Developmental Genetics in the Zebrafish Danio rerio

斑马鱼斑马鱼的发育遗传学

基本信息

批准号：
9339257
负责人：
Igor B Dawid
金额：
$ 63.06万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9339257
关键词：
5&apos Splice Site Affect Alanine Animals Anterior Area BTB/POZ Domain Binding Biological Assay Biological Models Biological Process Brain Branchial arch structure Cell Differentiation process Cell physiology Cells Chimeric Proteins Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Co-Immunoprecipitations Collaborations Complex Craniofacial Abnormalities Cultured Cells Defect Development Developmental Cell Biology Developmental Delay Disorders Dimerization Embryo Embryonic Development Endocrine Exons Family Fishes Frameshift Mutation Gene Targeting Genes Genotype Gland Human Inherited Institutes Introns Laboratories Left Ligands Light Lysine Methodology Methods Modeling Modification Molecular Genetics Mutation Nature Neural Crest Neural Crest Cell Neurons Notch Signaling Pathway Nuclear Pancreas Pancreatic Bud Pathway interactions Phenotype Pigments Population Post-Translational Protein Processing Process Production Proline Property Proteins RNA RNA Splicing Reagent Regulation Regulatory Pathway Reporter Role Sensory Site Skeleton Staging Stem cells Structure System TFAP2A gene Time Ubiquitin Vertebrates Work Zebrafish base cell type craniofacial developmental genetics feeding forward genetics gene discovery gene function genome editing hatching human disease in vivo inhibitor/antagonist interest knock-down malformation migration multicatalytic endopeptidase complex mutant neural plate notch protein novel null mutation numb protein overexpression paralogous gene precursor cell programs protein degradation research study transcription activator-like effector nucleases transcription factor transcriptome ubiquitin ligase ubiquitin-protein ligase zebrafish development

项目摘要

A focus of interest in this laboratory has been the study of genes that are involved in the formation of the neural crest and of its derivatives such as pigment cells, pharyngeal arches, and others. We found that the BTB domain-containing protein Kctd15 that is first expressed in the embryo at the neural plate border, may have a role in regulating the domain in which neural crest forms as overexpression of Kctd15 strongly inhibits neural crest specification. We have found that Kctd15 inhibits the activity of transcription factor AP-2, a regulatory molecule known to be critical for the specification of the neural crest in different animals. Mechanistic studies on the interaction between AP-2 and Kctd15 have been carried out. Ap-2 is a major regulator of neural crest formation, being involved in the initial induction of neural crest precursor cells as well as in later stages such as their migration and differentiation into multiple derivatives. Kctd15 is capable of binding AP-2 in co-immunoprecipitation experiments, and inhibits the activation of an AP-2 reporter both in cultured cells and in zebrafish embryos. In studying the mechanism of this inhibition we found that Kctd15 does not affect the level of AP-2 in the cell, does not inhibit dimerization of AP-2 which is required for activity, and does not affect nuclear localization of AP-2. Also, AP-2 remains bound to its cognate sites in chromatin in the presence of Kctd15. To probe the mechanism further we studied the activation domain of AP-2. Kctd15 binds to the activation domain, and specifically requires proline 59 for this interaction. An AP-2 mutant in which this proline is changed to alanine (P59A) is active in the reporter assay but cannot bind Kctd15, and its activity is not effectively inhibited by Kctd15. We conclude that Kctd15 inhibits AP-2 activity by specific binding to its activation domain which precludes its function. In further studies on Kctd15 we found that this protein is SUMOylated in vivo, both by SUMO1 and SUMO2/3. We identified the site of SUMO attachment and showed that mutation of the relevant lysine residue abolished SUMOylation. Non-SUMOylated Kctd15 was fully effective in inhibiting AP-2 function, but a stable fusion protein of Kctd15 and SUMO was not effective. We conclude that the non-SUMOylated form of Kctd15 functions in AP-2 inhibition, but leave open the possibility that the SUMOylated form has a role in a different biological function of Kctd15. To further analyze the function of Kctd15 in development we generated frameshift mutations in both Kctd15 paralogs with the use of the TALEN methodology. Homozygous Kctd15a and Kctd15b mutant fish are viable and fertile and show no overt phenotype. However, zebrafish mutant for both Kctd15a and Kctd15b, while viable and fertile, show a developmental delay and small size phenotype. These mutants also appear to have certain malformations in the craniofacial skeleton as well as some abnormalities in brain structure. The work on brain structure and function is a collaboration with the group of Harold Burgess in the Institute. We are currently studying the possibility that the small size phenotype may be due to inefficient feeding by the mutant fish. This might be due to the craniofacial abnormalities or to sensory deficits that potentially correlated with the observed changes in brain structure. In another approach at characterization of the Kctd15 mutant fish we entered a collaboration with the group of Anand Swaroop at NEI to compare the wild type and mutant transcriptomes by RNA-Seq. Sequencing was carried out at several stages of development, with results currently being analyzed. In our project on the function of E3 ubiquitin ligases in development we studied the role of the Lnx2 family in the initial specification of the pancreas in zebrafish. We found that the gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A TALEN induced frameshift null mutation in Lnx2a did not mimic this phenotype, being phenotypically normal. However, knockdown of the Lnx2b gene in the Lnx2a null mutant background mimicked the splice morpholino phenotype. Further and most compellingly, a mutation that removed the exon 2 splice donor site, which is targeted by the splice morpholino, effectively mimicked the exocrine suppression phenotype. From these findings we conclude that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. To analyze the mechanism of Lnx2 function in the pancreas in further detail we started from the earlier observation by others that human LNX1 destabilizes Numb. We could show that inhibition of Numb expression rescues the Lnx2a/b deficient phenotype, consistent with the hypothesis that Numb is a target for Lnx2 in the pancreas. Numb is a known inhibitor of the Notch signaling pathway, and Notch is known to have a critical role in pancreas differentiation. Further we found that inhibition of Lnx2a/b leads to a reduction in the number of Notch active cells in the pancreas. Thus we suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. An additional result of this study is to cast light on the complex relationships that may exist between genotype, phenotype, and morpholino effects. In the present example, disagreement between the consequences of a morpholino knockdown and a null mutation is not evidence of nonspecific effects of the morpholino but rather of a more complex relationship that could be revealed only by additional study. This conclusion is relevant to the ongoing discussion about the potential pitfalls of morpholino use as compared to the validity of conclusions based on morpholino reagents.

