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.

Effectiveness of auriculotherapy on stress reduction in health workers: a controlled randomized clinical trial.

OBJECTIVE: to assess the effectiveness of auriculotherapy in reducing occupational stress among Family Health Strategy workers during the COVID-19 pandemic. METHOD: a controlled randomized clinical trial divided into two groups, namely: auriculotherapy for stress group and placebo group. The Shapiro-Wilk test was used to assess data normality. The ANOVA test for repeated measures and the Tukey post-hoc test were applied to the group with normal samples. In turn, the Friedman and Durbin-Conover tests were employed in the group with non-normal distribution. Cohen's d index was calculated for the therapy effect size. A 95% significance level and p<0.05 were considered. RESULTS: the auriculotherapy group presented 16.3% and 23.7% reductions in occupational stress after the third and sixth auriculotherapy sessions, with Cohen's d indices of 1.12 (large effect) and 1.82 (very large effect), respectively. CONCLUSION: auriculotherapy proved to be effective in reducing occupational stress among Family Health Strategy workers during the COVID-19 pandemic. It is suggested that new studies are developed both during and after the pandemic in order to improve health workers' Quality of Life. ReBEC registration: RBR - 38hjyt3.

Effectiveness of auriculotherapy on stress reduction in health workers: a controlled randomized clinical trial / Efetividade da auriculoterapia na redução de estresse em trabalhadores de saúde: ensaio clínico controlado randomizado / Efectividad de la auriculoterapia para la reducción del estrés de trabajadores de la salud: ensayo clínico controlado aleatorizado

Abstract Objective: to assess the effectiveness of auriculotherapy in reducing occupational stress among Family Health Strategy workers during the COVID-19 pandemic. Method: a controlled randomized clinical trial divided into two groups, namely: auriculotherapy for stress group and placebo group. The Shapiro-Wilk test was used to assess data normality. The ANOVA test for repeated measures and the Tukey post-hoc test were applied to the group with normal samples. In turn, the Friedman and Durbin-Conover tests were employed in the group with non-normal distribution. Cohen's d index was calculated for the therapy effect size. A 95% significance level and p<0.05 were considered. Results: the auriculotherapy group presented 16.3% and 23.7% reductions in occupational stress after the third and sixth auriculotherapy sessions, with Cohen's d indices of 1.12 (large effect) and 1.82 (very large effect), respectively. Conclusion: auriculotherapy proved to be effective in reducing occupational stress among Family Health Strategy workers during the COVID-19 pandemic. It is suggested that new studies are developed both during and after the pandemic in order to improve health workers' Quality of Life. ReBEC registration: RBR - 38hjyt3.

Resumo Objetivo: avaliar a efetividade da auriculoterapia na redução do estresse ocupacional em trabalhadores de saúde da Estratégia de Saúde da Família durante a pandemia da COVID-19. Método: ensaio clínico controlado randomizado em dois grupos: grupo auriculoterapia para o estresse e grupo placebo. Foi utilizado o teste de Shapiro-Wilk para avaliar a normalidade dos dados. O teste ANOVA de medidas repetidas e o teste post-hoc Tukey foram aplicados para o grupo com amostras normais. Já o teste de Friedman e de Durbin-Conover foram utilizados no grupo com distribuição não normal. Para o tamanho do efeito da terapia, foi calculado o índice d de Cohen. Considerou-se o nível de significância de 95% e valor p<0,05. Resultados: o grupo auriculoterapia apresentou redução do estresse ocupacional de 16,3 e 23,7% após a terceira e sexta sessões de auriculoterapia, com índices d de Cohen de 1,12 (grande efeito) e 1,82 (efeito muito grande), respectivamente. Conclusão: a auriculoterapia mostrou-se efetiva na redução do estresse ocupacional em trabalhadores de saúde da Estratégia Saúde da Família durante a pandemia da COVID-19. Sugere-se que novos estudos sejam desenvolvidos durante e após a pandemia de maneira a melhorar a qualidade de vida dos trabalhadores de saúde. Registro ReBEC: RBR - 38hjyt3.

