Start Of Gastrulation Research Articles

TRNA expression and modification landscapes, and their dynamics during zebrafish embryo development.

tRNA genes exist in multiple copies in the genome of all organisms across the three domains of life. Besides the sequence differences across tRNA copies, extensive post-transcriptional modification adds a further layer to tRNA diversification. Whilst the crucial role of tRNAs as adapter molecules in protein translation is well established, whether all tRNAs are actually expressed, and whether the differences across isodecoders play any regulatory role is only recently being uncovered. Here we built upon recent developments in the use of NGS-based methods for RNA modification detection and developed tRAM-seq, an experimental protocol and in silico analysis pipeline to investigate tRNA expression and modification. Using tRAM-seq, we analysed the full ensemble of nucleo-cytoplasmic and mitochondrial tRNAs during embryonic development of the model vertebrate zebrafish. We show that the repertoire of tRNAs changes during development, with an apparent major switch in tRNA isodecoder expression and modification profile taking place around the start of gastrulation. Taken together, our findings suggest the existence of a general reprogramming of the expressed tRNA pool, possibly gearing the translational machinery for distinct stages of the delicate and crucial process of embryo development.

Molecular anatomy of emerging Xenopus left-right organizer at successive developmental stages.

Vertebrate left-right symmetry breaking is preceded by formation of left-right organizer. In Amphibian, this structure is formed by gastrocoel roof plate, which emerges from superficial suprablastoporal cells. GRP is subdivided into medial area, which generates leftward flow by rotating monocilia and lateral Nodal1 expressing areas, which are involved in sensing of the flow. After successful symmetry breaking, medial cells are incorporated into a deep layer where they contribute to the axial mesoderm, while lateral domains join somitic mesoderm. Here, we performed detailed analysis of spatial and temporal gene expression of important markers and the corresponding morphology of emerging GRP. Endodermal marker Sox17 and markers of superficial mesoderm display complementary patterns at all studied stages. At early stages, GRP forms Tekt2 positive epithelial domain clearly separated from underlying deep layers, while at later stages, this separation disappears. Marker of early somitic mesoderm MyoD1 was absent in emerging GRP and was induced together with Nodal1 during early neurulation. Decreasing morphological separation is accompanied by lateral to medial covering of GRP by endoderm. Our data supports continuous link between superficial mesoderm at the start of gastrulation and mature GRP and suggests late induction of somitic fate in lateral GRP.

