Generation of specialized blood vessels via lymphatic transdifferentiation.

The lineage and developmental trajectory of a cell are key determinants of cellular identity. In the vascular system, endothelial cells (ECs) of blood and lymphatic vessels differentiate and specialize to cater to the unique physiological demands of each organ1,2. Although lymphatic vessels were shown to derive from multiple cellular origins, lymphatic ECs (LECs) are not known to generate other cell types3,4. Here we use recurrent imaging and lineage-tracing of ECs in zebrafish anal fins, from early development to adulthood, to uncover a mechanism of specialized blood vessel formation through the transdifferentiation of LECs. Moreover, we demonstrate that deriving anal-fin vessels from lymphatic versus blood ECs results in functional differences in the adult organism, uncovering a link between cell ontogeny and functionality. We further use single-cell RNA-sequencing analysis to characterize the different cellular populations and transition states involved in the transdifferentiation process. Finally, we show that, similar to normal development, the vasculature is rederived from lymphatics during anal-fin regeneration, demonstrating that LECs in adult fish retain both potency and plasticity for generating blood ECs. Overall, our research highlights an innate mechanism of blood vessel formation through LEC transdifferentiation, and provides in vivo evidence for a link between cell ontogeny and functionality in ECs.

VEGFC/FLT4-induced cell-cycle arrest mediates sprouting and differentiation of venous and lymphatic endothelial cells.

The formation of new vessels requires a tight synchronization between proliferation, differentiation, and sprouting. However, how these processes are differentially activated, often by neighboring endothelial cells (ECs), remains unclear. Here, we identify cell cycle progression as a regulator of EC sprouting and differentiation. Using transgenic zebrafish illuminating cell cycle stages, we show that venous and lymphatic precursors sprout from the cardinal vein exclusively in G1 and reveal that cell-cycle arrest is induced in these ECs by overexpression of p53 and the cyclin-dependent kinase (CDK) inhibitors p27 and p21. We further demonstrate that, in vivo, forcing G1 cell-cycle arrest results in enhanced vascular sprouting. Mechanistically, we identify the mitogenic VEGFC/VEGFR3/ERK axis as a direct inducer of cell-cycle arrest in ECs and characterize the cascade of events that render "sprouting-competent" ECs. Overall, our results uncover a mechanism whereby mitogen-controlled cell-cycle arrest boosts sprouting, raising important questions about the use of cell cycle inhibitors in pathological angiogenesis and lymphangiogenesis.

Publisher Correction: Prokineticin receptor 2 affects GnRH3 neuron ontogeny but not fertility in zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Knocking-down of the Prokineticin receptor 2 affects reveals its complex role in the regulation of the hypothalamus-pituitary-gonadal axis in the zebrafish model.

Prokineticin receptors (PROKR1 and PROKR2) are G protein-coupled receptors which control human central and peripheral reproductive processes. Importantly, allelic variants of PROKR2 in humans are associated with altered migration of GnRH neurons, resulting in congenital hypogonadotropic hypogonadism (CHH), a heterogeneous disease characterized by delayed/absent puberty and/or infertility. Although this association is established in humans, murine models failed to fully recapitulate the reproductive and olfactory phenotypes observed in patients harboring PROKR2 mutations. Here, taking advantage of zebrafish model we investigated the role of prokr1b (ortholog of human PROKR2) during early stages of GnRH neuronal migration. Real-Time PCR and whole mount in situ hybridization assays indicate that prokr1b spatial-temporal expression is consistent with gnrh3. Moreover, knockdown and knockout of prokr1b altered the correct development of GnRH3 fibers, a phenotype that is rescued by injection of prokr1b mRNA. These results suggest that prokr1b regulates the development of the GnRH3 system in zebrafish. Analysis of gonads development and mating experiments indicate that prokr1b is not required for fertility in zebrafish, although its loss determine changes also at the testis level. Altogether, our results support the thesis of a divergent evolution in the control of vertebrate reproduction and provide a useful in vivo model for deciphering the mechanisms underlying the effect of PROKR2 allelic variants on CHH.

