Regional requirements for Dishevelled signaling during Xenopus gastrulation: separable effects on blastopore closure, mesendoderm internalization and archenteron formation

During amphibian gastrulation, the embryo is transformed by the combined actions of several different tissues. Paradoxically, many of these morphogenetic processes can occur autonomously in tissue explants, yet the tissues in intact embryos must interact and be coordinated with one another in order to accomplish the major goals of gastrulation: closure of the blastopore to bring the endoderm and mesoderm fully inside the ectoderm, and generation of the archenteron. Here, we present high-resolution 3D digital datasets of frog gastrulae, and morphometrics that allow simultaneous assessment of the progress of convergent extension, blastopore closure and archenteron formation in a single embryo. To examine how the diverse morphogenetic engines work together to accomplish gastrulation, we combined these tools with time-lapse analysis of gastrulation, and examined both wild-type embryos and embryos in which gastrulation was disrupted by the manipulation of Dishevelled (Xdsh) signaling. Remarkably, although inhibition of Xdsh signaling disrupted both convergent extension and blastopore closure, mesendoderm internalization proceeded very effectively in these embryos. In addition, much of archenteron elongation was found to be independent of Xdsh signaling, especially during the second half of gastrulation. Finally, even in normal embryos, we found a surprising degree of dissociability between the various morphogenetic processes that occur during gastrulation. Together, these data highlight the central role of PCP signaling in governing distinct events of Xenopus gastrulation, and suggest that the loose relationship between morphogenetic processes may have facilitated the evolution of the wide variety of gastrulation mechanisms seen in different amphibian species.     

Development of the dendrobatid frog Colostethus machalilla

To provide a developmental correlate with other frogs, we prepared a normal table of development for the dendrobatid, Colostethus machalilla and analyzed the morphology of its early development. This frog reproduces in captivity and deposits moderately sized eggs (1.6 mm in diameter) in terrestrial nests. The father guards the embryos until tadpole hatching. We divided development until hatching into 25 stages and implemented methods for in vitro culture of the embryos. The external and internal morphology of embryos were evaluated by observations in whole mount and in sections. Neural, notochord and somite specific antibodies were used to analyze gene expression patterns by immunostaining of embryos. Embryonic development of C. machalilla is slow and deviates from Xenopus laevis. In C. machalilla the elongation of the notochord, neural plate and somite formation occur after blastopore closure, possibly due to a delay in the dorsal convergence and extension movements. The gastrula of C. machalilla also deviates from X. laevis. The archenteron remains small until blastopore closure, where small cells accumulate at the blastopore lips. Simultaneously, the blastocoel roof thins until it becomes a monolayer of cells. Although C. machalilla does not form an embryonic disk, its thick blastopore lips resemble the embryonic disk of the marsupial frog Gastrotheca riobambae and represent an interesting deviation from the gastrulation pattern observed in X. laevis.     

Light-sheet microscopy-based 3D single-cell tracking reveals a correlation between cell cycle and the start of endoderm cell internalization in early zebrafish development

Controlling the initiation of cell migration plays a fundamental role in shaping the tissue during embryonic development. During gastrulation in zebrafish, some mesendoderm cells migrate inward to form the endoderm as the innermost germ layer along the yolk syncytial layer. However, how the initiation of inward migration is regulated is poorly understood. In this study, we performed light-sheet microscopy-based 3D single-cell tracking consisting of (a) whole-embryo time-lapse imaging with light-sheet microscopy and (b) three-dimensional single cell tracking in the zebrafish gastrula in which cells are marked with histone H2A-mCherry (nuclei) and the sox17:EGFP transgene (expressed in endoderm cells). We analyzed the correlation between the timing of cell internalization and cell division. Most cells that differentiated into endoderm cells began to internalize during the first half of the cell cycle, where the length of a cell cycle was defined by the period between two successive cell divisions. By contrast, the timing of other internalized cells was not correlated with a certain phase of the cell cycle. These results suggest the possibility that cell differentiation is associated with the relationship between cell cycle progression and the start of internalization. Moreover, the 3D single-cell tracking approach is useful for further investigating how cell migration is integrated with cell proliferation to shape tissues in zebrafish embryos.     