1001 – MAKING BLOOD BEFORE A BLOOD STEM CELL

Erythroid cells comprise a critical component of the cardiovascular network, which constitutes the first functional organ system of the mammalian embryo. In the beginning of the last century, it was recognized that distinct “primitive” and “definitive” red cell populations circulate in the embryo. Building on pioneering studies by Metcalf mapping myeloid progenitors in embryonic time and space, we determined that overlapping waves of primitive (EryP-CFC) and definitive (BFU-E) erythroid progenitors emerge in the yolk sac of pre-circulation murine embryos. EryP-CFC generate a synchronous wave of cells that comprises ∼40% of all cells in the mouse embryo, which undergo rapid hemoglobin accumulation, maturational globin switching, complex metabolic reprogramming, and ultimately enucleation, indicating that primitive erythropoiesis is indeed ‘mammalian’ in character. While EPO regulates the terminal maturation of primitive erythroblasts, it has remained unclear what factors regulate their initial emergence. Our recent studies indicate that Stat3 signaling, activated independently of EPO, may serve this function. Unlike EryP-CFC, the BFU-E that arise in the yolk sac are clonally associated with multiple myeloid lineages, and go on to seed the early fetal liver to serve as a critical bridge between primitive hematopoiesis and HSC-derived blood cell production. Interestingly, definitive, but not primitive, erythroblasts can self-renew when cultured ex vivo, through a Bmi1-regulated process. Overexpression of BMI1 in adult murine and human erythroblasts increases their in vitro self-renewal capacity, potentially providing a means of generating sufficient cultured red cells to meet future blood banking and transfusion needs. In summary, survival and growth of the mammalian embryo relies upon a complex and highly regulated process of robust red cell production from 2 distinct erythroid lineages that are initiated independently of HSCs soon after the start of gastrulation. Erythroid cells comprise a critical component of the cardiovascular network, which constitutes the first functional organ system of the mammalian embryo. In the beginning of the last century, it was recognized that distinct “primitive” and “definitive” red cell populations circulate in the embryo. Building on pioneering studies by Metcalf mapping myeloid progenitors in embryonic time and space, we determined that overlapping waves of primitive (EryP-CFC) and definitive (BFU-E) erythroid progenitors emerge in the yolk sac of pre-circulation murine embryos. EryP-CFC generate a synchronous wave of cells that comprises ∼40% of all cells in the mouse embryo, which undergo rapid hemoglobin accumulation, maturational globin switching, complex metabolic reprogramming, and ultimately enucleation, indicating that primitive erythropoiesis is indeed ‘mammalian’ in character. While EPO regulates the terminal maturation of primitive erythroblasts, it has remained unclear what factors regulate their initial emergence. Our recent studies indicate that Stat3 signaling, activated independently of EPO, may serve this function. Unlike EryP-CFC, the BFU-E that arise in the yolk sac are clonally associated with multiple myeloid lineages, and go on to seed the early fetal liver to serve as a critical bridge between primitive hematopoiesis and HSC-derived blood cell production. Interestingly, definitive, but not primitive, erythroblasts can self-renew when cultured ex vivo, through a Bmi1-regulated process. Overexpression of BMI1 in adult murine and human erythroblasts increases their in vitro self-renewal capacity, potentially providing a means of generating sufficient cultured red cells to meet future blood banking and transfusion needs. In summary, survival and growth of the mammalian embryo relies upon a complex and highly regulated process of robust red cell production from 2 distinct erythroid lineages that are initiated independently of HSCs soon after the start of gastrulation.

Molecular anatomy of the pre-primitive-streak chick embryo.

The early stages of development of the chick embryo, leading to primitive streak formation (the start of gastrulation), have received renewed attention recently, especially for studies of the mechanisms of large-scale cell movements and those that position the primitive streak in the radial blastodisc. Over the long history of chick embryology, the terminology used to define different regions has been changing, making it difficult to relate studies to each other. To resolve this objectively requires precise definitions of the regions based on anatomical and functional criteria, along with a systematic molecular map that can be compared directly to the functional anatomy. Here, we undertake these tasks. We describe the characteristic cell morphologies (using scanning electron microscopy and immunocytochemistry for cell polarity markers) in different regions and at successive stages. RNAseq was performed for 12 regions of the blastodisc, from which a set of putative regional markers was selected. These were studied in detail by in situ hybridization. Together this provides a comprehensive resource allowing the community to define the regions unambiguously and objectively. In addition to helping with future experimental design and interpretation, this resource will also be useful for evolutionary comparisons between different vertebrate species.

Developmental effects in fish embryos exposed to oil dispersions – The impact of crude oil micro-droplets

During accidental crude oil spills and permitted discharges of produced water into the marine environment, a large fraction of naturally occurring oil components will be contained in micron-sized oil droplets. Toxicity is assumed to be associated with the dissolved fraction of oil components, however the potential contribution of oil droplets to toxicity is currently not well known. In the present work we wanted to evaluate the contribution of oil droplets to effects on normal development of Atlantic cod (Gadus morhua) through exposing embryos for 96 h to un-filtered (dispersions containing droplets) and filtered (water soluble fractions) dispersions in a flow-through system at dispersion concentrations ranging from 0.14 to 4.34 mg oil/L. After exposure, the embryos were kept in clean seawater until hatch when survival, development and morphology were assessed. The experiment was performed at two different stages of embryonic development to cover two potentially sensitive stages (gastrulation and organogenesis). Exposure of cod embryos to crude oil dispersions caused acute and delayed toxicity, including manifestation of morphological deformations in hatched larvae. Oil droplets appear to contribute to some of the observed effects including mortality, larvae condition (standard length, body surface, and yolk sac size), spinal deformations as well as alterations in craniofacial and jaw development. The timing of exposure may be essential for the development of effects as higher acute mortality was observed when embryos were exposed from the start of gastrulation (Experiment 1) than when exposed during organogenesis (Experiment 2). Even though low mortality was observed when exposed during organogenesis, concentration-dependent mortality was observed during recovery.

