HNF1(beta) is required for mesoderm induction in the Xenopus embryo

XHNF1(&bgr;) is a homeobox-containing gene initially expressed at the blastula stage in the vegetal part of the Xenopus embryo. We investigated its early role by functional ablation, through mRNA injection of an XHNF1(beta)/engrailed repressor fusion construct (XHNF1(beta)/EngR). Dorsal injections of XHNF1(beta)/EngR mRNA abolish dorsal mesoderm formation, leading to axial deficiencies; ventral injections disrupt ventral mesoderm formation without affecting axial development. XHNF1(beta)/EngR phenotypic effects specifically depend on the DNA-binding activity of its homeodomain and are fully rescued by coinjection of XHNF1(beta) mRNA. Vegetal injection of XHNF1(beta)/EngR mRNA blocks the mesoderm-inducing ability of vegetal explants. Both B-Vg1 and VegT maternal determinants trigger XHNF1(beta) expression in animal caps. XHNF1(beta)/EngR mRNA blocks B-Vg1-mediated, but not by eFGF-mediated, mesoderm induction in animals caps. However, wild-type XHNF1(beta) mRNA does not trigger Xbra expression in animal caps. We conclude that XHNF1(beta) function is essential, though not sufficient, for mesoderm induction in the Xenopus embryo.     

The RADICLELESS1 gene is required for vascular pattern formation in rice

The molecular mechanisms through which the complex patterns of plant vascular tissues are established are largely unknown. The highly ordered, yet simple, striate array of veins of rice leaves represents an attractive system to study the dynamics underlying pattern formation. Here we show that mutation in the RADICLELESS1 (RAL1) gene results in distinctive vascular pattern defects. In ral1 embryonic scutella, secondary veins are absent and in the prematurely aborted and discontinuous primary veins, cells are misaligned to each other. In ral1 leaves, longitudinal and commissural (transverse) veins display altered spacing and the commissural veins additionally show atypical branching and interruptions in their continuity. The vascular pattern alterations of ral1 occur in the context of normally shaped leaf primordia. Anatomical inspection and analysis of the expression of the procambium specification marker Oshox1-GUS and of the auxin-inducible reporter DR5-GUS demonstrates that all the vascular patterning aberrations of ral1 originate from defects in the procambium, which represents the earliest identifiable stage of vascular development. Furthermore, the ral1 mutant is unique in that procambium formation in leaf primordium development is delayed. Finally, the ral1 vascular patterning distortions are associated with a defective response to auxin and with an enhanced sensitivity to cytokinin. ral1 is the first mutant impaired in both procambium development and vascular patterning to be isolated in a monocot species.     

The Drosophila ecdysone receptor (EcR) gene is required maternally for normal oogenesis

Oogenesis in Drosophila is regulated by the steroid hormone ecdysone and the sesquiterpenoid juvenile hormone. Response to ecdysone is mediated by a heteromeric receptor composed of the EcR and USP proteins. We have identified a temperature-sensitive EcR mutation, EcR(A483T), from a previously isolated collection of EcR mutations. EcR(A483T) is predicted to affect all EcR protein products (EcR-A, EcR-B1, and EcR-B2) since it maps to a common exon encoding the ligand-binding domain. In wild-type females, we find that both EcR-A and EcR-B1 are expressed in nurse cells and follicle cells throughout oogenesis. EcR mutant females raised at permissive temperature and then shifted to restrictive temperature exhibit severe reductions in fecundity. Oogenesis in EcR mutant females is defective, and the spectrum of oogenic defects includes the presence of abnormal egg chambers and loss of vitellogenic egg stages. Our results demonstrate a requirement for EcR during female reproduction and suggest that EcR is required for normal oogenesis.     

The mouse Cer1 (Cerberus related or homologue) gene is not required for anterior pattern formation.

Cer1 is the mouse homologue of the Xenopus Cerberus gene whose product is able to induce development of head structures during embryonic development. The Cer1 protein is a member of the cysteine knot superfamily and is expressed in anterior regions of the mouse gastrula. A segmental pattern of expression with nascent and newly formed somites is also seen. This suggests an additional role in development of the axial skeleton, musculature, or peripheral nervous system. Xenopus animal cap assays and mouse germ-layer explant recombination experiments indicate that the mouse protein can act as a patterning molecule for anterior development in Xenopus, including induction of Otx2 expression, and suggest it may have a similar role in mouse development. However, we present here genetic data that demonstrate that Cer1 is not necessary for anterior patterning, Otx2 expression, somite formation, or even normal mouse morphogenesis.     

