QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 25 August 2004
doi: 10.1242/dev.01370

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tsarovina, K. Articles by Rohrer, H. Search for Related Content PubMed PubMed Citation Articles by Tsarovina, K. Articles by Rohrer, H.

Essential role of Gata transcription factors in sympathetic neuron development

Konstantina Tsarovina¹, Alexandre Pattyn², Jutta Stubbusch¹, Frank Müller¹, Jacqueline van der Wees³, Carolin Schneider^1,*, Jean-Francois Brunet² and Hermann Rohrer^1,

¹ Max-Planck-Institut für Hirnforschung, Abteilung Neurochemie, Deutschordenstr. 46, 60528 Frankfurt/Main, Germany
² CNRS UMR 8542, Département de Biologie, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France
³ Department of Cell Biology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands

View larger version (32K): [in a new window]   Fig. 1. Expression of Gata2 and Gata3 in chick sympathetic ganglia. Frozen sections at the brachial level of E6 chick embryos were analysed for the expression of Gata2 and Gata3 by in-situ hybridisation. Sympathetic ganglia display a strong hybridisation signal for Gata2 (black arrowheads), whereas Gata3 expression was not detected (white arrowheads). In the spinal cord both Gata2 and Gata3 are expressed (black arrows). da, dorsal aorta.

View larger version (138K): [in a new window]   Fig. 5. Lack of Gata2 and Gata3 expression in Phox2b–/– mice. The expression of Gata2 and Gata3 was studied in E13.5 Phox2bLacZ/+ and Phox2bLacZ/LacZ mouse embryos. In the Phox2b knockout Gata2 (B) and Gata3 (D) were not detectable, by contrast to the heterozygotes (A,C). Sympathetic ganglion precursors could be detected at the dorsal aorta in both Phox2b+/– (E) and Phox2b–/– (F) embryos by LacZ staining.

View larger version (101K): [in a new window]   Fig. 2. The onset of Gata2 expression in sympathetic ganglia in relation to the expression of Phox2b, Hand2, Phox2a and Th. Frozen sections from the brachial region of stage 18/19 chick embryos, staged according to the number of somites, were analysed for the expression of Phox2b, Hand2, Phox2a, Gata2 and Th. Gata2 expression was first observed in 34-somite embryos, after the onset of expression of Phox2b, Hand2 and Phox2a, but before Th, which was first observed in 35-somite embryos. Black and white arrowheads indicate the presence or absence, respectively, of gene expression in primary sympathetic ganglia. Gata2 expression was additionally detected in the ventral part of the dorsal aorta (da) (I) and in the spinal cord (D,I,N). The results are summarised in Table 1.

View larger version (33K): [in a new window]   Fig. 3. Gata2 expression in sympathetic ganglion primordia is blocked by the Hand2 inhibitor noggin. Chick embryos were treated with BSA (A,B) or noggin (C,D) at stage 14 and analysed at stage 19 for the expression of Sox10 (A,C) and Gata2 (B,D) in sympathetic ganglion primordia close to the dorsal aorta (da). Arrowheads point to primary sympathetic ganglia. (E) Sox10 and Gata2 expression is quantified by determining the area of Sox10- and Gata2-expressing cells. The mean±s.e.m. of at least three embryos is shown. ** significantly different from BSA controls (P<0.01) (Student's t-test).

View larger version (86K): [in a new window]   Fig. 4. Gata2 is induced in peripheral nerve precursors by ectopic expression of Phox2b and BMP24. Chick embryos were infected at E2 with RCAS-Phox2b and RCAS-BMP4 and analysed at E8 for the presence of ectopic neurons in the brachial nerve. In response to Phox2b and BMP24 neurons were generated that express noradrenergic properties: Th (A,C) and Gata2 (B,D). These cells also coexpress neuronal characteristics (Stanke et al., 1999; Howard et al., 2000).