Gene expression analysis of bovine embryonic disc, trophoblast and parietal hypoblast at the start of gastrulation.

In cattle early gastrulation-stage embryos (Stage 5), four tissues can be discerned: (i) the top layer of the embryonic disc consisting of embryonic ectoderm (EmE); (ii) the bottom layer of the disc consisting of mesoderm, endoderm and visceral hypoblast (MEH); (iii) the trophoblast (TB); and (iv) the parietal hypoblast. We performed microsurgery followed by RNA-seq to analyse the transcriptome of these four tissues as well as a developmentally earlier pre-gastrulation embryonic disc. The cattle EmE transcriptome was similar at Stages 4 and 5, characterised by the OCT4/SOX2/NANOG pluripotency network. Expression of genes associated with primordial germ cells suggest their presence in the EmE tissue at these stages. Anterior visceral hypoblast genes were transcribed in the Stage 4 disc, but no longer by Stage 5. The Stage 5 MEH layer was equally similar to mouse embryonic and extraembryonic visceral endoderm. Our data suggest that the first mesoderm to invaginate in cattle embryos is fated to become extraembryonic. TGFβ, FGF, VEGF, PDGFA, IGF2, IHH and WNT signals and receptors were expressed, however the representative members of the FGF families differed from that seen in equivalent tissues of mouse embryos. The TB transcriptome was unique and differed significantly from that of mice. FGF signalling in the TB may be autocrine with both FGFR2 and FGF2 expressed. Our data revealed a range of potential inter-tissue interactions, highlighted significant differences in early development between mice and cattle and yielded insight into the developmental events occurring at the start of gastrulation.

Morphological changes and germ layer formation in the porcine embryos from days 7–13 of development

Morphogenesis and identification of embryonic differentiation in porcine embryos are crucial issues for developmental biology and laboratory animal science. The current paper presents a study on the asynchronous development of hatched porcine embryos from days 7 to 13 post-insemination. Examination of semi-thin sections of the hypoblast showed that it had characteristics similar to those of the mouse anterior visceral endoderm during embryonic disc formation. Also, a cavity appeared in the epiblast, which was similar to a mouse proamniotic cavity. With the gradual disappearance of Rauber's layer, the cavity opened and contacted the external environment directly, all of which formed the embryonic disc. To confirm the differentiation characteristics, we performed immunohistochemical analyses and showed that GATA6 was detected clearly in parietal endoderm cells during embryonic disc establishment. OCT4 was expressed in the inner cell mass (ICM) and trophoblast of hatched blastocysts and in the epiblast during formation of the embryonic disc. However, OCT4 showed comparatively decreased expression in the posterior embryonic disc, primitive streak and migrating cells. SOX2 was present in the ICM and epiblast. Therefore, both SOX2 and OCT4 can be used as markers of pluripotent cells in the porcine embryonic disc. At the start of gastrulation, staining revealed VIMENTIN in the posterior of the embryonic disc, primitive streak and in migrating cells that underlay the embryonic disc and was also expressed in epiblast cells located in the anterior primitive streak. Together with serial sections of embryos stained by whole mount immunohistochemistry, the mesoderm differentiation pattern was shown as an ingression movement that took place at the posterior of the embryonic disc and with bilateral migration along the embryonic disc borders.

