Dmrt2 and Hmx2 direct intercalated cell diversity in the mammalian kidney through antagonistic and supporting regulatory processes.

Abstract

Intercalated cells (ICs) in the mammalian kidney regulate circulatory pH through IC subtype-restricted actions of bicarbonate transporters: pH is elevated by Slc4a1 restricted to type A-ICs (A-ICs) and depressed by Slc26a4 in type B-IC (B-ICs). NonA-nonB-ICs (nA/nB-ICs) also produce Slc26a4 though their function is unclear. Though both nephron and ureteric progenitor lineages generate A-ICs, the former also generates nA/nB-ICs and the latter B-ICs. Lineage and cell type restricted transporter gene expression in the mouse and human kidney is preceded by expression of the transcriptional regulators <i>Dmrt2/DMRT2</i> in A-ICs, and either, or both, <i>Hmx2/HMX2</i> and <i>Hmx3/HMX3</i> in B- and nA/nB ICs. CRISPR/Cas9-directed removal of <i>Dmrt2</i> and the linked <i>Hmx2</i>/<i>Hmx3 genes</i> resulted in IC-subtype switching. A-ICs adopted an <i>Hmx2⁺/Slc26a4⁺</i> B-IC cell fate on Dmrt2 removal while B-ICs initiated a Dmrt2<i>⁺</i>/Slc4a1<i>⁺</i> A-IC program on <i>Hmx2/Hmx3</i> removal. Triple knockout of <i>Dmrt2</i>, <i>Hmx2</i>, and <i>Hmx3</i> resulted in hybrid ICs expressing both <i>Slc4a1</i> and <i>Slc26a4</i>. Thus, restricted expression of these regulators is essential for specifying IC subtypes. To explore these mechanisms, <i>Hmx2</i> and <i>Dmrt2</i> were activated ectopically in ureteric organoid cultures. Introduction of <i>Foxi1</i>-a pan determinant of ICs-activated early <i>Dmrt2⁺</i> A-IC development while cointroduction of <i>Hmx2</i> silenced Foxi1-dependent <i>Dmrt2</i> expression and led to an upregulation of Slc26a4. In contrast, coexpression of <i>Foxi1</i> and <i>Dmrt2</i> upregulated <i>Slc4a1</i>. These data support a model in which mutually repressive interactions between <i>Dmrt2</i> and <i>Hmx2/3</i> establish distinct IC identities and ongoing activity of these factors supports gene regulatory programs specific to each IC subtype.