October 1, 1996;
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
-associated serpentine receptor.
[+] show captions
Fig. 4. Localization of X-mr gene expression by whole mount in situ hybridization. (A) Vegetal view of a gastrula embryo. Note the strong labeling located in a horseshoe ring corresponding to involuting marginal zone, but the absence of labeling in the dorsal mesoderm above the blastopore lip (bl). (B) Lateral view of a neurula showing labeling in a ventral region close to the cement gland (cg), and in the dorsal part of the posterior region. A faint staining is observed in the lateral region, under the neural plate (np). (C,D) Lateral views of early larval stages with the head on the left and the back on the top. (E) Magnified view of an uncleared larva at stage 32-33. Note that the X-msr transcript is localized in vascular structures such as the anterior cardinal vein (acv), aortic arch (aa), intersomitic arteries (ia), the vascular vitelline network (vvn) and the posterior cardinal vein (pcv). (F) This larva is the one shown in E after clarification. Localization of the (G) and (H) magnifications are white-boxed. (G) Magnification of cardiac region. Note the labeling in the procardiac tube (pt) and its vascular extensions: ventral aorta (va) on the left and both vitelline veins (vv) on the right. (H) Magnitication of the lateral region, showing both lateral posterior cardinal veins (pcv), branched intersomitic arteries (ia) and dorsal aorta (da).
Fig 5. Embryonic origin of the cells expressing X-mu transcript. (A,B) Vegetal view of whole mount in situ hybridization of gastrulas from UV- ventralized (A) and lithium-dorsal&d (B) embryos, respectively. Note that the staining is localized all around the yolk plug in the ventralized embryo while it is absent in dorsalized embryo. (C) Thin transverse section from the anterior part of late neurula. Note that the labeling is exclusively localized in the lateral plate mesoderm (Ipm), and absent in axial and paraxial derivatives such as neural tube (nt), notochord (not), neural crest cells (nc) and somitic mesoderm (som). (D) Transverse section inside the branchial arches of a stage 32-33 embryo showing the labeling in the forming aortic arch (aa). Note also the staining in the retinal artery (ra) and in the anterior cardinal vein (acv). (E) Magnification of the branchial arches from (D), showing that cells of the visceral arches originating from the mesoderm are specifically labeled by the X-mu probe, whereas those deriving from endoderm (En) or neural crest (NCd) are unstained. Abbreviations: ar, archenteron; ph. pharynx; bv, brain vesicle.
Fig 6. Localization of X-msr transcript in the endothelial layer. (A) Parasagittal section of a larva at stage 32-33 showing the labeling in the procardiac tube (pt) and some primary blood vessels, such as the anterior cardinal vein (acv), aortic arches (aa) and intersomitic arteries (ia). (B) Transverse sec- tion at the level of the procardiac tube (pt). The lateral staining is in the aortic arch (aa). (C,D) Magnification of longitudinal and transverse procardiac tube sections. Note the exclusive labeling in the endocardium (En); myocardium (Myo) and pericardium (Per) am unstained. (E) Transverse section at the trunk level showing expression in both posterior cardinal veins (pcv). (F) Magnification of a transverse section at the trunk level. Note that the labeling is localized in the inner endothelial layer (white arrowhead). Abbreviations: e, eye; ph, pharynx; nt, neural tube; ov, otic vesicle; pc, pericar- dial cavity; not, notochord.
Fig. 3. Kinetics of X-msr gene expression. (A). Northern blot analysis. Ten pg of total RNA from oocytes (ovo) or staged embryos were loaded and
hybridized as described in Section 4. Note the presence of a single X-msr transcript, the size of which was estimated at 2.5 kb. (B) Determination of Xmsr
RNA levels by RNase protection assay. Total RNA from four unfertilized eggs (ovo), staged embryos or 4yg of yeast tRNA (lane DP) were subjected
to RNase protection. Lane P corresponds to l/IO undigested probe (0.284 kb). A protected fragment (0.238 kb) was obtained from stage 8-l I.
Note that no transcript could be detected before stage 8.