Spatially distinct epithelial and mesenchymal cell subsets along progressive lineage restriction in the branching embryonic mammary gland.

How cells coordinate morphogenetic cues and fate specification during development remains a fundamental question in organogenesis. The mammary gland arises from multipotent stem cells (MaSCs), which are progressively replaced by unipotent progenitors by birth. However, the lack of specific markers for early fate specification has prevented the delineation of the features and spatial localization of MaSC-derived lineage-committed progenitors. Here, using single-cell RNA sequencing from E13.5 to birth, we produced an atlas of matched mouse mammary epithelium and mesenchyme and reconstructed the differentiation trajectories of MaSCs toward basal and luminal fate. We show that murine MaSCs exhibit lineage commitment just prior to the first sprouting events of mammary branching morphogenesis at E15.5. We identify early molecular markers for committed and multipotent MaSCs and define their spatial distribution within the developing tissue. Furthermore, we show that the mammary embryonic mesenchyme is composed of two spatially restricted cell populations, and that dermal mesenchyme-produced FGF10 is essential for embryonic mammary branching morphogenesis. Altogether, our data elucidate the spatiotemporal signals underlying lineage specification of multipotent MaSCs, and uncover the signals from mesenchymal cells that guide mammary branching morphogenesis.

Exploring the principles of embryonic mammary gland branching morphogenesis.

Branching morphogenesis is a characteristic feature of many essential organs, such as the lung and kidney, and most glands, and is the net result of two tissue behaviors: branch point initiation and elongation. Each branched organ has a distinct architecture customized to its physiological function, but how patterning occurs in these ramified tubular structures is a fundamental problem of development. Here, we use quantitative 3D morphometrics, time-lapse imaging, manipulation of ex vivo cultured mouse embryonic organs and mice deficient in the planar cell polarity component Vangl2 to address this question in the developing mammary gland. Our results show that the embryonic epithelial trees are highly complex in topology owing to the flexible use of two distinct modes of branch point initiation: lateral branching and tip bifurcation. This non-stereotypy was contrasted by the remarkably constant average branch frequency, indicating a ductal growth invariant, yet stochastic, propensity to branch. The probability of branching was malleable and could be tuned by manipulating the Fgf10 and Tgfß1 pathways. Finally, our in vivo data and ex vivo time-lapse imaging suggest the involvement of tissue rearrangements in mammary branch elongation.

Fgf10 mutant newts regenerate normal hindlimbs despite severe developmental defects.

In amniote limbs, Fibroblast Growth Factor 10 (FGF10) is essential for limb development, but whether this function is broadly conserved in tetrapods and/or involved in adult limb regeneration remains unknown. To tackle this question, we established Fgf10 mutant lines in the newt Pleurodeles waltl which has amazing regenerative ability. While Fgf10 mutant forelimbs develop normally, the hindlimbs fail to develop and downregulate FGF target genes. Despite these developmental defects, Fgf10 mutants were able to regenerate normal hindlimbs rather than recapitulating the embryonic phenotype. Together, our results demonstrate an important role for FGF10 in hindlimb formation, but little or no function in regeneration, suggesting that different mechanisms operate during limb regeneration versus development.

DNA damage to bone marrow stromal cells by antileukemia drugs induces chemoresistance in acute myeloid leukemia via paracrine FGF10-FGFR2 signaling.

