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First published online 30 August 2006
doi: 10.1242/dev.02553

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Gata3 participates in a complex transcriptional feedback network to regulate sympathoadrenal differentiation

¹ Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan.
² Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-0616, USA.
³ Department of Biochemistry, University of Calgary, Canada.
⁴ Department of Pathology and Laboratory Medicine, Children's Memorial Hospital, Feinberg School of Medicine, Northwestern University, Chicago, IL 60614-3394, USA.
⁵ Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba 305-8575, Japan.
⁶ Laboratory Animal Resource Center, University of Tsukuba, Tsukuba 305-8575, Japan.

View larger version (69K): [in a new window]   Fig. 1. A human DBH promoter-directed transgene recapitulates endogenous Gata3 expression in the developing sympathoadrenal system. (A) Diagrammatic representations of the mouse Gata3 gene, the Gata3 knockout (Pandolfi et al., 1995), the Gata3 nuclear lacZ knock-in (van Doorninck et al., 1999) and the hDBH-Gata3 expression transgene (TghDBH-G3) and co-injected hDBH-GFP transgene. X, XhoI restriction enzyme site. (B) Genomic Southern blot showing the diagnostic XhoI genomic DNA fragments corresponding to wild-type (WT), Gata3/lacZ knock-in (KI), Gata3 knock-out (KO) and hDBH-Gata3 transgene (TghDBH-G3) alleles. (C-K) Anti-GFP and anti-ß-galactosidase immunoreactivities in the adrenal medulla (AM), thoracic paravertebral sympathetic ganglia (SG) and organ of Zuckerkandl (OZ) in E18.5 Gata3Z/+:TghDBH-G3 embryos. Scale bars: 100 µm.

View larger version (69K): [in a new window]   Fig. 2. Developmental deficiencies in TghDBH-G3-rescued Gata3-/- embryos. (A-H) E18.5 TghDBH-G3-rescued mutants have characteristic deficiencies in the lower jaw, parathyroid gland, thymus and kidney (Lim et al., 2000). The control is a wild-type (WT) littermate. The red circle (C) indicates a wild-type parathyroid gland. (I,J). The hearts of E18.5 TghDBH-G3-rescued mutants have normal cardiac wall thickness that is indistinguishable from wild-type littermates.

View larger version (51K): [in a new window]   Fig. 3. Th and Dbh expression is restored in the sympathetic ganglia of TghDBH-G3-rescued Gata3-/- embryos. (A) mRNA levels in the trunk region of individual E10.5 embryos (normalized to GAPDH mRNA) were assayed by real-time Q-PCR. Data were collected from individual E10.5 embryos of Gata3-/- (5), Gata3-/-:TghDBH-G3 (4) or wild-type (3) genotypes. Data are presented as mean±s.e.m. The statistical significance of differences between Gata3+/+ and Gata3-/- are indicated (**P<0.01; Student's t-test). (B) E10.5 wild-type embryos display strong Th expression in the sympathetic ganglia (SG; arrowhead) as well as weaker Th immunoreactivity in the ventrolateral neural tube (NT; arrow). (C) In E10.5 Gata3 mutant embryos, Th expression is significantly reduced in both the SG and NT. (D) The hDBH promoter directs transgenic eGFP expression in the SG and the NT in E10.5 embryos. (E) Th immunoreactivity was significantly restored both in the SG and NT of E10.5 transgenic rescued Gata3-null mutants. (F) E12.5 wild-type embryos display strong Th expression solely in the SG, and not in the NT. (G) Th expression is absent in E12.5 Gata3 mutant SG. (H) Transgenic eGFP expression was strong in the SG, but much weaker at E12.5 than at E10.5 in the NT. (I) Th expression was restored solely in the SG of E12.5 Tg-rescued Gata3 mutant embryos. White lines indicate the outer margin of the spinal cord and dorsal root ganglia. DA, dorsal aorta; V, vertebrae.

View larger version (48K): [in a new window]   Fig. 4. Phox2b and Th expression in thoracic paravertebral sympathetic ganglia is suppressed in E18.5 Gata3 mutant embryos. (A-C) Whole-mount X-gal staining of E18.5 Gata3Z/+ and Gata3Z/- embryos with and without TghDBH-G3 transgene. ß-Gal+ sympathetic chain was shrunken in the pharmacologically rescued Gata3Z/- embryos. (D,F) The size of sympathetic ganglion is reduced in the pharmacologically rescued Gata3-/- embryos in HE sections. In Gata3Z/-:TghDBH-G3 embryos, anti-Phox2b and anti-Th staining and sympathetic ganglion size were restored (B,E,H,K); the control animals used were Gata3Z/+ (A) and Gata3+/+ (WT; wild-type) littermates (D,G,J). Broken lines indicate outer margins of sympathetic ganglia. (I,L) In Gata3-/- embryos, Phox2b and Th expression was significantly reduced in thoracic paravertebral sympathetic ganglia. Scale bar: 100 µm. (M,N) At least four serial sections of each of the four examined ganglia from two different embryos of each genotype were evaluated. Phox2b- and Th-expressing cells (M) and the total surface areas (N) of the thoracic paravertebral sympathetic ganglia in control and Gata3 mutant embryos with and without TghDBH-G3 were quantified and represented as histogram. Data are presented as the mean±s.e.m. The statistical significance of the differences between Gata3-/-:TghDBH-G3 and Gata3-/- are indicated (*P<0.05; **P<0.01; Student's t-test).

