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Characterization of Tg(Etv4-GFP) and Etv5RFP Reporter Lines in the Context of Fibroblast Growth Factor 10 Signaling During Mouse Embryonic Lung Development

Members of the PEA3 transcription factors are emerging as bone fide targets for fibroblast growth factor (FGF) signaling. Among them, ETV4 and ETV5 appear to mediate FGF10 signaling during early embryonic lung development. In this paper, recently obtained Tg(Etv4-GFP) and Etv5CreERT2−RFP fluorescent reporter lines were generally characterized during early embryonic development and in the context of FGF10 signaling, in particular. We found that both Tg(Etv4-GFP) and Etv5CreERT2−RFP were primarily expressed in the epithelium of the lung during embryonic development. However, the expression of Etv5CreERT2−RFP was much higher than that of Tg(Etv4-GFP), and continued to increase during development, whereas Tg(Etv4-GFP) decreased. The expression patterns of the surrogate fluorescent protein GFP and RFP for ETV4 and ETV5, respectively, agreed with known regions of FGF10 signaling in various developing organs, including the lung, where ETV4-GFP was seen primarily in the distal epithelium and to a lesser extent in the surrounding mesenchyme. As expected, ETV5-RFP was restricted to the lung epithelium, showing a decreasing expression pattern from distal buds to proximal conducting airways. FGF10 inhibition experiments confirmed that both Etv4 and Etv5 are downstream of FGF10 signaling. Finally, we also validated that both fluorescent reporters responded to FGF10 inhibition in vitro. In conclusion, these two reporter lines appear to be promising tools to monitor FGF10/FGFR2b signaling in early lung development. These tools will have to be further validated at later stages and in other organs of interest.


INTRODUCTION
PEA3 transcription factors are a subfamily of the E26 transformation-specific (ETS) transcription factor family consisting of three members: ETV1 (also known as Er81), ETV4 (also known as PEA3), and ETV5 (also known as ERM) (reviewed by Sharrocks et al., 1997). Evidence from multiple studies has demonstrated that ETV4 and ETV5 are primary mediators of fibroblast growth factor (FGF) signaling via fibroblast growth factor receptor 2b (FGFR2b), and play overlapping roles in the patterning, morphogenesis, differentiation, and homeostasis of multiple organs and structures. For example, ETV4 and ETV5 have been shown to repress Shh expression in mouse limb buds, thus promoting bud outgrowth and proper anterior-posterior patterning (Mao et al., 2009;Zhang et al., 2009); studies in the mouse lung have shown that ETV4-and ETV5-mediated induction of Shh appear to regulate branching morphogenesis (Herriges et al., 2015); furthermore, ETV5 was shown to maintain alveolar type 2 (AT2) cell identity during mouse lung homeostasis and repair after injury, and has been implicated in lung tumorigenesis (Zhang et al., 2017); during development of the lacrimal gland in mice, it was demonstrated that PEA3 transcription factors control epithelial cell fate determination (Garg et al., 2018); in the kidney as well, research has shown that ETV4 and ETV5 play a wide range of functions within multiple signaling pathways, including FGF signaling, to regulate Wolffian duct and ureteric bud morphogenesis (Kuure et al., 2010); finally, a study in zebrafish has found that knocking down Etv4 and Etv5 resulted in embryonic abnormalities similar to a loss of FGF signaling, including cardiac, and left/right patterning defects (Znosko et al., 2010).
Lung organogenesis is a complex process involving a number of signaling pathways, and is divided into distinct stages comprising embryonic (E) and postnatal (P) development. In the mouse, these stages include the pseudoglandular stage (E9.5-16.5), the canalicular stage (E16.5-17.5), the saccular stage (E17.5-P5), and the alveolar stage (P5-P30) (reviewed in Chao et al., 2015). During each of the embryonic and postnatal stages, FGF signaling plays critical roles. For instance, during the pseudoglandular stage of lung development, the majority of the lung architecture is established via branching morphogenesis, and also most of the epithelial and mesenchymal cell types are formed (reviewed in El Agha and Bellusci, 2014). Disruption of FGFR2b signaling during this stage leads to morphological and differentiation defects (Bellusci et al., 1997b), while either Fgf10or Fgfr2b-null embryos display complete lung agenesis (Sekine et al., 1999;De Moerlooze et al., 2000).
Another critical signaling pathway during pseudoglandular lung development is the sonic hedgehog (SHH) pathway. SHH is a negative regulator of Fgf10 expression in the distal mesenchyme, and serves to limit the action of FGF10/FGFR2b signaling (Bellusci et al., 1997a). Recent studies propose that FGF10 acts on SHH via ETV4 and ETV5, creating an FGF10/ETV4/ETV5/SHH axis required for orchestrating proper branching morphogenesis (Herriges et al., 2015). It is unclear, however, how FGF10 acts on ETV4 and ETV5; whether it acts directly to regulate the expression of these transcription factors, for example, or whether these are regulated indirectly via another pathway.
To aid the study of the role of ETV4 and ETV5 during organogenesis, two transgenic reporter mice have recently been developed, Tg(Etv4-GFP) (Lamballe et al., 2011) and Etv5 CreERT2−RFP (also referred to as Etv5 RFP ). These lines report, via green and red fluorescent markers, the expression of Etv4 and Etv5, respectively. Furthermore, Etv5 CreERT2−RFP can be used to drive inducible genetic recombination in Etv5 positive cells, upon administration of tamoxifen. These lines can, therefore, potentially be used as invaluable research tools to study the role of ETV4 and ETV5 during the development of multiple organs, especially in the context of FGF signaling.
In this paper, we characterize these two reporter mouse lines during early embryogenesis (up to E16.5). We report the areas and organs of expression of ETV4-GFP and ETV5-RFP in the mouse embryo. We then focus on the expression of Etv4 and Etv5 in isolated embryonic lung mesenchyme and epithelium as well as the dynamic expression of the ETV4-GFP and ETV5-RFP protein during pseudoglandular development. Finally, various FGF10 inhibition experiments were carried out in vitro to determine the impact on the expression of ETV4-GFP and ETV5-RFP activity.

