Yap induces osteoblast differentiation by modulating Bmp signalling during zebrafish caudal fin regeneration.

Osteoblast differentiation is a key process for bone homeostasis and repair. Multiple signalling pathways have been associated with osteoblast differentiation, yet much remains unknown on how this process is regulated in vivo Previous studies have proposed that the Hippo pathway transcriptional co-activators YAP and TAZ (also known as YAP1 and WWTR1, respectively) maintain progenitor stemness and inhibit terminal differentiation of osteoblasts, whereas others suggest they potentiate osteoblast differentiation and bone formation. Here, we use zebrafish caudal fin regeneration as a model to clarify how the Hippo pathway regulates de novo bone formation and osteoblast differentiation. We demonstrate that Yap inhibition leads to accumulation of osteoprogenitors and prevents osteoblast differentiation in a cell non-autonomous manner. This effect correlates with a severe impairment of Bmp signalling in osteoblasts, likely by suppressing the expression of the ligand bmp2a in the surrounding mesenchymal cells. Overall, our findings provide a new mechanism of bone formation through the Hippo-Yap pathway, integrating Yap in the signalling cascade that governs osteoprogenitor maintenance and subsequent differentiation during zebrafish caudal fin regeneration.

Diversity in cell motility reveals the dynamic nature of the formation of zebrafish taste sensory organs.

Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud. We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly.

Identification of Dmrt2a downstream genes during zebrafish early development using a timely controlled approach.

BACKGROUND: Dmrt2a is a zinc finger like transcription factor with several roles during zebrafish early development: left-right asymmetry, synchronisation of the somite clock genes and fast muscle differentiation. Despite the described functions, Dmrt2a mechanism of action is unknown. Therefore, with this work, we propose to identify Dmrt2a downstream genes during zebrafish early development. RESULTS: We generated and validated a heat-shock inducible transgenic line, to timely control dmrt2a overexpression, and dmrt2a mutant lines. We characterised dmrt2a overexpression phenotype and verified that it was very similar to the one described after knockdown of this gene, with left-right asymmetry defects and desynchronisation of somite clock genes. Additionally, we identified a new phenotype of somite border malformation. We generated several dmrt2a mutant lines, but we only detected a weak to negligible phenotype. As dmrt2a has a paralog gene, dmrt2b, with similar functions and expression pattern, we evaluated the possibility of redundancy. We found that dmrt2b does not seem to compensate the lack of dmrt2a. Furthermore, we took advantage of one of our mutant lines to confirm dmrt2a morpholino specificity, which was previously shown to be a robust knockdown tool in two independent studies. Using the described genetic tools to perform and validate a microarray, we were able to identify six genes downstream of Dmrt2a: foxj1b, pxdc1b, cxcl12b, etv2, foxc1b and cyp1a. CONCLUSIONS: In this work, we generated and validated several genetic tools for dmrt2a and identified six genes downstream of this transcription factor. The identified genes will be crucial to the future understanding of Dmrt2a mechanism of action in zebrafish.

Control of tissue growth by Yap relies on cell density and F-actin in zebrafish fin regeneration.

Caudal fin regeneration is characterized by a proliferation boost in the mesenchymal blastema that is controlled precisely in time and space. This allows a gradual and robust restoration of original fin size. However, how this is established and regulated is not well understood. Here, we report that Yap, the Hippo pathway effector, is a chief player in this process: functionally manipulating Yap during regeneration dramatically affects cell proliferation and expression of key signaling pathways, impacting regenerative growth. The intracellular location of Yap is tightly associated with different cell densities along the blastema proximal-distal axis, which correlate with alterations in cell morphology, cytoskeleton and cell-cell contacts in a gradient-like manner. Importantly, Yap inactivation occurs in high cell density areas, conditional to F-actin distribution and polymerization. We propose that Yap is essential for fin regeneration and that its function is dependent on mechanical tension, conferred by a balancing act of cell density and cytoskeleton activity.

Notch signalling regulates left-right asymmetry through ciliary length control.