Chemoresistance remains a major challenge in the current treatment of acute myeloid leukemia (AML). The bone marrow microenvironment (BMM) plays a complex role in protecting leukemia cells from chemotherapeutics, and the mechanisms involved are not fully understood. Antileukemia drugs kill AML cells directly but also damage the BMM. Here, we determined antileukemia drugs induce DNA damage in bone marrow stromal cells (BMSCs), resulting in resistance of AML cell lines to adriamycin and idarubicin killing. Damaged BMSCs induced an inflammatory microenvironment through NF-κB; suppressing NF-κB with small molecule inhibitor Bay11-7082 attenuated the prosurvival effects of BMSCs on AML cell lines. Furthermore, we used an ex vivo functional screen of 507 chemokines and cytokines to identify 44 proteins secreted from damaged BMSCs. Fibroblast growth factor-10 (FGF10) was most strongly associated with chemoresistance in AML cell lines. Additionally, expression of FGF10 and its receptors, FGFR1 and FGFR2, was increased in AML patients after chemotherapy. FGFR1 and FGFR2 were also widely expressed by AML cell lines. FGF10-induced FGFR2 activation in AML cell lines operates by increasing P38 MAPK, AKT, ERK1/2, and STAT3 phosphorylation. FGFR2 inhibition with small molecules or gene silencing of FGFR2 inhibited proliferation and reverses drug resistance of AML cells by inhibiting P38 MAPK, AKT, and ERK1/2 signaling pathways. Finally, release of FGF10 was mediated by ß-catenin signaling in damaged BMSCs. Our data indicate FGF10-FGFR2 signaling acts as an effector of damaged BMSC-mediated chemoresistance in AML cells, and FGFR2 inhibition can reverse stromal protection and AML cell chemoresistance in the BMM.

Craniofacial anomalies in a murine model of heterozygous fibroblast growth factor 10 gene mutation.

OBJECTIVE: Dysregulation of Fibroblast Growth Factor 10 (FGF10), a member of the family of Fibroblast Growth Factor (FGF) proteins, has been implicated in craniofacial and dental anomalies, including craniosynostosis, cleft palate, and Lacrimo-Auriculo-Dento-Digital Syndrome. The aim of this murine study was to assess the craniofacial and dental phenotypes associated with a heterozygous FGF10 gene (FGF10+/- ) mutation at skeletal maturity. METHODS: Skulls of 40 skeletally mature mice, comprising two genotypes (heterozygous FGF10+/- mutation, n = 22; wildtype, n = 18) and two sexes (male, n = 23; female, n = 17), were subjected to micro-computed tomography. Landmark-based linear dimensions were measured for the cranial vault, maxilla, mandible, and first molar teeth. Multivariate analysis of variance was performed to assess whether there were significant differences in the craniofacial and dental structures between genotypes and sexes. RESULTS: The craniomaxillary skeleton and the first molar teeth were smaller in the FGF10+/- mice (P < .05), but the mandible was unaffected. Sex did not have a significant effect on these structures (P > .05). Cranial sutural defects were noted in 5/22 (22.7%) mutant versus 2/18 (11.1%) wildtype mice, and cleft palate in only one (4.5%) mutant mouse. None of the mice displayed craniosynostosis, expansive bony lesions, bifid condyles, or impacted teeth. CONCLUSION: The FGF10+/- mutation was associated with craniomaxillary skeletal hypoplasia that probably arose from deficient (delayed) intramembranous ossification of the sutured bones. Overall, the skeletal and dental data suggest that the FGF10 gene plays an important role in the aetiology of craniofacial dysmorphology and malocclusion.

Clinical efficacy of blood derivatives on wound healing: A systematic review and network meta-analysis.

