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Background Understanding the mechanisms that regulate hair cell (HC) differentiation in the organ of Corti (OC) is essential to designing genetic therapies for hearing loss due to HC loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in HC and supporting cell differentiation in the mouse OC. To investigate the genetic landscape regulated by FGF20 signaling in OC progenitors, we employ Translating Ribosome Affinity Purification combined with Next Generation RNA Sequencing (TRAPseq) in the Fgf20 lineage. Results We show that TRAPseq targeting OC progenitors effectively enriched for RNA from this rare cell population. TRAPseq identified differentially expressed genes (DEGs) downstream of FGF20, including Etv4, Etv5, Etv1, Dusp6, Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, Sall3, and cell cycle regulators such as Cdc20. Analysis of Cdc20 conditional-null mice identified decreased cochlea length, while analysis of Sall1-null and Sall1-ΔZn2-10 mice, which harbor a mutation that causes Townes-Brocks syndrome, identified a decrease in outer hair cell number. Conclusions We present two datasets: genes with enriched expression in OC progenitors, and DEGs downstream of FGF20 in the embryonic day 14.5 cochlea. We validate select DEGs via in situ hybridization and in vivo functional studies in mice.

Mammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that translatome analysis in Tsc1+/− nephron progenitor cells from mice with elevated nephron numbers reveals how differential translation of Wnt antagonists over agonists tips the balance between self-renewal and differentiation. Wnt agonists are poorly translated in young niches, resulting in an environment with low R-spondin and high Fgf20 promoting self-renewal. In older niches we find increased translation of Wnt agonists, including R-spondin and the signalosome-promoting Tmem59, and low Fgf20, promoting differentiation. This suggests that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and possibly clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving synchronized differentiation. As predicted by these findings, removing one Rspo3 allele in nephron progenitors delays cessation and increases nephron numbers in vivo., The cessation of nephrogenesis determines nephron endowments in mammals. Here the authors show that differential translation of Wnt antagonists versus agonists determines the tipping point from self-renewal to differentiation of nephron progenitors during nephrogenesis, using mutant mice with elevated nephron numbers.

