Gestational diabetes (GDM), or hyperglycemia that first manifests during pregnancy, worsens pregnancy outcomes and long-term health risks for both a mother and her offspring. As with all types of diabetes, a relative insufficiency of functional pancreatic ß-cells is a fundamental defect contributing to GDM. Normally, ß-cells adapt to the metabolic challenges of pregnancy by expanding ß-cell mass. This expanded mass regresses in the postpartum period. Thus, pregnancy is a unique physiologic condition that occurs in a fully-developed adult and requires rapid, dynamic changes in ß-cell mass. Unfortunately, the mechanisms of normal gestational ß-cell adaptation and the defects underlying GDM are poorly understood. Our long-term goal is to understand the mechanisms regulating ß-cell proliferation and mass during pregnancy, in order to leverage that knowledge for therapeutic expansion of ß-cells in all types of diabetes. Building on our work establishing that loss of prolactin receptor (PRLR) signaling in ß-cells results in GDM, we recently identified novel PRLR differentially expressed genes (PRLR-DEGs) and key transcriptional regulators of PRLR-DEG expression. The objective of this grant is to precisely define how PRLR regulates ß-cell gene expression during pregnancy and the postpartum period. We propose the central hypothesis that PRLR signaling orchestrates an anticipatory transcriptional program of ß-cell mass expansion during gestation and survival of adequate ß-cell mass during postpartum regression. We will test this hypothesis with the following Specific Aims: (1) elucidate transcriptional mechanisms regulating PRLR-DEGs within ß-cells during pregnancy. To do so, we will use ChIP-seq and ATAC-seq to examine how PRLR-DEGs are regulated in mouse and human islets during pregnancy or in response to prolactin stimulation. In Aim (2) we will define PRLR signaling-dependent and -independent ß-cell subpopulations during pregnancy using single-cell RNA sequencing, lineage tracing and colocalization studies. For Aim (3) we will identify mechanisms of ß-cell survival during ß-cell mass regression in the early postpartum period through pulse-chase labeling and lineage tracing of ß-cells that proliferated during pregnancy, as well as examine how inducible loss of PRLR specifically within the postpartum period affects ß-cell mass. Together, results from these studies will reveal transcriptional mechanisms downstream of PRLR and illuminate unique aspects of gestational proliferation (Aim 1), define ß-cell subpopulations spatially and temporally across pregnancy (Aim 2), and establish a new role for PRLR signaling in regulation of ß-cells postpartum (Aim 3). Our research is significant because these findings would clarify mechanisms of gestational ß-cell adaptation and expand our understanding of how PRLR activation regulates transcription. At a fundamental level, these studies will expand our understanding of the mechanisms regulating dynamic changes in ß-cell mass, which may identify novel strategies to promote ß-cell expansion for therapeutic purposes.