Upregulation of HB-EGF, Msx.1, and miRNA Let-7a by administration of calcitonin through mTOR and ERK1/2 pathways during a window of implantation in mice.

BACKGROUND: Successful implantation of embryos requires endometrial receptivity. Calcitonin is one of the factors influencing the implantation window. This study aimed to evaluate calcitonin effects on endometrial receptivity. To this end, the effects of calcitonin on the implantation window in the ovarian stimulation and the normal ovarian cycle were investigated by the morphological study of the endometrium as well as the expression of MSX.1, HB-EGF, and micro-RNA (miRNA) Let-7a; then the mechanisms of calcitonin effects were studied through the mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways. MATERIALS AND METHODS: A total of 64 Bulb/c mice were divided into two groups: Normal ovarian cycle and ovarian stimulation. Each group consisted of four subgroups: Ctrl, CT, PP242, and CT + PP242. Calcitonin and PP242 were injected on the fourth day of pregnancy and 24 hr later all the mice were killed. Uterine tissue samples were used for morphological analysis and the endometrial epithelial and the stromal cells were isolated from myometrium for evaluation of gene and protein expression. RESULTS: Ovarian stimulation increased the phosphorylation levels of mTOR and ERK1/2 and the expression of miRNA Let-7a. Calcitonin injection increased the expression of HB-EGF, Msx.1, and miRNA Let-7a in a normal ovarian cycle and in ovarian-stimulated mice. It also increased eukaryotic initiation factor 4E-binding protein 1 and ERK1/2 phosphorylation in normal ovarian cycles. CONCLUSION: Calcitonin improved the receptivity of the uterine endometrium by upregulation of the HB-EGF, Msx.1, and miRNA Let-7a likely through mTOR and ERK1/2 signaling pathway.

Msh homeobox 1 (Msx1)- and Msx2-overexpressing bone marrow-derived mesenchymal stem cells resemble blastema cells and enhance regeneration in mice.

Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes, such as Msh homeobox (Msx) genes, are absent in this animal group. The lack of BCs and positional information in other cells is therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema-like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx-regulated genes (Bmp4, Fgf8, and keratin 14 (K14)) in BlC groups, including MSX1, MSX2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced the proliferation rate and osteogenesis of BlCs compared with mBMSCs and BCs via activation of Fgf8 and Bmp4 Histological analyses indicated full regrowth of digit tips in the Msx-overexpressing groups, particularly in MSX1/2, through endochondral ossification 6 weeks post-injection. In contrast, mBMSCs and BCs formed abnormal bone and nail. Full digit tip was regenerated only in the MSX1/2 group and was related to boosted Bmp4, Fgf8, and K14 gene expression and to limb-patterning properties resulting from Msx1 and Msx2 overexpression. We propose that Msx-transduced cells that can regenerate epithelial and mesenchymal tissues may potentially be utilized in limb regeneration.

Expression of Dlx-5 and Msx-1 in Craniofacial Skeletons and Ilia of Rats Treated With Zoledronate.

PURPOSE: Because of the different embryologic origins of the craniofacial skeleton and ilium, differences in gene expression patterns have been observed between the jaw bones and ilium. Distal-less homeobox (Dlx) genes and Msh homeobox genes, particularly Dlx-5 and Msx-1, play major roles in cell differentiation and osteogenesis. The purpose of this study was to investigate the effects of zoledronate (ZOL) on the craniofacial skeleton and ilium by detecting changes in Dlx-5 and Msx-1 expression at both the protein and messenger RNA levels. MATERIALS AND METHODS: A total of 24 female Sprague-Dawley rats were randomly divided into 2 groups: ZOL group (n = 12), in which the rats were injected intraperitoneally with zoledronic acid for 12 weeks, and control group (n = 12), in which the rats were injected with saline solution for 12 weeks. By use of immunohistochemistry, Western blotting, and real-time reverse transcription polymerase chain reaction, the expression levels of Dlx-5 and Msx-1 in the craniofacial skeleton (including the maxilla, mandible, and parietal bone) and ilium were examined. RESULTS: Dlx-5 expression in the maxilla and mandible was increased at the protein and messenger RNA levels in the ZOL group compared with the control group (P < .01). In addition, Msx-1 expression in the maxilla and mandible was decreased in the ZOL group (P < .01). Furthermore, Dlx-5 and Msx-1 expression in the ilium was decreased in the ZOL group (P < .05). However, no significant difference in Dlx-5 or Msx-1 expression in the parietal bone was observed between the 2 groups (P > .05). CONCLUSIONS: Site-specific differences in the effects of ZOL on the craniofacial skeleton and ilium could be explained by differently altered tendencies in Dlx-5 and Msx-1 expression. The jaw bones were more susceptible to the effects of ZOL than the parietal bone and ilium.

