Nie S and Chang C (2006), Regulation of early Xenopus development by ErbB...

XB-ART-1168

Dev Dyn 2006 Feb 01;2352:301-14. doi: 10.1002/dvdy.20623.

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Regulation of early Xenopus development by ErbB signaling.

Nie S , Chang C .

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ErbB signaling has long been implicated in cancer formation and progression and is shown to regulate cell division, migration, and death during tumorigenesis. The functions of the ErbB pathway during early vertebrate embryogenesis, however, are not well understood. Here we report characterization of ErbB activities during early frog development. Gain-of-function analyses show that EGFR, ErbB2, and ErbB4 induce ectopic tumor-like cell mass that contains increased numbers of mitotic cells. Both the muscle and the neural markers are expressed in these ectopic protrusions. ErbBs also induce mesodermal markers in ectodermal explants. Loss-of-function studies using carboxyl terminal-truncated dominant-negative ErbB receptors demonstrate that blocking ErbB signals leads to defective gastrulation movements and malformation of the embryonic axis with a reduction in the head structures in early frog embryos. These data, together with the observation that ErbBs are expressed early during frog embryogenesis, suggest that ErbBs regulate cell proliferation, movements, and embryonic patterning during early Xenopus development.

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Species referenced: Xenopus laevis
Genes referenced: egfr en2 erbb2 erbb3 erbb4 myod1 sox2 tbxt
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	Figure 1. ErbB receptors are expressed during early Xenopus embryogenesis. A: Alignment of the four Xenopus ErbBs in the conserved domain shows that they are homologous to each other, with 65 to 88% identical residues in this region. Shaded residues are identical in all four ErbBs. B: The Xenopus ErbB fragments are highly homologous to their human counterparts in the conserved region in the tyrosine kinase domain, with 94, 93, 82, and 96% similarity between Xenopus EGFR, ErbBs 2, 3, 4, and their corresponding human homologs, respectively. C: RT-PCR analysis shows that all ErbBs are expressed maternally, and the expression persists to at least tailbud stages during early Xenopus development. D: Expression of XErbB2 detected by in situ hybridization. XErbB2 was expressed widely in both ectodermal and mesodermal regions during gastrula and early neurula stages (i-iii). During neurulation (iv) and early tailbud stages (v), its expression was elevated in the neural tissues. Starting from early tailbud stages, its transcripts were also seen in myotomal muscles in addition to neural and neural crest-derived structures, such as brain, spinal cord, eyes, and branchial arches (v-viii). This expression pattern persists to at least tadpole stages (ix). i and ii are animal and vegetal views of mid-gastrula embryos, respectively; iii. iv, and vii are dorsal views of late gastrula, neurula, and tailbud embryos, respectively; v, vi, viii, and ix are lateral views of tailbud and tadpole stage embryos. E: Xenopus EGFR (i-v), ErbB3 (vi-x), and ErbB4 (xi-xv) are expressed widely during early development. At gastrula stages, they were expressed uniformly in ectodermal and mesodermal regions (i, ii, vi, vii, xi, xii), and the expression was increased in the neural tissues during neurulation (iii, viii, and xiii). At tailbud stages, the expression was seen in the brain, spinal cord, brachial arches, somites, eyes, and otic vesicles (iv, v, ix, x, xiv, xv). The embryos were viewed from animal (i, vi, xi) or vegetal (ii, vii, xii) sides at gastrula stages; and from lateral (iii, v, x, xiv, xv) or dorsal (iv, viii, ix, xiii) sides with the anterior to the left for neurula and tailbud embryos.
	egfr (epidermal growth factor receptor) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	erbb2 (erb-b2 receptor tyrosine kinase 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	erbb3 (erb-b2 receptor tyrosine kinase 3 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	erbb4 (erb-b2 receptor tyrosine kinase 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior left, dorsal up.
	Figure 2. Overexpression of EGFR, ErbB2, and ErbB4 induces ectopic protrusions. RNAs encoding ErbBs were injected into the animal poles of 2-cell stage embryos and the resulting tadpoles were examined at stages 34–38. A: EGFR (1 ng), ErbB2 (500 pg), and ErbB4 (500 pg), but not the kinase-defective ErbB3 (2 ng), induced ectopic protrusions when overexpressed in the ectoderm of early frog embryos. B: Lineage tracing with nuclear beta-galactosidase (200 pg RNA) indicated that ErbBs induced ectopic structures cell-autonomously. Black arrows point to the ectopic structures. All embryos were oriented with anterior to the left.
	Fig. 3. ErbBs stimulate cell proliferation in the ectopic protrusions. RNAs encoding ErbBs were injected into the animal poles of 2-cell stage embryos as in Figure 2, and the resulting tadpoles (stages 32–36) were analyzed by whole mount immunohistochemistry with anti- phosphorylated histone H3 antibody for detection of mitotic cells. Transverse microtomal sections of the embryos are shown here, with the dorsal side up. While most mitotic cells were scattered in wild type embryos, a dramatic increase in mitotic cells was seen in the ectopic protrusions induced by overexpression of EGFR, ErbB2, and ErbB4 (compare B, C, and D with A; black arrows, ectopic structures). Cell counting indicated that there was an average of 8-fold increase in mitotic cells in the protrusions.
