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.)