In the previous article, by summarizing the research progress of FGF20, we revealed how researchers have linked variations in FGF20 and Parkinson’s disease (PD) in certain populations. In future studies, FGF20 may become a hot target for researchers of Parkinson’s disease. Genes perform a variety of functions, and FGF20 is no exception. In the mouse models, what new discoveries have researchers made? New ideas for treating thinning hair? New treatment for hearing loss? Explore the mystery of genes together with Cyagen: follow our technical bulletins and discover more interesting research news.
Fgf20 Mouse Models Phenotype
Guard mouse hair formation
Fgf20Cre knock-in mice were generated using the reported method, as follows. Briefly, exon1 of Fgf20 was replaced with a Cre-EGFP–FRT-neomycin-FRT cassette to generate Fgf20Cre(neo)/+ mice. The neomycin gene was eliminated by mating with CAG-FLPe mice to generate Fgf20Cre/+ mice. Mice were maintained on a 129X1/SvJ;C57BL/6J mixed background. To determine whether Fgf20 is functionally important for hair follicle formation, researchers examined hair from the back skin of adult Fgf20 βGal/ βGal mice. Of the four morphologically distinct hair types, guard hairs were missing in Fgf20 βGal/ βGal mice (Fig. 1A), while the awl, auchene, and zigzag hairs were readily identified and showed normal shaft morphology (Fig. 1B). To determine whether lack of Fgf20 affected the development of secondary and tertiary hair types, numbers of hairs from 3-wk-old Fgf20 βGal/+ and Fgf20 βGal/ βGal mice were quantified. In heterozygous Fgf20 βGal/+ mice, guard hairs represented 2.2% 6 1.1% of the total population. In Fgf20 βGal/ βGal mice, guard hairs were not detected (Fig. 1C). Interestingly, the percentage of awl and auchene hairs was significantly reduced from 9.2% 6 2.2% and 9.5% 6 3.1%, respectively, in Fgf20 βGal/+ mice to 1.8% 6 1.9% and 2.8% 6 1.7%, respectively, in Fgf20 βGal/ βGal mice (P < 0.002 and P < 0.009, n = 4, respectively) (Fig. 1C). The percentage of zigzag hairs was increased from 79% 6 3.5% in Fgf20 βGal/+ mice to 95.4% 6 3.5% in Fgf20 βGal/ βGal mice (P < 0.003) (Fig. 1C). Thus, Fgf20 is required for the formation of primary and most of the secondary hairs in mice.
Figure 1. Loss of Fgf20 results in guard hair agenesis. (A) Image of 4-wk-old mice showing guard hair shafts sprouting from back skin in a Fgf20 βGal/+ mouse (arrow, left) but not in a Fgf20 βGal/ βGal mouse (right). (B) Image of hairs from back skin of a Fgf20 βGal/+ (left) or Fgf20 βGal/ βGal (right) mouse. (C) Quantification of hair types from 3-wk-old mice. Fgf20 βGal/ βGal mice show complete loss of guard hairs and decreased awl and auchene hairs. (D) Scanning electron micrograph showing primary hair follicle primordial as round protrusions in a Fgf20 βGal/+ embryo, but the surface of a Fgf20 βGal/ βGal embryo appears flat. (E) Histology of E14.5, E15.5, E16.5, and E18.5 skin from Fgf20 βGal/+ and Fgf20 βGal/ βGal embryos. Arrows indicate dermal condensations. (Bottom right panel) Note the bifurcated hair follicle. (au) Auchene; (g) guard; (aw) awl; (z) zigzag. Bar, 100 mm.
