Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans.

Simple 3D culture of dissociated kidney mesenchyme mimics nephron progenitor niche and facilitates nephrogenesis Wnt-independently.

Kidney mesenchyme (KM) and nephron progenitors (NPs) depend on WNT activity, and their culture in vitro requires extensive repertoire of recombinant proteins and chemicals. Here we established a robust, simple culture of mouse KM using a combination of 3D Matrigel and growth media supplemented with Fibroblast Growth Factor 2 (FGF2) and Src inhibitor PP2. This allows dissociated KM to spontaneously self-organize into spheres. To reassess the requirement of WNT activity in KM self-organization and NPs maintenance, cells were cultured with short pulse of high-dose GSK3ß inhibitor BIO, on a constant low-dose or without BIO. Robust proliferation at 48 hours and differentiation at 1 week were observed in cultures with high BIO pulse. Importantly, dissociated KM cultured without BIO, similarly to that exposed to constant low dose of BIO, maintained NPs up to one week and spontaneously differentiated into nephron tubules at 3 weeks of culture. Our results show that KM is maintained and induced to differentiate in a simple culture system. They also imply that GSK3ß/WNT-independent pathways contribute to the maintenance and induction of mouse KM. The robust and easy 3D culture enables further characterization of NPs, and may facilitate disease modeling when applied to human cells.

Trim37-deficient mice recapitulate several features of the multi-organ disorder Mulibrey nanism.

Mulibrey nanism (MUL) is a rare autosomal recessive multi-organ disorder characterized by severe prenatal-onset growth failure, infertility, cardiopathy, risk for tumors, fatty liver, and type 2 diabetes. MUL is caused by loss-of-function mutations in TRIM37, which encodes an E3 ubiquitin ligase belonging to the tripartite motif (TRIM) protein family and having both peroxisomal and nuclear localization. We describe a congenic Trim37 knock-out mouse (Trim37(-/-)) model for MUL. Trim37(-/-) mice were viable and had normal weight development until approximately 12 months of age, after which they started to manifest increasing problems in wellbeing and weight loss. Assessment of skeletal parameters with computer tomography revealed significantly smaller skull size, but no difference in the lengths of long bones in Trim37(-/-) mice as compared with wild-type. Both male and female Trim37(-/-) mice were infertile, the gonads showing germ cell aplasia, hilus and Leydig cell hyperplasia and accumulation of lipids in and around Leydig cells. Male Trim37(-/-) mice had elevated levels of follicle-stimulating and luteinizing hormones, but maintained normal levels of testosterone. Six-month-old Trim37(-/-) mice had elevated fasting blood glucose and low fasting serum insulin levels. At 1.5 years Trim37(-/-) mice showed non-compaction cardiomyopathy, hepatomegaly, fatty liver and various tumors. The amount and morphology of liver peroxisomes seemed normal in Trim37(-/-) mice. The most consistently seen phenotypes in Trim37(-/-) mice were infertility and the associated hormonal findings, whereas there was more variability in the other phenotypes observed. Trim37(-/-) mice recapitulate several features of the human MUL disease and thus provide a good model to study disease pathogenesis related to TRIM37 deficiency, including infertility, non-alcoholic fatty liver disease, cardiomyopathy and tumorigenesis.

Expression of Foxi3 is regulated by ectodysplasin in skin appendage placodes.

BACKGROUND: Foxi3 is a member of the large forkhead box family of transcriptional regulators, which have a wide range of biological activities including manifold developmental processes. Heterozygous mutation in Foxi3 was identified in several hairless dog breeds characterized by sparse fur coat and missing teeth. A related phenotype called hypohidrotic ectodermal dysplasia (HED) is caused by mutations in the ectodysplasin (Eda) pathway genes. RESULTS: Expression of Foxi3 was strictly confined to the epithelium in developing ectodermal appendages in mouse embryos, but no expression was detected in the epidermis. Foxi3 was expressed in teeth and hair follicles throughout embryogenesis, but in mammary glands only during the earliest stages of development. Foxi3 expression was decreased and increased in Eda loss- and gain-of-function embryos, respectively, and was highly induced by Eda protein in embryonic skin explants. Also activin A treatment up-regulated Foxi3 mRNA levels in vitro. CONCLUSIONS: Eda and activin A were identified as upstream regulators of Foxi3. Foxi3 is a likely transcriptional target of Eda in ectodermal appendage placodes suggesting that HED phenotype may in part be produced by compromised Foxi3 activity. In addition to hair and teeth, Foxi3 may have a role in nail, eye, and mammary, sweat, and salivary gland development.

