|Title:||Single cell RNA analysis of the left-right organizer transcriptome reveals potential novel heterotaxy genes|
Bellechambers, Helen Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine; Amruta R. Phatak, 1Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine; Mardi J. Nenni, Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center; Maria B. Padua, Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine; Hongyu Gao, Department of Medical and Molecular Genetics, Indiana University School of Medicine; Yunlong Liu, Department of Medical and Molecular Genetics, Indiana University School of Medicine; Stephanie M. Ware, Herman B Wells Center for Pediatric Research, Department of Pediatrics, Department of Medical and Molecular Genetics, Indiana University School of Medicine
Background/Significance/Rationale: Heterotaxy is a rare syndrome characterized by organ laterality defects including complex heart defects. Heterotaxy arises early in development, prior to organogenesis, due to defects in formation and/or function of a transient structure called the left-right organizer (LRO). We aimed to use single cell RNA sequencing (scRNA-seq) to define the LRO transcriptome.
Methods: Mouse embryos were collected at the 0-1 somites stage of development, during establishment of LR patterning. Three embryos were dissociated to form single cell suspensions and processed via the 10x genomics pipeline to generate transcriptional profiles of 23,533 cells. Cell clusters and LRO specific genes were identified with the R package Seurat.
Results/Findings: By examining the expression of previously identified LRO specific genes such as Noto and Shh we were able to identify 238 LRO cells and show that these cells formed a distinct cluster separate from related cell types such as the notochord and definitive endoderm. We identified 196 genes with LRO specific expression, of which 127 were potential novel LRO genes.
Conclusions/Discussion: Study of heterotaxy has historically been technically challenging due to the transient nature of the LRO, a limited cell number, and difficulties in early embryonic manipulations. Single cell technology has enabled transcriptomic analysis at the single cell level, overcoming these issues. We utilized this technology to generate a novel LRO specific gene list; a useful resource for researchers interested in genes potentially involved either in LR patterning or the origins of heterotaxy.
Translational/Human Health Impact: The genetic cause of heterotaxy is currently diagnosed in less than 40% of the cases by clinical genetic testing. The list of novel LRO genes will be a useful resource for further studies on the genetic causes of heterotaxy and thus improve genetic diagnosis rate in patients.