p68, a DEAD-box RNA helicase, is expressed in chordate embryo neural and mesodermal tissues

The p68 DEAD-box RNA helicases have been identified in diverse organisms, including yeast, invertebrates, and mammals. DEAD-box RNA helicases are thought to unwind duplexed RNAs, and the p68 family may participate in initiating nucleolar assembly. Recent evidence also suggests that they are developmentally regulated in chordate embryos. bobcat, a newly described member of this gene family, has been found in eggs and developing embryos of the ascidian urochordate, Molgula oculata. Antisense RNA experiments have implicated this gene in establishing basic chordate features, including the notochord and neural tube in ascidians (Swalla et al. 1999). We have isolated p68 homologs from chick and Xenopus in order to investigate their possible role in vertebrate development. We show that embryonic expression of p68 in chick, frog, and ascidian embryos is high in the developing brain and spinal cord as well as in the sensory vesicles. In frog embryos, p68 expression also marks the streams of migrating cranial neural crest cells throughout neural tube development and in tailbud stages, but neural crest expression is faint in chick embryos. Ascidian embryos also show mesodermal p68 expression during gastrulation and neurulation, and we document some p68 mesodermal expression in both chick and frog. Thus, as shown in these studies, p68 is expressed in early neural development and in various mesodermal tissues in a variety of chordate embryos, including chick, frog, and ascidian. Further functional experiments will be necessary to understand the role(s) p68 may play in vertebrate development.     

Spatially and temporally controlled electroporation of early chick embryos

The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.     

An anterior signalling centre in Xenopus revealed by the homeobox gene XHex.

BACKGROUND: Signals from anterior endodermal cells that express the homeobox gene Hex initiate development of the most rostral tissues of the mouse embryo. The dorsal/anterior endoderm of the Xenopus gastrula, which expresses Hex and the putative head-inducing gene cerberus, is proposed to be equivalent to the mouse anterior endoderm. Here, we report the origin and signalling properties of this population of cells in the early Xenopus embryo. RESULTS: Xenopus anterior endoderm was found to derive in part from cells at the centre of the blastocoel floor that express XHex, the Xenopus cognate of Hex. Like their counterparts in the mouse embryo, these Hex-expressing blastomeres moved to the dorsal side of the Xenopus embryo as gastrulation commenced, and populated deep endodermal adjacent to Spemann's organiser. Experiments involving the induction of secondary axes confirmed that XHex expression was associated with anterior development. Ventral misexpression of XHex induced ectopic cerberus expression and conferred anterior signalling properties to the endoderm. Unlike the effect of misexpressing cerberus, these signals could not neuralise overlying ectoderm. CONCLUSIONS: XHex expression reveals the unexpected origin of an anterior signalling centre in Xenopus, which arises in part from the centre of the blastula and localises to the deep endoderm adjacent to Spemann's organiser. Signals originating from these endodermal cells impart an anterior identity to the overlying ectoderm, but are insufficient for neural induction. The anterior movement of Hex-expressing cells in both Xenopus and mouse embryos suggests that this process is a conserved feature of vertebrate development.     

Differential gene expression in the anterior neural plate during gastrulation of Xenopus laevis

We have isolated three cDNA clones that are preferentially expressed in the cement gland of early Xenopus laevis embryos. These clones were used to study processes involved in the induction of this secretory organ. Results obtained show that the induction of this gland coincides with the process of neural induction. Genes specific for the cement gland are expressed very early in the anterior neural plate of stage-12 embryos. This suggests that the anteroposterior polarity of the neural plate is already established during gastrulation. At later stages of development, two of the three genes have secondary sites of expression. The expression of these genes can be induced in isolated animal caps by incubation in 10 mM-NH4Cl, a treatment that is known to induce cement glands.     

Protocadherin-19 is essential for early steps in brain morphogenesis

One of the earliest stages of brain morphogenesis is the establishment of the neural tube during neurulation. While some of the cellular mechanisms responsible for neurulation have been described in a number of vertebrate species, the underlying molecular processes are not fully understood. We have identified the zebrafish homolog of protocadherin-19, a member of the cadherin superfamily, which is expressed in the anterior neural plate and is required for brain morphogenesis. Interference with Protocadherin-19 function with antisense morpholino oligonucleotides leads to a severe disruption in early brain morphogenesis. Despite these pronounced effects on neurulation, axial patterning of the neural tube appears normal, as assessed by in situ hybridization for otx2, pax2.1 and krox20. Characterization of embryos early in development by in vivo 2-photon timelapse microscopy reveals that the observed disruption of morphogenesis results from an arrest of cell convergence in the anterior neural plate. These results provide the first functional data for protocadherin-19, demonstrating an essential role in early brain development.     

