LGR4 deficiency results in delayed puberty through impaired Wnt/ß-catenin signaling.

The initiation of puberty is driven by an upsurge in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. In turn, GnRH secretion upsurge depends on the development of a complex GnRH neuroendocrine network during embryonic life. Although delayed puberty (DP) affects up to 2% of the population, is highly heritable, and is associated with adverse health outcomes, the genes underlying DP remain largely unknown. We aimed to discover regulators by whole-exome sequencing of 160 individuals of 67 multigenerational families in our large, accurately phenotyped DP cohort. LGR4 was the only gene remaining after analysis that was significantly enriched for potentially pathogenic, rare variants in 6 probands. Expression analysis identified specific Lgr4 expression at the site of GnRH neuron development. LGR4 mutant proteins showed impaired Wnt/ß-catenin signaling, owing to defective protein expression, trafficking, and degradation. Mice deficient in Lgr4 had significantly delayed onset of puberty and fewer GnRH neurons compared with WT, whereas lgr4 knockdown in zebrafish embryos prevented formation and migration of GnRH neurons. Further, genetic lineage tracing showed strong Lgr4-mediated Wnt/ß-catenin signaling pathway activation during GnRH neuron development. In conclusion, our results show that LGR4 deficiency impairs Wnt/ß-catenin signaling with observed defects in GnRH neuron development, resulting in a DP phenotype.

Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage.

Mouse fetal intestinal progenitors lining the epithelium prior to villogenesis grow as spheroids when cultured ex vivo and express the transmembrane glycoprotein Trop2 as a marker. Here, we report the characterization of Trop2-expressing cells from fetal pre-glandular stomach, growing as immortal undifferentiated spheroids, and their relationship with gastric development and regeneration. Trop2(+) cells generating gastric spheroids differed from adult glandular Lgr5(+) stem cells, but appeared highly related to fetal intestinal spheroids. Although they shared a common spheroid signature, intestinal and gastric fetal spheroid-generating cells expressed organ-specific transcription factors and were committed to intestinal and glandular gastric differentiation, respectively. Trop2 expression was transient during glandular stomach development, being lost at the onset of gland formation, whereas it persisted in the squamous forestomach. Undetectable under homeostasis, Trop2 was strongly re-expressed in glands after acute Lgr5(+) stem cell ablation or following indomethacin-induced injury. These highly proliferative reactive adult Trop2(+) cells exhibited a transcriptome displaying similarity with that of gastric embryonic Trop2(+) cells, suggesting that epithelium regeneration in adult stomach glands involves the partial re-expression of a fetal genetic program.

Autonomous adenomas caused by somatic mutations of the thyroid-stimulating hormone receptor in children.

In adults, autonomous adenomas of the thyroid causing hyperthyroidism are relatively common and are most often due to somatic mutations that increase the constitutive activity of the thyroid-stimulating hormone receptor (TSHR). By contrast, autonomous adenomas in hyperthyroid children are exceptional and reports of their clinical and molecular characteristics are few. We reviewed papers describing 16 autonomous adenomas due to a somatic mutation activating the TSHR and diagnosed in patients younger than 18 years, to which we added two of our own unpublished observations in a 4- and 8-year-old with the same TSHR mutation (c.CAG>CAC; p.Asp633His). This revealed that (a) autonomous adenomas occur more often in the right lobe (11 of 14 with available information) and the associated hyperthyroidism tends to be more severe, possibly reflecting the richer vascular supply of the right thyroid lobe, and (b) mutations found in benign adenomas in children have been associated with cancer in adults, suggesting that malignancy requires a second 'hit' at a later age.

Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium.

Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid transcriptome is strikingly different from that of adult intestinal stem cells, with minimal overlap of Wnt target gene expression. The receptor LGR4, but not LGR5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the embryonic-day-14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, qualifying as a fetal type of intestinal stem cell.

Leydig cell tumors in children: contrasting clinical, hormonal, anatomical, and molecular characteristics in boys and girls.

