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- Fig. 1.
Expression of Insm1 in the developing peripheral nervous system. To analyze Insm1 expression, we took advantage of the Insm1lacZ allele in which lacZ sequences replace the Insm1-coding sequence. Insm1lacZ/+ animals were analyzed at the indicated developmental stages, using antibodies directed against the lacZ gene product β-galactosidase (A,C-E) or X-gal staining (B). (A) Immunohistochemical analysis using anti-β-galactosidase (green) and anti-p75 (red) antibodies demonstrates expression of β-galactosidase in the primary sympathetic ganglion chain located lateral of the dorsal aorta (arrowhead), in the spinal cord and in condensing dorsal root ganglia at E9.5. (B) At E10.5, X-gal staining is detected in the entire primary sympathetic ganglion chain (arrowhead), as well as in sensory ganglia and in the central nervous system. (C,D) Immunohistochemical analyses of the primary sympathetic ganglion chain using antibodies directed against β-galactosidase (green), Phox2b (red in C) and Mash1 (red in D) indicate that the majority of β-galactosidase+ cells lateral of the dorsal aorta co-express Phox2b, and some β-galactosidase+ cells also express Mash1. (E) Immunohistochemical analysis of the adrenal gland at E13.5 using anti-β-galactosidase (green) and anti-Th (red) antibodies demonstrates Insm1 expression in chromaffin cells of the adrenal medulla. Scale bars: 100 μm in A,C,E; 500 μm in B.
- Fig. 2.
Reduced proliferation of sympatho-adrenal precursor cells causes a smaller size of primary sympathetic ganglia in Insm1 mutant mice. Phox2b and β-galactosidase expression was analyzed in the sympathetic ganglion chain at E10.5 (A-F), E11.5 (G-J) and E12.5 (K-N) of Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice by in situ hybridization or immunohistochemical analyses. (A,B,G,H,K,L) In situ hybridization using a Phox2b-specific probe. (C-F,I,J,M,N) Immunohistochemistry using antibodies against (C-F,I,J,M,N)β -galactosidase (green) and (E,F) Phox2b (red). The number of cells in the primary sympathetic ganglion chain is reduced in the mutant at E11.5 and E12.5. (O) Cell proliferation, as determined by BrdU injections at the indicated time points in the primary sympathetic ganglion chain of Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice. Shown is the proportion (%) of β-galactosidase+ cells that had incorporated BrdU 2 hours after the injection. Double asterisks indicate P<0.01. Scale bars: 100 μm in A,G,K.
- Fig. 3.
Delayed differentiation of sympatho-adrenal precursor cells in Insm1 mutant mice. The differentiation of the sympatho-adrenal precursor cells in Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice was assessed at the indicated stages using immunohistochemical analysis or in situ hybridization. (A-P) Analysis of E10.5 embryos. (A-D) Immunohistochemistry using antibodies against β-galactosidase (red), (A,B) Phox2b (green) and (C,D) Phox2a (green). (E-J) In situ hybridization using probes specific for (E,F) Hand2, (G,H) Gata3 and (I,J) Ret. (K,L) Immunohistochemistry using antibodies against β-galactosidase (red) and Th (green). (M,N) In situ hybridization using a Dbh-specific probe. (O,P) Immunohistochemistry using antibodies againstβ -galactosidase (red) and Tuj1 (green). There is a pronounced reduction in the number of sympatho-adrenal precursor cells that express Phox2a, Hand2, Gata3, Ret, Th or Dbh at E10.5 in Insm1lacZ/Insm1lacZ mice. (Q,R) Analysis of E12.5 embryos by immunohistochemistry using antibodies againstβ -galactosidase (red) and Th (green). (S) Quantification ofβ -galactosidase+ sympatho-adrenal precursor cells that co-express Th in Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice at various developmental stages. (T,U) Analysis of E12.5 embryos by immunohistochemistry using antibodies against β-galactosidase (red) and Phox2a (green). (V) Quantification of the β-galactosidase+ cells in sympathetic ganglia that co-express Phox2a at various developmental stages in Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice. (W,X) Analysis of E12.5 embryos by immunohistochemistry using antibodies againstβ -galactosidase (red) and Tuj1 (green). (Y) Quantification ofβ -galactosidase+ sympatho-adrenal precursor cells that co-express Tuj1 in Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice at various developmental stages. Single or double asterisks in S,V,Y indicate P<0.05 and P<0.01, respectively. Scale bars: 50 μm in A; 100 μm in C,Q.
- Fig. 4.
