Mid-gastrula Embryos Research Articles

Mechanical Tensions Regulate Gene Expression in the Xenopus laevis Axial Tissues.

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation.

Wnt proteins can direct planar cell polarity in vertebrate ectoderm.

The coordinated orientation of cells across the tissue plane, known as planar cell polarity (PCP), is manifested by the segregation of core PCP proteins to different sides of the cell. Secreted Wnt ligands are involved in many PCP-dependent processes, yet whether they act as polarity cues has been controversial. We show that in Xenopus early ectoderm, the Prickle3/Vangl2 complex was polarized to anterior cell edges and this polarity was disrupted by several Wnt antagonists. In midgastrula embryos, Wnt5a, Wnt11, and Wnt11b, but not Wnt3a, acted across many cell diameters to orient Prickle3/Vangl2 complexes away from their sources regardless of their positions relative to the body axis. The planar polarity of endogenous Vangl2 in the neuroectoderm was similarly redirected by an ectopic Wnt source and disrupted after depletion of Wnt11b in the presumptive posterior region of the embryo. These observations provide evidence for the instructive role of Wnt ligands in vertebrate PCP.

Identification of new regulators of embryonic patterning and morphogenesis in Xenopus gastrulae by RNA sequencing

During early vertebrate embryogenesis, cell fate specification is often coupled with cell acquisition of specific adhesive, polar and/or motile behaviors. In Xenopus gastrulae, tissues fated to form different axial structures display distinct motility. The cells in the early organizer move collectively and directionally toward the animal pole and contribute to anterior mesendoderm, whereas the dorsal and the ventral-posterior trunk tissues surrounding the blastopore of mid-gastrula embryos undergo convergent extension and convergent thickening movements, respectively. While factors regulating cell lineage specification have been described in some detail, the molecular machinery that controls cell motility is not understood in depth. To gain insight into the gene battery that regulates both cell fates and motility in particular embryonic tissues, we performed RNA sequencing (RNA-seq) to investigate differentially expressed genes in the early organizer, the dorsal and the ventral marginal zone of Xenopus gastrulae. We uncovered many known signaling and transcription factors that have been reported to play roles in embryonic patterning during gastrulation. We also identified many uncharacterized genes as well as genes that encoded extracellular matrix (ECM) proteins or potential regulators of actin cytoskeleton. Co-expression of a selected subset of the differentially expressed genes with activin in animal caps revealed that they had distinct ability to block activin-induced animal cap elongation. Most of these factors did not interfere with mesodermal induction by activin, but an ECM protein, EFEMP2, inhibited activin signaling and acted downstream of the activated type I receptor. By focusing on a secreted protein kinase PKDCC1, we showed with overexpression and knockdown experiments that PKDCC1 regulated gastrulation movements as well as anterior neural patterning during early Xenopus development. Overall, our studies identify many differentially expressed signaling and cytoskeleton regulators in different embryonic regions of Xenopus gastrulae and imply their functions in regulating cell fates and/or behaviors during gastrulation.

A Rapid Transcriptome Response Is Associated with Desiccation Resistance in Aerially-Exposed Killifish Embryos

Delayed hatching is a form of dormancy evolved in some amphibian and fish embryos to cope with environmental conditions transiently hostile to the survival of hatchlings or larvae. While diapause and cryptobiosis have been extensively studied in several animals, very little is known concerning the molecular mechanisms involved in the sensing and response of fish embryos to environmental cues. Embryos of the euryhaline killifish Fundulus heteroclitus advance dvelopment when exposed to air but hatching is suspended until flooding with seawater. Here, we investigated how transcriptome regulation underpins this adaptive response by examining changes in gene expression profiles of aerially incubated killifish embryos at ∼100% relative humidity, compared to embryos continuously flooded in water. The results confirm that mid-gastrula embryos are able to stimulate development in response to aerial incubation, which is accompanied by the differential expression of at least 806 distinct genes during a 24 h period. Most of these genes (∼70%) appear to be differentially expressed within 3 h of aerial exposure, suggesting a broad and rapid transcriptomic response. This response seems to include an early sensing phase, which overlaps with a tissue remodeling and activation of embryonic development phase involving many regulatory and metabolic pathways. Interestingly, we found fast (0.5–1 h) transcriptional differences in representatives of classical “stress” proteins, such as some molecular chaperones, members of signalling pathways typically involved in the transduction of sensor signals to stress response genes, and oxidative stress-related proteins, similar to that described in other animals undergoing dormancy, diapause or desiccation. To our knowledge, these data represent the first transcriptional profiling of molecular processes associated with desiccation resistance during delayed hatching in non-mammalian vertebrates. The exceptional transcriptomic plasticity observed in killifish embryos provides an important insight as to how the embryos are able to rapidly adapt to non-lethal desiccation conditions.

