Neurogenic Defects Occur in LRIG2-Associated Urinary Bladder Disease.

Introduction: Urofacial, or Ochoa, syndrome (UFS) is an autosomal recessive disease featuring a dyssynergic bladder with detrusor smooth muscle contracting against an undilated outflow tract. It also features an abnormal grimace. Half of individuals with UFS carry biallelic variants in HPSE2, whereas other rare families carry variants in LRIG2.LRIG2 is immunodetected in pelvic ganglia sending autonomic axons into the bladder. Moreover, Lrig2 mutant mice have abnormal urination and abnormally patterned bladder nerves. We hypothesized that peripheral neurogenic defects underlie LRIG2-associated bladder dysfunction. Methods: We describe a new family with LRIG2-associated UFS and studied Lrig2 homozygous mutant mice with ex vivo physiological analyses. Results: The index case presented antenatally with urinary tract (UT) dilatation, and postnatally had urosepsis and functional bladder outlet obstruction. He had the grimace that, together with UT disease, characterizes UFS. Although HPSE2 sequencing was normal, he carried a homozygous, predicted pathogenic, LRIG2 stop variant (c.1939C>T; p.Arg647∗). Lrig2 mutant mice had enlarged bladders. Ex vivo physiology experiments showed neurogenic smooth muscle relaxation defects in the outflow tract, containing the urethra adjoining the bladder, and in detrusor contractility. Moreover, there were nuanced differences in physiological outflow tract defects between the sexes. Conclusion: Putting this family in the context of all reported UT disease-associated LRIG2 variants, the full UFS phenotype occurs with biallelic stop or frameshift variants, but missense variants lead to bladder-limited disease. Our murine observations support the hypothesis that UFS is a genetic autonomic neuropathy of the bladder affecting outflow tract and bladder body function.

Expanding the HPSE2 Genotypic Spectrum in Urofacial Syndrome, A Disease Featuring a Peripheral Neuropathy of the Urinary Bladder.

Urofacial (also called Ochoa) syndrome (UFS) is an autosomal recessive congenital disorder of the urinary bladder featuring voiding dysfunction and a grimace upon smiling. Biallelic variants in HPSE2, coding for the secreted protein heparanase-2, are described in around half of families genetically studied. Hpse2 mutant mice have aberrant bladder nerves. We sought to expand the genotypic spectrum of UFS and make insights into its pathobiology. Sanger sequencing, next generation sequencing and microarray analysis were performed in four previously unreported families with urinary tract disease and grimacing. In one, the proband had kidney failure and was homozygous for the previously described pathogenic variant c.429T>A, p.(Tyr143*). Three other families each carried a different novel HPSE2 variant. One had homozygous triplication of exons 8 and 9; another had homozygous deletion of exon 4; and another carried a novel c.419C>G variant encoding the missense p.Pro140Arg in trans with c.1099-1G>A, a previously reported pathogenic splice variant. Expressing the missense heparanase-2 variant in vitro showed that it was secreted as normal, suggesting that 140Arg has aberrant functionality after secretion. Bladder autonomic neurons emanate from pelvic ganglia where resident neural cell bodies derive from migrating neural crest cells. We demonstrated that, in normal human embryos, neuronal precursors near the developing hindgut and lower urinary tract were positive for both heparanase-2 and leucine rich repeats and immunoglobulin like domains 2 (LRIG2). Indeed, biallelic variants of LRIG2 have been implicated in rare UFS families. The study expands the genotypic spectrum in HPSE2 in UFS and supports a developmental neuronal pathobiology.

Envisioning treating genetically-defined urinary tract malformations with viral vector-mediated gene therapy.

Human urinary tract malformations can cause dysfunctional voiding, urosepsis and kidney failure. Other affected individuals, with severe phenotypes on fetal ultrasound screening, undergo elective termination. Currently, there exist no specific treatments that target the primary biological disease mechanisms that generate these urinary tract malformations. Historically, the pathogenesis of human urinary tract malformations has been obscure. It is now established that some such individuals have defined monogenic causes for their disease. In health, the implicated genes are expressed in either differentiating urinary tract smooth muscle cells, urothelial cells or peripheral nerve cells supplying the bladder. The phenotypes arising from mutations of these genes include megabladder, congenital functional bladder outflow obstruction, and vesicoureteric reflux. We contend that these genetic and molecular insights can now inform the design of novel therapies involving viral vector-mediated gene transfer. Indeed, this technology is being used to treat individuals with early onset monogenic disease outside the urinary tract, such as spinal muscular atrophy. Moreover, it has been contended that human fetal gene therapy, which may be necessary to ameliorate developmental defects, could become a reality in the coming decades. We suggest that viral vector-mediated gene therapies should first be tested in existing mouse models with similar monogenic and anatomical aberrations as found in people with urinary tract malformations. Indeed, gene transfer protocols have been successfully pioneered in newborn and fetal mice to treat non-urinary tract diseases. If similar strategies were successful in animals with urinary tract malformations, this would pave the way for personalized and potentially curative treatments for people with urinary tract malformations.

