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First published online February 18, 2004
doi: 10.1242/10.1242/dev.01024

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Gata2 specifies serotonergic neurons downstream of sonic hedgehog

¹ Department of Molecular Biology, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA
² Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA
³ Rinat Neuroscience, 3155 Porter Drive, Palo Alto, California 94304, USA

View larger version (77K): [in a new window]   Fig. 1. Endogenous expression of Gata, Pet1 and Lmx1b. Whole-mount in situ hybridization of chick hindbrain showing expression of (A) Gata3 at HH18-19, (B) Pet1 at HH20-21, (C) Lmx1b at HH21-22, (D) Gata3 (blue), (E) Pet1 (blue), and (F) Lmx1b (blue) with Nkx2.2 (red) at HH24. The midbrain (MB), mid-hindbrain boundary (MHB, arrow), and the 5-HT progenitors (5-Htpr, arrowhead) are marked; the boundary between rhombomere1 (r1) and r2 is indicated by a horizontal black line.

View larger version (97K): [in a new window]   Fig. 2. Ectopic generation of 5-HT neurons by Gata2 in r1. (A,B) Whole-mount immunofluorescence of chick hindbrain 4 days after electroporation of Gata2, with antibodies against 5-HT (red, A,B) and 3C2, a viral envelope marker (B, green). Endogenous 5-HT neurons (en.) versus ectopic 5-HT neurons (ect.) are bracketed (A). An arrowhead indicates the lack of 5-HT neurons in the midbrain (MB) despite the presence of exogenous Gata2 (B, green). The midbrain (MB) and mid-hindbrain boundary (MHB, arrow) are marked. (C) Immunofluorescence for 5-HT on thin (10-12 µm) sections through r1 of the hindbrain. The dorsal roof plate (rp) and ventral floor plate (fp) are marked; endogenous 5-HT neurons (en. 5-HT) versus ectopic 5-HT neurons (ect. 5-HT), and unelectroporated side (control side) are marked by brackets. (D-G) Higher magnification of ectopic 5-HT positive neurons (red; D,E,G) from C, showing cell bodies and processes (D, arrows) and co-localization (E-G, arrows) with 3C2 (F,G, green). Scale bars: C, 75 µm; D, 12.5 µm; G, 8 µm.

View larger version (35K): [in a new window]   Fig. 3. Gata2 re-specifies dorsal neural progenitors independently of HD proteins. (A-E) Whole-mount in situ hybridization of chick hindbrains 3-4 days after electroporation of Gata2, showing (A) Pet1 with ectopic (ect.) expression bracketed, (B) Chx10, (C) Lim1, (D) Nkx2.2, and (E) Nkx6.1 in which the electroporated (ect.) side is bracketed. The midbrain (MB) and mid-hindbrain boundary (MHB, arrow) are marked.

View larger version (74K): [in a new window]   Fig. 4. Gata2 is necessary, and Gata3 is dispensable, for the development of rostral 5-HT neurons. (A,B) Whole-mount immunofluorescence for 5-HT of (A) Gata3-/+ and (B) Gata3-/- embryos at E11. (C,D) Whole-mount in situ hybridization for Pet1 on Gata2-/+ (C), and Pet1 (D, left) and Gata3 (D, top right) on Gata2-/- (D) embryos at E10.5. A close up of double in situ hybridization for Gata3 (blue) and Nkx2.2 (yellow) in r1 at the ventral midline is shown on the bottom right of D. The floor plate (fp), midbrain (MB), mid-hindbrain boundary (MHB, arrow), 5-HT immunoreactivity (A,B; arrowheads), and the presence (C) versus absence (D) of Pet1 transcript (arrowheads) are marked. (E-H) Immunofluorescence staining on explant cultures from Gata2-/+ (E,G) versus Gata2-/- (F,H) embryos, at the equivalent of E13, with antibodies against tyrosine hydroxylase (E,F; TH, red) or Isl1 (G,H, red), compared with 5-HT (E-H, green). (H) In situ hybridization for Gata3 on Gata2-/- explant cultures (inset, blue).

View larger version (62K): [in a new window]   Fig. 5. Shh induces 5-HT neurons via Gata2. (A-I) Whole-mount immunofluorescence, thin section immunofluorescence or whole-mount in situ hybridization on chick hindbrain 3-4 days after electroporation of Smo-M2.IRES.GFP. (A,B) Whole-mount immunofluorescence for 5-HT (A,B; red), merged with GFP (B, green); only the electroporated side is shown in B. (C,D) Immunofluorescence on thin (10-12 µm) sections through r1 for 5-HT (C,D; red), merged with GFP (D, green). Arrow (A,B) shows corresponding point in both panels. (E,F) Whole-mount in situ hybridization for Gata2 (E) and Lmx1b (F). Endogenous 5-HT neurons (en.) versus ectopic 5-HT neurons (ect.) are bracketed (A,E). (G-I) Immunofluorescence on thin (25 µm) sections through r1, with antibodies against Gata2 (red); higher magnification through the width of the neural tube shows co-localization of Gata2 with GFP (H, green) and 5HT (I, blue). (J-M) Immunofluorescence 3 days after electroporation of dominant-negative Gata2 (DN-G2), either with Smo-M2 (J,K) or alone (L-M). (J,K) Whole-mount immunofluorescence for 5-HT (J,K; red), merged with GFP (J, green). Only the electroporated side of the hindbrain is shown in J and an arrow indicates the absence of ectopic neurons. (L,M) Immunofluorescence on thin (10-12 µm) sections through the ventral neural tube at r1 of the hindbrain for 5-HT (L,M; red), merged with 3C2 (M, green). Arrows indicate the population of 5-HT neurons on the control (left) side and their absence on the electroporated (right) side. An arrowhead indicates the absence of 5-HT (L, red) in cells expressing DN-G2 (L, green) on the control side. The midbrain (MB), mid-hindbrain boundary (MHB, arrow) are indicated on wholemounts. fp, floor plate; rp, roof plate.