Mesenchyme cells can function to induce epithelial cell proliferation in starfish embryos

Here, we show that mesenchyme cells have a novel morphogenetic function in epithelial cell proliferation in starfish embryos. Blastula embryos were injected with pure populations of mesenchyme cells and the total cell numbers in the treated embryos were subsequently determined at different developmental stages. When a total of 40-50 mesenchyme cells was injected, total cells numbers in mid-gastrula embryos and 3-day-old bipinnaria larvae increased significantly (by 1.3-fold) compared with controls, with no indication of any mitotic activity in the injected mesenchyme cells. However, injection of more than 150 mesenchyme cells failed to induce proliferation of the epithelial cells and, moreover, interfered with normal morphogenesis. These developmental abnormalities occurred concomitantly with a severe condensation of the fibrous component of the extracellular matrix. Our data suggest that epithelial cell proliferation is induced by an appropriate number of mesenchyme cells in concert with the fibrous component of the extracellular matrix.

Xenopus fibrillin regulates directed convergence and extension

Fibrillin-based human diseases such as Marfan syndrome and congenital contractural arachnodactyly implicate fibrillins in the function and homeostasis of multiple adult tissues. Fibrillins are also expressed in embryos, but no early developmental role has been described for these proteins. We use three independent methods to reveal a role for Xenopus fibrillin (XF) at gastrulation. First, expressing truncated forms of XF in the embryo leads to failure of gastrulation concomitant with a dominant-negative effect on native fibrillin fibril assembly. Expressing truncated XF also inhibits normal progression of the patterned, polarized cell motility that drives convergence and extension at gastrulation and perturbs directed extension in cultured explants of dorsal mesoderm. Second, injection of a synthetic peptide encoding a cell-binding domain of XF into midgastrula embryos causes acute failure of gastrulation associated with defective fibrillin fibril assembly. These injections also reveal a critical role for this peptide in the fibril assembly process. Third, morpholino-mediated knockdown of translation of XF in the embryo also perturbs normal gastrulation and directed extension. Together, these data show that native Xenopus fibrillin is essential for the process of directed convergent extension in presumptive notochord at gastrulation.

Novel gene ashwin functions in Xenopus cell survival and anteroposterior patterning

The novel gene ashwin was isolated in a differential display screen for genes activated or up-regulated early in neural specification. ashwin is expressed maternally and zygotically, and it is up-regulated in the neural ectoderm after the midgastrula stage. It is expressed in the neural plate and later in the embryonic brain, eyes, and spinal cord. Overexpression of ashwin in whole embryos leads to anterior truncations and other defects. However, a second Organizer does not form, and the secondary axial structures may result from splitting of the Organizer, rather than axis duplication. Morpholino oligonucleotide-mediated reduction in ashwin expression leads to lethality or abnormalities in gastrulation, as well as significant apoptosis in midgastrula embryos. Apoptosis is also observed in midgastrula embryos overexpressing ashwin. Coexpression of ashwin with the bone morphogenetic protein-4 antagonist noggin has a synergistic effect on neural-specific gene expression in isolated animal cap ectoderm. Ashwin has no previously characterized domains, although two nuclear localization signals can be identified. Orthologues have been identified in the human, mouse, chicken, and pufferfish genomes. Our results suggest that ashwin regulates cell survival and anteroposterior patterning.

