DEV009225 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 - Fig. S1. Defects in muscle patterning and innervation were observed in Hoxc10<^b>⁻<^/b>^/<^b>⁻<^/b>/Hoxd10<^b>⁻<^/b>^/<^b>⁻ double mutants but not in Hoxc10<^b>⁻<^/b>^/<^b>⁻ or Hoxd10<^b>⁻<^/b>^/<^b>⁻ single mutants. Cross sections through mid-thigh and mid-shank of embryos stained for muscle (myosin) and nerve (β-III tubulin). In the Hoxc10^−/−/Hoxd10^−/− mutant embryo shown, all thigh muscles were present, but the extensor hallucis longus in the anterior group (A) and two muscles in the lateral group (L) (arrows) were missing from the shank. The muscles that were lost in double mutants were smaller than normal in Hoxc10^−/− or Hoxd10^−/− single-mutant embryos. However, serial sections through the limb revealed that all muscles were present in both the thigh and shank of single mutants. Furthermore, innervation was completely lost from the anterior and lateral groups of shank muscles in this double mutant, whereas muscle innervation was apparently normal in Hoxc10^−/− and Hoxd10^−/− single mutants. Scale bar: 200 µm.

• Supplemental Figure S2 -

  Fig. S2. Cross sections through the middle of the thoracic (T) and lumbar (L) spinal cord segments, showing Hoxd9 expression in control and Hoxc10^−/−/Hoxd10^−/− double-mutant embryos. Hoxd9 transcripts are strongly expressed in thoracic and the four most-rostral lumbar segments in control embryos, and this is unchanged in double-mutant embryos at this stage. Cartoon illustrates the region of spinal cord shown in each panel. Scale bar: 100 µm.

• Supplemental Figure S3 -

  Fig. S3. Cross sections through the middle of lumbar spinal cord segments L2-L5, triple-labeled for Hoxd11, Isl1 and Lim1. (A) In control embryos Hoxd11 transcripts are expressed in L3 and more-caudal segments but not in L2 or more-rostral segments. No obvious change in Hoxd11 expression was detected in Hoxc10^−/−/Hoxd10^−/− double-mutant embryos (B) as compared with control embryos . Scale bar: 100 µm.

• Supplemental Figure S4 -

  Fig. S4. Cross sections of spinal cord segments L2-L4 from Hoxc10<^b>⁻<^/b>^/<^b>⁻ or Hoxd10<^b>⁻<^/b>^/<^b>⁻ single-mutant embryos stained for Isl1 to detect MMC and mLMC neurons, and for Lim1 to detect lLMC neurons. The number and location of Lim1⁺ lLMC neurons (enclosed by dotted line) were normal in segments L2-L4 in single mutants. Scale bar: 100 µm.

• Supplemental Figure S5 -

  Fig. S5. Cross sections through the middle of lumbar segments L2-L4 of E11.5 and E12.5 spinal cord of control and Hoxc10^−/−/Hoxd10^−/− double-mutant embryos stained for Isl1 to detect MMC and mLMC neurons and for Lim1 to detect lLMC neurons. (A) E11.5, (B) E12.5. Note that Lim1⁺ lLMC neurons were missing from these early stages in Hoxc10^−/−/Hoxd10^−/− mutant embryos. Scale bar: 100 µm.

• Supplemental Figure S6 -

  Fig. S6. TUNEL and Hb9 staining in cross sections through the middle of lumbar segments L1-L4 of E13.5 spinal cord from control and Hoxc10^−/−/Hoxd10^−/− double-mutant embryos. Note that the number of TUNEL⁺ cells is not increased in the motoneuron area in Hoxc10^−/−/Hoxd10^−/− mutant embryos. Scale bar: 100 µm.