The importance of cilia in embryonic development and adult physiology is emphasized by human ciliopathies. Despite its relevance, molecular signalling pathways behind cilia formation are poorly understood. We show that Notch signalling is a key pathway for cilia length control. In deltaD zebrafish mutants, cilia length is reduced in Kupffer's vesicle and can be rescued by the ciliogenic factor foxj1a. Conversely, cilia length increases when Notch signalling is hyperactivated. Short cilia found in deltaD mutants reduce the fluid flow velocity inside Kupffer's vesicle, thus compromising the asymmetric expression of the flow sensor charon. Notch signalling brings together ciliary length control and fluid flow hydrodynamics with transcriptional activation of laterality genes. In addition, our deltaD mutant analysis discloses an uncoupling between gut and heart laterality.

terra is a left-right asymmetry gene required for left-right synchronization of the segmentation clock.

To establish the vertebrate body plan, it is fundamental to create left-right asymmetry in the lateral-plate mesoderm to correctly position the organs. However, it is also crucial to maintain symmetry between the left and the right sides of the presomitic mesoderm, ensuring the allocation of symmetrical body structures, such as the axial skeleton and skeletal muscles. Here, we show that terra is an early left-sided expressed gene that links left-right patterning with bilateral synchronization of the segmentation clock.

A regeneration-triggered metabolic adaptation is necessary for cell identity transitions and cell cycle re-entry to support blastema formation and bone regeneration.

Regeneration depends on the ability of mature cells at the injury site to respond to injury, generating tissue-specific progenitors that incorporate the blastema and proliferate to reconstitute the original organ architecture. The metabolic microenvironment has been tightly connected to cell function and identity during development and tumorigenesis. Yet, the link between metabolism and cell identity at the mechanistic level in a regenerative context remains unclear. The adult zebrafish caudal fin, and bone cells specifically, have been crucial for the understanding of mature cell contribution to tissue regeneration. Here, we use this model to explore the relevance of glucose metabolism for the cell fate transitions preceding new osteoblast formation and blastema assembly. We show that injury triggers a modulation in the metabolic profile at early stages of regeneration to enhance glycolysis at the expense of mitochondrial oxidation. This metabolic adaptation mediates transcriptional changes that make mature osteoblast amenable to be reprogramed into pre-osteoblasts and induces cell cycle re-entry and progression. Manipulation of the metabolic profile led to severe reduction of the pre-osteoblast pool, diminishing their capacity to generate new osteoblasts, and to a complete abrogation of blastema formation. Overall, our data indicate that metabolic alterations have a powerful instructive role in regulating genetic programs that dictate fate decisions and stimulate proliferation, thereby providing a deeper understanding on the mechanisms regulating blastema formation and bone regeneration.

Yap Regulates Müller Glia Reprogramming in Damaged Zebrafish Retinas.

Vertebrates such as zebrafish have the outstanding ability to fully regenerate their retina upon injury, while mammals, including humans, do not. In zebrafish, upon light-induced injury, photoreceptor regeneration is achieved through reprogramming of Müller glia cells, which proliferate and give rise to a self-renewing population of progenitors that migrate to the lesion site to differentiate into the new photoreceptors. The Hippo pathway effector YAP was recently implicated in the response to damage in the retina, but how this transcription coactivator is integrated into the signaling network regulating Müller glia reprogramming has not yet been explored. Here, we show that Yap is required in Müller glia to engage their response to a lesion by regulating their cell cycle reentry and progenitor cell formation, contributing to the differentiation of new photoreceptors. We propose that this regulation is accomplished through a lin28a-ascl1a-dependent mechanism, bona fide Müller glia-reprogramming factors. Overall, this study presents Yap as a key regulator of zebrafish Müller glia reprogramming and consequently retina regeneration upon injury.

Left-right function of dmrt2 genes is not conserved between zebrafish and mouse.