OBJECTIVE: To analyze the clinical, hormonal, anatomical, and molecular characteristics of Leydig cell tumors, a very rare cause of progressive hyperandrogenism in children. STUDY DESIGN: Description of a 9-year-old boy with isosexual precocious pseudopuberty, and of a 12-year-old girl with rapidly progressive virilization, both due to a pure Leydig cell tumor. Review of all cases of pediatric Leydig cell tumors published since 1999 (when the first somatic mutations of the luteinizing hormone receptor were described) and reporting hormonal and/or molecular data. RESULTS: Boys (n = 24) are younger than girls (n = 12) at diagnosis (median 6.5 vs 13.0 years, P = .04). Plasma gonadotrophins are more often completely suppressed in boys (6 cases) than in girls (2 cases). Pure Leydig cell tumors are exceedingly rare in girls (2 cases), who most often have Sertoli-Leydig tumors. These tumors affect either testis equally (11 left, 13 right) but occur more often in the left ovary (8 left, 3 right). Activating mutations of the alpha-subunit of the G(s) stimulatory protein have not been found in either boys or girls and activating mutations of the luteinizing hormone receptor have only been found in boys. CONCLUSIONS: Leydig cell tumors in children display clinical, hormonal, anatomical, and molecular sexual dimorphism.

Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo.

Gene inactivation of the orphan G protein-coupled receptor LGR4, a paralogue of the epithelial-stem-cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, LGR4-deficient crypts or progenitors, but not LGR5-deficient progenitors, die rapidly with marked downregulation of stem-cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists. Our results identify LGR4 as a permissive factor in the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy.

Genotypic and gene expression studies in congenital melanocytic nevi: insight into initial steps of melanotumorigenesis.

Large congenital melanocytic nevi (CMNs) are said to have a higher propensity to malignant transformation compared with acquired nevi. Thus, they represent a good model for studying initial steps of melanotumorigenesis. We have performed genotypic (karyotype, fluorescence in situ hybridization, and mutational analyses) and differential expression studies on a large cohort of medium (n=3) and large (n=24) CMN. Chromosomal abnormalities were rare and single, a feature probably reflecting the benignity of these lesions. Mutational screening showed a high frequency of NRAS mutations in our series (19/27 cases, 70%), whereas BRAF mutations were less common (4/27 cases, 15%). Differential did not show significant alterations of cellular processes such as cell proliferation, cell migration/invasion, angiogenesis, apoptosis, and immune/inflammatory responses. However, significant downregulation of genes involved in pigmentation and upregulation of genes playing a role in DNA protection were observed. Lastly, our microarrays displayed upregulation of genes mediating chemoresistance in cancer. As alteration of pigmentation mechanisms can trigger oxidative damage, increased expression of genes involved in maintenance of DNA integrity might reflect the ability of nevocytic cells to self-protect against cellular stress. Furthermore, the observed alterations linked to chemoresistance might partially account for the well-known inefficacy of chemotherapy in malignant melanoma.

A family with a novel TSH receptor activating germline mutation (p.Ala485Val).

Autosomal dominant nonautoimmune hyperthyroidism (ADNAH) is caused by gain of function mutations in the TSH receptor (TSHr) gene and characterized by toxic thyroid hyperplasia with a variable age of onset in the absence of thyroid antibodies and clinical symptoms of autoimmune thyroid disease in at least two generations. We report here a Turkish family with a novel TSHr gene mutation with distinct features all consistent with ADNAH. Thyroid function tests of the proband were as follows: free T3: 13.1 pg/ml (N: 1.8-4.6); free T4: 5.1 ng/dl (N: 0.9-1.7); TSH: 0.01 microIU/ml (N: 0.2-4.2); and TSH receptor antibody: 2 IU/ml (N: 0-10). A heterozygous missense mutation in exon 10 of the TSHr gene (c.1454C>T) resulting in the substitution of valine for alanine at codon 485 (p.Ala485Val) was found in the father and his son and daughter. This mutation had arisen de novo in the father. Functional studies of the novel TSHr germline mutation demonstrated a higher constitutive activation of adenyl cyclase than wild type without any effect on phospholipase C activity. In conclusion, our data indicate that gain of function germline mutations in the TSHr gene should be investigated in families with members suffering from thyrotoxicosis and progressive growth of goiter, but without clinical and biochemical evidence of autoimmune thyroid disease. In addition, patients harboring the same mutation of the TSHr gene may show wide phenotypic variability with respect to the age at onset, and severity of hyperthyroidism and thyroid growth.