A novel GUSB mutation in Brazilian terriers with severe skeletal abnormalities defines the disease as mucopolysaccharidosis VII.

Hundreds of different human skeletal disorders have been characterized at molecular level and a growing number of resembling dysplasias with orthologous genetic defects are being reported in dogs. This study describes a novel genetic defect in the Brazilian Terrier breed causing a congenital skeletal dysplasia. Affected puppies presented severe skeletal deformities observable within the first month of life. Clinical characterization using radiographic and histological methods identified delayed ossification and spondyloepiphyseal dysplasia. Pedigree analysis suggested an autosomal recessive disorder, and we performed a genome-wide association study to map the disease locus using Illumina's 22K SNP chip arrays in seven cases and eleven controls. A single association was observed near the centromeric end of chromosome 6 with a genome-wide significance after permutation (p(genome)= 0.033). The affected dogs shared a 13-Mb homozygous region including over 200 genes. A targeted next-generation sequencing of the entire locus revealed a fully segregating missense mutation (c.866C>T) causing a pathogenic p.P289L change in a conserved functional domain of ß-glucuronidase (GUSB). The mutation was confirmed in a population of 202 Brazilian terriers (p = 7,71×10(-29)). GUSB defects cause mucopolysaccharidosis VII (MPS VII) in several species and define the skeletal syndrome in Brazilian Terriers. Our results provide new information about the correlation of the GUSB genotype to phenotype and establish a novel canine model for MPS VII. Currently, MPS VII lacks an efficient treatment and this model could be utilized for the development and validation of therapeutic methods for better treatment of MPS VII patients. Finally, since almost one third of the Brazilian terrier population carries the mutation, breeders will benefit from a genetic test to eradicate the detrimental disease from the breed.

The expression of Visinin-like 1 during mouse embryonic development.

Visinin like 1 (Vsnl1) encodes a calcium binding protein which is well conserved between species. It was originally found in the brain and its biological functions in central nervous system have been addressed in several studies. Low expression levels have also been found in some peripheral organs, but very little information is available regarding its physiological roles in non-neuronal tissues. Except for the kidney, the expression pattern of Vsnl1 mRNA and protein has not yet been addressed during embryogenesis. By in situ hybridization and immunolabeling we have extensively analyzed the expression pattern of Vsnl1 during murine development. Vsnl1 specifies the cardiac primordia and its expression becomes restricted to the atrial myocardium after heart looping. However, in the adult heart, Vsnl1 is expressed by all four cardiac chambers. It also serves as a specific marker for the cardiomyocyte-derived structures in the systemic and pulmonary circulation. Vsnl1 is dynamically expressed also by many other organs during development e.g. taste buds, cochlea, thyroid, tooth, salivary and adrenal gland. The stage specific expression pattern of Vsnl1 makes it a potentially useful marker particularly in studies of cardiac and vascular morphogenesis.

Missing-in-metastasis MIM/MTSS1 promotes actin assembly at intercellular junctions and is required for integrity of kidney epithelia.

