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First published online May 16, 2007
doi: 10.1242/10.1242/dev.02850

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Regulation of ventral midbrain patterning by Hedgehog signaling

Roy D. Bayly¹, Minhtran Ngo^2,*, Galina V. Aglyamova² and Seema Agarwala^1,2,3,

¹ Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712-0248, USA.
² Section of Neurobiology and University of Texas at Austin, Austin, TX 78712-0248, USA.
³ Institute for Neuroscience, University of Texas at Austin, Austin, TX 78712-0248, USA.

View larger version (92K): [in this window] [in a new window]   Fig. 1. HH signaling is necessary for cell-fate specification in the ventral midbrain. For orientation and embryonic ages, see Materials and methods. (A) At E5, EGFP-electroporated (right side) controls do not show disruptions in the rostral floor plate (rFP; SHH+, brown) or in midbrain arc pattern formation. Midbrain arcs are marked by the homeobox (HX, blue) gene expression of PHOX2A in the first arc (1), PAX6 (P6) and EVX1 (E1). (B) Blockade of FOXA2 (brown) expression following unilateral Ptc1loop2 (blue) electroporation (right side). (C) Re-specification of ventral cell fates (marked by HX genes, blue) into dorsal (PAX7+, brown, arrowhead) cell fates. (D,E). Blockade and bi-directional spread of PHOX2A+ (brown) oculomotor complex neurons following bilateral electroporation (E) of Ptc1loop2 (blue). Compare E with EGFP-electroporated controls (D). D and E are photographed at the same magnification. (E) Note the lack (caudally, arrowhead) of overlap between PHOX2A and Ptc1loop2 transgene expression. Rostral cells (arrow) have extinguished their requirement for HH signaling by this stage (see text). (F,G) Reduced expression and spread of tyrosine hydroxylase (F, dopaminergic neurons), PAX6 (G, brown) and EVX1 (G, blue) following unilateral HH blockade. (H) Cross-section demonstrating the non-autonomous spread of PAX6+ (brown, arrowhead) cells following unilateral Ptc1loop2 (blue) electroporation. Note the presence of ectopic PAX6+/Ptc1loop2+ cells (arrow, see text). (I,J) E8 whole mounts electroproated at H&H stage 10, demonstrating that, compared to EGFP controls (I), cell spread following HH blockade (J) increases with time (compare with the E5 brains in D and E) and is multidirectional. Blue, TH (arrowheads); brown, ISL1+ motor neurons; 1, first arc; III, third ventricle; bi, bilateral electroporation; E1, EVX1; EP, electroporated; P6, PAX6; HX, homeobox expression of PHOX2A, PAX6, EVX1; MHB, midbrain-hindbrain boundary; rFP, rostral floor plate; TH, tyrosine hydroxylase; tec, tectum.

View larger version (97K): [in this window] [in a new window]   Fig. 2. HH blockade prevents differentiation and promotes proliferation in the ventral midbrain. (A,B) Increased cyclin B2 (A) and cyclin D1 (B) expression following unilateral misexpression of Ptc1loop2. (C) BrdU labeling shown in a cross-section through an EGFP-electroporated embryo, in which it is confined to proliferating cells of the ventricular layer. (D) Massive increase in BrdU labeling (blue) following Ptc1loop2 electroporation. Note that the increased thickness of the ventricular layer is associated with a reduction of the mantle layer, where differentiated neurons normally reside (compare double-headed arrows in C and D, which were photographed at the same magnification). (E) Cross-section through the ventral midbrain of a Ptc1loop2-electroporated embryo, showing a reduction in class III ß-tubulin expression (brown, asterisk) following HH blockade. (E, inset) Whole-mount view of the cross-section in E. (F,G) SHH (brown) overexpression results in reduced cyclin D1 (blue) expression. The same embryo is presented in F (before) and G (after) the detection of SHH. (H) Embryos bilaterally electroporated with either SHH (light embryos, upper) or Ptc1loop2 (dark embryos, lower) at H&H stage 9. Note the reduced size of SHH-electroporated embryos compared with Ptc1loop2-electroporated embryos. Embryos are shown in sagittal view, with rostral to the left. III, third ventricle; bi, bilateral electroporation; Di, diencephalon; EP, electroporated; HB, hindbrain; rFP, rostral floor plate.

