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BMP signaling patterns the dorsal and intermediate neural tube via regulation of homeobox and helix-loop-helix transcription factors

Molecular Biology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA

View larger version (61K): [in a new window]   Fig. 1. BMP signaling regulates Pax gene expression in dorsal and intermediate domains. (A) Identification of virally infected cells by immunofluorescence using an antibody to the viral gag protein. (B-E) Electroporation constructs generate different levels of BMP signaling. Electroporations into the left side of the neural tube included a GFP expression plasmid to mark transfected cells. (B,C) Low levels of activated BMPR expression generate weak BMP target gene activation. (B) Expression of activated BMPR-Ib driven by the EGFP vector is barely detectable over endogenous expression of BMPR-Ib (stage 24). An alternate section (C) shows correspondingly weak activation of Msx protein expression, a target of BMP signaling. Inset shows GFP, which labels transfected cells, indicating that the construct is present. (D,E) High levels of activated BMPR expression generate strong BMP target gene activation. (D) Expression of activated BMPR-Ib driven by pMiW-III vector results in expression readily detected above the endogenous expression pattern (stage 24 embryo, detection reaction stopped prior to clear detection of endogenous BMPR-Ib expression). This results in a correspondingly robust MSX response in alternate sections (E). (F-H) Pax7 expression is activated by BMP signaling. (F) Wild-type Pax7 expression in a stage 23 embryo. Ectopic Pax7 expression (arrowheads) is apparent in virally infected (G, stage 24) and electroporated (H, stage 24) embryos. The unusual morphology in H was caused by apoptosis (see Materials and Methods). (I-M) Expression of Pax6 is regulated by BMP signaling. The vertical bar denotes the normal region of high-level Pax6 expression in the intermediate region of the neural tube. (I) Wild-type Pax6 expression in a stage 24 sample. (J) Pax6 expression is repressed by high levels of BMP signaling (arrowheads; inset shows that Pax6 repression is limited to transfected cells). High signaling was generated by electroporation of pMiWIII-activated BMPR-Ib; sample is stage 24. (K,L) High level expression of Pax6 in intermediate cells is downregulated by moderate levels of BMP signaling. Moderate signaling was generated by electroporation of pCAGGS-activated BMPR-Ia; sample is stage 24, insets show correlation of alterations with transfected cells. (M) Ventral Pax6 expression is upregulated in response to BMP signaling (arrowhead; viral infection, stage 23).

View larger version (91K): [in a new window]   Fig. 2. BMP signaling regulates the expression of homeobox genes in the neural tube. (A-D) Expression of Msx genes is upregulated by BMP signaling. (A,B) Msx2 RNA expression. A stage 24 wild-type sample. (B) Expanded Msx2 expression following electroporation of activated BMPR-Ib (stage 23). (C,D) Msx1 RNA expression. (C) Stage 24 wild-type embryo. (D) Upregulation of Msx1 expression in an electroporated embryo expressing activated BMPR-Ib (stage 23). Expression is detected in the ventral neural tube and at higher levels within the endogenous expression domain. (E-H) Dbx protein expression is repressed by BMP signaling. (E,F) Dbx1 protein expression. (E) Stage 23 wild-type sample. (F) Cells expressing activated BMPR cease to express Dbx1 (stage 23, viral infection; arrowhead denotes region of BMP-driven repression). Inset shows viral infection in green; the absence of Dbx1 correlates with viral infection. (G,H) Dbx2 protein expression. (G) Stage 24 wild-type sample. (H) Repression of Dbx2 expression in a stage 23 sample expressing activated BMPR-Ib following viral infection. Arrowhead indicates a group of cells that no longer express Dbx2. (I-L) Differentiated cell types of the intermediate neural tube are altered in response to BMP signaling. (I,J) En1 protein expression, which marks the interneurons derived from the ventral-most Dbx2-expressing cells. (I) Wild-type expression in a stage 24 sample. (J) Arrowhead indicates that this population is greatly reduced in samples expressing activated BMPR (stage 24, electroporation). Inset shows that the absence of En1 expression correlates with extensive transfection (marked by GFP). (K,L) Evx protein expression, which marks the interneurons derived from Dbx1+, Dbx2+, Pax7– cells. (K) Wild-type expression in a stage 24 sample. (L) Arrowhead indicates that this population is greatly reduced in samples expressing activated BMPR (stage 24, electroporation). GFP fluorescence indicates extensive transfection of cells in the region that would normally express Evx.

View larger version (104K): [in a new window]   Fig. 3. Msx1 mediates the repression of Dbx2 by BMPs. (A,B) Dbx2 and Msx1 expression domains are juxtaposed. (A) Expression of Dbx2 and Msx1 in a stage 24 wild-type sample. Confocal microscopy reveals that cells do not co-express these proteins. (B) The mutually exclusive expression of these proteins is retained in samples expressing activated BMPR (viral infection, stage 24). Arrowhead indicates cells expressing Msx1 ventral to its normal expression border. Confocal microscopy reveals that these cells do not express Dbx2. (C-E) Mis-expression of Dbx2 does not affect Msx1 expression. Samples were generated by electroporation of viral DNA that drives Dbx2 expression and analyzed at stage 23. Brackets indicate a region of cells expressing Dbx2 within the normal Msx1 domain; bars represent endogenous Dbx2 expression domain. Virally infected cells express Dbx2 at levels equal to or higher than those in the endogenous domain. (C) Expression of Msx1 is unaltered by ectopic expression Dbx2. Co-expressing cells are apparent in D; E shows that many cells in the endogenous Msx1 domain are expressing Dbx2. (F,G) Mis-expression of Msx1 represses Dbx2 expression. Embryos were injected with an Msx1-expressing virus and analyzed at stage 24. Arrowhead denotes a region of ectopic Msx1 expression. (F) Gaps in the Dbx2 expression domain are apparent in the Msx1-infected samples. (G,H) These gaps correspond precisely with those cells that ectopically express Msx1.

