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First published online November 21, 2006
doi: 10.1242/10.1242/dev.02694

Development 133, 4945-4955 (2006)
Published by The Company of Biologists 2006
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T-Box transcription factor Tbx20 regulates a genetic program for cranial motor neuron cell body migration

Mi-Ryoung Song1, Ryuichi Shirasaki1,*, Chen-Leng Cai2, Esmeralda C. Ruiz1,{dagger}, Sylvia M. Evans2, Soo-Kyung Lee1,{ddagger} and Samuel L. Pfaff1,§

1 Gene Expression Laboratory, The Salk Institute, La Jolla, CA 92037, USA.
2 Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.


Figure 1
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  		Fig. 1. Tbx20 is expressed by branchiomotor and visceromotor neurons. (A-D) In situ hybridization analysis of Tbx20 expression in flat-mounted hindbrains between E10.5 and E13.5. Tbx20 is expressed in ventral columns of motor neurons flanking the midline at E10.5 (A). Trigeminal neurons are labeled at r2 (black arrowhead) and facial neurons at r4 (white arrowhead). (E-H) Isl1 (red) and Phox2b (green) co-expression (yellow) defines the location of BM and VM neurons in flat-mounted hindbrains. The expression of Tbx20 mirrors the pattern of Isl1 and Phox2b double-labeling. (I-L) Summary depicting the cell body migration patterns and transcription factor profiles of hindbrain motor neurons. At present, it is unclear whether all or only a subset of vestibuloacoustic neurons (blue) cross the midline. (M-P) Immunohistochemistry of E11.5 hindbrain transverse sections. Facial neurons co-express Isl1, Tbx20 and Phox2b. Vestibuloacoustic neurons (white arrowhead) express Gata2 and Isl1. (P) Abducens (VI) SM neurons at r5 lack Tbx20; the dashed line indicates the border between facial (VII) and abducens (VI) neurons. Scale bars: in H, 500 µm for A-H; in P, 50 µm for M-P.

 

Figure 2
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  		Fig. 2. Facial motor neuron specification is normal in Tbx20 conditional mutants. Gene expression profile analysis in transverse sections at r4 (A-N) or flat-mounted hindbrains (O-R). (A,B) Tbx20 cKO mutant mice lack Tbx20 protein in the neural tube. (C,D) Tbx20-null cells are viable and detected by their expression of the non-functional Tbx20 transcript. (E-N) Hoxb1, Math3, Mash1, Isl1, Nkx6.1 and Phox2a are expressed normally in Tbx20 mutants. (O,P) Gata2 expression is normal in Tbx20 mutants, although medial GATA2 labeling in r4 is reduced (dashed line marks the r4/r5 border). (Q,R) Phox2b expression (bracketed) is confined to r4 in Tbx20 mutants, whereas controls exhibit labeling in r4 and r5. The altered Phox2b labeling pattern indicates a cell migration defect. Data are representative of at least twelve sections from at least three embryos of each genotype. Scale bars: in B, 40 µm for A,B; in L, 80 µm for C-L; in N, 160 µm for M,N; in R, 300 µm for O-R.

 

Figure 3
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  		Fig. 3. BM/VM cells lacking Tbx20 do not convert their fate into somatic motor neurons. (A,B) The SM neuron marker Hb9 is expressed normally in E11.5 flat-mounted hindbrains from Tbx20 cKOs. (C,D) Isl1 (red) and Phox2b (green) expression in r8 transverse sections from E10.5 Tbx20 mutants and controls. Hypoglossal (XII) SM cells express Isl1 (red) whereas spinal accessory (XI) BM neurons co-express Isl1 and Phox2b (yellow). (E) Quantification of the number of SM neurons (Hb9+ or Isl1+/Phox2b-) in Tbx20 mutants and controls at r5 (E11.5) and r8 (E10.5). Each bar represents the average of at least eight sections collected from three different embryos; mean±s.e.m. Scale bars: in B, 500 µm for A,B; in D, 30 µm for C,D.

 

Figure 4
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  		Fig. 4. Tangential, mediolateral and trans-median defects in cell body migration occur in Tbx20 mutants. (A,B) Isl1 (green) and Hb9 (red) expression in flat-mounted hindbrains from E11.5 Tbx20 mutant and control. Trigeminal neurons (Isl1+/Hb9-) at r2 fail to migrate laterally and radially in Tbx20 mutants. Facial BM neurons (Isl1+/Hb9-) at r4 do not migrate caudally past the Isl1+/Hb9+ abducens cells (yellow, r5) in Tbx20 mutants. (C-H) Comparison of Isl1 (green) and Phox2b (red) expression in transverse sections between E11.5 Tbx20 mutants and controls (in r2, r4 and r6). Dashed line in C,D indicates the border between medial (m) and lateral (l) regions of the hindbrain used for quantification. (I) Quantification of mediolateral (M-L) cell movement determined by measuring the ratio of BM neurons (Isl1+/Phox2b+) located in the medial (m) versus lateral (l) region. Cell counts were performed on transverse sections taken at r2 levels containing trigeminal neurons at E11.5 in Tbx20 mutants and controls. Each bar represents the average of at least six sections from three different embryos; mean±s.e.m. (J) Quantification of rostrocaudal (R-C) migration of facial neurons marked by Isl1/Phox2b double labeling (yellow). Cell counts were taken from transverse sections at r4 and r6 in Tbx20 mutants and controls. Each bar represents the average of at least eight sections from three different embryos; mean±s.e.m. (K,L) The SE1::gfp transgenic reporter was crossed into the Tbx20 mutant background to reveal vestibuloacoustic (VIII) motor neuron cell bodies and axons. In E11.5 control embryos, GFP-labeled cells and axons cross the r4 midline (bracket). In Tbx20 mutants, midline crossing fails to occur. (M,N) Summary diagram comparing the cell body movements of trigeminal (V), vestibuloacoustic (VIII) and facial (VII) motor neuron migration in Tbx20 mutants and controls. Images shown are representative of four or more embryos of each genotype. Scale bars: in B, 400 µm for A,B; in H, 80 µm for C-H; in L, 160 µm for K,L.

