Role of bone morphogenetic proteins on cochlear hair cell formation: Analyses of Noggin and Bmp2 mutant mice

The mammalian organ of Corti of the inner ear is a highly sophisticated sensory end organ responsible for detecting sound. Noggin is a secreted glycoprotein, which antagonizes bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4). The lack of this antagonist causes increased rows of inner and outer hair cells in the organ of Corti. In mice, Bmp2 is expressed transiently in nascent cochlear hair cells. To investigate whether Noggin normally modulates the levels of Bmp2 for hair cell formation, we deleted Bmp2 in the cochlear hair cells using two cre strains, Foxg1^cre/+ and Gfi1^cre/+. Bmp2 conditional knockout cochleae generated using these two cre strains show normal hair cells. Furthermore, Gfi1^cre/+;Bmp2^lox/? mice are viable and have largely normal hearing. The combined results of Noggin and Bmp2 mutants suggest that Noggin is likely to regulate other Bmps in the cochlea such as Bmp4. Developmental Dynamics 239:505–513, 2010. Published 2010 Wiley‐Liss, Inc.     

人Noggin基因神经细胞特异性表达载体的构建

目的构建具有神经细胞表达特异性的人Noggin基因的真核表达载体，进一步研究Noggin基因在神经系统发育成熟、功能机制方面的作用。方法根据人Noggin基因的cDNA序列，设计合成一对5’端分别含有HindⅢ和XbaⅠ酶切位点的特异性引物，运用RT-PCR方法克隆人Noggin基因的cDNA序列；回收PCR产物，并将其与pMD18 simple T载体连接，进行HindⅢ和XbaⅠ双酶切鉴定和DNA测序鉴定；真核表达载体pCS2 [Tal]-GFP带有神经细胞特异性启动子(alpha1-微管蛋白启动子)，用HindⅢ和XbaⅠ双酶切后琼脂糖凝胶电泳回收带有特异性启动子的片断和T载体上人Noggin基因，在T4DNA连接酶作用下将二者连接，并筛选鉴定。结果RT-PCR产物含有人Noggin基因，DNA测序结果显示重组的pMD18-Noggin载体中含有正确的人Noggin基因序列，重组的pCS2 [Ted]-Noggin载体中含有人Noggin基因的cDNA序列以及alpha1-微管蛋白启动子。结论成功构建了人Noggin基因的神经细胞特异性真核表达载体pCS2 [Ted]-Noggin。     

SVZa neural stem cells differentiate into distinct lineages in response to BMP4

Neural stem cells (NSCs) reside in the anterior portion of the forebrain subventricular zone (SVZa) and generate the progenitors which will differentiate into neurons, and via a tangential migratory pathway, known as the rostral migratory stream (RMS), migrate to the olfactory bulbs (OB). Bone morphogenetic proteins (BMPs) play significant roles in neural development at different stages and locations, but their roles have not been determined in the SVZa. To explore possible roles of BMPs in SVZa NSCs, BMP4 at various concentrations were tested for their capacity to induce SVZa NSCs. The expression of BMP4 was also examined in living cells using a reportor vector, in which the BMP4 promotor was conjugated with red fluorescent protein (RFP). In the meantime, the differentiation of SVZa NSCs was dynamically monitored by using reportor vectors of the Nestin enhancer and the promoters of TH and GFAP. In the OB, high expression of BMP4 was found using both promoter activity analysis and in situ hybridization. However, low BMP4 expression was found in the RMS and only moderate expression of BMP4 was displayed in the SVZa. The results also demonstrated that low concentrations (1-5 ng/ml) of BMP4 promoted the proliferation of SVZa NSCs but high concentrations (10-100 ng/ml) of BMP4 inhibited this proliferation. BMP4 enhanced neuron commitment before 4 days but inhibited it after 4 days. As the antagonist of BMP4, Noggin almost completely blocked all these BMP4 responses. Thus, our findings indicate that BMP4 promotes the exit from the cell cycle and triggers the differentiation of neuron progenitors in the OB. BMP4 also promotes the proliferation of the committed neuron progenitors in the RMS, but in the SVZa, BMP4 may facilitate the commitment of NSCs into astrocytes.     

