Differential gene and transcript expression in E18.5 murine maxillo-mandibular complex

Melissa A. Morawski

doi:10.25820/etd.007188

Objectives: Most individuals demonstrate some degree of facial asymmetry, varying from mild to severe right-left asymmetries. While studies have been performed that investigate gene expression during facial development, there is limited information about the differential gene expression between the right and left sides of the craniofacial complex during various stages of embryogenesis. The purpose of this study is to investigate the right and left differential gene and transcript expression in the wildtype E18.5 murine maxilla-mandibular complex. It is hypothesized that there will be differential gene and transcript expression between the right and left side of the craniofacial complex. This study will provide a baseline of normal differential gene expression, which can be used in future studies to analyze differential gene expression in mice with significant craniofacial asymmetries. Additionally, this investigation may be further used to analyze differential gene expression in humans and give insight into the development of human craniofacial anomalies. Methods: Three (n=3) E18.5 C57BL6 murine embryos were studied in this investigation. The maxilla-mandibular complex of each embryo was sectioned in the mid-palatal plane. RT-PCR analysis was completed to confirm equal housekeeping genes in the right and left samples. RNA samples were isolated with Qiazol mRNA easy kit, and mRNA sequencing was completed at LC Sciences in Houston, Texas. Gene and transcript analysis was completed, selecting for >1 or <-1 log fold change, a q-score < 0.05, and at least two FPKM values greater than 0.5. Other studies on the genetic contribution to craniofacial microsomia and asymmetry studies were referenced. The Mouse Genome Informatics (MGI) database was referenced to identify and narrow down genes and transcripts associated with known murine craniofacial phenotypes, and the Online Mendelian Inheritance in Man (OMIM) database was utilized to identify human craniofacial phenotypes associated with these genes and transcripts. GnomAD constraint scores were utilized to analyze the differentially expressed genes and transcripts with constraint scores of interest in humans. Finally, functional enrichment analysis was performed to determine what roles the genes and transcripts with murine craniofacial phenotypes play in biological processes. Results: Data analysis resulted in the identification of numerous upregulated and downregulated genes and transcripts with known murine craniofacial phenotypes. More specifically, genes with known murine craniofacial phenotypes include upregulated gene Hmx1, and downregulated genes Krt36 and Hand2. Upregulated transcripts filtered for murine craniofacial phenotypes include Hmx1, Fes, Hps1, Lmna, Mast4, Npepps, Ptch1, and Satb2. Downregulated transcripts with murine craniofacial phenotypes include Krt36, Hand2, Acan, Acp4, Aff4, Amelx, Cdkn1c, Chl1, Crkl, Ctnnb1, Prr3, Plcb1, Pomgnt1, Rnf146, Rps6ka3, Sirt1, Zeb2, and Frmd4a (Frmd4a craniofacial phenotype only found in OMIM, not MGI). Human craniofacial phenotypes were also determined for these differentially expressed genes and transcripts. To analyze how important these differentially expressed genes and transcripts are with regards to the human phenotypes, constraint scores were determined. That is, genes and transcripts were selected for those with loss of function (LOF) scores 0.31 and between 0.31-0.60, and missense scores >3.0 since these genes and transcripts would result in more significant phenotypes. Transcripts with LOF  0.31 include upregulated transcripts (Lmna, Npepps, Ptch1, and Satb2) and downregulated transcripts (Acan, Aff4, Ctnnb1, Plcb1, Rps6ka3, Zeb2, and Frmd4a). Transcripts with LOF scores between 0.31-0.60 include upregulated transcript Mast4 and downregulated transcripts (Cdkn1c, Rnf146, and Sirt1). Finally, transcripts with missense scores >3.0 include upregulated transcripts (Npepps and Satb2) and downregulated transcripts (Ctnnb1, Plcb1, Rps6ka3, and Zeb2). Conclusions: Differential right and left gene and transcript expression was investigated in wildtype E18.5 murine craniofacial complex. It was hypothesized that differential gene and transcript expression would be found in the 3 subjects studied. Differentially expressed genes and transcripts were identified in the murine embryos, many of which were also previously identified in the E14.5 murine craniofacial complex. Many differentially expressed genes and transcripts are also associated with the cleft lip and palate phenotype, as well as genes previously associated with craniofacial microsomia. Several genes and transcripts identified are also linked to syndromes that have characteristic craniofacial phenotypes and anomalies. Many of the differentially expressed genes and transcripts also have clinically significant constraint scores in humans, likely resulting in more significant phenotypes. The differential expression found in the wildtype E18.5 murine samples likely relates to differential growth of the right versus left sides of the maxilla-mandibular complex since E18.5 is past the stage of embryonic facial development in mice. This data can be used as a baseline in future studies to identify normal versus abnormal differential expression, and possibly identify a genetic explanation to craniofacial microsomia and significant right-left facial asymmetries in humans.

Differential gene and transcript expression in E18.5 murine maxillo-mandibular complex

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