Mash1 and Dll1 are upregulated in sympatho-adrenal precursor cells of Insm1 mutant mice. Analysis of Mash1 expression by in situ hybridization and immunohistochemistry at E10.5 (A,B,E-H) and E12.5 (I,J), and of Dll1 expression at E10.5 (C,D) by in situ hybridization of Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice. In situ hybridization using a probe specific for Mash1 (A,B,I,J) and Dll1 (C,D). Immunohistochemistry using antibodies against (E,F) β-galactosidase (red) and Mash1 (green); (G,H) Phox2a (red) and Mash1 (green). Scale bars: 100 μm in A,I; 50 μm in E.
- Fig. 5.
Reduced size of secondary sympathetic ganglia in Insm1 mutant mice. Secondary sympathetic ganglia of Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice (E18.5) were analyzed by (A,B) X-gal staining and by (C-H) immunohistochemistry. Antibodies used were directed against: (C,D) Tuj1 (red) and Th (green), (E,F) Tuj1 (red) and NPY (green); (G,H) Phox2a (red) and Tlx3 (green). Arrowheads in A and B indicate superior cervical and stellate ganglia. Scale bars: 500 μm in A; 100 μm in C.
- Fig. 6.
Changed differentiation of chromaffin cells in Insm1 mutant mice. Adrenal glands of Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice (E14.5) were analyzed by (A,B) X-gal staining, (C,D) in situ hybridization using a Mash1-specific probe and (E,F) immunohistochemistry using antibodies against β-galactosidase (red) and Th (green). Adrenal glands of Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice (E18.5) were analyzed by (G,H) immunohistochemistry using antibodies againstβ -galactosidase (red) and PNMT (green), and by in situ hybridization using probes specific for (I,J) Th, (K,L) Dbh, (M,N) chromogranin A and (O,P) chromogranin B. (Q) Quantification of total numbers of β-galactosidase+ cells in adrenal glands at various developmental stages in Insm1lacZ/+ and Insm1lacZ/Insm1lacZ mice. Double asterisks indicate a P<0.01. Scale bars: 100 μm (A,G).
- Table 1.
Comparison of gene expression in the adrenal gland of heterozygous and homozygous Insm1lacZ mutant mice
Gene symbol Gene name FC P Hormone processing and secretion Akr1c18 Aldo-keto reductase family 1, member C18 −39.7 0.001 Pnmt * Phenylethanolamine-N-methyltransferase −28.1 0.001 Th * Tyrosine hydroxylase −6.7 0.001 Dbh * Dopamine beta hydroxylase −6.9 0.001 Chga * Chromogranin A −13.3 0.001 Chgb * Chromogranin B −8.7 0.001 Scg2 Secretogranin II −4.0 0.001 Scg3 Secretogranin III −4.1 0.001 Sgne1 Secretogranin V −4.8 0.001 Slc18a1 Vesicular monoamine Transporter (VMAT1) −4.2 0.001 Slc7a8 Cationic amino acid transporter (LAT2) − 4.1 0.001 Transcription factors Gata2 GATA binding protein 2 −3.4 0.001 Gata3 * GATA binding protein 3 −3.4 0.001 Phox2a * Paired-like homeobox 2a −2.9 0.001 Phox2b * Paired-like homeobox 2b −1.9 0.001 Hand2 * Hand2 −1.8 0.001 Mash1 * Achaete-scute complex homolog-like 1 (Ascl 1) 2.6 0.001 Ebf1 Early B-cell factor 1 2.1 0.001 Other factors Resp18 Regulated endocrine-specific protein 18 −10.5 0.001 Disp2 Dispatched homolog 2 −9.2 0.001 Sez612 Seizure related 6 homolog like 2 −4.2 0.001 Igf1 Insulin-like growth factor 1 4.5 0.001 Dkk2 Dickkopf homolog 2 (Xenopus laevis) 4.4 0.001 Nefl * Neurofilament 68 2.1 0.001 -
Systematic analysis of gene expression in control and Insm1lacZ/Insm1lacZ mice using Affymetrix oligonucleotide microarrays. The average signal fold change is shown. We selected the following genes for display: (1) genes that are downregulated at least fourfold; (2) genes of known function during chromaffin cell development; (3) genes that were upregulated at least twofold.
FC, fold change.
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↵* Also analyzed by immunohistochemistry or by in situ hybridization.
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- Fig. 7.
Epistasis between Insm1, Phox2b and Mash1 in the differentiation of sympatho-adrenal precursor cells. Analysis of the primary sympathetic ganglion chain in wild-type (A,B), Phox2b (C,D) and Mash1 (E,F) mutant mice at E10.5 (A,C,E) and E12.5 (B,D,F) by in situ hybridization using a probe specific for Insm1. (G) Schematic drawing of the epistatic relationship between Mash1, Phox2b and Insm1. Scale bars: 100 μm in A,B.
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