Much is known about the genetic and developmental basis of mutant phenotypes in the laboratory. However, much less is known about the mechanisms that control naturally evolved phenotypes in the wild. Are specific types of genes or mutations preferred? How are developmental programs modified to generate altered phenotypes? Finally, in cases where the same phenotype has evolved in independent lineages (convergent evolution), are the same genes or mutations used?The threespine stickleback fish (Gasterosteus aculeatus) is an emerging model organism in which the approaches and tools of developmental biology, genetics, and genomics can be applied to understand evolutionary changes. Marine sticklebacks have repeatedly colonized and adapted to new freshwater environments throughout the Northern Hemisphere. Freshwater sticklebacks have evolved many changes in their skeleton relative to marine fish. For example, freshwater sticklebacks have evolved changes to their defensive armor in response to decreased predation and changes in bones used for feeding in response to differences in diet. Despite their phenotypic differences, marine and freshwater sticklebacks can be bred in the lab. Crosses between marine and freshwater sticklebacks can be used to decipher the genetic basis of their evolved phenotypic differences.In chapter 2 of this thesis, I present the results of a large quantitative genetic study of stickleback skeletal evolution. I mapped regions of the genome, known as quantitative trait loci (QTL), affecting a wide range of skeletal traits. Over 100 QTL were mapped, and a meta-analysis of their properties was performed. Using these QTL, several general questions in evolutionary genetics were addressed. First, I explored the dominance of the QTL. The dominance of a QTL is a genetic property that might affect likelihood of being used during adaptation. Surprisingly, a large proportion of skeletal QTL had an additive (intermediate) dominance. Second, I investigated the modularity of QTL. To do this, I examined serially repeating skeletal elements such as vertebrae. Most (76%) QTL that influenced such traits had specific, modular effects on just a subset of the possible domains of the phenotype. Finally, I examined whether the QTL clustered in the genome. Many large-effect skeletal QTL were clustered on chromosomes 4, 20, and 21. Inheritance of these clusters of linked adaptive alleles might allow multiple aspects of skeletal morphology to evolve rapidly and simultaneously. In summary, the large set of skeletal QTL was used to explore the genetic properties of evolved mutations. These QTL are a starting point for the identification of the molecular basis of evolved changes in the vertebrate skeleton.In chapter 3 of this thesis, I focus on one skeletal trait, gill raker number. Gill rakers are periodically patterned bones in a fish’s throat important for feeding. In response to a change in diet in freshwater populations, hundreds of freshwater stickleback populations have independently evolved a reduction in gill raker number. This phenomenon is known as gill raker reduction. I found that heritable marine/freshwater differences in gill raker number were present early in development. The gill raker number differences were due to a difference in the spacing between adjacent gill rakers. The marine/freshwater number and spacing differences were present at the earliest detectable point of gill raker specification. This result suggests that gill raker reduction is caused by an early-acting change to a lateral inhibition process controlling raker bud spacing. Next, I performed linkage mapping in F2 fish from crosses with three independently derived freshwater populations. In all three crosses, gill raker QTL mapped to chromosomes 4 and 20, suggesting a similar genetic basis. Finally, the chromosome 4 and 20 QTL affected the early spacing of gill raker buds. Collectively, these data demonstrated that parallel developmental genetic features underlie the convergent evolution of gill raker reduction in freshwater sticklebacks. These results suggest that even highly polygenic adaptive traits can have a predictable developmental genetic basis.In chapter 4 of this thesis, I present an economical method that uses next-generation sequencing to perform genome-wide genotyping of hundreds of multiplexed samples. This method was used to genotype two large marine by freshwater F2 crosses of over 350 fish each. The resulting maps significantly improved the stickleback genome assembly by making over 100 changes to the order and orientation of the scaffolds that compose the genome assembly. In the revised genome assembly, 95% of the assembly was anchored to a chromosome, compared to 87% in the original assembly. To assess linkage map quality, I mapped quantitative trait loci (QTL) controlling lateral plate number. Plate number mapped as expected to a 200 kilobase genomic region containing Ectodysplasin. In addition, plate number mapped to a chromosome 7 QTL overlapping a previously identified modifier QTL. Finally, I examined gill raker length. I found that the two freshwater populations convergently evolved shorter gill rakers relative to marine fish. I mapped eight QTL controlling gill raker length in the two crosses. However, none of the QTL were overlapping between the two crosses. Thus, the convergent evolution gill raker length reduction has a surprisingly non-parallel genetic basis.In chapter 5 of this thesis, I present additional experiments to determine the genetic basis of evolved reduction in gill raker number. Using the genotyping method developed in chapter 4, I mapped QTL controlling raker number and spacing in two large marine by freshwater F2 crosses of over 350 fish each. Overlapping QTL on chromosomes 4, 16, and 20 were found in both crosses. In addition, 14 unique modifier QTL were mapped. Together these results indicate that a combination of similar and different genetic basis underlies the convergent evolution of gill raker number. Using crosses with recombinant chromosomes, I fine-mapped the chromosome 4 and 20 QTL, substantially narrowing the interval sizes. Surprisingly, the chromosome 4 QTL in the two crosses mapped to non-overlapping regions. Therefore, even though the chromosome 4 QTL appeared to map to similar genomic regions in the two crosses, a different genetic basis was actually used. An excellent candidate gene, Fibroblast Growth Factor 20 (Fgf20) was located within one of the fine-mapped chromosome 4 QTL. I mutated the coding region of Fgf20 with a genome-editing technology called Transcription Activator-Like Effector Nucleases (TALENs). Induced loss of function mutations in Fgf20 resulted in a decrease in gill raker number. The Fgf20 mutants had a concomitant increase in gill raker spacing, similar to the evolved freshwater phenotype. Induced mutations in a second gene, Smad5, also resulted in a raker phenotype. However, Smad5 was excluded from the fine-mapped QTL intervals, so it likely does not underlie gill raker evolution. No coding changes in Fgf20 likely cause evolved gill raker reduction. These results suggest a model in which cis-regulatory mutations that affect Fgf20 expression levels or spatial patterns contribute to evolved gill raker reduction.Together, this work makes significant progress towards understanding the genetic and developmental basis of skeletal evolution in stickleback fish. The results have broad implications for understanding the process of how genetic variation contributes to adaptive evolution.

Background Recently, the possibility of tumour classification based on genetic data has been investigated. However, genetic datasets are difficult to handle because of their massive size and complexity of manipulation. In the present study, we examined the diagnostic performance of machine learning applications using imaging-based classifications of oral squamous cell carcinoma (OSCC) gene sets. Methods RNA sequencing data from SCC tissues from various sites, including oral, non-oral head and neck, oesophageal, and cervical regions, were downloaded from The Cancer Genome Atlas (TCGA). The feature genes were extracted through a convolutional neural network (CNN) and machine learning, and the performance of each analysis was compared. Results The ability of the machine learning analysis to classify OSCC tumours was excellent. However, the tool exhibited poorer performance in discriminating histopathologically dissimilar cancers derived from the same type of tissue than in differentiating cancers of the same histopathologic type with different tissue origins, revealing that the differential gene expression pattern is a more important factor than the histopathologic features for differentiating cancer types. Conclusion The CNN-based diagnostic model and the visualisation methods using RNA sequencing data were useful for correctly categorising OSCC. The analysis showed differentially expressed genes in multiwise comparisons of various types of SCCs, such as KCNA10, FOSL2, and PRDM16, and extracted leader genes from pairwise comparisons were FGF20, DLC1, and ZNF705D.

