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First published online December 7, 2007
doi: 10.1242/10.1242/dev.009225

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Hoxc10 and Hoxd10 regulate mouse columnar, divisional and motor pool identity of lumbar motoneurons

Yuanyuan Wu^1,*, Guoying Wang^2,*, Sheryl A. Scott² and Mario R. Capecchi^1,3,

¹ Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA.
² Department of Neurobiology and Anatomy, University of Utah School of Medicine, 20 North 1900 East, Salt Lake City, UT 84132, USA.
³ Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112, USA.

View larger version (96K): [in this window] [in a new window]   Fig. 1. The expression pattern of Hox10 paralogs. (A) Schematic showing the columnar organization, peripheral targets and transcription factor expression of motoneurons in the thoracic and lumbar spinal cord of E13.5 mouse embryos (Kania et al., 2000; Sharma et al., 2000; Sharma et al., 1998; Thaler et al., 2004; Tsuchida et al., 1994). (B) Whole-mount in situ images showing Hoxa10, Hoxc10 and Hoxd10 expression in the spinal cord of E13.5 embryos (ventral view, rostral is toward the top). T9, thoracic segment 9; L1 and L5, lumbar segments 1 and 5. (C) Double labeling of E11 spinal cord with in situ probes for Hox10 genes and anti-Isl1, which labels all motoneurons and sensory neurons at this stage. Note that at this early stage, Hoxc10 and Hoxd10 are expressed throughout the motoneuron domain, whereas Hoxa10 is expressed only in a very focal ventral region, most likely the V3 interneuron domain (arrow). Panels showing Hoxa10, Hoxc10 and Hoxd10 are from adjacent sections of mid-lumbar cord. D, dorsal root ganglion; M, motoneuron domain. (D,E) Cross-sections through the middle of lumbar segments L2-L5 of E13.5 spinal cord, triple labeled for Hoxc10 (D) or Hoxd10 (E), Isl1 and Lim1. By this stage, Isl1 labels mMMC (arrow; compare with Lim3 staining in Fig. 4B) and mLMC (m) motoneurons, Lim1 labels motoneurons in the lLMC (l), and Hoxc10 and Hoxd10 are expressed broadly throughout much of the ventral two-thirds of the spinal cord. Although the overall expression patterns of Hoxc10 and Hoxd10 are similar, they appear to be in different subpopulations of motoneurons. Both genes, however, are expressed more strongly in lLMC and mMMC than in mLMC. In situ images were pseudocolored blue on a white background in the left columns, and blue on a black background in the merged images. Images in D and E are from adjacent sections. Scale bars: 100 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 2. Generation of Hoxc10 and Hoxd10 knockout alleles. (A) Schematic showing the wild-type (WT) genomic loci for Hoxc10 and Hoxd10, as well as targeted alleles for Hoxc10 and Hoxd10. (B) Southern blot analysis of DNA from ES cell clones for Hoxc10RFP and Hoxd10hrGFP. For Hoxc10RFP, ScaI digestion and a 3' flanking probe were used. The WT band is 11.5 kb and the correctly targeted band is 5.8 kb. For Hoxd10hrGFP, EcoRI digestion and a 3' flanking probe were used. The corresponding WT band is 12.4 kb, and the mutant band is 9.6 kb. (C) A typical hindlimb posture for Hoxc10-/-/Hoxd10-/- mutants and WT control.