BACKGROUND: Members of the Dmrt family, generally associated with sex determination, were shown to be involved in several other functions during embryonic development. Dmrt2 has been studied in the context of zebrafish development where, due to a duplication event, two paralog genes dmrt2a and dmrt2b are present. Both zebrafish dmrt2a/terra and dmrt2b are important to regulate left-right patterning in the lateral plate mesoderm. In addition, dmrt2a/terra is necessary for symmetric somite formation while dmrt2b regulates somite differentiation impacting on slow muscle development. One dmrt2 gene is also expressed in the mouse embryo, where it is necessary for somite differentiation but with an impact on axial skeleton development. However, nothing was known about its role during left-right patterning in the lateral plate mesoderm or in the symmetric synchronization of somite formation. METHODOLOGY/PRINCIPAL FINDINGS: Using a dmrt2 mutant mouse line, we show that this gene is not involved in symmetric somite formation and does not regulate the laterality pathway that controls left-right asymmetric organ positioning. We reveal that dmrt2a/terra is present in the zebrafish laterality organ, the Kupffer's vesicle, while its homologue is excluded from the mouse equivalent structure, the node. On the basis of evolutionary sub-functionalization and neo-functionalization theories we discuss this absence of functional conservation. CONCLUSIONS/SIGNIFICANCE: Our results show that the role of dmrt2 gene is not conserved during zebrafish and mouse embryonic development.

[Prevalence of pterygium in Botucatu city - São Paulo State, Brazil]. / Prevalência de pterígio no município de Botucatu - Estado de São Paulo, Brasil.

PURPOSE: To evaluate the prevalence of pterygium in a population-based sample at Botucatu City - São Paulo State, Brazil. METHODS: A population-based cross-sectional study with randomized clustered sampling of households was conducted in the urban area of the Botucatu City - São Paulo State, Brazil and 85.1% of the intended sample was evaluated. All participants were submitted to ophthalmologic examination and the data were statistically analyzed. RESULTS: The prevalence of pterygium lesion in Botucatu City was 8.12% (7.0% < CI < 9.2%), affecting mainly males (10.4% males X 6.5% females - 8.5% < CI < 12.3% for males and 5.1% < CI < 7.8% for females) with 49.6 ± 14.9 years old in average; 32.18% of the pterygium carriers aged between 40 and 50 years. CONCLUSIONS: The prevalence of pterygium at Botucatu is 8.12%, affecting most frequently 40-50 year-old males.

[Impact on colostrum retinol levels of immediate postpartum supplementation with retinyl palmitate]. / Impacto da suplementação com retinil palmitato no pós-parto imediato sobre os níveis de retinol do colostro.

OBJECTIVE: To assess the effect of retinyl palmitate supplementation on colostrum retinol levels, investigating the influence of maternal variables (age, type of delivery, and biochemical nutritional status) on these levels. METHOD: The study included 33 mothers receiving care at the Januário Cicco Maternity School, in the city of Natal, Rio Grande do Norte, Brazil. In the first hours after delivery, blood and colostrum samples were collected. Another colostrum sample was collected six hours after maternal supplementation with 200,000 IU of retinyl palmitate. Serum and colostrum retinol levels were determined by high-performance liquid chromatography. RESULTS: The mean retinol level in colostrum before the supplementation was 110.8 +/- 82.3 microg/dL, and after supplementation it was 164.4 +/- 106.5 microg/dL (P < 0.025). Of the 33 mothers, 12 of them either did not respond to supplementation or had an increase of less than 10% in colostrum retinol levels; serum retinol in these women was significantly lower as compared to the responders (P = 0.024). In comparison to women with a normal delivery, the mothers who underwent cesarean delivery (64%) had lower serum retinol levels, but not lower colostrum retinol levels (P = 0.036). Maternal age did not influence retinol levels in either serum or colostrum. CONCLUSION: The increase in colostrum retinol levels following vitamin A supplementation was sufficient to guarantee double the retinol requirements of a newborn infant.

Analysis of retinol concentrations in bovine liver and its habitual consumption by pregnant women.