A lack of thyroid hormones rather than excess thyrotropin causes abnormal skeletal development in hypothyroidism.

By proposing TSH as a key negative regulator of bone turnover, recent studies in TSH receptor (TSHR) null mice challenged the established view that skeletal responses to disruption of the hypothalamic-pituitary-thyroid axis result from altered thyroid hormone (T(3)) action in bone. Importantly, this hypothesis does not explain the increased risk of osteoporosis in Graves' disease patients, in which circulating TSHR-stimulating antibodies are pathognomonic. To determine the relative importance of T(3) and TSH in bone, we compared the skeletal phenotypes of two mouse models of congenital hypothyroidism in which the normal reciprocal relationship between thyroid hormones and TSH was intact or disrupted. Pax8 null (Pax8(-/-)) mice have a 1900-fold increase in TSH and a normal TSHR, whereas hyt/hyt mice have a 2300-fold elevation of TSH but a nonfunctional TSHR. We reasoned these mice must display opposing skeletal phenotypes if TSH has a major role in bone, whereas they would be similar if thyroid hormone actions predominate. Pax8(-/-) and hyt/hyt mice both displayed delayed ossification, reduced cortical bone, a trabecular bone remodeling defect, and reduced bone mineralization, thus indicating that the skeletal abnormalities of congenital hypothyroidism are independent of TSH. Treatment of primary osteoblasts and osteoclasts with TSH or a TSHR-stimulating antibody failed to induce a cAMP response. Furthermore, TSH did not affect the differentiation or function of osteoblasts or osteoclasts in vitro. These data indicate the hypothalamic-pituitary-thyroid axis regulates skeletal development via the actions of T(3).

Pseudodominant inheritance of goitrous congenital hypothyroidism caused by TPO mutations: molecular and in silico studies.

CONTEXT AND OBJECTIVE: Most cases of goitrous congenital hypothyroidism (CH) from thyroid dyshormonogenesis 1) follow a recessive mode of inheritance and 2) are due to mutations in the thyroid peroxidase gene (TPO). We report the genetic mechanism underlying the apparently dominant inheritance of goitrous CH in a nonconsanguineous family of French Canadian origin. DESIGN, SETTING, AND PARTICIPANTS: Two brothers identified by newborn TSH screening had severe hypothyroidism and a goiter with increased (99m)Tc uptake. The mother was euthyroid, but the father and two paternal uncles had also been diagnosed with goitrous CH. After having excluded PAX8 gene mutations, we hypothesized that the underlying defect could be TPO mutations. RESULTS: Both compound heterozygous siblings had inherited a mutant TPO allele carried by their mother (c.1496delC; p.Pro499Argfs2X), and from their father, one brother had inherited a missense mutation (c.1978C-->G; p.Gln660Glu) and the other an insertion (c.1955insT; p.Phe653Valfs15X). The thyroid gland of one uncle who is a compound heterozygote for TPO mutations (p.Phe653Valfs15X/p.Gln660Glu) was removed because of concurrent multiple endocrine neoplasia type 2A. Immunohistochemistry revealed normal TPO staining, implying that Gln660Glu TPO is expressed properly. Modeling of this mutant in silico suggests that its three-dimensional structure is conserved, whereas the electrostatic binding energy between the Gln660Glu TPO and its heme group becomes repulsive. CONCLUSION: We report a pedigree presenting with pseudodominant goitrous CH due to segregation of three different TPO mutations. Although goitrous CH generally follows a recessive mode of inheritance, the high frequency of TPO mutations carriers may lead to pseudodominant inheritance.

Evolutionary conservation of bursicon in the animal kingdom.