Disruption of the blood-brain barrier (BBB) and the cerebral inflammatory response occurring after traumatic brain injury (TBI) facilitate further brain damage, which leads to long-term complications of TBI. Fibroblast growth factor 20 (FGF20), a neurotrophic factor, plays important roles in brain development and neuronal homeostasis. The aim of the current study was to assess the protective effects of FGF20 on TBI via BBB maintenance. In the present study, recombinant human FGF20 (rhFGF20) reduced neurofunctional deficits, brain edema, Evans blue extravasation and neuroinflammation in a TBI mouse model. In anin vitroTNF-α-induced human brain microvascular endothelial cell (HBMEC) model of BBB disruption, rhFGF20 reduced paracellular permeability and increased trans-endothelial electrical resistance (TEER). Both in the TBI mouse model andin vitro, rhFGF20 increased the expression of proteins composing in BBB-associated tight junctions (TJs) and adherens junctions (AJs), and decreased the inflammatory response, which protected the BBB integrity. Notably, rhFGF20 preserved BBB function by activating the AKT/GSK3β pathway and inhibited the inflammatory response by regulating the JNK/NFκB pathway. Thus, FGF20 is a potential candidate treatment for TBI that protects the BBB by upregulating junction protein expression and inhibiting the inflammatory response.

BackgroundUnderstanding the mechanisms that regulate hair cell (HC) differentiation in the organ of Corti (OC) is essential to designing genetic therapies for hearing loss due to HC loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in HC and supporting cell differentiation in the mouse OC. To investigate the genetic landscape regulated by FGF20 signaling in OC progenitors, we employ Translating Ribosome Affinity Purification combined with Next Generation mRNA Sequencing (TRAPseq) in theFgf20lineage.ResultsWe show that TRAPseq targeting OC progenitors effectively enriched for mRNA within this rare cell population. TRAPseq identified differentially expressed genes downstream of FGF20, includingEtv4, Etv5, Etv1, Dusp6, Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, Sall3, and cell cycle regulators such asCdc20. Analysis ofCdc20conditional-null mice identified decreased cochlea length, while analysis ofSall1-ΔZn2-10mice, which harbor a mutation that causes Townes-Brocks syndrome, identified a decrease in outer hair cell number.ConclusionsWe present two datasets: genes with enriched expression in OC progenitors, and genes regulated by FGF20 in the embryonic day 14.5 cochlea. We validate select differentially expressed genes via in situ hybridization and in vivo functional studies in mice.Key findingsTranslating Ribosome Affinity Purification (TRAP) with Fgf20-Cre enriches for prosensory cell mRNATRAP combined with RNAseq identifies genes downstream of FGF20 during prosensory cell differentiationFGF20 regulates Sall1, gene implicated in human sensorineural hearing lossGrant Sponsor and NumberNational Institute on Deafness and Other Communication Disorders – DC017042 (DMO) Washington University Institute of Clinical and Translational Sciences and National Center for Advancing Translational Sciences – CTSA grant UL1TR002345 (JIT471 to DMO) March of Dimes – 6-FY13-127 (MR)

Mammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that in young niches, cellular Wnt agonists are poorly translated, Fgf20 levels are high and R-spondin levels are low, resulting in a pro self-renewal environment. By contrast, older niches are low in Fgf20 and high in R-spondin, with increased cellular translation of Wnt agonists, including the signalosome-promoting Tmem59. This suggests a hypothesis that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving differentiation. We show Tsc1 hemizygosity differentially promoted translation of Wnt antagonists over agonists, expanding a transitional (Six2+, Cited1+, Wnt4+) state and delaying the tipping point. As predicted by these findings, reducing Rspo3 dosage in nephron progenitors or Tmem59 globally increased nephron numbers in vivo.

Segmentation of the vertebrate hindbrain leads to the formation of rhombomeres, each with a distinct anteroposterior identity. Specialised boundary cells form at segment borders that act as a source or regulator of neuronal differentiation. In zebrafish, there is spatial patterning of neurogenesis in which non-neurogenic zones form at boundaries and segment centres, in part mediated by Fgf20 signalling. To further understand the control of neurogenesis, we have carried out single cell RNA sequencing of the zebrafish hindbrain at three different stages of patterning. Analyses of the data reveal known and novel markers of distinct hindbrain segments, of cell types along the dorsoventral axis, and of the transition of progenitors to neuronal differentiation. We find major shifts in the transcriptome of progenitors and of differentiating cells between the different stages analysed. Supervised clustering with markers of boundary cells and segment centres, together with RNA-seq analysis of Fgf-regulated genes, has revealed new candidate regulators of cell differentiation in the hindbrain. These data provide a valuable resource for functional investigations of the patterning of neurogenesis and the transition of progenitors to neuronal differentiation., Summary: A single cell transcriptome at three stages of zebrafish hindbrain development provides a valuable resource for investigations of the patterning of neurogenesis and transition of progenitors to neuronal differentiation.

Patterns of mitotic cells’ distribution and activation of the MAP-kinase cascade during the regeneration of Xenopus laevis tadpole tails were studied before and during the refractory period. It is known that the tadpoles of Xenopus laevis are able to fully restore the full structure of the tail after amputation. However, in the refractory period (stage 45–47), the ability to regenerate is significantly reduced, until its complete absence. The mechanisms of this phenomenon are still poorly understood. We conducted a comparative analysis of the average number of mitotic cells on 0–4 days post amputation in normally regenerating tails and in tails amputated during the refractory period. A significant decrease in the number of proliferating cells throughout the surface of the tail in the refractory period compared with their sharp increase in the blastema area in normally regenerating tadpoles was shown. In addition, we detected activation of the MAP-kinase cascade (dpERK1/2) during normal regeneration and demonstrated its full inhibition during the refractory period. At the same time, in the distal part of the tail amputated in the refractory period, activation of the expression of the regenerative marker gene Fgf20 was not detected. Thus, we can conclude that the blocking of the regenerative capacity in tadpoles during the refractory period is accompanied by a sharp suppression of the mitotic activity of the cells and a misregulation of the activation of the Fgf–MAP-kinase cascade in the tail after amputation.

