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First published online 3 May 2006
doi: 10.1242/dev.02377

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The GATA2 transcription factor negatively regulates the proliferation of neuronal progenitors

Abeer El Wakil^*, Cédric Francius^*,, Annie Wolff, Jocelyne Pleau-Varet and Jeannette Nardelli^,

UMR CNRS 7000, Cytosquelette et Développement, Faculté de Médecine Pitié-Salpêtrière, 105 boulevard de l'Hôpital, 75013 Paris, France and Université Pierre et Marie Curie, Paris 6, France.

View larger version (88K): [in a new window]   Fig. 1. GATA2 expression is turned on in newborn progenitors and is sufficient to arrest proliferation. (A) Transverse section of the caudal hindbrain of a E10.5 mouse embryo, first hybridised with Gata2 anti-sense RNA (green), then stained with antibodies against Isl1 (red), a pan-marker of motoneurons. The presumptive domain of V2 precursors, which express Gata2, is located between the respective domains of the motoneuron and V1 interneuron precursors; in the hindbrain, Gata2 is also activated to a lesser extent in the p3 domain, indicated by the white arrow. (B,C) Transverse section of the spinal cord of a E10.5 mouse embryo injected with BrdU. Hybridisation with Gata2 antisense RNA (B) was followed by BrdU immunostaining (C). (D-F) Higher magnification of the area included in the white rectangle in C. (G-L) Transverse sections of the spinal cord of chick embryos 24 hours after electroporation with the pAdRSV-GATA2HA plasmid, double-stained with anti-HA antibodies (G,J) and either with anti-phospho-Histone3 (pH3) (H) or anti-BrdU antibodies (K), and analysed by confocal microscopy. (I,L) Superimpositions of G,H (I) and J,K (L). (M,N) The percentages of phospho-Histone3- and BrdU-positive cells in the control (red, M) and the transfected sides (blue, N) are compared. (O,P) Adjacent sections of chick embryos misexpressing GATA2-HA, stained with anti-HA-antibodies and hybridised with chick Sox2 anti-sense RNA. Scale bar: in A, 70 µm in A-C; in D, 20 µm in D-F; in G, 20 µm in G-L; in O, 50 µm in O,P.

View larger version (101K): [in a new window]   Fig. 2. Analysis of the expression of cell cycle regulators in the context of GATA2 loss- and gain-of-function. (A-E) transverse sections of the spinal cord of E10.5 mouse embryos, wild type (A,C,D,E) or Gata2–/– (B); in situ hybridisation was performed with RNA anti-sense probes for cyclin D1 (A,B,E), cyclin D2 (C) and cyclin D3 (D) genes. In E, further staining, performed with anti-islet 1 antibodies (green), shows that the domain of higher concentration of cyclin D1 transcripts abuts the dorsal limit of motoneurons and overlaps V2 and V1 precursors. Broken lines in A,C-E delineate the presumptive limit between the ventricular and the marginal zones. Brackets in A,B indicate the domain where cyclin D1 transcription is upregulated. (F-H,J-L) Transverse sections of the spinal cord of electroporated chick embryos, hybridised with a chick cyclin D1 (G) or Gata2 (K) antisense RNA probe, then immunostained with anti-HA antibodies (F,J). (H,L) superimposition of E-G (H) and J-K (L). (I,M-T) Confocal analysis of double immunofluorescent staining performed on transverse spinal cord sections of wild-type (I,M-P) or Gata2–/– (Q-T) E10.5 mouse embryos. Anti-Kip2/p57 antibodies (I,M,Q, green) were coupled with either anti-Isl1 (I, red) or anti-Kip1/p27 (N,R). (O,S) Superimpositions of M,N (O) and Q,R (S). (P,T) Double immunostaining with anti-p27 and anti-Isl1 antibodies, respectively visualised with anti-mouse IgG1 coupled to Alexa Fluor 546 and anti mouse IgG2b coupled to Alexa Fluor 488. The respective presumptive domains of V1, V2 and motor (MN) neurons are indicated in I and P. White arrows in I indicate that the V2 presumptive domain does not express p57/Kip2, and in R,S,T, that the same domain lacks p27 expression in Gata2–/– embryos. Scale bar: 70 µm in A-D; 60 µm in E-L; 80 µm in M-T.

View larger version (88K): [in a new window]   Fig. 3. GATA2-HA misexpression can induce the expression of postmitotic differentiation markers 24 hours after electroporation. (A-L) Confocal analysis of adjacent spinal cord sections of a chick embryo misexpressing GATA2-HA 24 hours after electroporation. HA immunostaining (A,D,G,J, green) was coupled with p27/Kip1 (B), Tuj1/ßIIItubulin (E), neurofilament (NF) (H) or NeuN (K) immunostaining. (C,F,I,L) Superimpositions of A,B (C), D,E (F), G,H (I) and J,K (L). Scale bar: 80 µm.

