Generation of Nkx2.2:lacZ mice using recombination-mediated cassette exchange technology

Abstract

Nkx2.2 encodes a homeodomain transcription factor required for the correct specification and/or differentiation of cells in the pancreas, intestine, and central nervous system (CNS). To follow the fate of cells deleted for Nkx2.2 within these tissues, we generated Nkx2.2:lacZ knockin mice using a recombination-mediated cassette exchange (RMCE) approach. Expression analysis of lacZ and/or β-galactosidase in Nkx2.2(lacZ/+) heterozygote embryos and adults demonstrates that lacZ faithfully recapitulates endogenous Nkx2.2 expression. Furthermore, the Nkx2.2(lacZ/lacZ) homozygous embryos display phenotypes indistinguishable from the previously characterized Nkx2.2(-/-) strain. LacZ expression analyses in the Nkx2.2(lacZ/lacZ) homozygous embryos indicate that Nkx2.2-expressing progenitor cells within the pancreas are generated in their normal numbers and are not mislocalized within the pancreatic ductal epithelium or developing islets. In the CNS of Nkx2.2(lacZ/lacZ) embryos, LacZ-expressing cells within the ventral P3 progenitor domain display different migration properties depending on the developmental stage and their respective differentiation potential.

Figure 1. Generation of the Nkx2.2^LCA ES cells and Nkx2.2:lacZ knockin allele

(a) Schematic of the strategy used to create the Nkx2.2^LCA allele in ES cells. P1 and P2 indicate the location of the 5′ and 3′ external southern probes. The blue boxes labeled “1” and “2” represent the two Nkx2.2 coding exons. “SA” indicates the short arm of homology and “LA” indicates the long arm of homology. (b) Schematic of the Nkx2.2:lacZ cassette and Nkx2.2:lacZ knockin allele. The nls-lacZ gene was cloned in frame at the Nkx2.2 ATG codon and just upstream of the stop codon, effectively replacing the Nkx2.2 coding sequence and intervening intron. The Nkx2.2:lacZ cassette replaces the Nkx2.2^LCA allele using RMCE. The Flpe-recombinase is used to remove the FRT-flanked hygromycin cassette. (c) Representative southern blot analysis of targeted ES cells using the 3′ (P2) probe. ES cell DNA was digested with Kpn1. The Nkx2.2 wild type allele is 17,026 bp and Nkx2.2^LCA is 15,504 bp. TL1 is the parent ES cell line; the remaining lanes show correctly targeted ES cells. The wild type band from the TL1 parental ES cells migrates at a slightly faster rate due to a difference in DNA abundance and salt concentration in ES cell DNA preparation. (d) Representative PCR amplification results of Nkx2.2^LCA ES cells containing Cre-mediated recombination of the Nkx2.2:nls-lacZ allele. For each RMCE ES cell clone, two sets of primers were used. Lanes labeled “a” indicate a PCR reaction for the targeted Nkx2.2:lacZ allele using primers 1 and 2 (606 bp) and lanes labeled “b” indicate the reaction using primers 3 and 4 (556 bp for the Nkx2.2:nlslacZ allele and 479 bp for the Nkx2.2 wild type allele). (e) Representative PCR amplification results of Nkx2.2:lacZ chimeric mice and one of the correctly targeted ES cell clones (4E9/4D2). Lanes are labeled “a” and “b” as above. Chimera #53004 and 53005 were correctly targeted.

Figure 2. The Nkx2.2:lacZ allele reproduces endogenous Nkx2.2 expression pattern

(a,b) X-gal staining in whole mount Nkx2.2^lacZ/+ embryos. The dotted line indicates the level of the section shown in c. (c) A transverse section through an Nkx2.2^lacZ/+ e12.5 embryo stained with X-gal. The level of the section in indicated by the dotted line in b. (d) An Nkx2.2 mRNA in situ performed on a section from an e12.5 wild type embryo. (e,f) Ventral (e) and dorsal (f) views showing X-gal staining of whole mount Nkx2.2^lacZ/+ brain indicates LacZ expression in the dorsal and ventral CNS. (g) X-gal staining of dissected e17.5 pancreas. (h) X-gal staining of dissected e17.5 intestine. (i) Transverse section at e10.5 indicates Nkx2.2:lacZ expression in the CNS and the dorsal and ventral pancreas in e10.5 embryos. Boxed regions are magnified in i′-i‴. (j) A section through an e12.5 pancreas; X-gal staining can be detected throughout the pancreatic epithelium. (k) Section through an adult pancreas;X-gal staining is restricted to pancreatic islet in adults. CNS, central nervous system; FB, forebrain; MB, midbrain; HB, hindbrain; SC, spinal cord; DP, dorsal pancreas; VP, ventral pancreas.

