Precommitment low-level Neurog3 expression defines a long-lived mitotic endocrine-biased progenitor pool that drives production of endocrine-committed cells - PubMed (original) (raw)
Precommitment low-level Neurog3 expression defines a long-lived mitotic endocrine-biased progenitor pool that drives production of endocrine-committed cells
Matthew E Bechard et al. Genes Dev. 2016.
Abstract
The current model for endocrine cell specification in the pancreas invokes high-level production of the transcription factor Neurogenin 3 (Neurog3) in Sox9(+) bipotent epithelial cells as the trigger for endocrine commitment, cell cycle exit, and rapid delamination toward proto-islet clusters. This model posits a transient Neurog3 expression state and short epithelial residence period. We show, however, that a Neurog3(TA.LO) cell population, defined as Neurog3 transcriptionally active and Sox9(+) and often containing nonimmunodetectable Neurog3 protein, has a relatively high mitotic index and prolonged epithelial residency. We propose that this endocrine-biased mitotic progenitor state is functionally separated from a pro-ductal pool and endows them with long-term capacity to make endocrine fate-directed progeny. A novel BAC transgenic Neurog3 reporter detected two types of mitotic behavior in Sox9(+) Neurog3(TA.LO) progenitors, associated with progenitor pool maintenance or derivation of endocrine-committed Neurog3(HI) cells, respectively. Moreover, limiting Neurog3 expression dramatically increased the proportional representation of Sox9(+) Neurog3(TA.LO) progenitors, with a doubling of its mitotic index relative to normal Neurog3 expression, suggesting that low Neurog3 expression is a defining feature of this cycling endocrine-biased state. We propose that Sox9(+) Neurog3(TA.LO) endocrine-biased progenitors feed production of Neurog3(HI) endocrine-committed cells during pancreas organogenesis.
Keywords: Neurog3; endocrine-biased; mitotic; progenitor.
© 2016 Bechard et al.; Published by Cold Spring Harbor Laboratory Press.
Figures
Figure 1.
The Neurog3Protein+ population is comprised of mitotic Neurog3pLO and post-mitotic Neurog3pHI cells. E12.5 (A) and E14.5 (B) pancreatic epithelium showing Muc1, Sox9, and Neurog3. White and red arrowheads indicate Sox9+ Muc1+ Neurog3pLO cells and Sox9− Muc1− Neurog3pHI, respectively. (C) Average fluorescence intensity of nuclear Neurog3 signal in Sox9+ Neurog3Protein+ versus Sox9− Neurog3Protein+ cells. n = 9 cryosections; N = 3 pancreata at E12.5 and E14.5. (*) P = 2 × 10−5; (**) P = 0.0002. (D) Percentage of Sox9+ Neurog3pLO versus Sox9− Neurog3pHI cells at E12.5 (n = 1114; N = 3), E14.5 (n = 3797; N = 3), and E16.5 (n = 4374; N = 3). (*) P = 0.0895; (**) P = 3 × 10−5; (***) P = 0.0001. E12.5 (E,G) and E14.5 (F,H) pancreatic epithelium showing pHH3, Neurog3, Muc1, and DAPI or pHH3, Neurog3, and E-cadherin. Red arrowheads indicate intraepithelial pHH3+ Neurog3pLO cells. (I) Mitotic index of Muc1+ Neurog3pLO cells versus Muc1− Neurog3pHI cells at E12.5 (n = 1546) and E14.5 (n = 10080). (J) Average fluorescence intensity of nuclear Neurog3 signal in Muc1+ Neurog3Protein+ pHH3+ cells versus Muc1− Neurog3Protein+ pHH3− cells at E12.5 and E14.5, calculated as in C. Data are mean ± SEM. (*) P = 0.0005; (**) P = 0.0087.
Figure 2.
_Neurog3_TA Sox9+ cells, comprising _Neurog3_TA.pLO and _Neurog3_TA.pUD cells, represent a mitotic _Neurog3_TA.LO endocrine-biased progenitor pool. E12.5 (A,C) and E14.5 (B,D) pancreatic epithelium showing EYFP, Neurog3, and Sox9 or EYFP, Neurog3, Muc1, and pHH3. (A,B) Red and green arrowheads indicate Sox9+ EYFP+ _Neurog3_TA.pLO cells and Sox9+ EYFP+ _Neurog3_TA.pUD cells, respectively. (C,D) Red and green arrowheads indicate pHH3+ Muc1+ _Neurog3_TA.pLO and pHH3+ Muc1+ _Neurog3_TA.pUD cells, respectively.
Figure 3.
The _Neurog3_RG1 transgenic reporter marks the mitotic _Neurog3_TA.LO progenitor population. E12.5 (A,C) and E14.5 (B,D) pancreatic epithelium showing H2BmCherry, GFPGPI, Sox9, and Neurog3 or H2BmCherry, GFPGPI, Muc1, and pHH3. (A,B) Red and green arrowheads indicate Sox9+ _Neurog3_TA.pLO and Sox9+ _Neurog3_TA.pUD cells (seen by low H2BmCherry signal from _Neurog3_RG1), respectively. (C,D) Red arrowheads indicate Muc1+ _Neurog3_RG1+ pHH3+ _Neurog3_TA.LO cells. (E) The percentage of _Neurog3_RG1+ Sox9+ _Neurog3_TA.LO versus _Neurog3_RG1+ Sox9− _Neurog3_TA.HI cells at E12.5 (n = 585; N = 3) and E14.5 (n = 6334; N = 3). Data are mean ± SEM. (*) P = 1 × 105.