Resumen Objetivo: evaluar la efectividad de la auriculoterapia para la reducción del estrés laboral en trabajadores de salud de la Estrategia Salud de la Familia durante la pandemia de COVID-19. Método: ensayo clínico controlado aleatorizado en dos grupos: grupo auriculoterapia para el estrés y grupo placebo. Se utilizó la prueba de Shapiro-Wilk para evaluar la normalidad de los datos. Al grupo con muestras normales se les aplicó la prueba ANOVA de medidas repetidas y la prueba post-hoc de Tukey. Se utilizaron las pruebas de Friedman y Durbin-Conover en el grupo con distribución no normal. Para el tamaño del efecto de la terapia se calculó el índice d de Cohen. Se consideró un nivel de significación del 95% y un valor de p <0,05. Resultados: el grupo auriculoterapia mostró una reducción del estrés laboral de 16,3 y 23,7% después de la tercera y sexta sesión de auriculoterapia, con índices d de Cohen de 1,12 (efecto grande) y 1,82 (efecto muy grande), respectivamente. Conclusión: la auriculoterapia demostró ser eficaz para la reducción del estrés laboral en trabajadores de la salud de la Estrategia Salud de la Familia durante la pandemia de COVID-19. Se sugiere desarrollar nuevos estudios durante y después de la pandemia con el fin de mejorar la calidad de vida de los trabajadores de la salud. Registro ReBEC: RBR - 38hjyt3.

The Lysine Methylase SMYD3 Modulates Mesendodermal Commitment during Development.

SMYD3 (SET and MYND domain containing protein 3) is a methylase over-expressed in cancer cells and involved in oncogenesis. While several studies uncovered key functions for SMYD3 in cancer models, the SMYD3 role in physiological conditions has not been fully elucidated yet. Here, we dissect the role of SMYD3 at early stages of development, employing mouse embryonic stem cells (ESCs) and zebrafish as model systems. We report that SMYD3 depletion promotes the induction of the mesodermal pattern during in vitro differentiation of ESCs and is linked to an upregulation of cardiovascular lineage markers at later stages. In vivo, smyd3 knockdown in zebrafish favors the upregulation of mesendodermal markers during zebrafish gastrulation. Overall, our study reveals that SMYD3 modulates levels of mesendodermal markers, both in development and in embryonic stem cell differentiation.

Glycogen storage in a zebrafish Pompe disease model is reduced by 3-BrPA treatment.

Pompe disease (PD) is an autosomal recessive muscular disorder caused by deficiency of the glycogen hydrolytic enzyme acid α-glucosidase (GAA). The enzyme replacement therapy, currently the only available therapy for PD patients, is efficacious in improving cardiomyopathy in the infantile form, but not equally effective in the late onset cases with involvement of skeletal muscle. Correction of the skeletal muscle phenotype has indeed been challenging, probably due to concomitant dysfunctional autophagy. The increasing attention to the pathogenic mechanisms of PD and the search of new therapeutic strategies prompted us to generate and characterize a novel transient PD model, using zebrafish. Our model presented increased glycogen content, markedly altered motor behavior and increased lysosome content, in addition to altered expression of the autophagy-related transcripts and proteins Beclin1, p62 and Lc3b. Furthermore, the model was used to assess the beneficial effects of 3-bromopyruvic acid (3-BrPA). Treatment with 3-BrPA induced amelioration of the model phenotypes regarding glycogen storage, motility behavior and autophagy-related transcripts and proteins. Our zebrafish PD model recapitulates most of the defects observed in human patients, proving to be a powerful translational model. Moreover, 3-BrPA unveiled to be a promising compound for treatment of conditions with glycogen accumulation.

Síndrome de Burnout em residentes multiprofissionais em saúde / Burnout syndrome in multi-professional healthcare residents / Síndrome de Burnout en residentes multiprofesionales en salud

Objetivo: estimar a prevalência da Síndrome de Burnout e identificar o perfil sociodemográfico, laboral, estilo de vida e saúde de residentes multiprofissionais de saúde de uma Universidade pública da Bahia, Brasil. Metodologia: estudo transversal, descritivo, realizado com 63 residentes. Utilizou-se o Maslach Burnout Inventory e um questionário contendo questões sociodemográficas, laborais, estilo de vida e saúde. Resultados: ao analisar as dimensões da síndrome, 82,5% apresentaram nível alto de exaustão emocional; 55,5% nível moderado de despersonalização e 88,8% nível alto de reduzida realização profissional. Destaca-se a maior ocorrência do Burnout em enfermeiros (50%), profissionais atuantes no âmbito hospitalar (71,4%) e em recém-formados (média 2,1 anos). 60,3% dos residentes consideraram não ter uma alimentação saudável e 29,5% apresentaram excesso de peso. Conclusão: os resultados apontam uma prevalência significativa de síndrome de burnout, sendo encontradas altas alterações nas três dimensões da síndrome em 22,2% dos residentes.

