Islet α-, β-, and δ-cell development is controlled by the Ldb1 coregulator, acting primarily with the islet-1 transcription factor

Abstract

Ldb1 and Ldb2 are coregulators that mediate Lin11-Isl1-Mec3 (LIM)-homeodomain (HD) and LIM-only transcription factor-driven gene regulation. Although both Ldb1 and Ldb2 mRNA were produced in the developing and adult pancreas, immunohistochemical analysis illustrated a broad Ldb1 protein expression pattern during early pancreatogenesis, which subsequently became enriched in islet and ductal cells perinatally. The islet-enriched pattern of Ldb1 was similar to pan-endocrine cell-expressed Islet-1 (Isl1), which was demonstrated in this study to be the primary LIM-HD transcription factor in developing and adult islet cells. Endocrine cell-specific removal of Ldb1 during mouse development resulted in a severe reduction of hormone⁺ cell numbers (i.e., α, β, and δ) and overt postnatal hyperglycemia, reminiscent of the phenotype described for the Isl1 conditional mutant. In contrast, neither endocrine cell development nor function was affected in the pancreas of Ldb2(-/-) mice. Gene expression and chromatin immunoprecipitation (ChIP) analyses demonstrated that many important Isl1-activated genes were coregulated by Ldb1, including MafA, Arx, insulin, and Glp1r. However, some genes (i.e., Hb9 and Glut2) only appeared to be impacted by Ldb1 during development. These findings establish Ldb1 as a critical transcriptional coregulator during islet α-, β-, and δ-cell development through Isl1-dependent and potentially Isl1-independent control.

FIG. 1.

Ldb1 is enriched in islet and ductal cells. A: Ldb1 (left) and Ldb2 (middle) mRNA expression was visualized by RNA ISH under identical conditions in E15.5 tissue. A higher-magnification view of pancreatic Ldb1 and Ldb2 expression is shown on the right. B: qPCR was performed to measure Ldb1, Ldb2, and Isl1 mRNA levels in E15.5 total pancreas (black bars) and 3-month-old isolated islets (gray bars). Expression levels are displayed relative to TATA-binding protein (TBP), which is set as onefold. Error bars represent ± SEM (n = 5). Ldb1 mRNA is significantly more abundant than Ldb2 in E15.5 and adult samples. C–K: Ldb1, Pdx1, Isl1, hormone (insulin, glucagon, and somatostatin), and ductal (DBA, CK-19) markers were visualized at E10.5, E18.5, P5, and P21 by coimmunofluorescence. Yellow dashed lines mark dorsal and ventral pancreas domains in C and D. Notably, only a few of the pancreatic Ldb1⁺ cells in D are copositive for Isl1 at this stage (some marked by white arrowheads). L: Immunohistochemical analysis illustrates enriched Ldb1 protein (brown) expression in adult human islet cells; the sample is eosin (pink) counterstained. *P < 0.05. D, dorsal pancreas; du, duct; G, gut tube; in, intestine; k, kidney; L, liver; M, mesenchyme; nt, neural tube; P, pancreas (outlined with red dashed line); st, stomach; V, ventral pancreas. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Analysis of LIM-HD and Lmo levels in the developing and adult pancreas. A: LIM-HD and Lmo mRNA expression was measured by qPCR in the E15.5 pancreas (black bars) and 3-month-old islets (gray bars). Values are relative to TATA-binding protein (TBP), set at onefold. Error bars represent ± SEM (n = 5). B: Lmo4 (brown) and insulin (red) staining was performed on E18.5 and P21 pancreas tissue. Lmo4 was not found in hormone⁺ cells. C: Lmo4 (brown) colocalized with the ductal CK-19 (red) marker in E18.5 and P6 pancreata. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Deletion of Ldb1 in endocrine hormone⁺ cells causes reduced pancreatic hormone production, postnatal islet loss, and hyperglycemia in vivo. A: Six-hour fasting blood glucose levels in littermate control and Pax6-Cre;Ldb1^F/F pups. Horizontal bars indicate mean blood glucose values within each genotype, and values were significantly different between control and mutants at all ages (P < 0.0001). Insulin (brown) and eosin (pink) staining at P6 and P21 in the control (B and C) and Pax6-Cre;Ldb1^F/F (D and E) pancreas. F: Islet hormone insulin, glucagon, and somatostatin (SST) mRNA levels are significantly reduced in E18.5 Ldb1 mutant pancreata (n = 4–6). Data are presented relative to littermate controls, which are set at onefold and marked by the dashed line. Error bars represent ± SEM. The number of pancreatic insulin⁺ (green), glucagon⁺ (red), and somatostatin⁺ (white) cells is greatly reduced between E18.5 control (G and H) and Pax6-Cre;Ldb1^F/F (I and J) tissue. The insets in H and J show a magnified view of the cell clusters. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Isl1-regulated MafA and Arx expression is greatly reduced in the E18.5 Ldb1 mutant pancreas. A: mRNA levels of islet-enriched transcription factors in E18.5 littermate control (blue bars) and Pax6-Cre;Ldb1^F/F (red bars) pancreas (n = 4–6). Littermate control mRNA level was set at onefold ± SEM. Immunostaining levels of β-cell MafA (red) (B) and α-cell Arx (red) (C) were greatly reduced in E18.5 Pax6-Cre;Ldb1^F/F pancreata. Arrowheads in C mark Arx⁺ glucagon⁺ (white, top) or Arx⁻ glucagon⁺ cells (yellow, bottom), with some magnified hormone⁺ cell clusters shown. D: ChIP analysis of Ldb1 binding to MafA Region 3 (top) as well as Arx Re1 and Re2 (bottom). The PEPCK promoter served as the negative background control. Dilute input as well as Ldb1- and IgG-enriched DNA were analyzed by PCR using βTC-3 and αTC-6 chromatin isolated from whole-cell extract (WCE) and/or nuclear extract (NE). H₂O control serves as a negative control for the PCR. E: Binding between endogenous Ldb1 and Isl1 were found in coimmunoprecipitation experiments using βTC-3 nuclear extracts, whereas Ldb1 and Isl1 did not bind to Pdx1, NeuroD1, Hnf1α, MafA, or Pax6. Diluted βTC-3 nuclear extract served as input positive control (1%), and immunoprecipitation (IP) results were compared with species-matched IgG treatments. F: Dominant-negative acting Ldb1ΔN significantly reduced MafA region 3-driven reporter expression in βTC-3 cells. Data are presented as mean fold reporter activity, with the empty pFox-Luc + CMV cotransfection set at onefold ± SEM; n = 3. *P < 0.05, **P < 0.01. Blot, immunoblot antibody probe. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Glp1r is a novel Ldb1- and Isl1-activated target gene. A: qPCR quantification of Isl1 ChIP-Seq candidates from Ldb1- (blue bars) and Isl1-deficient (red bars) E18.5 pancreata (n = 4–6). Data are presented as fold of the littermate control, which was set at 1 (marked by the dashed line), ± SEM. B and C: Immunostaining of Glp1r (brown) and insulin (red) at P6 illustrates reduced Glp1r protein levels in insulin⁺ cells lacking Ldb1 or Isl1. D: βTC-3 ChIP-Seq pictograph demonstrating the four distal 5′ peaks of Isl1 occupancy near Glp1r. The red line denotes the Glp1r locus. E: ChIP enrichment of peak 1 Glp1r 5′ DNA in Isl1 (top panel) and Ldb1 βTC-3 immunoprecipitates (bottom panel) as compared with IgG control-treated DNA. H₂O serves as a negative control for the PCR. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