AIMS: The objectives of this study were to analyze retinol concentrations in two groups of bovine liver, to estimate the habitual consumption of bovine liver among pregnant women, and, based on these data, to evaluate the women's ingestion of vitamin A. METHOD: 60 samples of bovine liver, 30 of the Nelore breed and 30 of undefined breed (UB), were analyzed using the high-performance liquid chromatography (HPLC) method for retinol dosage. 100 women under care at the University Maternity Hospital Januário Cicco, UFRN, in Natal, RN, Brazil, completed the Frequency of Nourishment Questionnaire. RESULTS: The mean +/- SD values for the retinol concentrations in the liver samples of the Nelore breed and the UB groups were 16,947.8 +/- 6,866.9 and 5,213.1 +/- 2,517.2 microg of retinol/100 g, respectively. A high number of women consumed liver (73%) and in 71% of the cases the advice for the consumption of this food was provided by qualified professionals. CONCLUSION: It is concluded that the daily consumption of the bovine liver is not recommended for pregnant women, regardless of the genetic origin of the animal. The study alert health professionals of the danger of recommending the ingestion of bovine liver during pregnancy.

Prevalência de pterígio no município de Botucatu - Estado de São Paulo, Brasil / Prevalence of pterygium in Botucatu city - São Paulo State, Brazil

OBJETIVO: Avaliar a prevalência de pterígio no município de Botucatu - São Paulo, Brasil, por meio de amostra populacional. MÉTODOS: No ano de 2004 foi feito estudo de prevalência de alterações oftalmológicas na cidade de Botucatu - SP, por amostragem domiciliar aleatória, tendo sido avaliados 85,1 por cento (2.554 indivíduos) da amostra pretendida. Todos os participantes do estudo foram submetidos a exame oftalmológico completo, sendo os dados obtidos submetidos à análise estatística. RESULTADOS: A prevalência de pterígio na população estudada foi de 8,12 por cento (7,0 por cento < IC < 9,2 por cento), sendo mais frequente em homens (10,4 por cento homens X 6,5 por cento mulheres - 8,5 por cento < IC < 12,3 por cento em homens e 5,1 por cento < IC < 7,8 por cento em mulheres). A média de idade dos portadores de pterígio foi de 49,6 ± 14,9 anos, havendo 32,18 por cento indivíduos da amostra na faixa etária entre os 40 e 50 anos de idade. CONCLUSÃO: A prevalência do pterígio na cidade de Botucatu é de 8,12 por cento, sendo a lesão mais prevalente em homens, entre 40 e 50 anos de idade.

PURPOSE: To evaluate the prevalence of pterygium in a population-based sample at Botucatu City - São Paulo State, Brazil. METHODS: A population-based cross-sectional study with randomized clustered sampling of households was conducted in the urban area of the Botucatu City -São Paulo State, Brazil and 85.1 percent of the intended sample was evaluated. All participants were submitted to ophthalmologic examination and the data were statistically analyzed. RESULTS: The prevalence of pterygium lesion in Botucatu City was 8.12 percent (7.0 percent < CI < 9.2 percent), affecting mainly males (10.4 percent males X 6.5 percent females - 8.5 percent < CI < 12.3 percent for males and 5.1 percent < CI < 7.8 percent for females) with 49.6 ± 14.9 years old in average; 32.18 percent of the pterygium carriers aged between 40 and 50 years. CONCLUSIONS: The prevalence of pterygium at Botucatu is 8.12 percent, affecting most frequently 40-50 year-old males.

Impacto da suplementação com retinil palmitato no pós-parto imediato sobre os níveis de retinol do colostro / Impact on colostrum retinol levels of immediate postpartum supplementation with retinyl palmitate