This study aims to evaluate the clinical effects of different blood derivatives on wound healing using network meta-analysis. PubMed, Embase, OVID, Web of Science, SCOPUS and Cochrane Central were searched to obtain studies about blood derivatives on wound healing until October 2023. R 4.2.0 and Stata 15.0 softwares were used for data analysis. Forty-four studies comprising 5164 patients were included. The results of network meta-analysis showed that the healing area from high to low was GF + ORCCB, ORCCB, GF, PRF, Unnas paste dressing, APG, PRP injection, PRP, PRP + thrombin gel, PPP, HPL, CT. The healing time from low to high was PRP + thrombin gel, GF, PRP, PC + K, PC, APG, PRF, CT, Silver sulfadiazine ointment. The number of patients cured from high to low was APG, PRP injection, PRP, Aurix, PRF, Leucopatch, HPL, Antimicrobial Ointment Dressing, CT, 60 µg/cm2 repifermin, 120 µg/cm2 repifermin, AFG, PPP. The order of analgesic effect from high to low was AFG, Aminogam gel, PRF, PRP, Oxidised oil, APG, GF, CT. The order of the number of wound infection cases from low to high is APG, 20 µg/cm2 repifermin, 60 µg/cm2 repifermin, PRP, LeucoPatch, CT, PPP, Antiseptic ointment dressing. Healing area: GF + ORCCB had the best effect; Healing time: PRP + thrombin gel took the shortest time. The number of cured patients and the reduction of wound infection: APG has the best effect. Analgesic effect: AFG has the best effect. More studies with large sample sizes are needed to confirm the above findings.

Adenogenesis Factors FGF7, FGF10, FGF23, IFN-τ and HGF in Endometriosis Tissue Respect to Eutopic Endometrium: An Immunohistochemical Study.

Endometriosis is a pathological condition defined by the occurrence of endometrial glandular and stromal structures in anatomical compartments different from the uterine cavity. Endometriosis is a genetic polymorphism, estrogen-dependent inflammatory disease. This very common pathological entity causes a high level of morbidity in patients; it is also considered one of the most important causes of infertility. We and others have proposed as a pathogenetic mechanism of endometriosis a modification in the fine tuning of the processes of organogenesis of the uterus. We have correlated the immunohistochemical expression in deep endometriotic lesions and in normal endometrial tissue of several molecular factors that are implicated in the embryonic development of the uterine glands. We noticed a significant higher expression both for epithelium and stroma in the controls respect to the endometriosis samples for FGF7, FGF-10 and HGF. Interestingly, regarding FGF-23 and IFN-τ, we observed a significant higher expression in the ectopic endometrial stroma compared to the eutopic endometrium, while thepithetlium expression did not display a significant differential expression in endometriosis tissues respect to normal endometrium. The data generated support the fact that endometriosis tissues, both the epithelial and stromal component, have a different phenotype respect to the eutopic endometrium and sustain the hypothesis that alterations in the molecular mechanisms in control for adenogenesis and survival of endometrial structures are linked to the genesis and survival of endometriosis lesions outside of the uterus.

Slc26a9P2ACre : a new CRE driver to regulate gene expression in the otic placode lineage and other FGFR2b-dependent epithelia.

Pan-otic CRE drivers enable gene regulation throughout the otic placode lineage, comprising the inner ear epithelium and neurons. However, intersection of extra-otic gene-of-interest expression with the CRE lineage can compromise viability and impede auditory analyses. Furthermore, extant pan-otic CREs recombine in auditory and vestibular brain nuclei, making it difficult to ascribe resulting phenotypes solely to the inner ear. We have previously identified Slc26a9 as an otic placode-specific target of the FGFR2b ligands FGF3 and FGF10. We show here that Slc26a9 is otic specific through E10.5, but is not required for hearing. We targeted P2ACre to the Slc26a9 stop codon, generating Slc26a9P2ACre mice, and observed CRE activity throughout the otic epithelium and neurons, with little activity evident in the brain. Notably, recombination was detected in many FGFR2b ligand-dependent epithelia. We generated Fgf10 and Fgf8 conditional mutants, and activated an FGFR2b ligand trap from E17.5 to P3. In contrast to analogous mice generated with other pan-otic CREs, these were viable. Auditory thresholds were elevated in mutants, and correlated with cochlear epithelial cell losses. Thus, Slc26a9P2ACre provides a useful complement to existing pan-otic CRE drivers, particularly for postnatal analyses.

Fibroblast growth factor 10 is a negative regulator of postnatal neurogenesis in the mouse hypothalamus.