View larger version (54K): [in this window] [in a new window]   Fig. 3. Defects in muscle development, innervation and axon projections in Hoxc10-/-/Hoxd10-/- double-mutant embryos. (A) Cross-sections through mid-thigh and mid-shank of embryos stained for muscle (MyoD) and nerve (neurofilament 165). Two muscles, the anterior head of the biceps in the thigh and the extensor hallucis longus in the shank (arrows) are missing in this Hoxc10-/-/Hoxd10-/- mutant embryo. In the shank of these mutants, innervation is completely lost from muscles of the anterior group (A) and greatly reduced in the lateral group (L). (B) Whole-mount neurofilament staining of axons in the hindlimb (lateral view, rostral is to the left). Top panels show the contribution of spinal segments to the lumbar (L) and sacral (S) plexii. In control embryos, segments L1-L3 contribute to the lumbar plexus and L3-L5 to the sacral plexus. In double-mutant embryos, axons in the two plexii were derived from more-caudal segments; L3 and L4 contributed to the lumbar plexus and L4 and L5 to the sacral plexus. Bottom panels are higher magnification images showing that the dorsal peroneal nerve (p) of the sacral plexus is missing in Hoxc10-/-/Hoxd10-/- mutant embryos, whereas the ventral tibial nerve (t) is still present. Arrowhead indicates a cutaneous nerve. (C) Cross-sections of lumbar spinal cord and hindlimb, through the sacral plexus, stained for β-III-tubulin. In control embryos, both the dorsal (peroneal, p) and ventral (tibial, t) branches were present, whereas in Hoxc10-/-/Hoxd10-/- mutants only the ventral branch was detected. Scale bars: 200 µm.

View larger version (112K): [in this window] [in a new window]   Fig. 4. Motoneurons in segments L1 and L2 assume identities of thoracic motoneurons in Hoxc10-/-/Hoxd10-/- double-mutant embryos. (A) Whole-mount ventral view of DiI-labeled axons from lumbar segments L1 and L2. In the control embryo, axons from segment L2 project to the limb (arrows), but in both double-mutant embryos these axons project to the body wall. Inset shows boxed area with axon terminations of L2 at higher magnification. (B) Cross-sections through ventral spinal cord segments T13-L2 of E13.5 control and Hoxc10-/-/Hoxd10-/- mutant embryos stained with anti-Lim3, which labels mMMC neurons, and with anti-Isl1, which labels the entire MMC and mLMC neurons. In control embryos, the pattern of labeled motoneurons differs significantly between thoracic and lumbar segments, whereas in Hoxc10-/-/Hoxd10-/- mutants the thoracic pattern extends into lumbar cord. Dashed lines encircle the MMC. Arrowhead, mLMC neurons. (C) Cross-sections through ventral spinal cord segments T13-L2 of E13.5 control and Hoxc10-/-/Hoxd10-/- mutant embryos stained with anti-nNOS (arrow) to label the PGC motor column in the intermediolateral spinal cord and with anti-Isl1 to label MMC and mLMC (arrowhead) motor columns in the ventral horn, as well as PGC motoneurons. In control embryos, nNOS is expressed in thoracic segments, with expression extending only to rostral L1. In Hoxc10-/-/Hoxd10-/- mutant embryos, however, nNOS expression extended beyond segment L2 (arrow). (D) Cross-section through lumbar segment L2 of Hoxc10-/-/Hoxd10-/- mutant embryo stained with anti-nNOS and anti-Isl1. Note that nNOS+ axons project to the sympathetic ganglia. Arrows point to axonal projection path. Arrowhead indicates sympathetic ganglion (SG). Scale bars: 100 µm for B-D.

View larger version (92K): [in this window] [in a new window]   Fig. 5. Expression patterns of Raldh2, Pea3 and ER81 are altered in Hoxc10-/-/Hoxd10-/- double-mutant embryos. (A-C') In situ hybridization of Raldh2, Pea3 and ER81 in (A-C) whole-mount spinal cord (ventral view, rostral is to the top) and (A'-C') cross-sections through ventral spinal cord (outlined in black), as indicated in the drawing; lateral is to the left in each panel. D, dorsal root ganglion, outlined in white. Note that in Hoxc10-/-/Hoxd10-/- mutant embryos, Raldh2 and Pea3 expression was restricted to segments L3-L5, being absent from segments L1 and L2. By contrast, ER81 expression extends from thoracic segments into segments L1 and L2, but is lost from segments L3 and L4 in mutants. Scale bar: 100 µm for A'-C'.