Characteristics of a nationwide cohort of patients presenting with isolated hypogonadotropic hypogonadism (IHH).

OBJECTIVE: Isolated hypogonadotropic hypogonadism (IHH) is a rare disorder with pubertal delay, normal (normoosmic-IHH, nIHH) or defective sense of smell (Kallmann syndrome, KS). Other reproductive and non-reproductive anomalies might be present although information on their frequency are scanty, particularly according to the age of presentation. DESIGN: Observational cohort study carried out between January 2008 and June 2016 within a national network of academic or general hospitals. METHODS: We performed a detailed phenotyping of 503 IHH patients with: (1) manifestations of hypogonadism with low sex steroid hormone and low/normal gonadotropins; (2) absence of expansive hypothalamic/pituitary lesions or multiple pituitary hormone defects. Cohort was divided on IHH onset (PPO, pre-pubertal onset or AO, adult onset) and olfactory function: PPO-nIHH (n = 275), KS (n = 184), AO-nIHH (n = 36) and AO-doIHH (AO-IHH with defective olfaction, n = 8). RESULTS: 90% of patients were classified as PPO and 10% as AO. Typical midline and olfactory defects, bimanual synkinesis and familiarity for pubertal delay were also found among the AO-IHH. Mean age at diagnosis was significantly earlier and more frequently associated with congenital hypogonadism stigmata in patients with Kallmann's syndrome (KS). Synkinesis, renal and male genital tract anomalies were enriched in KS. Overweight/obesity are significantly associated with AO-IHH rather than PPO-IHH. CONCLUSIONS: Patients with KS are more prone to develop a severe and complex phenotype than nIHH. The presence of typical extra-gonadal defects and familiarity for PPO-IHH among the AO-IHH patients indicates a common predisposition with variable clinical expression. Overall, these findings improve the understanding of IHH and may have a positive impact on the management of patients and their families.

The zebrafish: an emerging animal model for investigating the hypothalamic regulation of reproduction.

Gonadotropin-releasing hormone (GnRH) neurons have a pivotal role in the physiological functions of hypotahlamic-pituitary-gonadal (HPG) axis. The pulsatile releasing of GnRH hormone into the hypophyseal portal circulation at the median eminence represent the first domino in the HPG cascade of events that regulate the development, fertility and aging in all vertebrates. These neurons principally originate in the olfactory placode and migrate during early embryonal stages into the hypothalamus. Alterations in developmental processes or in the releasing of GnRH hormone lead to a rare and complex disorder of the reproductive axis called congenital hypogonadotropic hypogonadism (CHH). Genetic screening of human patients and the use of model systems have led to the identification of several genes involved in the CHH pathogenesis underlying its oligogenic nature. Nevertheless CHH remains, for a large cohort of patients, idiopathic and GnRH neurogenesis processes not fully understood. This is due to intrinsic difficulties that exist in the analysis of earliest embryonic developmental stages and in the methodologies developed to study the CHH-causing genes. In this regard, zebrafish embryos, on account of its external fertilization and development, allow a real-time analysis that could overcome some of the above mentioned limitations. Moreover, the recent availability of several transgenic zebrafish reporter lines makes it an excellent model for the study of the oligogenic mechanisms leading to CHH.