Conserved Cis-Regulatory Modules Control Robustness in Msx1 Expression at Single-Cell Resolution.

The process of transcription is highly stochastic leading to cell-to-cell variations and noise in gene expression levels. However, key essential genes have to be precisely expressed at the correct amount and time to ensure proper cellular development and function. Studies in yeast and bacterial systems have shown that gene expression noise decreases as mean expression levels increase, a relationship that is controlled by promoter DNA sequence. However, the function of distal cis-regulatory modules (CRMs), an evolutionary novelty of metazoans, in controlling transcriptional robustness and variability is poorly understood. In this study, we used live cell imaging of transfected reporters combined with a mathematical modelling and statistical inference scheme to quantify the function of conserved Msx1 CRMs and promoters in modulating single-cell real-time transcription rates in C2C12 mouse myoblasts. The results show that the mean expression-noise relationship is solely promoter controlled for this key pluripotency regulator. In addition, we demonstrate that CRMs modulate single-cell basal promoter rate distributions in a graded manner across a population of cells. This extends the rheostatic model of CRM action to provide a more detailed understanding of CRM function at single-cell resolution. We also identify a novel CRM transcriptional filter function that acts to reduce intracellular variability in transcription rates and show that this can be phylogenetically separable from rate modulating CRM activities. These results are important for understanding how the expression of key vertebrate developmental transcription factors is precisely controlled both within and between individual cells.

LIM homeobox transcription factor Lhx2 inhibits skeletal muscle differentiation in part via transcriptional activation of Msx1 and Msx2.

LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the -2097 to -1189 genomic region of Msx1 and two additional sites in the -536 to +73 genomic region of Msx2. These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors.

Mononuclear cells from dedifferentiation of mouse myotubes display remarkable regenerative capability.

Certain lower organisms achieve organ regeneration by reverting differentiated cells into tissue-specific progenitors that re-enter embryonic programs. During muscle regeneration in the urodele amphibian, postmitotic multinucleated skeletal myofibers transform into mononucleated proliferating cells upon injury, and a transcription factor-msx1 plays a role in their reprograming. Whether this powerful regeneration strategy can be leveraged in mammals remains unknown, as it has not been demonstrated that the dedifferentiated progenitor cells arising from muscle cells overexpressing Msx1 are lineage-specific and possess the same potent regenerative capability as their amphibian counterparts. Here, we show that ectopic expression of Msx1 reprograms postmitotic, multinucleated, primary mouse myotubes to become proliferating mononuclear cells. These dedifferentiated cells reactivate genes expressed by embryonic muscle progenitor cells and generate only muscle tissue in vivo both in an ectopic location and inside existing muscle. More importantly, distinct from adult muscle satellite cells, these cells appear both to fuse with existing fibers and to regenerate myofibers in a robust and time-dependent manner. Upon transplantation into a degenerating muscle, these dedifferentiated cells generated a large number of myofibers that increased over time and replenished almost half of the cross-sectional area of the muscle in only 12 weeks. Our study demonstrates that mammals can harness a muscle regeneration strategy used by lower organisms when the same molecular pathway is activated.

