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, 12 (12), 1825-36

The ErbB2 and ErbB3 Receptors and Their Ligand, neuregulin-1, Are Essential for Development of the Sympathetic Nervous System

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The ErbB2 and ErbB3 Receptors and Their Ligand, neuregulin-1, Are Essential for Development of the Sympathetic Nervous System

S Britsch et al. Genes Dev.

Abstract

Neuregulins (NDF, heregulin, GGF ARIA, or SMDF) are EGF-like growth and differentiation factors that signal through tyrosine kinase receptors of the ErbB family. Here, we report a novel phenotype in mice with targeted mutations in the erbB2, erbB3, or neuregulin-1 genes. These three mutations cause a severe hypoplasia of the primary sympathetic ganglion chain. We provide evidence that migration of neural crest cells to the mesenchyme lateral of the dorsal aorta, in which they differentiate into sympathetic neurons, depends on neuregulin-1 and its receptors. Neuregulin-1 is expressed at the origin of neural crest cells. Moreover, a tight link between neuregulin-1 expression, the migratory path, and the target site of sympathogenic neural crest cells is observed. Sympathetic ganglia synthesize catecholamines in the embryo and the adult. Accordingly, catecholamine levels in mutant embryos are severely decreased, and we suggest that the lack of catecholamines contributes to the embryonal lethality of the erbB3 mutant mice. Thus, neuregulin-1, erbB2, and erbB3 are required for the formation of the sympathetic nervous system; the block in development observed in mutant mice is caused by a lack of neural crest precursor cells in the anlage of the primary sympathetic ganglion chain. Together with previous observations, these findings establish the neuregulin signaling system as a key regulator in the development of neural crest cells.

