FUCCI tracking shows cell-cycle-dependent Neurog3 variation in pancreatic progenitors

Abstract

During pancreas organogenesis, Neurog3^HI endocrine-committing cells are generated from a population of Sox9⁺ mitotic progenitors with only a low level of Neurog3 transcriptional activity (Neurog3^TA.LO ). Low-level Neurog3 protein, in Neurog3^TA.LO cells, is required to maintain their mitotic endocrine-lineage-primed status. Herein, we describe a Neurog3-driven FUCCI cell-cycle reporter (Neurog3^P2A.FUCCI ) derived from a Neurog3 BAC transgenic reporter that functions as a loxed cassette acceptor (LCA). In cycling Sox9⁺ Neurog3^TA.LO progenitors, the majority of cells in S-G₂ -M phases have undetectable levels of Neurog3 with increased expression of endocrine progenitor markers, while those in G₁ have low Neurog3 levels with increased expression of endocrine differentiation markers. These findings support a model in which variations in Neurog3 protein levels are coordinated with cell-cycle phase progression in Neurog3^TA.LO progenitors with entrance into G₁ triggering a concerted effort, beyond increasing Neurog3 levels, to maintain an endocrine-lineage-primed state by initiating expression of the downstream endocrine differentiation program prior to endocrine-commitment.

Keywords: endocrine-biased; lineage priming; progenitor.

Figure 1

Peptide-2A single-transgene FUCCI transgene. (A) Diagram indicating phases of the cell cycle marked by the components of the FUCCI reporter: mVenus-hGem^(1/110) (S-G₂-M) and mKO2-hCdt1^(30/120) (G₁) (Sakaue-Sawano et al., 2008). (B) Top, Diagram of CMV^P2A.FUCCI expression plasmid. Bottom, Immunofluorescence images showing CMV^P2A.FUCCI reporter expression in HeLa cells at appropriate stages of the cell cycle. Green arrowhead indicates mitotic chromosomes. Scale bars, 20 μm.

Figure 2

Generation of an LCA-capable BAC transgenic Neurog3^RG mESC line. (A) Schematic detailing the generation of transgenic mES cell lines carrying a single copy of a Neurog3^RG BAC transgene designed to serve as an LCA in future RMCE reactions. The Neurog3^RG BAC transgenic mESCs were previously used to generate Neurog3^RG reporter mice (Bechard et al., 2016). Neurog3 5′ untranslated region (UTR) represents the region 5′ of the start codon containing cis regulatory elements and the Neurog3 5′/UTR. (B) Table and graph of a standard curve, generated via a qPCR-based assay (see methods and materials), that relates transgene copy number to a specific C_T value. (C) Table and graph depicting the estimated Neurog3^RG BAC transgene copy number present in Neurog3^RG1 and Neurog3^RG2 mESC lines. Each data point represents the average of the indicated numbers of qPCR runs ± SEM.

Figure 3

Neurog3 protein levels vary according to cell-cycle phase. (A) E14.5 pancreatic epithelium showing Sox9, Neurog3, hCdt1^mKO2 and hGem^mVenus. Red arrowheads indicate Neurog3^P2A.FUCCI+ cells that are mKO2⁺ and thus in G₁, green arrowheads indicate Neurog3^P2A.FUCCI+ cells that are mVenus⁺ and thus in S-G₂-M phase. Asterisk indicates Sox9⁺ Neurog3^TA.LO cells, arrows with no asterisk indicate Sox9⁻ Neurog3^TA.HI cells. (B) Left, percentage of Sox9⁺ Neurog3^TA.LO and Sox9⁻ Neurog3^TA.HI cells in S-G₂-M versus G₁ phase. Right, percentage of Sox9⁺ Neurog3^TA.pLO and Sox9⁺ Neurog3^TA.pUD in S-G₂-M versus G₁ phase. (n = 1600, N = 3). (*) P = 0.0072; (**) P = 4 x 10⁻⁶; (***) P = 0.0607; (****) P = 0.0039. Each data point is an average of N = 3 with error bars representing ± SEM. Scale bars, 20 μm.

Figure 4

Neurog3 promotes low-level activation of downstream targets during G₁ in the mitotic Neurog3^TA.LO progenitor state. (A) flow cytometry plot detailing capture of lumen-apposed (Muc1⁺) Neurog3^P2A.FUCCI+ cells in S-G₂-M (mKO2⁻ mVenus⁺) (Blue population) or G₁ (mKO2⁺ mVenus⁻) (red population) from E14.5 Neurog3^P2A.FUCCI pancreata. Flow-sorted cells were collected into TRIzol for RNA isolation and cDNA synthesis. (B) Relative expression level (y-axis), normalized to Gapdh, of Sox9, Neurog3, Hes1, NeuroD1, Insm1, glucagon, and Insulin for E14.5 flow captured Muc1⁺ Neurog3^P2A.FUCCI+ mKO2⁻ mVenus⁺ (blue bars) and Muc1⁺ Neurog3^P2A.FUCCI mKO2⁺ mVenus⁻ (red bars) cells. Unless where noted differently on the figure, data points are an average of n = 6 technical replicates with error bars representing ±SEM. See supplemental table 1 for a list of primers used.