Lymphatic Defects in Zebrafish sox18 Mutants Are Exacerbated by Perturbed VEGFC Signaling, While Masked by Elevated sox7 Expression.

Mutations in the transcription factor-coding gene SOX18, the growth factor-coding gene VEGFC and its receptor-coding gene VEGFR3/FLT4 cause primary lymphedema in humans. In mammals, SOX18, together with COUP-TFII/NR2F2, activates the expression of Prox1, a master regulator in lymphatic identity and development. Knockdown studies have also suggested an involvement of Sox18, Coup-tfII/Nr2f2, and Prox1 in zebrafish lymphatic development. Mutants in the corresponding genes initially failed to recapitulate the lymphatic defects observed in morphants. In this paper, we describe a novel zebrafish sox18 mutant allele, sa12315, which behaves as a null. The formation of the lymphatic thoracic duct is affected in sox18 homozygous mutants, but defects are milder in both zygotic and maternal-zygotic sox18 mutants than in sox18 morphants. Remarkably, in sox18 mutants, the expression of the closely related sox7 gene is elevated where lymphatic precursors arise. Sox7 could thus mask the absence of a functional Sox18 protein and account for the mild lymphatic phenotype in sox18 mutants, as shown in mice. Partial knockdown of vegfc exacerbates lymphatic defects in sox18 mutants, making them visible in heterozygotes. Our data thus reinforce the genetic interaction between Sox18 and Vegfc in lymphatic development, previously suggested by knockdown studies, and highlight the ability of Sox7 to compensate for Sox18 lymphatic dysfunction.

Targeted Next-Generation Sequencing Indicates a Frequent Oligogenic Involvement in Primary Ovarian Insufficiency Onset.

Primary ovarian insufficiency (POI) is one of the major causes of female infertility associated with the premature loss of ovarian function in about 3.7% of women before the age of 40. This disorder is highly heterogeneous and can manifest with a wide range of clinical phenotypes, ranging from ovarian dysgenesis and primary amenorrhea to post-pubertal secondary amenorrhea, with elevated serum gonadotropins and hypoestrogenism. The ovarian defect still remains idiopathic in some cases; however, a strong genetic component has been demonstrated by the next-generation sequencing (NGS) approach of familiar and sporadic POI cases. As recent evidence suggested an oligogenic architecture for POI, we developed a target NGS panel with 295 genes including known candidates and novel genetic determinants potentially involved in POI pathogenesis. Sixty-four patients with early onset POI (range: 10-25 years) of our cohort have been screened with 90% of target coverage at 50×. Here, we report 48 analyzed patients with at least one genetic variant (75%) in the selected candidate genes. In particular, we found the following: 11/64 patients (17%) with two variants, 9/64 (14%) with three variants, 9/64 (14%) with four variants, 3/64 (5%) with five variants, and 2/64 (3%) with six variants. The most severe phenotypes were associated with either the major number of variations or a worse prediction in pathogenicity of variants. Bioinformatic gene ontology analysis identified the following major pathways likely affected by gene variants: 1) cell cycle, meiosis, and DNA repair; 2) extracellular matrix remodeling; 3) reproduction; 4) cell metabolism; 5) cell proliferation; 6) calcium homeostasis; 7) NOTCH signaling; 8) signal transduction; 9) WNT signaling; 10) cell death; and 11) ubiquitin modifications. Consistently, the identified pathways have been described in other studies dissecting the mechanisms of folliculogenesis in animal models of altered fertility. In conclusion, our results contribute to define POI as an oligogenic disease and suggest novel candidates to be investigated in patients with POI.

Fundamental role of BMP15 in human ovarian folliculogenesis revealed by null and missense mutations associated with primary ovarian insufficiency.