Pax9 regulates a molecular network involving Bmp4, Fgf10, Shh signaling and the Osr2 transcription factor to control palate morphogenesis.

Cleft palate is one of the most common birth defects in humans. Whereas gene knockout studies in mice have shown that both the Osr2 and Pax9 transcription factors are essential regulators of palatogenesis, little is known about the molecular mechanisms involving these transcription factors in palate development. We report here that Pax9 plays a crucial role in patterning the anterior-posterior axis and outgrowth of the developing palatal shelves. We found that tissue-specific deletion of Pax9 in the palatal mesenchyme affected Shh expression in palatal epithelial cells, indicating that Pax9 plays a crucial role in the mesenchyme-epithelium interactions during palate development. We found that expression of the Bmp4, Fgf10, Msx1 and Osr2 genes is significantly downregulated in the developing palatal mesenchyme in Pax9 mutant embryos. Remarkably, restoration of Osr2 expression in the early palatal mesenchyme through a Pax9(Osr2KI) allele rescued posterior palate morphogenesis in the absence of Pax9 protein function. Our data indicate that Pax9 regulates a molecular network involving the Bmp4, Fgf10, Shh and Osr2 pathways to control palatal shelf patterning and morphogenesis.

Three-dimensional epithelial and mesenchymal cell co-cultures form early tooth epithelium invagination-like structures: expression patterns of relevant molecules.

Epithelium invagination is the key feature of early tooth development. In this study, we built a three-dimensional (3D) model to represent epithelium invagination-like structure by tissue engineering. Human normal oral epithelial cells (OECs) and dental pulp stem cells (DPSCs) were co-cultivated for 2-7 weeks on matrigel or collagen gel to form epithelial and mesenchymal tissues. The histological change and gene expression were analyzed by HE staining, immunostaining, and quantitative real-time RT-PCR (qRT-PCR). After 4 weeks of cultivation, OECs-formed epithelium invaginated into DPSCs-derived mesenchyme on both matrigel and collagen gel. OEC-DPSC co-cultures on matrigel showed typical invagination of epithelial cells and condensation of the underlying mesenchymal cells. Epithelial invagination-related molecules, CD44 and E-cadherin, and mesenchymal condensation involved molecules, N-cadherin and Msx1 expressed at a high level in the tissue model, suggesting the epithelial invagination is functional. However, when OECs and DPSCs were co-cultivated on collagen gel; the invaginated epithelium was transformed to several epithelial colonies inside the mesenchyme after long culture period. When DPSCs were co-cultivated with immortalized human OECs NDUSD-1, all of the above-mentioned features were not presented. Immunohistological staining and qRT-PCR analysis showed that p75, BMP2, Shh, Wnt10b, E-cadherin, N-cadherin, Msx1, and Pax9 are involved in initiating epithelium invagination and epithelial-mesenchymal interaction in the 3D OEC-DPSC co-cultures. Our results suggest that co-cultivated OECs and DPSCs on matrigel under certain conditions can build an epithelium invagination-like model. This model might be explored as a potential research tool for epithelial-mesenchymal interaction and tooth regeneration.

[Immunohistochemical study of Dlx1 and Msx1 expression during cephalic development of Dumbo and Wistar rats. Correlation with morphological data]. / Étude immuno-histochimique de l'expression des protéines Dlx1 et Msx1 durant la céphalogenèse des rats Wistar et Dumbo Corrélation avec les données morphologiques.

The Dumbo rat is characterized by a short snout, low ears and relative hypoplasia of maxillar and zygomatic bones. It corresponds to an autosomal recessive genotype. Previous study demonstrated a global deficit of Dlx1 and Msx1 genes expression in comparison to Wistar embryos as considered as control animals. We performed a histological study of cephalic development of Dumbo rats compared to Wistar embryos and an immunohistochemical analysis of Dlx1 and Msx1 protein expression during cephalogenesis. Our data indicate that the pattern of expression of both genes is similar in both strains, but that quantitative differences in gene expression can be the result of delayed organogenesis in Dumbo rat in comparison to Wistar. Some data about gene expressions are discussed at the light of the postulated function of Dlx1 and Msx1 in cephalic development.

