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.

This study is part of a larger project examining the role of Prdm16, a transcriptional cofactor involved in TGF‐β signaling pathways, on craniofacial development. Prdm16 is expressed in various tissues, including cranial neural crest and the brain. Two, Prdm16 mutant mouse models, cleft secondary palate‐1 on a C57Bl6/J background strain (csp1) and Prdm16 conditional gene trap on an FVB/NJ background strain (cGT), display non‐syndromic clefting of the secondary palate and micrognathia due to loss of Prdm16 function. Our earlier pilot project on adult cGT and csp1 mice revealed that cGT heterozygous mutants have a relatively narrower skulls compared to their wild type (wt) littermates and that csp1 heterozygous mutants have relatively wider skulls than their wt counterparts. Homozygous mutants of both strains die shortly after birth. The aim of this study is to quantitatively assess variation in bony neurocranial development in newborn het and mut csp1 and cGT mice and contrast this with the wt morphology to determine if variation in degree of bone development in these strains is apparent at the newborn stage. Crania of newborn heterozygous mutant, homozygous mutant, and wt csp1 and cGT mice were microCT scanned and the resulting 2D images were volume‐merged to create a 3D model for each specimen. The neurocranial bones from each 3D model were segmented into separate images for analysis. For each specimen, we calculated the surface area and volume of each neurocranial bone and statistically compared these values among the genotype groupings. Smaller bone surface areas and volumes were interpreted as indicating less bone development. Homozygous mutant mice of both strains had significantly less developed neurocranial bones than their wt counterparts. The heterozygous mutant mice in both strains displayed substantial variation in degree of bone development. The calvarial bones were especially under‐developed in homozygous mutant csp1 and cGT mice, but the occipital and alisphenoid bones were well‐developed in most specimens. Our results suggest that reduced Prdm16 expression in newborn homozygous mutant csp1 and cGT mice correlates with overall underdevelopment of the neurocranium compared to wt mice. Bone development patterns in the heterozygous mutant mice are less apparent and require additional analysis. These findings complement our previous research on adult csp1 and cGT mice and additional insight on the downstream effects of Prdm16 expression on craniofacial development. Support or Funding Information NIH/NIDCR (R15DE023982) and Midwestern University Core Facility

Orofacial clefts are the most common congenital craniofacial anomaly affecting humans. Non‐syndromic clefts may result from decreased integration of the palate during fetal facial growth. This study will investigate whether this pattern is detectable in individuals who do not have a cleft, but do have a child with an orofacial cleft (and therefore carry genetic risk factors for clefting). In order to examine this, we hypothesize that 1) facial shape differences exist between unaffected parents of children with clefts (cases) compared to adults with no history of clefting (controls), and 2) cases will have lower facial integration values compared to controls.For this study, 3D facial scans were obtained from the University of Pittsburgh Center for Craniofacial and Dental Genetics (CCDG). 34 landmarks were placed on n=230 case faces of unaffected parents from the Pitt Orofacial Cleft Study and n=490 control faces from the Pitt 3D Facial Norms Study. Landmarks were aligned using Generalized Procrustes Analysis (GPA) and geometric morphometrics were used for quantifying facial phenotypes. Facial shape differences as a function of age were removed prior to all analyses using linear regression. Canonical Variate Analysis (CVA) was employed to examine shape differences in three different subsets of facial landmarks: a midfacial nose and mouth dataset, a lower facial mouth and jaw dataset and a third dataset consisting of the nose and jaw. Two Block Partial Least Squares analysis (2B PLS) was used to measure the strength of integration by comparing RV coefficients between the case and control populations.Results from the CVA identify significant differences in facial morphology, including jaw width, chin projection, nasal height and mouth width, between cases and controls across all datasets (nose/mouth midfacial set p<0.001, mouth/jaw lower facial set p<0.001, and nose/jaw dataset p=0.028). 2B PLS results, however, showed overall equivalent or higher integration levels in case faces compared to the controls. When comparing levels of integration within the midface (nose and mouth dataset), case samples demonstrate significant (p<0.001) overall higher integration levels (RV=0.352) compared to control samples (RV=0.301). This pattern is also seen within the nose and jaw dataset, with cases (RV=0.614) showing increased integration compared to controls (RV=0.554). Interestingly, integration levels within the lower facial mouth and jaw dataset were nearly identical in both case samples (RV=0.444) and controls (RV=0.447).While facial shape differences exist between unaffected cleft relatives and controls with no family history of clefting, integration values were actually higher in case samples compared to control samples. This indicates that integration in facial soft tissue structures may not serve as a good indicator of cleft risk. Future studies will examine skeletal structures (such as the maxilla and mandible) to test if differences in integration are quantifiable in the facial skeleton only.Support or Funding InformationNIDCR: R01‐DE016148, U01‐DE020078This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.