Bursicon bioactivity is essential for tanning of the exoskeleton and for wing spreading behavior that occur in newly emerged adult insects. Previously, we demonstrated that in the fruit fly, Drosophila melanogaster, bursicon exists as a heterodimeric cystine knot protein that activates the leucine-rich repeats containing G protein-coupled receptor 2 (DLGR2). By performing similarity based in silico searches in genomic and complementary DNA databases, we identified bursicon homologous sequences in several protostomian as well as deuterostomian invertebrates. In the genome of the honeybee, Apis mellifera, the coding regions for bursicon cystine knot subunits are organized in a genomic locus of approximately 4 kilobase pairs. Reverse transcription PCR analysis indicates that this region likely codes for two distinct bursicon cystine knot subunits. Our results illustrate the remarkable conservation of bursicon in invertebrate species and provide an avenue for functional analyses of this hormone in a wide range of animal species.

Chromosomal translocations as a mechanism of BRAF activation in two cases of large congenital melanocytic nevi.

Genetic studies of melanocytic tumors have mainly demonstrated activation of oncogenes such as NRAS or BRAF through point mutations. In two cases of large congenital melanocytic nevi, we observed a chromosomal translocation involving the BRAF oncogene on chromosome 7q34, resulting in both cases in removal of the auto-inhibitory N-terminal regulatory domain (hence the Ras-guanosine triphosphate binding domain) of BRAF from its protein kinase domain. This is early evidence of BRAF activation through chromosomal translocation in melanocytic tumors. Because BRAF point mutations are rather rare in congenital melanocytic nevi and melanoma arising in non-sun-exposed area, the molecular mechanism of oncogenic activation as described here could be a recurrent molecular feature in these groups of melanocytic tumors.

Lack of minocycline efficiency in genetic models of Huntington's disease.

According to the recent controversy regarding the effects of minocycline in the R6/2 transgenic model of Huntington's disease (HD), this tetracycline has been re-evaluated in another model, the N171-82Q strain. Ten miligrams per kilogram minocycline was given daily from the age of 2 mo, corresponding to an early symptomatic stage. We did not observe improvement in survival, weight loss, or motor function in treated transgenic mice. In addition, minocycline failed to mitigate the ventricle enlargement as well as the striatal and cortical atrophies induced by the transgene. Using high-performance liquid chromatography, it was observed that minocycline was similarly present in the plasma and the brain of both wild-type and N171-82Q mice following 14 daily injections. Using Western blot, we show that the increased expression of procaspase-1 induced by the transgene in the cortex was significantly reduced by the antibiotic. Combining together these data support that despite minocycline crosses blood-brain barrier in N171-82Q mice and displays an expected effect on procaspase-1 expression, it does not provide protection in this HD model. These in vivo results are in accordance with in vitro data, since minocycline failed to protect against mutated Huntingtin in an inducible PC12-clone expressing exon1 of mutated Huntingtin103Q. Altogether, the present data does not support minocycline as a beneficial drug for HD.

Defective postnatal development of the male reproductive tract in LGR4 knockout mice.

The final outcome of tube elongation and branching is to maximize the epithelial exchange surfaces in tubular organs. The molecular and cellular basis of these processes is actively studied in model organs such as mammary glands, liver and kidney, but they remain almost unexplored in the male reproductive tract. Here, we report that the orphan G protein-coupled receptor LGR4/GPR48 plays a role in the postnatal tissue remodeling needed for elongation and convolution of the efferent ducts and epididymis. In LGR4 knockout male mice, tube elongation fails, resulting in a hypoplastic and poorly convoluted tract. Cell proliferation is dramatically reduced in KO affected tissues, providing an explanation to the observed phenotype. Detailed analysis showed that LGR4 inactivation manifests differently in the affected organs. In efferent ducts, immune cells infiltrate the epithelium and reach the lumen, blocking the transit of sperm and testicular fluid. In addition, the hypoplasia and low convolution result in a reduction of the epithelial area involved in liquid reabsorption. Both phenomena contribute in tissue swelling upstream the blockade due to liquid and sperm accumulation, with secondary damaging effects on the germinal epithelium. In the epididymis, the thin and highly convoluted duct is replaced by a large cystic tube which is surrounded by a thick condensation of mesenchymal cells. The abnormal organization of the cellular compartments in and around the ducts suggests that LGR4 might play a role in epithelial-mesenchymal interactions. Altogether, our data identify LGR4 as an important signaling molecule implicated in the tube morphogenesis of the male reproductive tract.

