FGF Signaling Research Articles

Early-life exposure to tobacco smoke alters airway signaling pathways and later mortality in D. melanogaster

Early life environmental influences such as exposure to cigarette smoke (CS) can disturb molecular processes of lung development and thereby increase the risk for later development of chronic respiratory diseases. Among the latter, asthma and chronic obstructive pulmonary disease (COPD) are the most common. The airway epithelium plays a key role in their disease pathophysiology but how CS exposure in early life influences airway developmental pathways and epithelial stress responses or survival is poorly understood.Using Drosophila melanogaster larvae as a model for early life, we demonstrate that CS enters the entire larval airway system, where it activates cyp18a1 which is homologues to human CYP1A1 to metabolize CS-derived polycyclic aromatic hydrocarbons and further induces heat shock protein 70. RNASeq studies of isolated airways showed that CS dysregulates pathways involved in oxidative stress response, innate immune response, xenobiotic and glutathione metabolic processes as well as developmental processes (BMP, FGF signaling) in both sexes, while other pathways were exclusive to females or males. Glutathione S-transferase genes were further validated by qPCR showing upregulation of gstD4, gstD5 and gstD8 in respiratory tracts of females, while gstD8 was downregulated and gstD5 unchanged in males. ROS levels were increased in airways after CS. Exposure to CS further resulted in higher larval mortality, lower larval-pupal transition, and hatching rates in males only as compared to air-exposed controls.Taken together, early life CS induces airway epithelial stress responses and dysregulates pathways involved in the fly's branching morphogenesis as well as in mammalian lung development. CS further affected fitness and development in a highly sex-specific manner.

Transcriptional profiling reveals roles of intercellular Fgf9 signaling in astrocyte maturation and synaptic refinement during brainstem development

Neural tissue maturation is a coordinated process under tight transcriptional control. We previously analyzed the kinetics of gene expression in the medial nucleus of the trapezoid body (MNTB) in the brainstem during the critical postnatal phase of its development. While this work revealed timed execution of transcriptional programs, it was blind to the specific cells where gene expression changes occurred. Here, we utilized single-cell RNA-Seq to determine transcriptional profiles of each major MNTB cell type. We discerned directional signaling patterns between neuronal, glial, and vascular-associated cells for VEGF, TGFβ, and Delta-Notch pathways during a robust period of vascular remodeling in the MNTB. Furthermore, we describe functional outcomes of the disruption of neuron-astrocyte fibroblast growth factor 9 (Fgf9) signaling. We used a conditional KO (cKO) approach to genetically delete Fgf9 from principal neurons in the MNTB, which led to an early onset of glial fibrillary acidic protein (Gfap) expression in astrocytes. In turn, Fgf9 cKO mice show increased levels of astrocyte-enriched brevican (Bcan), a component of the perineuronal net matrix that ensheaths principal neurons in the MNTB and the large calyx of Held terminal, while levels of the neuron-enriched hyaluronan and proteoglycan link protein 1 (Hapln1) were unchanged. Finally, volumetric analysis of vesicular glutamate transporters 1 and 2 (Vglut1/2), which serves as a proxy for terminal size, revealed an increase in calyx of Held volume in the Fgf9 cKO. Overall, we demonstrate a coordinated neuron-astrocyte Fgf9 signaling network that functions to regulate astrocyte maturation, perineuronal net structure, and synaptic refinement.

Preventing ovariectomy-induced weight gain decreases tumor burden in rodent models of obesity and postmenopausal breast cancer

