E. Bryan Crenshaw

β-Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system

beta-Catenin is an essential component of the canonical Wnt signaling system that controls decisive steps in development. We employed here two conditional beta-catenin mutant alleles to alter beta-catenin signaling in the central nervous system of mice: one allele to ablate beta-catenin and the second allele to express a constitutively active beta-catenin. The tissue mass of the spinal cord and brain is reduced after ablation of beta-catenin, and the neuronal precursor population is not maintained. In contrast, the spinal cord and brain of mice that express activated beta-catenin is much enlarged in mass, and the neuronal precursor population is increased in size. beta-Catenin signals are thus essential for the maintenance of proliferation of neuronal progenitors, controlling the size of the progenitor pool, and impinging on the decision of neuronal progenitors to proliferate or to differentiate.

Epithelial Bmpr1a regulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development

Bone morphogenetic protein (BMP) signaling is thought to perform multiple functions in the regulation of skin appendage morphogenesis and the postnatal growth of hair follicles. However, definitive genetic evidence for these roles has been lacking. Here, we show that Cre-mediated mutation of the gene encoding BMP receptor 1A in the surface epithelium and its derivatives causes arrest of tooth morphogenesis and lack of external hair. The hair shaft and hair follicle inner root sheath (IRS) fail to differentiate, and expression of the known transcriptional regulators of follicular differentiation Msx1, Msx2, Foxn1 and Gata3 is markedly downregulated or absent in mutant follicles. Lef1 expression is maintained, but nuclearβ -catenin is absent from the epithelium of severely affected mutant follicles, indicating that activation of the WNT pathway lies downstream of BMPR1A signaling in postnatal follicles. Mutant hair follicles fail to undergo programmed regression, and instead continue to proliferate, producing follicular cysts and matricomas. These results provide definitive genetic evidence that epithelial Bmpr1a is required for completion of tooth morphogenesis, and regulates terminal differentiation and proliferation in postnatal hair follicles.

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

During spinal cord development, distinct classes of interneurons arise at stereotypical locations along the dorsoventral axis. In this paper, we demonstrate that signaling through bone morphogenetic protein (BMP) type 1 receptors is required for the formation of two populations of commissural neurons, DI1 and DI2, that arise within the dorsal neural tube. We have generated a double knockout of both BMP type 1 receptors, Bmpr1a and Bmpr1b, in the neural tube. These double knockout mice demonstrate a complete loss of D1 progenitor cells, as evidenced by loss of Math1 expression, and the subsequent failure to form differentiated DI1 interneurons. Furthermore, the DI2 interneuron population is profoundly reduced. The loss of these populations of cells results in a dorsal shift of the dorsal cell populations, DI3 and DI4. Other dorsal interneuron populations, DI5 and DI6, and ventral neurons appear unaffected by the loss of BMP signaling. The Bmpr double knockout animals demonstrate a reduction in the expression of Wnt and Id family members, suggesting that BMP signaling regulates expression of these factors in spinal cord development. These results provide genetic evidence that BMP signaling is crucial for the development of dorsal neuronal cell types.

Regulation of outgrowth and apoptosis for the terminal appendage: External genitalia: Development by concerted actions of BMP signaling

Extra-corporal fertilization depends on the formation of copulatory organs: the external genitalia. Coordinated growth and differentiation of the genital tubercle (GT), an embryonic anlage of external genitalia, generates a proximodistally elongated structure suitable for copulation, erection, uresis and ejaculation. Despite recent progress in molecular embryology, few attempts have been made to elucidate the molecular developmental processes of external genitalia formation. Bone morphogenetic protein genes (Bmp genes) and their antagonists were spatiotemporally expressed during GT development. Exogenously applied BMP increased apoptosis of GT and inhibited its outgrowth. It has been shown that the distal urethral epithelium (DUE), distal epithelia marked by the Fgf8 expression, may control the initial GT outgrowth. Exogenously applied BMP4 downregulated the expression of Fgf8 and Wnt5a, concomitant with increased apoptosis and decreased cell proliferation of the GT mesenchyme. Furthermore, noggin mutants and Bmpr1a conditional mutant mice displayed hypoplasia and hyperplasia of the external genitalia respectively. noggin mutant mice exhibited downregulation of Wnt5a and Fgf8 expression with decreased cell proliferation. Consistent with such findings, Wnt5a mutant mice displayed GT agenesis with decreased cell proliferation. By contrast, Bmpr1a mutant mice displayed decreased apoptosis and augmented Fgf8 expression in the DUE associated with GT hyperplasia. These results suggest that some of the Bmp genes could negatively affect proximodistally oriented outgrowth of GT with regulatory functions on cell proliferation and apoptosis. The DUE region can be marked only until 14.0 dpc (days post coitum) in mouse development, while GT outgrowth continues thereafter. Possible signaling crosstalk among the whole distal GT regions were also investigated.

Wnt signaling is sufficient to perturb oligodendrocyte maturation.

