The origin of renal fibroblasts/myofibroblasts and the signals that trigger fibrosis.

Renal fibrosis is a common characteristic of chronic kidney disease (CKD). Aberrant and excessive depositions of extracellular matrix (ECM) proteins in both glomeruli and interstitial regions are typical hallmarks of renal fibrosis and amplify the severity of kidney injury. To date, an approved therapy specifically targeted to renal fibrosis is needed to mitigate or even retard renal fibrosis. Recent findings have identified a unique population of myofibroblasts as a primary source of ECM in scar tissue formation. However, the origin of myofibroblasts in renal fibrosis remains the subject of controversial debates. The advancement in lineage tracing and immunofluorescent microscopy technologies have suggested that myofibroblasts may arise from a number of sources such as activated renal fibroblasts, pericytes, epithelial-to-mesenchymal transition (EMT), endothelial-to-mesenchymal transition (EndoMT), bone marrow derived cells and fibrocytes. Recent studies also indicate that multiple ligands of TGF-ß/Smads are the direct mediators for renal fibrosis. Consistently, inhibition of the TGF-ß/Smads signaling pathway using various strategies significantly reduce renal fibrotic lesions and ameliorate kidney injury, suggesting that targeting the TGF-ß/Smads signaling pathway could be a new strategy for effective therapies. In this review, we will briefly discuss the diverse origins of myofibroblasts and molecular pathways triggering renal fibrosis. Prospective therapeutic approaches based on those molecular mechanisms will hopefully offer exciting insights in the development of new therapeutic interventions for patients in the near future.

Validation of a Three-Dimensional Method for Counting and Sizing Podocytes in Whole Glomeruli.

Podocyte depletion is sufficient for the development of numerous glomerular diseases and can be absolute (loss of podocytes) or relative (reduced number of podocytes per volume of glomerulus). Commonly used methods to quantify podocyte depletion introduce bias, whereas gold standard stereologic methodologies are time consuming and impractical. We developed a novel approach for assessing podocyte depletion in whole glomeruli that combines immunofluorescence, optical clearing, confocal microscopy, and three-dimensional analysis. We validated this method in a transgenic mouse model of selective podocyte depletion, in which we determined dose-dependent alterations in several quantitative indices of podocyte depletion. This new approach provides a quantitative tool for the comprehensive and time-efficient analysis of podocyte depletion in whole glomeruli.

Cauli: a mouse strain with an Ift140 mutation that results in a skeletal ciliopathy modelling Jeune syndrome.

Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.

The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

Transforming growth factor-ß1 (TGF-ß1)/Smad signaling has a central role in the pathogenesis of renal fibrosis. Smad3 and Smad4 are pro-fibrotic, while Smad2 is anti-fibrotic. However, these Smads form heterogeneous complexes, the functions of which are poorly understood. Here we studied Smad complex function in renal fibrosis using the mouse model of unilateral ureteric obstruction. Mice heterozygous for Smad3/4 (Smad3/4+/-) exhibited substantial protection from renal fibrosis through day 7 of obstruction, whereas Smad2/3+/- and Smad2/4+/- mice showed only modest protection. Formation of Smad3/Smad4/CDK9 complexes was an early event following obstruction in wild-type mice, which involved nuclear phosphorylation of the linker regions of Smad3. Significantly, Smad3 or Smad4 deficiency decreased the formation of Smad4/CDK9 or Smad3/CDK9 complex, Smad3 linker phosphorylation, and fibrosis but at different degrees. In vitro, TGF-ß1 stimulation of collagen I promoter activity involved formation of Smad3/Smad4/CDK9 complexes, and overexpression of each component gave additive increases in collagen promoter activity. Co-administration of a CDK9 inhibitor and Smad3-specific inhibition achieved better protection from TGF-ß1-induced fibrotic response in vitro and renal interstitial fibrosis in vivo. Thus formation of Smad3/Smad4/CDK9 complex drives renal fibrosis during ureteral obstruction. Formation of this complex represents a novel target for antifibrotic therapies.

Smad3 deficiency protects mice from obesity-induced podocyte injury that precedes insulin resistance.