OBJETIVO: Avaliar o efeito da suplementação com retinil palmitato sobre os níveis de retinol no colostro, investigando a influência de variáveis maternas (idade, tipo de parto e estado nutricional bioquímico) sobre esses níveis. MÉTODO:Participaram do estudo 33 nutrizes atendidas na Maternidade Escola Januário Cicco, Estado do Rio Grande do Norte, Brasil. Foram coletadas, nas primeiras horas após o parto, uma amostra de sangue e uma de colostro. Uma nova amostra de colostro foi coletada 6 horas após suplementação materna com 200 000 UI de retinil palmitato. O retinol no sangue e colostro foi determinado por cromatografia líquida de alta eficiência. RESULTADOS: Os níveis médios de retinol no colostro antes da suplementação foram de 110,8 ± 82,3 mug/dL, tendo atingido 164,4 ± 106,5 mug/dL após a suplementação (P < 0,025). Doze puérperas não responderam à suplementação ou tiveram um aumento inferior a 10 por cento nos níveis de retinol no colostro; o nível de retinol sérico dessas mulheres foi significativamente mais baixo do que o daquelas que responderam à suplementação (P = 0,024). Puérperas com parto cesáreo (64 por cento) tiveram níveis menores de retinol no soro (P = 0,036), mas não no leite, em comparação a mulheres com parto normal. A idade não influenciou os níveis de retinol, nem no soro nem no leite. CONCLUSÕES: A suplementação com retinil palmitato foi eficaz na elevação dos níveis de retinol no colostro, garantindo o fornecimento de quantidade suficiente de vitamina A para satisfazer o dobro das necessidades de retinol do recém-nascido.

OBJECTIVE: To assess the effect of retinyl palmitate supplementation on colostrum retinol levels, investigating the influence of maternal variables (age, type of delivery, and biochemical nutritional status) on these levels. METHOD: The study included 33 mothers receiving care at the Januário Cicco Maternity School, in the city of Natal, Rio Grande do Norte, Brazil. In the first hours after delivery, blood and colostrum samples were collected. Another colostrum sample was collected six hours after maternal supplementation with 200 000 IU of retinyl palmitate. Serum and colostrum retinol levels were determined by high-performance liquid chromatography. RESULTS: The mean retinol level in colostrum before the supplementation was 110.8 ± 82.3 mug/dL, and after supplementation it was 164.4 ± 106.5 mug/dL (P < 0.025). Of the 33 mothers, 12 of them either did not respond to supplementation or had an increase of less than 10 percent in colostrum retinol levels; serum retinol in these women was significantly lower as compared to the responders (P = 0.024). In comparison to women with a normal delivery, the mothers who underwent cesarean delivery (64 percent) had lower serum retinol levels, but not lower colostrum retinol levels (P = 0.036). Maternal age did not influence retinol levels in either serum or colostrum. CONCLUSIONS: The increase in colostrum retinol levels following vitamin A supplementation was sufficient to guarantee double the retinol requirements of a newborn infant.

Prevalência de Hipovitaminose A em puérperas da Maternidade Escola Januário Cicco - Natal/RN / Prevalence of hypovitaminosis A in puerperal women at the University Maternity Januário Cicco – Natal, RN

As mães sociais que cuidam de crianças e adolescentes de abrigos filantrópicos de São Paulo, influenciam a elaboração dos cardápios e o modo de conduzir as refeições, de acordo com sua formação e cultura. Conhecer alguns aspectos sociais da alimentação desse grupo foi o objetivo deste trabalho. Foram feitas entrevistas com onze mães sociais e três diretoras de abrigos, analisadas por meio da metodologia do Discurso do Sujeito Coletivo. O estudo resultou em 32 categorias de discursos, por meio dos quais foram verificados valores e hábitos presentes nas refeições, que devem ser levados em conta quando se pretende desenvolver orientações e treinamentos na área da alimentação para esse público. Destaca-se a preocupação das mães com rituais familiares: comer “todos juntos ao redor da mesa” e manter silêncio durante as refeições são práticas impostas pela maioria das mães. A tarefa de servir os pratos praticamente é assumida pela mãe, não dando muitas oportunidades para as crianças aprenderem a se servir e escolher sozinhas os alimentos. O ensino de boas maneiras à mesa é uma preocupação diária e constante das mães, que não querem passar vergonha pelos maus modos das crianças, que precisam aprender a se comportar “lá fora”. Crianças, adolescentes e mães sociais de abrigos constituem uma alternativa de família, com dinâmicas e soluções próprias. As idéias centrais extraídas das entrevistas apontam um perfil dessas mães. Conhecer como pensam e sentem a respeito da alimentação é essencial para o aprimoramento da qualidade das refeições por meio de orientações e formação apropriadas