Animal Husbandry and Experimental Embryos
All animals were housed in a specific-pathogen-free (SPF) facility in accordance with local, state, and national laws. Timed pregnancies were set-up to obtain experimental embryos for analysis at the desired embryonic stage (from E9.5 to E18.5). Noon on the day of the vaginal plug was considered E0.5.
DNA was extracted from tails and limbs following basic lab procedures and prepared for PCR-based genotyping. The Tg(Etv4-GFP) gene was detected using the following primer sequences: Forward−5 ′ -GGA ATC TTG GGC CTT GAG AAC AGC-3 ′ ; reverse−5 ′ -CGC TGA ACT TGT GGC CGT TTA CG-3 ′ . The cycling protocol was as follows: denaturation at 94 • C for 3 min; 40 cycles of denaturation at 94 • C for 15 s, annealing at 60 • C for 30 s, and extension at 72 • C for 1 min; finish with a final extension at 72 • C for 5 min. The Etv5 CreERT2−RFP gene as well as the wild type Etv5, was detected using the following primer sequences: Etv5 CreERT2−RFP forward−5 ′ -TCG ATG CAA CGA GTG ATG AG-3 ′ ; Etv5 CreERT2−RFP reverse−5 ′ -TTC GGC TAT ACG TAA CAG GGT-3 ′ ; Etv5 forward−5 ′ -AAA GAG GAA CGC GGT CTG AG-3 ′ ; Etv5 reverse−5 ′ -CCA GCT GAG TCT CGT GTG AT-3 ′ . The cycling protocol was as follows: denaturation at 95 • C for 3 min; 30 cycles of denaturation at 94 • C for 30 s, annealing at 60 • C for 30 s, and elongation at 72 • C for 80 s. Product bands were detected by capillary gel electrophoresis.