Figure 3. Nkx2.2:lacZ spatio-temporal expression analysis in Nkx2.2^lacZ/+ embryos

(a–f) At e12.5 adjacent sections in the pancreatic epithelium show beta-galactosidase staining in the pancreatic domain marked by E-cadherin and Pdx1. (b–f) Beta-galactosidase is extensively co-expressed with Pdx1, except for a population of Nkx2.2:LacZ^high expressing cells that co-stain with glucagon. The small insets in b and c highlight the fields magnified in d and e respectively. (g–k) At e15.5, beta-galactosidase is co-expressed with insulin-, glucagon- and ghrelin. By this stage the exocrine lineage and LacZ-expressing cells are mutually exclusive. (k) Nkx2.2:lacZ is also expressed in Ngn3-expressing endocrine progenitor cells. Filled arrowhead indicates Ngn3⁺/beta-galactosidase⁺ and empty arrowhead indicates Ngn3⁺/beta-galactosidase⁻. (l–p) At e18.5, beta-galactosidase is expressed in most of the endocrine cell populations within the forming islet. (q–u) In adult islets, beta-galactosidase is restricted to α, β and PP cells and in the majority of Pdx1-expressing cells. l–u are confocal images. DAPI staining was used to visualize the nuclei in a,f,g,h,I,k,l,o,p, and t. Magnification 20X otherwise indicated. Insets show higher magnification. All staining was performed in Nkx2.2^lacZ/+ embryos and animals.

Figure 4. Number and location of Nkx2.2:lacZ expressing cells in the developing pancreas is indistinguishable between Nkx2.2^lacZ/lacZand Nkx2.2^lacZ/+ littermates

(a)Immunofluorescence analysis beta-galactosidase in the Nkx2.2^lacZ/lacZembryos shows that LacZ is expressed in glucagon-producing cells at e10.5. (b–e) At e12.5 in the Nkx2.2^lacZ/lacZembryos, beta-galactosidase is co-expressed with Pdx1 within the pancreatic epithelium, with the exception of a population of beta-galactosidase^high expressing cells that co-express with glucagon, as seen in Nkx2.2^lacZ/+. (f–j) By e15.5, beta-galactosidase is co-expressed with Ngn3 in the ductal epithelium and the endocrine hormones; beta-galactosidase is not detected in the amylase-expressing exocrine tissue. (k) Quantification of the hormone producing cells co-stained with beta-galactosidase in e15.5 Nkx2.2^lacZ/+ and Nkx2.2^lacZ/lacZ embryos. (l) The number of beta-galactosidase positive cells is unchanged in e15.5 Nkx2.2^lacZ/lacZcompared to Nkx2.2^lacZ/+. White and black bars represent Nkx2.2^lacZ/+ and Nkx2.2^lacZ/lacZ respectively. * p≤0.05. Magnification 20X. Insets show higher magnification. DAPI staining was used to visualize the nuclei in a,c,f,h, and i.

Figure 5. mRNA expression of endocrine markers is altered in Nkx2.2^lacZ/− embryos

(a) lacZ mRNA expression at different time points during pancreas development is unchanged in Nkx2.2^lacZ/− and Nkx2.2^lacZ/+ embryos, suggesting that the number of lacZ-expressing cells is unchanged in Nkx2.2^lacZ/− and Nkx2.2^lacZ/+ embryos. (b) Nkx2.2 function is required for maximum expression of Ngn3 at e12.5 and e15.5. White and black bars represent Nkx2.2^lacZ/+ and Nkx2.2^lacZ/− respectively. * p≤0.05. (c) Hormone mRNA quantification in e15.5 Nkx2.2^lacZ/− embryos compared to Nkx2.2^lacZ/+.

Figure 6. . Nkx2.2-lacZ is expressed in the absence of Ngn3

(a–d) Co-immunostaining of beta-galactosidase and amylase, marker of acinar cells or DBA, marker of pancreatic ducts in Ngn3^tTA/+ (a, c) and Ngn3^tTA/tTA (b, d) e18.5 embryos. Arrows in a’ indicate the rare beta-galactosidase positive, Nkx2.2-expressing cells present in the ducts of wild type embryos. Arrowheads in b’ indicate beta-galactosidase expression in amylase-expressing cells in Ngn3 null mice. (e) Nkx2.2 mRNA expression in wild type (white bar, n=3), Ngn3^tTA/tTA Nkx2.2^+/+ (grey bar, n=5) and Ngn3^tTA/tTA;Nkx2.2^lacZ/+ (black bar, n=5) e16.5 embryos. * p≤0.05 vs wild type. There is no statistical difference (p=0.1544) in Nkx2.2 expression between the Ngn3^tTA/tTA Nkx2.2^+/+ and Ngn3^tTA/tTA;Nkx2.2^lacZ/+ embryos (two-tailed student’s t test). Magnification 20X. DAPI staining was used to visualize the nuclei in a, c, and d.

Figure 7. A subset of Nkx2.2-lacZ expressing neural progenitor cells fail to migrate out of the ventricular zone in the rostral spinal cord of Nkx2.2^lacZ/lacZ mice

(a,d) At e10.5, beta-galatosidase-expressing cells migrate laterally out of the ventricular zone in both Nkx2.2^lacZ/+ and Nkx2.2^lacZ/lacZ embryos. Immunofluorescent staining of Olig2 (red), which is normally repressed by Nkx2.2, shows co-expression of Olig2 and beta-galactosidase in the Nkx2.2^lacZ/lacZ embryos, confirming this embryo has lost Nkx2.2 activity, despite the normal migration of progenitor cells. (b,e) At e12.5, beta-galactosidase cells migrate laterally out of the ventricular zone of the hindbrain in Nkx2.2^lacZ/+ embryos, but not in Nkx2.2^lacZ/lacZ littermates. (c,f)By e15.5, a large number of beta-galactosidase cells continue to migrate away from the midline in the Nkx2.2^lacZ/+ embryos, but little migration can be observed in the Nkx2.2^lacZ/lacZ embryos. DAPI staining was used to visualize the nuclei in all panels.