The actin cytoskeleton is required for maintenance of posterior pole plasm components in the Drosophila embryo.

Localization of mRNAs is one of many aspects of cellular organization that requires the cytoskeleton. In Drosophila, microtubules are known to be required for correct localization of developmentally important mRNAs and proteins during oogenesis; however, the role of the actin cytoskeleton in localization is less clear. Furthermore, it is not known whether either of these cytoskeletal systems are necessary for maintenance of RNA localization in the early embryo. We have examined the contribution of the actin and microtubule cytoskeletons to maintenance of RNA and protein localization in the early Drosophila embryo. We have found that while microtubules are not necessary, the actin cytoskeleton is needed for stable association of nanos, oskar, germ cell-less and cyclin B mRNAs and Oskar and Vasa proteins at the posterior pole in the early embryo. In contrast, bicoid RNA, which is located at the anterior pole, does not require either cytoskeletal system to remain at the anterior.     

Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo

A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human muscular dystrophies. Here, we show that one of these mutations, sapje, disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases.     

Decrease in DNA methylation 1(DDM1) is required for the formation of ~mCHH islands in maize

DNA methylation plays a crucial role in suppressing mobilization of transposable elements and regulation of gene expression. A number of studies have indicated that DNA methylation pathways and patterns exhibit distinct properties in different species, including Arabidopsis, rice, and maize.Here, we characterized the function of DDM1 in regulating genome-wide DNA methylation in maize. Two homologs of Zm DDM1 are abundantly expressed in the embryo and their simultaneous disruption caused embryo lethality with abnormalities in cell proliferation from the early stage of kernel development. We establish that Zm DDM1 is critical for DNA methylation, at CHG sites, and to a lesser extent at CG sites, inheterochromatic regions, and unexpectedly, it is required for the formation ofmCHH islands. In addition, Zm DDM1 is indispensable for the presence of 24-nt si RNA, suggesting its involvement in the Rd DM pathway. Our results provide novel insight into the role of Zm DDM1 in regulating the formation of mCHH islands, via the Rd DM pathway maize, suggesting that,in comparison to Arabidopsis, maize may have adopted distinct mechanisms for regulating ~mCHH.     

Krapfen/dMyd88 is required for the establishment of dorsoventral pattern in the Drosophila embryo

In Drosophila, the dorsoventral axis is set up by the action of the dorsal group of genes and cactus, which have been ordered genetically in a linear pathway. We have identified and characterised krapfen (kra) as a new member of the dorsal-group genes. kra encodes for the Drosophila homologue of MyD88, an adapter protein operating in the mammalian IL-1 pathway. Epistasis experiments reveal that kra acts between the receptor Toll and the cytoplasmic factor Tube. We show that there is a direct interaction between Kra and Tube presumably mediated by the death domains present in both proteins. Tube in turn interacts with its downstream effector Pelle through death domain association. We therefore suggest that upon Toll activation, Kra associates with Pelle and Tube, in an heterotrimeric complex.     

The Toxoplasma gondii kinetochore is required for centrosome association with the centrocone (spindle pole)

The kinetochore is a multi‐protein structure assembled on eukaryotic centromeres mediating chromosome attachment to spindle microtubules. Here we identified the kinetochore proteins Nuf2 and Ndc80 in the apicomplexan parasite Toxoplasma gondii. Localization revealed that kinetochores remain clustered throughout the cell cycle and colocalize with clustered centromeres at the centrocone, a structure containing the spindle pole embedded in the nuclear envelope. Pharmacological disruption of microtubules resulted in partial loss of some kinetochore and centromere clustering, indicating microtubules are necessary but not strictly required for kinetochore clustering. Generation of a TgNuf2 conditional knock‐down strain revealed it is essential for chromosome segregation, but dispensable for centromere clustering. The centromeres actually remained associated with the centrocone suggesting microtubule binding is not required for their interaction with the spindle pole. The most striking observation upon TgNuf2 depletion was that the centrosome behaved normally, but that it lost its association with the centrocone. This suggests that microtubules are essential to maintain contact between the centrosome and chromosomes, and this interaction is critical for the partitioning of the nuclei into the two daughter parasites. Finally, genetic complementation experiments with mutated TgNuf2 constructs highlighted an apicomplexan‐specific motif with a putative role in nuclear localization.     