Experimental long-term diabetes mellitus alters the transcriptome and biomechanical properties of the rat urinary bladder.

Diabetes mellitus (DM) is the leading cause of chronic kidney disease and diabetic nephropathy is widely studied. In contrast, the pathobiology of diabetic urinary bladder disease is less understood despite dysfunctional voiding being common in DM. We hypothesised that diabetic cystopathy has a characteristic molecular signature. We therefore studied bladders of hyperglycaemic and polyuric rats with streptozotocin (STZ)-induced DM. Sixteen weeks after induction of DM, as assessed by RNA arrays, wide-ranging changes of gene expression occurred in DM bladders over and above those induced in bladders of non-hyperglycaemic rats with sucrose-induced polyuria. The altered transcripts included those coding for extracellular matrix regulators and neural molecules. Changes in key genes deregulated in DM rat bladders were also detected in db/db mouse bladders. In DM rat bladders there was reduced birefringent collagen between detrusor muscle bundles, and atomic force microscopy showed a significant reduction in tissue stiffness; neither change was found in bladders of sucrose-treated rats. Thus, altered extracellular matrix with reduced tissue rigidity may contribute to voiding dysfunction in people with long-term DM. These results serve as an informative stepping stone towards understanding the complex pathobiology of diabetic cystopathy.

Dysfunctional bladder neurophysiology in urofacial syndrome Hpse2 mutant mice.

AIMS: Urofacial syndrome (UFS) is an autosomal recessive disease characterized by detrusor contraction against an incompletely dilated outflow tract. This dyssynergia causes dribbling incontinence and incomplete voiding. Around half of individuals with UFS have biallelic mutations of HPSE2 that encodes heparanase 2, a protein found in pelvic ganglia and bladder nerves. Homozygous Hpse2 mutant mice have abnormal patterns of nerves in the bladder body and outflow tract, and also have dysfunctional urinary voiding. We hypothesized that bladder neurophysiology is abnormal Hpse2 mutant mice. METHODS: Myography was used to study bladder bodies and outflow tracts isolated from juvenile mice. Myogenic function was analyzed after chemical stimulation or blockade of key receptors. Neurogenic function was assessed by electrical field stimulation (EFS). Muscarinic receptor expression was semi-quantified by Western blot analysis. RESULTS: Nitrergic nerve-mediated relaxation of precontracted mutant outflow tracts was significantly decreased vs littermate controls. The contractile ability of mutant outflow tracts was normal as assessed by KCl and the α1-adrenoceptor agonist phenylephrine. EFS of mutant bladder bodies induced significantly weaker contractions than controls. Conversely, the muscarinic agonist carbachol induced significantly stronger contractions of bladder body than controls. CONCLUSIONS: The Hpse2 model of UFS features aberrant bladder neuromuscular physiology. Further work is required to determine whether similar aberrations occur in patients with UFS.

Heparanase 2 and Urofacial Syndrome, a Genetic Neuropathy.

Urofacial syndrome (UFS) is a rare but potentially devastating autosomal recessive disease. It comprises both incomplete urinary bladder emptying and a facial grimace upon smiling. A subset of individuals with the disease has biallelic mutations of HPSE2, coding for heparanase-2. Heparanase-2 and the classical heparanase are both detected in nerves in the maturing bladder, and mice mutant for Hpse2 have UFS-like bladder voiding defects and abnormally patterned bladder nerves. Other evidence suggests that the heparanase axis plays several roles in the peripheral and central nervous systems, quite apart from UFS-related biology. Some individuals with UFS lack HPSE2 mutations and instead carry biallelic variants of LRIG2, encoding leucine-rich-repeats and immunoglobulin-like-domains 2. Like heparanase-2, LRIG2 is detected in bladder nerves, and mutant Lrig2 mice have urination defects and abnormal patterns of bladder nerves. Further work is now needed to define the precise roles of heparanase-2 and LRIG2 in normal and abnormal neural differentiation.