Maternal gene expression in Atlantic halibut (Hippoglossus hippoglossus L.) and its relation to egg quality

BackgroundThe commercial production of Atlantic halibut (Hippoglossus hippoglossus L.) suffers from a major bottleneck due to the low success of producing juveniles for on-growing. Atlantic halibut females are routinely hand-stripped and incorrect timing of stripping can result in low quality eggs due to post-ovulatory aging. Post-ovulatory aging leads to compositional changes in eggs that include maternally provided proteins and RNAs. There have been few studies of the maternally provided mRNA transcripts that control early development in commercially important fish species. The present study aimed to study maternal gene expression in Atlantic halibut and its relation to egg quality parameters including blastomere symmetry and hatching success.ResultsA maternal EST library containing 2341 sequences was constructed by suppressive subtractive hybridisation. Thirty genes were selected for expression studies; 23 novel genes and 7 genes with documented roles in early development. The expressions of twenty-one selected genes were measured by qPCR from fertilization to the 10-somite stage. Three genes were identified as strictly maternal genes that were expressed until the start of gastrulation; askopos (kop), si:dkey-30j22.9 (Tudor family member), and Tudor 5 protein (Tdrd5). The expressions of 18 genes at the 8-cell stage were correlated with egg quality parameters. The majority of genes showed either no or very minor correlations with egg quality parameter. However, two genes correlated positively with hatching success (r> 0.50, HHC00353: r = 0.58, p < 0.01; HHC01517: r = 0.56, p < 0.01) and one gene (HHC00255) was negatively correlated with the percentage of normal blastomeres (r = -0.62, p < 0.05).ConclusionsDuring this study we have related maternal levels of gene expression to hatching success in fish. Poor hatching success was not correlated with a general decrease in transcript abundance but with low transcript levels of some specific genes. Thus, the molecular mechanisms leading to low Atlantic halibut egg quality cannot be entirely explained by post-ovulatory aging.

Axial differentiation and early gastrulation stages of the pig embryo

Differentiation of the principal body axes in the early vertebrate embryo is based on a specific blueprint of gene expression and a series of transient axial structures such as Hensen's node and the notochord of the late gastrulation phase. Prior to gastrulation, the anterior visceral endoderm (AVE) of the mouse egg-cylinder or the anterior marginal crescent (AMC) of the rabbit embryonic disc marks the anterior pole of the embryo. For phylogenetic and functional reasons both these entities are addressed here as the mammalian anterior pregastrulation differentiation (APD). However, mouse and rabbit show distinct structural differences in APD and the molecular blueprint, making the search of general rules for axial differentiation in mammals difficult. Therefore, the pig was analysed here as a further species with a mammotypical flat embryonic disc. Using light and electron microscopy and in situ hybridisation for three key genes involved in early development ( sox17, nodal and brachyury), two axial structures of early gastrulation in the pig were identified: (1) the anterior hypoblast (AHB) characterised by increased cellular height and density and by sox17 expression, and (2) the early primitive streak characterised by a high pseudostratified epithelium with an almost continuous but unusually thick basement membrane, by localised epithelial–mesenchymal transition, and by brachyury expression in the epiblast. The stepwise appearance of these two axial structures was used to define three stages typical for mammals at the start of gastrulation. Intriguingly, the round shape and gradual posterior displacement of the APD in the pig appear to be species-specific (differing from all other mammals studied in detail to date) but correlate with ensuing specific primitive streak and extraembryonic mesoderm development. APD and, hence, the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraembryonic membranes and, possibly, the specific adult body form.

Notch signaling modulates the nuclear localization of carboxy-terminal-phosphorylated smad2 and controls the competence of ectodermal cells for activin A

Loss of mesodermal competence (LMC) during Xenopus development is a well known but little understood phenomenon that prospective ectodermal cells (animal caps) lose their competence for inductive signals, such as activin A, to induce mesodermal genes and tissues after the start of gastrulation. Notch signaling can delay the onset of LMC for activin A in animal caps [Coffman, C.R., Skoglund, P., Harris, W.A., Kintner, C.R., 1993. Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell 73, 659–671], although the mechanism by which this modulation occurs remains unknown. Here, we show that Notch signaling also delays the onset of LMC in whole embryos, as it did in animal caps. To better understand this effect and the mechanism of LMC itself, we investigated at which step of activin signal transduction pathway the Notch signaling act to affect the timing of the LMC. In our system, ALK4 (activin type I receptor) maintained the ability to phosphorylate the C-terminal region of smad2 upon activin A stimulus after the onset of LMC in both control- and Notch-activated animal caps. However, C-terminal-phosphorylated smad2 could bind to smad4 and accumulate in the nucleus only in Notch-activated animal caps. We conclude that LMC was induced because C-terminal-phosphorylated smad2 lost its ability to bind to smad4, and consequently could not accumulate in the nucleus. Notch signal activation restored the ability of C-terminal-phosphorylated smad2 to bind to smad4, resulting in a delay in the onset of LMC.