Prior to primary hair placode formation (E13.5), the skin of Fgf20 βGal/ βGal embryos was histologically indistinguishable from Fgf20 βGal/+ embryos (data not shown). At E14.5, scanning electron microscope analysis suggested the absence of primary hair follicle primordial in Fgf20 βGal/ βGal embryos (Fig. 1D), yet epithelial thickenings (placodes) were histologically evident in both Fgf20 βGal/+ and Fgf20 βGal/ βGal embryos (Fig. 1E). Strikingly, there was no histological evidence of dermal condensation formation in Fgf20 βGal/ βGal embryos (Fig. 1E). At E15.5, two types of hair follicles could be identified in Fgf20 βGal/ βGal embryos—small and flat placodes (the majority) and follicles that had grown deeper into the dermis (occasional)— but none of them were associated with dermal condensations (Fig. 1E). At E16.5 in Fgf20 βGal/+ embryos, the primary hair follicles reached the peg stage, and secondary hair placodes were formed. In contrast, in Fgf20 βGal/ βGal embryos, most hair follicles were very small and were not associated with dermal condensations. However, sporadic primary hair placodes extended into the dermis to form a hair peg and were associated with a very small mesenchymal condensation (Fig. 1E). At E18.5 in Fgf20 βGal/ βGal embryos, some primary hair follicles were observed and were occasionally bifurcated, indicating an additional defect in hair follicle development (Fig. 1E). Tertiary placodes formed in both genotypes (Fig. 1E). These data show that Fgf20 is necessary for the formation of dermal condensations in primary hair follicles.
New Research Progress of Fgf20 Gene
Fgf20 are expressed in the developing cochlea
Research found that mice homozygous for a knock-out allele exhibit imapired cochlear lateral compartment differentiation and deafness without loss of vestibular function. Since previous analysis of mice lacking Fgf20 did not reveal any function for Fgf20 at this stage of development, researchers have generated a Fgf20Cre knock-in mice model to research. Fgf20Cre knock-in mice were generated using a reported method. Briefly, exon1 of Fgf20 was replaced with a Cre-EGFP–FRT-neomycin-FRT cassette to generate Fgf20Cre(neo)/+ mice. The neomycin gene was eliminated by mating with CAG-FLPe mice to generate Fgf20Cre/+ mice. Mice were maintained on a 129X1/SvJ;C57BL/6J mixed background.
Figure 2. Fgf9 and Fgf20 are expressed in distinct regions of the otic vesicle. (A) βGal activity in an Fgf9lacZ/+ embryo at E10.5 visualized with xGal staining. (B, C) βGal (red) and Sox2 (green) co-immunostaining showing that Fgf9 (B) is expressed in Sox2- non-sensory epithelium and Fgf20 (C) is expressed in Sox2+ sensory epithelium at E11.5. (D) Schematic diagram of FGF9, FGF20, and Sox2 immunostaining showing that FGF9 and FGF20 are expressed in distinct domains in the otic vesicle. ov, otic vesicle, scale bars, 100 μm.
They first examined the expression domain of Fgf9 relative to Sox2-expressing sensory progenitors(and Fgf20) using a new Fgf9-βGal reporter allele (Fgf9lacZ) in which a splice acceptor-lacZ gene was inserted into the first intron of Fgf9. At E10.5, βGal activity was detected in the otic vesicle epithelium (Figure 2A). Co-staining of βGal and Sox2 at E11.5 showed no overlap, indicating that Fgf9 is expressed in the non-sensory epithelium of the otic vesicle (Figure 2B). Taken together with previous Fgf20 expression analysis, Fgf9 and Fgf20 are both expressed in the otic vesicle, but in non-overlapping domains in the otic epithelium (Figure 2D).
Fgf20Cre/βgal Mice Will Allow Enrichment for Prosensory Cell RNA
Fgf20-null allele, Fgf20βgal, was made by targeted insertion of a sequence encoding β-Galactosidase replacing exon 1 of Fgf20.9 We combined the Fgf20Cre and Fgf20βgal alleles to generate Fgf20−/− mice (Fgf20Cre/βgal), which maintained the same dosage of Cre as control mice (Fgf20Cre/+). Importantly, based on double fluorescence expression from the ROSAmTmG Cre-reporter allele, the Fgf20Cre lineage (green) did not change in Fgf20Cre/βgal compared to Fgf20Cre/+ cochleae (Figure 3C). Based on these results, we concluded that Fgf20Cre/+; ROSAfsTRAP/+ and Fgf20Cre/βgal; ROSAfsTRAP/+ mice will allow enrichment for prosensory cell RNA, increasing the sensitivity of RNAseq to identify changes in gene expression within these cells in the absence of FGF20 signaling.
