Characterization of a novel fibroblast growth factor 10 (Fgf10) knock-in mouse line to target mesenchymal progenitors during embryonic development - PubMed (original) (raw)
Characterization of a novel fibroblast growth factor 10 (Fgf10) knock-in mouse line to target mesenchymal progenitors during embryonic development
Elie El Agha et al. PLoS One. 2012.
Abstract
Fibroblast growth factor 10 (Fgf10) is a key regulator of diverse organogenetic programs during mouse development, particularly branching morphogenesis. Fgf10-null mice suffer from lung and limb agenesis as well as cecal and colonic atresia and are thus not viable. To date, the Mlcv1v-nLacZ-24 transgenic mouse strain (referred to as Fgf10(LacZ)), which carries a LacZ insertion 114 kb upstream of exon 1 of Fgf10 gene, has been the only strain to allow transient lineage tracing of Fgf10-positive cells. Here, we describe a novel Fgf10(Cre-ERT2) knock-in line (Fgf10(iCre)) in which a Cre-ERT2-IRES-YFP cassette has been introduced in frame with the ATG of exon 1 of Fgf10 gene. Our studies show that Cre-ERT2 insertion disrupts Fgf10 function. However, administration of tamoxifen to Fgf10(iCre); Tomato(flox) double transgenic embryos or adult mice results in specific labeling of Fgf10-positive cells, which can be lineage-traced temporally and spatially. Moreover, we show that the Fgf10(iCre) line can be used for conditional gene inactivation in an inducible fashion during early developmental stages. We also provide evidence that transcription factors located in the first intron of Fgf10 gene are critical for maintaining Fgf10 expression over time. Thus, the Fgf10(iCre) line should serve as a powerful tool to explore the functions of Fgf10 in a controlled and stage-specific manner.
Conflict of interest statement
Competing Interests: The authors have the following conflicts: Co-author Dr. Bellusci is a PLoS ONE Editorial Board member. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Figures
Figure 1. Generation of recombinant Fgf10iCre locus.
(A, B) Cre-ERT2-IRES-eYFP-Neo construct was introduced in frame with ATG of exon 1 by homologous recombination. Genes coding for Neomycin resistance (Neo) and Diphtheria Toxin A were used for positive and negative selection respectively. (C) Chimeras carrying the Neo cassette were crossed with C57BL/6J mice ubiquitously expressing Flp recombinase to generate heterozygous knock-in mice lacking the Neo cassette (D). Primers used for genotyping are shown as black arrows. Note that primers P1 and P2 detect the wild-type locus (297 bp band) whereas primers P3 and P4 detect the recombined locus (271 bp band). C: control; DTA: Diphtheria Toxin A; pGK: Phosphoglycerate kinase promoter.
Figure 2. Fgf10iCre is a null allele for Fgf10. E12.5 Fgf10iCre/iCre embryos show agenesis of the limbs (A, A’) and lung (B, B’) as well as cecal and colonic atresia (C, C’).
Arrowheads indicate sites of limb agenesis and dashed lines mark the epithelium in the cecum. Fgf10iCre/+ embryos were used as controls. (D) Fgf10 relative mRNA levels as quantified by qPCR. Fgf10iCre/iCre embryos (n = 4) express minimal Fgf10 levels compared to Fgf10iCre/+ (n = 5) and Fgf10+/+ embryos (n = 5). Data are shown as average values ± SEM. * P<0.05; *** P<0.0001. Ce: cecum; Co: colon.
Figure 3. Tomato expression in E18.5 Fgf10iCre/+; Tomatoflox/+ embryos.
Recombination was induced at E15.5 by a single IP injection of tamoxifen. Note the absence of Tomato expression in Fgf10+/+; Tomatoflox/+ embryos (A–D). Tomato-positive cells are detected in the ear, skin, limbs and cecum (A’–D’). (A”–D”) Higher magnifications of dotted boxes in A’, B’, C’, D’. n = 3. Tam: tamoxifen.
Figure 4. Tomato expression in E18.5 Fgf10iCre/+; Tomatoflox/+ lungs.
Recombination was induced at E15.5 by a single IP injection of tamoxifen. Note the absence of Tomato expression in Fgf10+/+; Tomatoflox/+ lungs (A; n = 3). The regions in the dotted boxes are magnified in (B) and (C). Tomato-positive cells are observed in the lung mesenchyme and interlobular septae of Fgf10iCre/+; Tomatoflox/+ lungs (white arrows) (A’, B’; n = 3). Inset in B’ shows high magnification of interlobular septae. Labeled cells in the trachea arrange in ring-like structures (black arrows) (C’; n = 3). (A”-C”; n = 8) X-Gal staining of Fgf10LacZ/+ lungs at E18.5. Inset in B” shows high magnification of interlobular septae. Tam: tamoxifen.
Figure 5. Inducible conditional Fgf10 inactivation using Fgf10iCre driver line.
Cre was activated by tamoxifen food from E8.5 to E14.5 in Fgf10iCre/flox; Tomatoflox/+ embryos. Labeled cells are present in the limbs, lung and cecum (D, G, H, L). Note the webbing of the digits in the forelimbs (arrows indicate webbing sites and dashed lines indicate peripheries) (H vs. E, M) and the hypomorph-like phenotype in the cecum (L vs. I, K). Fgf10iCre/flox; Tomatoflox/+ lungs show a deformed shape (C vs. B) as well as branching simplification (G vs. F, J). Dashed lines indicate the epithelium in the lung (F, G) and cecum (I, L). n = 3. Data are shown as average values ± SEM. * P<0.05; **** P<0.0001; Tam: tamoxifen.
Figure 6. Mismatch between Cre and Fgf10 expression levels in Fgf10iCre/+ embryos.
(A) Fgf10 and Cre relative mRNA levels as determined by qPCR. For RNA preparation, whole embryos were used at E11.5 (n = 5) and E13.5 (n = 3) whereas lungs were used at E18.5 (n = 7), P2 (n = 2), P12 (n = 4) and P161 (n = 3). Fgf10 expression levels increase throughout development and are maintained postnatally while Cre levels slowly increase throughout development and are minimal postnatally. (B) Induction in P1 Tomatoflox/+ pups reveals no recombination at P10 in the lung (n = 2). The area in the dotted box is magnified in (C). Inset in C shows high magnification with unlabeled cells. (D-G; n = 4) Induction in P1 and P4 Fgf10iCre/+; Tomatoflox/+ pups reveals recombination at P6 and P60 in the lung respectively. Insets in E and G show high magnification with labeled cells. (H, I; n = 3) Tamoxifen-induced recombination in lungs from Fgf10iCre/+; Tomatoflox/+ adult mice. Labeled cells are detected in the trachea and lung lobes. Inset in I shows high magnification with labeled cells. Data are shown as average values ± SEM. * _P<_0.05; ** _P<_0.01; *** _P<_0.001; **** _P<_0.0001.
Figure 7. Bioinformatic screening for putative regulatory elements 3 kb downstream of Fgf10 ATG.
(A) The screening reveals regions that are rich in transcription factor binding sites conserved between mouse and human (as predicted by rVista;
). Lung-related transcription factor binding sites within the 3′ end of the deleted sequence are shown. (B) Screening for lung-related H3K4me3 modification sites (as predicted by UCSC Genome Browser;
). A dense H3K4me3 modification site is predicted in the region overlapping exon 1-intron 1 boundary of Fgf10 gene. UTR: Untranslated region; ECRs: Evolutionary conserved regions.
References
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