Cytoplasmic TSC-22 (transforming growth factor-beta-stimulated clone-22) markedly enhances the radiation sensitivity of salivary gland cancer cells

We transfected a salivary gland cancer cell line, TYS, with three different forms of TSC-22 (transforming growth factor-beta-stimulated clone-22) gene: full-length TSC-22 (TSC-22FL) containing nuclear export signal, TSC-box and leucine zipper, truncated TSC-22 (TSC-22LZ) containing only TSC-box and leucine zipper, and truncated TSC-22 with nuclear localization signal (NLS-TSC-22LZ). High expression of TSC-22FL in the cytoplasm markedly enhanced the radiation-sensitivity of TYS cells, while, moderate expression of TSC-22FL marginally affected the radiation-sensitivity. TSC-22LZ, which was expressed in the cytoplasm and the nucleus, enhanced the radiation-sensitivity of TYS cells irrespective to its expression level. NLS-TSC-22LZ, which was expressed only in the nucleus, marginally affected the radiation-sensitivity of the cells even at high expression level. Interestingly, cytoplasmic TSC-22 translocates to nucleus concomitant with radiation-induced apoptosis. These results suggest that cytoplasmic localization of TSC-22 and translocation of TSC-22 from cytoplasm to nucleus is important for regulating the cell death signal after irradiation-induced DNA damage.     

Cleavage and gastrulation in the Kuruma shrimp Penaeus (Marsupenaeus) japonicus (Bate): A revised cell lineage and identification of a presumptive germ cell marker

A previous study suggested that mesendoderm (ME) cell arrest occurred at the 64‐cell stage and a ring of eight presumptive naupliar mesoderm cells or crown cells surrounded the blastopore in the Kuruma shrimp Penaeus (Marsupenaeus) japonicus. Since this varied from the pattern observed in other penaeoidean shrimp, cleavage and gastrulation was re‐examined in P. japonicus using the nucleic acid stain Sytox Green and confocal microscopy. In contrast to the earlier study, cleavage and gastrulation followed the pattern observed in other penaeoidean shrimp. The ME cells arrested at the 32‐cell stage, ingressed into the blastocoel, and resumed division after a three cell cycle delay. Nine naupliar mesoderm or crown cells surrounded the blastopore and their descendants invaginated during gastrulation. An intracellular body (ICB) was detected by Sytox Green and SYTO RNASelect staining to be segregated to one ME cell in P. japonicus, as described previously in Penaeus monodon. Staining of the ICB was eliminated by pre‐treatment with RNase but not DNase. The ICB was also found in two other penaeoidean shrimp, Penaeus vannamei (Family Penaeidae) and Sicyonia ingentis (Family Sicyoniidae). The results support the hypothesis that the ICB is a germ granule found in the Dendrobranchiata.     

Pericardin,a Drosophila type IV collagen-like protein is involved in the morphogenesis and maintenance of the heart epithelium during dorsal ectoderm closure

The steps that lead to the formation of a single primitive heart tube are highly conserved in vertebrate and invertebrate embryos. Concerted migration of the two lateral cardiogenic regions of the mesoderm and endoderm (or ectoderm in invertebrates) is required for their fusion at the midline of the embryo. Morphogenetic signals are involved in this process and the extracellular matrix has been proposed to serve as a link between the two layers of cells. Pericardin (Prc), a novel Drosophila extracellular matrix protein is a good candidate to participate in heart tube formation. The protein has the hallmarks of a type IV collagen alpha-chain and is mainly expressed in the pericardial cells at the onset of dorsal closure. As dorsal closure progresses, Pericardin expression becomes concentrated at the basal surface of the cardioblasts and around the pericardial cells, in close proximity to the dorsal ectoderm. Pericardin is absent from the lumen of the dorsal vessel.Genetic evidence suggests that Prc promotes the proper migration and alignment of heart cells. Df(3)vin6 embryos, as well as embryos in which prc has been silenced via RNAi, exhibit similar and significant defects in the formation of the heart epithelium. In these embryos, the heart epithelium appears disorganized during its migration to the dorsal midline. By the end of embryonic development, cardial and pericardial cells are misaligned such that small clusters of both cell types appear in the heart; these clusters of cells are associated with holes in the walls of the heart. A prc transgene can partially rescue each of these phenotypes, suggesting that prc regulates these events. Our results support, for the first time, the function of a collagen-like protein in the coordinated migration of dorsal ectoderm and heart cells.     