New neurons are generated in the postnatal rodent hypothalamus, with a subset of tanycytes in the third ventricular (3V) wall serving as neural stem/progenitor cells. However, the precise stem cell niche organization, the intermediate steps and the endogenous regulators of postnatal hypothalamic neurogenesis remain elusive. Quantitative lineage-tracing in vivo revealed that conditional deletion of fibroblast growth factor 10 (Fgf10) from Fgf10-expressing ß-tanycytes at postnatal days (P)4-5 results in the generation of significantly more parenchymal cells by P28, composed mostly of ventromedial and dorsomedial neurons and some glial cells, which persist into adulthood. A closer scrutiny in vivo and ex vivo revealed that the 3V wall is not static and is amenable to cell movements. Furthermore, normally ß-tanycytes give rise to parenchymal cells via an intermediate population of α-tanycytes with transient amplifying cell characteristics. Loss of Fgf10 temporarily attenuates the amplification of ß-tanycytes but also appears to delay the exit of their α-tanycyte descendants from the germinal 3V wall. Our findings suggest that transience of cells through the α-tanycyte domain is a key feature, and Fgf10 is a negative regulator of postnatal hypothalamic neurogenesis.

Fibroblast growth factor 10 reverses cigarette smoke- and elastase-induced emphysema and pulmonary hypertension in mice.

BACKGROUND: COPD is an incurable disease and a leading cause of death worldwide. In mice, fibroblast growth factor (FGF)10 is essential for lung morphogenesis, and in humans, polymorphisms in the human FGF10 gene correlate with an increased susceptibility to develop COPD. METHODS: We analysed FGF10 signalling in human lung sections and isolated cells from healthy donor, smoker and COPD lungs. The development of emphysema and PH was investigated in Fgf10+/- and Fgfr2b+/- (FGF receptor 2b) mice upon chronic exposure to cigarette smoke. In addition, we overexpressed FGF10 in mice following elastase- or cigarette smoke-induced emphysema and pulmonary hypertension (PH). RESULTS: We found impaired FGF10 expression in human lung alveolar walls and in primary interstitial COPD lung fibroblasts. In contrast, FGF10 expression was increased in large pulmonary vessels in COPD lungs. Consequently, we identified impaired FGF10 signalling in alveolar walls as an integral part of the pathomechanism that leads to emphysema and PH development: mice with impaired FGF10 signalling (Fgf10+/- and Fgfr2b+/- ) spontaneously developed lung emphysema, PH and other typical pathomechanistic features that generally arise in response to cigarette smoke exposure. CONCLUSION: In a therapeutic approach, FGF10 overexpression successfully restored lung alveolar and vascular structure in mice with established cigarette smoke- and elastase-induced emphysema and PH. FGF10 treatment triggered an initial increase in the number of alveolar type 2 cells that gradually returned to the basal level when the FGF10-mediated repair process progressed. Therefore, the application of recombinant FGF10 or stimulation of the downstream signalling cascade might represent a novel therapeutic strategy in the future.

FGF10 Triggers De Novo Alveologenesis in a Bronchopulmonary Dysplasia Model: Impact on Resident Mesenchymal Niche Cells.

Bronchopulmonary dysplasia (BPD) is a neonatal lung disease developing in premature babies characterized by arrested alveologenesis and associated with decreased Fibroblast growth factor 10 (FGF10) expression. One-week hyperoxia (HYX) exposure of newborn mice leads to a permanent arrest in alveologenesis. To test the role of Fgf10 signaling to promote de novo alveologenesis following hyperoxia, we used transgenic mice allowing inducible expression of Fgf10 and recombinant FGF10 (rFGF10) protein delivered intraperitoneally. We carried out morphometry analysis, and IF on day 45. Alveolospheres assays were performed co-culturing AT2s from normoxia (NOX) with FACS-isolated Sca1Pos resident mesenchymal cells (rMC) from animals exposed to NOX, HYX-PBS, or HYX-FGF10. scRNAseq between rMC-Sca1Pos isolated from NOX and HYX-PBS was also carried out. Transgenic overexpression of Fgf10 and rFGF10 administration rescued the alveologenesis defects following HYX. Alveolosphere assays indicate that the activity of rMC-Sca1Pos is negatively impacted by HYX and partially rescued by rFGF10 treatment. Analysis by IF demonstrates a significant impact of rFGF10 on the activity of resident mesenchymal cells. scRNAseq results identified clusters expressing Fgf10, Fgf7, Pdgfra, and Axin2, which could represent the rMC niche cells for the AT2 stem cells. In conclusion, we demonstrate that rFGF10 administration is able to induce de novo alveologenesis in a BPD mouse model and identified subpopulations of rMC-Sca1Pos niche cells potentially representing its cellular target.