View larger version (107K): [in this window] [in a new window]   Fig. 3. Ptc1loop2 affects cell-fate specification in a radial manner. (A) `Haloes' of PHOX2A-negative/Ptc1loop2-negative (uncolored) cells surround Ptc1loop2+/PHOX2A-negative (blue) cells. (B) Cross-section through whole-mount shown in A, demonstrating that the `haloes' are columns of PHOX2A-negative/Ptc1loop2-negative cells radially associated with more-pially located Ptc1loop2+ cells. (C) Ptc1loop2-electroporated embryos at E4 display midbrain columns in cross-section. (C, inset) Magnified view of a single column of cells (indicated by arrowhead). Individual cells are marked by asterisks. (D) Cross-section through an E4 embryo electroporated with low concentrations of EGFP, displaying bipolar-radial glia-like midbrain progenitors. Note that, when multiple cells are present in a single column, they are cytoplasmically continuous (arrowhead). (E) Close-up of boxed area in D, highlighting the radial glial-like morphology of the midbrain progenitors, including the presence of end-feet at the ventricular surface. (F) Demonstration of dye-coupling through gap junctions among three ventral midbrain cells following the injection of Alexa-Fluor 488 into the central cell (*). H&H stage 10 explant presented in whole-mount view (rostral is to the top and ventricular surface faces the viewer; orientation is the same as in Fig. 1A). Each cell is approximately 7.5 µm across and the cells are spaced approximately 5 µm apart. The central cell is ventricular with respect to the other two cells. H&H, embryonic stages according to Hamburger and Hamilton (Hamburger and Hamilton, 1951); rFP, rostral floor plate.

View larger version (114K): [in this window] [in a new window]   Fig. 4. Spatiotemporal regulation of HH requirement in the ventral midbrain. (A) Bilateral EGFP (blue) misexpression does not perturb the expression of rFP genes (FOXA2, brown). (B,C) Caudal-medial and lateral, but not antero-medial (arrowhead), regions of the rFP (FOXA2+, brown) can be disrupted following bilateral electroporation of Ptc1loop2 (blue) at H&H stages 6-9. (C) Cross-section of B at the level indicated by the line in B. (A-C) Note the meager number of Ptc1loop2+ cells at the midline (arrowheads, B,C) compared with controls (A). (D) HH blockade disrupts lateral rFP specification at H&H stages 15-16. (E) E6 embryo electroporated with Ptc1loop2 (blue) between H&H stage 9 and 11, demonstrating the uniform blockade of cell-fate specification in all midbrain arcs, assayed by HX gene expression (brown). Note the extensive cell mixing and disruption of the arc pattern. (F) Greater caudal perturbation of the PHOX2A+ (1, brown, arrowhead) first arc following Ptc1loop2 electroporation (blue) at H&H stages 10-12. The rostral expression of PHOX2A (arrow) is largely unaffected despite the higher bilateral expression of the Ptc1loop2 transgene in this region. (G) E6 embryo electroporated between H&H stages 17 and 20, demonstrating that midbrain cell fates (brown) are independent of HH signaling, except in lateral regions of the rFP and cells associated with it (e.g. arc 2). (H) Close-up of boxed area in G, demonstrating that midbrain progenitors within the lateral region of the rFP and the cells associated with it (e.g. arc 2; 2) can be re-specified to more-dorsal (PAX6+) cell fates in association with Ptc1loop2+ cells (arrow). In addition, dorsal cells (PAX6+) can move into this region non-autonomously (arrowhead). 1, first arc; 2, arc 2; III, third ventricle; bi, bilateral electroporation; EP, electroporated; P6, PAX6; H&H, embryonic stages according to Hamburger and Hamilton (Hamburger and Hamilton, 1951); HX, homeobox expression of PHOX2A, PAX6, EVX1; MHB, midbrain-hindbrain boundary; rFP, rostral floor plate.

