-) embryos at gastrulation and neural-fold stages. In these regions, Myc expression was absent, indicating CNBP targets Myc in rostral head formation. Our findings demonstrate that Cnbp is essential for the forebrain induction and specification.     

Mitochondrial malate-aspartate shuttle regulates mouse embryo nutrient consumption

Pyruvate has been considered the sole substrate that can support development of the mouse zygote to the two-cell stage, with lactate able to support development from the two-cell stage. This study has determined for the first time that mitochondrial reducing equivalent shuttles regulate metabolism in the early embryo. Activity of the malate-aspartate shuttle was found to be essential for the metabolism of lactate in the two-cell embryo. Furthermore, the inability of the mouse zygote to use lactate as an energy source was a result of a lack of malate-aspartate shuttle activity. The mRNA for the four enzymes for shuttle activity were detected at all stages of development. It was determined that aspartate was a rate-limiting factor in the activity of the malate-aspartate shuttle in mouse zygotes probably due to the high K(m) of the cytoplasmic aspartate aminotransferase. Addition of high concentrations of exogenous aspartate to the culture medium enabled mouse zygotes to utilize lactate in the absence of pyruvate and develop normally to the blastocyst stage as well as produce normal viable offspring. This study determined that the malate-aspartate shuttle is a key regulator of embryo metabolism and therefore viability and is the first report that mouse zygotes can develop normally to term in the absence of pyruvate.     

Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo

This study was an investigation of the interaction of lactate on pyruvate and glucose metabolism in the early mouse embryo. Pyruvate uptake and metabolism by mouse embryos were significantly affected by increasing the lactate concentration in the culture medium. In contrast, glucose uptake was not affected by lactate in the culture medium. At the zygote stage, the percentage of pyruvate taken up and oxidized was significantly reduced in the presence of increasing lactate, while at the blastocyst stage, increasing the lactate concentration increased the percentage of pyruvate oxidized. Lactate oxidation was determined to be 3-fold higher (when lactate was present at 20 mM) at the blastocyst stage compared to the zygote. Analysis of the kinetics of lactate dehydrogenase (LDH) determined that while the V(max) of LDH was higher at the zygote stage, the K(m) of LDH was identical for both stages of development, confirming that the LDH isozyme was the same. Furthermore, the activity of LDH isolated from both stages was reduced by 40% in the presence of 20 mM lactate. The observed differences in lactate metabolism between the zygote and blastocyst must therefore be attributed to in situ regulation of LDH. Activity of isolated LDH was found to be affected by nicotinamide adenine dinucleotide(+) (NAD(+)) concentration. In the presence of increasing concentrations of lactate, zygotes exhibited an increase in autofluorescence consistent with a depletion of NAD(+) in the cytosol. No increase was observed for later-stage embryos. Therefore it is proposed that the differences in pyruvate and lactate metabolism at the different stages of development are due to differences in the in situ regulation of LDH by cytosolic redox potential.