Orofacial clefting is one of the most common human birth defects (1-2 per 1000 live births). The social impact of these disorders is substantial with great financial and psychological costs to individuals. Approximately 70% of all clefting cases are isolated non-syndromic cases which exhibit complex, multifactorial inheritance with both multigenic and environmental contributions underlying its etiology. Pierre Robin Sequence (PRS)-type cleft palate (CP) is a common palate-extrinsic mechanism of clefting in which mandibular hypoplasia initiates a developmental sequence of events that culminates in failed palate shelf elevation and fusion and CP. Model organisms with gene mutations that result in craniofacial defects provide powerful in-roads into the cellular, molecular and developmental mechanisms at play during normal craniofacial development. We have shown that the Prdm16 transcription factor gene is required for normal palatogenesis in a novel recessive ENU- induced CP mutant, cleft secondary palate 1 (csp1). These mutants exhibit anterior-specific mandible hypoplasia and palate shelf elevation defects that model PRS-type CP. We have since generated and characterized a novel """"""""conditional"""""""" gene trap null Prdm16 allele upon which this proposal is based. Previous studies and preliminary mandible-specific Prdm16 ablation studies support our hypothesis that palate- extrinsic Prdm16 function in the mandible is necessary and sufficient to allow normal regulation of downstream genes'expression required for formation of an intact secondary palate. Prdm16 has leukemogenic potential, a role in the maintenance of hematopoietic and neuronal stem cell progenitors and a well-established role in promoting brown adipose tissue identity versus skeletal muscle. Its paralog Mds1 and Evi1 complex locus (Mecom) is critical for mouse embryogenesis beyond embryonic day (E) 10.5 and hematopoietic stem cell function. A role for Mecom during mouse embryonic palate and mandible development has not been studied, although a loss-of-function Mecom allele results in widespread hypocellularity that, in part, affects neural crest-derived craniofacial prominences. In zebrafish prdm16 and prdm3 interact during development of the neurocranium and viscerocranium (jaw). Since Prdm16 and Mecom transcript isoforms are paralogous, and they are co-expressed in developing mandible and palate, we hypothesize that Prdm16 AND Mecom function together to regulate or """"""""fine-tune"""""""" signaling pathways critical during mouse embryonic craniofacial development. To this end, we will assess the mechanism of clefting in Prdm16 null mutants (Aim 1). We also aim to identify tissue-specific Prdm16-dependent genes and direct transcriptional targets using comparative transcriptional profiling and targeted chromatin Immunoprecipitation-qPCR (ChIP- qPCR) studies in wild type versus null mutant embryonic mandible tissue (Aim 2). Finally, we will test our hypothesis that """"""""Prdm16 and Mecom genetically interact"""""""" through double heterozygous intercross breeding of mice that carry Prdm16 and Mecom null alleles to generate embryos with varying gene dosage to be assessed for CP and abnormal craniofacial phenotypes (Aim 3).
Orofacial clefts, including non-syndromic cleft palate, are one of the most common human birth defects, occurring in approximately 1-2 per 1000 live births on average. We have demonstrated through our studies of chemically-induced and loss-of-function Prdm16 mouse mutants that normal palate development is dependent upon Prdm16 function in the mandible, where Prdm16 is required for its proper growth and patterning and subsequently, palate shelf elevation and fusion. We aim to further characterize the molecular and developmental consequences of Prdm16 loss through 1) mandible- and palate shelf-specific inactivation of Prdm16 at the earliest stages of mandible and palate formation;2) identification of altered expression of genes and pathways between control and Prdm16 null mutant embryos prior to the onset of cleft palate and 3) characterization of a role for the Prdm16 paralog, Mecom, during embryonic craniofacial development and genetic interactions between them. These studies will contribute to our general knowledge of the mandible and palate development and likely uncover additional genetic contributors to the etiology of human clefting.
|Strassman, Alexander; Schnütgen, Frank; Dai, Qi et al. (2017) Generation of a multipurpose Prdm16 mouse allele by targeted gene trapping. Dis Model Mech 10:909-922|