Key Developmental Signaling Pathways Research Articles

FLT4 causes developmental disorders of the cardiovascular and lymphovascular systems via pleiotropic molecular mechanisms.

Rare, deleterious genetic variants in FLT4 are associated with Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease. The distinct genetic variants in FLT4 are also an established cause of Milroy disease, the most prevalent form of primary hereditary lymphoedema. The phenotypic features of these two conditions are non-overlapping, implying pleiotropic cellular mechanisms during development. In this study, we show that FLT4 variants identified in patients with TOF, when expressed in primary human endothelial cells, cause aggregation of FLT4 protein in the perinuclear endoplasmic reticulum, activating proteostatic and metabolic signalling, whereas lymphoedema-associated FLT4 variants and wild-type (WT) FLT4 do not. FLT4 TOF variants display characteristic gene expression profiles in key developmental signalling pathways, revealing a role for FLT4 in cardiogenesis distinct from its role in lymphatic development. Inhibition of proteostatic signalling abrogates these effects, identifying potential avenues for therapeutic intervention. Depletion of flt4 in zebrafish caused cardiac phenotypes of reduced heart size and altered heart looping. These phenotypes were rescued with coinjection of WT human FLT4 mRNA, but incompletely or not at all by mRNA harbouring FLT4 TOF variants. Taken together, we identify a pathogenic mechanism for FLT4 variants predisposing to TOF that is distinct from the known dominant negative mechanism of Milroy-causative variants. FLT4 variants give rise to conditions of the two circulatory subdivisions of the vascular system via distinct developmental pleiotropic molecular mechanisms.

Global ubiquitinome profiling identifies NEDD4 as a regulator of Profilin 1 and actin remodelling in neural crest cells

The ubiquitin ligase NEDD4 promotes neural crest cell (NCC) survival and stem-cell like properties to regulate craniofacial and peripheral nervous system development. However, how ubiquitination and NEDD4 control NCC development remains unknown. Here we combine quantitative analysis of the proteome, transcriptome and ubiquitinome to identify key developmental signalling pathways that are regulated by NEDD4. We report 276 NEDD4 targets in NCCs and show that loss of NEDD4 leads to a pronounced global reduction in specific ubiquitin lysine linkages. We further show that NEDD4 contributes to the regulation of the NCC actin cytoskeleton by controlling ubiquitination and turnover of Profilin 1 to modulate filamentous actin polymerization. Taken together, our data provide insights into how NEDD4-mediated ubiquitination coordinates key regulatory processes during NCC development.

Concepts in Multifactorial Etiology of Developmental Disorders: Gene-Gene and Gene-Environment Interactions in Holoprosencephaly.

Many common developmental disorders are thought to arise from a complex set of genetic and environmental risk factors. These factors interact with each other to affect the strength and duration of key developmental signaling pathways, thereby increasing the possibility that they fail to achieve the thresholds required for normal embryonic patterning. One such disorder, holoprosencephaly (HPE), serves as a useful model system in understanding various forms of multifactorial etiology. Genomic analysis of HPE cases, epidemiology, and mechanistic studies of animal models have illuminated multiple potential ways that risk factors interact to produce adverse developmental outcomes. Among these are: 1) interactions between driver and modifier genes; 2) oligogenic inheritance, wherein each parent provides predisposing variants in one or multiple distinct loci; 3) interactions between genetic susceptibilities and environmental risk factors that may be insufficient on their own; and 4) interactions of multiple genetic variants with multiple non-genetic risk factors. These studies combine to provide concepts that illuminate HPE and are also applicable to additional disorders with complex etiology, including neural tube defects, congenital heart defects, and oro-facial clefting.

Paths and pathways that generate cell-type heterogeneity and developmental progression in hematopoiesis.