BackgroundObesity and adult weight gain are linked to increased breast cancer risk and poorer clinical outcomes in postmenopausal women, particularly for hormone-dependent tumors. Menopause is a time when significant weight gain occurs in many women, and clinical and preclinical studies have identified menopause (or ovariectomy) as a period of vulnerability for breast cancer development and promotion.MethodsWe hypothesized that preventing weight gain after ovariectomy (OVX) may be sufficient to prevent the formation of new tumors and decrease growth of existing mammary tumors. We tested this hypothesis in a rat model of obesity and carcinogen-induced postmenopausal mammary cancer and validated our findings in a murine xenograft model with implanted human tumors.ResultsIn both models, preventing weight gain after OVX significantly decreased obesity-associated tumor development and growth. Importantly, we did not induce weight loss in these animals, but simply prevented weight gain. In both lean and obese rats, preventing weight gain reduced visceral fat accumulation and associated insulin resistance. Similarly, the intervention decreased circulating tumor-promoting growth factors and inflammatory cytokines (i.e., BDNF, TNFα, FGF-2), with greater effects in obese compared to lean rats. In obese rats, preventing weight gain decreased adipocyte size, adipose tissue macrophage infiltration, reduced expression of the tumor-promoting growth factor FGF-1 in mammary adipose, and reduced phosphorylated FGFR indicating reduced FGF signaling in tumors.ConclusionsTogether, these findings suggest that the underlying mechanisms associated with the anti-tumor effects of weight maintenance are multi-factorial, and that weight maintenance during the peri-/postmenopausal period may be a viable strategy for reducing obesity-associated breast cancer risk and progression in women.

Abstract 2405: Expression of the low affinity neurotrophin receptor (p75NTR) correlates with perineural invasion in adenoid cystic carcinoma of the lacrimal gland

Abstract Lacrimal gland adenoid cystic carcinoma (LGACC) is a rare but highly lethal indolent cancer originating in the cells of secretory glands, primarily of the head and neck. With the 10-year survival rate of 20%, the only life extending technique is to remove the eye and surrounding socket contents followed by extensive chemoradiation. Despite radical treatment, LGACC has a poorer prognosis than head and neck adenoid cystic carcinoma in part due to its propensity for perineural invasion (PNI) and intracranial spread. Analysis of LGACC tumors has revealed signatures enriched for pathways known to be involved in normal LG development, suggesting LGACC tumor cells re-engage or hijack developmental programs during its progression. This project sought to evaluate if developmental pathways involved in embryologic LG innervation were similarly recapitulated by LGACC tumors, and whether any of those markers could be correlated to perineural invasion phenotypes. We performed microarray analysis of embryonic and adult murine lacrimal glands and generated a differential expression signature associated with normal LG developmental pathways. Among other pathways, Notch and FGF signaling are significantly upregulated during LG organogenesis and our group and others have shown that activating mutations in these pathways are correlated with poor prognosis in LGACC. Interestingly, among the highest differentially expressed genes in the embryonic LG was the low-affinity nerve growth factor receptor (p75NTR). We thus subsequently evaluated the expression of p75NTR in LGACC tumors and its correlation to PNI. Our immunofluorescent imaging of histologically confirmed tumors with PNI demonstrated that p75NTR is highly correlated to cancer cells that have invaded the nerve. This correlation of p75NTR expression in nerve infiltrating LGACC cells can inform our investigation into the mechanisms for LGACC PNI and elucidate possible therapeutic targets to limit this aggressive manifestation in LGACC. Citation Format: Acadia H. Moeyersoms, Ryan A. Gallo, Madison Weiss, Michelle M. Maeng, David T. Tse, Daniel Pelaez. Expression of the low affinity neurotrophin receptor (p75NTR) correlates with perineural invasion in adenoid cystic carcinoma of the lacrimal gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2405.

Nucleolin loss of function leads to aberrant Fibroblast Growth Factor signaling and craniofacial anomalies.

ABSTRACTRibosomal RNA (rRNA) transcription and ribosome biogenesis are global processes required for growth and proliferation of all cells, yet perturbation of these processes in vertebrates leads to tissue-specific defects termed ribosomopathies. Mutations in rRNA transcription and processing proteins often lead to craniofacial anomalies; however, the cellular and molecular reasons for these defects are poorly understood. Therefore, we examined the function of the most abundant nucleolar phosphoprotein, Nucleolin (Ncl), in vertebrate development. ncl mutant (ncl−/−) zebrafish present with craniofacial anomalies such as mandibulofacial hypoplasia. We observed that ncl−/− mutants exhibited decreased rRNA synthesis and p53-dependent apoptosis, consistent with a role in ribosome biogenesis. However, we found that Nucleolin also performs functions not associated with ribosome biogenesis. We discovered that the half-life of fgf8a mRNA was reduced in ncl−/− mutants, which perturbed Fgf signaling, resulting in misregulated Sox9a-mediated chondrogenesis and Runx2-mediated osteogenesis. Consistent with this model, exogenous FGF8 treatment significantly rescued the cranioskeletal phenotype in ncl−/− zebrafish, suggesting that Nucleolin regulates osteochondroprogenitor differentiation. Our work has therefore uncovered tissue-specific functions for Nucleolin in rRNA transcription and post-transcriptional regulation of growth factor signaling during embryonic craniofacial development.