Signaling by TGF-ß/Smad3 plays a key role in renal fibrosis. As obesity is one of the major risk factors of chronic and end-stage renal disease, we studied the role of Smad3 signaling in the pathogenesis of obesity-related renal disease. After switching to a high fat diet, the onset of Smad3 C-terminal phosphorylation, increase in albuminuria, and the early stages of peripheral and renal insulin resistance occurred at 1 day, and 4 and 8 weeks, respectively, in C57BL/6 mice. The loss of synaptopodin, a functional marker of podocytes, and phosphorylation of the Smad3 linker region (T179 and S213) appeared after 4 weeks of the high fat diet. This suggests a temporal pattern of Smad3 signaling activation leading to kidney injury and subsequent insulin resistance in the development of obesity-related renal disease. In vivo, Smad3 knockout attenuated the high fat diet-induced proteinuria, renal fibrosis, overall podocyte injury, and mitochondrial dysfunction in podocytes. In vitro palmitate caused a rapid activation of Smad3 in 30 min, loss of synaptopodin in 2 days, and impaired insulin signaling in 3 days in isolated mouse podocytes. Blockade of either Smad3 phosphorylation by SIS3 (a Smad3 inhibitor) or T179 phosphorylation by flavopiridol (a CDK9 inhibitor) prevented the palmitate-induced loss of synaptopodin and mitochondrial function in podocytes. Thus, Smad3 signaling plays essential roles in obesity-related renal disease and may be a novel therapeutic target.

Regulation of renal fibrosis by Smad3 Thr388 phosphorylation.

Transforming growth factor-ß (TGF-ß) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-ß1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-ß-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-ß/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease.

Copy-number variation associated with congenital anomalies of the kidney and urinary tract.

BACKGROUND: The most common cause of end-stage renal disease in children can be attributed to congenital anomalies of the kidney and urinary tract (CAKUT). Despite this high incidence of disease, the genetic mutations responsible for the majority of CAKUT cases remain unknown. METHODS: To identify novel genomic regions associated with CAKUT, we screened 178 children presenting with the entire spectrum of structural anomalies associated with CAKUT for submicroscopic chromosomal imbalances (deletions or duplications) using single-nucleotide polymorphism (SNP) microarrays. RESULTS: Copy-number variation (CNV) was detected in 10.1 % (18/178) of the patients; in 6.2 % of the total cohort, novel duplications or deletions of unknown significance were identified, and the remaining 3.9 % harboured CNV of known pathogenicity. CNVs were inherited in 90 % (9/10) of the families tested. In this cohort, patients diagnosed with multicystic dysplastic kidney (30 %) and posterior urethral valves (24 %) had a higher incidence of CNV. CONCLUSIONS: The genes contained in the altered genomic regions represent novel candidates for CAKUT. This study has demonstrated that a significant proportion of patients with CAKUT harbour submicroscopic chromosomal imbalances, warranting screening in clinics for CNV.

Congenital anomalies of the kidney and urinary tract genetics in mice and men.

The most common cause of paediatric end-stage kidney disease results from congenital anomalies of the kidney and urinary tract (CAKUT). Genetic manipulation in mice has provided insight into the developmental events that give rise to the broad spectrum of malformations associated with CAKUT. Despite the increase in the number of identified CAKUT-causing genes, the underlying genetic cause for the majority of patients with CAKUT remains unknown. In this mini-review, we provide an overview of the genetic causes of CAKUT based on current mouse mutant models, as well as next-generation sequencing approaches in humans that are helping to bridge the gaps in our understanding.

A protocol for the identification and validation of novel genetic causes of kidney disease.

