Nkx2.2:Cre knock-in mouse line: a novel tool for pancreas- and CNS-specific gene deletion

Abstract

Nkx2.2 is a homeodomain-containing transcriptional regulator necessary for the appropriate differentiation of ventral neuronal populations in the spinal cord and hindbrain, and endocrine cell populations in the pancreas and intestine. In each tissue, Nkx2.2 inactivation leads to reciprocal cell fate alterations. To confirm the cell fate changes are due to respecification of Nkx2.2-expressing progenitors and to provide a novel tool for lineage tracing in the pancreas and CNS, we generated an Nkx2.2:Cre mouse line by knocking in a Cre-EGFP cassette into the Nkx2.2 genomic locus and inactivating endogenous Nkx2.2. The R26R-CAG-LSL-tdTomato reporter was used to monitor the specificity and efficiency of Nkx2.2:Cre activity; the tomato reporter faithfully recapitulated endogenous Nkx2.2 expression and could be detected as early as embryonic day (e) 9.25 in the developing CNS and was initiated shortly thereafter at e9.5 in the pancreas. Lineage analyses in the CNS confirmed the cell populations thought to be derived from Nkx2.2-expressing progenitor domains. Furthermore, lineage studies verified Nkx2.2 expression in the earliest pancreatic progenitors that give rise to all cell types of the pancreas; however they also revealed more robust Cre activity in the dorsal versus ventral pancreas. Thus, the Nkx2.2:Cre line provides a novel tool for gene manipulations in the CNS and pancreas.

Keywords: CNS; Cre-lox; Nkx2.2; lineage; pancreas.

FIG. 1

Generation of the Nkx2.2:CreEGFP knockin allele. (a) Schematic of the strategy used to create the Nkx2.2:CreEGFP knockin allele. Nkx2.2^LCA refers to the RMCE Nkx2.2 acceptor allele integrated into the genomic locus of the 4H9 ES cell line (Arnes et al., 2012). The RMCE lox sites are indicated by colored arrows. The targeting vector includes a FRT flanked hygromycin cassette for positive selection of the targeting event. The CreEGFP gene was recombinered in frame at the Nkx2.2 ATG codon and deleted most of coding exon 1. ExI’ signifies the truncated region of Nkx2.2 coding exon 1 and ExII refers to the Nkx2.2 coding exon 2. The CreEGFP cassette replaces the Nkx2.2^LCA allele using RMCE. P1 and P2 indicate the 5′ PCR primers that verify integration of the hygromycin cassette into the correct genomic locus. P3 and P4 indicate the 3′ primers that distinguish the wild-type Nkx2.2 allele and the newly integrated Nkx2.2:CreEGFP allele containing the 2722 lox site. The Flpe-recombinase is used to remove the FRT-flanked hygromycin cassette. P5 and P6 refer to the genotyping primer set used to determine for the presence of the Nkx2.2:CreEGFP allele. (b) Representative PCR amplification results of Nkx2.2^LCA ES cells containing RMCE-mediated recombination of the Nkx2.2:CreEGFP allele. For each RMCE ES cell clone, two sets of primers (P1/P2 and P3/P4) were used. Primers 1 and 2 detect a 606 bp product. Primers 3 and 4 detect a 556 bp for the Nkx2.2:CreEGFP allele and 479 bp for the Nkx2.2 wild-type allele. (c) Representative PCR amplification results of Nkx2.2:CreEGFP mice and wild-type littermates. Primer P5 and P6 amplify a 326 bp band from only the mice carrying the Nkx2.2:CreEGFP allele.

FIG. 2

Characterization of the Nkx2.2^CreEGFP; R26R:Tomato mice. (a,b) Pancreata derived from Nkx2.2^CreEGFP/+; R26R:Tomato e18.5 embryos and Nkx2.2^+/+; R26R:Tomato littermate controls were sectioned and immunolabeled with antibodies against amylase (blue) and insulin (green). Tomato reporter expression (red; direct fluorescence) could only be visualized in the embryos containing the Nkx2.2:CreEGFP allele and not the Nkx2.2^+/+ littermate controls, verifying the Tomato reporter is only activated in the presence of Cre recombinase. Magnification: 20×; Scale bar = 100 μm. Insets are 40×. (c-h) Pancreata derived from Nkx2.2^CreEGFP/+; R26R:Tomato e18.5 embryos and Nkx2.2^CreEGFP/EGFP; R26R:Tomato littermate controls were sectioned and immunolabeled with antibodies against endocrine hormones as indicated. Similar to the phenotype of the Nkx2.2^−/− mice, insulin+ cells are absent, glucagon+ cells are reduced and ghrelin+ cells are elevated. Magnification: 40×; Scale bar = 500 μm.

