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Gessert S
,
Maurus D
,
Brade T
,
Walther P
,
Pandur P
,
Kühl M
.
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Vertebrate heart development requires specification of cardiac precursor cells, migration of cardiac progenitors as well as coordinated cell movements during looping and septation. DM-GRASP/ALCAM/CD166 is a member of the neuronal immunoglobulin domain superfamily of cell adhesion molecules and was recently suggested to be a target gene of non-canonical Wnt signalling. Loss of DM-GRASP function did not affect specification of cardiac progenitor cells. Later during development, expression of cardiac marker genes in the first heart field of Xenopus laevis such as Tbx20 and TnIc was reduced, whereas expression of the second heart field marker genes Isl-1 and BMP-4 was unaffected. Furthermore, loss of DM-GRASP function resulted in defective cell adhesion and cardiac morphogenesis. Additionally, expression of DM-GRASP can rescue the phenotype that results from the loss of non-canonical Wnt11-R signalling suggesting that DM-GRASP and non-canonical Wnt signalling are functionally coupled during cardiac development.
Fig. 1. Spatio-temporal expression of DM-GRASP II during Xenopus laevis development. (A) Schematic representation of the overall structure of X. laevis DM-GRASP. (B) Temporal expression of DM-GRASP as monitored by semi-quantitative RT-PCR. (C) Expression of DM-GRASP in different adult tissues. H4 was used as loading control in panels B and C. − RT serves as negative control. (D–P) Spatial expression of DM-GRASP as determined by whole mount in situ hybridization. (D) At stage 12.5, expression of DM-GRASP starts in the anterior region of the embryo (arrow). (E) At stage 13, DM-GRASP expression is stronger in the anterior neural plate (arrow). Dotted line indicates level of sagittal sections shown in panels N and O. (F) At stage 21, DM-GRASP staining is detected in the neural tube (black arrow), the mid- (red arrow) and hindbrain (white arrow), the eye (black arrowhead) and the profundal-trigeminal placodal area (pPrV). The expression in the neural tube persists until late tailbud stages. (G) Ventral view of a stage 25 embryo indicates expression of DM-GRASP in the bilateral heart anlage (arrow). (H) At stage 23, placodal staining is reinforced in two neurogenic placodes, the anterodorsal lateral line placode (pAD) and the profundal-trigeminal placodal area (pPrV). Expression in the dorsal spinal cord persists (arrow). DM-GRASP expression in the eye has decreased but becomes visible in the pronephros (arrowhead). (I) At stage 25, DM-GRASP expression becomes detectable in the heart (arrow). (J) Lateral view of an embryo at stage 30. DM-GRASP can be visualized in the midbrain–hindbrain boundary (isthmus, red arrow), the hindbrain (white arrow), the pronephros (arrowhead) and the heart (black arrow). (K) Lateral view of an embryo at stage 38. Expression of DM-GRASP in the isthmus (red arrow) and the heart (black arrow). (L) Close up view of an embryo at stage 30. DM-GRASP is expressed in diverse cranial ganglia like the trigeminal ganglion (gV), the profundal ganglion (gPr), the anterodorsal lateral line ganglion (gAD), the middle lateral line ganglion (gM), the facial epibranchial ganglion (egVII), the glossopharyngeal epibranchial ganglion (egIX), and the first vagal epibranchial ganglion (egXI). (M) Closeup view of an embryo at stage 38. Expression of DM-GRASP can be visualized in the anterodorsal lateral line ganglion (gAD), the middle lateral line ganglion (gM), the posteriorlateral line ganglion (gP), the glossopharyngeal epibranchial ganglion (egIX), the first vagal epibranchial ganglion (egXI) and the cells contributing to the vagal and posteriorlateral line ganglion (gVPL). (N + O) Sagittal sections of embryos at stage 13. DM-GRASP is expressed in the sensorial layer of neuroectoderm (sne) and the anterior part of neural plate (np). The epithelial layer of neuroectoderm (e) and somitogenic mesoderm (m) are devoid of DM-GRASP staining. (P) Transverse section of an embryo at stage 34. DM-GRASP is expressed in the myocardium of the heart (arrow).