MIM/MTSS1 is a tissue-specific regulator of plasma membrane dynamics, whose altered expression levels have been linked to cancer metastasis. MIM deforms phosphoinositide-rich membranes through its I-BAR domain and interacts with actin monomers through its WH2 domain. Recent work proposed that MIM also potentiates Sonic hedgehog (Shh)-induced gene expression. Here, we generated MIM mutant mice and found that full-length MIM protein is dispensable for embryonic development. However, MIM-deficient mice displayed a severe urinary concentration defect caused by compromised integrity of kidney epithelia intercellular junctions, which led to bone abnormalities and end-stage renal failure. In cultured kidney epithelial (MDCK) cells, MIM displayed dynamic localization to adherens junctions, where it promoted Arp2/3-mediated actin filament assembly. This activity was dependent on the ability of MIM to interact with both membranes and actin monomers. Furthermore, results from the mouse model and cell culture experiments suggest that full-length MIM is not crucial for Shh signaling, at least during embryogenesis. Collectively, these data demonstrate that MIM modulates interplay between the actin cytoskeleton and plasma membrane to promote the maintenance of intercellular contacts in kidney epithelia.

ß-catenin causes renal dysplasia via upregulation of Tgfß2 and Dkk1.

Renal dysplasia, defined by defective ureteric branching morphogenesis and nephrogenesis, is the major cause of renal failure in infants and children. Here, we define a pathogenic role for a ß-catenin-activated genetic pathway in murine renal dysplasia. Stabilization of ß-catenin in the ureteric cell lineage before the onset of kidney development increased ß-catenin levels and caused renal aplasia or severe hypodysplasia. Analysis of gene expression in the dysplastic tissue identified downregulation of genes required for ureteric branching and upregulation of Tgfß2 and Dkk1. Treatment of wild-type kidney explants with TGFß2 or DKK1 generated morphogenetic phenotypes strikingly similar to those observed in mutant kidney tissue. Stabilization of ß-catenin after the onset of kidney development also caused dysplasia and upregulation of Tgfß2 and Dkk1 in the epithelium. Together, these results demonstrate that elevation of ß-catenin levels during kidney development causes dysplasia.

The GDNF target Vsnl1 marks the ureteric tip.

Glial cell line-derived neurotrophic factor (GDNF) is indispensable for ureteric budding and branching. If applied exogenously, GDNF promotes ectopic ureteric buds from the Wolffian duct. Although several downstream effectors of GDNF are known, the identification of early response genes is incomplete. Here, microarray screening detected several GDNF-regulated genes in the Wolffian duct, including Visinin like 1 (Vsnl1), which encodes a neuronal calcium-sensor protein. We observed renal Vsnl1 expression exclusively in the ureteric epithelium, but not in Gdnf-null kidneys. In the tissue culture of Gdnf-deficient kidney primordium, exogenous GDNF and alternative bud inducers (FGF7 and follistatin) restored Vsnl1 expression. Hence, Vsnl1 characterizes the tip of the ureteric bud epithelium regardless of the inducer. In the tips, Vsnl1 showed a mosaic expression pattern that was mutually exclusive with ß-catenin transcriptional activation. Vsnl1 was downregulated in both ß-catenin-stabilized and ß-catenin-deficient kidneys. Moreover, in a mouse collecting duct cell line, Vsnl1 compromised ß-catenin stability, suggesting a counteracting relationship between Vsnl1 and ß-catenin. In summary, Vsnl1 marks ureteric bud tips in embryonic kidneys, and its mosaic pattern demonstrates a heterogeneity of cell types that may be critical for normal ureteric branching.

Conservation, expression, and knockdown of zebrafish plxnb2a and plxnb2b.

In mice lacking Plexin B2, a receptor of the axon guidance molecules Semaphorin 4C and Semaphorin 4D, the closure of the neural tube and structural organization of the cerebellum are severely impaired. We cloned two Plexin B2 orthologs, plxnb2a and plxnb2b, in zebrafish, which is a widely used model for the development of the vertebrate central nervous system (CNS). The predicted proteins, Plexin B2a and Plexin B2b, contain all the conserved and functional domains of the plexin B-subfamily. During embryonic development, plxnb2a is expressed, e.g., in pharyngeal arches while plxnb2b expression is more confined to neuronal structures like the cerebellum. However, both plxnb2a and plxnb2b are expressed at the midbrain-hindbrain boundary, in the otic vesicles, facial ganglia, and pectoral fins. Knockdown of both plxnb2a and plxnb2b simultaneously (>95% and 45%, respectively) resulted in normal CNS structure, axon guidance and swimming performance of the morphants.