Mechanistic studies of Drosophila lymph gland hematopoiesis are limited by the availability of cell-type-specific markers. Using a combination of bulk RNA-Seq of FACS-sorted cells, single-cell RNA-Seq, and genetic dissection, we identify new blood cell subpopulations along a developmental trajectory with multiple paths to mature cell types. This provides functional insights into key developmental processes and signaling pathways. We highlight metabolism as a driver of development, show that graded Pointed expression allows distinct roles in successive developmental steps, and that mature crystal cells specifically express an alternate isoform of Hypoxia-inducible factor (Hif/Sima). Mechanistically, the Musashi-regulated protein Numb facilitates Sima-dependent non-canonical, and inhibits canonical, Notch signaling. Broadly, we find that prior to making a fate choice, a progenitor selects between alternative, biologically relevant, transitory states allowing smooth transitions reflective of combinatorial expressions rather than stepwise binary decisions. Increasingly, this view is gaining support in mammalian hematopoiesis.

Circular RNA ACTN4 promotes intrahepatic cholangiocarcinoma progression by recruiting YBX1 to initiate FZD7 transcription

Intrahepatic cholangiocarcinoma (ICC) is a primary liver cancer with high aggressiveness and extremely poor prognosis. The role of circular RNAs (circRNAs) in ICC carcinogenesis and progression remains to be determined. CircRNA microarray was performed to screen significantly upregulated circRNAs in paired ICC and non-tumor tissues. Colony formation, transwell, and xenograft models were used to examine the role of circRNAs in ICC proliferation and metastasis. RNA pulldown, mass spectrometry, chromatin immunoprecipitation, RNA-binding protein immunoprecipitation, chromatin isolation by RNA purification, electrophoretic mobility shift assay, and luciferase reporter assays were used to explore the molecular sponge role of the circRNA (via miRNA binding), and the interaction between circRNA and RNA-binding proteins. Hsa_circ_0050898, which originated from exon 1 to exon 20 of the ACTN4 gene (named circACTN4), was significantly upregulated in ICC. High circACTN4 expression was associated with enhanced tumor proliferation and metastasis invitro and invivo, as well as a worse prognosis following ICC resection. In addition, circACTN4 upregulated Yes-associated protein 1 (YAP1) expression by sponging miR-424-5p. More importantly, circACTN4 also recruited Y-box binding protein 1 (YBX1) to stimulate Frizzled-7 (FZD7) transcription. Furthermore, circACTN4 overexpression in ICC cells enhanced the interaction between YAP1 and β-catenin, which are the core components of the Hippo and Wnt signaling pathways, respectively. CircACTN4 was upregulated in ICC and promoted ICC proliferation and metastasis by acting as a molecular sponge of miR-424-5p, as well as by interacting with YBX1 to transcriptionally activate FZD7. These results suggest that circACTN4 is a potential prognostic marker and therapeutic target for ICC. Intrahepatic cholangiocarcinoma is a primary liver cancer associated with aggressiveness and extremely poor prognosis. It is essential for therapeutic development that we uncover relevant pathogenic pathways. Herein, we showed that a circular RNA (circACTN4) was highly expressed in intrahepatic cholangiocarcinoma and was positively associated with tumor growth and metastasis through key developmental signaling pathways. Thus, circACTN4 could be a prognostic biomarker and therapeutic target for intrahepatic cholangiocarcinoma.

Effect of electro-acupuncture at ST 36 on maternal food restriction-induced lung phenotype in rat offspring.

Maternal food restriction (MFR) during pregnancy leads to pulmonary dysplasia in the newborn period and increases susceptibility to diseases, such as asthma and chronic lung disease, later in life. Previous studies have shown that maternal electro-acupuncture (EA) applied to "Zusanli" (ST 36) could prevent the abnormal expression of key lung developmental signaling pathways and improve the lung morphology and function in perinatal nicotine exposed offspring. There is a significant overlap in lung developmental signaling pathways affected by perinatal nicotine exposure and MFR during pregnancy; however, whether maternal EA at ST 36 also blocks the MFR-induced lung phenotype is unknown. Here, we examined the effects of EA applied to maternal ST 36 on lung morphology and function and the expression of key lung developmental signaling pathways, and the hypercorticoid state associated with MFR during pregnancy. These effects were compared with those of metyrapone, an intervention known to block MFR-induced offspring hypercorticoid state and the resultant pulmonary pathology. Like metyrapone, maternal EA at ST 36 blocked the MFR-induced changes in key developmental signaling pathways and protected the MFR-induced changes in lung morphology and function. These results offer a novel and safe, nonpharmacologic approach to prevent MFR-induced pulmonary dysplasia in offspring.