Background ：Blood-brain barrier (BBB) disruption and the cerebral inflammatory response are two reciprocal mechanisms that work together to mediate the degree of brain edema, which is responsible for the majority of deaths after traumatic brain injury (TBI), and facilitate further brain damage, which leads to long-term TBI complications. Fibroblast growth factor 20 (FGF20), a neurotrophic factor, plays important roles in the development of dopaminergic neurons in Parkinson disease (PD). However, little is known about the role of FGF20 in TBI. The aim of the current study was to assess the protective effects of FGF20 in TBI through protecting the BBB. Methods: We explored the relationship between FGF20 and BBB function in controlled cortical impact (CCI)-induced TBI mice model and TNF-α-induced human brain microvascular endothelial cell (HBMEC) in vitro BBB disruption model. We also explored the mechanisms of these interactions and the signaling processes involved in BBB function and neuroinflammation. Results: In this study, we demonstrate that recombinant human FGF20 (rhFGF20) reduced neurofunctional deficits, brain edema and Evans Blue penetration in vivo after TBI. In an in vitro BBB disruption model of, rhFGF20 could reverse changes to TNF-α-induced HBMEC morphology, reduce Transwell permeability, and increase transendothelial electrical resistance (TEER). In both a TBI mouse model and in vitro , rhFGF20 upregulated the expression of BBB-associated tight junction (TJ) protein and adherens junction (AJ) protein via the AKT/GSK3β pathway. In addition, rhFGF20 inhibited the cerebral inflammatory response through regulating the JNK/NFκB pathway and further protected the function of the BBB. Conclusions : Our results contribute to a new treatment strategy in TBI research. FGF20 is a potential candidate to treat TBI as it protects the BBB via regulating the AKT/GSK3β and JNK/NFκB signaling pathways.

Background Parkinson’s disease (PD) is the second most common neurodegenerative disease and has a complex etiology. Single nucleotide polymorphisms in the 3′-untranslated region of Fibroblast growth factor 20 (FGF 20) have been reported to be associated with PD; however, the results are controversial. Although FGF20 enhances the survival of dopaminergic neurons, it may also result in PD susceptibility by altering alpha-synuclein expression. Materials and methods To identify and characterize genetic risk variants in FGF 20 in Eastern Indian PD patients, 2 SNPs of FGF 20 (rs1721100 and rs2720208) were genotyped in 336 PD cases and 313 ethnically matched controls by PCR-RFLP. Results We observed statistically significant differences in genotypic and allelic frequencies of rs1721100 between PD cases and controls but not for rs12720208. Haplotype G-C showed a significant protective effect against PD. A functional assay revealed that the risk allele C at rs1721100 has little or no effect on relative luciferase activity from a reporter construct in the presence of miR-3189-3p, whereas allele G results in significant dose-dependent reduction. Conclusion Our results suggest that FGF 20 is a susceptibility gene for PD in Eastern Indians.

The regulation of final nephron number in the kidney is poorly understood. Cessation of nephron formation occurs when the self-renewing nephron progenitor population commits to differentiation. Transcription factors within this progenitor population, such as SIX2, are assumed to control expression of genes promoting self-renewal such that homozygous Six2 deletion results in premature commitment and an early halt to kidney development. In contrast, Six2 heterozygotes were assumed to be unaffected. Using quantitative morphometry, we found a paradoxical 18% increase in ureteric branching and final nephron number in Six2 heterozygotes, despite evidence for reduced levels of SIX2 protein and transcript. This was accompanied by a clear shift in nephron progenitor identity with a distinct subset of downregulated progenitor genes such as Cited1 and Meox1 while other genes were unaffected. The net result was an increase in nephron progenitor proliferation, as assessed by elevated EdU (5-ethynyl-2′-deoxyuridine) labeling, an increase in MYC protein, and transcriptional upregulation of MYC target genes. Heterozygosity for Six2 on an Fgf20 -/- background resulted in premature differentiation of the progenitor population, confirming that progenitor regulation is compromised in Six2 heterozygotes. Overall, our studies reveal a unique dose response of nephron progenitors to the level of SIX2 protein in which the role of SIX2 in progenitor proliferation versus self-renewal is separable.