Inactivation of Palb2 gene leads to mesoderm differentiation defect and early embryonic lethality in mice.

Mutations of the PALB2 tumor suppressor gene in humans are associated with hereditary predisposition to breast and also some other cancers. In the present study, we have characterized mice deficient in Palb2. The data show that the Palb2((+/-)) mice are normal and fertile, and lack macroscopic tumors when followed up till the age of 8 months. Homozygous (HO) Palb2((-/-)) mice present with embryonic lethality and die at E9.5 at the latest. The mutant embryos are smaller in size, developmentally retarded and display defective mesoderm differentiation after gastrulation. In Palb2((-/-)) embryos, the expression of cyclin-dependent kinase inhibitor p21 is increased, and Palb2((-/-)) blastocysts show a growth defect in vitro. Hence, the phenotype of the Palb2((-/-)) mice in many regards resembles those previously reported for Brca1 and Brca2 knockout mice. The similarity in the phenotypes between Palb2, Brca1 and Brca2 knockout mice further supports the functional relationship shown in vitro for these three proteins. Accordingly, our data in vivo suggest that a key function for PALB2 is to interact with and to build up appropriate communication between BRCA1 and BRCA2, thereby licensing the successful performance of the physiological tasks mediated by these two proteins, particularly in homologous recombination and in proper DNA damage response signaling.

Hydroxysteroid (17{beta}) dehydrogenase 12 is essential for mouse organogenesis and embryonic survival.

Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) have a significant role in steroid metabolism by catalyzing the conversion between 17-keto and 17beta-hydroxysteroids. However, several studies in vitro have shown that some of these enzymes may also be involved in other metabolic pathways. Among these enzymes, HSD17B12 has been shown to be involved in both the biosynthesis of estradiol and the elongation of the essential very long fatty acids in vitro and in vivo. To investigate the function of mammalian HSD17B12 in vivo, we generated mice with a null mutation of the Hsd17b12 gene (HSD17B12KO mice) by using a gene-trap vector, resulting in the expression of the lacZ gene of the trapped allele. The beta-galactosidase staining of the heterozygous HSD17B12KO mice revealed that Hsd17b12 is expressed widely in the embryonic day (E) 7.5-E9.5 embryos, with the highest expression in the neural tissue. The HSD17B12KO mice die at E9.5 at latest and present severe developmental defects. Analysis of the knockout embryos revealed that the embryos initiate gastrulation, but organogenesis is severely disrupted. As a result, the E8.5-E9.5 embryos were void of all normal morphological structures. In addition, the inner cell mass of knockout blastocysts showed decreased proliferation capacity in vitro, and the amount of arachidonic acid was significantly decreased in heterozygous HSD17B12 ES cells. This, together with the expression pattern, suggests that in mouse, the HSD17B12 is involved in the synthesis of arachidonic acid and is essential for normal neuronal development during embryogenesis.

Hydroxysteroid (17beta) dehydrogenase 7 activity is essential for fetal de novo cholesterol synthesis and for neuroectodermal survival and cardiovascular differentiation in early mouse embryos.