BACKGROUND: Genetic renal diseases (GRD) are a heterogeneous and incompletely understood group of disorders accounting for approximately 10 % of those diagnosed with kidney disease. The advent of Next Generation sequencing and new approaches to disease modelling may allow the identification and validation of novel genetic variants in patients with previously incompletely explained or understood GRD. METHODS/DESIGN: This study will recruit participants in families/trios from a multidisciplinary sub-specialty Renal Genetics Clinic where known genetic causes of GRD have been excluded or where genetic testing is not available. After informed patient consent, whole exome and/or genome sequencing will be performed with bioinformatics analysis undertaken using a customised variant assessment tool. A rigorous process for participant data management will be undertaken. Novel genetic findings will be validated using patient-derived induced pluripotent stem cells via differentiation to renal and relevant extra-renal tissue phenotypes in vitro. A process for managing the risk of incidental findings and the return of study results to participants has been developed. DISCUSSION: This investigator-initiated approach brings together experts in nephrology, clinical and molecular genetics, pathology and developmental biology to discover and validate novel genetic causes for patients in Australia affected by GRD without a known genetic aetiology or pathobiology.

High nephron endowment protects against salt-induced hypertension.

While low nephron number is associated with increased risk of developing cardiovascular and renal disease, the functional consequences of a high nephron number are unknown. We tested the hypothesis that a high nephron number provides protection against hypertensive and renal insults. Mean arterial pressure (MAP) and renal function were characterized in male wild-type (WT) and transforming growth factor-ß2 heterozygous (Tgfb2(+/-)) mice under basal conditions and following a chronic high-salt diet. Kidneys were collected for unbiased stereological analysis. Baseline MAP and renal function were indistinguishable between genotypes. The chronic high-salt diet (5% NaCl for 4 wk followed by 8% NaCl for 4 wk) led to similar step-wise increases in urine volume, Na(+) excretion, and albuminuria in the genotypes. The 5% NaCl diet induced modest and similar increases in MAP (3.5 ± 1.6 and 3.4 ± 0.8 mmHg in WT and Tgfb2(+/-), respectively). After the step up to the 8% NaCl diet, MAP increased further in WT (+15.9 ± 5.1 mmHg), but not Tgfb2(+/-) (-0.1 ± 1.0 mmHg), mice. Nephron number was 30% greater in Tgfb2(+/-) than WT mice and was not affected by the chronic high-salt diet. Mean glomerular volume was lower in Tgfb2(+/-) than WT mice, and the chronic high-salt diet induced significant glomerular hypertrophy. In a separate cohort of mice, an acute, 7-day, 8% NaCl diet induced similar rises in MAP in the genotypes. This is the first study to examine the physiological characteristics of a model of high nephron number, and the findings are consistent with this phenotype providing protection against chronic, but not acute, hypertensive insults.

Expression patterns and roles of periostin during kidney and ureter development.

PURPOSE: Periostin is a secreted extracellular matrix protein that is differentially expressed in the developing kidney. We analyzed the temporal-spatial expression of periostin in the developing kidney and ureter as well as its roles in ureter branching morphogenesis, nephrogenesis and ureter development. MATERIALS AND METHODS: RNA in situ hybridization and immunofluorescence histochemistry were used to investigate the expression of periostin, αv integrin and α-smooth muscle actin during mouse renal and ureteral development. Metanephric explants were cultured in the presence of recombinant periostin, and ureteral branch points/tips and the glomerular number were quantified. Explants were also cultured in the presence of exogenous bone morphogenetic protein 4 and the effect on periostin mRNA levels was determined by quantitative real-time polymerase chain reaction. RESULTS: Periostin expression was observed in the mesenchyme surrounding the kidney and ureter, renal stroma, metanephric mesenchyme, ureter epithelium and developing nephrons. At embryonic day 15.5 periostin and αv integrin, a common subunit of periostin receptors, were co-expressed in smooth muscle cells of the ureter, renal artery and intrarenal arteries. Bone morphogenetic protein 4 up-regulated periostin mRNA expression and exogenous periostin inhibited branching morphogenesis and glomerular number. CONCLUSIONS: Bone morphogenetic protein 4 which inhibits ureteral branching morphogenesis and promotes smooth muscle cell migration in the ureter up-regulated periostin mRNA expression in the developing kidney. Ureteral smooth muscle cells express periostin and αv integrin. Periostin inhibited ureteral branching morphogenesis and glomerular number. Together these results suggest that periostin and bone morphogenetic protein 4 may have a role in branching morphogenesis, nephrogenesis and possibly smooth muscle cell migration.

