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First published online November 10, 2005
doi: 10.1242/10.1242/dev.02094

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Directed differentiation of neural cells to hypothalamic dopaminergic neurons

¹ Department of Biomedical Science, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
² National Cancer Institute, National Institute of Health, MD, 20892, USA

View larger version (65K): [in a new window]   Fig. 1. Expression patterns of HD proteins and Shh in the tuberal hypothalamus. Whole-mount lateral views (A,B) or transverse sections through the tuberal hypothalamus (C-Q) of stage 10-15 embryos. (A,B) Double-label immunofluoresence to detect Shh (green) and Nkx2.1 (red) at stage15. In the diencephalon, Nkx2.1+/Shh+ progenitor cells occupy the anterior ventral hypothalamus (asterisk) and lateral tuberal hypothalamus (arrow) (see R). These populations have previously been defined as `anterior-dorsal' hypothalamic cells (Mathieu et al., 2002). Blue shows DAPI counterstain. Telencephalic cells express only Nkx2.1 at this stage. (C-E,G-I) Double-label immunofluorescence reveals expression of Nkx2.1 and Shh at stage 10 (C), stage 13 (D) and stage 15 (E,G-I). From stage 15, Shh and Nkx2.1 are co-expressed by cells in the germinal zone I (G-I). (F) Shh mRNA expression in the germinal zone. (J-M) Double-label immunofluoresence shows expression of Lim1 and Nkx2.1. Lim1 is strongly expressed in lateral hypothalamic cells and overlaps extensively with Nkx2.1. (N,O) Nkx2.2 overlaps with Nkx2.1 and then extends more dorsally. (P,Q) Nkx2.2 expression is complementary to Pax6 expression. (R,S) Summary diagrams showing hypothalamic nomenclature, HD protein codes and Shh expression. Lim1* denotes high expression in lateral tuberal hypothalamic cells. For simplicity, weak Lim1 expression in more dorsal region is not shown in this diagram. AH, anterior hypothalamus; TuH, tuberal hypothalamus; MH, mammilary hypothalamus. Scale bars: 300 µm in A; 130 µm in B; 65 µm in C-F,J,N,P; 30 µm in G-I,O,Q; 25 µm in K-M.

View larger version (76K): [in a new window]   Fig. 2. Nkx2.1+/Th+ hypothalamic DA neurons are generated from Nkx2.1+/Shh+ progenitors in the lateral tuberal hypothalamus. (A,B) Transverse sections through embryonic day 6 (E6) tuberal hypothalamus (shown in A'), immunolabelled with Th (green) (A), Shh (red) (B) or both (B'). Th and Shh show extensive overlap. Blue shows DAPI counterstain. (C-F) Fate mapping of germinal zone of lateral tuberal hypothalamus. (C) Schematic, showing DiI labelling at stage 15. (D-F) Transverse sections through DiI-labelled embryos, fixed immediately (D) or developed to E6 (E,F). Immunolabelling with anti-Shh confirms that lateral tuberal cells are targeted at stage 15 (arrow, D) and remain in situ at E6 (arrow, E). Immunolabelling with anti-Th shows that DiI-labelled cells (red) co-label with anti-Th antibody (green; arrows in F). (G-I) Transverse sections through E6 tuberal hypothalamus, immunolabelled with Th (green) and Nkx2.1 (red) (G,H) or Th (red) and Lim1 (green) (I). Many Th+ neurons co-express Nkx2.1. Likewise, extensive co-expression of Th and Lim1 is detected. (J-L) Confocal image showing overlapping expression of Nkx2.1 and Msx1 in the lateral tuberal hypothalamus. Scale bars: 140 µm in A,B,G; 60 µm in D; 120 µm in E; 20 µm in F; 30 µm in H; 25 µm in J-L.

View larger version (72K): [in a new window]   Fig. 3. Induction of Nkx2.1+/Shh+ cells and Nkx2.1+/Msx/2+/Th+ DA neurons can be triggered by prechordal mesoderm in a contact-independent manner. (A-D) Stage 4-5 LNP explants (blue box, Fig. 4F) cultured alone express Lim1/2, Pax6 and Pax7 but not Nkx2.1 or Shh. (E-H) In stage 4-5 LNP explants co-cultured in contact with stage 5+ prechordal mesoderm, Nkx2.1+/Foxa2+/Bmp7+ ventral tuberal hypothalamic cells are detected (white brackets) at the boundary with prechordal mesoderm (broken line). Nkx2.1+/Shh+/Lim1+ lateral tuberal hypothalamic progenitor cells are detected adjacent to these. (I-L) In stage 5 LNP explants co-cultured at a distance from HH stage 5+ prechordal mesoderm, Nkx2.1+/Shh+/Lim1+ lateral tuberal hypothalamic cells are detected, but no Nkx2.1+/Foxa2+/Bmp7+ ventral tuberal cells are found. (M,N) Th+ neurons, but not Nkx2.1+/Th+ neurons, differentiate in a stage 5 pHyp explant (green box, Fig. 4F) cultured alone (M). Nkx2.1+/Msx1/2+/Th+ neurons differentiate in stage 5 pHyp explant co-cultured with prechordal mesoderm (N). (O) Transverse section of stage 10 embryo showing expression of Bmp7 in the ventral tuberal hypothalamus (arrow) and prechordal mesoderm (arrowhead). Scale bar: 40 µm in A-L; 30 µm in M,N.

