Duplicated zebrafish insulin‐like growth factor binding protein‐5 genes with split functional domains: evidence for evolutionarily conserved IGF binding, nuclear localization, and transactivation activity

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154240/1/fsb2fj09149435.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154240/2/fsb2fj09149435-sup-0001.pd

Duplication and Diversification of the Hypoxia-Inducible IGFBP-1 Gene in Zebrafish

Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes.We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1). IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker.These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions

追いつき成長における神経堤細胞のインスリン様活性の生理的意義の解明

金沢大学理工研究域生命理工学系本研究では、個体発生を支える神経堤細胞（NCCs）が個体成長を促進する インスリン・インスリン様シグナリング（IIS）の働きをどのように利用し胚の成長遅滞や追いつき成長がおこるのかゼブラフィッシュを用いた実験系により調べた。その結果NCCsでは低酸素状態でもIISの活性が最低限維持されており、このことがNCCsの生存及び追いつき成長の達成に必要であることが分かった。また、次世代シーケンサーを用いた解析から、胚の成長遅滞と追いつき成長で発現が変動する新しい成長関連遺伝子を見出した。これらの成果は、胎生期の成長阻害に端を発する様々な生理的変化を理解することの一助となることが期待される。Most animals retard growth in response to adverse conditions; however, upon the removal of unfavorable factors, they often show quick growth restoration, which is known as “catch-up” growth. In this study, by using zebrafish embryo model of hypoxia/reoxygenation-induced catch-up growth, I aimed to know how the neural crest cells (NCCs) and insulin/insulin-like growth factor signaling (IIS) play roles in this phenomenon. I found that the viability of NCCs was maintained under hypoxia with the activation of IIS at certain level, and it was seemingly crucial for the survival of these cells during the stunting period. In addition, by next generation sequencing analysis, previously unreported “catch-up growth”-related genes were discovered in this study. These results could be an aid for decoding the mechanism underlying various physiological changes including adult onset disease induced by growth arrest and by subsequent growth restoration at the early stages of animal growth.研究課題/領域番号:15K18799, 研究期間(年度):2015-04-01 - 2017-03-3

初期胚に見出された新規ホスファチジルイノシトール3キナーゼ結合分子の機能解析

金沢大学理工研究域生命理工学系本研究では、正常な発生に必要不可欠な分子であるホスファチジルイノシトール３キナーゼの触媒サブユニットβ（Pik3cb）の働きが初期胚の細胞でどのように調節されているのか調べる為、マウス胚性幹細胞（mESCs）やゼブラフィッシュ胚を用いてPik3cbとその結合分子に関する解析を行った。その結果、mESCsでは成体組織とは異なるタイプのPik3cbや結合分子が複合体を形成し、その活性が調節されることが示唆された。また、ゼブラフィッシュにおいてPik3cbと複合体を形成する分子を多数クローニングする事もできた。得られた成果は初期胚のPik3cbの働きを理解する上で有用な知見となる事が期待される。Phosphatidylinositol 3-kinase catalytic beta (Pik3cb) plays crucial role in early embryonic survival and development; however, yet the detailed molecular mechanism regulating its embryonic action has been poorly understood. Here the embryonic Pik3cb and its associating proteins were studied in mouse embryonic stem cells (mESCs) and zebrafish embryo. The cDNA sequencing and subsequent biochemical analyses revealed that mESCs express a unique splicing variant of Pik3cb, which can interact with embryo specific associating protein (Pik3cbAP) only when growth factor signaling is activated. The embryonic cell specific Pik3cb-Pik3cbAP complex is seemingly important for activating the enzymatic function of Pik3cb. Furthermore, cDNAs encoding other potential Pik3cb associating molecules were cloned and their embryonic actions were tested in zebrafish embryo. These data will help to decipher the molecular mechanisms governing embryonic Pik3cb function and developmental competency.研究課題/領域番号:25850220, 研究期間(年度):2013-04-01 - 2015-03-3