Bone morphogenetic protein 15 (BMP15) encodes an oocyte factor with a relevant role for folliculogenesis as homodimer or cumulin heterodimer (BMP15-GDF9). Heterozygous BMP15 variants in the precursor or mature peptide had been associated with primary ovarian insufficiency (POI), but the underlying mechanism remains elusive and a double dose of BMP15 was suggested to be required for adequate ovarian reserve. We uncovered two homozygous BMP15 null variants found in two girls with POI and primary amenorrhea. Both heterozygous mothers reported physiological menopause. We then performed western blot, immunofluorescence, and reporter assays to investigate how previously reported missense variants, p.Y235C and p.R329C, located in the precursor or mature domains of BMP15, may affect protein function. The p.R329C variant demonstrates an impaired colocalization with growth/differentiation factor 9 (GDF9) at confocal images and diminished activation of the SMAD pathways at western blot and reporter assays in COV434 follicular cell line. In conclusion, BMP15 null mutations cause POI only in the homozygous state, thus discarding the possibility that isolated BMP15 haploinsufficiency can cause evident ovarian defects. Alternatively, heterozygous BMP15 missense variants may affect ovarian function by interfering with cumulin activity. Our data definitely support the fundamental role of BMP15 in human ovarian folliculogenesis.

A light-gated potassium channel for sustained neuronal inhibition.

Currently available inhibitory optogenetic tools provide short and transient silencing of neurons, but they cannot provide long-lasting inhibition because of the requirement for high light intensities. Here we present an optimized blue-light-sensitive synthetic potassium channel, BLINK2, which showed good expression in neurons in three species. The channel is activated by illumination with low doses of blue light, and in our experiments it remained active over (tens of) minutes in the dark after the illumination was stopped. This activation caused long periods of inhibition of neuronal firing in ex vivo recordings of mouse neurons and impaired motor neuron response in zebrafish in vivo. As a proof-of-concept application, we demonstrated that in a freely moving rat model of neuropathic pain, the activation of a small number of BLINK2 channels caused a long-lasting (>30 min) reduction in pain sensation.

Characterization and expression analysis of mcoln1.1 and mcoln1.2, the putative zebrafish co-orthologs of the gene responsible for human mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder caused by mutations in the MCOLN1 gene coding for mucolipin-1 (TRPML1). TRPML1 belongs to a transient receptor potential channels (TRP) subfamily, which in mammals includes two other members: mucolipin-2 (TRPML2) and mucolipin-3 (TRPML3). Bioinformatic analysis of the Danio rerio (zebrafish) genome and trascriptome revealed the presence of five different genes related to human mucolipins: mcoln1.1, mcoln1.2, mcoln2, mcoln3.1 and mcoln3.2. We focused our efforts on the characterization of the two putative zebrafish MCOLN1 co-orthologs. Transient-expression experiments in human HeLa cells demonstrated that fish Mcoln1.1 and Mcoln1.2, similarly to TRPML1, localize to late endosomal/lysosomal compartments. Real-Time PCR (RT-PCR) experiments showed that both genes are maternally expressed and transcribed at different levels during embryogenesis. RT-PCR analysis in different zebrafish tissues displayed ubiquitary expression for mcoln1.1 and a more tissue-specific pattern for mcoln1.2. Spatial and temporal expression studies using whole-mount in situ hybridization confirmed that both genes are maternally expressed and ubiquitously transcribed during gastrulation and early somitogenesis. Notably, in the next developmental stages they are more expressed in neural regions and in retina layers, tissues affected in MLIV. Interestingly, mcoln1.1 is detected, from 10 somite-stage until to 36 hpf, in the yolk syncytial layer (YSL) and in the intermediate cell mass (ICM), the earliest site of hematopoiesis. Overall, the redundancy of mucolipins together with their expression profile support the biological relevance of this class of proteins in zebrafish. The data herein presented indicate that Danio rerio could be a suitable vertebrate model for the study of some aspects of MLIV pathogenesis.

Sox18 genetically interacts with VegfC to regulate lymphangiogenesis in zebrafish.

OBJECTIVE: Lymphangiogenesis is regulated by transcription factors and by growth factor pathways, but their interplay has not been extensively studied so far. We addressed this issue in zebrafish. APPROACH AND RESULTS: Mutations in the transcription factor-coding gene SOX18 and in VEGFR3 cause lymphedema, and the VEGFR3/Flt4 ligand VEGFC plays an evolutionarily conserved role in lymphangiogenesis. Here, we report a strong genetic interaction between Sox18 and VegfC in the early phases of lymphatic development in zebrafish. Knockdown of sox18 selectively impaired lymphatic sprouting from the cardinal vein and resulted in defective lymphatic thoracic duct formation. Sox18 and the related protein Sox7 play redundant roles in arteriovenous differentiation. We used a novel transgenic line that enables inducible expression of a dominant-negative mutant form of mouse Sox18 protein. Our data led us to conclude that Sox18 is crucially involved in lymphangiogenesis after arteriovenous differentiation. Combined partial knockdown of sox18 and vegfc, using subcritical doses of specific morpholinos, revealed a synergistic interaction in both venous and lymphatic sprouting from the cardinal vein and greatly impaired thoracic duct formation. CONCLUSIONS: This interaction suggests a previously unappreciated crosstalk between the growth factor and transcription factor pathways that regulate lymphangiogenesis in development and disease.

