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CNE_22803_sm_suppifofig1.tif1659KSupporting Information Figure 1. The development of the A13 nucleus occurs in two successive phases. This figure is a magenta-green-blue version of the Fig.1 in the main text. (A-D′) Immunofluorescence detection of tyrosine hydroxylase (TH) (magenta) and Islet-1 (Isl1) (green) on transverse (A) or coronal (B,C,D) sections in the diencephalon of wildtype embryos at e10.5 (A), e11.5 (B), e12.5 (C) or e18.5 (D). A schematic representation of A, B, C and D is shown (A′,B′,C′,D′) to illustrate the displacements of the A13 neurons. Anterior (A) or dorsal (B-D) is to the top. The third ventricle is to the left. Dotted lines delineate the lumen of the third ventricle and the pial surface. Dashed lines delineate the A13 neurons. (A,A′) At e10.5, the presumptive A13 neurons migrate from the ventricular zone and accumulate beneath the pial surface. (B,B′) At e11.5, the A13 neurons form a thick mantle layer beneath the pial surface. However, the displacement of some cells toward the third ventricle is initiated. (C,C′) One day later, the A13 neurons are detected inside the wall of the medial hypothalamus. (D,D′) At e18.5, the A13 nucleus is constituted by a compact cluster of dopaminergic cells located close to the third ventricle. (E-F) Whole-mount immunofluorescence labeling for TH on e12.5 (E) or e18.5 (F) wildtype embryos. View is from the top of the embryos. Anterior is to the top of the pictures. Dotted lines delineate the lumen of the third ventricle. (E) At e12.5, the A13 cells (dashed lines) constitute a longitudinally elongated population of dopaminergic neurons located inside the wall of the medial hypothalamus. (F) At e18.5, the A13 nucleus (dashed lines) displays a laterally elongated oval shape and is directly adjacent to the lumen of the third ventricle. (G-H′) 3D-reconstructions of the A13 population in e12.5 (G-G′) or e18.5 (H-H′) wildtype embryos. Top (G,H), Y-axis 45° rotation (G′) or X-axis 90° rotation (H′) views of the reconstructions. Third ventricle is in the middle. Movies 1 and 2 showing respectively horizontal or vertical rotation of the 3D-reconstructions are available as Supporting Information. (G,G′) During their displacement within the wall of the diencephalon, the A13 neurons (arrows) form a longitudinally elongated population. (H,H′) In contrast, when located at their final position, they gather as a compact laterally elongated nucleus. III: third ventricle, CP: caudoputamen, ML: mantle layer, nsp: nigrostriatal pathway, PA: preoptic area, SNc: substantia nigra pars compacta , VTA: ventral tegmental area, VZ: Ventricular zone. Scale bars are 50μm (A-D) or 400μm (E,H′).
CNE_22803_sm_suppifofig2.tif3577KSupporting Information Figure 2. Onecut (OC) transcription factors are dynamically and differentially expressed in A13 neurons throughout embryonic development. This figure is a magenta-green-blue version of the Fig.2 in the main text. (A-P) The OC factors are expressed from the initial steps of A13 neuron differentiation. Coimmunofluorescence (A-L, N-P) or combined in situ hybridization and immunofluorescence (M) for the OC factors and for markers of A13 neurons on transverse sections in the diencephalon of e10.5 wildtype embryos. Anterior is to the top. The third ventricle is to the left. Dotted lines delineate the lumen of the third ventricle and the pial surface. Dashed lines delineate the A13 neurons. Insets in A-D and M show higher magnifications of A-D and M, respectively. The OC factors HNF-6 (A-D), OC-2 (E-H) or OC-3 (I-L) (magenta) are detected in a subset of cells expressing βIII-tubulin (A,E,I) and Isl1 (B,F,J) (green) and in a majority of cells expressing high levels of Pax6 (C,G,K) and TH (D,H,L) (blue). (M) The cells containing HNF-6 (blue) still express the early A13 marker Dlx2 (black). (N,O,P) Coimmunofluorescence for the OC proteins. The distribution of HNF-6 (N,P) (green) is broader than that of OC-2 (N,P) (blue), which is broader than that of OC-3 (O,P) (magenta). (Q-T) The OC factors remain expressed during the second phase of A13 development. Coimmunofluorescence for the OC factors and the dopaminergic marker TH on coronal sections in the medial hypothalamus of e12.5 (Q,R) or e18.5 (S,T) wildtype embryos. Dorsal is to the top. The third ventricle is to the left, and is delineated by dotted lines. Dashed lines delineate the A13 neurons. (Q-Q′′) At e12.5, the OC factors (magenta) are present in most of the A13 neurons (blue) and (R) the expression domain of HNF-6 (green), OC-2 (blue) and OC-3 (magenta) are partially overlapping. (S,S′′) At e18.5, HNF-6 and OC-3 (magenta) are still present in the A13 neurons (blue), while (S′,T) OC-2 is barely detectable in very few of these cells. nsp: nigrostriatal pathway. Scale bars are 50μm.