Elimination of heparan sulfate by heparitinases induces abnormal mesodermal and neural formation in Xenopus embryos

The expression of heparan sulfate glycosaminoglycan (HS-GAG) was examined in Xenopus embryos during the developmental stages. Chemical analysis showed the existence of HS-GAG in the ³⁵S-labeled embryos. By western blot analysis using a specific anti-HS monoclonal antibody, HS-GAG related epitope was found after the neurulation on two protein bands, whose molecular weights were approximately 90 kDa and 100 kDa, respectively. Immunohistochemistry revealed that HS-GAG occurred exclusively in the animal hemisphere in early gastrulae, and then appeared predominantly on the sheath of the neural tube, the notochord and epithelium. To address whether HS-GAG chains contribute to Xenopus embryonic development, we eliminated the embryonic HS-GAG by injecting purified Flavobacterium heparitinases (HSase) into their blastocoels. Most of the injected embryos were aberrant in mesodermal and neural formation, and became acephalic. Histological examination showed that these embryos were completely devoid of the central nervous system and the mesodermal tissues. Neither heat-inactivated heparitinase nor chondroitinase showed such abnormality. The HS-GAG-eliminated embryos showed decreased expression of both muscular and neural-specific markers. These results suggest that HS-GAG plays an indispensable role in establishing the fundamental body plan during early Xenopus development.     

Retinoic acid causes abnormal development and segmental patterning of the anterior hindbrain in Xenopus embryos.

Retinoic acid is a very potent teratogen and has also been implicated as an endogenous developmental signalling molecule in vertebrate embryos. One of the regions of the embryo reliably affected by exogenously applied RA is the hindbrain. In this paper, we describe in detail the hindbrain of Xenopus laevis embryos briefly treated with various levels of RA at gastrula stages. Such treatments lead to development of embryos with loss of anterior structures. In addition, RA has a general effect on rhombomere morphology and specific effects on the development of the anterior rhombomeres. This effect is demonstrated using neurofilament antibodies, HRP staining and in situ hybridisation using a probe for expression of the Xenopus Krox-20 gene. Anatomically it is evident that the development of the hindbrain normally anterior to the otocyst (rhombomeres 1-4) is abnormal following RA treatment. Sensory and motor axons of cranial nerves V and VII form a single root and the peripheral paths of V and VII and IX and X are also abnormal, as is the more anterior location of the otocyst. These anatomical changes are accompanied by changes in the pattern of expression for the gene XKrox-20, which normally expresses in rhombomeres 3 and 5, but is found in a single band in the anterior hindbrain of treated embryos which standardly fail to generate the normal external segmental appearance. The results are discussed in terms of both the teratogenic and possible endogenous roles of RA during normal development of the central nervous system. We conclude that low doses of RA applied during gastrulation have specific effects on the anterior Xenopus hindbrain which appear to be evolutionarily conserved in the light of similar recent findings in zebrafish.     

Regulation of Hex gene expression and initial stages of avian hepatogenesis by Bmp and Fgf signaling

The vertebrate liver and heart arise from adjacent cell layers in the anterior lateral (AL) endoderm and mesoderm of late gastrula embryos, and the earliest stages of liver and heart development are interrelated through reciprocal tissue interactions. Although classical embryological studies performed several decades ago in chick and quail defined the timing of hepatogenic induction in birds and the important role for cardiogenic mesoderm in this process, almost nothing is known about the molecular aspects of avian liver development. Here we use in vivo and explantation assays to investigate tissue interactions and signaling pathways regulating Hex, a homeobox gene required for liver development, and the earliest stages of hepatogenesis in the chick embryo. We find that explants of late gastrula anterior lateral endoderm plus mesoderm, which have been used extensively for studies relating to heart development, also produce albumin-expressing hepatoblasts. Expression of Hex, the earliest known molecular marker for the hepatogenic endoderm, and albumin, indicative of early committed hepatoblasts, requires both autocrine Bmp signaling and a specific paracrine signal from the cardiogenic (anterior lateral) mesoderm. Endodermal expression of Fox2a, in contrast, requires the mesoderm but is independent of Bmp signaling. In vivo induction assays show that the ability of BMP2 to activate Hex expression in the endoderm is restricted to a region that is only slightly larger than the endogenous domain of Hex expression. Although Fgfs can substitute for the cardiogenic mesoderm to support the expression of Hex and albumin in the endoderm, several Fgf genes are expressed in the anterior lateral endoderm but an Fgf expressed predominantly in the mesoderm was not identified. Studies also showed that Fgf gene expression in the endoderm does not require a signal from the mesoderm. Mechanisms regulating endodermal signaling pathways activated by Fgfs may therefore be more complex than previously appreciated.     

Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod

We report the identification of two distinct noggin genes in the tetrapod Xenopus tropicalis. Noggin functions to antagonize BMP signaling in many developmental contexts, and much work has explored its role in early vertebrate development. We have identified two noggin genes in the tropical clawed frog, X. tropicalis, a diploid anuran which is being explored for its potential as a genetic model system for early vertebrate development. Here we report the cloning and characterization of the Xenopus tropicalis noggin1 and noggin2 genes, which have distinct expression domains in the early embryo with one overlapping domain in the anterior neural tissue. X. tropicalis noggin1 expression is very similar to that of noggin in Xenopus laevis, with expression beginning in the blastula organizer region and continuing through gastrulation and neurulation in the organizer and notochord. Later, it is also expressed in the anterior neural ridge and subsequent forebrain; noggin1 is also expressed in the pharyngeal arches after neural tube closure. At the tadpole stage expression is maintained in the dorsal neural tube and is present in the otic vesicle. However, the expression of noggin2 is much more similar to the expression of noggin2 in D. rerio with expression in the forebrain, hindbrain, and somites, but unlike D. rerio, X. tropicalis noggin2 is expressed in the heart by stage 28. This work presents the first example of a tetrapod with at least two noggin genes.     

Examination of the expression of the heat shock protein gene, hsp110, in Xenopus laevis cultured cells and embryos

Eukaryotic organisms respond to various stresses with the synthesis of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is part of the HSP70/DnaK superfamily. In this study, we have examined, for the first time, the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. Sequence analysis revealed that the protein encoded by the hsp110 cDNA exhibited 74% identity with its counterparts in mammals and only 27-29% with members of the Xenopus HSP70 family. Hsp110 mRNA and/or protein was detected constitutively in A6 kidney epithelial cells and was inducible by heat shock, sodium arsenite, and cadmium chloride. However, treatment with ethanol or copper sulfate had no detectable effect on hsp110 mRNA levels. Similar results were obtained for hsp70 mRNA except that it was inducible with ethanol. In Xenopus embryos, hsp110 mRNA was present constitutively during development. Heat shock-inducible accumulation of hsp110 mRNA occurred only after the midblastula stage. Whole mount in situ hybridization analysis revealed that hsp110 mRNA accumulation in control and heat shocked embryos was enriched in selected tissues. These studies demonstrate that Xenopus hsp110 gene expression is constitutive and stress inducible in cultured cells and developmentally- and tissue specifically-regulated during early embryogenesis.     

An amphibian with ambition: a new role for Xenopus in the 21st century

Much of our knowledge about the mechanisms of vertebrate early development comes from studies using Xenopus laevis. The recent development of a remarkably efficient method for generating transgenic embryos is now allowing study of late development and organogenesis in Xenopus embryos. Possibilities are also emerging for genomic studies using the closely related diploid frog Xenopus tropicalis.     

Hsp70 expression and function during embryogenesis

This review focuses on the expression and function of 70-kDa heat shock proteins (Hsp70s) during mammalian embryogenesis, though many features of embryogenesis and the developmental expression of Hsp70s are conserved between mammals and other vertebrates. A variety of Hsp70s are expressed from the point of zygotic gene activation in cleavage-stage embryos, through blastulation, implantation, gastrulation, neurulation, organogenesis, and on throughout fetal maturation. The regulation and patterns of hsp70 gene expression and the known and putative Hsp70 protein functions vary from constitutive and metabolic housekeeping to stress-inducible and embryo-protective roles. Understanding the genetic regulation and molecular function of Hsp70s has been pursued by developmental biologists interested in the control of gene expression in early embryos as well as reproductive toxicologists and teratologists interested in how Hsp70s protect embryos from the adverse effects of environmental exposures. These efforts have also been joined by those interested in the chaperone functions of Hsp70s, and this confluence of effort has yielded many advances in our understanding of Hsp70s during critical phases of embryonic development and cellular differentiation.