Bone morphogenetic proteins are involved in fetal kidney tissue transplantation-induced neuroprotection in stroke rats

Both bone morphogenetic proteins (BMPs) and glial cell line-derived neurotrophic factor (GDNF) reduce ischemia-induced cerebral injury in rats. Intracerebral transplantation of fetal kidney tissue, which normally expresses BMPs and GDNF during development, reduces ischemic injury in cerebral cortex. In this study, we tested the hypothesis that BMP is involved in this neuroprotective response. Fetal kidney tissue was cut into small pieces and transplanted into cortical areas adjacent to the right middle cerebral artery (MCA) in adult rats. In situ hybridization of brain indicated that these fetal kidney transplants contained high levels of BMP-7 mRNA three days after grafting. Immunohistochemical analysis of grafted brain showed co-localization of BMP-7 and PAX-2 immunoreactivity in the graft, suggesting that these transplants contained BMP protein. Some animals were grafted with fetal kidney tissue after intraventricular administration (ICV) of the BMP antagonist noggin (1 micro g) or after vehicle, followed by MCA ligation for 60 min. Animals receiving fetal kidney tissue transplantation developed significantly less body asymmetry, as compared to stroke animals that either did not receive transplantation or received fetal kidney grafts and noggin pretreatment. Analysis of these brains after triphenyltetrazolium chloride staining showed that fetal kidney tissue transplantation reduced the volume of infarction in the cerebral cortex. Noggin pretreatment reduced the protection induced by fetal kidney grafting, although noggin itself did not cause increase in cerebral infarction. Eight hours after ischemia, brain homogenates were obtained from grafted and control animals to assay caspase-3 enzymatic activity. This analysis demonstrated that fetal kidney grafts significantly reduced ischemia-induced caspase-3 activity. Reduction of caspase-3 activity could also be antagonized by noggin pretreatment. In conclusion, our data suggest that fetal kidney transplantation reduces ischemia/reperfusion-induced cortical infarction and behavioral deficits in adult rats, which are, at least partially, mediated through the effect of BMPs from the transplants.     

Further delineation of the GDF6 related multiple synostoses syndrome

A mutation in GDF6 was recently found to underlie a multiple synostoses syndrome. In this report, we describe the second family with GDF6‐related multiple synostoses syndrome (SYNS4), caused by a novel c.1287C>A/p.Ser429Arg mutation in GDF6. In addition to synostoses of carpal and/or tarsal bones, at least 6 of 10 affected patients in this family have been diagnosed with mild to moderate hearing loss. In four of them otosclerosis was said to be present, one patient had hearing loss due to severe stapes fixation at the age of 6 years, providing evidence that hearing loss in the GDF6‐related multiple synostoses syndrome can be present in childhood. Two others had surgery for stapes fixation at adult age. We hypothesize that, identical to the recently published GDF6‐related multiple synostoses family, the p.Ser429Arg mutation also leads to a gain of function. The previously reported c.1330T>A/pTyr444Asn mutation was located in a predicted Noggin and receptor I interacting domain and the gain of function was partly due to resistance of the mutant GDF6 to the BMP‐inhibitor Noggin. The results in our family show that mutations predicting to affect the type II receptor interface can lead to a similar phenotype and that otosclerosis presenting in childhood can be part of the GDF6‐related multiple synostoses syndrome.     