Effect of vitrification on biogenesis pathway and expression of development-related microRNAs in preimplantation mouse embryos

Vitrification of embryos has been known as the most efficient cryopreservation method in assisted reproductive technology clinics. Vitrification of preimplantation embryo might be associated with altered gene expression profile and biochemical changes of vitrified embryos. Stringent regulation of gene expression in early embryonic stages is very critical for normal development. In the present study, we investigated the effect of vitrification on the canonical miRNA biogenesis pathway, and also the expression of developmental related miRNAs, in 8-cell and blastocyst mouse embryos. Although the expression pattern of the miRNA biogenesis pathway genes differed between 8-cell and blastocyst mouse embryos, vitrification did not affect the expression level of these genes in preimplantation embryos. The expression levels of miR-21 and let-7a were significantly decreased in vitrified 8-cell embryos and fresh blastocysts when compared with fresh 8-cell embryos. The expression of Stat3 was significantly reduced in blastocysts after vitrification. The alteration in the expression pattern of miRNAs, due to their mode of action, can affect broad downstream key developmental signaling pathways. Therefore, the blastocyst stage is the preferred point for embryo vitrification as they are less susceptible to cryo-damages regarding the stability of miRNAs related to the developmental and implantation competence of embryo.

Deletion of the Dishevelled family of genes disrupts anterior-posterior axis specification and selectively prevents mesoderm differentiation

The Dishevelled proteins transduce both canonical Wnt/β-catenin and non-canonical Wnt/planar cell polarity (PCP) signaling pathways to regulate many key developmental processes during embryogenesis. Here, we disrupt both canonical and non-canonical Wnt pathways by targeting the entire Dishevelled family of genes (Dvl1, Dvl2, and Dvl3) to investigate their functional roles in the early embryo. We identified several defects in anterior-posterior axis specification and mesoderm patterning in Dvl1+/−; Dvl2−/−; Dvl3−/− embryos. Homozygous deletions in all three Dvl genes (Dvl TKO) resulted in defects in distal visceral endoderm migration and a complete failure to induce mesoderm formation. To identify potential mechanisms that lead to the defects in the developmental processes preceding gastrulation, we generated Dvl TKO mouse embryonic stem cells (mESCs) and compared the transcriptional profile of these cells with wild-type (WT) mESCs during germ lineage differentiation into 3D embryoid bodies (EBs). While the Dvl TKO mESCs displayed similar morphology, self-renewal properties, and minor transcriptional variation from WT mESCs, we identified major transcriptional dysregulation in the Dvl TKO EBs during differentiation in a number of genes involved in anterior-posterior pattern specification, gastrulation induction, mesenchyme morphogenesis, and mesoderm-derived tissue development. The absence of the Dvls leads to specific down-regulation of BMP signaling genes. Furthermore, exogenous activation of canonical Wnt, BMP, and Nodal signaling all fail to rescue the mesodermal defects in the Dvl TKO EBs. Moreover, endoderm differentiation was promoted in the absence of mesoderm in the Dvl TKO EBs, while the suppression of ectoderm differentiation was delayed. Overall, we demonstrate that the Dvls are dispensable for maintaining self-renewal in mESCs but are critical during differentiation to regulate key developmental signaling pathways to promote proper axis specification and mesoderm formation.

BMP- and neuropilin 1-mediated motor axon navigation relies on spastin alternative translation.