CNE_22803_sm_suppifofig3.tif1144KSupporting Information Figure 3. The OC transcription factors are dispensable for the first phase of A13 nucleus development. This figure is a magenta-green-blue version of the Fig.3 in the main text. (A-H) Immunofluorescence for markers of A13 differentiating neurons on transverse sections in the diencephalon of e10.5 control (A,C,E,G) or Hnf6/Oc2-/- (B,D,F,H) embryos. Anterior is to the top. The third ventricle is to the left. Dotted lines delineate the lumen of third ventricle and the pial surface. Dashed lines delineate the A13 neurons. The distribution of Pax6, βIII-tubulin, Isl1, Nkx2.2, TH and Lhx1/5 is similar in the A13 neurons and adjacent neuroepithelium of control and of mutant embryos, as is the location of the A13 cells. Scale bars are 100μm. (I) Quantification (n=4) of the number of A13 neurons in e10.5 control or Hnf6/Oc2-/- embryos. No significant difference is detected (p=0.856).
CNE_22803_sm_suppifofig4.tif3705KSupporting Information Figure 4. Figure S4. The OC transcription factors are required for the second phase of A13 nucleus development. This figure is a magenta-green-blue version of the Fig.4 in the main text. (A-C) Coimmunofluorescence for TH (magenta) and Isl1 (green) (A,B) on coronal sections in the medial hypothalamus of e12.5 control (A), Hnf6/Oc2-/- (B) or Hnf6-/- (C) embryos. Dorsal is to the top. The third ventricle is to the left, and is delineated by dotted lines. Dashed lines delineate the A13 neurons. Insets show higher magnification of the A13 area. (A,C) In control or Hnf6-/- embryos, A13 neurons form a well-defined population located inside the wall of the medial hypothalamus. (B) In mutant embryos, dopaminergic neurons are present but scattered (compare insets) and are located farther from the third ventricle. (D-F′) 3D-reconstructions of whole-mount immunofluorescence for TH on e12.5 control (D,D′), Hnf6/Oc2-/- (E,E′) or Hnf6-/- (F,F′) embryos. Top (D-F) or Y-axis 45° rotation (D′-F′) views of the reconstructions. Third ventricle is in the middle. Movies 3 to 5 showing horizontal rotation of the 3D-reconstructions are available as Supporting Information. (D-D′) In control embryos, the A13 dopaminergic neurons (arrows) form a well-defined longitudinally elongated population located inside the wall of the medial hypothalamus. (E,E′) In Hnf6/Oc2-/- mutant embryos, no compact A13 population is observed (asterisks) and only scattered dopaminergic neurons are detected in a very lateral position. (F,F′) In Hnf6-/- embryos, as observed in control embryos, the A13 neurons (arrows) form a longitudinally elongated population located inside the wall of the medial hypothalamus. (G-J) Quantifications (n=3) of the distribution area (G), the cell density (H) and the mean distance from the third ventricle (I) of the A13 population, and of the thickness of the wall of the medial hypothalamus (J) in control, Hnf6/Oc2-/- or Hnf6-/- embryos. In Hnf6/Oc2-/- embryos, the A13 population spreads over a significantly larger area (p=0.035) and is more scattered (p<0.001) than in control embryos. In addition, it is located farther from the third ventricle (p=0.049), although the thickness of the wall of the medial hypothalamus is comparable in control and in Hnf6/Oc2-/- samples (p=0.958). In contrast, no significant difference is detected in Hnf6-/- embryos compared to control embryos. * : p<0.05; *** : p<0.001. (K-R) Immunofluorescence for Pax6 (K,O), Lhx1/5 (L,P), R-Cadherin (R-Cadh) (M,Q) and coimmunofluorescence for 5-bromo-2′-deoxyuridine (BrdU) injected at e10.5 (green) and TH (blue) (N,R) on coronal sections in the medial