Expression of Na+, K+-ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na⁺, K⁺-ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na⁺, K⁺-ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na⁺, K⁺-ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na⁺, K⁺-ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8–16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li⁺ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na⁺, K⁺-ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.     

Axial patterning interactions in the sea urchin embryo: suppression of nodal by Wnt1 signaling

Wnt and Nodal signaling pathways are required for initial patterning of cell fates along anterior-posterior (AP) and dorsal-ventral (DV) axes, respectively, of sea urchin embryos during cleavage and early blastula stages. These mechanisms are connected because expression of nodal depends on early Wnt/β-catenin signaling. Here, we show that an important subsequent function of Wnt signaling is to control the shape of the nodal expression domain and maintain correct specification of different cell types along the axes of the embryo. In the absence of Wnt1, the posterior-ventral region of the embryo is severely altered during early gastrulation. Strikingly, at this time, nodal and its downstream target genes gsc and bra are expressed ectopically, extending posteriorly to the blastopore. They override the initial specification of posterior-ventral ectoderm and endoderm fates, eliminating the ventral contribution to the gut and displacing the ciliary band dorsally towards, and occasionally beyond, the blastopore. Consequently, in Wnt1 morphants, the blastopore is located at the border of the re-specified posterior-ventral oral ectoderm and by larval stages it is in the same plane near the stomodeum on the ventral side. In normal embryos, a Nodal-dependent process downregulates wnt1 expression in dorsal posterior cells during early gastrulation, focusing Wnt1 signaling to the posterior-ventral region where it suppresses nodal expression. These subsequent interactions between Wnt and Nodal signaling are thus mutually antagonistic, each limiting the range of the other's activity, in order to maintain and stabilize the body plan initially established by those same signaling pathways in the early embryo.     

Effects of Inducers on Inner and Outer Gastrula Ectoderm Layers of Xenopus laevis

Abstract. Gastrula ectoderm, isolated from Xenopus laevis , was cultured in Holtfreter solution or modified Leibovitz medium (L-15) by the sandwich-method with or without inducer. The ectoderm (SD cell layers) consists of two cell sheets, representing a superficial (S) and a deep (D) layer. In the L-15 medium rather than in Holtfreter solution, the two cell layers separate out into distinct cell masses. This difference in cell affinity under certain experimental conditions could indicate that the deep layer contains endodermal cells. However, an endodermal character of the deep layer can be ruled out by induction experiments with vegetalizing factor or dorsal blastopore lip as inducers. Under the influence of vegetalizing factor the outer as well as the inner ectoderm layer differentiated into mesodermal derivatives such as notochord and somites. The results of the experiments with dorsal blastopore lip as inducer indicate that both inner and outer ectoderm layers are responsive to the neural stimulus. The lower neural competence of the outer ectoderm layer observed by several authors in normogenesis is discussed with regard to the hypothesis about short distance diffusion of the neuralizing factor and/or close cell-to-cell contact between inducing tissue and ectodermal target cells.     