Abstract 3241: IC50-Seeding Density Slope (ISDS)-measurement as a standardized methodology for assessing anti-cancer therapeutic activity in vitro

Abstract The 50% inhibitory concentration (IC50) is a widely used measure of how effective a given anti-cancer therapeutic is at reducing cancer cell viability in vitro. It is known that IC50 of a given therapeutic agent is dependent on the seeding density of the treated cells (in that more densely-seeded cells tend to be more resistant to therapeutic agents). We performed experiments using the CellTiter Glo cell viability assay to assess the cytotoxicity of various chemotherapeutic drugs towards various cancer cell lines. To elucidate the role the extracellular environment of densely-seeded cells plays in cancer cell chemoresistance, we tested conditioned media from cells cultured at high density on cells seeded at low densities. The resulting impact on the IC50 of the low-density cells was determined. The IC50 of the various therapeutic agents tested was cell density-dependent in each cancer cell line examined. Based on this analysis, we propose the IC50-seeding density slope (ISDS), which relates seeding density to the effectiveness of a drug and that we believe could be a standardized quantitative measurement of how a given cancer cell line responds to a given therapeutic treatment. Our results from the conditioned media experiments consistently indicated that the environment of densely-seeded tumor cells conferred chemoresistance to sparsely-seeded cells, suggesting that extracellular cytokines and growth factors may be involved in density-dependent chemoresistance mechanisms. We are currently exploring the effect of serum deprivation on in vitro chemoresistance, in order to further understand how the presence of extracellular nutrients impacts observed IC50 trends. In addition, we are examining the role that cell-cell contact, integrin and FGF signaling pathways play in giving rise to density-dependent chemoresistance. Moreover, to analyze the manner in which conditioned media gives rise to chemoresistance and how this effect relates to intracellular survival pathways, we are performing cytokine profiling experiments. Overall, we describe density-dependent IC50 variations as a hallmark of cancer cells and propose that ISDS may serve as a standardized method of assessing chemosensitivity. Citation Format: Ujwal Punyamurtula, Shengliang Zhang, Thomas Brown, Arielle De La Cruz, Alexander Raufi, Jillian Strandberg, Lindsey Carlsen, Lanlan Zhou, Wafik El-Deiry. IC50-Seeding Density Slope (ISDS)-measurement as a standardized methodology for assessing anti-cancer therapeutic activity in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3241.

Single-cell transcriptomics reveals the natural product Shi-Bi-Man promotes hair regeneration by activating the FGF pathway in dermal papilla cells

BackgroundFinasteride and minoxidil are two commonly used drugs for the treatment of hair loss. However, these two drugs have certain side effects. Thus, the further elucidation of treatments for hair loss, including those using Chinese herbal medicine, remains important clinically. Shi-Bi-Man (SBM) is a hair health supplement that darkens hair and contains ginseng radix, tea polyphenols, polygonum multiflorum, radix angelicae sinensis, aloe, linseed, and green tea extract. PurposeThis study aimed to find potential effective monomer components to promote hair regeneration from SBM and to explore the mechanism of SBM to promote hair regeneration. MethodsSupplementation with the intragastric administration or smear administration of SBM in artificially shaved C57BL/6 mice, observe its hair growth. UPLC/MS and UPLC/LTQ-Orbitrap-MS detect the main components in SBM and the main monomers contained in the skin after smearing, respectively. A network pharmacology study on the main components of SBM and single-cell RNA sequencing was performed to explore the role of SBM for hair regeneration. ResultsSBM significantly induced hair growth compared with a control treatment. TSG and EGCG were the main monomers in the skin after SBM smearing. The results of single-cell sequencing revealed that after SBM treatment, the number of hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs) increased significantly. Cell interactions and volcano dots show that the interaction of the FGF signaling pathway was significantly enhanced, in which Fgf7 expression was especially upregulated in DPCs. In addition, the Wnt signaling pathway also had a partially enhanced effect on the interactions between various cells in the skin. The network pharmacology study showed that the promotion of the FGF and Wnt pathways by SBM was also enriched in alopecia diseases. ConclusionWe report that SBM has a potential effect on the promotion of hair growth by mainly activating the FGF signaling pathway. The use of SBM may be a novel therapeutic option for hair loss.

