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First published online February 18, 2004
doi: 10.1242/10.1242/dev.00994

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Distinct activities of Msx1 and Msx3 in dorsal neural tube development

Center for Basic Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA

View larger version (106K): [in a new window]   Fig. 1. Early expression of Bmp receptors and Msx1 but not Msx3 repress neuronal differentiation. (A-F) Immunofluorescence labeling with the antibody Tuj1 in embryos electroporated at HH10-12 and assayed 48 hours post electroporation at HH20-22. Electroporated constructs are indicated. Broken lines indicate the midline of the neural tube. Horizontal lines indicate the width of the control (left) versus the electroporated (right) side of the neural tube. (A'-F') Immunofluorescence labeling with the Msx antibody showing the endogenous Msx expression on the control side and the ectopic expression of Msx on the electroporated side by Bmp signaling induction (A' and B') or overexpression of Msx constructs (C'-F'). (G-J) Immunofluorescence labeling of neuronal differentiation markers Lhx2/9 (red) and Lhx1/5 (green) (G,H), and Isl1 (red) and Pax2 (green) (I,J) in neural tubes electroporated with Msx1 or Msx1a. (K) Schematic showing the dorsoventral boundaries of the ventricular zone markers Msx, Cath1, chick Ngn1, Cash1, and the differentiation markers Lhx2/9, Lhx1/5, Brn3a, Isl1, Pax2 and Lmx1b. dI1-6, dorsal interneuron populations 1-6; MN, motor neuron; V0-1, ventral interneuron populations 0 and 1. In all panels left is the control side and right is the electroporated side. Scale bar: 150 µm.

View larger version (56K): [in a new window]   Fig. 3. Early expression of Bmp receptors and Msx1 induce apoptosis and decrease proliferation. TUNEL labeling (A-D) and BrdU incorporation (F-I) in embryos electroporated at HH10-12 with ca-Bmpr1a/b or Msx1 as indicated and viewed at 24 (A,B,F,G) or 48 (C,D,H,I) hpe. (E,J) Graphs showing number of TUNEL+ (E) or BrdU+ (J) cells/section (mean±s.e.m., n=16 sections from at least four embryos; ***P<0.001). hpe, hour post-electroporation. In all panels, left is the control side and right is the electroporated side. Scale bar: 150 µm.

View larger version (47K): [in a new window]   Fig. 2. Msx1 represses the expression of neural progenitor genes. Neural progenitor markers Cath1 (A,B) and Pax7 (I,J), which are detected by immunofluorescence labeling; as well as Cash1 (C,D), chick Ngn1 (E,F), chick Ngn2 (G,H), which are detected by in situ hybridization, are repressed by Msx1 (A,C,E,G,I) but not Msx1a (B,D,F,H,J). Arrows in C and E indicate the disruption of dorsoventral patterns of Cash1 and chick Ngn1 expression. In all panels, left is the control side and right is the electroporated side.

View larger version (56K): [in a new window]   Fig. 4. Neural crest marker Dlx is induced by Bmp signaling, Msx1 and Msx3. Immunofluorescence labeling showing an increase in number of migrating Dlx+ cells induced by overexpression of ca-Bmpr1 (A), Msx1 (B) and Msx3 (C). Arrows indicate migrating Dlx+ cells. ca-Bmpr1 also induces ectopic Dlx expression within the neural tube (A). (D) Quantification of Dlx+ cells/section on the control side and the electroporated side of the neural tubes shown in A-C. Data shown as mean±s.e.m., n=16 sections from at least four embryos; ***P<0.001; **P<0.005. (E-G) The expression of another neural crest marker, Slug, detected by in situ hybridization, is induced within the neural tube by ca-Bmpr1 (E), but not Msx1 (F) or Msx3 (G). DA, dorsal aorta; DRG, dorsal root ganglia; NC, notochord. In all panels, left is the control side and right is the electroporated side. Scale bars: 150 µm.

View larger version (100K): [in a new window]   Fig. 5. Roof-plate markers are induced by Bmp signaling and Msx1 but not Msx3. In situ hybridization of Bmp4 (A-E), Wnt1 (F-J) and immunofluorescence labeling of Lmx1 (K-O) on transverse sections of HH15-18 (A-C,F-H,K-M) or HH20-22 (D-E,IJ,N-O) neural tubes electroporated with ca-Bmpr1, Msx1 or Msx3 at stages indicated. Brackets indicate the size of each population on the control (left) and the electroporated (right) sides. Arrowheads indicate the midline in each panel. Insets in (A-E) show the expression of the GFP control plasmid on the electroporated side of the neural tube. (P) Quantification of Lmx+ cells/section on the control side and the electroporated side of the neural tubes shown in K-O. Data shown as mean±s.e.m., n=10 sections from at least four embryos; ***P<0.001. dI1,5, dorsal interneuron populations 1 or 5; FP, floor plate; hpe, hour post-electroporation; RP, roof plate. In all panels left is the control side and right is the electroporated side. Scale bar: 150 µm.

View larger version (57K): [in a new window]   Fig. 6. Msx3 induces neural progenitor cells to adopt a dorsal cell fate. (A-F) Immunofluorescence labeling of Cath1 in HH20-22 neural tubes. Electroporation of Msx3 in HH10-12 (A,B) or HH14-16 (D,E) embryos induces ventral ectopic expression of Cath1 at HH20-22. (B,E) Higher magnification images of the electroporated (right) side of the neural tubes in A and D showing Cath1 (red) and Msx (green) double positive cells (yellow, arrows) and Cath1+Msx- cells (red, arrowheads). Overexpression of Msx3a does not induce ectopic Cath1 expression (C,F). (G) Bar graph indicating number of Cath1+ cells/section in Msx3-versus Msx3a-electroporated embryos (late indicates electroporation at HH14-16; early indicates electroporation at HH10-12) (mean±s.e.m., n=16 sections from at least four embryos; ***P<0.001, **P<0.005). In all panels, left is the control side and right is the electroporated side. Scale bars: 150 µm in A,C,D,F; 75 µm in B,E.

View larger version (95K): [in a new window]   Fig. 7. Msx3 and late activation of Bmp signaling induce dorsal interneuron cell fates at the expense of ventral cell fates. (A-D) Lhx2/9 (red) and Lhx1/5 (green) immunofluorescence labeling shows a ventral expansion of Lhx2/9+ dI1 population in embryos overexpressing Msx3 (A,B), ca-Bmpr1a/b (D) but not Msx3a (C). (E-H) Isl1 (red) and Pax2 (green) labeling shows a ventral expansion of Isl1+ dI3 population in embryos electroporated with Msx3 (E,F), ca-Bmpr1a/b (H) but not Msx3a (G). (I-L) Mnr2 (red) and Pax2 (green) labeling shows no significant dorsal expansion of Mnr2+ motoneurons. dI1-6, dorsal interneuron populations 1-6; MN, motoneuron; V0-1, ventral interneuron populations 0 and 1. Brackets outline the size of each neuronal population indicated. Arrowheads in A,E,I indicate the dorsal midline. In all panels, left is the control side and right is the electroporated side. Scale bars: 150 µm in A,E,I; 75 µm in B-D,F-H,J-L.