	Fig. 4. ErbBs promote expression of differentiated cell markers in the ectopic protrusions. Tadpoles injected with ErbBs (same as in Fig. 2) were analyzed with immunohistochemistry studies using antibodies against a muscle epitope (12/101) and a neural epitope (Xen2). In the ectopic structures induced by EGFR, ErbB2, and ErbB4, both markers were ex- pressed; the new sites of marker expression were often separated from the endogenous ex- pression domains, suggesting a de novo induc- tion of these differentiated genes in the protru- sions. Black arrows point to the ectopic structures
	Fig. 5. ErbBs induce mesodermal markers in Xenopus ectodermal explants. RNAs encoding ErbB receptors were injected into the animal poles of two-cell stage embryos. Animal caps were dissected at blastula stages and incu- bated to gastrula (stage 11) or tailbud (stages 28–32) stages before total RNA was extracted for RT-PCR assay. Though the kinase-defective ErbB3 did not significantly induce any mesodermal markers, EGFR, ErbB2, and ErbB4 induced the expression of different mesodermal markers with different efficiencies in animal caps. While 1 ng of ErbB2 and ErbB4 RNAs were sufficient to induce multiple mesodermal genes, 4 ng of EGFR RNA was required to induce a subset of the mesodermal markers.
	Fig. 6. Blocking ErbB signaling with dominant negative ErbB receptors induces gastrulation and axial patterning defects. A: Schematic representation of the truncated ErbB receptors (DN-ErbBs) and the evidence supporting their actions in a dominant negative fashion to inhibit ErbB signaling. Two nanograms ErbB2 RNA was coinjected with 4 ng RNAs encoding DN-ErbBs into both animal poles of 2-cell stage embryos. Animal caps were explanted at blastula stages and RNA was extracted at gastrula stage (stage 11) for RT-PCR assay. Coexpression of any of the DN-ErbBs with ErbB2 (lanes 3 to 6) efficiently blocked the mesodermal induction by ErbB2 (lane 2), suggesting that DN-ErbBs function as dominant negative receptors. B: Inhibition of ErbB signaling leads to gastrulation and axial patterning defects. RNAs (250 pg to 1 ng) encoding DN-ErbBs were injected into the marginal zones of 2- to 4-cell stage embryos, and the morphology of the injected embryos was observed at gastrula (stages 10 –12) or tadpole (stages 34–38) stages. Embryos injected with 1 ng DN-ErbBs are shown. Though the onset of gastrulation was normal in embryos injected with DN-ErbBs (stage 10, a– e), the closure of the blastopore was severely delayed during gastrulation (stage 11, f–j; stage 12, k– o). At tadpole stages (stages 34 –38), embryos expressing DN-ErbBs showed a variety of defects, including open back (r, u, x, a’), reduced head structures (r to c’), shortened and curved body axis (s, t, v, w, y, z, b’, c’), and somite malformation (t, w, z, c’). C: Microtomal sections (horizontal sections, a–c; parasagittal sections, d–f) of the embryos showed that while muscle differentiation occurred in the absence of ErbB signaling, somite segmentation was impaired in embryos lacking ErbB signals. The arrows point to the segmented and the continuous somites in wild type and DN-ErbB embryos. The embryos were orientated with the head to the left.
	Fig. 8. Blocking the ErbB pathway affects the pattern of marker gene expression. RNAs encoding DN-ErbB2 or DN-ErbB4 (total 2ng) were injected into the marginal zones of 4-cell stage embryos, and marker gene expression was examined by in situ hybridization or immunohistochemistry at gastrula (stages 11–12) or tadpole (stages 36–40) stages. A: Inhibition of ErbB signaling does not lead to repression of early pan-mesodermal gene Brachyury (Xbra) expression, though the delay in gastrulation results in larger blastopore in embryos injected with DN-ErbBs. The embryos are at stages 11–12 and viewed from the vegetal side. B: The muscle marker 12/101 epitope was expressed in embryos expressing DN-ErbBs, but the repeated expression pattern of the epitope in distinct somites was not apparent. C: In situ hybridization with the MyoD probe illustrated that expression of MyoD in embryos injected with DN-ErbBs was reduced, and the reiterated pattern of expression in each somite was indistinct. D: The neural marker Xen2 epitope was expressed in the trunk region in embryos expressing DN-ErbBs, but the forebrain and the brachial arch neuronal expression domains were missing. E: The pan-neural marker Sox2 was expressed at normal levels in embryos injected with DN-ErbBs at gastrula stages (not shown); at tadpole stages, its expression in the head region was severely reduced, though the trunk expression was relatively normal. The embryos in B–E are orientated with the head on the left side.
	Figure 7. Inhibition of ErbB signaling in the head ectoderm leads to malformation of the head structures. RNAs encoding DN-ErbBs (1 ng) were coinjected with the lineage tracer nβGal (200 pg) into one animal blastomere of 16- to 32-cell stage embryos. This targeted expression of DN-ErbBs in the ectoderm greatly reduced the incidence of gastrulation defects. The resulting tadpoles (stages 32–36) were stained with the Red-Gal substrate and the embryos with the labeling in the head were scored for their head morphology. While the majority of nβGal-injected embryos showed normal head phenotype, more than half of the embryos injected with DN-ErbBs displayed malformation of the head structures, including reduction of the eyes and the forebrain. The data suggest that ErbBs may regulate head development directly independent of their effects on gastrulation.

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Alaoui-Jamali, Regulation of multiple tumor microenvironment markers by overexpression of single or paired combinations of ErbB receptors. 2003, Pubmed