Msx1 and Msx2 gene expression is downregulated in the cadmium-induced omphalocele in the chick model.

PURPOSE: The administration of cadmium (Cd) induces an omphalocele phenotype in the chick embryo. The molecular mechanism by which Cd acts still remains unclear. Msx1 and Msx2 are expressed in the developing body wall and regulate cellular proliferation and differentiation. It has been reported that Msx1/Msx2 double-mutant mice display an omphalocele phenotype. We hypothesized that gene expression levels of Msx1 and Msx2 are downregulated in the Cd chick model during the critical period of embryogenesis. METHODS: After 60 hours of incubation, chick embryos were exposed to either Cd or saline and harvested at 1 hour (1H), 4H, and 8H after treatment. Chicks were divided into 2 groups: control and Cd (n = 8 for each group at each time-point). Real-time polymerase chain reaction was performed to evaluate the messenger RNA levels of Msx1 and Msx2 in the Cd-induced omphalocele chick model and analyzed statistically. Immunohistochemistry was also performed to examine protein expression of Msx1 and Msx2 at each time-point. RESULTS: Messenger RNA expression levels of Msx1 and Msx2 at 1H were significantly decreased in the Cd group compared with controls (P < .01), whereas there were no significant differences at the other time-points. Immunoreactivity of Msx1 and Msx2 at 1H was remarkably decreased in Cd group compared with controls. CONCLUSION: Downregulation of Msx1 and Msx2 gene expression during the narrow window of early embryogenesis may cause an omphalocele by disrupting cellular proliferation and differentiation in the developing body wall.

Expression of muscle segment homeobox genes in the developing myocardium.

Msx1 and Msx2 are essential for the development of many organs. In the heart, they act redundantly in development of the cardiac cushions. Additionally, Msx2 is expressed in the developing conduction system. However, the exact expression of Msx1 has not been established. We show that Msx1 is expressed in the cardiac cushions, but not in the myocardium. In Msx2-null mice, Msx1 is not ectopically expressed in the myocardium. The absence of myocardial defects in the Msx2 knock-out can therefore not be attributed to a redundant action of Msx1 in the myocardium.

Hepatocyte growth factor exerts promoting functions on murine dental papilla cells.

Hepatocyte growth factor (HGF) is a potent mitogenic and angiogenic factor involved in the development of many tissues including the teeth. We previously reported that dental pulp cells express the HGF receptor c-met and that HGF can enhance cell proliferation and differentiation. The purpose of this study was to determine whether HGF could stimulate proliferation and/or differentiation of murine dental papilla cells (MDPCs). MDPCs were isolated from neonatal mice, and the mitogenic potential of HGF on MDPCs was measured by 3-(4, 5-dimethyl-thyazol-2-yl)-2, 5-diphenyltetrazolium and flow cytometry. Differentiation of MDPCs in the presence of HGF was characterized by mineralization assay, alkaline phosphatase (ALP) activity and reverse-transcription polymerase chain reaction. We reported that MDPCs expressed both HGF and c-met and that HGF enhanced proliferation of MDPCs by concentrations ranging from 5 ng/mL to 20 ng/mL, with 10 ng/mL as the optimal concentration (p < 0.05). HGF prolonged S phase and shortened G1 phase of MDPCs, increased ALP activity significantly over the control (p < 0.05), and enhanced the formation of mineral nodules. ALP, dentin sialophosphoprotein, and dentin matrix protein-1 expression were significantly increased in the presence of HGF (p < 0.05), as were the expression of Msx1, Runx2, and Pax9. Our data suggest that HGF plays an important role in tooth development, by promoting the proliferation and differentiation of MDPCs.

Msx and dlx homeogene expression in epithelial odontogenic tumors.

