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Hoxd10 induction and regionalization in the developing lumbosacral spinal cord

¹ Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
² Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, IL 60637, USA

View larger version (132K): [in a new window]   Fig. 1. Expression of Hoxd10 in normal chick embryos from stage 15 to stage 25. In situ hybridization on (A,C,E) whole mounts, or (B,D,F) transverse sections. Asterisks in A, C and E mark the approximate levels of the transverse sections. (A,B) Dorsal and transverse views of posterior neural tube regions at stage 16 (A) and stage 15 (B). At stages 15-16, Hoxd10 is diffusely expressed in the tail bud, the neural tube through midLS levels, and LS lateral plate mesoderm (arrow in B). The neural tube at future anterior LS levels shows little if any Hoxd10 expression (arrow in A). (C,D) Ventral and transverse views at stage 21. A ventral laminectomy and hemisection were performed on the embryo pictured in C, revealing one half of the ventral surface of the spinal cord. High expression is visible in the stage 21 ventral LS neural tube, in the region of the future motor columns. Diffuse Hoxd10 expression is still visible at the posterior end of the embryo (arrow in C). (E,F) Ventral and transverse views at stage 25. In the embryo pictured in E, a ventral laminectomy was performed prior to processing. Hoxd10 expression is widespread in postmitotic neurons in LS segments, but not in adjacent segments. Cells lining LS ventral roots (arrow, F) also show expression. Bars, 250 µm.

View larger version (102K): [in a new window]   Fig. 2. Expression of Hoxd10 in normal chick embryos at stage 29. (A) Ventral view of posterior spinal regions after whole-mount in situ hybridization. (B-E) Transverse sections through T7 (B), LS1 (C), LS2 (D), and LS3 (E) after section in situ processing. Hoxd10 is highly expressed in the LS spinal cord, sensory ganglia and spinal nerves (A). Expression is graded on the AP axis, being lowest in LS1 and highest in middle and posterior LS segments (A, C-E). Within LS1 and LS2, two dark strips of Hoxd10 expression are visible in A. In transverse sections (C,D), these strips represent high expression in cells at the lateral edge of the LMC and in intermediate cord regions. Cells within middle and dorsomedial motor column regions show less expression (arrow, E). Slight Hoxd10 expression is visible in T7 but not in more anterior T segments (A,B). Bars, 250 µm.

View larger version (67K): [in a new window]   Fig. 3. Hoxd10 expression in whole-mount embryos after reversals of neural tube segments at the T-LS boundary. (Left) Schematics of stage 13 and 15 embryos. Anterior CNS regions are shown in blue; prospective LS regions, in yellow. The boxed areas in the drawings of normal whole embryos correspond to the expanded diagrams showing reversal surgeries. (A-D) Ventral views of posterior spinal cord regions from control (A) and experimental embryos (B-D) at stages 27-34. Transplant regions are delineated by brackets in this and all subsequent figures. Following reversals of either T6-LS3 (B) or T6-LS2 (C) at stage 15, the manipulated segments show a reversal of the normal Hoxd10 expression pattern. The spinal cord shown in C exhibits lower Hoxd10 expression in the LMC (arrow) than that shown in B, in accord with its older stage of sacrifice (B, stage 27; C, stage 34). Following reversals of T6-LS2 at stage 13, Hoxd10 expression is low in both reversed T and LS segments at stage 29. Only the originally posterior-most segment in the reversal region (the original LS2) shows Hoxd10 expression above that found in a normal mid-T segment. st, stage. Bars, 250 µm.

View larger version (77K): [in a new window]   Fig. 4. Hoxd10 expression in whole-mount embryos after transplantation of LS neural tube segments to T regions. On the left are schematics of the transplantation of donor LS segments (yellow) into T regions (blue). On the right are ventral views of experimental embryos at stages 30-31. In each case, transplanted LS segments had been positioned in a normal AP orientation. (A) Following transplantation of stage 15 donor LS segments into posterior T regions of a stage 14 host embryo, high Hoxd10 expression develops in the transplant region in accord with its LS origin. (B, C) Results were variable following transplantation of stage 13 donor LS segments into T regions of stage 12-13 host embryos. In a transplant placed in anterior T regions (B), Hoxd10 expression is only detected at the posterior end of the transplant and the level of expression is very low. In a transplant extending into posterior T regions (C), expression of Hoxd10 in the transplanted LS segments is equivalent to that in the host’s LS segments. Bars, 250 µm.

View larger version (19K): [in a new window]   Fig. 5. Summary of the levels of Hoxd10 expression in whole-mount embryos after transplantation of LS neural tube segments to T regions. Hoxd10 expression was visually scored in donor and host regions of each experimental embryo. This histogram expresses Hoxd10 levels in transplant regions as mean percent of levels (± s.e.m.) in host LS regions (see text). The number of embryos in different experimental groups are given in parentheses. Values are given separately for embryos with transplants placed into anterior and posterior T regions. Stage 14-15 transplant groups (black bars) show high values, irrespective of position. Stage 13 transplant groups (white and gray bars) differ; transplants in anterior T positions show low expression; transplants in posterior T regions show high levels. Within the stage 13 anterior transplant group, transplants + LS3 show a higher mean level value than transplants –LS3. The number of cases are low because the precise level of origin could only be determined for a subset of embryos (see text). Transplants of LS neural tissue + adjacent paraxial mesoderm (gray bar) show a value similar to that of transplants of LS neural tissue alone.