Understanding ovarian hyperstimulation syndrome.

The ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of ovarian stimulation treatments. Severe forms are characterized by a massive ovarian enlargement with the formation of multiple ovarian cysts associated with extravascular fluid shifts resulting in the development of ascites, pleural and/or pericardial effusion. The pathophysiology of the syndrome has not been completely elucidated yet. The vascular fluid leakage is thought to result from an increased capillary permeability of mesothelial surfaces under the action of one or several vasoactive ovarian factor(s) produced by the multiple corpora lutea. The paper focuses on the recent identification of mutations in the FSH receptor gene that display an increased sensitivity to hCG and are responsible for the development of spontaneous OHSS occurring during pregnancy. These findings have shed light for the first time on the molecular basis of the pathophysiology of the spontaneous form of the syndrome. As spontaneous and iatrogenic OHSS share similar pathophysiological sequences including massive recruitment and growth of ovarian follicles, extensive luteinization provoked by hCG, and oversecretion of vasogenic molecules by the corpora lutea, they have also opened new research perspectives for the understanding of the much more frequent iatrogenic OHSS.

Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity.

The monomeric model of rhodopsin-like G protein-coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo- and heterodimers, via interactions involving primarily their heptahelical domains. The large hormone-binding ectodomains were dispensable for dimerization but modulated protomer interaction. Dimerization was not affected by agonist binding. Observed functional complementation indicates that TSHr dimers may function as a single functional unit. Finally, heterologous binding-competition studies, performed with heterodimers between TSHr and LH/CG-TSHr chimeras, demonstrated the unsuspected existence of strong negative cooperativity of hormone binding. Tracer desorption experiments indicated an allosteric behavior in TSHr and, to a lesser extent, in LH/CGr and FSHr homodimers. This study is the first report of homodimerization associated with negative cooperativity in rhodopsin-like GPCRs. As such, it may warrant revisitation of allosterism in the whole GPCR family.

Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2.

Bursicon is a neurohumoral agent responsible for tanning and hardening of the cuticle and expansion of the wings during the final phase of insect metamorphosis. Although the hormonal activity was described more than 40 years ago, the molecular nature of bursicon has remained elusive. We identify here Drosophila bioactive bursicon as a heterodimer made of two cystine knot polypeptides. This conclusion was reached in part from the unexpected observation that in the genome of the honey bee, the orthologs of the two Drosophila proteins are predicted to be fused in a single open reading frame. The heterodimeric Drosophila protein displays bursicon bioactivity in freshly enclosed neck-ligated flies and is the natural agonist of the orphan G protein-coupled receptor DLGR2.

An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor.

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.

Identification and characterization of an endogenous chemotactic ligand specific for FPRL2.

Chemotaxis of dendritic cells (DCs) and monocytes is a key step in the initiation of an adequate immune response. Formyl peptide receptor (FPR) and FPR-like receptor (FPRL)1, two G protein-coupled receptors belonging to the FPR family, play an essential role in host defense mechanisms against bacterial infection and in the regulation of inflammatory reactions. FPRL2, the third member of this structural family of chemoattractant receptors, is characterized by its specific expression on monocytes and DCs. Here, we present the isolation from a spleen extract and the functional characterization of F2L, a novel chemoattractant peptide acting specifically through FPRL2. F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases through the G(i) class of heterotrimeric G proteins. When tested on monocytes and monocyte-derived DCs, F2L promotes calcium mobilization and chemotaxis. Therefore, F2L appears as a new natural chemoattractant peptide for DCs and monocytes, and the first potent and specific agonist of FPRL2.