QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00346

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pabst, O. Articles by Arnold, H.-H. Search for Related Content PubMed PubMed Citation Articles by Pabst, O. Articles by Arnold, H.-H.

Targeted disruption of the homeobox gene Nkx2.9 reveals a role in development of the spinal accessory nerve

Department of Cell and Molecular Biology, Institute of Biochemistry and Biotechnology, Technical University of Braunschweig, 38106 Braunschweig, Germany

View larger version (44K): [in a new window]   Fig. 1. The targeting strategy to inactivate the Nkx2.9 gene. (A) Illustrates the genomic organization of the Nkx2.9 locus (wt), the targeting vector, and the mutated allele after homologous recombination (mut). Most of the exons (red boxes), including the homeodomain contained in exon 2, were replaced by the IRES-lacZ reporter gene (small green/blue box) and the neomycin selection cassette (large green box). IRES-lacZ-coding sequence was inserted at the translation initiation codon. The transcriptional orientations of both inserted genes are indicated by arrows. The following sites for restriction endonucleases have been mapped: H, HindIII; C, ClaI; E, EcoRI; S, SacI; K, KpnI. Hybridization probes used for genotyping were obtained from sequences flanking the vector insert on both sites as indicated. (B) A typical Southern blot analysis with DNA taken from a litter produced by heterozygous parents. DNA was digested with EcoRI and hybridized with the 3' probe. Wild type and mutant alleles are represented by 7.0 (top, arrowhead) and 4.0 (bottom, arrowhead) kb restriction fragments, respectively. (C) RNA isolated from brain and muscle of wild-type, heterozygous and homozygous adult mice was analyzed using RT-PCR (top). Nkx2.9-specific transcripts were detected in brain of wild-type and heterozygous mutant mice but not in homozygous mutants. RNA from muscle contained no Nkx2.9 transcripts and served as control for PCR specificity (bottom). RNA loading was controlled by RT-PCR for the constitutively expressed ribosomal protein L7.

View larger version (63K): [in a new window]   Fig. 2. Expression of the Nkx2.9-lacZ reporter gene in heterozygous (A-E) and homozygous (F) Nkx2.9 mutants. lacZ staining of whole-mount embryos at E8.5 (A), E9.5 (B), E11.5 (C) and E12.5 (D). Insets in A and B show whole-mount in situ hybridization with Nkx2.9-specific probe. Note the slightly delayed onset of lacZ activity compared with endogenous Nkx2.9 transcription in the E8.5 embryo (A) but a comparable expression in the E9.5 embryo (B). lacZ activity continues to be present in Nkx2.9 domains of embryos at E11.5 (C) and E12.5 (D). Comparison of lacZ staining in E10.5 heterozygous (E) and homozygous (F) mutants reveals identical activity patterns except for the enlarged domain in hindbrain of homozygous Nkx2.9 mutants at the level of rhombomeres 3 and 4 (arrows in E,F). Inset in E,F shows lacZ staining on transverse sections of spinal cord at the forelimb level.

View larger version (106K): [in a new window]   Fig. 3. Dorsoventral patterning in spinal cord is not affected by the Nkx2.9 mutation. Immunohistochemistry on transverse sections of spinal cord at forelimb (A-D,G,H) and hindlimb level (E,F) demonstrates the expression of marker genes for distinct subpopulations of neuronal progenitors in E10.5 heterozygous (A,C,E,G) and homozygous (B,D,F,H) Nkx2.9 mutant embryos. Immunofluorescence staining for HNF3ß marks the floorplate (A,B), Nkx2.2 (red in C-H) and Isl1 (green in C-F) label V3 interneurons and motoneurons, respectively. Pax6 (green in G,H) labels all neuronal precursors except V3 neurons and floorplate. No major differences are seen between heterozygous and homozygous animals. Note, however, individual Isl1-positive cells within the Nkx2.2 domain of the neural tube at hindlimb level (E,F). In situ hybridization with Sim1- (I,J) and Dbx2- (K,L) specific probes on transverse sections illustrates normal production of V3 neurons and dorsoventral patterning of the V1 progenitor domain in neural tube of Nkx2.9-deficient embryos, respectively. (I-L) Heterozygous (I,K) and homozygous (J,L) mutant embryos.

View larger version (85K): [in a new window]   Fig. 4. Neurofilament staining reveals nerve defects in hindbrain of Nkx2.9 mutants. Whole-mount preparations of wild type (A,C,E,G) and homozygous Nkx2.9 mutant (B,D,F,H) embryos at E10.5 (A-D) and E11.5 (E-H) were stained with anti-neurofilament antibody. Note the considerably reduced length and thickness of the spinal accessory nerve in mutants (compare arrows in C,D and G,H). (D) Partial fusion of the N. vagus (X) with the N. glossopharyngeus (IX) were frequently observed in homozygous mutant animals (arrowhead). In addition, the axon bundle of the vagal nerve appears reduced.

View larger version (76K): [in a new window]   Fig. 5. Branchial motoneurons of the spinal accessory nerve are reduced in Nkx2.9-deficient mouse embryos. Transversal serial sections through the neural tube at C4-C3 level of E 10.5 wild-type (A,C) and homozygous mutant (B,D) embryos were stained with Isl1-specific antibody (green) together with Nkx2.2 antibody (red in A,B) or Lim3 antibody (red in C,D). Note the drastic reduction of dorsolaterally migrating Isl1-positive cells (compare arrows in A and B) and the markedly increased number of Isl1/Lim3-positive somatic motoneurons (yellow cells indicated by arrows in C,D) in the mutant. Quantification of migrating Isl1-positive and Isl1/Lim3 double-positive cells was obtained by computer-aided determination of total pixel area with color information for the respective cell types. (C',D') A typical example of at least five independent determinations on level-matched sections of embryos of both genotypes. Changes in cell numbers of approximately 60% were consistently found.

View larger version (104K): [in a new window]   Fig. 6. Phox2b-expressing progenitors of visceral and branchial motoneurons in hindbrain of E10.5 wild-type and Nkx2.9 mutant mouse embryos. Transverse sections through hindbrain at the level of rhombomere 4 (A,B,E,F) and rhombomere 7 (C,D,G,H) were immunostained with Phox2b-(A,E,C,G) and Isl1-specific antibodies (B,F,D,H). Sections of wild-type (A-D) and mutant embryos (E-H) reveal similar patterns of Phox2b expression in mitotic and postmitotic cells of the ventral and lateral domain, and in the mantel layer of the dorsal domain at r4 level. At r7 only postmitotic cells of the ventral domain and a dorsoventral stripe of presumably migratory cells express Phox2b. Isl1-positive cells are found only in postmitotic cells of the ventral domain both at r4 and r7 levels. Note that the expression of both markers appears essentially unchanged in wild-type and mutant embryos.

View larger version (96K): [in a new window]   Fig. 7. Phox2b expression in the dmnX and nucleus ambiguus (NA) of adult wild-type (A,B,F,G) and homozygous Nkx2.9 mutant (C,D,H,I) mice. Nissl-stained transverse sections through hindbrains (A,C,F,H) reveal normal morphology of the dmnX (A,C) and Nucleus ambiguus (F,H). (B,D,G,I) Parallel sections immunostained with Phox2b-specific antibody. Note the drastically reduced number of Phox2b-positive cells in the NA (G,I) but not in the dmnX (B,D). Graphs illustrate the ratio of Nissl-positive cell counts (total) and the ratio of Phox2b-positive cells (Phox2b+) from mutants versus wild-type animals in dmnX (E) and NA (J). Each column represents the mean of five slides counted from three individual animals for each genotype.