High-level production of keratinocyte growth factor 2 in Escherichia coli.

Recombinant human keratinocyte growth factor 2 (KGF-2), also known as repifermin, is used in various therapeutic applications. However, KGF-2 production has not been optimized for facilitating large-scale production. Therefore, we attempted to attain high-level production of bioactive KGF-2. KGF-2 was fused with 6HFh8 (6HFh8-KGF-2) at the tobacco etch virus protease cleavage site. The 6HFh8-KGF-2 was expressed in Escherichia coli with high expression levels of approximately 33% and 20% of soluble protein in flask culture and 5 L fermentation, respectively. 6HFh8-KGF-2 was purified via nickel affinity chromatography. To maintain a stable form of KGF-2, the conditions of the cleavage reaction were optimized based on the isoelectric point. KGF-2 was purified via ion-exchange chromatography to high purity (>99%) with an optimal purification yield (91%). Circular dichroism spectroscopy demonstrated that purified KGF-2 had a secondary structure and thermal stability similar to that of commercial KGF-2. Bioactivity assays indicated that purified KGF-2 could induce MCF-7 cell proliferation in the same manner as commercial KGF-2. These results demonstrate that bioactive KGF-2 was overexpressed in E. coli and purified to high quality. Our findings indicated that bioactive KGF-2 can be produced in large quantities in E. coli.

FGF10 mitigates doxorubicin-induced myocardial toxicity in mice via activation of FGFR2b/PHLDA1/AKT axis.

Doxorubicin is a common chemotherapeutic agent in clinic, but myocardial toxicity limits its use. Fibroblast growth factor (FGF) 10, a multifunctional paracrine growth factor, plays diverse roles in embryonic and postnatal heart development as well as in cardiac regeneration and repair. In this study we investigated the role of FGF10 as a potential modulator of doxorubicin-induced cardiac cytotoxicity and the underlying molecular mechanisms. Fgf10+/- mice and an inducible dominant negative FGFR2b transgenic mouse model (Rosa26rtTA; tet(O)sFgfr2b) were used to determine the effect of Fgf10 hypomorph or blocking of endogenous FGFR2b ligands activity on doxorubicin-induced myocardial injury. Acute myocardial injury was induced by a single injection of doxorubicin (25 mg/kg, i.p.). Then cardiac function was evaluated using echocardiography, and DNA damage, oxidative stress and apoptosis in cardiac tissue were assessed. We showed that doxorubicin treatment markedly decreased the expression of FGFR2b ligands including FGF10 in cardiac tissue of wild type mice, whereas Fgf10+/- mice exhibited a greater degree of oxidative stress, DNA damage and apoptosis as compared with the Fgf10+/+ control. Pre-treatment with recombinant FGF10 protein significantly attenuated doxorubicin-induced oxidative stress, DNA damage and apoptosis both in doxorubicin-treated mice and in doxorubicin-treated HL-1 cells and NRCMs. We demonstrated that FGF10 protected against doxorubicin-induced myocardial toxicity via activation of FGFR2/Pleckstrin homology-like domain family A member 1 (PHLDA1)/Akt axis. Overall, our results unveil a potent protective effect of FGF10 against doxorubicin-induced myocardial injury and identify FGFR2b/PHLDA1/Akt axis as a potential therapeutic target for patients receiving doxorubicin treatment.