Role of cell interactions in ascidian muscle and pigment cell specification

Summary Muscle and brain pigment cell specification was studied by disrupting cell adhesion, cell dissociation, and reaggregation in embryos of the ascidianStyela clava. Treatment of embryos with Ca²⁺-free sea water between the 2-cell and gastrula stages disrupted blastomere adhesion but did not prevent acetylcholinesterase or muscle actin expression in presumptive muscle cells. Similar treatments initiated between the 2- and 32-cell stages caused more ectoderm cells to express tyrosinase and develop pigment granules than expected from the cell lineage. Whereas 2 pigment cells become the otolith and ocellus sensory organs in normal embryos, up to 33 pigment cells could differentiate in embryos after disruption of cell adhesion. Replacement of Ca²⁺-free sea water with normal sea water restored cell adhesion and usually resulted in development of embryos containing the conventional number of pigment cells. Dissociation of embryos into single cells between the 2- and 64-cell stages and culture of these cells beyond the fate restricted stage had no effect on the accumulation of muscle actin mRNA and muscle actin synthesis, but blocked pigment cell differentiation. Reaggregation of the dissociated cells did not enhance the number of cells that developed muscle features, but rescued pigment cell development. The results indicate that ascidian muscle cell specification occurs by an autonomous mechanism, whereas pigment cell specification occurs by a conditional mechanism involving cell interactions. In addition, the results suggest that negative cell interactions may restrict the potential for pigment cell development in the ectoderm of cleaving ascidian embryos.     

Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo

The transition from early (E) to late (L) histone gene expression in developing sea urchin (Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L/E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.     

Constitutive and stress‐inducible expression of the endoplasmic reticulum heat shock protein 70 gene family member,immunoglobulin‐binding protein (BiP), during Xenopus laevis early development

We have characterized the constitutive and stress‐inducible pattern of immunoglobulin‐binding protein (BiP) gene expression during Xenopus early development. Whole mount in situ hybridization analysis revealed that BiP mRNA was detected in unfertilized eggs, cleavage and blastula stage embryos. In gastrulae, BiP mRNA was present across the surface of the embryo, while in neurulae BiP mRNA was enriched in the neural plate, neural fold, and around the blastopore. In early and late tailbud embryos, BiP mRNA was found primarily in the dorsal region. Tunicamycin and A23187, the calcium ionophore, enhanced BiP mRNA accumulation first at the neurula stage, while heat shock induced BiP mRNA accumulation first at the gastrula stage. Compared to control, A23187‐ and heat shock‐treated neurulae displayed relatively high levels of BiP mRNA in selected tissues, including the neural plate, neural folds, around the blastopore, and ectoderm. At the early tailbud stage, A23187 and heat shock enhanced BiP mRNA accumulation primarily in the head, somites, tail, and along the spinal cord. A similar situation was found with A23187‐ and heat shock‐treated late tailbud embryos, except that heat‐shocked embryos also displayed enhanced BiP mRNA accumulation in the epidermis. These studies demonstrate a preferential accumulation of BiP mRNA in selected tissues during development and in response to stress. Dev. Genet. 25:31–39, 1999. © 1999 Wiley‐Liss, Inc.     

Difference in the maternal and zygotic contributions of tumorhead on embryogenesis

Tumorhead (TH) is a maternally expressed gene in Xenopus laevis, that when overexpressed, increased proliferation of ectodermal derivatives and inhibited neural and epidermal differentiation. However, injection of anti-TH antibodies inhibited cleavage of all blastomeres, not only those contributing to the ectoderm. The injection of TH morpholino antisense oligonucleotide (TH-MO), which inhibits translation of TH mRNA, did not affect early cleavage but inhibited cell division in both the neural field and epidermis. This was accompanied by the inhibition of neural and epidermal markers. TH-MO did not affect the formation and differentiation of mesoderm and endoderm derivatives. Our overexpression and loss-of-function studies demonstrated that TH plays an important role in differentiation of the ectoderm by regulating cell proliferation. They also supported the conclusion that the maternal component of TH may affect the cell cycle in all cells, while the zygotic component has a germ layer-specific effect on the ectoderm.     