Overactivity or blockade of transforming growth factor-ß each generate a specific ureter malformation.

Transforming growth factor-ß (TGFß) has been reported to be dysregulated in malformed ureters. There exists, however, little information on whether altered TGFß levels actually perturb ureter development. We therefore hypothesised that TGFß has functional effects on ureter morphogenesis. Tgfb1, Tgfb2 and Tgfb3 transcripts coding for TGFß ligands, as well as Tgfbr1 and Tgfbr2 coding for TGFß receptors, were detected by quantitative polymerase chain reaction in embryonic mouse ureters collected over a wide range of stages. As assessed by in situ hybridisation and immunohistochemistry, the two receptors were detected in embryonic urothelia. Next, TGFß1 was added to serum-free cultures of embryonic day 15 mouse ureters. These organs contain immature smooth muscle and urothelial layers and their in vivo potential to grow and acquire peristaltic function can be replicated in serum-free organ culture. Such organs therefore constitute a suitable developmental stage with which to define roles of factors that affect ureter growth and functional differentiation. Exogenous TGFß1 inhibited growth of the ureter tube and generated cocoon-like dysmorphogenesis. RNA sequencing suggested that altered levels of transcripts encoding certain fibroblast growth factors (FGFs) followed exposure to TGFß. In serum-free organ culture exogenous FGF10 but not FGF18 abrogated certain dysmorphic effects mediated by exogenous TGFß1. To assess whether an endogenous TGFß axis functions in developing ureters, embryonic day 15 explants were exposed to TGFß receptor chemical blockade; growth of the ureter was enhanced, and aberrant bud-like structures arose from the urothelial tube. The muscle layer was attenuated around these buds, and peristalsis was compromised. To determine whether TGFß effects were limited to one stage, explants of mouse embryonic day 13 ureters, more primitive organs, were exposed to exogenous TGFß1, again generating cocoon-like structures, and to TGFß receptor blockade, again generating ectopic buds. As for the mouse studies, immunostaining of normal embryonic human ureters detected TGFßRI and TGFßRII in urothelia. Collectively, these observations reveal unsuspected regulatory roles for endogenous TGFß in embryonic ureters, fine-tuning morphogenesis and functional differentiation. Our results also support the hypothesis that the TGFß up-regulation reported in ureter malformations impacts on pathobiology. Further experiments are needed to unravel the intracellular signalling mechanisms involved in these dysmorphic responses. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Congenital Disorders of the Human Urinary Tract: Recent Insights From Genetic and Molecular Studies.

The urinary tract comprises the renal pelvis, the ureter, the urinary bladder, and the urethra. The tract acts as a functional unit, first propelling urine from the kidney to the bladder, then storing it at low pressure inside the bladder which intermittently and completely voids urine through the urethra. Congenital diseases of these structures can lead to a range of diseases sometimes associated with fetal losses or kidney failure in childhood and later in life. In some of these disorders, parts of the urinary tract are severely malformed. In other cases, the organs appear grossly intact yet they have functional deficits that compromise health. Human studies are beginning to indicate monogenic causes for some of these diseases. Here, the implicated genes can encode smooth muscle, neural or urothelial molecules, or transcription factors that regulate their expression. Furthermore, certain animal models are informative about how such molecules control the development and functional differentiation of the urinary tract. In future, novel therapies, including those based on gene transfer and stem cell technologies, may be used to treat these diseases to complement conventional pharmacological and surgical clinical therapies.

Lrig2 and Hpse2, mutated in urofacial syndrome, pattern nerves in the urinary bladder.

Mutations in leucine-rich-repeats and immunoglobulin-like-domains 2 (LRIG2) or in heparanase 2 (HPSE2) cause urofacial syndrome, a devastating autosomal recessive disease of functional bladder outlet obstruction. It has been speculated that urofacial syndrome has a neural basis, but it is unknown whether defects in urinary bladder innervation are present. We hypothesized that urofacial syndrome features a peripheral neuropathy of the bladder. Mice with homozygous targeted Lrig2 mutations had urinary defects resembling those found in urofacial syndrome. There was no anatomical blockage of the outflow tract, consistent with a functional bladder outlet obstruction. Transcriptome analysis revealed differential expression of 12 known transcripts in addition to Lrig2, including 8 with established roles in neurobiology. Mice with homozygous mutations in either Lrig2 or Hpse2 had increased nerve density within the body of the urinary bladder and decreased nerve density around the urinary outflow tract. In a sample of 155 children with chronic kidney disease and urinary symptoms, we discovered novel homozygous missense LRIG2 variants that were predicted to be pathogenic in 2 individuals with non-syndromic bladder outlet obstruction. These observations provide evidence that a peripheral neuropathy is central to the pathobiology of functional bladder outlet obstruction in urofacial syndrome, and emphasize the importance of LRIG2 and heparanase 2 for nerve patterning in the urinary tract.