Epithelial odontogenic tumors are rare jaw pathologies that raise clinical diagnosis and prognosis dilemmas notably between ameloblastomas and clear cell odontogenic carcinomas (CCOCs). In line with previous studies, the molecular determinants of tooth development-amelogenin, Msx1, Msx2, Dlx2, Dlx3, Bmp2, and Bmp4-were analyzed by RT-PCR, ISH, and immunolabeling in 12 recurrent ameloblastomas and in one case of CCOC. Although Msx1 expression imitates normal cell differentiation in these tumors, other genes showed a distinct pattern depending on the type of tumor and the tissue involved. In benign ameloblastomas, ISH localized Dlx3 transcripts and inconstantly detected Msx2 transcripts in epithelial cells. In the CCOC, ISH established a lack of both Dlx3 and Msx2 transcripts but allowed identification of the antisense transcript of Msx1, which imitates the same scheme of distribution between mesenchyme and epithelium as in the cup stage of tooth development. Furthermore, while exploring the expression pattern of signal molecules by RT-PCR, Bmp2 was shown to be completely inactivated in the CCOC and irregularly noticeable in ameloblastomas. Bmp4 was always expressed in all the tumors. Based on the established roles of Msx and Dlx transcription factors in dental cell fates, these data suggest that their altered expression is a proposed trail to explain the genesis and/or the progression of odontogenic tumors.

Short-period effects of zirconia and titanium on osteoblast microRNAs.

BACKGROUND: MicroRNAs (miRNAs) are a class of small, functional, noncoding RNAs of 19 to 23 nucleotides which induce degradation of specific messenger RNAs (mRNAs), thus controlling the translational process (ie, synthesis of proteins from mRNAs). In addition, mRNAs regulate the promoter of specific miRNAs activating an autoregulatory feedback loop. PURPOSE: Titanium and zirconium dioxide ceramics (ZDCs) are used to make dental implants. Because the molecular mechanism by which ZDC and Ti act on osteoblasts is incompletely understood, we attempted to get more information by comparing the effect of ZDC and Ti on osteoblast miRNAs. MATERIALS AND METHODS: By using miRNA microarray technique, we identified in osteoblast-like cell line (MG63) grown on grade 3 Ti and ZDC disks several miRNAs whose expression was modified. We collected mRNAs after 24 hours of cell culturing to better understand molecular events related to early bone healing around inserted implants. An mRNA microarray technique was then performed as a control. RESULTS: There were six up- and four down-regulated miRNAs. Because every miRNA regulates hundreds of genes, we focused only on those related to bone formation. Among them, the most notable are BMP4 and 7, which are both up-regulated in osteoblasts cultured on Ti disks. CONCLUSION: The detected miRNAs differentially expressed in osteoblast-like cells grown on ZDC versus Ti act on a limited number of miRNAs and bone-related genes. The most notable are BMP4 and 7, which are more expressed in osteoblasts exposed to Ti surface. Consequently, we suggest that Ti surfaces could provide some advantages to immediate load implantology.

Developmentally regulated expression of MSX1, MSX2 and Fgfs in the developing mouse cranial base.

OBJECTIVE: To examine the expression pattern of the Fgf and Msx genes in cranial base development. MATERIALS AND METHODS: To detect the expression of these genes, antisense riboprobes were synthesized by in vitro transcription. Radioactive in situ hybridization was performed on parasagittal sections of embryonic mouse heads. RESULTS: Msx2 was observed in the underlying perichondrium at restricted stages. Msx1 was not observed in cranial base development. Fgf1 was localized in osteogenic cells from the time of ossification; Fgf10 was highly expressed in the occipital-vertebral joint during E13 to E14; Fgf2, Fgf7, and Fgf18 were localized in the perichondria; Fgf12 was transitorily expressed at early chondrocranium; Fgf9 was seen in the hypertrophic chondrocytes. CONCLUSIONS: The Fgf and Msx gene expression in the cranial base was different from that of other skeletons.