Rice globular embryo 4 (gle4) mutant is defective in radial pattern formation during embryogenesis

In higher plants, the main elements of the fundamental body plan, the apical-basal and radial patterns, are established during embryogenesis. We have isolated several globular embryo (gle) mutants of rice that fail to develop any embryonic organs. We expected that these gle mutants might include mutants defective in their radial pattern formation ability. We developed two markers specifically staining the L2 and L3 layers (OsSCR and OsPNH1, respectively) and characterized the gle mutants by using these markers in addition to the already developed markers Roc1 (marker for the L1 layer), Ramy1A (marker of the L1 layer of the epithelium), and OSH1 (marker of the apical region). One of the gle mutants, gle4, expressed Roc1 and Ramy1A at the normal positions, but other markers exhibited an abnormal expression pattern; that is, both OsPNH1 and OsSCR were expressed in the central region of the embryo and OSH1 expression was not observed. Calli from the gle4 epithelium regenerated plants with abnormal morphologies. These results indicate that the GLE4 gene is involved in radial pattern formation during rice embryogenesis to differentiate the L2 and L3 layers, but is not involved in the establishment of the L1 layer or in the formation of embryonic organs.     

Coronin-1 function is required for phagosome formation

Coronin-1 is an actin-associated protein whose function in actin dynamics has remained obscure. All coronin proteins have a variable N-terminal domain, followed by WD repeats and a C-terminal coiled-coil dimerization domain. Transfection of coronin-1-GFP into RAW 264.7 cells revealed that coronin rapidly and transiently associates with the phagosome. To determine if coronin is involved in mammalian phagocytosis we used a dominant-negative approach by expressing only the central WD domains. However, this caused cell rounding and dissociation from the substratum, hampering analysis of their phenotype. We therefore developed TAT-fusion constructs of coronin-1 WD domains to acutely introduce the recombinant protein fragment into live cells. We show that although TAT-WD has no effect on binding of opsonized RBCs to RAW 264.7 cells, receptor clustering or several downstream signaling events, lamellipodial extensions, and actin accumulation at the base of the bound particle were diminished. Furthermore, Arp3 accumulation at the phagosome was impaired after TAT-WD treatment. Interestingly, whereas coronin-1 also accumulates at the sites of actin remodeling associated with Salmonella invasion, TAT-WD had no effect on this process. Together, our data demonstrates that coronin-1 is required for an early step in phagosome formation, consistent with a role in actin polymerization.     

ACOS5 is required for primexine formation and exine pattern formation during microsporogenesis in Arabidopsis

Pollen exine, mainly composed of sporopollenin, plays important roles during microspore development. It has been reported that Acyl-CoA Synthetase5 (ACOS5) is required for sporopollenin biosynthesis in Arabidopsis. Here we show that ACOS5 is essential for primexine formation during Arabidopsis microspore development. Through genetic screen, we identified a point mutation of ACOS5 allele, acos5-2, showing abnormal microspore development. Its microspores were degenerated and aborted after released from the tetrads. Transmission electron microscopy showed that primexine formation was reduced in acos5-2 mutant as compared to that of the wild-type. Consequently, sporopollenin was aggregated and randomly deposited on the microspores. In situ hybridization indicated that the key regulators of tapetum development, DYT1 and TDF1, are required for the expression of ACOS5 in tapetum. Furthermore, the GUS reporter showed that the 593-bp promoter sequence was sufficient for the expression of ACOS5 in the anther. Our data provide evidence that ACOS5 is required for primexine formation and sporopollenin deposition during microspore development.     

Annexin 11 is required for midbody formation and completion of the terminal phase of cytokinesis

Annexins are Ca(2+)-binding, membrane-fusogenic proteins with diverse but poorly understood functions. Here, we show that during cell cycle progression annexin 11 translocates from the nucleus to the spindle poles in metaphase and to the spindle midzone in anaphase. Annexin 11 is recruited to the midbody in late telophase, where it forms part of the detergent-resistant matrix that also contains CHO1. To investigate the significance of these observations, we used RNA interference to deplete cells of annexin 11. A combination of confocal and video time-lapse microscopy revealed that cells lacking annexin 11 fail to establish a functional midbody. Instead, daughter cells remain connected by intercellular bridges that contain bundled microtubules and cytoplasmic organelles but exclude normal midbody components such as MKLP1 and Aurora B. Annexin 11-depleted cells failed to complete cytokinesis and died by apoptosis. These findings demonstrate an essential role for annexin 11 in the terminal phase of cytokinesis.     