View larger version (82K): [in a new window]   Fig. 4. Cells misexpressing GATA2-HA can fail to further progress into the differentiation pathway. (A-L) Confocal analysis of double immunofluorescent stainings performed on adjacent sections of spinal cord of chick embryos 48 hours after electroporation. HA immunostaining (A,D,G,J, green) was coupled with p27/Kip1 (B), Tuj1/ßIIItubulin (E), neurofilament (NF) (H) or NeuN (K) immunostaining. (C,F,I,L) Superimpositions of A,B (C), D,E (F), G,H (I) and J,K (L). Scale bar: 80 µm in A-F; 90 µm in G-L.

View larger version (96K): [in a new window]   Fig. 5. The Notch pathway is shut off in the context of Gata2 misexpression. (A-I) Spinal cord cross-sections of chick embryos 24 hours after electroporation, hybridised with chick Dll1 (B), Serrate1/Jag1 (C), Cash1 (E), Ngn2 (F), Notch1 (H) and Hes5 (I) antisense RNA probes, or stained with anti-HA antibodies (A,D,G). (B,C,E,F,H,I) Adjacent sections to A,D,G. Scale bar: 70 µm.

View larger version (110K): [in a new window]   Fig. 6. Gata2 function does not require proneural activity. (A-J) Spinal cord sections of chick embryos 24 hours after electroporation either with pAdRSV-GATA2HA and pACGGScId2 (A-F) or with pACGGScId2 and pAdRSV-GFP (G-J). (A,C,E) Immunostaining with anti-HA antibodies and (G) with anti-GFP antibodies. In situ hybridisation was performed with chick RNA antisense probes for Cash1 (B,H), Hes5 (D,I) and Ngn2 (F,J). (A,B; C,D; E,F; G-J) Adjacent sections. Scale bar: 70 µm in A-F; 60 µm in G-J.

View larger version (53K): [in a new window]   Fig. 7. GATA2 inhibits the proliferation of mouse embryonic neuroepithelial cells in culture. (A-F) Wild-type (A,B) and Gata2–/– (C-F) embryonic mouse neuroepithelial cells (ENC); immunostaining with anti-GATA2 (A), anti-nestin (B,C, green), anti Sox2 (D, green) and anti-Tuj1/ßIII-tubulin (F, red) antibodies. Nuclei were stained with DAPI (blue) in B,C,E,F. Wild-type (B) or Gata2–/– (C) ENCs express Nestin. Sox2 (D), but no Tuj1/ßIII-Tubulin (F), can be detected in Gata2–/– cells, as in wild-type cells (not shown); D-F correspond to the same field. (G) Comparison of the percentage of serum-starved cells reaching the S phase at different time intervals after cell cycle release in the presence of serum and BrdU. (H) Comparison of the transcription level of cyclin D1, cyclin D2, cyclin D3 and cyclin E1, p27 and Notch1 genes by semi-quantitative RT-PCR. PCR products were separated on BEt agarose gels, then analysed and quantified with GelDoc camera and software (BioRad). Scale bar: 40 µm in A-C; 20 µm in D-F.

View larger version (94K): [in a new window]   Fig. 8. Gata2-deficient ENC complemented with Gata2 exhibit much more active cyclin D1 and cyclin D2 transcription, while Gata2 transient overexpression blocks the proliferation of mouse neuroblastoma cells. (A-D) Gata2–/– ENC were transfected either with pAdRSV-GATA2HA and pAdRSV-ßGal plasmids (A,C) or with pAdRSV-ßGal alone, then treated for X-Gal colouration and in situ hybridisation with cyclin D1 (A,B) or cyclin D2 (C,D) antisense RNA probes. (E-J) NB2a mouse neuroblastoma cells, either control (E-G) or transiently transfected with pAdRSV-GATA2HA (H-J). BrdU was added in the culture medium 30 hours after transfection, cells were recovered 15 hours later, processed for BrdU staining (F,I) and further HA-immunostained (I, red); nuclei were stained with DAPI (E,H) before mounting. (G,J) Superimpositions of E,F (G) and H,I (J). In the control culture (E-G), cells have not stopped growing and are all BrdU positive; whereas in transfected cultures (H-J), cells have not reached the same density and, especially GATA2-HA expressing cells, have not incorporated BrdU (J). Scale bar: 45 µm in A-D; 15 µm in E-J.