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First published online 6 October 2004
doi: 10.1242/dev.01379

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Signaling through BMP type 1 receptors is required for development of interneuron cell types in the dorsal spinal cord

¹ Mammalian Neurogenetics Group, Center for Childhood Communication, 712 Abramsom Research Center, The Children's Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, PA 19104, USA
² Neuroscience Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
³ Department of Otorhinolaryngology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
⁴ Laboratory of Reproductive and Developmental Toxicology, National Institutes of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
⁵ Department of Orthopaedic Surgery, Department of Molecular, Cellular and Developmental Biology and Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA

View larger version (20K): [in a new window]   Fig. 1. Mating scheme to generate BMP type 1 receptor (Bmpr) double knockouts. (A) Parental genotypes required to generate mutant embryos with a BMP type 1 receptor double knockout. Parent 1 expresses the Bcre32 transgene and is heterozygous for the Bmpr1b knockout allele (Bmpr1bKO). Bmpr1aKO is a Bmpr1a receptor null allele produced by classical knockout technology (Mishina et al., 1995). Parent 2 is homozygous for the floxed Bmpr1a allele (Bmpr1aflox) and heterozygous at the Bmpr1b locus. (B) Four classes of embryos are generated by the parents in A. The genotypes of animals is depicted below the names of each classes, and refer to the genetic composition of the neural tube. Normal embryos have at least one functional allele of Bmpr1a and Bmpr1b genes in all tissues and do not show any phenotype. The top row of the table depicts the status of the Bmpr1a gene in each class. The middle row depicts the status of the Bmpr1b gene. The bottom row depicts the expected Mendelian ratios of each phenotypes.

View larger version (56K): [in a new window]   Fig. 2. Spatial and temporal expression of Bcre-32 using the ROSA reporter demonstrates Cre-mediated recombination in the neural tube. (A) lacZ expression at 8.5 dpc in the anlage of the diencephalon and mesencephalon. (B) By 9.75 dpc, Bcre32-mediated recombination has targeted the rostral neural tube, including the developing brain and the rostral spinal cord, but largely excluding the telencephalon. (C) lacZ expression throughout the neural tube by 10.5 dpc indicating widespread Bcre32-mediated recombination, except in the dorsomedial telencephalon and regions of the ventral forebrain (detail not shown). (D) At 9.75 dpc, lacZ expression demonstrates Bcre32-mediated recombination throughout the neural ectoderm.

View larger version (79K): [in a new window]   Fig. 3. Loss of BMP signaling in dorsal neural tube of Bmpr double knockout mice. (A) Immunostaining for phoshorylated-SMAD1 (phospho-SMAD1) in a normal embryo at 10.0 dpc. Immunoreactive cells are found in the roof plate and the adjacent dorsal neural tube (arrowhead). (B) Phospho-SMAD1 immunostaining in double mutant embryos demonstrates loss of immunoreactivity in the dorsal neural tube (arrowhead). A few phospho-SMAD1 positive cells remain in the roof plate (arrow). (C) Msx2 immunostaining in the dorsal neural tube of a 10.0 dpc normal embryo. (D) Msx2 immunostaining is intact at 10.0 dpc in Bmpr double knockout embryos. (E) Msx2 expression in the roof plate (arrowhead) and epidermal ectoderm of normal animals. (F) Msx2 expression is lost in the roof plate of mutant animals (arrowhead), although expression in the epidermal ectoderm is intact (arrow). (G) Bmp6 is expressed in the roof plate and immediately adjacent tissue at 10.5 dpc in normal embryos. (H) Bmp6 expression is intact in the Bmpr double knockout animals.

View larger version (147K): [in a new window]   Fig. 4. Expression of bHLH factors shows loss of Math1 expression, and a dorsal shift of Ngn2 and Mash1 expression in Bmpr double knockout animals. (A) Math1 is expressed in the ventricular zone of the dorsal neural tube (arrow) in normal animals at 10.5 dpc. (B) Math1 expression is lost in Bmpr double knockout animals. (C) Ngn2 expression in a subset of neural progenitors (arrow) is found ventral to Math1 expression in normal animals and (D) is intact but dorsally shifted (arrowhead) in Bmpr double knockout animals at 11.0 dpc. (E) In normal animals, Mash1 expression marks the remainder of the dorsal ventricular zone (arrow) at 11.0 dpc. (F) In mutant animals, Mash1 expression is dorsally shifted (arrowhead).

View larger version (127K): [in a new window]   Fig. 5. Loss of DI1 interneurons in Bmpr double knockout animals. (A) At 10.5 dpc, Lhx2 is expressed by the dorsalmost, DI1A, population of sensory interneurons, arising adjacent to the roof plate in normal animals. (B) This population is absent in Bmpr double mutant animals, as shown by loss of Lhx2 expression. (C) Lhx9 marks the DI1B population of sensory interneurons in the dorsalmost neural tube at 10.5 dpc. (D) This population is completely absent in the Bmpr double knockouts, as shown by loss of Lhx9 expression. (E,F) TUNEL staining (red) at 10.5 dpc, shows no difference in TUNEL-positive cells in the dorsal neural tube of normal (E) and Bmpr double knockout (F) animals.