FACS-based Isolation of Epithelial and Mesenchymal Cells
Lungs from embryonic mice were dissected at desired time points (E14.5, E16.5, and E18.5), and transferred to ice-cold Hank's Balanced Salt Solution (HBSS). Lungs were finely chopped with a sterile razor blade on a glass plate. The tissue was then added to a falcon tube and digested in 0.5% collagenase at 37 • C for 45 min, with constant mixing. Single cell suspensions were made by successively flushing the samples through 18, 20, and 24 g grade needles and then filtering the samples through 70 and 40 µm nylon strainers (BD Biosciences). The cell suspensions were diluted with 5 ml HBSS and centrifuged at 12,000 rpm for 5 min. and the supernatant was discarded. The pellet was resuspended in 10 µl blocking buffer and the following antibodies were added: 488-CD31 (1:50); FITC-CD45 (1:50); and Apc Cy7 EpCam (Epithelial cell adhesion molecule) (1:50) (Biolegend), for 20 min at 4 • C. The samples were washed 2x with 100 µl FACS buffer and centrifuged at 12,000 rpm for 5 min at 4 • C. The supernatant was discarded. The pellet was resuspended in 100 µl FACS buffer.
Cell sorting and isolation was performed using the FACSAria TM III (BD Biosciences) cell sorter. Alveolar epithelial cells were identified as CD45 −ve /CD31 −ve / Epcam +ve and mesenchymal cells as CD45 −ve /CD31 −ve / Epcam −ve . Cells were sorted through a flow chamber with a 100-µm nozzle tip under 25 psi sheath fluid pressure. Isolated cells were used for RNA isolation. As a main criterion for gating, we used the settings allowing to capture 98% of the cells in the isotype control and then we applied these gating conditions to the stained cells.

RT-qPCR
Either FACS-isolated cells, or embryonic lungs were lysed in Qiazol lysis reagent, and total RNA isolated using a miRNeasy mini RNA extraction kit according to the manufacturer's instructions (Qiagen). Please note that from cell dissociation to RNA isolation, the process takes 3-4 h. Our results indicate that there is very little variability between samples from the same time point and same group (epithelium or mesenchyme).
Up to 1 µg RNA was reverse-transcribed using the QuantiTect reverse transcription kit (Qiagen). cDNA was used to specifically amplify the desired DNA sequence by quantitative PCR (qPCR). Primers were designed to amplify specific mature mRNAs using NCBI's primer-BLAST option (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (last accessed, 01-08-2018). Primers were further validated by PCR-based gel electrophoresis. The following primer sequences were used: qPCR reaction mixtures were set up using the PowerUp SYBR Green Master Mix kit following the manufacturer's instructions (Thermo Fisher). Samples were run with three technical replicates on a LightCycler 480II (Roche) using the following protocol: UDG activation at 50 • C for 2 min; DNA polymerase activation at 95 • C for 2 min; and 40 cycles of denaturation at 95 • C for 15 s, annealing at 60 • C for 15 s, and extension at 72 • C for 1 min. To validate amplification specificity, a dissociation step was also included for each sample. Threshold cycles (Ct) were calculated and used for relative expression analyses, using mouse Hprt as the reference gene.
Ct and Ct values were calculated according to the following formulas: Note, this equation accounts for the fact that Ct is proportional to the -log of gene expression. Ct is therefore positively related to the expression of the gene of interest.
Expression relative to Hprt = 2 Ct Unpaired two-tailed Student's t-tests were performed on the Ct values, which can be assumed to be normally distributed. Number of "n" form of graphical representation, and significance level is indicated either in the figures or in the figure legends.

Lung Explant Culture for in vitro Experiments
Embryonic lungs used for in vitro experiments were obtained either from genetically modified embryos generated as described above, or from C57BL/6 wild-type embryos.
Doxycycline was administered via intraperitoneal injection to timed-pregnant females, as previously described (Danopoulos et al., 2013).

Imaging Acquisition and Measurements
Brightfield images of lungs from in vivo and in vitro experiments were captured either on a Leica MZ 125 stereoscopic dissecting microscope using a Spot Insight 2.0 Mp Color Mosaic camera and Spot 4.5.9 imaging software, or were obtained from live imaging experiments using a Leica DM6000B inverted microscope, DFC 305FX camera, and Leica Application Suite Advanced Fluorescence imaging software. Fluorescence intensities were quantified using FIJI software (version 2.0.0-rc-68/1.52 g). Branching was quantified by counting and averaging the distal tips of the left lobe of control and experimental samples at 0 and 24 h. These groups were analyzed using a two-factorial quasi-Poisson model, with the dispersion parameter estimated as 0.317. Differences were considered statistically significant at p < 0.05.