Species-specific cusp patterns result from the iterative formation of enamel knots, the epithelial signaling centers, at the future cusp positions. The expressions of fibroblast growth factors (FGFs), especially Fgf4, in the secondary enamel knots in the areas of the future cusp tips are generally used to manifest the appearance of species-specific tooth shapes. However, the mechanism underlying the predictive role of FGFs in species-specific cusp patterns remains obscure. Here, we demonstrated that gerbils, which have a lophodont pattern, exhibit a striped expression pattern of Fgf4, whereas mice, which have a bunodont pattern, have a spotted expression pattern, and these observations verify the predictive role of Fgf4 in species-specific cusp patterns. By manipulating FGFs’ signaling in the inner dental epithelium of gerbils, we provide evidence for the intracellular participation of FGF signaling, specifically FGF4 and FGF20, in Rac1- and RhoA-regulated cellular geometry remolding during the determination of different cusp patterns. Our study presents a novel explanation of how different FGF expression patterns produce different cusp patterns and implies that a conserved intracellular FGF-GTPase signaling module might represent an underlying developmental basis for evolutionary changes in cusp patterns.

Fibroblast growth factor-20 (FGF20) is a paracrine member of the FGF family that is preferentially expressed in the substantia nigra pars compacta (SNpc). Previous studies have demonstrated that FGF20 enhances the survival of dopaminergic neurons suggesting the potential use of FGF20 to treat Parkinson’s disease (PD). However, the reduced solubility of the bacterial recombinant human FGF20 (rhFGF20) and the absence of efficient strategies to transport rhFGF20 across the blood–brain barrier (BBB) have halted its clinical application. In the present study, we have examined the efficiency of fuzing a small ubiquitin-related modifier (SUMO) to rhFGF20 to enhance its soluble expression and further investigated the efficacy of FUS-guided, rhFGF20-liposome transport across the BBB. We also examined the bioavailability and behavioral improvement in a 6-hydroxydopamine-lesioned rat model of PD following 2 weeks’ FUS-liposomal combinatorial treatment. Our results showed that, in contrast with rhFGF20 or LIP-FGF20, the FUS-LIP-rhFGF20 treatment could significantly improve the apomorphine-induced rotations by protecting against the loss of dopaminergic neurons in the SNpc. Our Results suggest that our combinatorial method would help overcome key challenges that hinder the currently available methods for the use of rhFGF20 in PD treatment.

The mouse organ of Corti, housed inside the cochlea, contains hair cells and supporting cells that transduce sound into electrical signals. These cells develop in two main steps: progenitor specification followed by differentiation. Fibroblast Growth Factor (FGF) signaling is important in this developmental pathway, as deletion of FGF receptor 1 (Fgfr1) or its ligand, Fgf20, leads to the loss of hair cells and supporting cells from the organ of Corti. However, whether FGF20-FGFR1 signaling is required during specification or differentiation, and how it interacts with the transcription factor Sox2, also important for hair cell and supporting cell development, has been a topic of debate. Here, we show that while FGF20-FGFR1 signaling functions during progenitor differentiation, FGFR1 has an FGF20-independent, Sox2-dependent role in specification. We also show that a combination of reduction in Sox2 expression and Fgf20 deletion recapitulates the Fgfr1-deletion phenotype. Furthermore, we uncovered a strong genetic interaction between Sox2 and Fgf20, especially in regulating the development of hair cells and supporting cells towards the basal end and the outer compartment of the cochlea. To explain this genetic interaction and its effects on the basal end of the cochlea, we provide evidence that decreased Sox2 expression delays specification, which begins at the apex of the cochlea and progresses towards the base, while Fgf20-deletion results in premature onset of differentiation, which begins near the base of the cochlea and progresses towards the apex. Thereby, Sox2 and Fgf20 interact to ensure that specification occurs before differentiation towards the cochlear base. These findings reveal an intricate developmental program regulating organ of Corti development along the basal-apical axis of the cochlea., Author summary The mammalian cochlea contains the organ of Corti, a specialized sensory epithelium populated by hair cells and supporting cells that detect sound. Hair cells are susceptible to injury by noise, toxins, and other insults. In mammals, hair cells cannot be regenerated after injury, resulting in permanent hearing loss. Understanding genetic pathways that regulate hair cell development in the mammalian organ of Corti will help in developing methods to regenerate hair cells to treat hearing loss. Many genes are essential for hair cell and supporting cell development in the mouse organ of Corti. Among these are Sox2, Fgfr1, and Fgf20. Here, we investigate the relationship between these three genes to further define their roles in development. Interestingly, we found that Sox2 and Fgf20 interact to affect hair cell and supporting cell development in a spatially-graded manner. We found that cells toward the outer compartment and the base of the cochlea are more strongly affected by the loss of Sox2 and Fgf20. We provide evidence that this spatially-graded effect can be partially explained by the roles of the two genes in the precise timing of two sequential stages of organ of Corti development, specification and differentiation.