ABSTRACTFunctional analyses of genes responsible for neurodegenerative disorders have unveiled crucial links between neurodegenerative processes and key developmental signalling pathways. Mutations in SPG4-encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes that couple microtubule severing to membrane remodelling. Two main spastin isoforms are synthesised from alternative translational start sites (M1 and M87). However, their specific roles in neuronal development and homeostasis remain largely unknown. To selectively unravel their neuronal function, we blocked spastin synthesis from each initiation codon during zebrafish development and performed rescue analyses. The knockdown of each isoform led to different motor neuron and locomotion defects, which were not rescued by the selective expression of the other isoform. Notably, both morphant neuronal phenotypes were observed in a CRISPR/Cas9 spastin mutant. We next showed that M1 spastin, together with HSP proteins atlastin 1 and NIPA1, drives motor axon targeting by repressing BMP signalling, whereas M87 spastin acts downstream of neuropilin 1 to control motor neuron migration. Our data therefore suggest that defective BMP and neuropilin 1 signalling may contribute to the motor phenotype in a vertebrate model of spastin depletion.

BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Defining Adult Stem Cells by Function, not by Phenotype.

Central to the classical hematopoietic stem cell (HSC) paradigm is the concept that the maintenance of blood cell numbers is exclusively executed by a discrete physical entity: the transplantable HSC. The HSC paradigm has served as a stereotypic template in stem cell biology, yet the search for rare, hardwired professional stem cells has remained futile in most other tissues. In a more open approach, the focus on the search for stem cells as a physical entity may need to be replaced by the search for stem cell function, operationally defined as the ability of an organ to replace lost cells. The nature of such a cell may be different under steady state conditions and during tissue repair. We discuss emerging examples including the renewal strategies of the skin, gut epithelium, liver, lung, and mammary gland in comparison with those of the hematopoietic system. While certain key housekeeping and developmental signaling pathways are shared between different stem cell systems, there may be no general, deeper principles underlying the renewal mechanisms of the various individual tissues.

Osteoblast Bioenergetics and Global Energy Homeostasis.

The emergence of the endochondral skeleton in terrestrial animals enabled ambulation against increased gravitational forces and provided a storage site for scarce minerals essential for life. This skeletal upgrade increased overall fuel requirements and altered global energy balance, prompting the evolution of endocrine networks to coordinate energy expenditure. Bone-forming osteoblasts require a large and constant supply of energy substrates to fuel bone matrix production and mineralization. When fuel demands are unmet, bone quality and strength are compromised. Recent studies suggest that key developmental signaling pathways are coupled to bioenergetic programs, accommodating changes in energy requirements at different stages of the osteoblast life cycle. Studies in genetically altered mice have confirmed a link between bone cells and global metabolism and have led to the identification of hormonal interactions between the skeleton and other tissues. These observations have prompted new questions regarding the nature of the mechanisms of fuel sensing and processing in the osteoblast and their contribution to overall energy utilization and homeostasis. Answers to such questions should advance our understanding of metabolic diseases and may ultimately improve treatments for patients with diabetes and osteoporosis.

Probing the missing mature \u03b2-cell proteomic landscape in differentiating patient iPSC-derived cells

MODY1 is a maturity-onset monogenic diabetes, caused by heterozygous mutations of the HNF4A gene. To date the cellular and molecular mechanisms leading to disease onset remain largely unknown. In this study, we demonstrate that insulin-positive cells can be generated in vitro from human induced pluripotent stem cells (hiPSCs) derived from patients carrying a non-sense HNF4A mutation, proving for the first time, that a human HNF4A mutation is neither blocking the expression of the insulin genes nor the development of insulin-producing cells in vitro. However, regardless of the mutation or diabetes status, these insulin-producing cells are immature, a common downfall off most current β-cell differentiation protocols. To further address the immature state of the cells, in vitro differentiated cells and adult human islets were compared by global proteomic analysis. We report the predicted upstream regulators and signalling pathways characterizing the proteome landscape of each entity. Subsequently, we focused on the molecular components absent or misregulated in the in vitro differentiated cells, to probe the components involved in the deficient in vitro maturation towards fully functional β-cells. This analysis identified the modulation of key developmental signalling pathways representing potential targets for improving the efficiency of the current differentiation protocols.

