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.     

Cortical anchorages and cell type segregations of maternal postplasmic/PEM RNAs in ascidians

Ascidian postplasmic/PEM RNAs constitute a large class of cortical maternal RNAs which include developmental determinants (macho-1 and pem-1). We have analyzed the localization, cortical anchorage and cell type segregation of postplasmic/PEM RNAs in Ciona intestinalis and Phallusia mammillata using very high-resolution fluorescent in situ hybridization. We also compared RNAs extracted from whole oocytes and from isolated cortices using microarrays and localized RNAs possessing clusters of xCACx motifs in their 3′UTRs.Based on these combined approaches we conclude that: (1) the vast majority of the 39 postplasmic/PEM RNAs (including vasa) are localized in the egg cortex. (2) Many postplasmic/PEM RNAs 3′UTR are enriched in xCACx motifs, allowing us to identify 2 novel postplasmic/PEM RNAs (PSD and MnK). (3) Postplasmic/PEM RNAs anchored to cortical Endoplasmic Reticulum (cER) and those associated with granules have different cell destinations.We propose that there are 2 distinct categories of postplasmic/PEM RNAs on the basis of their cortical anchorages and cell destinations: (1) macho-1-like postplasmic/PEM RNAs anchored to cER which segregate into somatic B8.11 cells. (2) vasa-like postplasmic/PEM RNAs associated with granules which in addition to B8.11 cells segregate into B8.12 germ cells.     

Expression of a muscle determinant gene, macho-1, in the anural ascidian Molgula tectiformis

In anural (tailless) ascidian species, functional embryonic muscle is not formed. In urodele (tailed) ascidians, macho-1 functions as a maternally supplied factor for embryonic muscle formation. The failure of embryonic muscle development in anural ascidians may be due to the suppression of macho-1 expression. In this paper, however, we report the expression of macho-1 in embryos of an anural ascidian, Molgula tectiformis. Although M. tectiformis has lost the developmental potential to form functional embryonic muscle, macho-1 was expressed in a very similar manner as in urodele ascidians. This result, together with those of previous studies, strongly suggests that in M. tectiformis the upstream genetic cascade responsible for muscle formation is intact, while the downstream cascade including the expression of muscle structural genes is severely affected.Electronic Supplementary Material Supplementary material is available for this article at     

The effect of aging on the neural competence of the presumptive ectoderm and the effect of aged ectoderm on the differentiation of the trunk organizer inCynops pyrrhogaster

Summary The effect of aging on the neural competence of the presumptive ectoderm of the early gastrula, and the effect of aged ectoderm on the differentiation of the still uninvaginated dorsal blastoporal lip at the small yolk-plug stage — representing the trunk organizer — were examined by the sandwich method inCynops pyrrhogaster.The presumptive ectoderm to be used as reaction system was taken from 0 to 36 h exogastrulae obtained by operation at the early gastrula stage and combined with trunk organizer. In the 0 to 12 h explants typical trunktail structures were formed. With further aging of the presumptive ectoderm a decrease in frequency of spinal cord, notochord, and muscle and a simultaneous increase in frequency of mesenchyme and mesothelium were observed. In the 30 and 36 h explants neural competence had largely disappeared, the frequency of notochord and muscle become very low and their differentiation very poor, whereas the frequency of mesenchyme and mesothelium reached very high levels.We infer a reciprocal relationship between the induced spinal cord and the differentiation of notochord and muscle, as well as a transformation of notochordal material into mesenchyme and mesothelium under the influence of the aged ectoderm. The mode of action of the trunk organizer in normal development is discussed.     

Establishment of animal-vegetal polarity during maturation in ascidian oocytes

Mature ascidian oocytes are arrested in metaphase of meiosis I (Met I) and display a pronounced animal-vegetal polarity: a small meiotic spindle lies beneath the animal pole, and two adjacent cortical and subcortical domains respectively rich in cortical endoplasmic reticulum and postplasmic/PEM RNAs (cER/mRNA domain) and mitochondria (myoplasm domain) line the equatorial and vegetal regions. Symmetry-breaking events triggered by the fertilizing sperm remodel this primary animal-vegetal (a-v) axis to establish the embryonic (D-V, A-P) axes. To understand how this radial a-v polarity of eggs is established, we have analyzed the distribution of mitochondria, mRNAs, microtubules and chromosomes in pre-vitellogenic, vitellogenic and post-vitellogenic Germinal Vesicle (GV) stage oocytes and in spontaneously maturing oocytes of the ascidian Ciona intestinalis. We show that myoplasm and postplasmic/PEM RNAs move into the oocyte periphery at the end of oogenesis and that polarization along the a-v axis occurs after maturation in several steps which take 3-4 h to be completed. First, the Germinal Vesicle breaks down, and a meiotic spindle forms in the center of the oocyte. Second, the meiotic spindle moves in an apparently random direction towards the cortex. Third, when the microtubular spindle and chromosomes arrive and rotate in the cortex (defining the animal pole), the subcortical myoplasm domain and cortical postplasmic/PEM RNAs are excluded from the animal pole region, thus concentrating in the vegetal hemisphere. The actin cytoskeleton is required for migration of the spindle and subsequent polarization, whereas these events occur normally in the absence of microtubules. Our observations set the stage for understanding the mechanisms governing primary axis establishment and meiotic maturation in ascidians.     

Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor)

Summary Recently the mesoderm-inducing effects of the transforming growth factor (TGF-) family of proteins have been widely examined. In an attemt to elucidate the functions of these proteins, porcine inhibin A and activin A (erythroid differentiation factor; EDF) were examined. Treatment of explants with activin A led to differentiation of mesodermal derivatives such as mesenchyme, notochord, blood cells and muscle, but inhibin A had a much lesser effect. The mesodermal differentiation induced by activin A was also comfirmed by analyses using a polyclonal antibody against muscle myosin. By indirect immunofluorescence analysis, the differentiation of muscle blocks was observed in the activin-A-treated explants, whereas no differentiation was observed in inhibin-A-treated and control explants. These findings confirm that this protein of the TGF- family has mesoderm-inducing ability.     

Developmental expression of the amphioxus <Emphasis Type="Italic">Tbx1</Emphasis>/<Emphasis Type="Italic">10</Emphasis> gene illuminates the evolution of vertebrate branchial arches and sclerotome

We have isolated an amphioxus T-box gene that is orthologous to the two vertebrate genes, Tbx1 and Tbx10, and examined its expression pattern during embryonic and early larval development. AmphiTbx1/10 is first expressed in branchial arch endoderm and mesoderm of developing neurulae, and in a bilateral, segmented pattern in the ventral half of newly formed somites. Branchial expression is restricted to the first three branchial arches, and disappears completely by 4 days post fertilization. Ventral somitic expression is restricted to the first 10–12 somites, and is not observed in early larvae except in the most ventral mesoderm of the first three branchial arches. No expression can be detected by 4 days post fertilization. Integrating functional, phylogenetic and expression data from amphioxus and a variety of vertebrate model organisms, we have reconstructed the early evolutionary history of the Tbx1/10 subfamily of genes within the chordate lineage. We conclude that Tbx1/10-mediated branchial arch endoderm and mesoderm patterning functions predated the origin of neural crest, and that ventral somite specification functions predated the origin of vertebrate sclerotome, but that Tbx1 was later co-opted during the evolution of developmental programs regulating branchial neural crest and sclerotome migration.Edited by M. Akam     

Different spatial distribution of mRNAs for activin receptors (type IIA and IIB) and follistatin in developing embryos of Xenopus laevis

Spatial distribution of mRNAs for activin receptors and follistatin was studied by Northern blot hybridization using RNAs from different parts of dissected Xenopus embryos. mRNAs of two activin receptors (type IIA and IIB) occurred uniformly in pre-gastrular embryos, but occurred in larger amounts in ectoderm (in gastrulae), neural plate (in neurulae) and anterior (head) regions (in tailbud embryos) than in other embryonic regions. By contrast, follistatin mRNA appeared almost exclusively in the dorsal mesoderm including invaginating organizer region at the gastrula stage, in notochord and in dorsal ectoderm at the neurula stage, then in anterior part at the tailbud stage. The localized patterns of the distribution of these mRNAs may be due to the regionally different zygotic expression of genes in embryos at later stages. From the relatively widespread pattern of distribution of their mRNAs, we assume that both type IIA and type IIB activin receptors have broad functions in ectodermal and neural differentiation. On the other hand, follistatin mRNA showed quite a restricted pattern of expression, and therefore, we assume that follistatin may have functions more specifically related to the sites of expression of its mRNA. Thus, follistatin may be involved in the differentiation of notochord itself and/or directly be responsible for organizer functions such as neural induction and subsequent differentiation of induced neural tissues at the gastrula and later stages.     