Morpholino phenocopies of sqt, oep, and ntl mutations.

We have used antisense morpholino oligonucleotides (MOs) to target three genes whose loss of function were previously known to cause embryonic phenotypes in zebrafish: squint (sqt), one-eyed pinhead (oep), and no tail (ntl). One-eyed pinhead is an EGF-CFC protein and Sqt is a Nodal-related protein (Feldman et al., 1998; Zhang et al., 1998). Embryos homozygous for oep or sqt mutations have cyclopia and axial mesendoderm defects (Heisenberg and Nusslein-Volhard, 1997; Schier et al., 1996). Homozygous mutants for ntl, the zebrafish homologue of brachyury, have tail truncations and notochord deficiencies (Halpern et al., 1993; Schulte-Merker et al., 1994). Embryos injected with moderate doses of a ntl MO displayed a classic ntl mutant phenotype (class I ntl morphants, Table 1 and Fig. 1b), as previously reported for a partially overlapping ntl MO (Nasevicius and Ekker, 2000). Additionally, morphants with higher doses of ntl MO have an apparent excess of cell death near the head (class II ntl morphants, Table 1). Antibody staining of mid-gastrula embryos injected with ntl MO or a control MO revealed a specific and complete loss of Ntl in ntl MO-injected embryos (Fig. 1i–j), as shown previously by Western blot (Nasevicius and Ekker, 2000). The sqt mutation has low penetrance (data not shown). Consistent with this, only a fraction of sqt MOinjected embryos display the sqt phenotype, suggesting frequent rescue by another factor such as Cyclops (Feldman et al., 1998). These morphants were morphologically identical to sqt mutants (Table 1 and Fig. 1f). We also co-injected ntl and sqt MOs. Consistent with individual MO injections, most embryos had the ntl phenotype and a fraction of these also displayed sqt defects (Table 1 and Fig. 1d). Injection of oep MOs yielded variable results. At low doses, cyclopia and mesendoderm defects, but not floor plate defects, were phenocopied (class I oep morphants, Table 1 and Fig. 1c). A second class of oep morphants (class II oep morphants) lacking eyes was also observed, and higher doses of oep MO increased class II frequency (Table 1 and Fig. 1e). The presence of floor plate—a later differentiating structure—in class I and II oep morphants suggests the oep MO loses effectiveness by the second day of development. Eyeless and cyclopean oep morphant phenotypes were previously reported by Nasevicius and Ekker, using an identical oep MO. The same authors reported a third class of morphants resembling maternal-zygotic oep embryos (Gritsman et al., 1999; Nasevicius and Ekker, 2000). We never obtained this phenotype, even when embryos were injected immediately after fertilization.

Inversion of left–right asymmetry in the formation of the adult rudiment in sea urchin larvae: removal of a part of embryos at the gastrula stage

At the late pluteus stage, sea urchin larvae form an adult rudiment left–right (LR) asymmetrically, on only the left side. Little is known about how the LR asymmetry of the adult rudiment is established in earlier stages during which the larval body is basically LR symmetric. To investigate how the different regions of the embryo function to establish LR asymmetry, we removed different regions at the gastrula stage and assessed the effects of the operation on the establishment of LR asymmetry of the adult rudiment.Surgery was performed on mid- to late-gastrula embryos of two indirect developing species: Scaphechinus mirabilis and Hemicentrotus pulcherrimus. The embryos were placed under a dissecting microscope (SMZ8, Leica) and ectodermal epithelium together with underlying mesenchyme cells was dissected out with a glass needle. The region along the midline of the embryo at the width of the archenteron was designated the ‘midline region’, and the region lateral to the ‘midline region’ was designated the ‘lateral region’. When the left and/or the right side was excised, the whole lateral region was precisely removed on the animal side, but the plane of incision deviated more laterally on the vegetal side to avoid the ventro-lateral cluster of primary mesenchyme cells, so that a part of the defined ‘lateral region’ was left on the vegetal side. The vertical excision was made by cutting mid-gastrula embryos vertically to the archenteron at a level just superior to the tip of the archenteron. In the sham operation, embryos were pressed with the needle as in the vertical excision, but the incision was stopped slightly before the animal and vegetal halves of the embryos were completely divided from each other. The operated embryos were examined through an optical microscope (Optiphoto, Nikon) to confirm that the excision was correct, and they were then cultured with a food supply (diatoms, Chaetoceros gracilis). The handedness of the adult rudiment was examined at the six-armed or eight-armed pluteus stage through an optical microscope (Table 1).