Intraflagellar transport 88 (IFT88) is crucial for craniofacial development in mice and is a candidate gene for human cleft lip and palate.

Ciliopathies are pleiotropic human diseases resulting from defects of the primary cilium, and these patients often have cleft lip and palate. IFT88 is required for the assembly and function of the primary cilia, which mediate the activity of key developmental signaling pathways. Through whole exome sequencing of a family of three affected siblings with isolated cleft lip and palate, we discovered that they share a novel missense mutation in IFT88 (c.915G > C, p.E305D), suggesting this gene should be considered a candidate for isolated orofacial clefting. In order to evaluate the function of IFT88 in regulating craniofacial development, we generated Wnt1-Cre;Ift88fl/fl mice to eliminate Ift88 specifically in cranial neural crest (CNC) cells. Wnt1-Cre;Ift88fl/flpups died at birth due to severe craniofacial defects including bilateral cleft lip and palate and tongue agenesis, following the loss of the primary cilia in the CNC-derived palatal mesenchyme. Loss of Ift88 also resulted in a decrease in neural crest cell proliferation during early stages of palatogenesis as well as a downregulation of the Shh signaling pathway in the palatal mesenchyme. Importantly, Osr2KI-Cre;Ift88fl/flmice, in which Ift88 is lost specifically in the palatal mesenchyme, exhibit isolated cleft palate. Taken together, our results demonstrate that IFT88 has a highly conserved function within the primary cilia of the CNC-derived mesenchyme in the lip and palate region in mice and is a strong candidate as an orofacial clefting gene in humans.

Abstract A66: Runx3 drives metastatic programs in pancreas cancer

Abstract The extreme proclivity of pancreatic ductal adenocarcinoma (PDA) for metastatic spread is the principal cause of mortality for this disease, even for patients who successfully undergo surgical resection of the primary tumor. We have engaged in a concerted effort to define the fundamental molecular and cellular drivers of PDA through the use of cognate genetically engineered mouse models that recapitulate the spectrum of human PDA. Recently, we incorporated conditional deletion of Smad4 into the KPC mouse model and noted a significant attenuation of metastatic burden in the resultant KPDC mice. Gene expression analysis identified the Runt family transcription factor Runx3 as highly expressed in metastatic KPC tumors, but minimally expressed in PDA from KPDC mice. We showed that aberrant expression of Runx3 strongly enhanced the metastatic potential of PDA through an orchestrated program that stimulated cancer cell invasion and migration and also helped generate a niche for disseminated cells to establish themselves in foreign tissues (such as the liver or lung) and form metastases. Runx3 expression additionally inhibited proliferation by upregulating expression of the cell cycle inhibitor Cdkn1a (p21), initiating a metastatic switch that favored PDA dissemination over local outgrowth. We have also identified influences of Runx3 expression on key developmental and immune-regulatory signaling pathways that are known to be aberrantly regulated in PDA. Because Runx3 has been shown to interact with Smad4 and p53, the genomic context of PDA expressing Runx3 critically modulates its function through de novo or altered protein complex formation. Defining the dichotomous, contextual roles of Runx3 as both a tumor “suppressor” and “promoter” may provide insight into the proximal cause of lethality in patients, thereby informing clinical prioritization of local versus systemic therapies for individual patients with PDA. Citation Format: Martin C. Whittle, Libing Feng, Ashley Dotson, Fiona Pakiam, Sunil R. Hingorani.{Authors}. Runx3 drives metastatic programs in pancreas cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A66.

Effect of Maternal Electroacupuncture on Perinatal Nicotine Exposure-Induced Lung Phenotype in Offspring.