The development of oligodendrocytes, the myelinating cells of the central nervous system, is temporally and spatially controlled by local signaling factors acting as inducers or inhibitors. Dorsal spinal cord tissue has been shown to contain inhibitors of oligodendrogliogenesis, although their identity is not completely known. We have studied the actions of one family of dorsal signaling molecules, the Wnts, on oligodendrocyte development. Using tissue culture models, we have shown that canonical Wnt activity through beta-catenin activation inhibits oligodendrocyte maturation, independently of precursor proliferation, cell death, or diversion to an alternate cell fate. Mice in which Wnt/beta-catenin signaling was constitutively activated in cells of the oligodendrocyte lineage had equal numbers of oligodendrocyte precursors relative to control littermates, but delayed appearance of mature oligodendrocytes, myelin protein, and myelinated axons during development, although these differences largely disappeared by adulthood. These results indicate that activating the Wnt/beta-catenin pathway delays the development of myelinating oligodendrocytes.

Neuron-specific alternative RNA processing in transgenic mice expressing a metallothionein-calcitonin fusion gene

Alternative RNA processing of the calcitonin/CGRP gene generates transcripts encoding predominantly calcitonin in thyroid C cells or CGRP in the nervous system. To examine the RNA processing choice of this gene in a wide variety of tissues, we created transgenic mice expressing the rat calcitonin/CGRP transcript from the mouse metallothionein-I promoter. Most cells that do not express the endogenous calcitonin/CGRP gene have the capability to make a clear splicing choice for calcitonin or CGRP transcript. In the majority of tissues studied, 90%-97% of the transgene mRNA encodes calcitonin. In contrast, both calcitonin and CGRP mRNAs were detected in the transgenic mice brains. Immunohistochemical and in situ RNA hybridization analyses show that CGRP transcripts are selectively expressed in a wide variety of neurons, while calcitonin is expressed predominantly in nonneuronal structures. Splicing choice operates independently of calcitonin/CGRP gene transcription. The data suggest that a specific regulatory machinery is required for the processing of CGRP transcripts and is restricted primarily to neurons.

BMP Signaling Mutant Mice Exhibit Glial Cell Maturation Defects

Bone morphogenetic proteins have been implicated in the development of oligodendrocytes and astrocytes, however, a role for endogenous BMP signaling in glial development has not been demonstrated in a genetic model. Using mice in which signaling via type I BMP receptors Bmpr1a and Bmpr1b have been inactivated in the neural tube, we demonstrate that BMP signaling contributes to the maturation of glial cells in vivo. At P0, mutant mice exhibited a 25-40% decrease in GFAP+ or S100beta+ astrocytes in the cervical spinal cord. The number of oligodendrocyte precursors and the timing of their emergence was unchanged in the mutant mice compared to the normals, however myelin protein expression and mature oligodendrocyte numbers were significantly reduced. These data indicate that BMP signaling promotes the generation of astrocytes and mature, myelinating oligodendrocytes in vivo but does not affect oligodendrocyte precursor development, thus suggesting tight regulation of BMP signaling to ensure proper gliogenesis.

Neuronal expression of chimeric genes in transgenic mice.

Gene expression may occur in unexpected ectopic sites when diverse genetic elements are juxtaposed as chimeric genes in transgenic mice. To determine the specific contribution of the promoter and reporter gene in ectopic expression, we have analyzed the expression of 14 different fusion genes in transgenic mice. Chimeric genes containing the mouse metallothionein-I promoter linked to either the rat or human growth hormone gene or the calcitonin/CGRP gene are expressed in a very similar pattern of neuronal regions. This ectopic expression is not a unique feature of the metallothionein promoter, since transferring the human growth hormone gene to four other heterologous promoters resulted in varying degrees of ectopic expression in overlapping subsets of cortical and hypothalamic neurons. The novel pattern of ectopic expression suggests that these otherwise unrelated neurons share a common developmental regulatory machinery for activation of gene transcription.

The closely related transcription factors Sox4 and Sox11 function as survival factors during spinal cord development

J. Neurochem. (2010) 115, 131–141.

Changes in the subcellular localization of the Brn4 gene product precede mesenchymal remodeling of the otic capsule.

To better understand the genetic mechanisms that regulate the formation of the temporal bone, we have characterized the developmental expression pattern of the mouse gene, Brn4/Pou3f4, which plays a central role in bony labyrinth formation. Expression of this gene is initially detected in the ventral aspect of the otic capsule at 10.5 days post coitus (dpc), and correlates with the onset of mesenchymal condensation in the otic capsule. As the otic capsule condenses further and surrounds the entire otic vesicle, the Brn4 gene product is detected throughout the inner ear in the mesenchyme of both the cochlear and vestibular aspects. Early in otic embryogenesis, the Brn4 gene product is localized to the nucleus of the vast majority of cells in which it is expressed. The Brn4 gene product remains nuclear in those regions of the otic capsule that eventually give rise to the mature bony labyrinth. However, the subcellular localization of the Brn4 gene product shifts from strictly nuclear to perinuclear in those regions of the otic capsule that will cavitate to form acellular regions in the temporal bone, such as the scala tympani, scala vestibuli, and the internal auditory meatus. These data provide a detailed analysis of the expression pattern of the Brn4 gene, and provide insight into the role of the Brn4 gene product and its regulation during otic capsule formation.