该实验室的关注点是对与神经rest及其衍生物（例如色素细胞，咽弓等）形成的基因的研究。我们发现，最初在神经板边界的胚胎中最初表达的含BTB结构域的蛋白KCTD15可能在调节该结构域中起作用，在调节该结构域中，神经rest形成KCTD15的过表达强烈抑制了神经rest rest的指定。我们发现，KCTD15抑制转录因子AP-2的活性，转录因子AP-2的活性对于不同动物中神经rest的规范至关重要。已经进行了有关AP-2和KCTD15之间相互作用的机械研究。 AP-2是神经rest形成的主要调节剂，参与了神经rest前体细胞的初步诱导以及在后期阶段，例如它们的迁移和分化为多个衍生物。 KCTD15能够在共免疫沉淀实验中结合AP-2，并抑制培养细胞和斑马鱼胚胎中AP-2报告剂的激活。在研究这种抑制作用的机制时，我们发现KCTD15不会影响细胞中的AP-2水平，不会抑制AP-2的二聚化，而AP-2的二聚化，并且不会影响AP-2的核定位。同样，在KCTD15存在下，AP-2仍与其染色质中的同源位点结合。为了进一步探测机制，我们研究了AP-2的激活结构域。 KCTD15与激活结构域结合，特别需要脯氨酸59进行这种相互作用。该脯氨酸变为丙氨酸（p59a）的AP-2突变体在报告基因测定中活跃，但不能结合KCTD15，并且KCTD15没有有效抑制其活性。我们得出的结论是，KCTD15通过特异性结合其活化域抑制了AP-2活性，从而抑制其功能。在对KCTD15的进一步研究中，我们发现该蛋白在SUMO1和SUMO2/3的体内被体内sumoyl。我们确定了相扑附着的部位，并表明相关的赖氨酸残基的突变废除了Sumoylation。非肿瘤化的KCTD15在抑制AP-2功能方面完全有效，但是KCTD15的稳定融合蛋白和SUMO无效。我们得出的结论是，KCTD15在AP-2抑制作用中的非湿润形式的功能，但请打开sumoymet形式在KCTD15的不同生物学功能中起作用的可能性。为了进一步分析KCTD15在开发中的功能，我们通过使用TALEN方法论在两个KCTD15旁系同源物中产生了移码突变。纯合KCTD15A和KCTD15B突变鱼是可行且肥沃的，没有明显的表型。但是，KCTD15A和KCTD15B的斑马鱼突变体虽然可行且肥沃，但显示出发育延迟和尺寸较小的表型。这些突变体在颅面骨骼中似乎也有某些畸形以及大脑结构的某些异常。关于大脑结构和功能的工作是与研究所中的哈罗德·伯吉斯（Harold Burgess）的合作。我们目前正在研究小尺寸表型可能是由于突变鱼效率低下的可能性。这可能是由于颅面异常或可能与观察到的大脑结构变化相关的感觉缺陷。在表征KCTD15突变鱼的另一种方法中，我们与NEI的Anand Swaroop组进行了合作，以比较RNA-Seq的野生型和突变转录组。测序是在多个发展阶段进行的，目前正在分析结果。在我们关于E3泛素连接酶在开发中的功能的项目中，我们研究了LNX2家族在斑马鱼中胰腺初始规范中的作用。我们发现编码Numb蛋白-X（LNX）2a的E3泛素连接酶配体的基因在斑马鱼胚胎的腹侧胰腺芽中表达，此外还表达了其在大脑中的表达。通过使用外显子2/内含子2剪接Morpholino敲低LNX2A导致特异性抑制了腹芽衍生的外分泌细胞类型的分化，对内分泌细胞类型的影响很小。 Talen诱导的LNX2A中的移码为空突变并不模仿表型，在表型上是正常的。然而，LNX2A无效突变体背景中LNX2B基因的敲低模仿了剪接形态的表型。进一步和最令人震惊的是，一种突变去除了外显子2剪接供体位点，该位点是由剪接Morpholino靶向的，实际上模仿了外分泌抑制表型。从这些发现中，我们得出的结论是，LNX2B的旁系同源物lnx2a以多余的方式发挥作用。 LNX2A外显子2/3剪接的抑制会导致外显子2跳动，并导致产生N截断的蛋白质，该蛋白充当干扰分子。因此，以抑制外分泌细胞分化为特征的表型需要灭活LNX2A和LNX2B。为了进一步分析胰腺中LNX2功能的机制，我们是从其他人的早期观察结果开始的，即人类LNX1破坏了麻木。我们可以证明，抑制Numb表达会挽救LNX2A/B缺乏表型，这与假设Numb是胰腺中LNX2的靶标的假设。 Numb是Notch信号通路的已知抑制剂，已知Notch在胰腺分化中具有关键作用。此外，我们发现抑制LNX2A/B会导致胰腺中缺口活性细胞的数量减少。因此，我们建议LNX2A/B的功能来微调在斑马鱼早期胰腺中细胞类型的分化中对Notch的调节。这项研究的另一个结果是阐明基因型，表型和形态效应之间可能存在的复杂关系。在本例中，形态敲低的后果与无效突变之间的分歧不是形态上的非特异性效应的证据，而是更复杂的关系，只能通过其他研究才能揭示。与基于形态的试剂的结论有效性相比，该结论与关于形态使用的潜在陷阱的持续讨论有关。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Cloning and developmental expression of zebrafish pdzrn3.