Gene Expression Patterns
To assess the expression patterns of genes in early stage embryonic lungs (E14.5), the online database genepaint.org was used (last accessed 01-08-2018). Each of the genes significantly regulated in our in vivo studies was entered in genepaint. The whole embryo section displaying the clearest gene expression in the lung, along with a magnification of the lung itself, was chosen for the figures.

Tg(Etv4-GFP) Expression During Early Embryonic Development
The Tg(Etv4-GFP) mouse line was created by Lamballe et al. (2011). These researchers used a bacterial artificial chromosome containing the Etv4 gene fused with a green fluorescent protein gene (GFP) in frame with exon 9, to generate the Tg(Etv4-GFP) transgenic mouse line ( Figure 1A). In the process, exons 10-12 of the Etv4 construct were deleted. Thus, the randomly inserted Tg(Etv4-GFP) transgene is non-functional, and does not interfere with endogenous Etv4 activity.
To characterize Tg(Etv4-GFP) expression in various organs during development, embryos were generated at different stages (E9.5, E11.5, E12.5, and E13.5). Tg(Etv4-GFP) was crossed with wild type mice to generate controls (Etv4 +/+ ) and transgenic embryos (Tg(Etv4-GFP)/+) at different developmental stages. Figure 1B shows the PCR-based strategy designed to detect the band corresponding to the presence of the transgene (see sequence of the primers in materials and methods). Figure 1C shows embryos negative for GFP (Figures 1Ca,g,n,u) and positive for GFP (Figures 1Cb,r,h,v) at different developmental stages. At E9.5, GFP was detected in the nasal placode, the mandibullary and maxillary processes, and at the midbrain/hindbrain junction (Figures 1Cc), the anterior limb bud and the mammary line (Figures 1Ce), the otic placode (Figures 1Cd), and the tail bud (Figures 1Cf). Similar expression sites were found at E11.5 (Figures 1Ch-l). Close examination of the lung indicated that GFP was expressed specifically at the tips in the epithelium and mesenchyme (Figures 1Cm). This expression overlaps with the previously reported endogenous Etv4 expression in the E12.5 lung (Yin et al., 2011). At E12.5, the external expression of GFP did not appear as specific. However, upon dissection of internal organs, GFP was enriched in the posterior (glandular) stomach (Figures 1Co), the developing kidneys (Figures 1Cq), the epithelium of the cecum (Figures 1Cs), and the lung epithelium and mesenchyme (Figures 1Ct, see also insert in figure 1t). At E13.5, GFP was found in similar places (Figures 1Cw,y,z). In addition, GFP was located at the tip of the forming digits (Figures 1Cx).