Pregnant women exposed to tobacco smoke predispose the offspring to many adverse consequences including an altered lung development and function. There is no effective therapeutic intervention to block the effects of smoke exposure on the developing lung. Clinical and animal studies demonstrate that acupuncture can modulate a variety of pathophysiological processes, including those involving the respiratory system; however, whether acupuncture affects the lung damage caused by perinatal smoke exposure is not known. To determine the effect of acupuncture on perinatal nicotine exposure on the developing lung, pregnant rat dams were administered (1) saline, (2) nicotine, or (3) nicotine+electroacupuncture (EA). Nicotine was administered (1mg/kg subcutaneously) once a day and EA was applied to both "Zusanli" (ST 36) points. Both interventions were administered from gestational day 6 to postnatal day 21 (PND21), following which pups were sacrificed. Lungs, blood, and brain were collected to examine markers of lung injury, repair, and hypothalamic pituitary adrenal (HPA) axis. Concomitant EA application blocked nicotine-induced changes in lung morphology, lung peroxisome proliferator-activated receptor γ and wingless-int signaling, two key lung developmental signaling pathways, hypothalamic pituitary adrenal axis (hypothalamic corticotropic releasing hormone and lung glucocorticoid receptor levels), and plasma β-endorphin levels. Electroacupuncture blocks the nicotine-induced changes in lung developmental signaling pathways and the resultant myogenic lung phenotype, known to be present in the affected offspring. We conclude that EA is a promising novel intervention against the smoke exposed lung damage to the developing lung.

Editor's Highlight: Hydroxyurea Exposure Activates the P53 Signaling Pathway in Murine Organogenesis-Stage Embryos.

Hydroxyurea, an anticancer agent and potent teratogen, induces oxidative stress and activates a DNA damage response pathway in the gestation day (GD) 9 mouse embryo. To delineate the stress response pathways activated by this drug, we investigated the effect of hydroxyurea exposure on the transcriptome of GD 9 embryos. Timed pregnant CD-1 mice were treated with saline or hydroxyurea (400 mg/kg or 600 mg/kg) on GD 9; embryonic gene and protein expression were examined 3 h later. Microarray analysis revealed that the expression of 1346 probe sets changed significantly in embryos exposed to hydroxyurea compared with controls; the P53 signaling pathway was highly affected. In addition, P53 related family members, P63 and P73, were predicted to be activated and had common and unique downstream targets. Western blot analysis revealed that active phospho-P53 was significantly increased in drug-exposed embryos; confocal microscopy showed that the translocation of phospho-P53 to the nucleus was widespread in the embryo. Furthermore, qRT-PCR showed that the expression of P53-regulated genes (Cdkn1A, Fas, and Trp53inp1) was significantly upregulated in hydroxyurea-exposed embryos; the concentration of the redox sensitive P53INP1 protein was also increased in a hydroxyurea dose-dependent fashion. Thus, hydroxyurea elicits a significant effect on the transcriptome of the organogenesis stage murine embryo, activating several key developmental signaling pathways related to DNA damage and oxidative stress. We propose that the P53 pathway plays a central role in the embryonic stress response and the developmental outcome after teratogen exposure.

Developmental signalling pathways in renal fibrosis: the roles of Notch, Wnt and Hedgehog.

Kidney fibrosis is a common histological manifestation of functional decline in the kidney. Fibrosis is a reactive process that develops in response to excessive epithelial injury and inflammation, leading to myofibroblast activation and an accumulation of extracellular matrix. Here, we describe how three key developmental signalling pathways - Notch, Wnt and Hedgehog (Hh) - are reactivated in response to kidney injury and contribute to the fibrotic response. Although transient activation of these pathways is needed for repair of injured tissue, their sustained activation is thought to promote fibrosis. Excessive Wnt and Notch expression prohibit epithelial differentiation, whereas increased Wnt and Hh expression induce fibroblast proliferation and myofibroblastic transdifferentiation. Notch, Wnt and Hh are fundamentally different signalling pathways, but their choreographed activation seems to be just as important for fibrosis as it is for embryonic kidney development. Decreasing the activity of Notch, Wnt or Hh signalling could potentially provide a new therapeutic strategy to ameliorate the development of fibrosis in chronic kidney disease.