Trb2, a mouse homolog of tribbles, is dispensable for kidney and mouse development

Glomeruli comprise an important filtering apparatus in the kidney and are derived from the metanephric mesenchyme. A nuclear protein, Sall1, is expressed in this mesenchyme, and we previously reported that Trb2, a mouse homolog of Drosophila tribbles, is expressed in the mesenchyme-derived tissues of the kidney by microarray analyses using Sall1-GFP knock-in mice. In the present report, we detected Trb2 expression in a variety of organs during gestation, including the kidneys, mesonephros, testes, heart, eyes, thymus, blood vessels, muscle, bones, tongue, spinal cord, and ganglions. In the developing kidney, Trb2 signals were detected in podocytes and the prospective mesangium of the glomeruli, as well as in ureteric bud tips. However, Trb2 mutant mice did not display any apparent phenotypes and no proteinuria was observed, indicating normal glomerular functions. These results suggest that Trb2 plays minimal roles during kidney and mouse development.     

Cortical and cytoplasmic flows driven by actin microfilaments polarize the cortical ER-mRNA domain along the a-v axis in ascidian oocytes

Cellular mechanisms generating the polarized redistribution of maternal Type I postplasmic/PEM mRNAs in ascidian oocytes remain unknown. We have previously shown that PEM-1 mRNA is associated with a network of rough cortical Endoplasmic Reticulum (cER) polarized along the animal-vegetal (a-v) axis forming a cER-mRNA domain in mature oocytes. We now investigate the a-v polarization of this cER-mRNA domain during meiotic maturation using H. roretzi and C. intestinalis. We show that the cER and Hr-PEM-1 aggregate as interconnected cortical patches at the cell periphery before maturation, which uniformly spread out during maturation and form a reticulated organization enriched in the vegetal hemisphere at the end of maturation. Time-lapse video recordings coupled with micromanipulations reveal that stereotyped surface, cortical and cytoplasmic flows accompany the vegetal shift of the cER-mRNA domain and mitochondria-rich myoplasm. Treatments with cytochalasin B and nocodazole indicate that both polarization of the cER-mRNA domain and mitochondria-rich myoplasm and cortical and cytoplasmic flows depend on actin cytoskeleton, but not microtubules. Using cortical fragments prepared from maturing oocytes coupled with high resolution immuno/in situ localization, we have further analyzed the effects of these inhibitors on the reorganizations the cER network and Hr-PEM-1 mRNA. We show that before maturation starts, Hr-PEM-1 mRNAs are already associated with the cER, and actin cytoskeleton inhibitors disturb their association. Finally, we hypothesize that Germinal Vesicle Break Down (GVBD) triggers an actomyosin-dependant cortical flow which directs the a-v polarization of ascidian oocytes.     

Inductive interactions required for mesodermal differentiation in Bufo arenarum gastrulae

 Dorso-marginal epithelium, a distinct crescent-shaped region in the early gastrula of Bufo arenarum, appears after involution as a narrow dorso-median strip of archenteric endoderm close to the notochord. In explant cultures, this layer showed an extreme dorsalizing activity, promoting the formation of notochordal structures from ventro-mesodermal cells. In the presence of ectoderm, this inductive activity was expanded resulting in a wide range of dorso-lateral components such as notochord, muscle, nephric tubules, mesothelium and mesenchyme. The mesodermal origin of these derivatives was confirmed by the use of FDA (fluorescein-dextran-amine)-labelled explants. Extensive mesodermal development in cultures seems to require cell contacts between the inner aspect of the dorso-marginal epithelium and mesodermal cells. When such contacts were prevented, cultures would only differentiate erythrocytes as a result of a purely ectodermal stimulus. Bisection of the dorso-marginal epithelium in whole embryos resulted in the development of a duplicated set of axial structures, clearly showing the role of the epithelium as a dorsal organizer. Received: 30 July 1996 / Accepted: 20 February 1997     

Induction of notochord by the organizer inXenopus

Summary One important step in understanding early development is to define the cell interactions involved in establishing tissue types. In amphibian embryos, one such interaction is the induction by the organizer region after the late blastula stage of lateral and ventral regions of the marginal zone (MZ) to form dorsal tissue types such as muscle. It is not known whether the organizer can also induce lateral MZ to form notochord after the late blastula stage. We find that this induction occurs under experimental conditions and plays a role in normalXenopus development. The ability to induce notochord is strongest at the center of the organizer along the dorsal midline and weaker at the lateral edges of the organizer. Organizer tissue along the dorsal midline, which would differentiate as notochord in normal development, can exhibit organizer functions such as the induction of the dorsolateral MZ to form notochord without later differentiating as notochord itself. Thus organizer activity can be dissociated from subsequent notochord formation.