View larger version (114K): [in a new window]   Fig. 6. Loss of DI2 interneurons in Bmpr double knockout animals. (A,B,E,F) Foxd3 expression at 11.0 dpc. (A) Foxd3 marks dorsal DI2 neurons (arrow), and ventral V1 neurons in normal animals. (B) Higher magnification of DI2 cells from A (arrowhead). (E) Dorsal Foxd3 expression is reduced in Bmpr double knockouts with a few cells found adjacent to the roof plate (arrow). Ventral expression is unaffected. (F) Magnification of remaining DI2 cells (arrowhead). (C) Lim1/2-positive, Pax2-negative cells of the dorsal neural tube indicate the DI2 (green) population. (D) These cells are markedly reduced in the mutant animals, and are found adjacent to the roof plate (arrow). Lim1/2-positive, Pax2-positive, DI4 (yellow) cells are dorsally expanded. (G,H) Sections from 10.5 dpc mouse embryo labeled with antibodies against Isl1 (green) and Pax2 (red). (G) Double immunostaining demonstrates that Isl1-positive DI3 cells (green) and Pax2-positive DI4 cells (red) are intermingled but represent separate populations. (H) In mutant animals, both of these populations are dorsally expanded, and are found adjacent to the roof plate (arrowhead). (I) Double knockout animals show a complete loss of DI1 neurons and a fourfold decrease in DI2 cells (P<0.001). DI3 and DI4 cells are increased (P<0.01). Single knockout animals do not demonstrate significant differences (data not shown). (J,K) Sections from 11.5 dpc mouse embryo labeled with antibody against phosphorylated histone H3 (phosphoH3, red) and DAPI (blue), demonstrating no change in cell proliferation in mutant animals (K).

View larger version (108K): [in a new window]   Fig. 7. Mid-dorsal and ventral populations of neurons are not affected in Bmpr double knockout animals. (A,B) Lmx1b expression labels the DI5 neurons of the dorsal spinal cord at 11.5 dpc normal (A) and mutant animals (B). (C) In normal animals, Lim1/2 is expressed by three populations of dorsal sensory neurons, DI2, DI4 and DI6, and by two populations of ventral neurons, V0 and V1. (D) In double mutant animals, Lim1/2 expression is unaffected in the DI6, V0 and V1 populations, but the number of DI2 cells is greatly reduced (arrow). (E,F) Sections from 11.5 dpc spinal cord, labeled with antibodies against Isl1/2 (green) and Pax2 (red). (E) In the neural tube, Isl1/2 marks dorsal DI3 cells and ventral motoneurons (MN). Pax2-positive cell groups include DI4, DI6, V0 and V1. (F) In double knockout animals, DI3 and DI4 cells are shifted dorsally toward the roof plate (*), but are otherwise normal. Other populations marked by Isl1 and Pax2 are unaffected. (G,H) Sections from 11.5 dpc spinal cord, showing expression of En1 in normal (G) and double knockout (H) animals, indicating V1 populations are unaffected.

View larger version (83K): [in a new window]   Fig. 8. Wnt1 and Wnt3a expression are decreased in Bmpr double knockout animals. (A) In normal animals, Wnt1 is expressed in the roof plate and adjacent neural ectoderm at 10.5 dpc. (B) In double mutant animals, Wnt1 expression is restricted to the roof plate. (C) In normal animals, Wnt3a is expressed in the roof plate and adjacent neural ectoderm. (D) Wnt3a in double mutants is not expressed in the neural ectoderm, but roof plate expression appears unaffected at 10.5 dpc.

View larger version (74K): [in a new window]   Fig. 9. Id gene expression is decreased in Bmpr double knockout animals. (A) Id1 expression in 10.0 dpc spinal cord in the roof plate and adjacent neural ectoderm of normal animals. There is also a region of ventral expression (arrowhead). (B) In double knockout animals, Id1 expression is greatly reduced in the dorsal neural tube (arrow). The ventral domain of expression (arrowhead) is unaffected. (C) In normal animals, Id2 is expressed diffusely throughout the dorsal half of the neural tube at 10.0 dpc. (D) Expression of Id2 appears unaffected at 10.0 dpc in mutant animals. (E) The roof plate and dorsal neural ectoderm express Id3 in normal animals. (F) In mutant animals, expression of Id3 in the neural ectoderm is diminished (arrow) at 10.0 dpc. (G) Id3 expression at 10.5 dpc in normal embryos. (H) By 10.5 dpc, Id3 expression is abolished in double mutant embryos.

View larger version (25K): [in a new window]   Fig. 10. Summary of the phenotype of dorsal neural tube development in Bmpr1a;Bmpr1b double knockout animals. (A) On the left, the normal distribution of dorsal interneurons, DI1-DI6, are schematically illustrated. Ventral subtypes, V0-V3 and MN are shown in grey. The neuron populations are characterized by expression of LIM-homeodomain factors, shown on the right. The ventricular zone of the developing spinal cord is subdivided by the expression domains of the bHLH factors, as shown. (B) In the Bmpr double knockout animals, the DI1 population is absent, as shown by the loss of Lhx2 and Lhx9. Furthermore, Math1 expression in the ventricular zone is also lost. The other bHLH domains are intact but shifted dorsally. Only a small number of DI2 cells remain with decreased expression of Foxd3 and Lim1/2. The DI3 and DI4 populations are shifted dorsally as well.