Exogenous transforming growth factor-ß1 enhances smooth muscle differentiation in embryonic mouse jejunal explants.

An ex vivo experimental strategy that replicates in vivo intestinal development would in theory provide an accessible setting with which to study normal and dysmorphic gut biology. The current authors recently described a system in which mouse embryonic jejunal segments were explanted onto semipermeable platforms and fed with chemically defined serum-free media. Over 3 days in organ culture, explants formed villi and they began to undergo spontaneous peristalsis. As defined in the current study, the wall of the explanted gut failed to form a robust longitudinal smooth muscle (SM) layer as it would do in vivo over the same time period. Given the role of transforming growth factor ß1 (TGFß1) in SM differentiation in other organs, it was hypothesized that exogenous TGFß1 would enhance SM differentiation in these explants. In vivo, TGFß receptors I and II were both detected in embryonic longitudinal jejunal SM cells and, in organ culture, exogenous TGFß1 induced robust differentiation of longitudinal SM. Microarray profiling showed that TGFß1 increased SM specific transcripts in a dose dependent manner. TGFß1 proteins were detected in amniotic fluid at a time when the intestine was physiologically herniated. By analogy with the requirement for exogenous TGFß1 for SM differentiation in organ culture, the TGFß1 protein that was demonstrated to be present in the amniotic fluid may enhance intestinal development when it is physiologically herniated in early gestation. Future studies of embryonic intestinal cultures should include TGFß1 in the defined media to produce a more faithful model of in vivo muscle differentiation. Copyright © 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

ACTB Loss-of-Function Mutations Result in a Pleiotropic Developmental Disorder.

ACTB encodes ß-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, ß-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic ß-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, ß-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development.

From gene discovery to new biological mechanisms: heparanases and congenital urinary bladder disease.

We present a scientific investigation into the pathogenesis of a urinary bladder disease. The disease in question is called urofacial syndrome (UFS), a congenital condition inherited in an autosomal recessive manner. UFS features incomplete urinary bladder emptying and vesicoureteric reflux, with a high risk of recurrent urosepsis and end-stage renal disease. The story starts from a human genomic perspective, then proceeds through experiments that seek to determine the roles of the implicated molecules in embryonic frogs and newborn mice. A future aim would be to use such biological knowledge to intelligently choose novel therapies for UFS. We focus on heparanase proteins and the peripheral nervous system, molecules and tissues that appear to be key players in the pathogenesis of UFS and therefore must also be critical for functional differentiation of healthy bladders. These considerations allow the envisioning of novel biological treatments, although the potential difficulties of targeting the developing bladder in vivo should not be underestimated.

Bridging the gap: functional healing of embryonic small intestine ex vivo.

The ability to grow embryonic organs ex vivo provides an opportunity to follow their differentiation in a controlled environment, with resulting insights into normal development. Additionally, similar strategies can be used to assess effects on organogenesis of physical and chemical manipulations. This study aimed to create an organ culture model with which to test physical manipulations to enhance healing of gut segments, thus generating a single functional organ. Embryonic mouse jejunum was isolated and cut into 2-3 mm tubes, which were placed in pairs, separated by a small gap, on semi-permeable supports. Each pair was linked by a nylon suture threaded through their lumens. After 3 days in organ culture fed by defined serum-free media, the rudiments differentiated to form tubes of smooth muscle surrounding a core of rudimentary villi. Of 34 such pairs, 74% had touching and well aligned proximate ends. Of these joined structures, 80% (59% of the total pairs) had a continuous lumen, as assessed by observing the trajectories of fluorescent dextrans injected into their distal ends. Fused organ pairs formed a single functional unit, as assessed by spontaneous contraction waves propagated along their lengths. In these healed intestines, peripherin(+) neurons formed a nexus in the zone of fusion, linking the rudiment pairs. In future, this system could be used to test whether growth factors enhance fusion. Such results should in turn inform the design of novel treatments for short bowel syndrome, a potentially fatal condition with a currently limited and imperfect range of therapies.