Integrating spatial transcriptomics with single-cell transcriptomics reveals a spatiotemporal gene landscape of the human developing kidney

BackgroundResearch on spatiotemporal gene landscape can provide insights into the spatial characteristics of human kidney development and facilitate kidney organoid cultivation. Here, we profiled the spatiotemporal gene programs of the human embryonic kidneys at 9 and 18 post-conception weeks (PCW) by integrating the application of microarray-based spatial transcriptomics and single-cell transcriptomics.ResultsWe mapped transcriptomic signatures of scRNA-seq cell types upon the 9 and 18 PCW kidney sections based on cell-type deconvolution and multimodal intersection analyses, depicting a spatial landscape of developing cell subpopulations. We established the gene characteristics in the medullary regions and revealed a strong mitochondrial oxidative phosphorylation and glycolysis activity in the deeper medullary region. We also built a regulatory network centered on GDNF-ETV4 for nephrogenic niche development based on the weighted gene co-expression network analysis and highlighted the key roles of Wnt, FGF, and JAG1-Notch2 signaling in maintaining renal branching morphogenesis.ConclusionsOur findings obtained by this spatiotemporal gene program are expected to improve the current understanding of kidney development.

TBIO-08. The molecular basis for rational targeting of FGFR-driven growth and invasiveness in pediatric brain tumors

Abstract The oncogenic activation of receptor tyrosine kinases (RTK) promotes growth, survival and dissemination in pediatric tumors including glioma, ependymoma and medulloblastoma (MB). Direct targeting of either the RTK or of downstream kinases can effectively block tumor promoting pathway functions. However, emergence of resistance is common. We hypothesized that alternative interference strategies that target protein-protein interactions (PPIs) instead of enzymatic activities could overcome the emergence of resistance. We characterized the molecular interactions downstream of the FGFR that regulate relevant growth and invasion-promoting mechanisms in MB cells, to identify potentially druggable PPIs. We found that the FRS2 protein is an essential up-stream effector of FGFR signaling towards invasiveness. Using a proteomics approach, we furthermore identified the Striatin 3 protein as a novel oncogenic effector of the FGFR pathway downstream of FRS2, as it integrates antagonistic growth and invasion signals downstream of FGFR. Mechanistically, Striatin 3 interacts with the Ser/Thr kinase MAP4K4, couples it to the protein phosphatase 2A, and thereby inactivates growth repressing activities of MAP4K4. In parallel, Striatin 3 enables MAP4K4-mediated phosphorylation of PKC-theta and VASP, which combined are necessary to promote tissue invasion. To selectively repress pro-invasive FGFR functions, we identified and functionally validated small molecule ligands of FRS2, that prevent FRS2 activation and downstream signaling. We demonstrate efficacy of these compounds in inhibiting invasion and growth promoting activities in vitro and in vivo, and identified potential off-target activities of the ligand using a proteome-wide interaction analysis. We propose inhibition of FRS2 by a small molecular PTB domain ligand as a strategy to repress FGF signaling in FGFR-driven tumors. The development of this ligand, and the de novo design of functional analogs thereof bear promise for further pre-clinical evaluation of these structures as anti-growth promoting and anti-metastatic therapeutics applicable to FGFR-driven tumors.

FGF/MAPK/Ets signaling in Xenopus ectoderm contributes to neural induction and patterning in an autonomous and paracrine manner, respectively.