An ancient dental gene set governs development and continuous regeneration of teeth in sharks

The evolution of oral teeth is considered a major contributor to the overall success of jawed vertebrates. This is especially apparent in cartilaginous fishes including sharks and rays, which develop elaborate arrays of highly specialized teeth, organized in rows and retain the capacity for life-long regeneration. Perpetual regeneration of oral teeth has been either lost or highly reduced in many other lineages including important developmental model species, so cartilaginous fishes are uniquely suited for deep comparative analyses of tooth development and regeneration. Additionally, sharks and rays can offer crucial insights into the characters of the dentition in the ancestor of all jawed vertebrates. Despite this, tooth development and regeneration in chondrichthyans is poorly understood and remains virtually uncharacterized from a developmental genetic standpoint. Using the emerging chondrichthyan model, the catshark (Scyliorhinus spp.), we characterized the expression of genes homologous to those known to be expressed during stages of early dental competence, tooth initiation, morphogenesis, and regeneration in bony vertebrates. We have found that expression patterns of several genes from Hh, Wnt/β-catenin, Bmp and Fgf signalling pathways indicate deep conservation over ~450 million years of tooth development and regeneration. We describe how these genes participate in the initial emergence of the shark dentition and how they are redeployed during regeneration of successive tooth generations. We suggest that at the dawn of the vertebrate lineage, teeth (i) were most likely continuously regenerative structures, and (ii) utilised a core set of genes from members of key developmental signalling pathways that were instrumental in creating a dental legacy redeployed throughout vertebrate evolution. These data lay the foundation for further experimental investigations utilizing the unique regenerative capacity of chondrichthyan models to answer evolutionary, developmental, and regenerative biological questions that are impossible to explore in classical models.

FGF9 and FGF18 in idiopathic pulmonary fibrosis promote survival and migration and inhibit myofibroblast differentiation of human lung fibroblasts in vitro.

Idiopathic pulmonary fibrosis (IPF) is characterized by an accumulation of extracellular matrix proteins and fibroblasts in the distal airways. Key developmental lung signaling pathways are reactivated in IPF. For instance, fibroblast growth factor 9 (FGF9) and FGF18, involved in epithelial-mesenchymal interactions, are critical for lung development. We evaluated the expression of FGF9, FGF18, and FGF receptors (FGFRs) in lung tissue from controls and IPF patients and assessed their effect on proliferation, survival, migration, and differentiation of control and IPF human lung fibroblasts (HLFs). FGF9, FGF18, and all FGFRs were present in the remodeled alveolar epithelium close to the fibroblast foci in IPF lungs. FGFR3 was generally detected in fibroblast foci by immunohistochemistry. In vitro, HLFs mainly expressed mesenchyme-associated FGFR isoforms (FGFR1c and FGFR3c) and FGFR4. FGF9 did not affect fibroblast proliferation, whereas FGF18 inhibited cell growth in control fibroblasts. FGF9 and FGF18 decreased Fas-ligand-induced apoptosis in control but not in IPF fibroblasts. FGF9 prevented transforming growth factor β1-induced myofibroblast differentiation. FGF9 and FGF18 increased the migratory capacities of HLF, and FGF9 actively modulated matrix metalloproteinase activity. In addition, FGFR3 inhibition by small interfering RNA impacted p-ERK activation by FGF9 and FGF18 and their effects on differentiation and migration. These results identify FGF9 as an antiapoptotic and promigratory growth factor on HLF, maintaining fibroblasts in an undifferentiated state. The biological effects of FGF9 and FGF18 were partially driven by FGFR3. FGF18 was a less potent molecule. Both growth factors likely contribute to the fibrotic process in vivo.