Role of the 2 zebrafish survivin genes in vasculo-angiogenesis, neurogenesis, cardiogenesis and hematopoiesis

<p>Abstract</p> <p>Background</p> <p>Normal growth and development of organisms requires maintenance of a dynamic balance between systems that promote cell survival and those that induce apoptosis. The molecular mechanisms that regulate these processes remain poorly understood, and thus further <it>in vivo </it>study is required. Survivin is a member of the inhibitor of apoptosis protein (IAP) family, that uniquely also promotes mitosis and cell proliferation. Postnatally, survivin is hardly detected in most tissues, but is upregulated in all cancers, and as such, is a potential therapeutic target. Prenatally, survivin is also highly expressed in several tissues. Fully delineating the properties of survivin <it>in vivo </it>in mice has been confounded by early lethal phenotypes following <it>survivin </it>gene inactivation.</p> <p>Results</p> <p>To gain further insights into the properties of survivin, we used the zebrafish model. There are 2 zebrafish <it>survivin </it>genes (<it>Birc5a </it>and <it>Birc5b</it>) with overlapping expression patterns during early development, prominently in neural and vascular structures. Morpholino-induced depletion of <it>Birc5a </it>causes profound neuro-developmental, hematopoietic, cardiogenic, vasculogenic and angiogenic defects. Similar abnormalities, all less severe except for hematopoiesis, were evident with suppression of <it>Birc5b</it>. The phenotypes induced by morpholino knockdown of one <it>survivin </it>gene, were rescued by overexpression of the other, indicating that the <it>Birc5 </it>paralogs may compensate for each. The potent vascular endothelial growth factor (VEGF) also entirely rescues the phenotypes induced by depletion of either <it>Birc5a </it>and <it>Birc5b</it>, highlighting its multi-functional properties, as well as the power of the model in characterizing the activities of growth factors.</p> <p>Conclusion</p> <p>Overall, with the zebrafish model, we identify survivin as a key regulator of neurogenesis, vasculo-angiogenesis, hematopoiesis and cardiogenesis. These properties of survivin, which are consistent with those identified in mice, indicate that its functions are highly conserved across species, and point to the value of the zebrafish model in understanding the role of this IAP in the pathogenesis of human disease, and for exploring its potential as a therapeutic target.</p

Early Depletion of Primordial Germ Cells in Zebrafish Promotes Testis Formation

As complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome

Integration of CNS survival and differentiation by HIF2α

Hypoxia-inducible factor (HIF) 1α and HIF2α and the inhibitor of apoptosis survivin represent prominent markers of many human cancers. They are also widely expressed in various embryonic tissues, including the central nervous system; however, little is known about their functions in embryos. Here, we show that zebrafish HIF2α protects neural progenitor cells and neural differentiation processes by upregulating the survivin orthologues birc5a and birc5b during embryogenesis. Morpholino-mediated knockdown of hif2α reduced the transcription of birc5a and birc5b, induced p53-independent apoptosis and abrogated neural cell differentiation. Depletion of birc5a and birc5b recaptured the neural development defects that were observed in the hif2α morphants. The phenotypes induced by HIF2α depletion were largely rescued by ectopic birc5a and birc5b mRNAs, indicating that Birc5a and Birc5b act downstream of HIF2α. Chromatin immunoprecipitation assay revealed that HIF2α binds to birc5a and birc5b promoters directly to modulate their transcriptions. Knockdown of hif2α, birc5a or birc5b reduced the expression of the cdk inhibitors p27/cdkn1b and p57/cdkn1c and increased ccnd1/cyclin D1 transcription in the surviving neural progenitor cells. The reduction in elavl3/HuC expression and enhanced pcna, nestin, ascl1b and sox3 expression indicate that the surviving neural progenitor cells in hif2α morphants maintain a high proliferation rate without terminally differentiating. We propose that a subset of developmental defects attributed to HIF2α depletion is due in part to the loss of survivin activity

Germ Cell Determinant Transmission, Segregation, and Function in the Zebrafish Embryo