View larger version (107K): [in this window] [in a new window]   Fig. 6. The lLMC is missing in Hoxc10-/-/Hoxd10-/- double-mutant embryos, leading to defects in axon projection and inappropriate innervation. (A) Cross-sections of ventral spinal cord segments L1-L4 of control and double-mutant embryos stained for Isl1 to identify MMC and mLMC neurons and for Lim1 to identify lLMC neurons (dashed outlines). Note that Lim1+ lLMC neurons are nearly eliminated in double-mutant embryos. (B) The number of Isl1+ motoneurons counted unilaterally from L3 to L5 in control and Hoxc10-/-/Hoxd10-/- mutant embryos (n=7 for each genotype). There was no significant difference between control and mutant embryos at E13.0-13.5; P>0.2 for each segment, t-test. (C) Cross-sections through lumbar segment L3 stained for Hb9 for motoneurons. Note that the number of Hb9+ cells was markedly reduced in Hoxc10-/-/Hoxd10-/- mutants. (D) DiI injected into spinal cord segments L3 and L4 labeled axons in the hindlimb. In control embryos, both the tibial (ventral view, arrow) and peroneal (dorsal, arrowhead) nerves were labeled. In double-mutant embryos, the ventral tibial nerve was present, but the peroneal nerve was missing. Some cutaneous axons were also labeled in dorsal views. (E) Cross-sections through ventral spinal cord segment L2 of control, and segment L3 of Hoxc10-/-/Hoxd10-/- mutant embryos. In control embryos, tetramethylrhodamine dextran injected into the quadriceps muscle retrogradely labeled a subgroup of Lim1+ lLMC neurons (arrow, yellow neurons) in L2, but did not label Isl1+ mLMC neurons. By contrast, in double-mutant embryos, injection of dextran into the quadriceps retrogradely labeled Isl1+ mLMC neurons in L3 (arrow). Scale bars: 100 µm in A,C; 50 µm in E.

View larger version (34K): [in this window] [in a new window]   Fig. 7. Motoneurons are generated normally in Hoxc10-/-/Hoxd10-/- double-mutant embryos. Cross-sections of lumbar spinal cord of control and double-mutant embryos stained for (A) Olig2, a marker for motoneuron progenitors, and (B) Isl1 to label newly generated postmitotic motoneurons. There were no apparent differences in the numbers of Olig2+ motoneuron progenitors or Isl1+ motoneurons between control and double-mutant embryos. D, dorsal root ganglion; M, motoneurons. Scale bars: 100 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 8. lLMC neurons are generated normally, but fail to migrate laterally, and are scattered throughout ventral horn in Hoxc10-/-/Hoxd10-/- double-mutant embryos. (A) Cross-sections of the ventral spinal cord of Hoxc10+/-/Hoxd10+/- and Hoxc10-/-/Hoxd10-/- embryos processed with an hrGFP in situ probe. In control embryos, hrGFP expression closely resembles Hoxd10 expression (see Fig. 1E). In Hoxc10-/-/Hoxd10-/- mutant embryos, however, hrGFP+ cells are scattered throughout the ventral horn. Dashed white lines outline the LMC; dashed red lines outline the region of the LMC that contains hrGFP+ cells. (B) To examine the fate of late-born motoneurons, timed pregnant animals were injected with BrdU at E10.5 and analyzed at E12.0. Cross-sections of the ventral spinal cord were labeled with anti-BrdU and either anti-Isl1 (top panels) or anti-Lim1 (bottom panels). In control embryos, most late-born BrdU+ motoneurons are laterally migrating Lim1+ lLMC neurons (arrows indicate double-labeled cells), whereas in Hoxc10-/-/Hoxd10-/- double-mutant embryos, most late-born BrdU+ motoneurons expressed neither Isl1 nor Lim1 and were scattered in ventral horn, intermixed with earlier-born Isl1+ motoneurons. Scale bars: 100 µm.

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