Fibroblast growth factor 10 ameliorates renal ischaemia-reperfusion injury by attenuating mitochondrial damage.

Ischaemia-reperfusion (I/R) injury is one of the leading causes of acute kidney injury (AKI). Its pathologic mechanism is quite complex, involving oxidative stress, inflammatory response, autophagy, and apoptosis. Fibroblast growth factor 10 (FGF10) and 5-hydroxydecanoate (5-HD) play essential roles in kidney injury. Rats were divided into four groups: (i) sham group, sham-operated animals with an unconstructed renal artery; (ii) I/R group, kidneys were subjected to 50 min of ischaemia followed by reperfusion for 2 days; (iii) I/R + FGF10 group, animals treated with 0.5 mg/kg FGF10 (i.p.) 1 h before ischaemia; and (iv) 5-HD group, animals treated with 5 mg/kg 5-HD (i.m.) 30 min before FGF10 treatment. Renal injury, apoptosis damage, mitochondrial oxidative damage, mitochondrial membrane potential (MMP), and expression of the ATP-sensitive K+ (KATP) channel subunit Kir6.2 were evaluated. FGF10 treatment significantly alleviated I/R-induced elevation in the serum creatinine level and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling-positive tubular cells in the kidney. In addition, FGF10 dramatically ameliorated renal mitochondrial-related damage, including reducing mitochondrial-dependent apoptosis, alleviating oxidative stress, maintaining the mitochondrial membrane potential, and opening the mitochondrial KATP channels. The protective effect of FGF10 was significantly compromised by the ATP-dependent potassium channel blocker 5-HD. Our data suggest that FGF10 offers effective protection against I/R and improves animal survival by attenuating mitochondrial damage.

Distinct effects of Fgf7 and Fgf10 on the terminal differentiation of murine bladder urothelium revealed using an organoid culture system.

BACKGROUND: Dysregulation of the terminal differentiation of bladder urothelium is associated with the pathogenesis of urinary tract disorders. Fibroblast growth factor (Fgf)7 and Fgf10 stimulate urothelial proliferation; however, their roles in cellular differentiation remain unclear. In this study, we used an organoid system to investigate the roles of these Fgfs in regulating bladder urothelium differentiation and identify their distribution patterns in the mouse bladder. METHODS: Adult bladder epithelia (AdBE) isolated from adult mouse bladder tissues (AdBTs) were used to culture adult bladder organoids (AdBOs) in the presence of Fgf7 and Fgf10. The differentiation status of the cells in AdBTs, AdBEs, AdBOs, and neonatal bladder tissues (NeoBTs) was analyzed via quantitative real-time-PCR for the presence of undifferentiated cell markers (Krt5, Trp63, and Krt14) and differentiated cell markers (Krt20, Upk1a, Upk2, and Upk3a). Organoid cell proliferation was assessed by counting cell numbers using the trypan blue method. The effects of Fgf7 and Fgf10 on organoid differentiation were assessed using different doses of Fgfs, and the involvement of peroxisome proliferator-activated receptor γ (PPARγ) signaling in these processes was tested by introducing a PPARγ agonist (Rosiglitazone) and antagonist (T0070907) to the culture. The expression patterns of Fgf7 and Fgf10 were examined via in situ hybridization of AdBTs. RESULTS: AdBOs showed higher expression of undifferentiated cell markers and lower expression of differentiated cell markers than AdBTs, NeoBTs, and AdBEs, indicating the relatively immature state of AdBOs. Differentiation of AdBOs was enhanced by Rosiglitazone and Fgf7, suggesting an interplay of intracellular signals between Fgf7 and PPARγ. Co-addition of T0070907 suppressed Fgf7-mediated differentiation, demonstrating that PPARγ is activated downstream of Fgf7 to promote cellular differentiation into umbrella cells. Furthermore, we found that Fgf7 is predominantly expressed in the umbrella cells of the urothelium, whereas Fgf10 is predominantly expressed in the urothelium and stroma of AdBTs. CONCLUSIONS: We demonstrated that unlike Fgf10, Fgf7 induces cellular differentiation via PPARγ activity and has a unique tissue distribution pattern in the adult bladder. Further studies on the Fgf7-PPARγ signaling axis would provide insights into the differentiation mechanisms toward functional umbrella cells and the pathogenesis of several urinary tract diseases.