Etv5 CreERT2−RFP/+ Expression During Early Embryonic Development
The Etv5 CreERT2−RFP/+ mouse line was recently generated by Barry Stripp (Cedars-Sinai Medical Center, Los Angeles, USA). In this line, a knock-in of CreERT2/RFP in the 3 ′ untranslated region of the Etv5 gene was carried in embryonic stem cells (Figure 1D). Recombinant clones were used to generate Etv5 CreERT2−RFP/+ knock-in mice. Figure 1E shows the PCR based strategy to detect the presence of the Etv5 RFP knock in allele in heterozygous or homozygous embryos (see sequence of the primers in materials and methods). We performed a similar characterization on this line as we did on the Tg(Etv4-GFP) mouse line.
Etv5 CreERT2−RFP/+ mice were crossed with wild type mice to generate controls (Etv5 +/+ ) and heterozygous embryos (Etv5 CreERT2−RFP/+ ) at different developmental stages. During the E9.5 and E11.5 stages, RFP expression was general throughout the embryo (Figures 1Fa,g). At E9.5, expression was seen in the heart (Figures 1Fb), otic placode (Figures 1Fd), and nasal placode (Figures 1Fc). Expression in the tail was seen at E9.5 and E11.5 (Figures 1Ff,k), and in limb buds, limbs and the tips of forming digits in all stages (Figures 1Ff,h,q,w). Expression was seen in whisker regions (Figures 1Fn,t). After dissection, RFP was found to be enriched in the anterior stomach (Figures 1Fp), kidneys (Figures 1Frx), and epithelium of the cecum (Figures 1Fj,v). The expression of RFP in the lung at E11.5 did not appear enriched in the epithelium (Figures 1Fl). However, in later stages, RFP expression did appear enriched in the epithelium (Figures 1Fo,s,u), not only distally, but throughout the epithelium, with the expression disappearing in the main bronchi and trachea (e. g., Figures 1Fo,s, see also  Figures 4, 6).
was extracted from the Epcam-positive (epithelial) and Epcamnegative (mesenchymal) cells and qPCR analysis was performed (Figure 2A). Etv4 expression was seen in both the mesenchyme and epithelium during embryonic development. At E14.5, Etv4 was more highly expressed in the epithelium than in the mesenchyme. However, at later stages (E16.5 and E18.5), Etv4 expression decreased in the epithelium while remaining relatively constant in the mesenchyme (Figure 2B).
Etv5 was likewise expressed in both the mesenchymal and epithelial compartments throughout embryonic development. However, its expression in the epithelium was much higher than in the mesenchyme (around 10 times higher at E14.5, and 15 times higher at E18.5). Etv5 epithelial expression decreased at E16.5 before greatly increasing at E18.5, compared to E14.5 ( Figure 2B).
The expression of Etv4 and Etv5 was also validated using the publicly available genepaint.org data base. While Etv4 expression was diffusely seen in the lung epithelium and surrounding mesenchyme, Etv5 showed very strong expression restricted to the epithelium (compare insets in Figure 2C).

Dynamic GFP Expression Reports FGF10 Signaling
To monitor the expression of Tg(Etv4-GFP) in the epithelial buds of pseudoglandular stage lungs in the context of FGF10 signaling, E12.5 Tg(Etv4-GFP) lungs were cultured and live imaged for 24 h, after which FGF10 signaling was blocked using an anti-FGF10 antibody for an additional 24 h (Figure 3A). During the first 24 h, ETV4-GFP was dynamically expressed, showing greater expression at the distal tips of growing buds, in likely regions of active FGF10 signaling (see Figures 3Ba-c and Supplementary Movie 1). During the FGF10 inhibition, ETV4-GFP expression was greatly reduced. Still images from multiple time points during the live imaging from three independent lungs were used to quantify these global changes in fluorescence intensity ( Figure 3C). We also confirmed that the loss of GFP expression of the E12.5 lungs after 24 h in culture was neither due to bleaching, nor to a normal decrease in ETV4-GFP expression. Supplementary Figure 1 shows that the expression of ETV4-GFP in E12.5 transgenic lungs was maintained in culture for at least 38 h. Furthermore, still images of individual buds were used to quantify the dynamic expression of ETV4-GFP in three regions of the bud during a branching event (Figures 3D,E). Intensity was measured during new branch formation at the tip (regions 4 and 6), stalk (regions 1-3; 7-9) and cleft (region 5). Tip ETV4-GFP fluorescence intensity initially increased before stabilizing (Figures 3Ea), whereas stalk and cleft fluorescence intensity increased before decreasing to initial levels (Figures 3Eb,c). The expression patterns of the stalk and cleft reflected the initial single bud branching into two buds.
These results suggest that the Tg(Etv4-GFP) mouse line can be used as a valid tool to report FGF10 signaling in the distal tips of lungs during pseudoglandular development. This conclusion is supported by the previously described dynamic expression pattern of Sprouty2 during the branching process (Mailleux et al., 2001). Sprouty2 is a well-accepted target of FGF10 signaling in the lung, and displays a remarkably similar expression pattern to what we found for Tg(Etv4-GFP).