Low proliferative and high migratory activity in the area of Brachyury expressing mesoderm progenitor cells in the gastrulating rabbit embryo.

General mechanisms initiating the gastrulation process in early animal development are still elusive, not least because embryonic morphology differs widely among species. The rabbit embryo is revived here as a model to study vertebrate gastrulation, because its relatively simple morphology at the appropriate stages makes interspecific differences and similarities particularly obvious between mammals and birds. Three approaches that centre on mesoderm specification as a key event at the start of gastrulation were chosen. (1) A cDNA fragment encoding 212 amino acids of the rabbit Brachyury gene was cloned by RT-PCR and used as a molecular marker for mesoderm progenitors. Whole-mount in situ hybridisation revealed single Brachyury-expressing cells in the epiblast at 6.2 days post conception, i.e. several hours before the first ingressing mesoderm cells can be detected histologically. With the anterior marginal crescent as a landmark, these mesoderm progenitors are shown to lie in a posterior quadrant of the embryonic disc, which we call the posterior gastrula extension (PGE), for reasons established during the following functional analysis. (2) Vital dye (DiI) labelling in vitro suggests that epiblast cells arrive in the PGE from anterior parts of the embryonic disc and then move within this area in a complex pattern of posterior, centripetal and anterior directions to form the primitive streak. (3) BrdU labelling shows that proliferation is reduced in the PGE, while the remaining anterior part of the embryonic disc contains several areas of increased proliferation. These results reveal similarities with the chick with respect to Brachyury expression and cellular migration. They differ, however, in that local differences in proliferation are not seen in the pre-streak avian embryo. Rather, rabbit epiblast cells start mesoderm differentiation in a way similar to Drosophila, where a transient downregulation of proliferation initiates mesoderm differentiation and, hence, gastrulation.

Xiro-1 controls mesoderm patterning by repressing bmp-4 expression in the Spemann organizer.

The Iroquois genes code for homeodomain proteins that have been implicated in the neural development of Drosophila and vertebrates. We show here for the first time that Xiro-1, one of the Xenopus Iroquois genes, is expressed in the Spemann organizer from the start of gastrulation and that its overexpression induces a secondary axis as well as the ectopic expression of several organizer genes, such as chordin, goosecoid, and Xlim-1. Our results also indicate that Xiro-1 normally functions as a transcriptional repressor in the mesoderm. Overexpression of Xiro-1 or a chimeric form fused to the repressor domain of Engrailed cause similar phenotypes while overexpression of functional derivatives of Xiro-1 fused with transactivation domains (VP16 or E1A) produce the opposite effects. Finally, we show that Xiro-1 works as a repressor of bmp-4 transcription and that its effect on organizer development is dependent on BMP-4 activity. We propose that the previously observed down regulation of bmp-4 in the dorsal mesoderm during gastrulation can be explained by the repressor activity of Xiro-1 described here. Thus, Xiro-1 seems to have at least two different functions: control of neural plate and organizer development, both of which could be mediated by repression of bmp-4 transcription.

Involvement of a urethane-sensitive system in timing the onset of gastrulation in Xenopus laevis embryos.