Figures

Figure 1
Figure 1
(a) Strategy applied to mutate the erbB2 gene. The genomic structure of the wild-type erbB2 allele is schematically shown at the top. The schematic structures of the targeting vector and of the mutant erbB2 locus are displayed. Exon sequences are indicated by boxes; exons indicated in white were mapped, whereas the position of the exons indicated in black were determined by sequence. In the targeting vector, lacZ was fused in frame to exon h. The inserted neomycin resistance gene (neo) is driven by the PGK promoter. The probe used for Southern hybridization and sizes of predicted fragments obtained after EcoRI digestion of genomic DNA containing the wild-type and mutant alleles are indicated. The structure of the predicted fusion protein encoded by the mutated erbB2 locus is shown at the very bottom. (b) Southern blot analysis of genomic DNA from wild-type embryos (lane 1), and embryos heterozygous (lane 2), or homozygous (lane 3) for the mutant erbB2 alleles; for hybridization, a genomic DNA fragment indicated above was used as probe. (c) Western blot analysis of the ErbB2 receptor on protein extracts of embryonal hearts from wild-type embryos (lane 1), heterozygous (lane 2), and homozygous (lane 3) mutant embryos.
Figure 2
Figure 2
Appearance of the sympathetic nervous system in embryos with mutant erbB2, erbB3, or neuregulin-1 genes. The anlage of the sympathetic nervous system was visualized by in situ hybridization with a Phox2a-specific probe on E10.5 embryos. Control embryos heterozygous for the erbB2 (a), the erbB3 (c), and the neuregulin-1 (e) mutations. Embryos homozygous for the erbB2 (b), erbB3 (d), and neuregulin-1 (f) mutations. (Arrowheads) Residual Phox2a-positive cells in the rostral portion of the sympathetic nervous system; (arrows) position of the caudal anlage of the sympathetic nervous system. Magnifications of a–d and e–f are identical, respectively.
Figure 3
Figure 3
Migrating neural crest cells visualized by in situ hybridization with erbB3-specific probes in embryos heterozygous (a,c,e) or homozygous (b,d,f) for the erbB2 mutation. Lateral view of the entire E9 embryos (a,b). Dorsal view of the embryos showing neural crest cells which emerged form the neural tube (c,d). Magnification of a lateral view on the forelimb level, showing streams of neural crest cells that migrate to the anlage of dorsal root and sympathetic ganglia (e,f). For comparison, the anlage of the sympathetic nervous system is visualized by in situ hybridization with Mash-I in embryos heterozygous (g) or homozygous for the mutant erbB2 (h) gene. Arrowheads in a and b indicate neural crest cells at the anlage of the trigeminal ganglion; arrowheads in c and d point to neural crest cells that have emerged from the neural tube; arrowheads in e–g indicate the anlage of the sympathetic nervous system; and arrows in e and f point toward the streams of neural crest cells that migrate to the anlage of the dorsal root ganglia. Magnifications in a, b, and c–h are identical, respectively.
Figure 4
Figure 4
Migration of neural crest cells and the anlage of the sympathetic ganglion chain in control (a,c,e,g), and erbB2 (b,d,f,h) mutant E9 embryos. Shown are 50-μm vibratome sections (a–f) or 5-μm frozen sections (g,h). Migrating neural crest cells were visualized by in situ hybridization with probes specific for ErbB3 (a,b) or for the low-affinity NGF receptor p75NTR (c,d). The anlage of the sympathetic nervous system was visualized by in situ hybridization with a Mash-1-specific probe (e,f). Distribution and cell death of neural crest cells were analyzed by immunohistochemistry using anti-p75NTR antibodies (red) combined with a TUNEL analysis of apoptotic cells (green) (g,h). The dorsal aorta (ao) is indicated. Magnifications in a–h are identical.
Figure 5
Figure 5
Numbers and incidence of cell death of neural crest cells located in distinct areas in control (wild-type or erbB2+/−) and mutant [erbB2−/−, erbB3−/−, and neuregulin-1−/− (nrg−/−)] embryos. Consecutive transverse sections of E9 embryos (20–22 somites) that together covered the entire forelimb were analyzed by immunohistochemistry by use of anti-p75NTR antibodies; this was combined with a TUNEL analysis to identify apoptotic cells. Neural crest cells identified by p75NTR immunofluorescence were counted and the average number per section was determined (a). Depicted are total numbers of neural crest cells (hatched bars), numbers of neural crest cells grouped in a dorsal position (dark gray), migrating farther ventrally (light gray) and located at the target site in the mesenchyme lateral of the dorsal aorta (white); numbers ± s.e.m. are displayed. In addition, the numbers of apoptotic neural crest cells identified by p75NTR immunofluorescence and TUNEL staining were determined (b). Shown are the percentages ± s.e.m. of neural crest cells undergoing apoptosis that are located in a dorsal position (dark gray) and neural crest cells that are either migrating or located at the target (light gray bars). n = 5 embryos (wild-type, erbB2+/−, erbB2−/−, and erbB3−/−) or n = 3 embryos (neuregulin-1−/−).
Figure 6
Figure 6
Expression of type I neuregulin-1 during migration of neural crest cells and the formation of the sympathetic nervous system. Whole-mount β-galactosidase staining of embryos heterozygous for the neuregulinlacZ allele on E9 (a,b) and E10 (c); cross sections of the embryo shown in a and b on a caudal (d) and upper forelimb level (e); cross section of the E10 embryo shown in c on the forelimb level (f). Lines in a indicate the level of the section shown in d and e. The arrowhead in b points to somite-associated neuregulin-1 expression; arrows in b and c point to the mesenchyme-associated neuregulin expression bilateral of the dorsal aorta (ao).
Figure 7
Figure 7
Appearance of the primary sympathetic ganglion chain and the anlage of the enteric nervous system in erbB3+/− (a,c,e) and erbB3−/− (b,d,f) embryos on E12.5. Phox-2a (a,b), or TH (c,d)-specific probes were used to visualize the primary sympathetic ganglion chain and a c-Ret (e,f)-specific probe to observe enteric ganglia by in situ hybridization. The embryos were cut at the midline prior to hybridization. Arrows in a and c point to cells that dissociate from the primary sympathetic ganglion chain and migrate to the mesentery and the anlage of the adrenal gland. Magnifications in a–d and e and f are identical, respectively.
Figure 8
Figure 8
Appearance of the superior cervical ganglion (a–d) and the celiac ganglion (e,f) in control (a,c,e) and erbB3 mutant (b,d,f) embryos on E17.5. Histological sections were stained with hematoxylin/eosin (a,b). Double immunofluorescence analysis on frozen sections with anti-TH (red) and anti-neurofilament 160 (green) antibodies (c–f). Superior cervical ganglia (scg), internal (ica) and external (eca) carotid artery, and the Xth cranial nerve (X) are indicated. Sectional levels were matched by the presence of the bifurcation of the carotid artery (a–d) and the origin of the mesenteric artery. Arrowheads point to the residue of the celiac ganglion of an erbB3 mutant embryo.
Figure 9
Figure 9
Appearance of the adrenal medulla in control (a,c) and erbB3 mutant (b,d) embryos on E17.5. Histological sections were stained with hematoxylin/eosin (a,b); (arrowhead) indicates islets of chromaffin cells within the adrenal gland. The arrow points to ordered columns of epithelial cells in the adrenal cortex. Double immunofluorescence analysis of frozen sections of the adrenal gland with anti-TH (red) and anti-neurofilament (green) antibodies (c,d).

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