Mesenchymal condensation is a critical step in organogenesis, yet the underlying molecular and cellular mechanisms remain poorly understood. The hair follicle dermal condensate is the precursor to the permanent mesenchymal unit of the hair follicle, the dermal papilla, which regulates hair cycling throughout life and bears hair inductive potential. Dermal condensate morphogenesis depends on epithelial Fibroblast Growth Factor 20 (Fgf20). Here, we combine mouse models with 3D and 4D microscopy to demonstrate that dermal condensates form de novo and via directional migration. We identify cell cycle exit and cell shape changes as early hallmarks of dermal condensate morphogenesis and find that Fgf20 primes these cellular behaviors and enhances cell motility and condensation. RNAseq profiling of immediate Fgf20 targets revealed induction of a subset of dermal condensate marker genes. Collectively, these data indicate that dermal condensation occurs via directed cell movement and that Fgf20 orchestrates the early cellular and molecular events., eLife digest All mammal hair springs from hair follicles under the skin. These follicles sit in the dermis, beneath the outermost skin layer, the epidermis. In the embryo, hair follicles develop from unspecialized cells in two tissues, the epithelium and the mesenchyme, which will later develop into the dermis and epidermis, respectively. As development progresses, the cells of these tissues begin to cluster, and signals passing back and forth between the epithelium and mesenchyme instruct the cells what to do. In the mesenchyme, cells called fibroblasts squeeze up against their neighbors, forming patches called dermal condensates. These mature into so-called dermal papillae, which supply specific molecules called growth factors that regulate hair formation throughout lifetime. Fibroblasts in the developing skin respond to a signal from the epithelium called fibroblast growth factor 20 (Fgf20), but we do not yet understand its effects. It is possible that Fgf20 tells the cells to divide, forming clusters of daughter cells around their current location. Or, it could be that Fgf20 tells the cells to move, encouraging them to travel towards one another to form groups. To address this question, Biggs, Mäkelä et al. examined developing mouse skin grown in the laboratory. They traced cells marked with fluorescent tags to analyze their behavior as the condensates formed. This revealed that the Fgf20 signal acts as a rallying call, triggering fibroblast movement. The cells changed shape and moved towards one another, rather than dividing to create their own clusters. In fact, they switched off their own cell cycle as the condensates formed, halting their ability to divide. A technique called RNA sequencing revealed that Fgf20 also promotes the use of genes known to be active in dermal condensates. Dermal papillae control hair growth, and transplanting them under the skin can form new hair follicles. However, these cells lose this ability when grown in the laboratory. Understanding how they develop could be beneficial for future hair growth therapy. Further work could also address fundamental questions in embryology. Condensates of cells from the mesenchyme also precede the formation of limbs, bones, muscles and organs. Extending this work could help us to understand this critical developmental step.

Purpose: To reveal the key genes and pathways involved in the metastasis of endometrial cancer remains. Methods: Microarray data GSE29436 consisting of 4 progressive endometrial cancer samples and 4 nonprogressive controls were downloaded from Gene Expression Omnibus database. Differentially Expressed Genes (DEGs) were screened out using Limma package in R, followed by hierarchical clustering. Gene Ontology and pathway enrichment analysis were performed for DEGs. Results: Bioinformatic analysis revealed a total of 65 DEGs between progressive and non-progressive samples. Functional annotation showed that those genes were mainly enriched in functions of cell proliferation, MPKA and TGF-beta signaling pathways, which involved with up-regulated genes pleiomorphic adenoma gene 1 (PLAG1), Insulin-Like Growth Factor 2 (IGF2), and down-regulated genes Fibroblast Growth Factor 20 (FGF20) and Thrombospondin 4 (THBS4). Conclusion: Sixty-five identified genes in progressive endometrial cancer samples were mainly associated with tumor metastasis possibly by enhancing cell proliferation and affecting MAPK and TGF-beta signaling pathways. High expression of PLAG1, IGF2 and low expression of FGF20, THBS4, therefore, appear to play an important role in tumorigenesis and progression of endometrial cancer.

SUMMARYCell fate transitions are essential for specialization of stem cells and their niches, but the precise timing and sequence of molecular events during embryonic development are largely unknown. Here, we show that dermal condensates (DC), signaling niches for epithelial progenitors in hair placodes, are specified before niche formation and function. With 3D/4D microscopy we identify unclustered DC precursors. With population-based and single-cell transcriptomics we define a molecular time-lapse of dynamic niche signatures and the developmental trajectory as the DC lineage emerges from fibroblasts. Co-expression of downregulated fibroblast and upregulated DC genes in niche precursors reveals a transitory molecular state following a proliferation shutdown. Waves of transcription factor and signaling molecule expression then consolidate DC niche formation. Finally, ablation of epidermal Wnt signaling and placode-derived FGF20 demonstrates their requirement for DC-precursor specification. These findings uncover a progenitor-dependent niche precursor fate and the transitory molecular events controlling niche formation and function.Graphical AbstractHIGHLIGHTSPrecursors of the hair follicle niche are specified before niche cluster formationBulk/single cell RNA-seq defines early niche fate at molecular transitional stateSuccessive waves of transcription factor/signaling genes mark niche fate acquisitionNiche fate acquisition is not “pre-programmed” and requires FGF20 from progenitors

SummaryDuring fetal development, nephrons of the metanephric kidney form from a mesenchymal progenitor population that differentiates en masse before or shortly after birth. We explored intrinsic and extrinsic mechanisms controlling progenitor lifespan in a transplantation assay that allowed us to compare engraftment of old and young progenitors into the same young niche. The progenitors displayed an age-dependent decrease in proliferation and concomitant increase in niche exit rates. Single-cell transcriptome profiling revealed progressive age-dependent changes, with heterogeneity increasing in older populations. Age-dependent elevation in mTor and reduction in Fgf20 could contribute to increased exit rates. Importantly, 30% of old progenitors remained in the niche for up to 1 week post engraftment, a net gain of 50% to their lifespan, but only if surrounded by young neighbors. We provide evidence in support of a model in which intrinsic age-dependent changes affect inter-progenitor interactions that drive cessation of nephrogenesis.