This paper describes success in delaying the onset of gastrulation in Xenopus laevis embryos without damage to their subsequent development by temporarily arresting cleavage with urethane. Exposure of X. laevis embryos to 150 mM urethane before gastrulation resulted in cleavage arrest and its removal led to cleavage resumption. During cleavage arrest, cyclic activities including nuclear replication and the M-phase-promoting factor cycle continued, although their duration was lengthened to nearly 1.8-fold that of the controls. Because of a 30-min time lag from removal of urethane to resumption of cleavage, as well as the retardation of cyclic activities during cleavage arrest, the development of embryos after a 60-min exposure to urethane lagged two cell cycles behind that of control embryos. Here, the two cell cycle delay is equivalent to 50 min at 22-23 degrees C. The start of gastrulation in exposed embryos was accordingly delayed about 50 min, although the delay in mid-blastula transition was as little as 20-25 min. Consistent results were obtained in embryos exposed to urethane for 90 or 120 min and those exposed to procaine or NH4Cl for 60 min. Although these results imply that delay in the start of gastrulation in exposed embryos is ascribed simply to delay in their development raised by cleavage arrest, at the same time they suggest that the onset of gastrulation is timed by systems sensitive to urethane, procaine and NH4Cl in X. laevis embryos.

Xenopus frizzled-7 morphant displays defects in dorsoventral patterning and convergent extension movements during gastrulation.

Wnt signaling has been implicated in the process of gastrulation in Xenopus laevis. Overexpression of Wnt molecules, such as a putative dominant-negative form of Wnt8 (Hoppler et al., 1996), Wnt11 (Tada and Smith, 2000), as well as wild type Wnt5A and Wnt11 proteins (Du et al., 1995; Gradl et al., 1999; Moon et al., 1993) interferes with convergent extension movements during gastrulation. Overexpression of some other Wnt signaling pathway members such as full-length and truncated forms of Xenopus frizzled-7 (Djiane et al., 2000; Sumanas et al., 2000), a truncated form of Xenopus disheveled (Sokol, 1996) also causes similar defects. However, evidence for the requirement of a specific Wnt pathway protein in the gastrulation process is still missing. Here we investigated the function of Xenopus frizzled-7 (Xfz7) utilizing the morpholino phosphorodiamidate antisense oligonucleotides approach (Heasman et al., 2000; Summerton, 1999). A mixture of two morpholinos designed against two different orthologous copies of Xfz7 was injected into unfertilized oocytes. The resulting embryos (Xfz7 morphants) failed to properly close the blastopore, whereas the more severely affected embryos exogastrulated. The gastrulation defect was caused by inhibition of convergent extension movements as demonstrated by an animal cap cell motility assay (Smith et al., 1990). Adding synthetic Xfz7 RNA alleviated the observed gastrulation defect arguing for the specificity of the morpholino-based targeting. At tadpole stages, Xfz7 morphants displayed loss or reduction of anterior structures. Molecular analysis showed that approximately half of Xfz7 morphants had reduced expression of a dorsal marker Xnr3 at the gastrula stage. Xnr3 is known to be the direct target of the maternal Wnt axis induction pathway (Smith et al., 1995; McKendry et al., 1997) and has previously been shown to be under maternal Xfz7 control (Sumanas et al., 2000). The results presented here argue for two different Xfz7 functions. They confirm our previous findings that Xfz7 is required for the dorsoventral Wnt/b-catenin axis induction pathway (Sumanas et al., 2000) and demonstrate a Xfz7 requirement in a zygotic Wnt pathway that regulates morphogenetic movements during gastrulation. This is the first evidence for a vertebrate Frizzled protein to act in two distinct Wnt-signaling pathways, as known to be the case for Drosophila Frizzled-1 (Adler, 1992; Bhanot et al., 1999; Chen and Struhl, 1999). To study Xfz7 function during development, we designed two different morpholinos against the 5’UTR of two distinct orthologous copies of the Xfz7 gene present due to the allotetraploid nature of Xenopus (Kobel and Du Pasquier, 1986). One ortholog has been reported by Djiane et al. (2000)., Medina et al. (2000), and Sumanas et al. (2000), and the other ortholog of Xfz7 was reported by Wheeler and Hoppler (1999). These two Xfz7 orthologs are 97% identical in amino acid sequences, but contain more significant differences in the UTR sequences (data not shown). Both orthologs have similar expression patterns at all stages reported. A mixture of the two different morpholinos (MO-1 and MO-2) was injected into unfertilized oocytes, and the oocytes were subsequently fertilized using the host–transfer method (Heasman et al., 1994). Injection of individual morpholinos up to 20 ng resulted in no significant developmental defects, whether delivered into early embryos or into oocytes followed by host transfer fertilization (data not shown). We used doses of 5 ng (2.5ng each MO) and 10 ng (5ng each MO) of the morpholino mixture to simultaneously knockdown both orthologous copies of Xfz7. Xfz7 morphants developed seemingly normally with only a slight developmental delay until the start of gastrulation. During gastrulation, Xfz7 morphants formed greatly enlarged blastopores, which subsequently failed to involute properly, resulting in exogastrulating embryos at higher doses of morpholino used (Fig. 1a–c, i). Microinjecting synthetic Xfz7 RNA into oocytes significantly reduced the severity of the phenotype, resulting in a greater percentage of normal and weakly affected embryos (Fig. 1d, i). At tailbud and tadpole stages, Xfz7 morphants displayed defects in dorsoanterior structures. They commonly had a reduced or missing head and cement gland