View larger version (104K): [in a new window]   Fig. 6. Hox10 and LIM patterns following transplantation of LS segments to T regions. (A-C) Adjacent sections from the LS2/3 region of a stage 28 control embryo. Hoxd10 expression (A) is high throughout the transverse plane of the spinal cord except in the ventricular zone and in a medial strip of motor column cells. Isl1/2+ staining (B) identifies all motoneurons and facilitates the identification of a large, laterally directed motor column region characteristic of the normal LS region. Lim 1/2+ staining (C) identifies motoneurons unique to the normal LS region, the LMCl (arrow). (J-L) Adjacent sections from mid-T regions of the same control embryo. Hoxd10 expression is virtually absent in the cord (J). Isl1/2+ staining (K) shows a motor column region that is much smaller than that in LS sections. In addition, Isl1/2+ cells of the Column of Terni are visible in intermediate regions, along the edge of the ventricular zone (arrowhead). (D-I) Transverse sections through the transplant regions of stage 28-30 experimental embryos. (D-F) Sections taken from an embryo in which stage 13 LS segments had been transplanted into posterior T region of a stage 12 host embryo. Hoxd10 expression (D), Isl1/2 staining (E), and Lim1/2 staining (F) patterns are LS-like in character. (G-I) Sections taken from an embryo in which stage 13 LS segments had been transplanted into anterior T regions of a stage 13 host embryo. Here, Hoxd10 expression (G) is present only at low levels. Isl1/2 (H) and Lim1/2 staining (I) patterns are mid T-like in character. Bars, 250 µm.

View larger version (54K): [in a new window]   Fig. 7. The development of regional character in T neural segments following transplantation into LS regions. Shown at top is a schematic of the transplant surgery. (A) Ventral view of the posterior spinal cord from a stage 29 experimental embryo. Stage 11 T segments had been placed in the LS region of a stage 13 host embryo. Hoxd10 expression is high in host LS segments, anterior and posterior to the T transplant, but low in donor T segments. Only the most posterior end of the transplant shows slight expression. (B-G) Sections from a stage 28 embryo in which transplanted T segments show higher levels of Hoxd10 expression. In this case, stage 13 donor T segments had been placed into the LS region of a stage 13 host. The axial level of placement was similar to that of the embryo shown in A. Hoxd10 expression patterns and LIM patterns (Isl1/2 and Lim1/2) are T-like in anterior transplant regions (B-D) but largely LS-like in posterior transplant regions (E-G). Bars, 250 µm.

View larger version (117K): [in a new window]   Fig. 8 The development of regional character in LS neural segments bordered by T paraxial mesoderm. (Top left) A schematic of the surgery. Paraxial mesoderm on one side of the anterior LS neural tube of a stage 13 chick embryo was replaced with stage 11-13 anterior T paraxial mesoderm from a chick or quail embryo. (A) Ventral view of the LS cord from one stage 29 embryo. Hoxd10 expression is high in LS neural tissue adjacent to the transplant region, although, the lateral strip of high Hoxd10-expressing cells is reduced. (B-D) Sections through the anterior LS region of a stage 29 embryo. (B) The experimental side (arrow) shows lower Hoxd10 expression in the mesoderm than the control side. In neural tissue, Hoxd10 expression (B) as well as Isl1/2 (C) and Lim1/2 (D) protein patterns are normal on the experimental side with the exception of a decrease in motor column size. (E-G) Sections through the anterior LS region in a stage 29 quail/chick chimera. The experimental side is marked with an arrow. Hoxd10 expression is equivalently high on both sides of the LS cord (E), despite the fact that the control side is bordered by chick myotomal cells of LS origin (F) and the experimental side is bordered by quail myotomal cells of T origin (G). Bars, (A-E) 250 µm; (F-G) 25 µm.

View larger version (73K): [in a new window]   Fig. 9. The development of Hoxd10 expression in T segments bordered by LS paraxial mesoderm and LS segments transplanted to T regions with adjacent paraxial mesoderm. On the left are schematics of the surgical procedures. Whole-mount in situ hybridization was done at stages 28-29. On the right are ventral views of T and LS spinal regions. (A) Hoxd10 is not induced in the T spinal cord after placement of stage 13 anterior LS paraxial mesoderm into the T region of a stage 11 embryo. (B) Stage 13 LS neural tissue + its adjacent paraxial mesoderm was transposed to anterior T regions in a stage 11 host. Hoxd10 expression is low in transplanted neural segments as it is following transposition of LS neural tissue alone (see Fig. 4B). Only the most posterior transplant regions show expression above that in adjacent host regions. (C) No expression is induced in anterior T neural segments despite transplantation of stage 14 posterior LS paraxial mesoderm into anterior T regions at stage 11. Hoxd10 expression is visible in the transplanted mesoderm (see bracket). (D) Hoxd10 expression is also not induced in posterior T neural segments following an equivalent transplant of posterior LS paraxial mesoderm into posterior T regions. Again expression is visible in transplanted mesoderm. Bars, 250 µm.

View larger version (31K): [in a new window]   Fig. 10. Schematics of a possible mode of Hoxd10 induction. At stage 13, a prepattern for Hoxd10 expression is present in the anterior LS neural tube. The most anterior LS segments (LS1-2) have less ability to develop high levels of Hoxd10 expression than more posterior segments (LS3+). This gradient of competence may reflect exposure to a very early gradient of inducing signals, in extreme posterior embryonic regions. At stage 13+, inducing signals may spread anteriorly, as far as posterior T regions, stabilizing and amplifying the LS prepattern. Associated with this process is an anterior extension of non-axial mesoderm and a posterior elongation of the LS neural tube (arrows, after Catala et al., 1995). The asterisks denote displacement of mesoderm at one axial level.