POPK-1/Sad-1 kinase is required for the proper translocation of maternal mRNAs and putative germ plasm at the posterior pole of the ascidian embryo

Maternal mRNAs localized to specific regions in eggs play important roles in the establishment of embryonic axes and germ layers in various species. Type I postplasmic/PEM mRNAs, which are localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, such as the muscle determinant macho-1 mRNA, play key roles in embryonic development. In the present study, we analyzed the function of the postplasmic/PEM RNA Hr-POPK-1, which encodes a kinase of Halocynthia roretzi. When the function of POPK-1 was suppressed by morpholino antisense oligonucleotides, the resulting malformed larvae did not form muscle or mesenchyme, as in macho-1-deficient embryos. Epistatic analysis indicated that POPK-1 acts upstream of macho-1. When POPK-1 was knocked down, localization of every Type I postplasmic/PEM mRNA examined, including macho-1, was perturbed, showing diffuse early distribution and eventual concentration into a smaller area. This is the probable reason for the macho-1 dysfunction. The postplasmic/PEM mRNAs such as macho-1 and Hr-PEM1 are co-localized with the cortical endoplasmic reticulum (cER) and move with it after fertilization. Eventually they become highly concentrated into a subcellular structure, the centrosome-attracting body (CAB), at the posterior pole of the cleaving embryos. The suppression of POPK-1 function reduced the size of the domain of concentrated cER at the posterior pole, indicating that POPK-1 is involved in the movement of postplasmic/PEM RNAs via relocalization of cER. The CAB also shrank. These results suggest that Hr-POPK-1 plays roles in concentration and positioning of the cER, as well as in the concentration of CAB materials, such as putative germ plasm, in the posterior blastomeres.     

Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-β superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.     

Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo

In Xenopus, the Mix/Bix family of homeobox genes has been implicated in mesendoderm development. Mixl1 is the only known murine member of this family. To examine the role of Mixl1 in murine embryogenesis, we used gene targeting to create mice bearing a null mutation of Mixl1. Homozygous Mixl1 mutant embryos can be distinguished from their littermates by a marked thickening of the primitive streak. By the early somite stage, embryonic development is arrested, with the formation of abnormal head folds, foreshortened body axis, absence of heart tube and gut, deficient paraxial mesoderm, and an enlarged midline tissue mass that replaces the notochord. Development of extra-embryonic structures is generally normal except that the allantois is often disproportionately large for the size of the mutant embryo. In chimeras, Mixl1(-/-) mutant cells can contribute to all embryonic structures, with the exception of the hindgut, suggesting that Mixl1 activity is most crucial for endodermal differentiation. Mixl1 is therefore required for the morphogenesis of axial mesoderm, the heart and the gut during embryogenesis.     

Sonic hedgehog maintains proliferation in secondary heart field progenitors and is required for normal arterial pole formation

The Sonic hedgehog (Shh)-null mouse was initially described as a phenotypic mimic of Tetralogy of Fallot with pulmonary atresia (Washington Smoak, I., Byrd, N.A., Abu-Issa, R., Goddeeris, M.M., Anderson, R., Morris, J., Yamamura, K., Klingensmith, J., and Meyers, E.N. 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev. Biol. 283, 357–372.); however, subsequent reports describe only a single outflow tract, leaving the phenotype and its developmental mechanism unclear. We hypothesized that the phenotype that occurs in response to Shh knockdown is pulmonary atresia and is directly related to the abnormal development of the secondary heart field. We found that Shh was expressed by the pharyngeal endoderm adjacent to the secondary heart field and that its receptor Ptc2 was expressed in a gradient in the secondary heart field, with the most robust expression in the caudal secondary heart field, closest to the Shh expression. In vitro culture of secondary heart field with the hedgehog inhibitor cyclopamine significantly reduced proliferation. In ovo, cyclopamine treatment before the secondary heart field adds to the outflow tract reduced proliferation only in the caudal secondary heart field, which coincided with the region of high Ptc2 expression. After outflow tract septation should occur, embryos treated with cyclopamine exhibited pulmonary atresia, pulmonary stenosis, and persistent truncus arteriosus. In hearts with pulmonary atresia, cardiac neural crest-derived cells, which form the outflow tract septum, migrated into the outflow tract and formed a septum. However, this septum divided the outflow tract into two unequal sized vessels and effectively closed off the pulmonary outlet. These experiments show that Shh is necessary for secondary heart field proliferation, which is required for normal pulmonary trunk formation, and that embryos with pulmonary atresia have an outflow tract septum.