Novel population of embryonic secondary mesenchyme cells in the keyhole sand dollar Astriclypeus manni

We have found a novel embryonic cell population in the keyhole sand dollar Astriclypeus manni, which we refer to as lucent fluorescent cells (LFCs). Live LFCs are transparent, but emit autofluorescence after formaldehyde fixation. LFCs become noticeable in the vegetal plate of early gastrulae immediately after the appearance of pigment cells. As development progresses, LFCs increase in number and migrate from the vegetal plate toward the animal pole in a manner similar to pigment cells. Notably, LFCs also migrate into the oral ectoderm, while pigment cells do not. In addition, we determined that there were nearly 300 LFCs per embryo, which greatly exceeds the number of pigment cells. Treatment with the Notch signaling inhibitor N‐[(3,5‐Difluorophenyl)acetyl]‐l ‐alanyl‐2‐phenyl]glycine‐1,1‐dimethylethyl ester (DAPT) resulted in a marked decrease in pigment cell number, but only a modest decrease in LFCs. In DAPT‐treated embryos, LFCs had a distribution pattern similar to pigment cells and were excluded from the oral ectoderm. Unlike other sea urchins, Nodal signaling was not involved in the specification of pigment cells and LFCs in these embryos. Pulse treatment and measurement of cell diameters revealed that LFCs underwent 13–15 cycles of cell division and were specified during the 11th cleavage, one cell cycle later than observed for pigment cells. At the pluteus stage, a cluster of LFCs was observed in the animal plate in addition to two rows of LFCs running along the ciliary band. In addition, dozens of LFCs aligned at the uppermost level of the stomodaeum. Therefore, though the two cell populations share some features, LFCs are considerably different from pigment cells.     

食管癌细胞诱导肥大细胞迁移的小鼠模型

目的建立小鼠肥大细胞（mast cell,MC）迁移模型,探讨化疗药物三氧化二砷对人食管癌EC109细胞株生长的杀伤机理。方法利用肥大细胞的特征性蛋白酶抗体及其免疫荧光标记MC和PI标记EC109细胞内DNA;以流式细胞术分析小鼠腹腔液中肥大细胞各亚型的百分率及肿瘤细胞周期变化;使用激光扫描共聚焦显微镜显示肥大细胞内分泌颗粒的分布。并通过组织化学方法,观察各种诱导处理后小鼠肠组织肥大细胞由肠道向腹腔移动的变化。结果①根据流式细胞仪点图分布分析,将小鼠肥大细胞分为：类胰蛋白酶阳性、类糜蛋白酶阴性（MC T）;类糜蛋白酶阳性、类胰蛋白酶阴性（MC C）和类胰蛋白酶阳性、类糜蛋白酶阳性（MC TC）三种亚型,且T型MC明显多余TC型和C型（P〈0.05）;以共聚焦显微镜显示三种亚型的MC均含有丰富的分泌颗粒并分布于细胞膜内侧,为其出芽突起形成储备的状态。②经组织切片观察到诱导处理后小鼠肠组织MC由肠道向腹腔移动,且胰酶对MC的诱导作用大于食管癌细胞和As2O3。③经诱导迁移入腹腔的MC可能与癌细胞周期由S期向G2/M期跨越相关;As2O3能延迟食管癌细胞的G0/G1期,阻碍细胞向S期跨越,从而抑制食管癌细胞的生长。结论食管癌细胞移入小鼠腹腔,主要诱导T型MC参与免疫反应。在生物机体内环境（这里指MC影响）的条件下,As2O3对肿瘤细胞生长的作用主要表现为促使癌细胞周期的G0/G1期向S期跨越延迟,或G2/M期进入细胞分裂的延迟。     

Cell proliferation in ectodermal explants from Xenopus embryos

Summary Dissociated prospective ectoderm cells from Xenopus laevis embryos divide autonomously up to the 17th division cycle of the embryo. To examine the requirements for the further proliferation of these cells, the continuation of cell division in compact ectodermal explants beyond the 17th division cycle has been studied. Such explants develop into aggregates of epidermal cells, as can be shown immunohistochemically with an anti-serum against Xenopus epidermal cytokeratin. Cell division in these explants is comparable to the in vivo proliferation rate at least during the first 24 h of cultivation, that is, well beyond the 17th division cycle. Thus, epidermal cells are provided with all the factors necessary for continued proliferation, but these can be effective only when the cells form tight aggregates. The long-term changes in cell number are complex. Mitotic figures are present until the explants disintegrate after 3–4 days. However, the total cell number per explant does not increase during later development. The production of cells by mitotic divisions is likely to be countered by the loss of cells due to cell death, which is indicated by the presence of pyknotic nuclei.