Sox factors transcriptionally regulate ROBO4 gene expression in developing vasculature in zebrafish.

Despite their importance as members of the Roundabout (Robo) family in the control of axonal and vascular patterning, the transcriptional regulation of these genes is poorly understood. In this study, we show that members of the Sry-related high mobility box (Sox) transcription factor family as being transcriptional regulators of roundabout4 (robo4), a Robo gene family member that participates in sprouting angiogenesis in vivo, in zebrafish. Double whole mount in situ hybridization analysis in zebrafish embryos revealed co-localization of the vascular relevant Sox factors sox7 or sox18 mRNA with robo4 transcripts in developing intersomitic vessels. A 3-kb human ROBO4 promoter element was able to drive reporter expression in zebrafish to recapitulate the endogenous temporal intersomitic vessel expression pattern of robo4. EMSA analysis confirmed binding of Sox18 to a canonical Sox binding site (from -1170 bp to -1176 bp) in the ROBO4 promoter (3 kb), and mutation analysis indicated that this site was partially responsible for ROBO4 promoter activity in ECs. A combination of gain- and loss-of-function analysis identified Sox7 and Sox18 co-regulation of robo4 but not fli1a transcripts in zebrafish. Finally, Sox-mediated robo4 transcriptional regulation is conserved across evolution. These studies imply Sox-mediated transcriptional regulation of Robo4 in the developing embryonic vasculature.

The HMGB protein gene family in zebrafish: Evolution and embryonic expression patterns.

The High-Mobility Group Box (HMGB) proteins are highly abundant proteins with both nuclear and extracellular roles in key biological processes. In mammals, three family members are present: HMGB1, HMGB2 and HMGB3. We characterized the HMGB family in zebrafish and report a detailed phylogenetic analysis of HMGB proteins. The B1, B2, and B3 subfamilies are present in cartilaginous fish, bony fish, and tetrapods, while jawless fish sequences emerge as basal to the gene family expansion. Two co-orthologs of each mammalian HMGB gene are present in zebrafish. All six zebrafish hmgb genes are maternally expressed, but huge differences in expression levels exist during embryonic development. The hmgb2a/hmgb2b genes are the most highly expressed, while hmgb3b is expressed at the lowest level. Remarkably, hmgb3 genes are not present in fugu, medaka, Tetraodon and stickleback. Our analysis highlights substantial overlaps, but also subtle differences and specificities in the expression patterns of the zebrafish hmgb genes.

Sox18 and Sox7 play redundant roles in vascular development.

Mutations in SOX18 cause the human hypotrichosis-lymphedema-telangiectasia (HLT) syndrome. Their murine counterparts are the spontaneous ragged mutants, showing combined defects in hair follicle, blood vessel, and lymphatic vessel development. Mice null for Sox18 display only mild coat defects, suggesting a dominant-negative effect of Sox18/ragged mutations and functional redundancy between Sox18 and other Sox-F proteins. We addressed this point in zebrafish. The zebrafish homologs of Sox18 and of Sox7 are expressed in angioblasts and in the endothelial component of nascent blood vessels in embryos. Knockdown of either gene, using moderate doses of specific morpholinos, had minimal effects on vessels. In contrast, simultaneous knockdown of both genes resulted in multiple fusions between the major axial vessels. With combined use of transgenic lines and molecular markers, we could show that endothelial cells are specified, but fail to acquire a correct arteriovenous identity. Venous endothelial cell differentiation was more severely affected than arterial. Thus, sox7 and sox18 play redundant but collectively essential roles in the establishment of proper arteriovenous identity in zebrafish. Our data suggest that a defect in arteriovenous identity could be responsible for the formation of telangiectases in patients with HLT.