The complex genetic basis of congenital hypogonadotropic hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) is a rare disease characterized by delayed/absent puberty and infertility due to an inadequate secretion or action of gonadotrophin-releasing hormone (GnRH), with an otherwise structurally and functionally normal hypothalamic-pituitary-gonadal (HPG) axis. CHH is genetically heterogeneous but, due to the infertility of affected individuals, most frequently emerges in a sporadic form, though numerous familial cases have also been registered. In around 50-60% of cases, CHH is associated with a variety of non-reproductive abnormalities, most commonly anosmia/hyposmia, which defines Kallmann Syndrome (KS) by its presence. Broadly-speaking, genetic defects that directly impact on hypothalamic secretion, regulation, or action of GnRH result in a pure neuroendocrine phenotype, normosmic CHH (nCHH), whereas genetic defects that impact of embryonic migration of GnRH neurons to the hypothalamus most commonly result in KS, though nCHH can also arise. Hence, the description of several pedigrees, comprising subjects exhibiting KS and others with nCHH. Although more than 24 genes have been described to be involved in CHH, molecular variants of these do not presently explain more than 35-45% of reported cases. Therefore, numerous other unidentified genes (or conceivably, epigenetic mechanisms) remain to be described to fully understand the pathogenesis of CHH, explaining the emergent idea that CHH is a complex genetic disease characterized by variable expressivity and penetrance. This review summarizes the current state of knowledge on the complex genetic basis of congenital hypogonadotropic hypogonadism and aims to be accessible to both researchers and clinicians.

A new variant in signal peptide of the human luteinizing hormone receptor (LHCGR) affects receptor biogenesis causing leydig cell hypoplasia.

The human luteinizing hormone/chorionic gonadotropin receptor (LHCGR) plays a fundamental role in male and female reproduction. In males, loss-of-function mutations in LHCGR have been associated with distinct degrees of impairment in pre- and postnatal testosterone secretion resulting in a variable phenotypic spectrum, classified as Leydig cell hypoplasia (LCH) type 1 (complete LH resistance and disorder of sex differentiation) and type 2 (partial LH resistance with impaired masculinization and fertility). Here, we report the case of an adolescent who came to the pediatric endocrinologist at the age of 12 years old for micropenis and cryptorchidism. Testis biopsy showed profound LCH and absent germinal line elements (Sertoli-only syndrome). The sequence analysis of the LHCGR gene showed the presence of a compound heterozygosity, being one variation, c.1847C>A p.S616Y, already described in association to Hypergonadotropic Hypogonadism, and the other, c.29 C>T p.L10P, a new identified variant in the putative signal peptide (SP) of LHCGR. Functional and structural studies provide first evidence that LHCGR have a functional and cleavable SP required for receptor biogenesis. Moreover, we demonstrate the pathogenic role of the novel p.L10P allelic variant, which has to be considered a loss-of-function mutation significantly contributing, in compound heterozygosity with p.S616Y, to the LCH type 2 observed in our patient.

Expression of a single siRNA against a conserved region of NP gene strongly inhibits in vitro replication of different Influenza A virus strains of avian and swine origin.

Influenza A virus is the principal agent responsible of the respiratory tract's infections in humans. Every year, highly pathogenic and infectious strains with new antigenic assets appear, making ineffective vaccines so far developed. The discovery of RNA interference (RNAi) opened the way to the progress of new promising drugs against Influenza A virus and also to the introduction of disease resistance traits in genetically modified animals. In this paper, we show that Madin-Darby Canine Kidney (MDCK) cell line expressing short hairpin RNAs (shRNAs) cassette, designed on a specific conserved region of the nucleoprotein (NP) viral genome, can strongly inhibit the viral replication of four viral strains sharing the target sequence, reducing the viral mRNA respectively to 2.5×10(-4), 7.5×10(-5), 1.7×10(-3), 1.9×10(-4) compared to the control, as assessed by real-time PCR. Moreover, we demonstrate that during the challenge with a viral strain bearing a single mismatch on the target sequence, although a weaker inhibition is observed, viral mRNA is still lowered down to 1.2×10(-3) folds in the shRNA-expressing clone compared to the control, indicating a broad potential use of this approach. In addition, we developed a highly predictive and fast screening test of siRNA sequences based on dual-luciferase assay, useful for the in vitro prediction of the potential effect of viral inhibition. In conclusion, these findings reveal new siRNA sequences able to inhibit Influenza A virus replication and provide a basis for the development of siRNAs as prophylaxis and therapy for influenza infection both in humans and animals.