FGF and anti-BMP signals from the Spemann organizer of mesodermal origin are essential for Xenopus neural development from gastrula ectoderm. However, the detailed cellular and molecular mechanisms of signaling, especially those underlying the neural induction process, are still controversial. We show here that the expression of early neural marker genes such as sox2 and otx2 is suppressed both in vivo and in vitro, when ectoderm cells are loaded with a dominant-negative construct of Ets transcription factors or a translation-blocking antisense FGF2 MO or FGF8 MO, respectively. This indicates that the expression of these FGF signaling molecules in ectoderm cells contributes significantly to neural induction, in contrast to the "neural default model" that has been emphasized, in which anti-BMP signaling from the organizer plays a major role in the neural induction in Xenopus. Our results indicate that cell-autonomous FGF signaling between ectoderm cells, rather than paracrine signaling from organizer cells, directly induces the expression of sox2 and otx2 via the FGF2, FGF8/MAPK/Ets pathway at very low signaling levels. This is independent of inhibiting BMP signaling via the FGF/MAPK/Smad pathway, which has been proposed as the primary pathway for FGF signaling in Xenopus neural induction. Using cultured ectoderm cells, we also obtained results suggesting that the mode of contribution of FGF signaling to neural development is altered during the subsequent neural patterning stage. As we increase the amounts of FGF in the culture medium, anterior neural genes activated at low FGF signaling levels are suppressed, and instead position-specific, more posterior neural genes are activated in a dose-dependent manner via the FGF/MAPK/Ets pathway. These results support the claim that morphogenic FGFs from the organizer diffuse in a paracrine manner through the plane of the induced neuroectoderm, causing anteroposterior patterning of neural tissue.

Delamination of trophoblast-like syncytia from the amniotic ectodermal analogue in human primed embryonic stem cell-based differentiation model.

Human primed embryonic stem cells (ESCs) are known to be converted to cells with several trophoblast properties, but it has remained controversial whether this phenomenon represents the inherent differentiation competence of human primed ESCs to trophoblast lineages. In this study, we report that chemical blockage of ACTIVIN/NODAL and FGF signals is sufficient to steer human primed ESCs into GATA3-expressing cells that give rise to placental hormone-producing syncytia analogous to syncytiotrophoblasts of the post-implantation stage of the human embryo. Despite their cytological similarity to syncytiotrophoblasts, these syncytia arise from the non-trophoblastic differentiation trajectory that recapitulates amniogenesis. These results provide insights into the possible extraembryonic differentiation pathway that is unique in primate embryogenesis.

Robust derivation of transplantable dopamine neurons from human pluripotent stem cells by timed retinoic acid delivery

Stem cell therapies for Parkinson’s disease (PD) have entered first-in-human clinical trials using a set of technically related methods to produce mesencephalic dopamine (mDA) neurons from human pluripotent stem cells (hPSCs). Here, we outline an approach for high-yield derivation of mDA neurons that principally differs from alternative technologies by utilizing retinoic acid (RA) signaling, instead of WNT and FGF8 signaling, to specify mesencephalic fate. Unlike most morphogen signals, where precise concentration determines cell fate, it is the duration of RA exposure that is the key-parameter for mesencephalic specification. This concentration-insensitive patterning approach provides robustness and reduces the need for protocol-adjustments between hPSC-lines. RA-specified progenitors promptly differentiate into functional mDA neurons in vitro, and successfully engraft and relieve motor deficits after transplantation in a rat PD model. Our study provides a potential alternative route for cell therapy and disease modelling that due to its robustness could be particularly expedient when use of autologous- or immunologically matched cells is considered.

Kub3 Deficiency Causes Aberrant Late Embryonic Lung Development in Mice by the FGF Signaling Pathway

As a Ku70-binding protein of the KUB family, Kub3 has previously been reported to play a role in DNA double-strand break repair in human glioblastoma cells in glioblastoma patients. However, the physiological roles of Kub3 in normal mammalian cells remain unknown. In the present study, we generated Kub3 gene knockout mice and revealed that knockout (KO) mice died as embryos after E18.5 or as newborns immediately after birth. Compared with the lungs of wild-type (WT) mice, Kub3 KO lungs displayed abnormal lung morphogenesis and pulmonary atelectasis at E18.5. No difference in cell proliferation or cell apoptosis was detected between KO lungs and WT lungs. However, the differentiation of alveolar epithelial cells and the maturation of type II epithelial cells were impaired in KO lungs at E18.5. Further characterization displayed that Kub3 deficiency caused an abnormal FGF signaling pathway at E18.5. Taking all the data together, we revealed that Kub3 deletion leads to abnormal late lung development in mice, resulting from the aberrant differentiation of alveolar epithelial cells and the immaturation of type II epithelial cells due to the disturbed FGF signaling pathway. Therefore, this study has uncovered an essential role of Kub3 in the prenatal lung development of mice which advances our knowledge of regulatory factors in embryonic lung development and provides new concepts for exploring the mechanisms of disease related to perinatal lung development.