Fig. 2. Expression of DM-GRASP in the FHF of Xenopus laevis. (A) Expression of different cardiac markers as determined by whole mount in situ hybridization at different stages as indicated. Arrows indicate cardiac progenitor cell population at stage 20, first heart field (FHF) at stage 24 and cells of the developing primary heart tube (PHT) at stage 29. Arrowheads indicate second heart field (SHF) at stage 24 and 29. The cement gland is highlighted by a dotted line. (B) Schematic representation of the location of cardiac progenitor cells, first heart field (FHF), second heart field (SHF) and forming primary heart tube (PHT) with the corresponding marker genes. DM-GRASP is not expressed in the cardiac progenitor cell population but in the FHF and the forming PHT at later stages. Note that at this stage, the linear heart tube has not yet formed.
Fig. 3. Loss of DM-GRASP function results in cardiac developmental defects. (A) Binding site of the DM-GRASP MO within the endogenous RNA. The start codon is indicated in green. δ5′-UTR is a mutant that lacks part of the 5′-UTR resulting in 9 mismatched bases (red asterisks). Coupled transcription and translation assays using 35S-radioactively labelled methionine indicate that the DM-GRASP MO but not a control MO blocks translation of DM-GRASP RNA. The 5′UTR deletion construct is not targeted by the DM-GRASP MO. (B) DM-GRASP depleted embryos have incompletely looped and smaller hearts at stage 43. oft: outflow tract, a: atrium, v: ventricle. (C) Quantitative presentation of observed phenotype in panel B. Error bars are standard errors of the means. N = number of examined embryos, n = number of independent experiments. (D) Histological analysis of DM-GRASP MO injected embryos and stained for Nkx2.5 expression by whole mount in situ hybridization indicates problems with morphogenetic movements in DM-GRASP depleted embryos. Embryos were overstained for histological analyses and expression levels of Nkx2.5 cannot be compared between DM-GRASP MO (N = 4) and control MO (N = 3) injected embryos.
Fig. 5. Loss of DM-GRASP affects marker gene expression in the FHF but not the SHF. (A) DM-GRASP MO was unilaterally injected at the 8-cell stage and expression of cardiac marker genes was monitored at stage 20. No changes for Tbx20, Isl-1 and Nkx2.5 could be detected. Anterior views of whole embryos are given. (B) DM-GRASP MO was unilaterally injected at the 8-cell stage and expression of cardiac marker genes as indicated was monitored at stage 28. Loss of DM-GRASP resulted in down-regulation of some marker genes of the FHF (arrows) but not SHF such as Isl-1 or BMP-4. Ventral views of embryos are given. (C) Quantitative presentation of observed phenotype in panel B. Error bars are standard errors of the means. N = number of examined embryos, n = number of independent experiments.
Fig. 6. ?5 -UTR DM-GRASP RNA can rescue the loss of marker gene expression in DM-GRASP MO injected embryos. (A) DM-GRASP MO was unilaterally injected at 8-cell stage together with RNA coding for ?5 UTR DM-GRASP or GFP. Expression of cardiac marker genes was monitored at stage 28. (B) Quantitative presentation of observed phenotype. Error bars are standard errors of the means. N = number of examined embryos, n = number of independent experiments.
Fig. 7. DM-GRASP and Wnt11-R are functionally linked. (A) Wnt11-R MO was unilaterally injected at the 8-cell stage and expression of cardiac marker genes was monitored at stage 28. Wnt11-R does not affect expression of the early cardiac marker genes Nkx2.5, Isl-1, and Tbx20, but markers of differentiated cardiomyocytes of the FHF such as TnIc, cActin, and MHCα. Bar graphs depict the quantitative presentation of the observed phenotype. Error bars are standard errors of the means. N = number of examined embryos, n = number of independent experiments. (B) Co-injection of RNA coding for DM-GRASP partially reverted the phenotype triggered by the Wnt11-R MO. Bar graphs depict the quantitative presentation of observed phenotype. Error bars are standard errors of the means. N = number of examined embryos, n = number of independent experiments.
