Generation of mice encoding a conditional allele of Nkx2.2

Abstract

Nkx2.2 is a homeobox transcription factor that is expressed in the pancreas, intestine and central nervous system (CNS) during embryogenesis and in the adult. In mice, global deletion of Nkx2.2 results in cell mis-specification in each of the tissues where it is expressed, and the null mice die as neonates with severe hyperglycemia. Although a whole body knockout demonstrates the importance of Nkx2.2 in cell specification and postnatal viability, it precludes assessment of the cell-autonomous and postnatal functions of Nkx2.2. In this study we report the generation and functional characterization of mice encoding a conditional allele of Nkx2.2. We demonstrate the functional integrity of the conditional Nkx2.2 allele and report successful in vivo deletion using a pancreas-specific Cre recombinase. The pancreas-specific deletion of Nkx2.2 results in similar defects found in the Nkx2.2 null pancreas and the mice die shortly after birth, demonstrating that the neonatal lethality associated with the null allele is caused by pancreatic islet dysfunction. The generation of a conditional Nkx2.2 allele provides an important tool for identifying the cell-autonomous and postnatal activities of Nkx2.2 in establishing and maintaining cell type identities and functions in the pancreas, intestine and CNS.

Fig. 1. Nkx2.2 conditional allele construct

(a) A loxP site (L83) and a FNFL cassette (FRT-Neo-FRT-LoxP) were engineered to flank exon 2 (E2) of the Nkx2.2 gene to generate the Nkx2.2 conditional targeting construct on a Bacterial Artificial Chromosome (BAC). The targeting construct was retrieved onto a vector carrying the diphtheria toxin alpha chain (DTA) negative selection marker. (b) Targeted ES cells were identified by PCR screening, producing a 2396pb product if the construct had incorporated. Male chimeras were bred to B6;SJL-Tg(ACTFLPe)9205Dym/J (ACTB^FLPe/FLPe; (Rodriguez et al. 2000)) female mice on a C57BL/6 background to both transmit the Nkx2.2^flox allele through the germline and to simultaneously remove the Neomycin cassette. (c) Mice carrying the Nkx2.2 conditional allele (Nkx2.2^flox) were genotyped for the presence of both loxP sites; the additional sequence added by the presence of the loxP site or the loxP and fragment of the FNFL cassette provided a sequence size difference in PCR product between the Nkx2.2^flox allele and the wildtype locus. Representative genotyping PCR amplification of wildtype, Nkx2.2^flox/+ and Nkx2.2^flox/flox mice, using both the L83 and FL146 primer sets.

Fig. 2. Nkx2.2^flox mice have normal growth and glucose tolerance

Body weight was measured in female (a) and male (b) mice from 3 weeks (weaning) until 12 weeks of age, and the presence of the Nkx2.2^flox allele did not affect mass. Glucose tolerance tests were performed on female (c) and male (d) mice at 3 weeks of age, and the Nkx2.2^flox allele did not alter glucose tolerance. For these mice there was no significant difference in area under the curve (e), body weight (f) and ad lib blood glucose (g).

Fig. 3. Islet cell hormone expression and morphology in the Nkx2.2^flox pancreas

The presence of hormone-expressing endocrine cells was assessed by immunofluorescence staining on pancreas tissue sections from Nkx2.2^flox/flox mice and wildtype control littermates. At E15.5 and P0, the presence of cells expressing glucagon (a, d, g, j), insulin (a–l), ghrelin (b, e, h, k), somatostatin or pancreatic polypeptide (c, f, i, l) appeared unchanged between genotypes. By 2 weeks of age (P14) there continued to be no difference in islet hormone expression and islet morphology appeared identical between wildtype (m – o) and Nkx2.2^flox/flox (p – r) mice.

Fig. 4. Nkx2.2^flox/flox;Pdx1-cre mutants recapitulate the Nkx2.2 null pancreatic phenotype

Using immunofluorescent staining for the expression of glucagon, the presence of alpha cells was observed in the pancreas from Nkx2.2^flox/+ (a) and Nkx2.2^flox/flox (b) embryos at E15.5, whereas a significant decrease of alpha cells was observed in the Nkx2.2^flox/flox;Pdx1-cre pancreas (c). The known pattern of expression for Nkx2.2 was observed in the control pancreas sections (d, e) compared with the efficient loss of Nkx2.2 in the mutant (f) due to Pdx1-cre mediated recombination of the Nkx2.2^flox allele. Similar to the phenotype observed in the Nkx2.2 null mice, pups were born and had severely elevated blood glucose (g). DAPI marks nuclei (a–c). Images are 20X

Fig. 5. Pancreatic hormone expression in the Nkx2.2^flox/flox;Pdx1-cre mutants is similar to Nkx2.2 null mice

(a) The relative expression of islet hormones was assessed in the Nkx2.2^flox/flox;Pdx1-cre mice and wildtype control littermates at P0. The expression of ghrelin (Ghr), insulin1 (Ins1), insulin2 (Ins2), glucagon (Gcg), pancreatic polypeptide (Ppy) and somatostatin (Sst) appeared similar to the expected pancreas phenotype of the Nkx2.2 null mice. Pdx1-cre mediated recombination of the Nkx2.2^flox allele was efficient in reducing Nkx2.2 transcript. E15.5 pancreas tissue from wildtype (b, c) and Nkx2.2^flox/flox;Pdx1-cre (d, e) embryos was subject to immunofluorescence for glucagon, ghrelin, insulin and amylase. DAPI marks nuclei. Images are 40X.