E18.5 Glut2 and Hb9 mRNA and protein expression is only compromised in Ldb1 mutant mice. Immunofluorescence analysis of Glut2 (red) and insulin (green) in the E18.5 control, mutant Ldb1 (A), and mutant Isl1 (B) pancreas. Insets show magnified insulin⁺ cell clusters. C: ChIP-Seq pictograph demonstrating Isl1 occupancy at distal Glut2 Re1 (5′) and Re2 (3′) domains in βTC-3 cells. The red line denotes the Glut2-coding region, whereas ChIP-tested proximal 5′ promoter region is represented by the red box. D: βTC-3 ChIP analysis of Isl1 (top panel) and Ldb1 (bottom panel) occupancy of Glut2 Re1, Re2, and the proximal domain compared with the PEPCK control (from top to bottom, respectively). H₂O serves as a negative PCR control. Results recapitulate observed Isl1 ChIP-Seq occupation of Glut2 Re1 and Re2, whereas Ldb1 also binds to the proximal domain. E: qPCR analysis of E18.5 Hb9 mRNA levels in pancreata from Ldb1- (blue bar) and Isl1-deficient (red bar) pancreata. Littermate control mRNA level was set at onefold (dashed line) ± SEM. F: E18.5 immunostaining analysis demonstrates that Hb9 protein (white) is maintained in the insulin⁺ (red) nuclei of Pdx1-Cre;Isl1^F/F pancreata as compared with littermate controls, as denoted by the white arrowheads. G: However, Hb9 is lost from most remaining insulin⁺ cells in the Pax6-Cre;Ldb1^F/F pancreata seen by comparing white arrowhead–labeled Hb9⁺ cells of control and mutant in F and G. The nuclear Hb9 signals are shown in the right panels. Yellow arrowheads in G illustrate autofluorescence from erythrocytes. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)