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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.02.006 Byline: Xuguang Nie (a), Chu-xia Deng (b), Qin Wang (c), Kai Jiao (a) Keywords: Smad4; Pharyngeal arch; Pharyngeal arch artery; Outflow tract; Facial primordia; Neural crest cells Abbreviations: NC, neural crest; NCCs, neural crest cells; OFT, outflow track; PA, pharyngeal arch; PAA, pharyngeal arch artery Abstract: TGF[beta]/BMP signaling pathways are essential for normal development of neural crest cells (NCCs). Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of TGF[beta]/BMP signaling. In this work, we sought to investigate the roles of Smad4 for development of NCCs. To overcome the early embryonic lethality of Smad4 null mice, we specifically disrupted Smad4 in NCCs using a Cre/loxP system. The mutant mice died at mid-gestation with defects in facial primordia, pharyngeal arches, outflow tract and cardiac ventricles. Further examination revealed that mutant embryos displayed severe molecular defects starting from E9.5. Expression of multiple genes, including Msx1, 2, Ap-2[alpha], Pax3, and Sox9, which play critical roles for NCC development, was downregulated by NCC disruption of Smad4. Moreover, increased cell death was observed in pharyngeal arches from E10.5. However, the cell proliferation rate in these areas was not substantially altered. Taken together, these findings provide compelling genetic evidence that Smad4-mediated activities of TGF[beta]/BMP signals are essential for appropriate NCC development. Author Affiliation: (a) Department of Genetics, Division of Genetic and Translational Medicine, The University of Alabama at Birmingham, Birmingham, AL 35294, USA (b) Genetics of Development and Disease Branch, NIDDK, NIH, Bethesda, MA 20892, USA (c) Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA Article History: Received 22 February 2007; Revised 31 January 2008; Accepted 5 February 2008

Background . SOX9 is a critical transcription factor for chondrogenesis and sex determination. Haploinsufficiency mutations of Sox9 in humans lead to campomelic dysplasia. Inactivation of Sox9 in the craniofacial region of mice results in an absence of endochondral bones and in malformation of other structures. This suggests that Sox9 plays multiple roles in craniofacial development and these remain to be elucidated. In order to study the functions of Sox9 in craniofacial development, a preliminary expression examination was performed. Material and Methods. To detect the expression of Sox9 mRNA, antisense riboprobe was synthesized by in vitro transcription. Radioactive in situ hybridization was performed on sagittal and coronal sections of mice head from organogenesis to the early postnatal stage. Results. It was found that Sox9 was expressed in multiple stages and distinct processes. Besides the expression in cartilage, it was seen in the fusing stage of palatogenesis. Sox9 was also present during differentiation and maturation of craniofacial muscles. In addition, it was observed in intramembranous skeletal elements at restricted sites and stage. Conclusions . The expression pattern suggests that Sox9 serves broad roles in craniofacial development. [ABSTRACT FROM AUTHOR]

The cranial base is formed by endochondral ossification and is characterized by the presence of the synchondrosis growth centers. The aim of this study was to describe the histological development of the mouse midsagittal cranial base area from embryonic day 10 (E10) to the postnatal age of 2 months. The Bmp family of signaling molecules serves important functions in embryo and bone development and may therefore play a significant role in the early formation of the cranial base. To investigate this, we analyzed the mRNA pattern of expression of Bmp2-6 in the mouse cranial base from E10 to 5 days postnatally using radioactive in situ hybridization. We found that the formation of the mouse cranial base corresponds to that of rat and proceeds in a caudorostral sequence. Moreover, all Bmps studied showed distinct and overlapping developmentally regulated expression domains. Bmp2, Bmp5, and Bmp6 were expressed in the early mesenchymal condensations. Later, Bmp2, Bmp3, Bmp4, and Bmp5 were detected in the perichondrium and in the adjacent mesenchyme. Subsequently, Bmp2 and Bmp6 expressions were confined to hypertrophic chondrocytes, while Bmp3, Bmp4, and Bmp5 were expressed in the osteoblasts of the trabecular bone and bone collar. Interestingly, Bmp3 was uniquely expressed postnatally in the resting zone of the synchondrosis growth center, suggesting a role in the regulation of cranial base growth. These results suggest that Bmp signaling may serve specific and synergistic functions at different key stages of cranial base development and growth., (Copyright (c) 2006 Wiley-Liss, Inc.)

The cranial base, located between the cranial vault and the facial bones, plays an important role in integrated craniofacial development and growth. Transgenic Shh and Sox9-deficient mice show similar defects in cranial base patterning. Therefore, in order to examine potential interactions of Shh, Ihh, another member of the Hh family, and Sox9 during cranial base development and growth, we investigated their cellular mRNA expression domains in the embryonic (E) and early postnatal (PN) cranial base from E10 to PN5 using sectional radioactive 35-S in situ hybridization. Of the Hhs, Shh was observed in the foregut epithelium and the notochord, while Sox9 showed broad expression in the loose mesenchyme of the cranial base area during E10-E11. Subsequently, from E12 onward, all genes were observed in the developing cranial base, and after birth the genes were prominently colocalized in the prehypertrophic chondrocytes of the synchondroses. Collectively, these data suggest that Hh-Sox9 auto- and paracrine signaling interactions may provide a critical mechanism for regulating the patterning of the cranial base as well as for its development and growth.