View larger version (58K): [in a new window]   Fig. 4. Progressive specification of Nkx2.1+/Msx1/2+/Th+ DA neurons. (A,F,K) Schematic of stage 4-6 embryos, showing regions dissected. Prospective hypothalamic (pHyp) regions (green) and anterior (blue) or posterior (purple) lateral neural plate are indicated. (B-E,G-J,L) pHyp explants, cultured for 2-3 days and analysed by immunofluorescent labelling. Co-expression of Shh and Lim1 is detected in HH stage 5 explants (H'). Nkx2.1 expression is detected only in stage 6 explants and is exclusive to Pax6 (L). From HH stage 6, Nkx2.1+/Lim1+, Nkx2.1+/Nkx2.2+, Nkx2.1+/Shh+, Nkx2.1+/Msx1/2+ cells are specified, as in vivo (M-O; see Fig. 1, Fig. 2L). (P-R) Immunofluorescent analysis of stage 6 pHyp explants cultured for 6-7 days. Many Nkx2.1+/Msx1/2+/Th+ neurons differentiate (arrows in P; inset in P). Th+ neurons (arrowheads) do not co-express En1 (Q) or Pax6 (R). (S) Widespread expression of Six 3 is detected within stage 6 pHyp explants after 40 hours culture. Scale bars: 40 µm.

View larger version (92K): [in a new window]   Fig. 5. Bmp7 induces hypothalamic identity in postmitotic cells that are ventralised by Shh. (A-C) Immunofluorescent and in situ hybridisation analyses of stage 4-5 aLNP explants, incubated with Shh. After 2 days, Lim1+/Shh+/Six3+, but not Nkx2.1+/Shh+ progenitor cells are detected (A,B). After 6 days, Th+ (arrowheads), but not Nkx2.1+/Th+ neurons are detected (C). (D) RT-PCR analysis of Shh mRNA in HH stage 5 and HH stage 6 prechordal mesoderm. No significant difference in Shh mRNA levels is detected (Ct=5.0±0.4, 4.8±0.5, respectively). (E,F) Recombinate explants of stage 4-5 pHyp and prechordal mesoderm, incubated alone (E) or with anti-Shh IgG (F). No Nkx2.1+ cells are detected in the presence of anti-Shh IgG. (G-I) Shh and Bmp7 cooperate to induce Nkx2.1+/Shh+progenitors and Nkx2.1+/Th+ neurons. Stage 4-5 aLNP explants, incubated with Shh and Bmp7. After 2 days, Nkx2.1+/Shh+ (G) and Nkx2.1+/Lim1+ (H) progenitors are detected. Msx+/Th+ neurons are detected on day 5 (I, inset) and Nkx2.1+/Th+ neurons detected on day 7 (I, white arrows). Th+ (blue arrowheads) neurons can also be detected. (J,K) Chordin blocks prechordal mesoderm-mediated specification of Nkx2.1+/Shh+ progenitors. (J) A stage 5 pHyp explant, co-cultured with prechordal mesoderm in the presence of chordin. Very few Nkx2.1+/Shh+ cells are detected. (K) Significantly more Nkx2.1+/Shh+ cells form in control cultures compared with cultures treated with chordin (control, 100%±23.2%; +Chd, 15.9%±4.3%, P<0.0001; unpaired t-test). (L) A stage 5 pHyp explant co-cultured with prechordal mesoderm and chordin for 6 days. Th+ cells differentiate (blue arrowheads), but these do not co-express Nkx2.1. (M) Stage 5 aLNP explants, incubated with Shh. After 22 hours, Shh but not Nkx2.1 (M, inset) was induced. (N) Stage 5 aLNP explants, incubated with Bmp7. After 22 hours, Msx/2 was detected but no Nkx2.1 was observed. (O) Stage 5 aLNP explants, transiently incubated with Bmp7 (day 0-1), then Shh (day1-7). On day 7, Th+ cells differentiate but they do not co-express Nkx2.1 (blue arrowheads). (P) Stage 5 aLNP explant, transiently incubated with Shh (day 0-1), then Bmp7 (days 1-7). On day 7, Nkx2.1+/Th+ cells differentiate (arrows). (Q) Stage 5 aLNP explant, transiently incubated with Shh (day 0-1), then transiently incubated with Bmp7 (day1-2). After 7 days, Nkx2.1+/Th+ cells differentiate (arrows). (R) Stage 5 aLNP explants, transiently incubated with Shh (day 0-1), then transiently exposed to Bmp7 (day 5-6). On day 7, Nkx2.1+/Th+ cells differentiate (arrows). (S) Stage 5 aLNP explants, transiently incubated with Shh (day 0-1), then transiently exposed to Bmp7 (day 6-7). On day 7, Th+ cells differentiate but they do not co-express Nkx2.1 (blue arrowheads). (T) Stage 5 pHyp explants, incubated with BrdU on day 5. On day 7, no BrdU+/Th+ cells are observed (blue arrowheads). Inset: stage 5 pHyp explants, incubated with BrdU on day 3. On day 7, BrdU+/Th+ cells are observed. (U) Stage 5 pHyp explants, transiently incubated with Bmp7 on day 5. On day 7, Nkx2.1+/Th+ cells differentiate (arrows). Scale bars: 40 µm in A,B,E-G,J,L,M-U,W; 30 µm in C,I.

