Laurent Paquereau

Expression of a new G protein-coupled receptor X-msr is associated with an endothelial lineage in Xenopus laevis.

In order to determine whether G protein-coupled receptors play a role in early embryogenesis, we looked for cDNA fragments amplified between primers located in consensus sequences of transmembrane segments. Using one such amplified fragment as a probe, we cloned a novel member of the G protein-coupled receptor superfamily in Xenopus. Alignment of the deduced protein sequence with that of other receptors discloses some homology with angiotensin receptors. A single transcript of 2.5 kb is detected at the late blastula stage and its expression increases during gastrulation. In situ hybridization reveals transcripts initially in the ventrolateral involuting marginal zone and later in the lateral plate mesoderm. At larval stages, the transcript is expressed in procardiac tube and forming blood vessels, where it is localized in the inner endothelial layer. Thus, this gene traces an endothelial lineage and represents a very early and unique marker in Xenopus of the specification of cardiac and vascular endothelia. We propose the name of X-msr for mesenchyme-associated serpentine receptor.

Primary structure of the rat gene encoding an inhibitor of the insulin receptor tyrosine kinase.

The gene (PP63) encoding the inhibitor (PP63) of the insulin receptor tyrosine kinase was isolated from a rat genomic library. The intron/exon organization was deduced from Southern-blot analysis and sequence data (i.e., the exons + the boundaries). The PP63 gene, which maps to chromosome 11, spans approx. 8 kb and contains seven exons separated by six introns of different sizes. All of the boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Primer extension and S1 mapping experiments were used to locate the transcription start point (tsp) 73 nt upstream from the translational initiator. Both in vitro transcription assays and transcription of a chimeric gene in intact hepatoma cells indicated that the sequence located immediately upstream from the tsp contained a promoter. Several putative cis-regulatory elements, including a TATA box and a C/EBP-binding site were found within the 250 bp preceding the tsp.

Early expression of a β1-adrenergic receptor and catecholamines in Xenopus oocytes and embryos

From a Xenopus stage 11 cDNA library, we have cloned a gene, termed X‐β 1AR, whose sequence is highly homologous to that of the human β1‐adrenergic receptor. As shown by RT‐PCR assay, X‐β 1AR RNA is present in the mature oocyte, decreases after fertilization up to stage 6 and then gradually increases during gastrulation. Binding studies performed with radiolabeled ligands reveal that X‐β 1AR RNA is translated into the receptor protein. Furthermore, noradrenaline and adrenaline are also detected in oocytes and early embryos. The concomitant presence of β1‐adrenergic receptors and catecholamines suggest that this ligand‐receptor couple could play a role in the very early stages of embryonic development.

The mRNA encoding a β subunit of heterotrimeric GTP-binding proteins is localized to the animal pole of Xenopus laevis oocyte and embryos

In order to provide evidence for a potential role of heterotrimeric GTP-binding proteins in the transduction of developmental signals, we prepared cDNAs from Xenopus laevis embryos and looked for fragments amplified between primers located in conserved sequences of the different subtypes of beta subunit. Using the amplified fragment as a probe, we cloned a member of the beta subunit family. The deduced protein sequence of the amphibian cDNA is highly homologous to the beta 1 subtype and, accordingly, we have named the Xenopus gene XG beta 1. In situ hybridization and RNase protection assay revealed that XG beta 1 mRNA is confined to the animal hemisphere of the mature oocyte. This localization of XG beta 1 mRNA is established at stage V during oogenesis. Following fertilization, the maternal mRNAs cosegregate with animal cells during cleavage stages. At gastrulation, transcripts are expressed in the dorsal ectoderm layer that will give rise to the central nervous system. Thus, XG beta 1 mRNA belongs to the small family of localized maternal mRNAs; as a transducing protein, its restriction to a subset of embryonic cells could mediate the distinct responsiveness which contributes to the patterning of the embryo.

A constitutively activated mutant of Gαq down-regulates EP-cadherin expression and decreases adhesion between ectodermal cells at gastrulation

We have examined the expression and function of the heterotrimeric GTP-binding protein Gq during early Xenopus embryogenesis. Abundant XGalphaq transcripts were detected in oocytes and early embryos by Northern blot analysis. In situ hybridization revealed that these transcripts are confined to the animal hemisphere of the mature oocyte and to the presumptive ectoderm of cleaving embryos. Microinjection at the two-cell stage of alphaq and Q209Lalphaq, a constitutively activated mutant, causes a disruption in ectodermal cell adhesion at late gastrulation. Dissociation/reaggregation experiments performed on animal cap explants clearly demonstrate that the Q209Lalphaq-induced phenotype occurs after reaggregation of the explants with a time-course similar to that observed in whole embryos. RT-PCR experiments performed on the explants from Q209Lalphaq-injected embryos revealed a selective decrease in the amount of EP-cadherin mRNA. Co-injection of EP-cadherin RNA, but also E-cadherin RNA, rescued the disaggregated phenotype. These data emphasize the functional link between Gq protein-coupled signalling pathways and cadherin molecules in the ectodermal layer during the morphogenetic movements of gastrulation.

GTP-binding proteins and early embryogenesis in Xenopus

During early embryogenesis the specification of body axes and the determination of cell subtypes proceeds through cell interactions and movements which involve the decoding of various signals in a spatial and temporal manner. An increasingly abundant literature has revealed the participation of growth factors and their receptors in the induction and regionalization of the mesoderm. The question therefore arises as to whether other signal transducing systems are expressed and play a role in early embryogenesis. In this mini review we describe the main developmental events occurring during early embryogenesis in Xenopus and the signalling pathways that are potentially involved; we then summarize the major properties of heterotrimeric GTP-binding proteins; finally, we present results suggesting that heterotrimeric GTP-binding proteins are expressed during early embryogenesis and discuss their potential function.