Animals specify primordial germ cells (PGCs) in two alternate modes: preformation and epigenesis. Epigenesis relies on signal transduction from the surrounding tissues to instruct a group of cells to acquire PGC identity. Preformation, thought to be the more derived PGC specification mode, is instead based on the maternal inheritance of germ cell-determining factors. We use the zebrafish as a model system, in which PGCs are specified through maternal inheritance of germ plasm, to study this process in vertebrates. In zebrafish, maternally inherited germ plasm ribonucleoparticles (RNPs) have co-opted the cytoskeletal machinery to reach progressive levels of multimerization, resulting in the formation of four large masses of aggregated germ plasm RNPs. At later stages, germ plasm masses continue to use components of the cell division machinery, such as the spindles, centrosomes, and/or subcellular organelles to segregate asymmetrically during cell division and subsequently induce germ cell fate. This chapter discusses the current knowledge of germ cell specification focusing on the zebrafish as a model system. We also provide a comparative analysis of the mechanism for germ plasm RNP segregation in zebrafish versus other known vertebrate systems of germ cell preformation, such as in amphibian and avian models

Transcriptome Analysis of Zebrafish Embryogenesis Using Microarrays

Zebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html)

Comparative Oncogenomic Analysis of Copy Number Alterations in Human and Zebrafish Tumors Enables Cancer Driver Discovery

The identification of cancer drivers is a major goal of current cancer research. Finding driver genes within large chromosomal events is especially challenging because such alterations encompass many genes. Previously, we demonstrated that zebrafish malignant peripheral nerve sheath tumors (MPNSTs) are highly aneuploid, much like human tumors. In this study, we examined 147 zebrafish MPNSTs by massively parallel sequencing and identified both large and focal copy number alterations (CNAs). Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs. We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes. For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs. We conclude that zebrafish-human comparative analysis represents a powerful, and broadly applicable, tool to enrich for evolutionarily conserved cancer drivers.Kathy and Curt Marble Cancer Research FundArthur C. MerrillNational Institutes of Health (U.S.) (Grant CA106416)National Institutes of Health (U.S.) (Grant ROI RR020833)National Institutes of Health (U.S.) (Grant 1F32GM095213-01

Activation of NF- B protein prevents the transition from juvenile ovary to testis and promotes ovarian development in Zebrafish

Testis differentiation in zebrafish involves juvenile ovary to testis transformation initiated by an apoptotic wave. The molecular regulation of this transformation process is not fully understood. NF-κB is activated at an early stage of development and has been shown to interact with steroidogenic factor-1 in mammals, leading to the suppression of anti-Müllerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-κB-mediated induction of anti-apoptotic genes could, therefore, also play a role in zebrafish gonad differentiation. The aim of this study was to examine the potential role of NF-κB in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-κB pathways and resulted in an increased ratio of females from 30 to 85%. Microarray and quantitative real-time-PCR analysis of gonads showed elevated expression of NF-κB-regulated genes. To confirm the involvement of NF-κB-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-κB or NF-κB activation inhibitor (NAI). Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and resulted in an increased proportion of females from 25 to 45%, whereas the inhibition of NF-κB using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-κB in gonadal differentiation of zebrafish and represents an important step toward the complete understanding of the complicated process of sex differentiation in this species and possibly other cyprinid teleosts as well

