Overexpression of Smad2 in Tgf-beta3-null mutant mice rescues cleft palate

Transforming growth factor (TGF)-beta3 is an important contributor to the regulation of medial edge epithelium (MEE) disappearance during palatal fusion. SMAD2 phosphorylation in the MEE has been shown to be directly regulated by TGF-beta3. No phospho-SMAD2 was identified in the MEE in Tgf-beta3-null mutant mice (Tgf-beta3-/-), which was correlated with the persistence of the MEE and failure of palatal fusion. In the present study, the cleft palate phenotype in Tgf-beta3-/- mice was rescued by overexpression of a Smad2 transgene in Keratin 14-synthesizing MEE cells following mating Tgf-beta3 heterozygous mice with Keratin 14 promoter directed Smad2 transgenic mice (K14-Smad2). Success of the rescue could be attributed to the elevated phospho-SMAD2 level in the MEE, demonstrated by two indirect evidences. The rescued palatal fusion in Tgf-beta3-/-/K14-Smad2 mice, however, never proceeded to the junction of primary and secondary palates and the most posterior border of the soft palate, despite phospho-SMAD2 expression in these regions at the same level as in the middle portion of the secondary palate. The K14-Smad2 transgene was unable to restore all the functional outcomes of TGF-beta3. This may indicate an anterior-posterior patterning in the palatal shelves with respect to TGF-beta3 signaling and the mechanism of secondary palatal fusion.     

Tgfb1 expressed in the Tgfb3 locus partially rescues the cleft palate phenotype of Tgfb3 null mutants

Although TGF-β isoforms (TGF-β1-3) display very similar biochemical characteristics in vitro, it has been determined that they demonstrate different or even opposing effects in vivo. During embryogenesis, TGF-βs play important roles in several developmental processes. Tgfb3 is strongly expressed in the prefusion palatal epithelium, and mice lacking Tgfb3 display a cleft of the secondary palate. To test whether the effect of TGF-β3 in palatogenesis is isoform-specific in vivo, we generated a knockin mouse by replacing the coding region of exon1 in the Tgfb3 gene with the full-length Tgfb1 cDNA, which resulted in the expression of Tgfb1 in the Tgfb3 expressing domain. The homozygote knockin mice display a complete fusion at the mid-portion of the secondary palate, while the most anterior and posterior regions fail to fuse appropriately indicating that in vivo replacement of TGF-β3 with TGF-β1 can only partially correct the epithelial fusion defect of Tgfb3 knockout embryos. Palatal shelves of Tgfb1 knockin homozygote mice adhere, intercalate, and form characteristic epithelial triangles. However, decreased apoptosis in the midline epithelium, slower breakdown of the basement membrane and a general delay in epithelial fusion were observed when compared to control littermates. These results demonstrate an isoform-specific role for TGF-β3 in the palatal epithelium during palate formation, which cannot be fully substituted with TGF-β1.     

Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse

Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx1(+/-) mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Here, we identify enhancer elements in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. We describe the generation of transgenic mice that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. We use these Tbx1-Cre mice to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which are derived from Tbx1-expressing cells. We test the hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery.     

Mutation and association analysis of the PVR and PVRL2 genes in patients with non-syndromic cleft lip and palate

Orofacial clefts (OFC; MIM 119530) are among the most common major birth defects. Here, we carried out mutation screening of the PVR and PVRL2 genes, which are both located at an OFC linkage region at 19q13 (OFC3) and are closely related to PVRL1, which has been associated with both syndromic and non-syndromic cleft lip and palate (nsCLP). We screened a total of 73 nsCLP patients and 105 non-cleft controls from the USA for variants in PVR and PVRL2, including all exons and encompassing all isoforms. We identified four variants in PVR and five in PVRL2. One non-synonymous PVR variant, A67T, was more frequent among nsCLP patients than among normal controls, but this difference did not achieve statistical significance.     