Murine primary hair follicle induction is driven by the communication between the mesenchyme and epithelium and mostly governed by signaling pathways including wingless-related integration site (WNT), ectodysplasin A receptor (EDAR), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF), as observed in genetically modified mouse models. Sheep skin may serve as a valuable system for hair research owing to the co-existence of sweat glands with wool follicles in trunk skin and asynchronized wool follicle growth pattern similar to that of human head hair follicles. However, the mechanisms underlying wool follicle development remain largely unknown. To understand how long non-coding RNAs (lncRNAs) and mRNAs function in primary wool follicle induction in carpet wool sheep, we conducted high-throughput RNA sequencing and revealed globally altered lncRNAs (36 upregulated and 26 downregulated), mRNAs (228 elevated and 225 decreased), and 80 differentially expressed novel transcripts. Several key signals in WNT (WNT2B and WNT16), BMP (BMP3, BMP4, and BMP7), EDAR (EDAR and EDARADD), and FGF (FGFR2 and FGF20) pathways, and a series of lncRNAs, including XLOC_539599, XLOC_556463, XLOC_015081, XLOC_1285606, XLOC_297809, and XLOC_764219, were shown to be potentially important for primary wool follicle induction. GO and KEGG analyses of differentially expressed mRNAs and potential targets of altered lncRNAs were both significantly enriched in morphogenesis biological processes and transforming growth factor-β, Hedgehog, and PI3K-Akt signaling, as well as focal adhesion and extracellular matrix-receptor interactions. The prediction of mRNA-mRNA and lncRNA-mRNA interaction networks further revealed transcripts potentially involved in primary wool follicle induction. The expression patterns of mRNAs and lncRNAs of interest were validated by qRT-PCR. The localization of XLOC_297809 and XLOC_764219 both in placodes and dermal condensations was detected by in situ hybridization, indicating important roles of lncRNAs in primary wool follicle induction and skin development. This is the first report elucidating the gene network of lncRNAs and mRNAs associated with primary wool follicle early development in carpet wool sheep and will shed new light on selective wool sheep breeding.

Mammary gland development begins with the appearance of epithelial placodes that invaginate, sprout, and branch to form small arborized trees by birth. The second phase of ductal growth and branching is driven by the highly invasive structures called terminal end buds (TEBs) that form at ductal tips at the onset of puberty. Ectodysplasin (Eda), a tumor necrosis factor-like ligand, is essential for the development of skin appendages including the breast. In mice, Eda regulates mammary placode formation and branching morphogenesis, but the underlying molecular mechanisms are poorly understood. Fibroblast growth factor (Fgf) receptors have a recognized role in mammary ductal development and stem cell maintenance, but the ligands involved are ill-defined. Here we report that Fgf20 is expressed in embryonic mammary glands and is regulated by the Eda pathway. Fgf20 deficiency does not impede mammary gland induction, but compromises mammary bud growth, as well as TEB formation, ductal outgrowth and branching during puberty. We further show that loss of Fgf20 delays formation of Eda-induced supernumerary mammary buds and normalizes the embryonic and postnatal hyperbranching phenotype of Eda overexpressing mice. These findings identify a hitherto unknown function for Fgf20 in mammary budding and branching morphogenesis.

Supplemental Digital Content is available in the text, To identify variants of the genes in fibroblast growth factors/fibroblast growth factor receptors (FGF/FGFR) signal pathway that predispose to mandibular prognathism (MP) in the general Chinese population systematically. Targeted sequencing of the FGF/FGFR genes was conducted in 176 MP individuals and 155 class I malocclusion controls. The associations of common and rare variants with MP as a categorical phenotype and also continuous malocclusion phenotypes generated by principal component (PC) analysis were analyzed. One common variant, rs372127537, located in the 3’-untranslated region of FGF7 gene, was significantly related to PC1 (P = 4.22 × 10–4), which explained 23.23% of the overall phenotypic variation observed and corresponded to vertical discrepancies ranging from short anterior face height to long anterior face height, after Bonferroni correction. Also, 15 other variants were associated with PC1–4, although not significant after multiple corrections (P

Many studies have investigated the association between fibroblast growth factor 20(FGF20) rs12720208(C/T) polymorphism and the susceptibility of Parkinson's disease (PD). However, published data are still controversial. Here, we performed a meta-analysis to evaluate the association of rs12720208 polymorphism with the risk of PD.Up to April 2016, Pubmed, EMbase, Web of science, the Chinese National Knowledge Infrastructure, and Wanfang Medicine were reviewed to identify appropriate documents. A total of seven papers involving 11 studies with 3360 PD cases and 3681 controls were included based on the strict inclusion and exclusion standards. And STATA 12.0 statistics software was used to calculate available data from each study. The pooled odds ratios (OR) and 95% confidence interval (CI) were calculated to assess the association between FGF20 rs12720208 polymorphism and PD risk.When all studies were pooled into this meta-analysis, neither the minor T allele frequencies nor the genotypic distributions were different between PD cases and controls. But the subgroup analysis stratified by ethnicity showed FGF20 rs12720208 polymorphism was associated with increased risk in the allele model (T vs. C:OR = 1.167, 95% CI = 1.020-1.335) and dominant model (TT + TC vs. CC:OR = 1.156, 95% CI = 1.001-1.335) in Caucasians but not in Asians.This meta-analysis indicates that rs12720208 C/T variant might be associated with PD susceptibility in Caucasians.