The role of Hippo pathway in Zebrafish (Danio rerio) caudal fin regeneration

Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2011O crescimento dos tecidos e um dos processos fundamentais que contribuem para o desenvolvimento embrionário, no entanto, e apesar da sua importância, pouco se sabe acerca dos mecanismos que determinam o tamanho dos órgãos e consequentemente do organismo, uma das questões clássicas da Biologia do Desenvolvimento. Em particular, como e que as células desencadeiam os mecanismos adequados que asseguram as proporções certas e que resultam num tamanho final determinado com precisão? O controlo do tamanho dos tecidos/órgãos e um processo altamente coordenado e complexo que envolve diferentes mecanismos em resposta a sinais fisiológicos, incluindo factores circulantes como por exemplo hormonas e factores de crescimento semelhantes a insulina. Estudos clássicos sugeriram pela primeira vez, atraves de experiências de transplantação em membros de salamandra, que os órgãos podem possuir informação intrinseca sobre o seu tamanho final, mas o mecanismo subjacente continua por identificar. Recentemente, foi descoberta a via de sinalização Hippo, uma cascata de cinases que culmina na regulação de um potente regulador do crescimento. Por isso, esta via tem-se vindo a afirmar como um dos mecanismos regulatórios mais pertinentes na abordagem da questão do controlo do crescimento. A via Hippo foi descoberta inicialmente em Drosophila melanogaster como uma potente cascata de fosforilação que envolve simultaneamente a coordenação da proliferação celular e da apoptose durante o desenvolvimento. Posteriormente, foi demonstrado que existem vários homólogos directos dos seus componentes em mamíferos e que a via assume um papel semelhante, indicando que esta altamente conservada ao longo da evolução e que pode funcionar como um mecanismo global de regulação do crescimento em Drosophila e em vertebrados. Os primeiros componentes desta via foram identificados em rastreios genéticos em Drosophila que tinham como objectivo encontrar genes supressores de tumores, aqueles que quando mutados com perda de função induzissem um crescimento exagerado dos tecidos. Os primeiros genes a serem identificados foram warts (wts) e salvador (sav), dois genes supressores de tumores, cujas proteinas interagem entre si. Foi demonstrado que a perda de função de qualquer um destes genes resulta num aumento da proliferação celular e redução da apoptose, o que constituiu a primeira evidencia de regulação destes processos por estes componentes. Um passo decisivo na descoberta desta via de sinalização foi a identificação do gene hippo (hpo), o terceiro componente em Drosophila. Este gene, e consequentemente a via, acabaram por ser chamados de hippo uma vez que quando esta mutado, o seu fenótipo é caracterizado por várias dobras de tecido com crescimento excessivo, fazendo lembrar as dobras na pele do hipopotamo. Até agora, já foi identificado um grande número de componentes da via, tanto em Drosophila como em mamíferos, fazendo emergir uma complexa rede de reguladores positivos e negativos, que foi dividida em três grandes grupos: os componentes centrais e os componentes a montante e a jusante dos componentes centrais. Em Drosophila, o grupo dos componentes centrais é formado pelas cinases Hpo e Wts, e pelas proteinas adaptadoras Sav e Mats, que interagem entre si, estabelecendo uma cascata de fosforilação. Quando a via é activada, a cascata de fosforilação inibe o co-activador de transcrição Yorkie (Yki), que quando fosforilado, fica retido no citoplasma sem conseguir migrar para o nucleo e activar a transcrição dos seus genes-alvo. Por outro lado, quando a via está inactiva, Yki não é fosforilado e é transferido para o núcleo, onde se liga a diferentes factores de transcrição, promovendo a expressão de genes especificos, que induzem a proliferação celular e inibem a apoptose. Desta forma, a sobreexpressão de yki induz um sobre-crescimento nos tecidos, mediado pela transcrição activa dos seus genes-alvo, o que faz com que este gene actue como um oncogene, enquanto os outros membros nucleares actuam como supressores de tumores, inibindo a sua actividade. Em mamíferos, foi demonstrado que todos os componentes nucleares da via identificados na mosca tem homólogos directos como Mst1/2 (homólogos de Hpo), Sav1 (homólogo de Sav), Lats 1/2 (homólogos de Wts); MOBKL1 A/B (colectivamente denominado Mob; homólogos de Mats) e dois co-activadores da transcrição YAP e o seu parálogo TAZ (homólogos de Yki), e que interagem entre si da mesma forma descrita anteriormente em Drosophila. O Peixe-zebra (Danio rerio), e um organismo frequentemente usado como modelo vertebrado para o estudo do desenvolvimento, uma vez que possui diversas caracteristicas atractivas, como o rápido desenvolvimento, facil reprodução e manutenção, e oferece varias possibilidades em termos de ferramentas genéticas, como por exemplo a criação de linhas transgénicas. Adicionalmente, o peixe-zebra é um modelo muito usado no estudo da regeneração pois, à semelhança de outros vertebrados inferiores, apresenta um