Figure 4.
Division of _Neurog3_TA.LO progenitors in real time. Division of a _Neurog3_TA.LO cell into two delaminating _Neurog3_TA.HI cells (A) or two _Neurog3_TA.LO progenitors (B). Images are screen captures from Fig. 4B;
Supplemental Movie 2
. White and red arrowheads indicate a _Neurog3_TA.LO division event and apical attachment, respectively.
Figure 5.
The mitotic _Neurog3_TA.LO population is prevalent under Neurog3 heterozygous and strong hypomorphic conditions. E12.5 (A,C,E,G) and E14.5 (B,D,F,H) pancreatic epithelium showing EGFP, Neurog3, and Sox9 or EGFP, Muc1, and pHH3 from _Neurog3_EGFP/+ and _Neurog3_EGFP/FL embryos. (A_–_D) Red and green arrowheads indicate Sox9+ _Neurog3_TA.pLO and Sox9+ _Neurog3_TA.pUD cells (marked by low EGFP signal from _Neurog3_EGFP), respectively. (E_–_H) Red arrowheads indicate Muc1+ EGFP+ pHH3+ _Neurog3_TA.LO cells.
Figure 6.
Limiting Neurog3 expression expands the _Neurog3_TA.LO population at the expense of _Neurog3_TA.HI cells. (A) The percentage of Sox9+ _Neurog3_TA.pLO versus Sox9+ _Neurog3_TA.pUD cells in wild-type (_Neurog3_RG1) (see Fig. 3E for n), _Neurog3_EGFP/+ (E12.5, n = 168, N = 3; E14.5, n = 2425, N = 3), and _Neurog3_EGFP/FL (E12.5, n = 1237, N = 3; E14.5, n = 5818, N = 3) pancreata. (*) P = 0.0523; (**) P = 0.00067; (***) P = 8.3 × 10−5; (****) P = 4 × 10−6; (*****) P = 2.5 × 10−5. (B) The percentage of Sox9+ _Neurog3_TA.LO versus Sox9− _Neurog3_TA.HI cells in _Neurog3_EGFP/+ and _Neurog3_EGFP/FL (see A for n) pancreata. (*) P = 8 × 10−5; (**) P = 8 × 10−8; (***) P = 2.4 × 10−7. (C) The percentage of Sox9+ cells that are _Neurog3_TA.LO in wild-type (_Neurog3_RG1) (E12.5, n = 4497, N = 3; E14.5, n = 11930, N = 4), _Neurog3_EGFP/+ (E12.5, n = 4718; E14.5, n = 15903), and _Neurog3_EGFP/FL (E12.5, n = 4656; E14.5, n = 15671) pancreata. (*) P = 0.00015; (**) P = 0.0009; (***) P = 0.0022; (****) P = 2 × 10−7; (*****) P = 1 × 10−6. Data are mean ± SEM.
Figure 7.
Reducing Neurog3 expression increases the mitotic index of Sox9+ _Neurog3_TA.LO progenitors. (A, left) The mitotic index (percentage pHH3+) of Muc1+ _Neurog3_TA.LO cells under wild-type (_Neurog3_RG1) (E12.5, n = 569, N = 6; E14.5, n = 2401, N = 6), _Neurog3_EGFP/+ (E12.5, n = 362, N = 3; E14.5, n = 904, N = 3), and _Neurog3_EGFP/FL (E12.5, n = 783, N = 4; E14.5, n = 3905, N = 3) conditions. (Right) The mitotic index of Muc1+ _Neurog3_− cells in wild-type (_Neurog3_RG1) (E12.5, n = 1200 cells, N = 3; E14.5, n = 7490, N = 3), _Neurog3_EGFP/+ (E12.5, n = 1003, N = 3; E14.5, n = 2737, N = 3) and _Neurog3_EGFP/FL (E12.5, n = 2226, N = 4; E14.5, n = 5217, N = 3) pancreata. Data are mean ± SEM. (*) P = 0.0138; (**) P = 0.0207; (***) P = 0.0018. (B) Model depicting the behavior of _Neurog3_TA.LO cells in the pancreatic epithelium.
References
- Bradford JA, Clarke ST. 2011. Dual-pulse labeling using 5-ethynyl-2′-deoxyuridine (Edu) and 5-bromo-2′-deoxyuridine (BrdU) in flow cytometry. Curr Protoc Cytom 55: 7.38.1–7.38.15. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- P30 CA068485/CA/NCI NIH HHS/United States
- U01 DK072473/DK/NIDDK NIH HHS/United States
- U01 DK089570/DK/NIDDK NIH HHS/United States
- P30 DK058404/DK/NIDDK NIH HHS/United States
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Molecular Biology Databases
Research Materials