View larger version (49K): [in a new window]   Fig. 6. Shh and Bmp7 are necessary and sufficient to induce Nkx2.1+/Shh+ cells and Nkx2.1+/Th+ neurons in a Six3-dependent manner in vivo. (A-D) Transverse section through tuberal hypothalamus (A-C) or anterior hypothalamus/dorsal telencephalon (D) of HH stage 15 embryos, immunolabelled to detect Nkx2.1 and Shh. (A) Control embryo showing Nkx2.1+/Shh+ cells in the lateral tuberal hypothalamus (arrow) and Nkx2.1+ ventral tuberal cells (arrowhead) after implantation of a control bead (bead has been displaced). (B) A chordin (Chd)-soaked bead implanted at stage 5, adjacent to the prechordal mesoderm/ventral tuberal hypothalamus. Nkx2.1 expression is abolished from lateral tuberal hypothalamic cells (arrow). Concomitantly, Shh expression is expanded (arrowhead in D). (C,D,D') Implanting a Shh/Bmp7-soaked bead at stage 5 (schematic above: bead displaced laterally) results in ectopic Nkx2.1+/Shh+ cells in the dorsal telencephalon (arrow in D,D'). Ventral cells in the anterior hypothalamus also co-express Shh and Nkx2.1. (C) Normal expression of Nkx2.1+/Shh+ lateral hypothalamic progenitors (arrow). (E,F) Electroporation of the RD Six3 construct results in the ectopic expression of Six3 in mesencephalic lateral neural tube explants (F, arrow). No Six3 expression is detected in control (RFP) electroporated explants (E). (G,H) Nkx2.1+/Th+ cells are induced in RD Six3-electroporated lateral mesencephalic explants, exposed to Shh and Bmp7 (G, arrows), whereas mock-electroporated explants give rise only to Nkx2.1–/Th+ cells after exposure to Shh and Bmp7 (H, blue arrowheads). (I-K) In AD Six3-electroporated mesencephalic tissue, exposed to Shh and Bmp7, Th+ cells do not co-express Nkx2.1 (I, blue arrowheads) but co-express En1 and Lmx (J,K, light blue arrows). Scale bars: 75 µm in A,B; 150 µm in C,D; 40 µm in G-K.

View larger version (74K): [in a new window]   Fig. 7. Shh and Bmp7 direct mouse ES cell-derived neural progenitor cells to an Nkx2.1+/Th+ DA fate. (A-E) Mouse ES cell-derived neural progenitor cells, cultured without (A-C) or with (D,E) Shh and Bmp7. In control cultures, Th+, Six3+, Nkx2.1+ and Shh+ (A-C) cells are observed but no Nkx2.1+/Th+ neurons are detected. In the presence of Shh and Bmp7, many Nkx2.1+/Th+ cells (arrows in E) are found (D,E). (F) Quantification of Nkx2.1+/Th+ neurons after directed ES cell differentiation. The average number of Nkx2.1+/Th+ cells in cultures shows a significant increase after exposure to Shh and Bmp7 (control 0.66±0.42 cells/2.56mm2; +Shh+Bmp7 51.44±7.27 cells/2.56mm2, P<0.0001; unpaired t-test), whereas the average of total number of Th+ cells does not show any significant difference (P>0.05, unpaired t-test; data not shown). Scale bar: 150 µm in A,D; 40 µm in B,C,E.

View larger version (9K): [in a new window]   Fig. 8. Model for the differentiation of hypothalamic DA neurons. Our data suggest a model for the specification of hypothalamic DA neurons. An early patterning step promotes expression of Six3+ cells, potentially affecting ambient levels of Wnt activity (Lagutin et al., 2003; Kapsimali et al., 2004). Shh acts to ventralise Six3+ cells, downregulating Pax6 and upregulating Shh. Cells at this point are specified to a forebrain-like/incomplete hypothalamic DA neurotransmitter identity, but do not exhibit definitive aspects of hypothalamic regional identity. Subsequently, Bmp7 acts to induce hypothalamic regional identity on Shh+ Six3+ cells, upregulating Nkx2.1 and Msx1/2.