hypothalamus of e12.5 control (K-N) or Hnf6/Oc2-/- (O,R) embryos. Dorsal is to the top. The third ventricle is to the left, and is delineated by dotted lines. Insets in N and R show magnifications of indicated areas. Distribution of Pax6, Lhx1/5 and R-Cadh,which label cell populations surrounding the A13 area, is similar in control and in Hnf6/Oc2-/- embryos. (N) In control embryos, some A13 neurons contain BrdU, while most of the other BrdU-positive cells are located in the mantle layer between the A13 area and the pial zone. (R) The distribution of BrdU is similar in Hnf6/Oc2-/- embryos. BrdU: 5-bromo-2′-deoxyuridine; nsp: nigrostriatal pathway; R-cadh: R-Cadherin. Scale bars are 100μm (A-C), or 200 μm (D-R).
CNE_22803_sm_suppifofig5.tif2189KSupporting Information Figure 5. The OC transcription factors are collectively required for maintenance of the A13 nucleus. This figure is a magenta-green-blue version of the Fig.5 in the main text. (A-C) Coimmunofluorescence for TH (magenta) and Isl1 (green) on coronal sections in the hypothalamus of e18.5 control (A), Hnf6/Oc2-/- (B) or Hnf6-/- embryos (C). Dorsal is to the top. The third ventricle is to the left, and is delineated by dotted lines. Dashed lines delineate the A13 nucleus. (A,B) Whereas a compact dopaminergic nucleus adjacent to the third ventricle is detected in control embryos, no TH expression is detected at this position (asterisk) in Hnf6/Oc2-/- mutants. (C) In Hnf6 -/- embryos, the A13 nucleus is smaller and abnormally shaped. (D-F) Whole-mount immunofluorescence for TH on control (D), Hnf6/Oc2-/- (E) or Hnf6-/- (F) embryos. View is from the top of the head. Anterior is to the top of the pictures. Dotted lines delineate the lumen of third ventricle. Boxes delineate the area shown in G-I′. (D) In control embryos, the A13 neurons (dashed lines) are clustered into a compact nucleus and are located in close vicinity of the third ventricle. (E) In Hnf6/Oc2-/- embryos, no dopaminergic neurons are observed at their expected location (asterisks). (F) In Hnf6-/- embryos, the A13 nucleus (dashed lines) is smaller and seems to be located farther from the third ventricle than in control embryos. (G-I′) 3D-reconstructions of the A13 nucleus of e18.5 control (G,G′), Hnf6/Oc2-/- (H,H′) or Hnf6-/- (I,I′) embryos. Top (G-I) or X-axis 90° rotation (G′-I′) views of the reconstructions. Third ventricle is in the middle. Movies 6 to 8 showing vertical rotation of the 3D-reconstructions are available as Supporting Information. (G,G′) In control embryos, the A13 nucleus is constituted by tightly clustered dopaminergic neurons adjacent to the third ventricle and can be divided into three subgroups: dorsal (A13d), lateral (A13l) or medial (A13m) portions of the A13 nucleus. (H,H′) In Hnf6/Oc2-/- embryos, dopaminergic neurons are barely detectable in this region of the medial hypothalamus. (I,I′) In Hnf6-/- embryos, the A13 nucleus is smaller and located farther from the third ventricle (compare the distance between the left and right A13 nuclei in control and in Hnf6-/- samples). (J-M) Immunofluorescence for Pax6 and R-Cadh on coronal sections in the hypothalamus of e18.5 control (J,L) or Hnf6/Oc2-/- (K,M) embryos. Dorsal is to the top. The third ventricle is to the left, and is delineated by dotted lines. The distribution of Pax6 (J,K) and R-Cadherin (L,M) is similar in control (J,L) and in Hnf6/Oc2-/- (K,M) embryos. nsp: nigrostriatal pathway. Scale bars are 100μm (A-C), 1000μm (D-F), 400μm (G-I′) or 50μm (J-M). A13d: dorsal portion of the A13 nucleus; A13l: lateral portion of the A13 nucleus; A13m: medial portion of the A13 nucleus.