DOI：
10.1387/ijdb.113437ld
发表时间：
2011
期刊：
The International journal of developmental biology
影响因子：
0
作者：
Dente L;Gestri G;Tsang M;Kudoh T;Wilson SW;Dawid IB;Andreazzoli M
通讯作者：
Andreazzoli M

Brd4 associates with mitotic chromosomes throughout early zebrafish embryogenesis.

Brd4 在整个早期斑马鱼胚胎发生过程中与有丝分裂染色体相关。

DOI：
10.1002/dvdy.21576
发表时间：
2008
期刊：
Developmental dynamics : an official publication of the American Association of Anatomists
影响因子：
0
作者：
Toyama,Reiko;Rebbert,MarthaL;Dey,Anup;Ozato,Keiko;Dawid,IgorB
通讯作者：
Dawid,IgorB

XIer2 is required for convergent extension movements during Xenopus development.

XIer2 是爪蟾发育过程中收敛伸展运动所必需的。

DOI：
10.1387/ijdb.113288sh
发表时间：
2011
期刊：
The International journal of developmental biology
影响因子：
0
作者：
Hong,Sung-Kook;Tanegashima,Kosuke;Dawid,IgorB
通讯作者：
Dawid,IgorB

Habenular commissure formation in zebrafish is regulated by the pineal gland-specific gene unc119c.

斑马鱼的缰核连合形成受到松果体特异性基因 unc119c 的调节。

DOI：
10.1002/dvdy.23994
发表时间：
2013
期刊：
Developmental dynamics : an official publication of the American Association of Anatomists
影响因子：
0
作者：
Toyama,Reiko;Kim,MiHa;Rebbert,MarthaL;Gonzales,John;Burgess,Harold;Dawid,IgorB
通讯作者：
Dawid,IgorB

Alpha2 macroglobulin-like is essential for liver development in zebrafish.