From cortical rotation to organizer gene expression: toward a molecular explanation of axis specification in Xenopus.

After fertilization of Xenopus eggs, the cortex rotates relative to the cytoplasm, resulting in the formation of a cytoplasmic and transplantable dorsal-determining activity opposite the sperm entry point. This activity induces the dorsal expression of regulatory genes, which in turn establishes the Spemann organizer at the start of gastrulation. There has been considerable debate as to whether Vg1, or components of the Wnt-1 signaling pathway, normally function as this early dorsal determinant. Experiments now support the hypothesis that beta-catenin, a component of the Wnt pathway, provides the initial dorsoventral polarity to the embryo, and that Vg1 functions at a subsequent step in development. Specifically, beta-catenin is required for formation of the endogenous axes, and it is expressed at greater levels in dorsal cells during the early cleavage stages. Moreover, on the dorsal side of the embryo, complexes of beta-catenin and Tcf-3 directly bind the promoter of the dorsal regulatory genes siamois and twin and facilitate their expression, thereby contributing to the subsequent formation of the Spemann organizer. On the ventral side of the embryo, Tcf-3 likely works in the absence of beta-catenin as a transcriptional repressor of siamois. These and other data are considered in the context of how the initial polarization of the fertilized egg by the localized accumulation of beta-catenin establishes a range of subsequent dorsoventral asymmetries in the embryo.

From cortical rotation to organizer gene expression: toward a molecular explanation of axis specification in Xenopus

After fertilization of Xenopus eggs, the cortex rotates relative to the cytoplasm, resulting in the formation of a cytoplasmic and transplantable dorsal-determining activity opposite the sperm entry point. This activity induces the dorsal expression of regulatory genes, which in turn establishes the Spemann organizer at the start of gastrulation. There has been considerable debate as to whether Vg1, or components of the Wnt-1 signaling pathway, normally function as this early dorsal determinant. Experiments now support the hypothesis that β-catenin, a component of the Wnt pathway, provides the initial dorsoventral polarity to the embryo, and that Vg1 functions at a subsequent step in development. Specifically, β-catenin is required for formation of the endogenous axes, and it is expressed at greater levels in dorsal cells during the early cleavage stages. Moreover, on the dorsal side of the embryo, complexes of β-catenin and Tcf-3 directly bind the promoter of the dorsal regulatory genes siamois and twin and facilitate their expression, thereby contributing to the subsequent formation of the Spemann organizer. On the ventral side of the embryo, Tcf-3 likely works in the absence of β-catenin as a transcriptional repressor of siamois. These and other data are considered in the context of how the initial polarization of the fertilized egg by the localized accumulation of β-catenin establishes a range of subsequent dorsoventral asymmetries in the embryo. BioEssays 20:536–545, 1998.© 1998 John Wiley & Sons Inc.

