The window period of NEUROGENIN3 during human gestation

Abstract

The basic helix-loop-helix transcription factor, NEUROG3, is critical in causing endocrine commitment from a progenitor cell population in the developing pancreas. In human, NEUROG3 has been detected from 8 weeks post-conception (wpc). However, the profile of its production and when it ceases to be detected is unknown. In this study we have defined the profile of NEUROG3 detection in the developing pancreas to give insight into when NEUROG3-dependent endocrine commitment is possible in the human fetus. Immunohistochemistry allowed counting of cells with positively stained nuclei from 7 wpc through to term. mRNA was also isolated from sections of human fetal pancreas and NEUROG3 transcription analyzed by quantitative reverse transcription and polymerase chain reaction. NEUROG3 was detected as expected at 8 wpc. The number of NEUROG3-positive cells increased to peak levels between 10 wpc and 14 wpc. It declined at and after 18 wpc such that it was not detected in human fetal pancreas at 35-41 wpc. Analysis of NEUROG3 transcription corroborated this profile by demonstrating very low levels of transcript at 35-41 wpc, more than 10-fold lower than levels at 12-16 wpc. These data define the appearance, peak and subsequent disappearance of the critical transcription factor, NEUROG3, in human fetal pancreas for the first time. By inference, the window for pancreatic endocrine differentiation via NEUROG3 action opens at 8 wpc and closes between 21 and 35 wpc.

Keywords: NEUROG3, Neurogenin-3; Neurogenin-3 (NEUROG3); SOX9, Sex-determining region Y-box 9; development; dpc, dayspost-conception; endocrine; fetal; human; pancreas; sex-determining region Y-box 9 (SOX9); wpc, weeks post-conception.

Figure 1.

Profile of immunohistochemistry for NEUROG3 during human development. (A–E) Brightfield images show NEUROG3 in brown counterstained with toluidine blue. Insets (A) and (E) show brown SOX9 staining in nearby sections from the same fetus. Arrowheads exemplify positively stained cells. Scale bar represents 50 μm in all panels.

Figure 2.

Quantification of NEUROG3 by cell-counting and mRNA analysis during human fetal development. (A) and (B) The NEUROG3 count was quantified relative to the total pancreatic epithelial cell population (A) and the SOX9-positive cell population (B). By either approach the NEUROG3 count at 10-11 wpc and at 12–14 wpc were statistically greater than at <8 wpc, 18–21 wpc and 35–41 wpc. When quantified relative to the SOX9-positive cell population, 10-11 wpc, 12–14 wpc and 15–17 wpc emerged as the period of peak NEUROG3 detection. In neither (A) nor (B), was there a significant difference between counts at 10–11, 12–14 and 15–17 wpc. *P < 0.05, **P < 0.01, ^P < 0.005, ^^P < 0.0001 by one-way ANOVA with post-hoc Tukey's test. The number of specimens in each group (n) is shown in parentheses. (C). Relative NEUROG3 expression by qRT-PCR demonstrated higher levels in samples from 12–16 wpc than at 35–41 wpc. *P < 0 .01 by 2-tailed unpaired Student's t-test.

Figure 3.

Immunofluoresence for NEUROG3 in human fetal pancreas. (A–E) Immunofluorescence for NEUROG3 at 14 wpc. (A) Arrowheads point to green PCNA-positive cells while arrows point to separate red NEUROG3-positive cells. (B) and (C) Arrows point to hormone-negative NEUROG3-positive cells. Arrowhead points to a very rare NEUROG3-positive cell with faint somatostatin (SS) staining. (D) and (E) Arrowheads point to NEUROG3-positive nuclei visible in (D) but not (E) following pre-incubation with full-length human NEUROG3 protein. Amplification of the red gain with DAPI counterstaining to investigate the loss of nuclear staining has introduced some background cytoplasmic staining. Scale bar represents 50 μm.