 

Figure 5
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  		Fig. 5. BM and VM neurons in Tbx20 mutants display axon guidance defects. (A-H) Axonal projections in flat-mounted hindbrains of SE1::gfp; Tbx20 mutants and controls. GFP marks BM/VM neurons in SE1::gfp transgenic mice (see Fig. S3 in the supplementary material). Axonal exit points of BM/VM neurons (white arrowheads) in hindbrains from Tbx20 mutants are normal. In Tbx20 mutants, the cell bodies of trigeminal neurons (green arrowheads in E-G) partially migrate dorsolaterally toward the exit point (white arrowheads), whereas those of facial neurons (red arrowhead in E-G) fail to migrate. These observations are summarized in D and H. (I-N) Whole-mount neurofilament staining of axonal projections in E11.5 Tbx20 mutants and controls. (I,L) Trigeminal axons project toward their correct peripheral targets in Tbx20 mutants. (J,M) High power view of the embryos shown in I and L revealing abnormal facial axon pathfinding (VII, red arrowhead). (K,N) High power view from I and L at a different focal plane revealing defasciculation and misrouting of vagal (X) axons (red arrowheads); the dashed line indicates the projectory of vagal axons. Scale bar: in G, 500 µm for A-C, E-G.

 

Figure 6
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  		Fig. 6. Migration defects in Tbx20 mutants are not due to developmental arrest. (A-D) Flat-mounted hindbrains from E11.5 SE1::gfp mice before (A) and after (B-D) organotypic culture. Facial neurons migrate caudally after 30 hours in vitro (compare white brackets in A and B). (E-H) Isl1 expression in flat-mounted hindbrain explants from E11.5 Tbx20 mutants or controls. Facial neurons from Tbx20 mutants do not migrate, in contrast to the controls. Asterisk in F marks abducens SM neurons at r5. Trigeminal neurons from Tbx20 mutants fail to migrate laterally; compare medial (yellow arrowhead) and lateral (red arrowhead) Isl1+ cells in Tbx20 mutants and controls. Scale bars: in F, 250 µm for A-F; in H, 320 µm for G,H.

 

Figure 7
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  		Fig. 7. A genetic interaction between Tbx20 and Tbx2. (A-F) In situ hybridization analysis on flat-mounted hindbrain preparations and transverse sections reveals that Tbx2 expression is absent in Tbx20 mutants. The {Delta}Tbx20 probe detects both full-length and mutated transcripts. Adjacent sections were used to locate Tbx20-null cells. (G-I) Mis-expression of Phox2a induces Isl1, Tbx20 and Tbx2 expression in chick embryonic spinal cord at HH stage 20. Scale bars: in D, 400 µm for A,D; in F, 250 µm for B,C,E,F; in I, 100 µm for G-I.

 

Figure 8
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  		Fig. 8. PCP pathways are impaired in Tbx20 mutants. (A-X) Ret, Fzd7, Wnt11, Vang1, Vang2, Pk1, Dsh3 and Celsr3 expression in flat-mounted hindbrains or transverse sections at r4 from E11.5 Tbx20 mutants or littermate controls. To localize facial cells at r4, {Delta}Tbx20 and Hoxb1 probes were used on adjacent sections (C,F,I,L, and see Fig. S6 in the supplementary material). (A-F) Ret is ectopically expressed at r4 in Tbx20 mutants. (G-R) Fzd7, Wnt11, Vang1 and Vang2 are absent at r4 in Tbx20 mutants, in constrast to controls. (S-X) Pk1 expression is reduced in Tbx20 mutants whereas Dsh3 and Celsr1 are unchanged. The boundaries of r4 are indicated by the white dashed lines; the blue dashed line encircles facial cells. (Y) Summary of genes examined in facial neurons from controls and Tbx20 mutants. Each bar represents the expression of an individual gene from r4 to r6. Striped bars represent genes expressed by facial neuron progenitors residing at r4. Genes expressed at similar stages are aligned next to each other. (Z) Summary of the genetic cascade required (but not necessarily sufficient) for facial motor neuron development and migration based on data presented here and elsewhere (reviewed by Chandrasekhar, 2004Go). It is unknown whether Tbx2 is required to activate PCP gene expression. Scale bars: in J, 400 µm for A,D,G,J; in F, 250 µm for B,C,E,F; in X, 110 µm for H,I,K-X.

 

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© The Company of Biologists Ltd 2006