Quantification of Dynamic ETV5-RFP Fluorescence
Similar to the analysis of ETV4-GFP expression in early lung development, the expression of ETV5-RFP in branching epithelial buds was assessed. E12.5 Etv5 CreERT2−RFP/+ lungs were cultured and live imaged for 40 h (Figure 4A). During this time ETV5-RFP was expressed throughout the epithelial tree at the exception of the primary bronchi and trachea. This was in contrast with the expression of ETV4-GFP, which was only at the tip. Interestingly, the more proximal regions of ETV5-RFP expression were not localized to known areas of FGF10 signaling. Close-up examination of ETV5-RFP indicated higher expression in distal tips compared to more proximal regions (see arrows in Figure 4B and Supplementary Movie 2). Finally, we quantified the fluorescence intensity of bud regions during a branching event using still images at multiple time points (Figures 4Ca-c). Fluorescence intensity at the tip (regions 1 and 2) increased before leveling off around 30 h, and then decreased slightly (Figures 4Da), whereas intensity at the cleft and stalk regions (regions 3 and 4-5, respectively) initially increased before decreasing to original levels (Figures 4Db,c). This temporal expression pattern was similar to what was found for Tg(Etv4-GFP) lungs, and is likely a consequence of the initial bud branching into two buds.  (Parsa et al., 2008(Parsa et al., , 2010. Upon administration of doxycycline, soluble FGFR2b is produced and secreted from cells, functionally inhibiting all FGFR2b ligands from properly signaling. We have previously shown that at E12.5, FGF10 is the predominant FGF ligand signaling in the lung (Bellusci et al., , and Etv5, in experimental lungs, demonstrating the usefulness of the Etv5 RFP line to report FGF10/FGFR2b signaling. Note, we only had one control Etv5 RFP lung and two experimental Etv5 RFP lungs, thus statistical analysis was not possible (Data are presented as mean ± SEM; **p < 0.01).
1997b). Therefore, induction of soluble FGFR2b at this time point inhibits FGF10 signaling. As Figure 5A shows, after only 9 h of FGF10 inhibition, experimental lungs were smaller, displayed simplified branching, and had fewer distal buds. Furthermore, the expressions of Etv4 and Etv5, as evaluated by RT-qPCR, were reduced ( Figure 5B).
A similar approach to inhibiting FGFR2b signaling was conducted in vitro. In this experiment, wild type E12.5 lungs were harvested and cultured with (experimental) or without (control) recombinant FGFR2b added to the culture medium for 24 h (Figure 5C). A similar phenotype to that seen in vivo was produced in experimental lungs, while the downregulation of Etv4 and Etv5 mRNA was even more pronounced ( Figure 5D).
Taken together, these results suggest that FGF10 signaling can be inhibited both in vitro and in vivo, and that Etv4 and Etv5 are regulated by FGF10.

ETV5-RFP Expression Reports FGF10 Signaling in vitro
Using our in vitro approach to inhibit FGF10/FGFR2b signaling in pseudoglandular stage lungs, we investigated whether the Etv5 CreERT2−RFP line could be used as a tool to report FGF10/FGFR2b signaling. E12.5 Etv5 CreERT2−RFP lungs were cultured and live imaged for 48 h with (experimental) or without (control) recombinant soluble FGFR2b ( Figure 6A). As can be seen in the still images, the soluble FGFR2b treated lungs showed arrest in branching over time, compared to the control ( Figure 6B). This phenotype is a hallmark of inhibited FGF10 signaling. Furthermore, demonstrating the usefulness of this line to report FGF10/FGFR2b signaling, the fluorescence intensity of the ETV5-RFP decreased over time in experimental lungs, while that of the controls remained constant. This finding is supported by RT-qPCR results after 48 h, which show a decrease in the expression of RFP, Etv4, and Etv5 in experimental lungs compared to controls ( Figure 6C).
These results not only confirm that soluble FGFR2b can be used to inhibit FGF10 signaling in Etv5 CreERT2−RFP lung explants, but they also suggest that the Etv5 CreERT2−RFP line responds to FGF10/FGFR2b signaling in the developing lung.

CONCLUSION
In conclusion, the Tg(Etv4-GFP) and Etv5 RFP reporter mouse lines appear to be promising tools to monitor FGF10/FGFR2b signaling in early lung development. These tools will have to be further validated at later stages and in other organs of interest.

ETHICS STATEMENT
Animal experiments, harvesting organs and tissues from wild type and mutant mice following euthanasia using pentobarbital was approved at Justus Liebig University Giessen by the federal authorities for animal research of the Regierungspraesidium Giessen, Hessen, Germany (Approved Protocol GI 20/10 Nr. G 84/2016).