Microtubule disruption reveals that Spemann's organizer is subdivided into two domains by the vegetal alignment zone.

Mediolateral cell intercalation is proposed to drive morphogenesis of the primary embryonic axis in Xenopus. Mediolateral intercalation begins in a group of cells called the vegetal alignment zone, a subpopulation of cells in Spemann's organizer, and spreads through much of the marginal zone. To understand the functions of the vegetal alignment zone during gastrulation and axis formation, we have inhibited its formation by disrupting microtubules with nocodazole in early gastrula embryos. In such embryos, mediolateral intercalation, involution and convergent extension of the marginal zone do not occur. Although cell motility continues, and the anterior notochordal and somitic mesoderm differentiate in the pre-involution marginal zone, posterior notochordal and somitic mesoderm do not differentiate. In contrast, microtubule depolymerization in midgastrula embryos, after the vegetal alignment zone has formed, does not inhibit mediolateral cell intercalation, involution and convergent extension, or differentiation of posterior notochord and somites. We conclude that microtubules are required only for orienting and polarizing at stage 101/2 the first cells that undergo mediolateral intercalation and form the vegetal alignment zone, and not for subsequent morphogenesis. These results demonstrate that microtubules are required to form the vegetal alignment zone, and that both microtubules and the vegetal alignment zone play critical roles in the inductive and morphogenetic activities of Spemann's organizer. In addition, our results suggest that Spemann's organizer contains multiple organizers, which act in succession and change their location and function during gastrulation to generate the anterior/posterior axis in Xenopus.

Induction of neuronal differentiation by planar signals in Xenopus embryos.

The induction of the central nervous system in amphibian embryos is mediated both by early planar signals produced by mesoderm at the dorsal lip and later vertical signals emanating from the dorsal mesoderm after involution. We have examined the role and spatial extent of planar signals in the induction of neuronal differentiation. Planar explants that included only the deep layer of the dorsal marginal zone, comprising both the dorsal mesoderm and the contiguous dorsal ectoderm, were isolated at the beginning of gastrulation. After removal of the epithelial layer, explants were maintained in modified Danilchik's medium until mid-neurula stages, when they were transferred to modified Danilchik's medium + 0.1% bovine serum albumin and cultured on laminin. Neurite outgrowth occurred in 90% of these planar explants. In contrast, little or no neuronal differentiation occurred in either ventral planar explants or explants of ectoderm alone. Video analysis of cell movements shows that large-scale cell mixing does not occur between mesoderm cells and ectoderm cells in planar explants. Retrograde labelling of neuronal cell bodies indicates that cells throughout the ectoderm undergo neuronal differentiation; neurons also differentiate in cultures of distal ectoderm isolated at early neurula stages from planar explants prepared at the beginning of gastrulation. These observations indicate that planar signals act over an extended range to induce neuronal differentiation. The inductive capacity of vertical signals was examined by recombining animal caps from ultra-violet (UV) irradiated embryos with involuted mesoderm from normal midgastrula embryos. Differentiation of either neurons or anterior neural structures occurred in 73% of vertical recombinates. Our results demonstrate that planar signals from the dorsal lip of the blastopore are capable of inducing neuronal differentiation over a considerable distance in the absence of epithelial confinement, convergence and extension, and mixing between the mesoderm and ectoderm.

Tubulin in sea urchin embryonic cilia: post-translational modifications during regeneration.