Heimler Syndrome Is Caused by Hypomorphic Mutations in the Peroxisome-Biogenesis Genes PEX1 and PEX6.

Heimler syndrome (HS) is a rare recessive disorder characterized by sensorineural hearing loss (SNHL), amelogenesis imperfecta, nail abnormalities, and occasional or late-onset retinal pigmentation. We ascertained eight families affected by HS and, by using a whole-exome sequencing approach, identified biallelic mutations in PEX1 or PEX6 in six of them. Loss-of-function mutations in both genes are known causes of a spectrum of autosomal-recessive peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome. PBDs are characterized by leukodystrophy, hypotonia, SNHL, retinopathy, and skeletal, craniofacial, and liver abnormalities. We demonstrate that each HS-affected family has at least one hypomorphic allele that results in extremely mild peroxisomal dysfunction. Although individuals with HS share some subtle clinical features found in PBDs, the diagnosis was not suggested by routine blood and skin fibroblast analyses used to detect PBDs. In conclusion, our findings define HS as a mild PBD, expanding the pleiotropy of mutations in PEX1 and PEX6.

Urinary tract effects of HPSE2 mutations.

Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs. HPSE2 mutations were absent in 23 non-neurogenic neurogenic bladder probands and, of 439 families with nonsyndromic vesicoureteric reflux, only one carried a putative pathogenic HPSE2 variant. Homozygous Hpse2 mutant mouse bladders contained urine more often than did wild-type organs, phenocopying human UFS. Pelvic ganglia neural cell bodies contained heparanase 1, heparanase 2, and leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2), which is mutated in certain UFS families. In conclusion, heparanase 2 is an autonomic neural protein implicated in bladder emptying, but HPSE2 variants are uncommon in urinary diseases resembling UFS.

Heparanase 2, mutated in urofacial syndrome, mediates peripheral neural development in Xenopus.

Urofacial syndrome (UFS; previously Ochoa syndrome) is an autosomal recessive disease characterized by incomplete bladder emptying during micturition. This is associated with a dyssynergia in which the urethral walls contract at the same time as the detrusor smooth muscle in the body of the bladder. UFS is also characterized by an abnormal facial expression upon smiling, and bilateral weakness in the distribution of the facial nerve has been reported. Biallelic mutations in HPSE2 occur in UFS. This gene encodes heparanase 2, a protein which inhibits the activity of heparanase. Here, we demonstrate, for the first time, an in vivo developmental role for heparanase 2. We identified the Xenopus orthologue of heparanase 2 and showed that the protein is localized to the embryonic ventrolateral neural tube where motor neurons arise. Morpholino-induced loss of heparanase 2 caused embryonic skeletal muscle paralysis, and morphant motor neurons had aberrant morphology including less linear paths and less compactly-bundled axons than normal. Biochemical analyses demonstrated that loss of heparanase 2 led to upregulation of fibroblast growth factor 2/phosphorylated extracellular signal-related kinase signalling and to alterations in levels of transcripts encoding neural- and muscle-associated molecules. Thus, a key role of heparanase 2 is to buffer growth factor signalling in motor neuron development. These results shed light on the pathogenic mechanisms underpinning the clinical features of UFS and support the contention that congenital peripheral neuropathy is a key feature of this disorder.

Urofacial syndrome: a genetic and congenital disease of aberrant urinary bladder innervation.

The urofacial, or Ochoa, syndrome is characterised by congenital urinary bladder dysfunction together with an abnormal grimace upon smiling, laughing and crying. It can present as fetal megacystis. Postnatal features include urinary incontinence and incomplete bladder emptying due to simultaneous detrusor muscle and bladder outlet contractions. Vesicoureteric reflux is often present, and the condition can be complicated by urosepsis and end-stage renal disease. The syndrome has long been postulated to have neural basis, and it can be familial when it is inherited in an autosomal recessive manner. Most individuals with urofacial syndrome genetically studied to date carry biallelic, postulated functionally null mutations of HPSE2 or, less commonly, of LRIG2. Little is known about the biology of the respective encoded proteins, heparanase 2 and leucine-rich repeats and immunoglobulin-like domains 2. Nevertheless, the observations that heparanase 2 can bind heparan sulphate proteolgycans and inhibit heparanase 1 enzymatic activity and that LRIG2 can modulate receptor tyrosine kinase growth factor signalling each point to biological roles relevant to tissue differentiation. Moreover, both heparanase 2 and LRIG2 proteins are detected in autonomic nerves growing into fetal bladders. The collective evidence is consistent with the hypothesis that urofacial syndrome genes code for proteins which work in a common pathway to facilitate neural growth into, and/or function within, the bladder. This molecular pathway may also have relevance to our understanding of the pathogenesis of other lower tract diseases, including Hinman-Allen syndrome, or non-neurogenic neurogenic bladder, and of the subset of individuals who have primary vesicoureteric reflux accompanied by bladder dysfunction.