View larger version (61K): [in a new window]   Fig. 4. Expression of activated BMPR alters the expression of bHLH proteins in the dorsal neural tube. (A,B) Cath1 expression expands ventrally in response to BMP signaling. (A) Wild-type expression of Cath1 protein occurs in cells immediately ventral to the roofplate (stage 23 sample). (B) Expression of Cath1 expands dramatically in response to expression of activated BMPR (stage 24, viral infection). Note that the expanded expression remains restricted to dorsal neural tissue. Inset shows viral antigen expression, indicating infected cells; expanded Cath1 correlates with areas of extensive viral infection. (C,D) Cash1 expression is repressed by BMP signaling. (C) Wild-type expression of Cash1 protein includes a broad band of cells in the dorsal neural tube (stage 24). (D) The Cash1 domain is reduced in samples expressing activated BMPR (stage 23 viral infection). (E,F) Dorsal Ngn2 expression is reduced by BMP signaling. (E) Wild-type expression of Ngn2 RNA is found in ventricular and subventricular cells in the dorsal half of the neural tube, as well as a large population of ventral cells (stage 24; arrowhead indicates dorsal expression domain). (F) Expression of Ngn2 in most dorsal cells is extinguished by the expression of activated BMPR (stage 24 electroporation); ventral expression remains unaffected. (G,H) Dorsal expression of Ngn1 is repressed by BMP signaling. (G) At stage 24, Ngn1 RNA is expressed by cells of the dorsal root ganglia (DRG), proliferating ventral cells and a narrow band of cells in the dorsal neural tube (arrowhead). (H) Ngn1 expression in the dorsal neural tube is absent in samples expressing activated BMPR (stage 24 electroporation), while ventral and DRG expression remains normal.

View larger version (74K): [in a new window]   Fig. 5. BMP signaling regulates the generation of terminally differentiated dorsal interneurons. (A) Ngn1 RNA expression is activated at a threshold of BMP signaling. Ngn1 is activated by low levels of BMP signaling. A construct that weakly expresses activated BMPR-Ib (EGFP vector) results in an increase in Ngn1-expressing cells, apparently by activating it in cells ventral to its normal domain (arrowhead, stage 24). (B,C) LH2A/B-expressing cells are generated throughout the dorsal neural tube in response to high levels of BMP signaling. (B) In stage 23 wild-type samples, LH2A/B-expressing cells are generated by those cells that express Cath1 and migrate ventrally. (C) In response to high level BMP signaling, LH2A/B+ cells are generated throughout the dorsal neural tube (arrowhead, stage 24). Inset shows co-localization of GFP and LH2A/B expression on the transfected side of the neural tube. (D,E) High levels of BMP signaling block the development of dorsal Lim1/2+ cells. (D) In wild-type samples, Lim1/2+ cells are produced in two locations within the dorsal neural tube (bars; stage 23). (E) Expression of activated BMPR reduces or eliminates the generation of the two dorsal populations of Lim1/2-expressing cells (arrowhead denotes normal region of their generation, stage 23). Inset shows GFP expression, indicating transfected cells. Note that suppression of Lim1/2 expression is specific to dorsal cells, as intermediate expression continues in transfected cells. (F-H) Isl1-expressing cells are formed within a narrow range of BMP signaling activity. (F) Samples with slight increases in BMP signaling (generated with the same vector as in A, EGFP-activated BMPR-Ib) generate Isl1-expressing cells over a broader region of the dorsal neural tube (bracketed; bar indicates endogenous domain, late stage 24). Inset shows GFP label of transfected cells. Note that Isl1 expansion is restricted to the region near its normal expression domain, despite extensive transfection. (G) In stage 24 wild-type samples, Isl1 expression marks a small population of dorsal interneurons (bar) as well as the DRG and motoneurons.(H) High levels of BMP signaling, however, abolish the generation of Isl1+ interneurons (arrowhead, stage24). Inset shows that the absence of Isl1 is correlated with the presence of transfected cells marked by GFP.

View larger version (18K): [in a new window]   Fig. 6. BMP signaling regulates many aspects of neural tube patterning. (A) BMP signaling regulates homeobox gene expression to define dorsal and intermediate cell fates. In cooperation with SHH signaling, BMPs set the expression domain boundaries of Pax6 and Pax7. The border between dorsal and intermediate cell fates, marked by the dorsal border of high level Pax6 expression is refined by the BMP-mediated activation of Msx1, which represses Dbx2 expression. (B) BMP signaling regulates the dorsal expression of bHLH proteins along a gradient of activity. bHLH protein expression boundaries are set by thresholds of BMP signaling. bHLH expression domains give rise to a limited number of types of terminally differentiated neurons. (C) BMP signaling promotes a diversity of intermediate cell fates. BMP regulation of Pax7 sets a dorsal limit on the generation of Evx1-expressing neurons. BMP regulation of the dorsal border of Dbx1-expressing cells may help divide the Pax2+, Lim1/2+ cells into two distinct progenitor populations.

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