Bone morphogenetic protein-induced MSX1 and MSX2 inhibit myocardin-dependent smooth muscle gene transcription.

During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22alpha and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription.

Waking-up the sleeping beauty: recovery of the ancestral bird odontogenic program.

Recent advances in molecular and developmental genetics have provided tools for understanding evolutionary changes in the nature of the epithelial-mesenchymal interactions regulating the patterned outgrowth of the tooth primordia. Tissue recombination experiments in mice have identified the oral epithelium as providing the instructive information for the initiation of tooth development. Teeth were lost in birds for more than 80 million years ago, but despite their disappearance, a number of gene products and the requisite tissue interactions needed for tooth formation are found in the avian oral region. It is believed that the avian ectomesenchyme has lost the odontogenic capacity, whilst the oral epithelium retains the molecular signaling required to induce odontogenesis. In order to investigate the odontogenic capacity of the neural crest-derived mesenchyme and its potential activation of the avian oral epithelium, we have realized mouse neural tube transplantations to chick embryos to replace the neural crest cells of chick with those of mouse. Teeth are formed in the mouse/chick chimeras, indicating that timing is critical for the acquisition of the odontogenic potential by the epithelium and, furthermore, suggesting that odontogenesis is initially directed by species-specific mesenchymal signals interplaying with common epithelial signals.

BMP-signaling regulates the generation of hair-cells.

Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, yet there is no understanding of the role of BMP4 in this process. The present work was aimed at exploring the effects of BMP-signaling on the development of hair-cells. For this purpose, we studied gene expression, cell proliferation and cell death in isolated chick otic vesicles that were grown in vitro in the presence of recombinant BMP4 or the BMP-inhibitor Noggin. Cath1 was used as a marker for hair-cell specification. BMP4 reduced the number of Cath1-cells and, conversely, Noggin increased the size of the sensory patches and the number of Cath1-positive cells. The effect of BMP4 was irreversible and occurred before hair-cell specification. Lfng and Fgf10 were expressed in the prosensory domain before Cath1, and their expression was expanded by Noggin. At these stages, modifications of BMP activity did not respecify non-sensory epithelium of the otic vesicle. The expression of Bmp4 at sensory patches was suppressed by BMP4 and induced by Noggin suggesting an autoregulatory loop. Analysis of BrdU incorporation during 6 and 18 h indicated that the effects of BMP4 were due to its ability to reduce the number of actively proliferating progenitors and inhibit cell fate specification. BMP4 induced cell death within the prosensory domain of the otic vesicle, along with the expression of Msx1, but not Msx2. On the contrary, BMP-inhibition with Noggin favored hair-cell specification without changes in the overall cell proliferation. We propose that about the stage of terminal division, the balance between BMP and BMP-inhibitory signals regulates survival and specification of hair-cell precursors, the final number of sensory hair-cells being limited by excess levels of BMPs. The final size of sensory patches would hence depend on the balance between BMP4 and opposing signals.

Transcription factor c-Myb is involved in the regulation of the epithelial-mesenchymal transition in the avian neural crest.

Multipotential neural crest cells (NCCs) originate by an epithelial-mesenchymal transition (EMT) during vertebrate embryogenesis. We show for the first time that the key hematopoietic factor c-Myb is synthesized in early chick embryos including the neural tissue and participates in the regulation of the trunk NCCs. A reduction of endogenous c-Myb protein both in tissue explants in vitro and in embryos in ovo, prevented the formation of migratory NCCs. A moderate over-expression of c-myb in naive intermediate neural plates triggered the EMT and NCC migration probably through cooperation with BMP4 signaling because (i) BMP4 activated c-myb expression, (ii) elevated c-Myb caused accumulation of transcripts of the BMP4 target genes msx1 and slug, and (iii) the reduction of c-Myb prevented the BMP4-induced formation of NCCs. The data show that in chicken embryos, the c-myb gene is expressed prior to the onset of hematopoiesis and participates in the formation and migration of the trunk neural crest.