Genetic and functional analyses of TBX4 reveal novel mechanisms underlying pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease, with approximately 10% of cases associated with genetic variants. Recent genetic studies have reported pathogenic variants in the TBX4 gene in patients with PAH, especially in patients with childhood-onset of the disease, but the pathogenesis of PAH caused by TBX4 variant has not been fully uncovered. METHODS: We analysed the TBX4 gene in 75 Japanese patients with sporadic or familial PAH using a PCR-based bidirectional sequencing method. Detected variants were evaluated using in silico analyses as well as in vitro analyses including luciferase assay, immunocytochemistry and chromatin immunoprecipitation (ChIP) whether they have altered function. We also analysed the function of TBX4 using mouse embryonic lung explants with inhibition of Tbx4 expression. RESULTS: Putative pathogenic variants were detected in three cases (4.0%). Our in vitro functional analyses revealed that TBX4 directly regulates the transcriptional activity of fibroblast growth factor 10 (FGF10), whereas the identified TBX4 variant proteins failed to activate the FGF10 gene because of disruption of nuclear localisation signal or poor DNA-binding affinity. Furthermore, ex vivo inhibition of Tbx4 resulted in insufficiency of lung morphogenesis along with specific downregulation of Tie2 and Kruppel-like factor 4 expression. CONCLUSION: Our results implicate variants in TBX4 as a genetic cause of PAH in a subset of the Japanese population. Variants in TBX4 may lead to PAH through insufficient lung morphogenesis by disrupting the TBX4-mediated direct regulation of FGF10 signalling and pulmonary vascular endothelial dysfunction involving PAH-related molecules.

Complex Compound Inheritance of Lethal Lung Developmental Disorders Due to Disruption of the TBX-FGF Pathway.

Primary defects in lung branching morphogenesis, resulting in neonatal lethal pulmonary hypoplasias, are incompletely understood. To elucidate the pathogenetics of human lung development, we studied a unique collection of samples obtained from deceased individuals with clinically and histopathologically diagnosed interstitial neonatal lung disorders: acinar dysplasia (n = 14), congenital alveolar dysplasia (n = 2), and other lethal lung hypoplasias (n = 10). We identified rare heterozygous copy-number variant deletions or single-nucleotide variants (SNVs) involving TBX4 (n = 8 and n = 2, respectively) or FGF10 (n = 2 and n = 2, respectively) in 16/26 (61%) individuals. In addition to TBX4, the overlapping ∼2 Mb recurrent and nonrecurrent deletions at 17q23.1q23.2 identified in seven individuals with lung hypoplasia also remove a lung-specific enhancer region. Individuals with coding variants involving either TBX4 or FGF10 also harbored at least one non-coding SNV in the predicted lung-specific enhancer region, which was absent in 13 control individuals with the overlapping deletions but without any structural lung anomalies. The occurrence of rare coding variants involving TBX4 or FGF10 with the putative hypomorphic non-coding SNVs implies a complex compound inheritance of these pulmonary hypoplasias. Moreover, they support the importance of TBX4-FGF10-FGFR2 epithelial-mesenchymal signaling in human lung organogenesis and help to explain the histopathological continuum observed in these rare lethal developmental disorders of the lung.

Wnt/Fgf crosstalk is required for the specification of basal cells in the mouse trachea.