Fgf10 dosage is critical for the amplification of epithelial cell progenitors and for the formation of multiple mesenchymal lineages during lung development

The key role played by Fgf10 during early lung development is clearly illustrated in Fgf10 knockout mice, which exhibit lung agenesis. However, Fgf10 is continuously expressed throughout lung development suggesting extended as well as additional roles for FGF10 at later stages of lung organogenesis. We previously reported that the enhancer trap Mlcv1v-nLacZ-24 transgenic mouse strain functions as a reporter for Fgf10 expression and displays decreased endogenous Fgf10 expression. In this paper, we have generated an allelic series to determine the impact of Fgf10 dosage on lung development. We report that 80% of the newborn Fgf10 hypomorphic mice die within 24 h of birth due to respiratory failure. These mutant mouse lungs display severe hypoplasia, dilation of the distal airways and large hemorrhagic areas. Epithelial differentiation and proliferation studies indicate a specific decrease in TTF1 and SP-B expressing cells correlating with reduced epithelial cell proliferation and associated with a decrease in activation of the canonical Wnt signaling in the epithelium. Analysis of vascular development shows a reduction in PECAM expression at E14.5, which is associated with a simplification of the vascular tree at E18.5. We also show a decrease in α-SMA expression in the respiratory airway suggesting defective smooth muscle cell formation. At the molecular level, these defects are associated with decrease in Vegfa and Pdgfa expression likely resulting from the decrease of the epithelial/mesenchymal ratio in the Fgf10 hypomorphic lungs. Thus, our results indicate that FGF10 plays a pivotal role in maintaining epithelial progenitor cell proliferation as well as coordinating alveolar smooth muscle cell formation and vascular development.     

Genetic interactions between Pax9 and Msx1 regulate lip development and several stages of tooth morphogenesis

Developmental abnormalities of craniofacial structures and teeth often occur sporadically and the underlying genetic defects are not well understood, in part due to unknown gene-gene interactions. Pax9 and Msx1 are co-expressed during craniofacial development, and mice that are single homozygous mutant for either gene exhibit cleft palate and an early arrest of tooth formation. Whereas in vitro assays have demonstrated that protein-protein interactions between Pax9 and Msx1 can occur, it is unclear if Pax9 and Msx1 interact genetically in vivo during development. To address this question, we compounded the Pax9 and Msx1 mutations and observed that double homozygous mutants exhibit an incompletely penetrant cleft lip phenotype. Moreover, in double heterozygous mutants, the lower incisors were consistently missing and we find that transgenic BMP4 expression partly rescues this phenotype. Reduced expression of Shh and Bmp2 indicates that a smaller “incisor field” forms in Pax9^+/−;Msx1^+/− mutants, and dental epithelial growth is substantially reduced after the bud to cap stage transition. This defect is preceded by drastically reduced mesenchymal expression of Fgf3 and Fgf10, two genes that encode known stimulators of epithelial growth during odontogenesis. Consistent with this result, cell proliferation is reduced in both the dental epithelium and mesenchyme of double heterozygous mutants. Furthermore, the developing incisors lack mesenchymal Notch1 expression at the bud stage and exhibit abnormal ameloblast differentiation on both labial and lingual surfaces. Thus, Msx1 and Pax9 interact synergistically throughout lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry. The data also suggest that a combined reduction of PAX9 and MSX1 gene dosage in humans may increase the risk for orofacial clefting and oligodontia.     

Doubleridge,a mouse mutant with defective compaction of the apical ectodermal ridge and normal dorsal-ventral patterning of the limb

doubleridge is a transgene-induced mutation characterized by polydactyly and syndactyly of the forelimbs. The transgene insertion maps to the proximal region of chromosome 19. During embryonic development of the mutant forelimb, delayed elevation and compaction of the apical ectodermal ridge (AER) produces a ridge that is abnormally broad and flat. Fgf8 expression persists in the ventral forelimb ectoderm of the mutant until E10.5. Strong expression of Fgf8 and other markers at the borders of the AER at E11.5 gives the appearance of a double ridge. At E11.5, apoptotic cells are distributed across the broadened ridge, but at E13.5, there is reduced apoptosis in the interdigital regions. The Shh expression domain is widely spaced at the posterior margin of the AER. The doubleridge AER is morphologically similar to that of En1 null mice, but the expression of En1 and Wnt7a is properly restricted in doubleridge, and the dorsal and ventral structures are correctly determined. doubleridge thus exhibits an unusual limb phenotype combining abnormal compaction of the AER with normal dorsal/ventral patterning.     