Hydroxysteroid (17beta) dehydrogenase 7 (HSD17B7) has been shown to catalyze the conversion of both estrone to estradiol (17-ketosteroid reductase activity) and zymosterone to zymosterol (3-ketosteroid reductase activity involved in cholesterol biosynthesis) in vitro. To define the metabolic role of the enzyme in vivo, we generated knockout mice deficient in the enzyme activity (HSD17B7KO). The data showed that the lack of HSD17B7 results in a blockage in the de novo cholesterol biosynthesis in mouse embryos in vivo, and HSD17BKO embryos die at embryonic day (E) 10.5. Analysis of neural structures revealed a defect in the development of hemispheres of the front brain with an increased apoptosis in the neuronal tissues. Morphological defects in the cardiovascular system were also observed from E9.5 onward. Mesodermal, endodermal, and hematopoietic cells were all detected by the histological analysis of the visceral yolk sac, whereas no organized vessels were observed in the knockout yolk sac. Immunohistological staining for platelet endothelial cell adhesion molecule-1 indicated that the complexity of the vasculature also was reduced in the HSD17B7KO embryos, particularly in the head capillary plexus and branchial arches. At E8.5-9.5, the heart development and the looping of the heart appeared to be normal in the HSD17B7KO embryos. However, at E10.5 the heart was dilated, and the thickness of the cardiac muscle and pericardium in the HSD17B7KO embryos was markedly reduced, and immunohistochemical staining for GATA-4 revealed that HSD17B7KO embryos had a reduced number of myocardial cells. The septum of the atrium was also defected in the knockout mice.

Integrin-linked kinase is an adaptor with essential functions during mouse development.

The development of multicellular organisms requires integrin-mediated interactions between cells and their extracellular environment. Integrin binding to extracellular matrix catalyses assembly of multiprotein complexes, which transduce mechanical and chemical signals that regulate many aspects of cell physiology. Integrin-linked kinase (Ilk) is a multifunctional protein that binds beta-integrin cytoplasmic domains and regulates actin dynamics by recruiting actin binding regulatory proteins such as alpha- and beta-parvin. Ilk has also been shown to possess serine/threonine kinase activity and to phosphorylate signalling proteins such as Akt1 and glycogen synthase kinase 3beta (Gsk3beta) in mammalian cells; however, these functions have been shown by genetic studies not to occur in flies and worms. Here we show that mice carrying point mutations in the proposed autophosphorylation site of the putative kinase domain and in the pleckstrin homology domain are normal. In contrast, mice with point mutations in the conserved lysine residue of the potential ATP-binding site of the kinase domain, which mediates Ilk binding to alpha-parvin, die owing to renal agenesis. Similar renal defects occur in alpha-parvin-null mice. Thus, we provide genetic evidence that the kinase activity of Ilk is dispensable for mammalian development; however, an interaction between Ilk and alpha-parvin is critical for kidney development.

Ancestral T-box mutation is present in many, but not all, short-tailed dog breeds.

Dogs differ greatly in their morphological characteristics including various tail phenotypes. Congenitally short-tailed dogs are present in many breeds; however, the causative mutation located in the T-box transcription factor T gene (C189G) had only been described in the bobtailed Pembroke Welsh Corgis. We investigated here the presence of the T gene mutation in 23 other breeds (360 dogs, including 156 natural short tailed) in which natural bobtailed dogs exist. In the 17 breeds in which the C189G mutation was observed, there was a perfect correlation between this mutation and the short-tail phenotype. However, 6 breeds did not carry the known substitution or any other mutations in the T gene coding regions. No dogs were found to be homozygous for the C189G mutation, suggesting that the homozygous condition is lethal. In order to study the effect of the T gene mutation on litter size, we compared the number of puppies born from short-tailed parents to that born from long-tailed parents. In the Swedish Vallhund breed, we observed a 29% decrease in the litter size when both parents were short tailed. Given that the T gene mutation is not present in all breeds of short-tailed dog, there must be yet other genetic factors affecting tail phenotypes to be discovered.

A mutation in hairless dogs implicates FOXI3 in ectodermal development.

Mexican and Peruvian hairless dogs and Chinese crested dogs are characterized by missing hair and teeth, a phenotype termed canine ectodermal dysplasia (CED). CED is inherited as a monogenic autosomal semidominant trait. With genomewide association analysis we mapped the CED mutation to a 102-kilo-base pair interval on chromosome 17. The associated interval contains a previously uncharacterized member of the forkhead box transcription factor family (FOXI3), which is specifically expressed in developing hair and teeth. Mutation analysis revealed a frameshift mutation within the FOXI3 coding sequence in hairless dogs. Thus, we have identified FOXI3 as a regulator of ectodermal development.