FGF-2 signaling in nasopharyngeal carcinoma modulates pericyte-macrophage crosstalk and metastasis.

Molecular signaling in the tumor microenvironment (TME) is complex, and crosstalk among various cell compartments in supporting metastasis remains poorly understood. In particular, the role of vascular pericytes, a critical cellular component in the TME, in cancer invasion and metastasis warrants further investigation. Here, we report that an elevation of FGF-2 signaling in samples from patients with nasopharyngeal carcinoma (NPC) and xenograft mouse models promoted NPC metastasis. Mechanistically, tumor cell–derived FGF-2 strongly promoted pericyte proliferation and pericyte-specific expression of an orphan chemokine (C-X-C motif) ligand 14 (CXCL14) via FGFR1/AHR signaling. Gain- and loss-of-function experiments validated that pericyte-derived CXCL14 promoted macrophage recruitment and polarization toward an M2-like phenotype. Genetic knockdown of FGF2 or genetic depletion of tumoral pericytes blocked CXCL14 expression and tumor-associated macrophage (TAM) infiltration. Pharmacological inhibition of TAMs by clodronate liposome treatment resulted in a reduction of FGF-2–induced pulmonary metastasis. Together, these findings shed light on the inflammatory role of tumoral pericytes in promoting TAM-mediated metastasis. We provide mechanistic insight into an FGF-2/FGFR1/pericyte/CXCL14/TAM stromal communication axis in NPC and propose an effective antimetastasis therapy concept by targeting a pericyte-derived inflammation for NPC or FGF-2hi tumors.

Xenopus Dusp6 modulates FGF signaling to precisely pattern pre-placodal ectoderm

Pre-placodal ectoderm (PPE), a horseshoe-shaped narrow region formed during early vertebrate development, gives rise to multiple types of sensory organs and ganglia. For PPE induction, a certain level of FGF signal activation is required. However, it is difficult to reproducibly induce the narrow region with variations in gene expression, including FGF, among individuals. An intracellular regulatory factor of FGF signaling, Dusp6, is expressed by FGF signal activation and inactivates a downstream regulator, ERK1/2, in adult tissues; however, its role in early development is not well known. Here, we reveal that Dusp6 is expressed in an FGF-dependent manner in Xenopus PPE. Gain- and loss-of-function experiments showed that Dusp6 is required for expression of a PPE gene, Six1, and patterning of adjacent regions, neural plate, and neural crest. To reveal the importance of Dusp6 in variable FGF production, we performed Dusp6 knockdown with FGF-bead implantation, which resulted in varying Six1 expression patterns. Taken together, these results suggest that Dusp6 is required for PPE formation and that it contributes to the robust patterning of PPE by mediating FGF signaling.

The product of BMP-directed differentiation protocols for human primed pluripotent stem cells is placental trophoblast and not amnion.

SummaryThe observation that trophoblast (TB) can be generated from primed pluripotent stem cells (PSCs) by exposure to bone morphogenetic protein-4 (BMP4) when FGF2 and ACTIVIN signaling is minimized has recently been challenged with the suggestion that the procedure instead produces amnion. Here, by analyzing transcriptome data from multiple sources, including bulk and single-cell data, we show that the BMP4 procedure generates bona fide TB with similarities to both placental villous TB and TB generated from TB stem cells. The analyses also suggest that the transcriptomic signatures between embryonic amnion and different forms of TB have commonalities. Our data provide justification for the continued use of TB derived from PSCs as a model for investigating placental development.

Critical roles of FGF, RA, and WNT signalling in the development of the human otic placode and subsequent lineages in a dish

IntroductionEfficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs.MethodsWe differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors.ResultsWe demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1.ConclusionsWe demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.

An epithelial signalling centre in sharks supports homology of tooth morphogenesis in vertebrates.