View larger version (18K): [in a new window]   Fig. 6. The expression of a dominant-negative variant of Gata2 results in a reduction of Th, Scg10, Dbh and Phox2b expression. Chick embryos were infected unilaterally at E2 with either RCAS-dnGata2 or RCAS-engrailed and RCAS-VP16-Gata2 as controls and analysed at E8 for the expression of Th (A), Scg10 (B), Dbh (C) and Phox2b (D) in infected sympathetic ganglia. The areas of Th-, Scg10-, Dbh- or Phox2b-expressing cells were quantified on alternate sections, both on the infected and the contralateral uninfected side. For the RCAS-engrailed and RCAS-VP16-Gata2 controls, only data of the infected ganglia are shown for simplicity; the data of the uninfected side are identical. The data represent the mean±s.e.m. of at least nine embryos. (A) * significantly different from engrailed-infected side, VP16-Gata2-infected side and dnGata2-uninfected side (P<0.001, P<0.005, P<0.005, respectively). (B) * significantly different from engrailed-infected side, VP16-Gata2-infected side and dnGata2-uninfected side (P<0,005, P<0,01, P<0.01, respectively). (C) * significantly different from dnGata2-uninfected side (P<0.005). (D) * significantly different from dnGata2-uninfected side (P<0.01) (all Student's t-tests).

View larger version (55K): [in a new window]   Fig. 7. Gene-expression pattern in E10.5 sympathetic ganglia of Gata3–/– mice. The expression of Phox2b (A,B), Mash1 (C,D), Phox2a/TuJ1 (E,F), Hand2 (G,H), Ret (I,J) and Dbh (K,L) is not affected in Gata3-deficient mice. By contrast, the ganglia are devoid of Gata2 expression (O,P) and display a significant reduction in Th expression levels (M,N).

View larger version (53K): [in a new window]   Fig. 8. Reduction in ganglion size and Th expression and increased apoptosis in sympathetic ganglia of Gata3–/– mice. (A,B) Immunostaining for ßIII-tubulin combined with in-situ hybridisation for Sox10 reveal a reduced ganglion size in mutant (B) compared with control (A) embryos at E11.5. (C,D) Cell death analysis by TUNEL staining showing an increased number of apoptotic cells. (E) Quantification (P<0,01; n=4). (F-O) Reduction in sympathetic ganglion size and Th expression in E13.5 sympathetic ganglia of Gata3–/– mice. In Gata3–/– mice the sympathetic stellate ganglion (G) is strongly reduced in size compared with wild type (F), as revealed by immunostaining for Phox2b antibody. The reduction, quantified in (H) is by about 88% (P<0.001; n=4). Also at thoracic levels (I,J,L,M) a strong reduction in ganglion size is evident on Phox2b immunostains (I,J), quantified in (K) at around 60% (P<0.01; n=4). While Dbh expression was somewhat reduced (L,M), Th expression was almost undetectable in the mutants (N,O).

View larger version (74K): [in a new window]   Fig. 9. Gata2 expression in peripheral nerve precursors results in the generation of neurons that display mostly a non-autonomic phenotype. Chick embryos were infected by RCAS-Gata2 at E2 and analysed at E8 for the expression of ectopic neurons in the brachial nerve. Whereas a large number of Scg10-positive cells was detected (A, overview; B, enlargement of the region of the nerve), only few cells expressing autonomic marker genes (C, Phox2b; E, Cash1) or noradrenergic markers (D, Th) were present. This is confirmed by double in-situ hybridisations for Scg10 (red, F) and Th (blue, G). Please note the low number of cells that coexpress Scg10 and Th (arrow) and of Th+, Scg10– cells (arrowheads).

View larger version (106K): [in a new window]   Fig. 10. Expression of Gata2 and Th in chick parasympathetic ganglia. Ciliary (A-C), sphenopalatine (D-F), submandibular (G-I) and cardiac (J-L) ganglia were analysed for the expression of Phox2b, Gata2 and Th. Whereas Gata2 is not detectable in the cranial ciliary and sphenopalatine ganglion, Gata2 is expressed at very low levels in submandibular ganglia, and strong expression is evident in cardiac ganglia. Low-level Th expression is detectable in the sphenopalatine ganglion, submandibular and cardiac ganglia and in a small number of cells in the ciliary ganglion (Müller and Rohrer, 2002), but not in the section shown in C. Expression was analysed on parallel sections.

View larger version (75K): [in a new window]   Fig. 11. Gata2 and Gata3 do not control the central noradrenergic phenotype. (A-C) The anlage of locus coeruleus (LC) visible in the dorsal metencephalon by anti-Phox2a immunohistochemistry (A) expresses neither Gata2 (B) nor Gata3 (C) at E10.5. (D-G) At E13.5 the anlage of the LC, visible by in-situ hybridisation for Dbh (F) expresses neither Gata2 nor Gata3 (arrowhead in D and E, respectively) and is intact in Gata3 mutants (G).