Cranial neural crest deletion of VEGFa causes cleft palate with aberrant vascular and bone development.

Cleft palate is among the most common craniofacial congenital anomalies. Up to 30% of patients with cleft palate also have associated cardiac and vascular defects. VEGFa, a critical growth factor involved in multiple developmental processes including angiogenesis and ossification, is also required for palate development. Conditional deletion of VEGFa in cranial neural crest (CNC) cells using Wnt1-Cre (VEGFaCKO) resulted in cleft palate in mice. The phenotype included reduced proliferation of cells within the palatal shelves, abnormal palatal shelf elongation and elevation, and the inability to undergo fusion. Vascularization of the VEGFaCKO palatal shelves was greatly reduced, suggesting a non-cell autonomous role of VEGFa signaling from the CNC-derived cells to the endothelium during vessel formation. Defective vascular development was coupled with deficient intramembranous ossification of maxillary and palatal mesenchyme. In vitro assessment of CNC-derived palatal mesenchymal cells from VEGFaCKO mice demonstrated normal ossification after BMP2 stimulation, suggesting that inadequate expression of Bmp2 in VEGFaCKO mice was, in part, responsible for reduced ossification. Taken together, these data demonstrate that VEGFa produced in the CNC-derived mesenchyme drives proliferation, vascularization, and ossification, all of which are critical for palate development.

Type III transforming growth factor beta receptor regulates vascular and osteoblast development during palatogenesis.

BACKGROUND: Cleft palate occurs in up to 1:1,000 live births and is associated with mutations in multiple genes. Palatogenesis involves a complex choreography of palatal shelf elongation, elevation, and fusion. Transforming growth factor ß (TGFß) and bone morphogenetic protein 2 (BMP2) canonical signaling is required during each stage of palate development. The type III TGFß receptor (TGFßR3) binds all three TGFß ligands and BMP2, but its contribution to palatogenesis is unknown. RESULTS: The role of TGFßR3 during palate formation was found to be during palatal shelf elongation and elevation. Tgfbr3(-) (/) (-) embryos displayed reduced palatal shelf width and height, changes in proliferation and apoptosis, and reduced vascular and osteoblast differentiation. Abnormal vascular plexus organization as well as aberrant expression of arterial (Notch1, Alk1), venous (EphB4), and lymphatic (Lyve1) markers was also observed. Decreased osteoblast differentiation factors (Runx2, alk phos, osteocalcin, col1A1, and col1A2) demonstrated poor mesenchymal cell commitment to the osteoblast lineage within the maxilla and palatal shelves in Tgfbr3(-) (/) (-) embryos. Additionally, in vitro bone mineralization induced by osteogenic medium (OM+BMP2) was insufficient in Tgfbr3(-) (/) (-) palatal mesenchyme, but mineralization was rescued by overexpression of TGFßR3. CONCLUSIONS: These data reveal a critical, previously unrecognized role for TGFßR3 in vascular and osteoblast development during palatogenesis.

Cell-autonomous and non-cell-autonomous roles for IRF6 during development of the tongue.

Interferon regulatory factor 6 (IRF6) encodes a highly conserved helix-turn-helix DNA binding protein and is a member of the interferon regulatory family of DNA transcription factors. Mutations in IRF6 lead to isolated and syndromic forms of cleft lip and palate, most notably Van der Woude syndrome (VWS) and Popliteal Ptyerigium Syndrome (PPS). Mice lacking both copies of Irf6 have severe limb, skin, palatal and esophageal abnormalities, due to significantly altered and delayed epithelial development. However, a recent report showed that MCS9.7, an enhancer near Irf6, is active in the tongue, suggesting that Irf6 may also be expressed in the tongue. Indeed, we detected Irf6 staining in the mesoderm-derived muscle during development of the tongue. Dual labeling experiments demonstrated that Irf6 was expressed only in the Myf5+ cell lineage, which originates from the segmental paraxial mesoderm and gives rise to the muscles of the tongue. Fate mapping of the segmental paraxial mesoderm cells revealed a cell-autonomous Irf6 function with reduced and poorly organized Myf5+ cell lineage in the tongue. Molecular analyses showed that the Irf6-/- embryos had aberrant cytoskeletal formation of the segmental paraxial mesoderm in the tongue. Fate mapping of the cranial neural crest cells revealed non-cell-autonomous Irf6 function with the loss of the inter-molar eminence. Loss of Irf6 function altered Bmp2, Bmp4, Shh, and Fgf10 signaling suggesting that these genes are involved in Irf6 signaling. Based on these data, Irf6 plays important cell-autonomous and non-cell-autonomous roles in muscular differentiation and cytoskeletal formation in the tongue.

Neurokinin B acts via the neurokinin-3 receptor in the retrochiasmatic area to stimulate luteinizing hormone secretion in sheep.

Recent data have demonstrated that mutations in the receptor for neurokinin B (NKB), the NK-3 receptor (NK3R), produce hypogonadotropic hypogonadism in humans. These data, together with reports that NKB expression increases after ovariectomy and in postmenopausal women, have led to the hypothesis that this tachykinin is an important stimulator of GnRH secretion. However, the NK3R agonist, senktide, inhibited LH secretion in rats and mice. In this study, we report that senktide stimulates LH secretion in ewes. A dramatic increase in LH concentrations to levels close to those observed during the preovulatory LH surge was observed after injection of 1 nmol senktide into the third ventricle during the follicular, but not in the luteal, phase. Similar increases in LH secretion occurred after insertion of microimplants containing this agonist into the retrochiasmatic area (RCh) in anestrous or follicular phase ewes. A low-dose microinjection (3 pmol) of senktide into the RCh produced a smaller but significant increase in LH concentrations in anestrous ewes. Moreover, NK3R immunoreactivity was clearly evident in the RCh, although it was not found in A15 dopaminergic cell bodies in this region. These data provide evidence that NKB stimulates LH (and presumably GnRH) secretion in ewes and point to the RCh as one important site of action. Based on these data, and the effects of NK3R mutations in humans, we hypothesize that NKB plays an important stimulatory role in the control of GnRH and LH secretion in nonrodent species.

Iatrogenic perinatal pharyngoesophageal injury: a disease of prematurity.

OBJECTIVE: Perinatal pharyngoesophageal instrumentation, including endotracheal intubation, oral suctioning, and feeding tube placement, is often necessary but risks tissue damage. Our objective was to estimate the incidence of iatrogenic perinatal pharyngoesophageal injury (IPPI) in preterm versus term infants in a children's hospital neonatal intensive care unit (NICU). A secondary goal was to explore the clinical characteristics and outcomes associated with these complications. METHODS: All NICU discharge summaries from 2004 to 2008 were searched for IPPI-related keywords. Highlighted records were reviewed and the incidence of complications calculated by gestational age and weight. RESULTS: Of 5910 total NICU discharges, 6 cases of IPPI were identified, for an overall incidence of 0.10%. All injuries occurred in infants less than 33 weeks gestational age and 1500g, with a trend towards higher incidence with increasing prematurity. The incidence of IPPI rose to 4/1321 (0.30%) at 27-32 weeks and 2/521 (0.38%) at less than 27 weeks gestation. Similarly, IPPI occurred in 3/675 (0.44%) babies born at 1000-1500g and 3/642 (0.47%) babies below 1000g. All affected infants survived with conservative management. CONCLUSIONS: IPPI is a rare but serious complication of perinatal airway instrumentation and is primarily a disease of prematurity. In this sizeable cohort, no complications occurred in term infants, and the incidence of injury increased with decreasing gestational age and weight. This increased propensity towards injury should prompt special care when performing even routine airway procedures on premature neonates.