Development of tooth shape is regulated by the enamel knot signalling centre, at least in mammals. Fgf signalling regulates differential proliferation between the enamel knot and adjacent dental epithelia during tooth development, leading to formation of the dental cusp. The presence of an enamel knot in non-mammalian vertebrates is debated given differences in signalling. Here, we show the conservation and restriction of fgf3, fgf10, and shh to the sites of future dental cusps in the shark (Scyliorhinus canicula), whilst also highlighting striking differences between the shark and mouse. We reveal shifts in tooth size, shape, and cusp number following small molecule perturbations of canonical Wnt signalling. Resulting tooth phenotypes mirror observed effects in mammals, where canonical Wnt has been implicated as an upstream regulator of enamel knot signalling. In silico modelling of shark dental morphogenesis demonstrates how subtle changes in activatory and inhibitory signals can alter tooth shape, resembling developmental phenotypes and cusp shapes observed following experimental Wnt perturbation. Our results support the functional conservation of an enamel knot-like signalling centre throughout vertebrates and suggest that varied tooth types from sharks to mammals follow a similar developmental bauplan. Lineage-specific differences in signalling are not sufficient in refuting homology of this signalling centre, which is likely older than teeth themselves.

Growth factor dependency in mammary organoids regulates ductal morphogenesis during organ regeneration

Signaling pathways play an important role in cell fate determination in stem cells and regulate a plethora of developmental programs, the dysregulation of which can lead to human diseases. Growth factors (GFs) regulating these signaling pathways therefore play a major role in the plasticity of adult stem cells and modulate cellular differentiation and tissue repair outcomes. We consider murine mammary organoid generation from self-organizing adult stem cells as a tool to understand the role of GFs in organ development and tissue regeneration. The astounding capacity of mammary organoids to regenerate a gland in vivo after transplantation makes it a convenient model to study organ regeneration. We show organoids grown in suspension with minimal concentration of Matrigel and in the presence of a cocktail of GFs regulating EGF and FGF signaling can recapitulate key epithelial layers of adult mammary gland. We establish a toolkit utilizing in vivo whole animal imaging and ultrasound imaging combined with ex vivo approaches including tissue clearing and confocal imaging to study organ regeneration and ductal morphogenesis. Although the organoid structures were severely impaired in vitro when cultured in the presence of individual GFs, ex vivo imaging revealed ductal branching after transplantation albeit with significantly reduced number of terminal end buds. We anticipate these imaging modalities will open novel avenues to study mammary gland morphogenesis in vivo and can be beneficial for monitoring mammary tumor progression in pre-clinical and clinical settings.

Downstream Branches of the Fibroblast Growth Factor Signaling Pathway Act Interdependently to Shape the Face

The musculoskeletal tissues of the head develop from primordia, composed of neural crest cells, that evaginate and fuse to form the structures of the face. This occurs through a complex process controlled not only by regional differences in cellular proliferation, apoptosis, and differentiation, but also directed cellular migration and oriented cellular behavior. Disruption of normal development of the facial primordia can either prevent apposition and fusion of craniofacial primordia, leading to malformations such as cleft lip and/or palate. Previously, we found that stimulating signaling by fibroblast growth factor (FGF) in neural crest cells of chick embryos slowed proximodistal expansion of the facial primordia and widened the midface. Activation of the FGF pathway decreased cell proliferation in the frontonasal process (FNP) and disrupted cellular orientation, which were correlated with shape of the FNP. Blocking FGF signaling with a chemical inhibitor caused severe truncation of the FNP and decreased proliferation. Our previous results confirm that FGF signaling affects craniofacial morphogenesis, seemingly by altering multiple cellular functions. FGF receptors are receptor tyrosine kinases which utilize many common downstream signaling cascades that direct cellular proliferation, apoptosis, differentiation, migration, and oriented cell behaviors, but it is not clear which downstream pathway explains our previous results. In this study we used chemical inhibitors to block three pathways downstream of FGF receptor activation to determine their effect on facial morphology and cellular activity in the FNP of the chick embryo. Blocking MAPK, PLCγ, and PI3K pathways individually caused similar mild truncations of proximo‐distal growth in the FNP over 24 and 72 hours, and similar reductions in cellular proliferation. Cellular orientation was not significantly affected by any treatment. Blocking all three downstream pathways together caused a more severe proximo‐distal truncation of growth of the FNP, but not as severe as blocking Fgf signaling entirely. These results suggest that the downstream branches of the FGF pathway act inter‐dependently and in a non‐linear fashion to shape the face. Soon we will measure cellular orientation in the inhibitor cocktail treated FNPs and quantify the changes in FNP shape using three‐dimensional geometric morphometrics.