grande potencial regenerativo, na medida em que consegue regenerar um elevado numero de tecidos, tal como a retina, o coração, a espinal medula, as escamas e todas as barbatanas. Em particular, a barbatana caudal e uma das estruturas de eleição para o estudo da regeneração, pois pode ser facilmente acedida para efectuar uma amputação e danificada sem comprometer a sobrevivência do animal. Além disso, após amputação, a barbatana caudal tem a capacidade de recuperar o seu tamanho entre 7 a 14 dias. É de salientar, que independentemente do número de amputações ou outros factores externos, a barbatana recupera sempre o mesmo tamanho original, o que sugere a existência de um mecanismo altamente controlado que determina o tamanho correcto da estrutura a regenerar. Tendo em conta o curto espaco de tempo que este processo regenerativo necessita para ficar concluído, existe um aumento significativo da proliferação celular, mas que ocorre de forma controlada. Um dos factores que determina essa taxa de proliferação diz respeito ao local de amputação, sendo que quanto mais proximal for o tecido amputado, ou seja, quanto mais tecido for amputado, maior será a taxa de proliferação e consequentemente mais rápida será a regeneração do tecido. Esta propriedade reflecte um mecanismo ao qual se chama memória posicional, que permite ao organismo reconhecer e regenerar apenas as estruturas/tecidos removidos na amputação. Dadas as potencialidades da via Hippo ao nivel da regulação do crescimento, esta apresenta-se como um bom candidato para abordar o estudo da regeneração no peixe-zebra. A sua grande conservação entre espécies já demonstrada nos diferentes modelos, faria prever que a via estivesse também presente no peixe-zebra, sendo este evolutivamente mais próximo dos mamíferos do que da mosca. Até agora, já foi evidenciado que alguns componentes tais como Mats, YAP e Lats estão conservados, no entanto, pouco se sabe ainda sobre as funções fisiológicas desta via neste organismo. Para mais conclusões, será necessário efectuar uma análise mais completa. Tendo em conta todos estes dados, este projecto tem como principal objectivo ajudar a perceber qual o mecanismo que regula o processo de regeneração na barbatana caudal do peixezebra, que permite à barbatana recuperar sempre o seu tamanho original. Para tentar responder a esta questão, uma das mais antigas na area da Biologia Regenerativa, fomos estudar o possivel envolvimento da via Hippo na regulação do tamanho final durante a regeneração. Nesse sentido, foi efectuada uma analise que abrange diversos componentes da via, atraves do estudo da expressão génica por hibridação in situ e RT-PCR não quantitativo. Esta análise permitiu-nos observar que vários componentes da via estão efectivamente a ser expressos durante a regeneração e os nossos resultados sugerem que a via Hippo pode desempenhar um papel importante na regulação deste processo de reparação. A via Hippo pode mesmo ser o regulador central que coordena o processo de crescimento da barbatana durante a regeneração, fornecendo as células sinais de proliferação ou apoptose, permitindo uma constante manutenção do tamanho da barbatana após amputação.The Hippo pathway is a recently discovered signaling pathway that has been shown to be a potent growth regulator, whereas its deregulation leads to dramatic tissue overgrowth. This pathway is highly conserved throughout evolution, since its components are present and have direct homology between Drosophila and mammals, indicating this pathway as a potential universal mechanism of growth control. This topic is of outmost interest since the mechanisms by which the organism controls growth to obtain its final size remain as a long-standing question in Developmental Biology. Zebrafish (Danio rerio), a vertebrate model frequently used in the study of development due to its several advantages such as the possibility of generating transgenic lines, has direct homologs to some of the components of the Hippo pathway, although the conservation of their function has not been shown. Additionally, zebrafish has a great capacity to regenerate several structures, such as the fins. The caudal fin is typically used to assess regeneration, since upon amputation it fully recovers the lost appendage with full functionality and restoring the original size, after 7-14 days. The original and final size of the fin is always restored regardless of the number of times it is amputated, which suggests the existence of a strict mechanism regulating growth during this repair process. Therefore, the Hippo pathway seems to be a promising approach to help understanding how the growth and final size are regulated during epimorphic regeneration. In this work, we perform a broad and systematic analysis of several components of the pathway and show that most of them are effectively expressed in the fin during regeneration. Our data thus suggest that the Hippo pathway might be one of the key regulators of size maintenance upon injury, instructing the cells with proliferation or apoptotic cues, a long-lasting question in the field of Regenerative Biology