View larger version (121K): [in a new window]   Fig. 6. Nkx2.2 and Nkx6.1 mediate Gata expression and 5-HT development in r1. (A,B) Chick hindbrain 3-4 days after electroporation of Nkx2.2, showing whole-mount immunofluorescence for 5-HT (A, red) and in situ hybridization (B) divided into r1 versus r2-3 for Gata3 (purple) and Nkx6.1 (orange). Ectopic (ect.) versus endogenous neurons (en.) are bracketed and rhombomere 1 (r1) is marked (A). Black arrowheads delineate the extent of Gata3 to Nkx6.1 expression on the electroporated (ect) versus control side (B, top). Orange arrowheads indicate the gradient of Nkx6.1 expression, whereas purple and orange arrowheads indicate the ectopic expression (ect. bracketed) of Nkx6.1 and Gata3, respectively (B, bottom). (C) Immunofluorescence on thin (25 µm) sections through r1 on the side of the neural tube electroporated with Nkx2.2 or Nkx6.1 (red), Gata2, (green) and 5-HT (blue), showing the higher expression of Nkx6.1 (arrows) where endogenous and ectopic (bracketed) Gata2 expression and 5-HT neurons develop. (D-F) Immunofluorescence on thin (25 µm) sections through r1 showing endogenous expression of Nkx6.1 (D-F, red), Gata2 (D,F, green), Nkx2.2 (E, inset green) and 5-HT (E,F, blue) at HH20 (D,E) and HH25 (F), with the higher Nkx6.1 expressing ventral region marked by white arrows. (D, inset) Co-localization between high Nkx6.1 and Gata2 in bracketed region. (E, inset) Overlay of Nkx2.2 with the Nkx6.1 and 5-HT expression shown in the main panel. (G,H) Whole-mount immunofluorescence for 5-HT (G, red), or in situ hybridization for Gata3 (purple) and Nkx6.1 (orange) (H), on chick hindbrain performed 3-4 days after electroporation of Nkx2.2 and Nkx6.1. Ectopic (ect.) versus endogenous neurons (en.) are bracketed (G); black arrowheads delineate the extent of Gata3 to Nkx6.1 expression on the electroporated (right) versus control side in (H). (I) Immunofluorescence on thin (25 µm) sections through r1 on the electroporated side of the neural tube, with antibodies against Gata2 (green) and 5-HT (blue). The approximate boundaries of the endogenous high expressing Nkx6.1 domain are marked by arrows, and the ectopic Gata2 and 5-HT positive neurons are bracketed. The midbrain (MB) and mid-hindbrain boundary (MHB, arrow) are indicated on wholemounts; the floor plate (fp) is indicated on sections.

View larger version (53K): [in a new window]   Fig. 7. Nkx6.1 is necessary for the development of 5-HT neurons in r1. (A,B) Whole-mount in situ hybridization of chick hindbrain 3 days after electroporation with morpholino-modified antisense oligonucleotides targeted against Nkx6.1 (6.1 MO), for (A) Gata2 and Gata3, or (B) Gata (purple) and Nkx2.2 (orange). Arrows indicate the loss of Gata expression of the electroporated side. The midbrain (MB) and mid-hindbrain boundary (MHB, arrow) are indicated (A). (C-E) Immunofluorescence on thin (10-12 µm) sections through the ventral neural tube in r1 of the hindbrain three days after electroporation with 6.1 MO (D, red), with antibodies against 5-HT (C,D, green; E, red) or Nkx2.2 (E, green). The location and absence of 5-HT neurons is indicated by arrows; the floor plate (fp) is marked.

View larger version (10K): [in a new window]   Fig. 8. The specification of rostral 5-HT neurons in the vertebrate hindbrain. Shh signaling in the ventral midline activates the type II HD protein Nkx2.2 and high levels of Nkx6.1 in the p3 domain of the rostral hindbrain. Nkx2.2 and Nkx6.1 are sufficient to activate expression of Gata2 and Gata3, which can positively regulate each other. Gata2, in turn, is necessary and sufficient to activate Lmx1b and Pet1, and to specify 5-HT neurons. Gata2 may activate additional transcription factors and/or may be required to directly cooperate with Lmx1b and Pet1 in 5-HT specification. By contrast, Gata3 is dispensable for the specification of rostral 5-HT neurons.

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