Snail family members and cell survival in physiological and pathological cleft palates

Palate fusion is a complex process that involves the coordination of a series of cellular changes including cell death and epithelial to mesenchymal transition (EMT). Since members of the Snail family of zinc-finger regulators are involved in both triggering of the EMT and cell survival, we decided to study their putative role in palatal fusion. Furthermore, Snail genes are induced by transforming growth factor beta gene (TGF-beta) superfamily members, and TGF-beta(3) null mutant mice (TGF-beta(3)-/-) show a cleft palate phenotype. Here we show that in the wild-type mouse at the time of fusion, Snail is expressed in a few cells of the midline epithelial seam (MES), compatible with a role in triggering of the EMT in a small subpopulation of the MES. We also find an intriguing relationship between the expression of Snail family members and cell survival associated to the cleft palate condition. Indeed, Snail is expressed in the medial edge epithelial (MEE) cells in TGF-beta(3)-/-mouse embryo palates, where it is activated by the aberrant expression of its inducer, TGF-beta(1), in the underlying mesenchyme. In contrast to Snail-deficient wild-type pre-adhesion MEE cells, Snail-expressing TGF-beta(3) mutant MEE cells survive as they do their counterparts in the chick embryo. Interestingly, Slug is the Snail family member expressed in the chick MEE, providing another example of interchange of Snail and Slug expression between avian and mammalian embryos. We propose that in the absence of TGF-beta(3), TGF-beta(1) is upregulated in the mesenchyme, and that in both physiological (avian) and pathological (TGF-beta(3)-/-mammalian) cleft palates, it induces the expression of Snail genes promoting the survival of the MEE cells and permitting their subsequent differentiation into keratinized stratified epithelium.     

The role of Fgf10 signaling in branching morphogenesis and gene expression of the rat prostate gland: lobe-specific suppression by neonatal estrogens

Brief exposure of rats to high-dose estrogen during the neonatal period interrupts prostate development in a lobe-specific manner and predisposes the gland to dysplasia with aging, a phenomenon referred to as developmental estrogenization. Our previous studies have revealed that these effects are initiated through altered steroid receptor expression; however, the immediate downstream targets remain unclear. We have recently shown that developmental expression of Shh-ptc-gli is downregulated in the dorsolateral prostate following estrogenization, and this is responsible, in part, for branching deficits observed in that prostatic region specifically. In the present study, we examine the role of Fgf10 signaling during rat prostate development and as a mediator of the developmental estrogenized phenotype. Fgf10 and FgfR2iiib localize to the distal signaling center of elongating and branching ducts in separate prostate lobes where they regulate the expression of multiple morphoregulatory genes including Shh, ptc, Bmp7, Bmp4, Hoxb13, and Nkx3.1. Ventral and lateral lobe organ cultures and mesenchyme-free ductal cultures demonstrate a direct role for Fgf10/FgfR2iiib in ductal elongation, branching, epithelial proliferation, and differentiation. Based on these findings, a model is proposed depicting the localized expression and feedback loops between several morphoregulatory factors in the developing prostate that contribute to tightly regulated branching morphogenesis. Similar to Shh-ptc-gli, neonatal estrogen exposure downregulates Fgf10, FgfR2iiib, and Bmp7 expression in the dorsolateral prostate while ventral lobe expression of these genes is unaffected. Lateral prostate organ culture experiments demonstrate that growth and branching inhibition as well as Fgf10/FgfR2iiib suppression are mediated directly at the prostatic level. Furthermore, exogenous Fgf10 fully rescues the growth and branching deficits due to estrogen exposure. Together, these studies demonstrate that alterations in Fgf10 signaling are a proximate cause of Shh-ptc-gli and Bmp7 downregulation that together result in branching inhibition of the dorsolateral prostate following neonatal estrogen exposure.     

Fgf8 expression in the Tbx1 domain causes skeletal abnormalities and modifies the aortic arch but not the outflow tract phenotype of Tbx1 mutants

Fgf8 and Tbx1 have been shown to interact in patterning the aortic arch, and both genes are required in formation and growth of the outflow tract of the heart. However, the nature of the interaction of the two genes is unclear. We have utilized a novel Tbx1(Fgf8) allele which drives Fgf8 expression in Tbx1-positive cells and an inducible Cre-LoxP recombination system to address the role of Fgf8 in Tbx1 positive cells in modulating cardiovascular development. Results support a requirement of Fgf8 in Tbx1 expressing cells to finely control patterning of the aortic arch and great arteries specifically during the pharyngeal arch artery remodeling process and indicate that the endoderm is the most likely site of this interaction. Furthermore, our data suggest that Fgf8 and Tbx1 play independent roles in regulating outflow tract development. This finding is clinically relevant since TBX1 is the candidate for DGS/VCFS, characterized clinically by variable expressivity and reduced penetrance of cardiovascular defects; Fgf8 gene variants may provide molecular clues to this variability.