BMP signaling regulates the fate of chondro‐osteoprogenitor cells in facial mesenchyme in a stage‐specific manner

Background : Lineage tracing has shown that most of the facial skeleton is derived from cranial neural crest cells. However, the local signals that influence postmigratory, neural crest‐derived mesenchyme also play a major role in patterning the skeleton. Here, we study the role of BMP signaling in regulating the fate of chondro‐osteoprogenitor cells in the face. Results : A single Noggin‐soaked bead inserted into stage 15 chicken embryos induced an ectopic cartilage resembling the interorbital septum within the palate and other midline structures. In contrast, the same treatment in stage 20 embryos caused a loss of bones. The molecular basis for the stage‐specific response to Noggin lay in the simultaneous up‐regulation of SOX9 and downregulation of RUNX2 in the maxillary mesenchyme, increased cell adhesiveness as shown by N‐cadherin induction around the beads and increased RA pathway gene expression. None of these changes were observed in stage 20 embryos. Conclusions : These experiments demonstrate how slight changes in expression of growth factors such as BMPs could lead to gain or loss of cartilage in the upper jaw during vertebrate evolution. In addition, BMPs have at least two roles: one in patterning the skull and another in regulating the skeletogenic fates of neural crest‐derived mesenchyme. Developmental Dynamics 245:947–962, 2016. © 2016 Wiley Periodicals, Inc.     

Snail regulates Nanog status during the epithelial–mesenchymal transition via the Smad1/Akt/GSK3β signaling pathway in non-small-cell lung cancer

The epithelial–mesenchymal transition (EMT), a crucial step in cancer metastasis, is important in transformed cancer cells with stem cell-like properties. In this study, we established a Snail-overexpressing cell model for non-small-cell lung cancer (NSCLC) and investigated its underlying mechanism. We also identified the downstream molecular signaling pathway that contributes to the role of Snail in regulating Nanog expression. Our data shows that high levels of Snail expression correlate with metastasis and high levels of Nanog expression in NSCLC. NSCLC cells expressing Snail are characterized by active EMT characteristics and exhibit an increased ability to migrate, chemoresistance, sphere formation, and stem cell-like properties. We also investigated the signals required for Snail-mediated Nanog expression. Our data demonstrate that {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, SB431542, LDN193189, and Noggin pretreatment inhibit Snail-induced Nanog expression during EMT. This study shows a significant correlation between Snail expression and phosphorylation of Smad1, Akt, and GSK3β. In addition, pretreatment with SB431542, LDN193189, or Noggin prevented Snail-induced Smad1 and Akt hyperactivation and reactivated GSK3β. Moreover, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 pretreatment prevented Akt hyperactivation and reactivated GSK3β without altering Smad1 activation. These findings provide a novel mechanistic insight into the important role of Snail in NSCLC during EMT and indicate potentially useful therapeutic targets for NSCLC.     

Shh maintains dermal papilla identity and hair morphogenesis via a Noggin-Shh regulatory loop

During hair follicle morphogenesis, dermal papillae (DPs) function as mesenchymal signaling centers that cross-talk with overlying epithelium to regulate morphogenesis. While the DP regulates hair follicle formation, relatively little is known about the molecular basis of DP formation. The morphogen Sonic hedgehog (Shh) is known for regulating hair follicle epithelial growth, with excessive signaling resulting in basal cell carcinomas. Here, we investigate how dermal-specific Shh signaling contributes to DP formation and hair growth. Using a Cre-lox genetic model and RNAi in hair follicle reconstitution assays, we demonstrate that dermal Smoothened (Smo) loss of function results in the loss of the DP precursor, the dermal condensate, and a stage 2 hair follicle arrest phenotype reminiscent of Shh(-/-) skin. Surprisingly, dermal Smo does not regulate cell survival or epithelial proliferation. Rather, molecular screening and immunostaining studies reveal that dermal Shh signaling controls the expression of a subset of DP-specific signature genes. Using a hairpin/cDNA lentiviral system, we show that overexpression of the Shh-dependent gene Noggin, but not Sox2 or Sox18, can partially rescue the dermal Smo knockdown hair follicle phenotype by increasing the expression of epithelial Shh. Our findings suggest that dermal Shh signaling regulates specific DP signatures to maintain DP maturation while maintaining a reciprocal Shh-Noggin signaling loop to drive hair follicle morphogenesis.