Objective: to estimate the prevalence of Burnout Syndrome, and identify the sociodemographic, occupational, lifestyle, and health profile of multi-professional healthcare residents at a public university in Bahia, Brazil. Methods: a descriptive, crosssectional study was conducted with 63 residents, using the Maslach Burnout Inventory and a questionnaire on sociodemographic, labor, lifestyle, and health issues. Results: on examining the dimensions of the syndrome, 82.5% were found to present high levels of emotional exhaustion; 55.5%, moderate levels of depersonalization; and 88.8%, high levels of diminished professional fulfilment. Burnout occurred most often in nurses (50%), professionals working in hospitals (71.4%) and recent graduates (mean 2.1 years), while 60.3% of the residents regarded their diet at unhealthy, and 29.5% were overweight. Conclusion: the results indicate a significant prevalence of Burnout Syndrome, and marked changes in the three dimensions of the syndrome were found in 22.2% of the residents.

Objetivo: estimar la prevalencia del Síndrome de Burnout e identificar el perfil sociodemográfico, laboral, estilo de vida y salud de residentes multiprofesionales de salud de una universidad pública de Bahía, Brasil. Método: estudio transversal, descriptivo, realizado junto a 63 residentes. Se utilizó el Maslach Burnout Inventory y un cuestionario que contenía cuestiones sociodemográficas, laborales, estilo de vida y salud. Resultados: al analizar las dimensiones del síndrome, el 82,5% presentó un alto nivel de agotamiento emocional; 55,5% nivel moderado de despersonalización y 88,8% nivel alto de reducida realización profesional. Se destaca la mayor ocurrencia de Burnout en enfermeros (50%), profesionales actuantes en el ámbito hospitalario (71,4%) y recién graduados (promedio 2,1 años). El 60,3% de los residentes consideró no tener una alimentación sana y el 29,5% presentó un exceso de peso. Conclusión: los resultados apuntan a una prevalencia significativa de síndrome de Burnout y se encontraron grandes alteraciones en las tres dimensiones del síndrome en el 22,2% de los residentes.

Advantages and Challenges of Cardiovascular and Lymphatic Studies in Zebrafish Research.

Since its introduction, the zebrafish has provided an important reference system to model and study cardiovascular development as well as lymphangiogenesis in vertebrates. A scientific workshop, held at the 2018 European Zebrafish Principal Investigators Meeting in Trento (Italy) and chaired by Massimo Santoro, focused on the most recent methods and studies on cardiac, vascular and lymphatic development. Daniela Panáková and Natascia Tiso described new molecular mechanisms and signaling pathways involved in cardiac differentiation and disease. Arndt Siekmann and Wiebke Herzog discussed novel roles for Wnt and VEGF signaling in brain angiogenesis. In addition, Brant Weinstein's lab presented data concerning the discovery of endothelium-derived macrophage-like perivascular cells in the zebrafish brain, while Monica Beltrame's studies refined the role of Sox transcription factors in vascular and lymphatic development. In this article, we will summarize the details of these recent discoveries in support of the overall value of the zebrafish model system not only to study normal development, but also associated disease states.

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.

ESCRT genes and regulation of developmental signaling.

ESCRT (Endosomal Sorting Complex Required for Transport) proteins have been shown to control an increasing number of membrane-associated processes. Some of these, and prominently regulation of receptor trafficking, profoundly shape signal transduction. Evidence in fungi, plants and multiple animal models support the emerging concept that ESCRTs are main actors in coordination of signaling with the changes in cells and tissues occurring during development and homeostasis. Consistent with their pleiotropic function, ESCRTs are regulated in multiple ways to tailor signaling to developmental and homeostatic needs. ESCRT activity is crucial to correct execution of developmental programs, especially at key transitions, allowing eukaryotes to thrive and preventing appearance of congenital defects.

Zebrafish Tmem230a cooperates with the Delta/Notch signaling pathway to modulate endothelial cell number in angiogenic vessels.

During embryonic development, new arteries, and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre-existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co-operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel-network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis.

SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development.