ANALYSIS OF THE IN VIVO FUNCTION OF HASPIN KINASE USING ZEBRAFISH AS A MODEL SYSTEM: KNOCKDOWN AND KNOCKOUT APPROACHES

The Haspin gene encodes an atypical serine/threonine mitotic kinase first discovered in mouse spermatocytes and preferentially expressed in tissues with a high rate of proliferating cells. Haspin acts at metaphase by phosphorylating threonine 3 of histone H3 (H3Thr3PH) and this modification allows the recruitment of the chromosomal passenger complex, a key factor required to orchestrate different steps of mitosis. In human cells, HASPIN depletion causes a decrease in H3Thr3 levels, resulting in premature loss of sister chromatid cohesion and in defects in chromosome alignment at metaphase. Haspin has been found in all eukaryotic organisms; however, up to know, its role during animal embryonic development has never been investigated. We decided to investigate its function and expression during zebrafish embryonic development and, to this aim, we took advantage of a morpholino (MO)-mediated knockdown approach and of the CRISPR-Cas9 knockout strategy. We identified and cloned the zebrafish haspin ortholog, together with a previously unknown splicing isoform, and we clarified its expression pattern during embryogenesis and in some adult tissues. We demonstrated a relevant maternal contribution for the haspin transcript and important levels of zygotic expression in tissues with a high rate of proliferating cells, such as the developing brain and hematopoietic tissues. We also detected haspin transcript in the adult gonads and found that its expression is significantly switched on after injury during adult fin tissue regeneration. Interestingly, after Haspin functional inactivation using two different MOs, a translation blocking (ATG MO) and a splicing one, we demonstrated that Haspin is involved in H3Thr3PH also in zebrafish. Moreover, microinjection of the haspin ATG MO results in high embryo mortality and severe defects during epiboly stages, indicating important alterations in cellular rearrangements and movements. A haspin stable mutant line was generated by using the CRISPR-Cas9 technology: we isolated three different mutant haspin alleles, all causing the formation of premature stop codons. Although they do not show evident phenotypic alterations during embryogenesis, embryos carrying a homozygous genotype for these mutations are not able to reach the adulthood stage, showing a high rate of mortality in the first three weeks of larval development, indicating that Haspin is fundamental for larval survival and growth. To conclude, we clarified various aspects of haspin expression pattern during zebrafish development and in adult organs. Even though we were not able yet to unambiguously define the phenotypic effect of Haspin functional inactivation by using a MO-mediated approach, we paved the way for the analysis of the effect of a complete haspin gene knockout during zebrafish development by generating a haspin stable KO line and by showing that this null mutant allele significantly affects larval survival and growth

Control of programmed cell death during zebrafish embryonic development

Programmed cell death (PCD) is a conserved cellular process, which is essential during embryonic development, morphogenesis and tissue homeostasis. PCD participates in the elimination of unwanted or potentially harmful cells, and contributes in this way to the precise shaping of the developing embryo. In this review the current knowledge related to the role of PCD during zebrafish development was described and an overview was provided about the main actors that induce, control and execute the apoptosis pathways during zebrafish development. Finally, we point out some important issues regarding the regulation of apoptosis during the early stages of zebrafish development