Figure 3. Fgf20Cre targets L10a-eGFP expression to the prosensory domain and Kölliker's organ. A, Schematic representing cross-sectional view through the E14.5 and P0 cochlear duct. At E14.5, the epithelium at the cochlear duct floor can be divided into three regions: outer sulcus (OS), prosensory domain (PD), and Kölliker's organ (KO). Cells from these three regions contribute to the lesser epithelial ridge (LER), organ of Corti (OC), and greater epithelial ridge (GER), respectively, at P0. Double-headed arrow indicates medial (neural) and lateral (abneural) directions. In all figures, sections through the cochlear duct are presented in this orientation. B, Sections through the middle turn of E14.5 and P0 Fgf20Cre/+; ROSAfsTRAP/+ cochlear ducts, showing L10a-eGFP (green) expression. At E14.5, L10a-eGFP is found in the prosensory domain (PD; bracket), Kölliker's organ and medial wall, and spiral ganglion (SG). At P0, it is found in the organ of Corti (OC; bracket), greater epithelial ridge, and medial wall. DAPI, nuclei (blue); scale bar, 100 μm. C, Section through the middle turn of E14.5 cochlear ducts from Fgf20Cre/+; ROSAmTmG/+ and Fgf20Cre/βgal; ROSAmTmG/+ embryos. Cells of the Fgf20Cre lineage express mGFP (mG, green); non-lineage cells express mTomato (mT, red). DAPI, nuclei (blue); scale bar, 100 μm. D, Schematic showing an overview of the TRAPseq protocol (see Experimental Procedures). (1) Ventral otocysts containing the cochlea were dissected from E14.5 embryos. (2) Otocysts from each litter were pooled according to genotype to increase RNA yield. (3) Otocysts were then homogenized and centrifuged to make polysomes (pre-TRAP samples were collected at this stage). This was followed by immunoprecipitation with anti-GFP antibodies to collect L10a-eGFP labeled polysomes. (4) This produced TRAP samples, which were then purified for RNA along with pre-TRAP samples and used for downstream applications. E, Schematic of a cross-sectional view of the E14.5 ventral otocyst, showing three turns of the cochlear duct, surrounded by periotic mesenchyme and otic capsule. Pre-TRAP RNA (representing total input tissue) comes from the cochlear duct epithelium, periotic mesenchyme, and otic capsule (gray). TRAP RNA (representing Fgf20Cre-lineage tissue, which expresses L10a-eGFP) comes from the prosensory domain, Kölliker's organ, medial wall of the cochlear duct, and some cells of the spiral ganglion (green). F, qRT-PCR showing fold change in Twist2 and Id2 expression (normalized to Gadph) in TRAP RNA samples compared to pre-TRAP samples from Fgf20Cre/+; ROSAmTmG/+ E14.5 cochleae. Each dot represents an RNA sample pooled from at least three embryos.
Constructing different types of mouse models is helpful for researchers to study the different functions of gene Fgf20. The understanding of genes is becoming increasingly clear: whether it is in the study of the pathways of hair growth or the study of cochlear formation and function. Cyagen wants to give you the most suitable mouse construction strategy according to your ideas. Our team of scientists can discuss with you any questions about mouse construction. You are welcome to contact us for a complimentary consultation, model generation strategy, and quote.
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 Huh S H, Warchol M E, Ornitz D M. Cochlear progenitor number is controlled through mesenchymal FGF receptor signaling[J]. Elife, 2015, 4: e05921.
 Yang L M, Stout L, Rauchman M, et al. Analysis of FGF20‐regulated genes in organ of Corti progenitors by translating ribosome affinity purification[J]. Developmental Dynamics, 2020, 249(10): 1217-1242.