Introdução: A doença de Parkinson é a segunda doença neurodegenerativa mais frequente e está relacionada à perda de neurônios dopaminérgicos na porção ventrolateral da substância nigra. A fisiopatologia da doença é pouco conhecida, mas fatores de risco genéticos e ambientais parecem ter um papel. O FGF20 é um fator neurotrófico preferencialmente expresso na substância nigra, e está relacionado com maior sobrevida de neurônios dopaminérgicos. O objetivo deste estudo é avaliar se polimorfismos genéticos do FGF20 está associado com a idade de início da doença de Parkinson. Métodos: Trata-se de um estudo transversal. O polimorfismo rs1989754 foi genotipado em 224 pacientes ambulatoriais com doença de Parkinson idiopática. Uma entrevista estruturada foi realizada com os pacientes e seus familiares, e a idade de início da doença de Parkinson foi definida como a idade de início dos sintomas motores. Resultados: O polimorfismo rs1989754 do gene FGF20 está associado com a idade de início da doença de Parkinson. Os portadores do alelo C tiveram uma idade de início mais precoce da doença tanto quando foi considerado um modelo dominante (56 ± 11.8 vs. 61.5 ± 11.6 anos, p=0.001), quando um modelo aditivo (55 ± 10.9 anos para o genótipo CC, 56.4 ± 11.2 anos para o genótipo CG e 61.5 ± 11.6 anos para o genótipo GG, p=0.002). Conclusão: Os achados do presente estudo sugerem que o polimorfismo rs1989754 do FGF20 está associado com idade de início mais precoce da doença de Parkinson, e dão apoio à hipótese de que a idade de início da doença de Parkinson é modulada por polimorfismos comuns de múltiplos genes, cada um com tamanho de efeito pequeno. Background: Parkinson’s disease is the second most prevalent neurodegenerative disorder and is related to loss of dopaminergic neurons in the ventrolateral substantia nigra. The physiopathology of the disease is poor understood, but both environmental and genetics risk factors are thought to play a role. FGF20 is a neurotrophic factor preferentially expressed in substantia nigra that enhances dopaminergic cells survival. The aim of this study is to evaluate if polymorphisms in FGF20 are associated with age of onset of Parkinson’s disease. Methods: This is a transversal study, in which the rs1989754 polymorphism was genotyped 224 outpatients with idiopathic PD. A structures interview was performed with patients and their relatives, and the age of onset of Parkinson’s disease was defined as the age of onset of the motor symptoms, as reported by the patients and their caregivers. Results: We found an association of the rs1989754 single nucleotide polymorphism with the age of onset of Parkinson’s disease. The carriers of the C allele had an earlier age of onset of the disease in both dominant (56 ± 11.8 vs. 61.5 ± 11.6 years, p=0.001) and additive models (55 ± 10.9 years for CC genotype, 56.4 ± 11.2 years for CG genotype and 61.5 ± 11.6 years for GG genotype, p=0.002). Conclusion: The findings of the present study suggest that FGF20 rs1989754 single nucleotide polymorphism is associated with younger age at onset of Parkinson’s disease, and also support the hypothesis that the age of onset of Parkinson’s disease is modulated by common polymorphisms of multiple risk genes with low effect size.

Fibroblast growth factor 20 (FGF20) is a neurotrophic factor which enhances the survival of rat midbrain dopamine neurons. Genetic variation in FGF20 may influence the risk of occurrence and development in Parkinson's diseases (PD). Many studies have evaluated the association between FGF20 rs1721100 C/G polymorphism and the risk of sporadic PD; however, published data are still controversial. The aim of the present meta-analysis was to evaluate the association of FGF20 rs1721100 C/G polymorphism with susceptibility of PD. The summary odds ratio (OR) with its 95 % confidence interval (CI) was calculated to estimate the association. Five case-control studies with a total of 3,463 sporadic PD cases and 4,606 controls were finally included into this meta-analysis. Neither the basic allele frequencies nor the genotypic distributions of rs1721100 C/G within FGF20 were different between two groups when all studies were pooled into the meta-analysis. Subgroup analysis by ethnicity showed FGF20 rs1721100 C/G polymorphism was significantly associated with increased risk in the heterozygote comparison model (CG versus GG: OR = 0.83, 95 % CI, 0.72-0.95, P = 0.009) in Asians but not in Caucasians. Overall, this meta-analysis suggests that FGF20 rs1721100 C/G polymorphism is associated with sporadic PD in Asians.