Commitment to Hematopoietic Fates Begins During Early Gastrulation in the Mammalian Embryo • 292

During mammalian embryogenesis, erythropoiesis occurs in two distinct waves. The first wave, primitive erythropoiesis, characterized by nucleated megaloblasts expressing embryonic globins, occurs in yolk sac blood islands. The second wave, definitive erythropoiesis, is characterized by adult globincontaining anucleate cells, that differentiate later in the fetal liver and bone marrow. To determine where and when embryonic cells become committed to hematopoietic fates, we previously examined the spatial and temporal expression of transcription factors critical to yolk sac hematopoiesis. SCL/tal-1 and LMO2/rbtn-2 were found exclusively in mesoderm cells as early as the mid-primitive streak stage, just 12 hours after the start of gastrulation. These findings suggest that commitment of mesoderm cells to blood cell fates occurs during early gastrulation. We tested this hypothesis by culturing cells from carefully staged gastrulating mouse embryos in methylcellulose with cytokines that support the growth of both primitive and definitive erythroid colonies. No hematopoietic progenitors were evident prior to, or at the onset of, primitive streak formation (E6.5). Blood cell progenitors, consisting of primitive erythroid and macrophage lineages, were first present at the mid-primitive streak stage (E7.0). Definitive erythroid progenitors first appeared at early somite pair stages (E8.5) exclusively within the yolk sac. We conclude that commitment of embryonic cells to hematopoietic fates occurs soon after the onset of mesoderm formation and that definitive erythroid potential is evident in the yolk sac prior to the development of the fetal liver. These findings suggest that hematopoietic stem cells with primitive and definitive hematopoietic potential arise during gastrulation in the yolk sac.

Identification of homeotic target genes in Drosophila melanogaster including nervy, a proto-oncogene homologue.

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes.

A Clonal Analysis of Secondary Mesenchyme Cell Fates in the Sea Urchin Embryo

The secondary mesenchyme cells (SMCs) give rise to most of the mesoderm of the sea urchin embryo. Although the early embryonic lineage of these cells has been described, the mechanisms that cause SMCs to become restricted to a particular mesodermal cell fate are unknown. To begin to address this question, we performed a clonal analysis of the fates of SMC precursors in the vegetal plate by labeling single cells with the fluorescent dye DiI(C18). Our data show that some presumptive SMCs remain pluripotent at the late blastula stage, since some cells labeled at this stage gave rise to more than one mesodermal cell type. Surprisingly, however, most labeled cells gave rise to homogeneous clones composed of a single cell type. This observation indicates that either many SMC precursors are restricted in their fate before the start of gastrulation or that all the progeny of a single vegetal plate cell are influenced by the same instructional signals during gastrulation, despite the cell divisions and extensive cell movements that occur during this time. The percentage of clones composed of a single cell type increased during the blastula stage, supporting the view that the process of SMC fate specification begins before the onset of gastrulation.

Inductive Events in the Patterning of the Xenopus laevis Hatching and Cement Glands, Two Cell Types Which Delimit Head Boundaries

In Xenopus, the hatching and cement glands form at important boundaries in the head. We have examined induction of these glands in order to see the basis for this ectodermal patterning. Ectoderm can be induced to form hatching and cement glands by dorsal mesoderm and by the neural plate. Because the neural plate is sufficient to induce hatching and cement glands and lies adjacent to presumptive hatching and cement glands at the time of induction, it is the best candidate for the in vivo inducer of these tissues. The cement gland is restricted to the front of the head in part because the anterior but not posterior neural plate is capable of inducing it. The hatching gland is also restricted to the head, but can be induced by both anterior and posterior neural plates. Therefore, some factor suppresses hatching gland differentiation in the trunk. Transplanted neural plate pieces induced hatching gland cells in the ectoderm of embryos, suggesting that the inductive signal is planar. Treatment with retinoic acid or lithium at the start of gastrulation caused a loss of head structures. The presence of hatching gland in lithium-treated embryos suggests that these two agents have distinct effects and supports the idea that the induction of hatching and cement glands involves different pathways.