Development of the human pancreas from foregut to endocrine commitment.

Knowledge of human pancreas development underpins our interpretation and exploitation of human pluripotent stem cell (PSC) differentiation toward a ß-cell fate. However, almost no information exists on the early events of human pancreatic specification in the distal foregut, bud formation, and early development. Here, we have studied the expression profiles of key lineage-specific markers to understand differentiation and morphogenetic events during human pancreas development. The notochord was adjacent to the dorsal foregut endoderm during the fourth week of development before pancreatic duodenal homeobox-1 detection. In contrast to the published data from mouse embryos, during human pancreas development, we detected only a single-phase of Neurogenin 3 (NEUROG3) expression and endocrine differentiation from approximately 8 weeks, before which Nirenberg and Kim homeobox 2.2 (NKX2.2) was not observed in the pancreatic progenitor cell population. In addition to revealing a number of disparities in timing between human and mouse development, these data, directly assembled from human tissue, allow combinations of transcription factors to define sequential stages and differentiating pancreatic cell types. The data are anticipated to provide a useful reference point for stem cell researchers looking to differentiate human PSCs in vitro toward the pancreatic ß-cell so as to model human development or enable drug discovery and potential cell therapy.

LRIG2 mutations cause urofacial syndrome.

Urofacial syndrome (UFS) (or Ochoa syndrome) is an autosomal-recessive disease characterized by congenital urinary bladder dysfunction, associated with a significant risk of kidney failure, and an abnormal facial expression upon smiling, laughing, and crying. We report that a subset of UFS-affected individuals have biallelic mutations in LRIG2, encoding leucine-rich repeats and immunoglobulin-like domains 2, a protein implicated in neural cell signaling and tumorigenesis. Importantly, we have demonstrated that rare variants in LRIG2 might be relevant to nonsyndromic bladder disease. We have previously shown that UFS is also caused by mutations in HPSE2, encoding heparanase-2. LRIG2 and heparanase-2 were immunodetected in nerve fascicles growing between muscle bundles within the human fetal bladder, directly implicating both molecules in neural development in the lower urinary tract.

Regulation of protein kinase D activity in adult myocardium: novel counter-regulatory roles for protein kinase Cepsilon and protein kinase A.

Protein kinase D (PKD) is activated downstream of protein kinase C (PKC) in many cell types, although little is known about the mechanisms that regulate PKD in adult myocardium. Exposure of cultured adult rat ventricular myocytes (ARVM) to phorbol 12-myristate 13-acetate (PMA; 100 nM for 5 min) activated PKD, as evidenced by significantly increased phosphorylation at Ser744/8 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). PKD activation occurred concomitantly with translocation of the enzyme from the cytosolic to the particulate fraction. The role of PKC was confirmed by pretreatment (15 min) of ARVM with the PKC inhibitors GF109203X (1 microM) and Ro31-8220 (1 microM), both of which prevented PKD phosphorylation on subsequent exposure to PMA. Exposure of ARVM to endothelin-1 (ET1; 100 nM for 10 min) also activated PKD by a PKC-dependent mechanism. To determine the PKC isoform(s) involved in the ET1-induced PKD activation, ARVM were infected with adenoviral vectors encoding dominant-negative (DN) mutants of PKCalpha, PKCdelta and PKCepsilon. Expression of DN-PKCalpha and DN-PKCdelta had little effect on ET1-induced PKD activation, whilst this was significantly attenuated by expression of DN-PKCepsilon, indicating that PKCepsilon plays a predominant role in the pertinent ET1 signaling pathway. Intriguingly, prior exposure to the adenylyl cyclase activator forskolin (1 microM for 5 min) or the beta-adrenergic agonist isoprenaline (100 nM for 5 min) markedly attenuated ET1-induced PKD activation, but not PMA-induced PKD activation. The ET1-induced response was rescued when protein kinase A (PKA) was inhibited (H89, 10 microM) before exposure to isoprenaline. These results show that ET1-induced PKD activation in ARVM is mediated by PKC, primarily the PKCepsilon isoform, and is suppressed by PKA activation.