FIG. 3

Characterization of the onset of Nkx2.2-Cre activity. (a) Whole mount bright field image of an ~9.25 (19 somites) Nkx2.2^CreEGFP/+; R26R:Tomato embryo with the somite numbers indicated. (b) Detection of the tomato reporter in the same embryo shown in (a) using direct fluorescence imaging. The reporter is visible along the A/P axis of the CNS. (c) Fluorescence imaging of an e9.5 Nkx2.2^CreEGFP/+; R26R:Tomato embryo. Tomato reporter expression is now visible in the pancreas. The inset shows higher magnification of the pancreas and spinal cord. (d,e) Fluorescence imaging of tomato expression in whole mount Nkx2.2^CreEGFP/+; R26R:Tomato embryos at successively later embryonic stages. The tomato reporter is also visible in the cells and axons derived from Nkx2.2-expressing progenitor domains in the CNS. Scale bars = 250 μm (E9.25); 450 μm (E9.5); 600 μm (E10.5); 1 mm (E11.5, E12.5).

FIG. 4

Assessment of Nkx2.2-Cre activity in the developing pancreas. Combined direct fluorescence (tomato reporter; red) and immunofluorescence of different pancreatic markers in Nkx2.2^CreEGFP/+; R26R:Tomato embryos to illustrate cell type specific Cre activity. (a) In a transverse section through an e9.5 embryo, tomato expression can be visualized in the dorsal pancreatic epithelium (DP) and not in the ventral pancreas epithelium (VP). In the dorsal pancreas, tomato is co-expressed with Pdx1 (blue) and glucagon (green). Scale bar = 25 μm. (a’) Higher magnification two channel image showing that tomato is co-expressed with a subset of Pdx1+ cells. (a”) Higher magnification two channel image showing tomato is co-expressed with glucagon+, Pdx1− cells. (b) Tomato is extensively co-expressed with Pdx1 (green) in the dorsal (DP) pancreatic bud at e12.5; only a small subset of Pdx1 cells does not express the tomato reporter. Scale bar = 75 μm. (c-e) In sections through E14.5 pancreas, tomato is co-expressed with Neurog3, insulin, glucagon, ghrelin, and amylase. Magnification 20×; Scale bars = 75 μm. (c’-e’) Higher power images showing co-expressing cells within the pancreas (40×).

FIG. 5

Nkx2.2-Cre activity in the postnatal pancreas and intestine. Combined direct fluorescence (tomato reporter; red) and immunofluorescence of in Nkx2.2^CreEGFP/+; R26R:Tomato mice. (a) The tomato reporter is expressed in the exocrine and endocrine compartments of the adult pancreas. Within the islet, tomato is co-expressed with insulin and glucagon. White squares indicate magnified fields in a’ (islet cells) and a” (acinar cells). (b) Tomato expression can be detected in enteroendocrine cells within the villi of the small intestine. Dapi staining (blue) labels all cell nuclei to help visualize the intestinal morphology. Magnification 20×; Scale bars = 100 μm.

FIG. 6

Nkx2.2-Cre activity in the central nervous system. Analysis of tomato expression in the CNS of Nkx2.2^CreEGFP/+; R26R:Tomato embryos. (a) Transverse section through the thoracic region of an e14.5 embryo. The tomato reporter is expressed in the ventral spinal cord in the P3 progenitor domain adjacent to the floor plate and in the more lateral V3 interneuron domain. Tomato expression can also be detected adjacent to the esophagus in the left (L) and right (R) vagal nerves. Dapi (blue) labels cell nuclei. Magnification 5×; Scale bar = 500 μm. The rectangle represents the region shown at higher magnification in b-d. (b-d) The tomato reporter is co-expressed with Tuj1 in motor neuron axons extending from Nkx2.2-derived visceral motor neurons in the hindbrain. Magnification 20×; Scale bar = 100 μm. (e) Transverse section of an e14.5 thoracic spinal cord showing co-expression of Tomato and Sox9 (red) in the ventricular zone containing neural precursors. Tomato expressing cells are also present in the more lateral domain containing differentiated interneuron populations. Magnification 20×; Scale bar = 100 μm. (f) Whole mount of dissected e12.5 visceral tissue from a Nkx2.2^CreEGFP/+; R26R:Tomato embryo. Tomato is expressed in the dorsal (DP) and ventral (VP) and in a subset of axons extending into the stomach. (g) Section through the distal stomach of an e14.5 Nkx2.2^CreEGFP/+; R26R:Tomato embryo. Tomato+ axons extend into the Tuj1 expressing neurons innervating the stomach. Magnification 20×; Scale bar = 100 μm.