Redirection of renal mesenchyme to stromal and chondrocytic fates in the presence of TGF-beta2.

Many members of the transforming growth factor-beta (TGF-beta) superfamily have been shown to be important regulators of metanephric development. In this study, we characterized the effect of TGF-beta2 on metanephric development. Rat and mouse metanephroi cultured in the presence of exogenous TGF-beta2 for up to 15 days were small, and contained rudimentary ureteric branches and few glomeruli. These metanephroi were mostly comprised of mesenchymal cells, with two cell populations (designated Type 1 and Type 2 cells) evident. Type 1 cells were only observed when TGF-beta2 was added from the commencement of culture, they resembled chondroblasts and were Alcian Blue and Col IIB positive. Type 2 cells were observed whenever TGF-beta2 was added to the media, formed a band at the periphery of the explants consisting of 5-10 layers of spindle-shaped cells, and were alpha-smooth muscle actin positive. Molecular and RNA in situ hybridization analysis of metanephroi cultured in the presence of TGF-beta2 for 6 days demonstrated that Type 1 and 2 cells were negative for Pax2, WT1, GDNF and FoxD1. Gene expression profiling demonstrated an upregulation of chondrocyte, myogenic and stromal genes, some of which were identified as markers of Type 1 and Type 2 cells. In addition, TGF-beta2 was capable of maintaining the survival of mouse isolated metanephric mesenchyme (iMM) in the absence of serum or inductive signals from the ureteric epithelium. TGF-beta2 also induced the differentiation of iMM into Type 1 and 2 cells. The presence of chondrocytes and muscle in these cultures is reminiscent of the cell types found in some Wilms' tumors. These studies demonstrate that TGF-beta2 is capable of differentiating metanephric mesenchyme away from a renal cell fate.

Sexual dimorphism in mouse metanephroi exposed to 17 beta-estradiol in vitro.

UNLABELLED: 17beta-Estradiol, the most potent circulating estrogen, has been shown to greatly impact on the development and formation of tissues of the urogenital tract. The adult kidney has previously been shown to be highly responsive to 17beta-estradiol stimulation. However, the direct effect of 17beta-estradiol on kidney development remains unclear. AIM: To investigate the direct effect of 17beta-estradiol on male and female metanephric kidney development. METHODS: Whole embryonic-day-12.5 (E12.5) C57Bl/6 male and female mouse metanephroi were cultured in the presence of varying concentrations of 17beta-estradiol (0.1-5.0 nM) for 72 h. Metanephric development was assessed using immunofluorescence labeling techniques. The real-time polymerase chain reaction was used to investigate estrogen receptor-alpha (ERalpha), glial-cell-line-derived neurotrophic factor (GDNF) and its associated receptor cRET, transforming growth factor-beta (TGFbeta1), TGFbeta2 and TGFbeta3 mRNA expression levels. RESULTS: ERalpha was present in developing metanephroi at E12.5; however, ERbeta was absent. No significant sex difference in ERalpha mRNA expression was observed. Significant increases in the number of ureteric branch points, terminal tips and developing glomeruli were observed in female metanephroi cultured in the presence of 1.0 and 5.0 nM 17beta-estradiol. Conversely, no significant effect was observed in male metanephroi cultured with 17beta-estradiol. GDNF and cRET mRNA expression was increased in both male and female metanephroi, whilst TGFbeta1 and TGFbeta2 mRNA expression was decreased following culture in the presence of 17beta-estradiol. CONCLUSION: This study is the first to establish that the mouse metanephros displays a sexual dimorphism in response to specific concentrations of estrogens.

Requirement of PDZ-containing proteins for cell cycle regulation and differentiation in the mouse lens epithelium.