CNE_22803_sm_suppifomov1.avi6295KSupporting Information Movie 1. Horizontal rotation of 3D-reconstruction of wildtype e12.5 embryo (corresponding to Fig. 1G) During the second phase of the development of the A13 nucleus, the A13 cells constituted a longitudinally elongated population of dopaminergic neurons. They were located inside the wall of the medial hypothalamus, between the fibers of the nigrostriatal pathway and the lumen of the third ventricle.
CNE_22803_sm_suppifomov2.avi2986KSupporting Information Movie 2. Vertical rotation of 3D-reconstruction of wildtype e18.5 embryo (corresponding to Fig.1H) At the end-stage of development, the A13 nucleus was constituted by a compact population of TH-positive cells adjacent to the lumen of the third ventricle and displayed a laterally elongated oval shape.
CNE_22803_sm_suppifomov3.avi5701KSupporting Information Movie 3. Horizontal rotation of 3D-reconstruction of control e12.5 embryo (corresponding to Fig.4D) In control embryos, during the second phase of development, the A13 nucleus corresponded to a well-defined longitudinally elongated population located inside the wall of the medial hypothalamus.
CNE_22803_sm_suppifomov4.avi4448KSupporting Information Movie 4. Horizontal rotation of 3D-reconstruction of Hnf6/Oc2-/- e12.5 embryo (corresponding to Fig.4E) In e12.5 Hnf6/Oc2-/- embryos, no compact A13 population was observed inside the wall of the medial hypothalamus. Instead, scattered dopaminergic neurons were detected in a very lateral position, close to the fibers of the nigrostriatal pathway.
CNE_22803_sm_suppifomov5.avi4437KSupporting Information Movie 5. Horizontal rotation of 3D-reconstruction of Hnf6-/- e12.5 embryo (corresponding to Fig.4F) In e12.5 Hnf6-/- embryos, the A13 neurons formed a longitudinally elongated population located inside the wall of the medial hypothalamus, as observed in control embryos.
CNE_22803_sm_suppifomov6.avi33483KSupporting Information Movie 6. Vertical rotation of 3D-reconstruction of control e18.5 embryo (corresponding to Fig.5G) In e18.5 control embryos, the A13 nucleus was adjacent to the third ventricle and was constituted by tightly clustered dopaminergic neurons. The A13 neurons formed a laterally elongated oval population.
CNE_22803_sm_suppifomov7.avi1950KSupporting Information Movie 7. Vertical rotation of 3D-reconstruction of Hnf6/Oc2-/- e18.5 embryo (corresponding to Fig.5H) In e18.5 Hnf6/Oc2-/- embryos, no A13 nucleus was detected.
CNE_22803_sm_suppifomov8.avi2720KSupporting Information Movie 8. Vertical rotation of 3D-reconstruction of Hnf6-/- e18.5 embryo (corresponding to Fig.5I) In Hnf6-/- embryos, the A13 nucleus was present. However, it was smaller, displayed an abnormal shape and was located farther from the third ventricle compared to control embryos.

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