Keystone genes of mammalian tooth patterning and quantification of their expression

Abstract

Organ development is regulated by complex interactions of multiple regulatory pathways. These pathways (Wnt, Tgfβ, Fgf, Hh, Eda, Notch) are becoming increasingly better known, with many identified genes having well-characterized effects on the phenotype. We classify genes required for normal organogenesis into different categories that range from essential to subtle modification of the phenotype. We focus on the mouse tooth development in which over 70 genes are known to be required for normal odontogenesis. These genes were classified into progression, shape, and tissue categories based on whether their null mutations cause early developmental arrests, altered morphologies, or hard tissue defects, respectively. Collectively, we call these here the developmental keystone genes. Additionally, we identified 100 developmental genes with no phenotypic effects on molars when null mutated, thereby providing the means to contrast expression dynamics between keystone and non-keystone genes. Transcriptome profiling using microarray and RNAseq analyses of patterning stage mouse molars show elevated expression levels for progression and shape genes, the former category showing the most significant upregulation. Single-cell RNAseq analyses reveal that even though the size of the expression domain, measured in number of cell, is the main driver of organ-level expression, the progression genes show high cell-level transcript abundances. Furthermore, progression genes, but not shape genes, are upregulated even when compared to the other genes in the pathways into which they belong. The lower upregulation of the shape category is found to be due to the high proportion of locally secreted ligands among the shape genes. We postulate that regulation of shape is highly dependent on spatially diffusing ligands, which are generally more locally expressed compared to their receptors and intracellular components. The combination of phenotypically defined gene categories and organ transcriptomes allow the characterization of the expression dynamics underlying different aspects of organogenesis.