Arterial specification and differentiation are influenced by a number of regulatory pathways. While it is known that the Vegfa-Notch cascade plays a central role, the transcriptional hierarchy controlling arterial specification has not been fully delineated. To elucidate the direct transcriptional regulators of Notch receptor expression in arterial endothelial cells, we used histone signatures, DNaseI hypersensitivity and ChIP-seq data to identify enhancers for the human NOTCH1 and zebrafish notch1b genes. These enhancers were able to direct arterial endothelial cell-restricted expression in transgenic models. Genetic disruption of SoxF binding sites established a clear requirement for members of this group of transcription factors (SOX7, SOX17 and SOX18) to drive the activity of these enhancers in vivo Endogenous deletion of the notch1b enhancer led to a significant loss of arterial connections to the dorsal aorta in Notch pathway-deficient zebrafish. Loss of SoxF function revealed that these factors are necessary for NOTCH1 and notch1b enhancer activity and for correct endogenous transcription of these genes. These findings position SoxF transcription factors directly upstream of Notch receptor expression during the acquisition of arterial identity in vertebrates.

Nfix Induces a Switch in Sox6 Transcriptional Activity to Regulate MyHC-I Expression in Fetal Muscle.

Sox6 belongs to the Sox gene family and plays a pivotal role in fiber type differentiation, suppressing transcription of slow-fiber-specific genes during fetal development. Here, we show that Sox6 plays opposite roles in MyHC-I regulation, acting as a positive and negative regulator of MyHC-I expression during embryonic and fetal myogenesis, respectively. During embryonic myogenesis, Sox6 positively regulates MyHC-I via transcriptional activation of Mef2C, whereas during fetal myogenesis, Sox6 requires and cooperates with the transcription factor Nfix in repressing MyHC-I expression. Mechanistically, Nfix is necessary for Sox6 binding to the MyHC-I promoter and thus for Sox6 repressive function, revealing a key role for Nfix in driving Sox6 activity. This feature is evolutionarily conserved, since the orthologs Nfixa and Sox6 contribute to repression of the slow-twitch phenotype in zebrafish embryos. These data demonstrate functional cooperation between Sox6 and Nfix in regulating MyHC-I expression during prenatal muscle development.

Optogenetics. Engineering of a light-gated potassium channel.

The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K(+)) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light-induced K(+) channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K(+) channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K(+) selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K(+) equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities.

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.

Ve-ptp modulates vascular integrity by promoting adherens junction maturation.

BACKGROUND: Endothelial cell junctions control blood vessel permeability. Altered permeability can be associated with vascular fragility that leads to vessel weakness and haemorrhage formation. In vivo studies on the function of genes involved in the maintenance of vascular integrity are essential to better understand the molecular basis of diseases linked to permeability defects. Ve-ptp (Vascular Endothelial-Protein Tyrosine Phosphatase) is a transmembrane protein present at endothelial adherens junctions (AJs). METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of Ve-ptp in AJ maturation/stability and in the modulation of endothelial permeability using zebrafish (Danio rerio). Whole-mount in situ hybridizations revealed zve-ptp expression exclusively in the developing vascular system. Generation of altered zve-ptp transcripts, induced separately by two different splicing morpholinos, resulted in permeability defects closely linked to vascular wall fragility. The ultrastructural analysis revealed a statistically significant reduction of junction complexes and the presence of immature AJs in zve-ptp morphants but not in control embryos. CONCLUSIONS/SIGNIFICANCE: Here we show the first in vivo evidence of a potentially critical role played by Ve-ptp in AJ maturation, an important event for permeability modulation and for the development of a functional vascular system.

The synaptic proteins ß-neurexin and neuroligin synergize with extracellular matrix-binding vascular endothelial growth factor a during zebrafish vascular development.

OBJECTIVE: The goal of this study was to determine the in vivo functions of the synaptic proteins neurexins and neuroligins in embryonic vascular system development using zebrafish as animal model. METHODS AND RESULTS: In the present study, we show that the knockdown of the α-form of neurexin 1a induces balance defects and reduced locomotory activity, whereas ß-neurexin 1a and neuroligin 1 morphants present defects in sprouting angiogenesis and vascular remodeling, in particular in the caudal plexus and subintestinal vessels. Coinjection of low doses of morpholinos for ß-neurexin 1a and neuroligin 1 together or in combination with morpholinos targeting the -heparin--binding isoforms of vascular endothelial growth factor A (encoded by the VEGFAb gene) recapitulates the observed abnormalities, suggesting synergistic activity of these molecules. Similar coinjection experiments with morpholinos, targeting the enzyme heparan sulfate 6-O-sulfotransferase 2, confirm the presence of a functional correlation between extracellular matrix maturation and ß-neurexin 1a or neuroligin 1. CONCLUSIONS: Our data represent the first in vivo evidence of the role of neurexin and neuroligin in embryonic blood vessel formation and provide insights into their mechanism of action.

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.