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First published online March 7, 2005
doi: 10.1242/10.1242/dev.01718

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Retinaldehyde dehydrogenase 2 and Hoxc8 are required in the murine brachial spinal cord for the specification of Lim1+ motoneurons and the correct distribution of Islet1+ motoneurons

¹ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, BP 10142, 67404 Illkirch Cedex, C.U. de Strasbourg, France
² Unité d'Embryologie Moléculaire, Institut Pasteur, Bat. J. Monod, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
³ E. Kennedy Shriver Center, University of Massachusetts Medical School, University of Massachusetts, Waltham, MA 02452, USA
⁴ Departments of Medicine and Molecular and Cellular Biology, Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

View larger version (77K): [in a new window]   Fig. 8. Loss of Hoxc8 function phenocopies the molecular defects observed in Raldh2L–/– spinal cord. Expression of Raldh2 protein (A-C) and selected molecular markers (D-O) on E12.5 flat-mounted spinal cords at brachial level. All the insets represent in-situ hybridization of transverse sections at the middle posterior LMC level (level of the horizontal bars in J-O). (C) In the Hoxc8–/– posterior LMC region, the distance between the ventral midline (dotted line) and the Raldh2+ cells is increased (compare horizontal bars in A and C). (D-O) Composite images in which Raldh2 protein is shown on the left side only. (D-F) Decreased RARß expression in the posterior medial LMC domain in the Hoxc8+/– spinal cord (brackets, D,E). (F) In Hoxc8–/– mutant, RARß expression is absent throughout the LMC. (G-I) Lim1 expression is diminished in the posterior LMC of Hoxc8+/– and Hoxc8–/– mutants (brackets). (J-L) The outline of the two Islet1+ motor columns is marked by arrowheads. (L) No segregation of motoneurons into columns is visible in Hoxc8–/– spinal cord. (M-O) Horizontal bars (white, red in insets) delineate the bigger distance between the ventral midline (dotted line) and the Pea3+ cells in Hoxc8+/– and Hoxc8–/– mutants compared with wild type. (O) In Hoxc8–/– mutant, Pea3 expression is greatly reduced throughout the LMC. (P-R) Gdnf transcripts on transverse sections at thoracic level and brachial plexus level (bp; insets). a, anterior; CM, cutaneous maximus; p, posterior.

View larger version (63K): [in a new window]   Fig. 2. Adult phenotype and Raldh2 protein analysis in Raldh2L–/– mutants. (A,B) Ventral (left) and profile (right) views of the forepaws of 2-month-old mice. Note the abnormal flexure of anterior digits (bracket) and partial fusion between digits 2, 3 and 4 (arrowheads). (C,J) Immunodetection of Raldh2 on whole-mount embryos (C,D), transverse sections at the level of the brachial spinal cord (E-H) and dissected spinal cords flattened into an `open book' configuration (I,J). Arrowheads (C,D) highlight the brachial region. 2,5, digit numbers; bp, brachial plexus; me, meningeal cells; ms, mesenchymal cells.

View larger version (104K): [in a new window]   Fig. 1. Expression of Raldh2 in the murine brachial LMC. (A-C) Immunodetection of Raldh2 (green) and Islet1/2 (red) proteins on transverse spinal cord sections. (E-F) Raldh2, Islet1 and Lim1 transcripts on adjacent transverse spinal cord sections, at the posterior LMC level. The Islet1 and Lim1 subpopulations within the LMC are marked by brackets and vertical lines, respectively.

View larger version (115K): [in a new window]   Fig. 3. Altered distribution of RA-responsive cells in Raldh2L–/– brachial spinal cord. (A-F) Immunodetection of ß-Gal (red) on transverse ventral spinal cord sections (A-D) and dissected spinal cords (E-F) in RARE_hsp68_lacZ transgenic mice. (C,D) Combined immunodetection of ß-Gal (red) and Islet1/2 (green). Arrows point to ß-Gal+/Islet1/2+ cells (orange). Brackets in (F) indicate abnormal ß-Gal distribution in a broad longitudinal band within the intermediate region. (G,H) Rarb transcript distribution (red) combined to Raldh2 immunofluorescence (green) on flat-mounted spinal cords. (G) Composite image in which the overlay of the two signals is shown on the left side only. The dotted lines in (G,H) point to the thoracic MMC. (I,L) Rarb transcripts on transverse ventral spinal cord sections at two different AP levels as indicated in E,F. Arrowheads point to Rarb+ motor columns and asterisks show a deficiency of Rarb+ cells in mutants compared with controls. i, intermediate region; vh, ventral horns.