Impaired meningeal development in association with apical expansion of calvarial bone osteogenesis in the Foxc1 mutant.

Loss of function of the mouse forkhead/winged helix transcription factor Foxc1 induces congenital hydrocephalus and impaired skull bone development due to failure of apical expansion of the bone. In this study we investigated meningeal development in the congenital hydrocephalus (ch) mouse with spontaneous loss of function mutant of Foxc1, around the period of initiation of skull bone apical expansion. In situ hybridization of Runx2 revealed active apical expansion of the frontal bone begins between embryonic day 13.5 and embryonic day 14.5 in the wild type, whereas expansion was inhibited in the mutant. Ultrastructural analysis revealed that three layers of the meninges begin to develop at E13.5 in the basolateral site of the head and subsequently progress to the apex in wild type. In ch homozygotes, although three layers were recognized at first at the basolateral site, cell morphology and structure of the layers became abnormal except for the pia mater, and arachnoidal and dural cells never differentiated in the apex. We identified meningeal markers for each layer and found that their expression was down-regulated in the mutant arachnoid and dura maters. These results suggest that there is a close association between meningeal development and the apical growth of the skull bones.

Canonical WNT/beta-catenin signaling is required for ureteric branching.

WNT/beta-catenin signaling has an established role in nephron formation during kidney development. Yet, the role of beta-catenin during ureteric morphogenesis in vivo is undefined. We generated a murine genetic model of beta-catenin deficiency targeted to the ureteric bud cell lineage. Newborn mutant mice demonstrated bilateral renal aplasia or renal dysplasia. Analysis of the embryologic events leading to this phenotype revealed that abnormal ureteric branching at E12.5 precedes histologic abnormalities at E13.5. Microarray analysis of E12.5 kidney tissue identified decreased Emx2 and Lim1 expression among a small subset of renal patterning genes disrupted at the stage of abnormal branching. These alterations are followed by decreased expression of genes downstream of Emx2, including Lim1, Pax2, and the ureteric tip markers, c-ret and Wnt 11. Together, these data demonstrate that beta-catenin performs essential functions during renal branching morphogenesis via control of a hierarchy of genes that control ureteric branching.

A proGDNF-related peptide BEP increases synaptic excitation in rat hippocampus.

The glial cell-derived neurotrophic factor (GDNF) precursor contains several putative sites for prohormone convertase-mediated excision of short peptides. Here, we show that one of the predicted peptides, named BEP (brain excitatory peptide), induces a substantial increase in the synaptic excitability in rat CA1 pyramidal neurons. The excitation is sensitive to N-ethylmaleimide, suggesting involvement of a G-protein-coupled receptor.

Glycogen synthase kinase-3 inactivation and stabilization of beta-catenin induce nephron differentiation in isolated mouse and rat kidney mesenchymes.

Wnt proteins are required for induction of nephrons in mouse metanephric kidneys, but the downstream pathways that mediate tubule induction and epithelial differentiation have remained obscure. The intracellular mechanisms by which Wnt signaling mediates nephron induction in embryonic kidney mesenchymes were studied. First is shown that transient exposure of isolated kidney mesenchymes to structurally different glycogen synthase kinase-3 (GSK3) inhibitors lithium or 6-bromoindirubin-3'-oxime results in abundant epithelial differentiation and full segregation of nephrons. Shown further by mice with genetically disrupted ureteric bud or Wolffian duct development is that this nephrogenic competence arises independent of the influence of Wolffian duct-derived epithelia. Analysis of the intracellular signaling cascades downstream of GSK3 inhibition revealed stabilization of beta-catenin and upregulation of Lef1 and Tcf1, both events that are associated with the active canonical Wnt signaling. Last, genetic evidence that metanephric mesenchyme-specific stabilization of beta-catenin is sufficient to induce nephron differentiation in isolated kidney mesenchymes, similar to that induced by GSK3 inhibitors, is provided. These data show that activation of canonical Wnt pathway is sufficient to induce nephrogenesis and suggest that this pathway mediates the nephron induction in murine kidney mesenchymes.