View larger version (73K): [in a new window]   Fig. 5. Repressed Phox2b and Th expression in Gata3 mutant chromaffin cells. Whole-mount X-gal staining (A-C), HE staining (D-F), anti-Phox2b, anti-Th and anti-chromogranin A staining (G-O) on adrenal gland sections of E18.5 control (A,D,G,J,M), Gata3 mutant embryos bearing TghDBH-G3 (B,E,H,K,N) or no transgene (C,F,I,L,O). ß-Gal+ adrenal medulla (arrow) and organ of Zuckerkandl (arrowhead) is significantly smaller in the pharmacologically rescued E18.5 GataZ/- embryos (C). The size of adrenal medullary region is reduced in the pharmacologically rescued Gata3-/- embryos in the HE sections (F). Expression of Phox2b, Th and chromogranin A is virtually absent in the Gata3-/- embryos (I,L,O). In the Tg-rescued homozygous mutant embryos, the adrenal chromaffin cell population and the expression of above-mentioned SA markers are partially restored (B,E,H,K,N). The control animals used were Gata3Z/+ (A) and Gata3+/+ (wild-type) littermates (D,G,J,M). (P-R) Co-localization of transgenic eGFP and restored Th expression in Gata3Z/-:TghDBH-G3-rescued mutants. (S) Relative Th- and Phox2b-positive chromaffin cell areas in serial sections of adrenal glands. At least four serial sections of each of the four examined ganglia from different two embryos of each genotype were evaluated. Data are presented as mean±s.e.m. Scale bars: 100 µm. The statistical significance of the differences between Gata3-/-:TghDBH-G3 and Gata3-/- are indicated (*P<0.05; **P<0.01; Student's t-test).

View larger version (76K): [in a new window]   Fig. 6. Structural and ultrastructural analyses of the adrenal glands of Gata3 mutant mice. (A) Electron micrograph of wild-type E18.5 adrenal glands reveals the presence of numerous large secretory granules (arrowheads) that are characteristics of mature chromaffin cells, whereas E18.5 Gata3-/- mutant adrenal glands (C) lack granules. (B) TghDBH-G3 bred into the Gata3 mutant background restores typical chromaffin cell ultrastructure. Scale bar: 2 µm. (D-F). Anti-Sf1-negative chromaffin cells were abundant in wild-type and Tg-rescued Gata3 mutant mice, whereas a normal, centrally located adrenal medulla was never observed in pharmacologically rescued Gata3 mutant adrenal gland. The size of the adrenal cortex appears unaltered in the Gata3 mutant adrenal gland. Scale bars: 100 µm.

View larger version (83K): [in a new window]   Fig. 7. Adrenal chromaffin cell number is reduced in Gata3 mutant embryos. lacZ expression (A-F) in the adrenal gland of E13.5 and E15.5 of Gata3z/+ (A,D), Gata3z/-:TghDBH-G3 (B,E) and Gata3Z/- (C,F) embryos. (G) Quantification of ß-gal+ chromaffin cells in embryos of various genotypes. At least four adrenal glands from two different embryos of each genotype were analyzed. Data are presented as mean±s.e.m. The statistical significance of the differences between Gata3-/-:TghDBH-G3 and Gata3-/- are indicated by (*P<0.05; Student's t-test). Scale bars: 100 µm.

View larger version (68K): [in a new window]   Fig. 8. Apoptosis in sympathoadrenal cells is partially rescued by SA tissue-specific Gata3 expression. (A-F) TUNEL (green) and anti-Th (red) staining was performed on the same adrenal gland sections. Scale bars: 100 µm. (G) Quantification of TUNEL-positive cells in the E13.5 and E15.5 embryonic adrenal glands of various genotypes. At least four adrenal glands from two different embryos of each genotype were analyzed. Data are presented as mean±s.e.m. The statistical significance of the differences between Gata3-/-:TghDBH-G3 and Gata3-/- are indicated (*P<0.05; Student's t-test).

View larger version (46K): [in a new window]   Fig. 9. Alteration in SA lineage-specific transcription factor expression in purified adrenal chromaffin cells. (A) Single-cell suspensions dissociated from E18.5 adrenal glands were analyzed by flow cytometry after staining with FDG (a fluorogenic substrate for ß-galactosidase) and PE-conjugated CD45 antibody. lacZ+CD45- (red quadrants) and lacZ-CD45- (blue quadrants) fractions were isolated. The percentages of cells in lacZ+CD45- fractions are indicated. (B) The mRNA level of Sf1, a cortex-specific nuclear receptor, was undetectable in the lacZ+CD45- fractions, indicating the high purity of chromaffin cells. (C) Gata3, Th, Dbh, Hand2, Phox2b and Mash1 mRNA levels in the lacZ+CD45- fractions (normalized to GAPDH mRNA) were assayed by real-time RT-PCR. Data were collected from E18.5 individual embryos of Gata3Z/- (5), Gata3Z/-:TghDBH-G3 (5) or Gata3Z/+ (5) genotypes. Data are presented as mean±s.e.m. The statistical significance of the differences between Gata3Z/- and Gata3Z/+ are indicated (*P<0.05; **P<0.01; Student's t-test).

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