The roles of PDZ domain-containing proteins such as Dlg and Scrib have been well described for Drosophila; however, their requirement for mammalian development is poorly understood. Here we show that Dlg, Scrib, MAGI1, MAGI3, and MPDZ are expressed in the mouse ocular lens. We demonstrate that the increase in proliferation and defects in cellular adhesion and differentiation observed in epithelia of lenses that express E6, a viral oncoprotein that can bind to several PDZ proteins, including the human homologs of Dlg and Scrib, is dependent on E6's ability to bind these proteins via their PDZ domains. Analyses of lenses from mice carrying an insertional mutation in Dlg (dlg(gt)) show increased proliferation and proliferation in spatially inappropriate regions of the lens, a phenotype similar to that of lenses expressing E6. The results from this study indicate that multiple PDZ domain-containing proteins, including Dlg and Scrib, may be required for maintaining the normal pattern of growth and differentiation in the lens. Furthermore, the phenotypic similarities among the Drosophila dlg mutant, the lenses of dlg(gt) mice, and the lenses of E6 transgenic mice suggest that Dlg may have a conserved function in regulating epithelial cell growth and differentiation across species.

Spatial gene expression in the T-stage mouse metanephros.

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.

Differential gene expression in the developing mouse ureter.

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations.

Imaging the embryonic kidney.

The structural and functional development of the permanent mammalian kidney or metanephros is a complex process involving the actions of thousands of gene products, complex cell movements and tissue patterning in three dimensions (3D). This review focuses on the recent advances made in imaging technology, processing and analysis combined with mouse genetics and the generation of protein-reporter mice which has enabled us to monitor the development and movement of defined cell populations within the developing kidney in 3D and over time (4D).

The where, what and why of the developing renal stroma.

In recent years, a great deal has been learnt about the molecular regulation of kidney development. While most research has focused on the molecular regulation of ureteric branching morphogenesis and nephron formation, significant insights into the definition and functions of the renal stroma have emerged. Many molecules expressed in the developing renal stroma are now known to play significant regulatory roles in kidney development. However, the term 'renal stroma' continues to have different meanings to different researchers. This review clarifies this situation and defines the derivation, location and functions of the stroma in the developing metanephros.

Genetic studies define MAGUK proteins as regulators of epithelial cell polarity.

Polarized epithelial cells play critical roles during early embryonic development and organogenesis. Multi-domain scaffolding proteins belonging to the membrane associated guanylate kinase (MAGUK) family are commonly found at the plasma membrane of polarized epithelial cells. Genetic studies in Drosophila melanogaster and Caenorhabditis elegans have revealed that MAGUK proteins regulate various aspects of the polarized epithelial phenotype, including cell junction assembly, targeting of proteins to the plasma membrane and the organisation of polarized signalling complexes. This review will focus on the genetic studies that have contributed to our understanding of the MAGUK family members, Dlg and Lin-2/CASK, in controlling these processes. In addition, our recent genetic analysis of mouse Dlg, in combination with genetic and biochemical studies of Lin-2/CASK by others suggests a model placing Dlg and Lin-2/CASK within the same developmental pathway.

A mouse splice-site mutant and individuals with atypical chromosome 22q11.2 deletions demonstrate the crucial role for crkl in craniofacial and pharyngeal development.

The 22q11.2 deletion syndrome (22q11DS) is thought to be a contiguous gene syndrome caused by haploinsufficiency for a variable number of genes with overlapping function during the development of the craniofacial, pharyngeal and cardiac structures. The complexity of genetic and developmental anomalies resulting in 22q11DS has made attributing causation to specific genes difficult. The CRKL gene resides within the common 3-Mb region, most frequently affected in 22q11DS, and has been shown to play an essential role in the development of tissues affected in 22q11DS. Here, we report the characterisation of a mouse strain we named 'snoopy', harbouring a novel Crkl splice-site mutation that results in a loss of Crkl expression. The snoopy strain exhibits a variable phenotype that includes micrognathia, pharyngeal occlusion, aglossia and holoprosencephaly, and altered retinoic acid and endothelin signalling. Together, these features are reminiscent of malformations occurring in auriculocondylar syndrome and agnathia-otocephaly complex, 2 conditions not previously associated with the CRKL function. Comparison of the features of a cohort of patients harbouring small 22q11.2 deletions centred over the CRKL gene, but sparing TBX1, highlights the role of CRKL in contributing to the craniofacial features of 22q11DS. These analyses demonstrate the central role of Crkl in regulating signalling events in the developing oropharyngeal complex and its potential to contribute to dysmorphology.