View larger version (57K): [in a new window]   Fig. 4. Abnormal distributions of Islet1+ and Lim1+ cells in Raldh2L–/– brachial spinal cord. (A) Quantitation of Islet1/2+ motoneurons in the brachial LMC of E11.5 control and Raldh2L–/– embryos (Y axis: number of motoneurons per hemisection; ct: 96.84±1.77; mut: 86.04±3; mean±s.e.m.; t-test: P=0.009; ten sections spanning the Raldh2 LMC domain were counted in nine embryos of each genotype). (B) Quantitation of Lim1/2/Islet2+ and Islet1+ motoneurons per hemisection in the brachial LMC of E11.5 control and Raldh2L–/– embryos. The number of Lim1/2/Islet2+ cells was significantly decreased in mutants (Lim1/2/Islet2: ct, 25.78±1.48; mut, 20.50±1.11; mean±s.e.m.; t-test P=0.024; ten sections were counted in five embryos of each genotype), whereas the number of Islet1+ cells was similar in mutants and controls (Islet1: ct, 80.36±2.68, mut, 76.28±2.94; mean±s.e.m.; t-test P=0.336). (C-F) Lim1 (C,D, red) and Islet1 (E,F, red) transcripts on flat-mounted spinal cords combined with Raldh2 immunofluorescence (C,E, green). (C,E) Composite images in which the overlay of the two signals is shown on the left side only. (E,F) The posterior domain of high Islet1 expression, marked by a bracket, is expanded anteriorly in the mutant. Lim1+ and Islet1+ motor columns are indicated by arrowheads. (G-I) Lim1+ (G,H) and Islet1+ (I,J) transcripts on transverse ventral spinal cord sections at levels indicated by horizontal arrows in C-F. The Islet1 and Lim1 subpopulations within the LMC are marked by brackets.

View larger version (128K): [in a new window]   Fig. 5. Alterations of specific motor pools in Raldh2L–/– brachial LMC at E12.5. (A-D) Ephb2 transcripts on flat-mounted spinal cords (A,B) and transverse sections (C,D) at levels indicated in A,B. (E-H) Epha4 transcripts on flat-mounted spinal cords (E,F) and transverse sections (G,H) at levels indicated in E,F. Brackets (E,F) indicate the extent of the Epha4 motor pools along the AP axis. The asterisk (H) points to decreased Epha4 signal in the Raldh2L–/– lateral LMC. (I-L) Pea3 transcripts on flat-mounted spinal cords (I,J) and transverse sections (K,L) at levels indicated in I,J. Horizontal bars (white in J and red in K,L) delineate the larger distance between the ventral midline (dotted lines, I,J) and the Pea3+ cells in mutants. Raldh2 immunofluorescence has been combined with in-situ hybridization on control spinal cords (A,E,I, green), the overlay of the two signals is shown on the left side only. (M,N) Gdnf transcripts on transverse sections at thoracic and brachial plexus (bp; insets) levels. (O,P) X-gal staining of RARE_hsp68_lacZ transgenic embryos. (P) The red arrowhead points to the almost complete absence of staining in the cutaneous maximus (CM) hypaxial muscle.

View larger version (64K): [in a new window]   Fig. 6. Impaired development of dorsal forelimb nerve branches in Raldh2L–/– embryos. (A-D) Combined immunodetection of Epha4 (green) and neurofilaments (red) on transverse sections of E11.5 embryos. (C,D) Epha4 signal alone. Insets show high magnifications of the extremities of the growing dorsal nerves. EphA4 is greatly diminished in the distal tip of the growing dorsal nerves in mutants (thin arrows). Epha4 level in the dorsal limb mesenchyme is not altered in the mutant (arrowheads). (E-H) Nerve patterns in the developing forelimb, using whole-mount anti-neurofilament IHC. (G,H) Details of the growing axons of the ramus profundus of the radialis nerve, viewed after removal of other nerve branches. The corresponding nerve from another, more severely affected mutant, is shown at the same magnification in an inset. nm, nerve medianus; nr: nerve radialis; nr(p), nr(s), profundus and superficialis ramii of the nerve radialis, respectively; nu, nerve ulnaris.

View larger version (115K): [in a new window]   Fig. 7. Decreased expression of Hoxc6 and Hoxc8 in Raldh2L–/– brachial LMC. (A-D) Combined Raldh2 immunofluorescence (green, left side) and Hoxc6 (A,B, red) or Hoxc8 (C,D, red) in-situ hybridization on dissected spinal cords. In Raldh2L–/– mutant, Hoxc6 expression is diminished preferentially within its central domain (A,B, brackets). (E,F) Combined Hoxc8 immunofluorescence (green) and Lim1 in-situ hybridization (red). A posterior shift of Hoxc8 protein is observed in Raldh2L–/– spinal cord, mostly within the Lim1+ motor column (red arrows). (G-L) Hoxc8 transcripts on serial transverse sections at levels indicated in C and D; 70 µm separate each section plane. The inset in L shows the following posterior section in which Hoxc8 transcript distribution in the mutant is comparable to the previous control section (compare with K). Two red arrows delineate the Lim1+ LMC domain. a, anterior; p, posterior.

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