Tubulin is the major protein found in the membrane/periaxonemal matrix fraction of mature sea urchin embryonic cilia but its distribution and possible function during ciliary assembly are unknown. Hypertonic salt may be used to deciliate the embryos, allowing synchronous regrowth of cilia and subsequent deciliation of the regenerating embryos at various times. During the earliest stages of regeneration, the amounts of tubulin in the axoneme and membrane/matrix fractions are nearly equal, but the proportion of tubulin in the axoneme fraction increases coincident with the quasi-linear growth phase while the membrane/matrix tubulin remains constant. Antibodies to tyrosinated and detyrosinated alpha-tubulin show that both the membrane/matrix and axonemal tubulin fractions are primarily unmodified (i.e. tyrosinated) at the earliest stages of regeneration but are progressively and equally detyrosinated coincident with regeneration, approaching a final level of 50% C-terminal Glu. A monoclonal antibody to acetylated alpha-tubulin reveals that both tubulin fractions are equally and maximally acetylated at relatively early stages of regeneration. In contrast, three-times-repolymerized tubulin from either unfertilized eggs or midgastrula embryos is primarily tyrosinated (greater than 97%) and not detectably acetylated. These data suggest that membrane/matrix tubulin is a precursor to axonemal tubulin and that acetylation and detyrosination may be involved in partitioning tubulin among cytoplasmic, ciliary membrane/matrix, and 9 + 2 compartments.

Characterization of protein kinase C in early Xenopus embryogenesis

Recently, we presented evidence that protein kinase C (PKC) is involved in mediating the endogenous signals that induced competent Xenopus ectoderm to differentiate to neural tissue. We report here that PKC is already strongly activated in neural-induced ectoderm from midgastrula embryos and that this activation runs parallel with an increase in the level of inositol phosphates. We further identify several proteins that are phosphorylated, both in natural neural-induced ectoderm and in TPA-treated ectoderm, suggesting that they are phosphorylated through the PKC route. We found no major changes in PKC activity among different pregastrula stages, including the unfertilized egg. However, PKC isolated from animal, ectodermal cells is highly sensitive to Ca2+ and can be activated by low concentrations, (6-25 microM) of arachidonic acid, while PKC isolated from vegetal, endodermal cells is more insensitive to Ca2+ and cannot be activated by arachidonic acid. These results suggest that different PKC isozymes are present in animal and vegetal cells.

An extracellular fibrillar matrix in gastrulating sea urchin embryos

Fine structural studies of fractured developing sea urchin embryos revealed the existence of a voluminous, fibrillar, extracellular matrix composed of fine filaments, twisting fibers and granules lining the blastocoel of midgastrula embryos. Glycine disaggregated embryos also exhibited this material. The fibrillar matrix is closely associated with the basal lamina of the ectodermal cells of the embryo and histochemical studies suggest it is composed mostly of sulfated glycosaminoglycans. The position of the matrix within the blastocoel as well as its organized association with embryonic cell surfaces is consistent with the hypothesis that it plays a major role in guiding the invaginating archenteron during gastrulation.

The synthesis and degradation of RNA containing polyriboadenylate during sea urchin embryogeny

The synthesis and degradation of polyriboadenylate [poly (A)] was studied during sea urchin embryogenesis in an effort to learn more about the role of poly (A) in the cell and its relationship to embryogeny. Midcleavage, midblastula, and midgastrula embryos were labeled with 3H-adenosine, and the kinetics of incorporation followed for 4 hr in both the entire cell and the cytoplasmic fraction. Cleaving embryos show linear incorporation of isotope. At the other stages isotope incorporation follows saturation kinetics indicative of turnover. RNA that contains poly (A) was shown to exhibit a type of incorporation kinetics similar to nonadenylylated RNA, although somewhat more of the RNA that contains poly (A) is exported to the cytoplasm than is the nonadenylylated portion. A considerable amount of the RNA that contains poly (A) is degraded in the nucleus without export to the cytoplasm. The poly (A) was isolated from the newly synthesized RNA that contains poly (A), and its size showed a broad range from about 80 to 200 nucleotides at all stages and for all labeling times. It is concluded that polyadenylylation is very active throughout embryogenesis, but that the stability of polyadenylylated molecules may be different in nucleus and cytoplasm, and from one developmental stage to another.