Cranial neural crest cells (CNCCs) delaminate from embryonic neural folds and migrate to pharyngeal arches, which give rise to most mid-facial structures. CNCC dysfunction plays a prominent role in the etiology of orofacial clefts, a frequent birth malformation. Heterozygous mutations in SPECC1L have been identified in patients with atypical and syndromic clefts. Here, we report that in SPECC1L-knockdown cultured cells, staining of canonical adherens junction (AJ) components, β-catenin and E-cadherin, was increased, and electron micrographs revealed an apico-basal diffusion of AJs. To understand the role of SPECC1L in craniofacial morphogenesis, we generated a mouse model of Specc1l deficiency. Homozygous mutants were embryonic lethal and showed impaired neural tube closure and CNCC delamination. Staining of AJ proteins was increased in the mutant neural folds. This AJ defect is consistent with impaired CNCC delamination, which requires AJ dissolution. Further, PI3K-AKT signaling was reduced and apoptosis was increased in Specc1l mutants. In vitro, moderate inhibition of PI3K-AKT signaling in wildtype cells was sufficient to cause AJ alterations. Importantly, AJ changes induced by SPECC1L-knockdown were rescued by activating the PI3K-AKT pathway. Together, these data indicate SPECC1L as a novel modulator of PI3K-AKT signaling and AJ biology, required for neural tube closure and CNCC delamination.

Gene trap mutagenesis is a powerful tool to create loss-of-function mutations in mice and other model organisms. Modifications of traditional gene trap cassettes, including addition of conditional features in the form of Flip-excision (FlEx) arrays to enable directional gene trap cassette inversions by Cre and Flpe site-specific recombinases, greatly enhanced their experimental potential. By taking advantage of these conditional gene trap cassettes, we developed a generic strategy for generating conditional mutations and validated this strategy in mice carrying a multipurpose allele of the Prdm16 transcription factor gene. We demonstrate that the gene trap insertion creates a null mutation replicating the Pierre Robin sequence-type cleft palate phenotype of other Prdm16 mutant mice. Consecutive breeding to Flpe and Emx1IREScre deleter mice spatially restricted Prdm16 loss to regions of the forebrain expressing the homeobox gene Emx1, demonstrating the utility of the technology for the analysis of tissue-specific gene functions.

SPECC1L mutations have been identified in patients with rare atypical orofacial clefts and with syndromic cleft lip and/or palate (CL/P). These mutations cluster in the second coiled-coil and calponin homology domains of SPECC1L and severely affect the ability of SPECC1L to associate with microtubules. We previously showed that gene-trap knockout of Specc1l in mouse results in early embryonic lethality. We now present a truncation mutant mouse allele, Specc1lΔC510, that results in perinatal lethality. Specc1lΔC510/ΔC510 homozygotes showed abnormal palate rugae but did not show cleft palate. However, when crossed with a gene-trap allele, Specc1lcGT/ΔC510 compound heterozygotes showed a palate elevation delay with incompletely penetrant cleft palate. Specc1lcGT/ΔC510 embryos exhibit transient oral epithelial adhesions at E13.5, which may delay shelf elevation. Consistent with oral adhesions, we show periderm layer abnormalities, including ectopic apical expression of adherens junction markers, similar to Irf6 hypomorphic mutants and Arhgap29 heterozygotes. Indeed, SPECC1L expression is drastically reduced in Irf6 mutant palatal shelves. Finally, we wanted to determine if SPECC1L deficiency also contributed to non-syndromic (ns) CL/P. We sequenced 62 Caucasian, 89 Filipino, 90 Ethiopian, 90 Nigerian and 95 Japanese patients with nsCL/P and identified three rare coding variants (p.Ala86Thr, p.Met91Iso and p.Arg546Gln) in six individuals. These variants reside outside of SPECC1L coiled-coil domains and result in milder functional defects than variants associated with syndromic clefting. Together, our data indicate that palate elevation is sensitive to deficiency of SPECC1L dosage and function and that SPECC1L cytoskeletal protein functions downstream of IRF6 in palatogenesis.

The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (ϳ53-kDa and ϳ47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a K m value for PABA-GLU of 60 ؎ 0.08 M and a specific activity of 63,300 ؎ 600 nmol min ؊1 mg ؊1 . Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.Reduced derivatives of folic acid are required for biosynthesis of DNA, RNA, amino acids, and other important cellular components (14). Folic acid is an essential dietary supplement for humans, while both microorganisms and plants can synthesize this vitamin de novo. The Escherichia coli folic acid biosynthetic pathway is composed of proteins encoded by genes scattered across the chromosome (9); these genes appear to be constitutively expressed at low levels (26,27). The genes and enzymes involved in folate catabolism in E. coli remain largely unidentified.The abg region of E. coli was first identified in a search for mutants able to grow on folic acid in order to circumvent paminobenzoate (PABA) auxotrophy; characterization showed that the auxotrophs were utilizing the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU), not folic acid ( Fig. 1) (11). The original mutations were point mutations in the intergenic region between abgA and abgR, and it was hypothesized that this resulted in increased expression of the abgABT genes. The abg region, named for enhanced growth on p-aminobenzoyl-glutamate, was mapped to 30 min and was found to consist of a potential operon including abgA, abgB, and abgT. Divergently oriented from abgABT, abgR encodes AbgR, which has homology to LysR-type regulatory proteins (21). Sequence analysis of the putative gene products revealed that AbgA and AbgB were similar to one another and to aminoacyl aminohydrolases and that AbgT was similar to transport proteins (11).Previously, we had found that wild-typ...

Objective To compare the effects of dietary fat and sex on murine oral squamous cell carcinoma pathology. Materials and methods Male and female C57Bl/6 mice (36/sex) received a low‐fat (10 kcal%) or high‐fat (60 kcal%) diet. Water (control), vehicle, or 4‐nitroquinoline‐1‐oxide in vehicle (50 μg/ml) was provided for 17 weeks followed by six additional weeks of water. Oral lesion development was recorded weekly. Histopathologic changes in tongues were examined, and T cells (CD3+), macrophages (CD68+), and neutrophils (Ly6+) were quantified. Results All 4‐nitroquinoline‐1‐oxide‐treated mice developed oral tumors. High‐fat diet exacerbated pathology, demonstrated by an increased final tumor burden (10.9 ± 4.5 vs. 7.9 ± 2.5, mm/mouse, p < .05; high‐fat diet vs. low‐fat diet, respectively), and a greater histopathology score. When dietary groups were combined, 4‐nitroquinoline‐1‐oxide‐treated males displayed higher histopathology scores than females (4.2 ± 0.3 vs. 3.6 ± 0.2, respectively, p < .05). Lymphoid cell infiltration was greater in the 4‐nitroquinoline‐1‐oxide mouse tongues than controls: T cells (14.0 vs. 0.96 cells/mm2), macrophages (3.6 vs. 1.8 cells/mm2), and neutrophils (12.0 vs. 0.38 cells/mm2). Conclusion High‐fat diet and male sex increased the pathology of 4‐nitroquinoline‐1‐oxide‐induced oral cancer. Elevated lymphoid cell infiltration contributed to disease pathology.