View larger version (131K): [in this window] [in a new window]   Fig. 5. Disruption and cell mixing at the chick MHB following HH blockade. (A,B) Unlike EGFP controls (A), bilateral electroporation of Ptc1loop2 (blue; B) disrupts WNT1 (brown, arrowhead) expression at the MHB. (C,D) Unlike controls (C), HH blockade (D) results in the broadening of FGF8 expression at the MHB (blue; compare the length of the double-headed arrows in C and D). (D) Note the ectopic mixing of FGF8+ (white arrowhead) and OTX2+ (brown, black arrowhead) cells. C and D were photographed at the same magnification. (E,F) Increased cyclin D1 expression within the MHB following bilateral Ptc1loop2 electroporations. E and F are photographs of the same embryo demonstrating that all FGF8+ cells (arrows, brown) are also cyclin D1+. However, all ectopic cyclin D1+ (arrowhead) cells are not FGF8+ (see Fig. 2A, left side, for normal cyclin D1+ expression). bi, bilateral electroporation; EP, electroporated; HB, hindbrain; MHB, midbrain-hindbrain boundary; rFP, rostral floor plate.

View larger version (152K): [in this window] [in a new window]   Fig. 6. HH blockade leads to a disruption of the DV boundary. (A) Ectopic PAX7 expression in the ventral midbrain after HH blockade. (B) Serrate 1 expression (blue), which is normally confined to the dorsal midbrain (tec) and to a thickening at the DV boundary (arrowhead), is perturbed in Ptc1loop2 electroporations. (C) Absence of PAX7+ (blue) cells in the ventral midbrain of EGFP (brown)-electroporated explants. Note the presence of PAX7 (blue) expression in the tectum (tec). (D) Bilateral Ptc1loop2 electroporation induces ectopic PAX7 expression in ventral midbrain explants with no associated tectal tissue. (E) EGFP misexpression (blue) near the DV boundary (broken line) fails to perturb PAX7 expression (brown). (F) Ptc1loop2 misexpression (blue) near the DV boundary (broken line) induces ectopic PAX7+ (brown) cells, some non-autonomously (arrowhead). Arrow points to the upregulation of PAX7 in association with Ptc1loop2 misexpression. III, third ventricle; bi, bilateral electroporation; EP, electroporated; HB, hindbrain; MHB, midbrain-hindbrain boundary; rFP, rostral floor plate; tec, tectum.

View larger version (34K): [in this window] [in a new window]   Fig. 7. Summary of HH function in the ventral midbrain. (A-D) Whole mounts; (E) cross-section. (A-C) A three-step temporal patterning of the ventral midbrain by HH blockade. (A) Electroporation at or before H&H stage 11: anterior-medial rFP patterning is complete or HH-independent. The caudo-medial rFP, lateral rFP and all other midbrain cell fates still require HH signaling for their specification. Increased cell spread is noted. (B) Electroporation at H&H stages 11-13: medial rFP specification is complete. Lateral rFP and arcuate cell fates, represented here by PHOX2A (blue), PAX6 (purple) and EVX1 (yellow), continue to be specified. The caudo-medial midbrain requires HH signaling for a longer time than does the rostral midbrain. HH is also required for forming a coherent arc pattern. (C) Electroporation after H&H stage 13: midbrain patterning is complete, with the exception of the lateral rFP and cell fates associated with this region (e.g. arc 2). Ectopic cell spread is noted only in this region. (D) HH signaling regulates midbrain boundaries. Disruption of the boundaries of the midbrain results in the non-autonomous spread of PAX7+ (green) cells at the DV boundary and of WNT1+ (blue) cells and FGF8+ (brown) cells at the MHB. Ectopic PAX7 expression (arrow) is also seen in ventral midbrain progenitors as a result of re-specification into dorsal phenotypes. (E) PHOX2A expression, demonstrating that Ptc1loop2 electroporations (right of the vertical line) result in increased cell proliferation (expanded ventricular layer) and in reduced differentiation compared with controls (left of the vertical line). The effects are seen within columns of cytoplasmically connected midbrain cells (white), which line up ventricular to the Ptc1loop2+ cells (blue circles). Non-autonomous cell spread (*) is also seen. 1, first arc; 2, arc 2; III, third ventricle; E1, EVX1; EP, electroporated; P6, PAX6; H&H, embryonic stages according to Hamburger and Hamilton (Hamburger and Hamilton, 1951); MHB, midbrain-hindbrain boundary; rFP: rostral floor plate; TEC, tectum.

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