Basal progenitor cells are crucial for the establishment and maintenance of the tracheal epithelium. However, it remains unclear how these progenitor cells are specified during foregut development. Here, we found that ablation of the Wnt chaperone protein Gpr177 (also known as Wntless) in mouse tracheal epithelium causes a significant reduction in the number of basal progenitor cells accompanied by cartilage loss in Shh-Cre;Gpr177loxp/loxp mutants. Consistent with the association between cartilage and basal cell development, Nkx2.1+p63+ basal cells are co-present with cartilage nodules in Shh-Cre;Ctnnb1DM/loxp mutants, which maintain partial cell-cell adhesion but not the transcription regulation function of ß-catenin. More importantly, deletion of Ctnnb1 in the mesenchyme leads to the loss of basal cells and cartilage, concomitant with reduced transcript levels of Fgf10 in Dermo1-Cre;Ctnnb1loxp/loxp mutants. Furthermore, deletion of Fgf receptor 2 (Fgfr2) in the epithelium also leads to significantly reduced numbers of basal cells, supporting the importance of Wnt/Fgf crosstalk in early tracheal development.

Hippo signaling promotes lung epithelial lineage commitment by curbing Fgf10 and ß-catenin signaling.

Organ growth and tissue homeostasis rely on the proliferation and differentiation of progenitor cell populations. In the developing lung, localized Fgf10 expression maintains distal Sox9-expressing epithelial progenitors and promotes basal cell differentiation in the cartilaginous airways. Mesenchymal Fgf10 expression is induced by Wnt signaling but inhibited by Shh signaling, and epithelial Fgf10 signaling activates ß-catenin signaling. The Hippo pathway is a well-conserved signaling cascade that regulates organ size and stem/progenitor cell behavior. Here, we show that Hippo signaling promotes lineage commitment of lung epithelial progenitors by curbing Fgf10 and ß-catenin signaling. Our findings show that both inactivation of the Hippo pathway (nuclear Yap) or ablation of Yap result in increased ß-catenin and Fgf10 signaling, suggesting a cytoplasmic role for Yap in epithelial lineage commitment. We further demonstrate redundant and non-redundant functions for the two nuclear effectors of the Hippo pathway, Yap and Taz, during lung development.

Cellular responses in the FGF10-mediated improvement of hindlimb regenerative capacity in Xenopus laevis revealed by single-cell transcriptomics.

Xenopus laevis tadpoles possess regenerative capacity in their hindlimb buds at early developmental stages (stages ~52-54); they can regenerate complete hindlimbs with digits after limb bud amputation. However, they gradually lose their regenerative capacity as metamorphosis proceeds. Tadpoles in late developmental stages regenerate fewer digits (stage ~56), or only form cartilaginous spike without digits or joints (stage ~58 or later) after amputation. Previous studies have shown that administration of fibroblast growth factor 10 (FGF10) in late-stage (stage 56) tadpole hindlimb buds after amputation can improve their regenerative capacity, which means that the cells responding to FGF10 signaling play an important role in limb bud regeneration. In this study, we performed single-cell RNA sequencing (scRNA-seq) of hindlimb buds that were amputated and administered FGF10 by implanting FGF10-soaked beads at a late stage (stage 56), and explored cell clusters exhibiting a differential gene expression pattern compared with that in controls treated with phosphate-buffered saline. The scRNA-seq data showed expansion of fgf8-expressing cells in the cluster of the apical epidermal cap of FGF10-treated hindlimb buds, which was reported previously, indicating that the administration of FGF10 was successful. On analysis, in addition to the epidermal cluster, a subset of myeloid cells and a newly identified cluster of steap4-expressing cells showed remarkable differences in their gene expression profiles between the FGF10- or phosphate-buffered saline-treatment conditions, suggesting a possible role of these clusters in improving the regenerative capacity of hindlimbs via FGF10 administration.