初期胚発生特異的ホスファチジルイノシトール3キナーゼ活性制御タンパク質の探索

本研究では、初期胚の品質とホスファチジルイノシトール3キナーゼ*(以下PI3K)活性がどのように連動し、どのような分子がそこに関与するか調べる為、主にゼブラフィッシュ胚やマウス胚性幹細胞を用いた解析を行った。まずPI3K触媒サブユニットの各サブタイプ(p110αおよびβ)の特異的阻害剤を用いた実験から、胚発生のごく初期においてはp110βの活性化が重要であることが示された。またマウス胚性幹細胞を用いた実験系においてp110βと結合する分子群の解析を行った。その結果、数種類の新たなp110β結合分子群を見出した。現在、新たに同定された分子がPI3Kの活性や胚の品質管理にどのような意味を持つのか更に詳しく調べている。用語説明*ホスファチジルイノシトール3キナーゼ(PI3K):細胞の生存に必須であり、同時に細胞増殖や分化、細胞運動にも重要な働きをもつことが知られているシグナル伝達タンパク質。重要な生体膜脂質であるホスファチジルイノシトールをリン酸化する酵素として知られている。Embryonic development is orchestrated by numerous growth factors and cytokines, including insulin-like growth factors. Phosphatidylinositol 3-kinase (PI3K) operates as a pivotal node in their cellular signaling cascades. Albeit its physiological relevance and importance is well acknowledged, little is known about the regulatory mechanisms of PI3K in early stages of embryogenesis. Here we examine the regulation of early embryonic PI3K by utilizing zebrafish embryo and mouse embryonic stem cell (mESC) models. Pharmacological blockade of PI3K activity in zebrafish embryo showed that the early embryonic PI3K particularly in the developmental stage preceding zygotic gene expression was prerequisite for embryonic survival whereas the inhibition at subsequent organogenesis stages caused developmental delay without causing severe lethality. In addition, the p85-bound PI3K activities in early zebrafish embryos were not limited simply by the p85 protein level nor by its bound phospho-tyrosine level. These data imply a previously unknown PI3K regulatory mechanism(s) in early embryogenesis which is important for embryonic survival. Indeed, LC-MS/MS analysis of p110 PI3K catalytic subunit immunoprecipitated from mESC lysate revealed several novel p110β associating molecules. Based on these results, we proposed an “embryo specific”PI3K regulatory mechanism, which is regulated by previously unknown PI3K-associated proteins.研究課題/領域番号:23880008, 研究期間(年度):2011-201

エネルギーセンサーが操るインスリン様シグナルと追いつき成長

金沢大学理工研究域生命理工学系『追いつき成長』は何らかの原因で一時的に成長遅滞に陥った個体が『遅滞要因の除去により成長を急加速させる現象』であり、様々な動物の胚や成長期の個体で観察される。本研究では、ゼブラフィッシュ胚を酸素の欠乏・再供給に供することで追いつき成長を誘導する独自の実験系を用いて「遅滞後の成長再開時には細胞のエネルギー感知の中核を担う脱アセチル化酵素サーチュイン（Sirt）が体成長を支えるインスリン・インスリン様成長因子シグナル（IIS）の下流に位置するMAPキナーゼの働きを修飾し、その働きを増強することが加速成長の誘導に必要になる」という新しい体成長の仕組みを示すことができた。Catch-up growth is a phenomenon in which an individual that has temporarily fallen into growth retardation for some reason rapidly accelerates its growth upon removal of the adverse condition, and is observed in embryos and growing individuals of various animals.In this study, using an original experimental system in which zebrafish embryos were subjected to oxygen deprivation and resupply to induce catch-up growth, we found that, when growth resumes after retardation, the deacetylase sirtuin (Sirt) that plays a central role in cellular energy sensing modified the insulin-like growth factor-MAPK signaling to induce the catch-up growth.研究課題/領域番号: 18K06014, 研究期間(年度):2018-04-01 - 2021-03-31出典：「エネルギーセンサーが操るインスリン様シグナルと追いつき成長Cellular energy-sensing mechanism modulates insulin-like growth factor signaling to facilitate catch-up growth.」研究成果報告書　課題番号18K06014（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-18K06014/18K06014seika/）を加工して作

Zebrafish IGF genes: gene duplication, conservation and divergence, and novel roles in midline and notochord development.

Insulin-like growth factors (IGFs) are key regulators of development, growth, and longevity. In most vertebrate species including humans, there is one IGF-1 gene and one IGF-2 gene. Here we report the identification and functional characterization of 4 distinct IGF genes (termed as igf-1a, -1b, -2a, and -2b) in zebrafish. These genes encode 4 structurally distinct and functional IGF peptides. IGF-1a and IGF-2a mRNAs were detected in multiple tissues in adult fish. IGF-1b mRNA was detected only in the gonad and IGF-2b mRNA only in the liver. Functional analysis showed that all 4 IGFs caused similar developmental defects but with different potencies. Many of these embryos had fully or partially duplicated notochords, suggesting that an excess of IGF signaling causes defects in the midline formation and an expansion of the notochord. IGF-2a, the most potent IGF, was analyzed in depth. IGF-2a expression caused defects in the midline formation and expansion of the notochord but it did not alter the anterior neural patterning. These results not only provide new insights into the functional conservation and divergence of the multiple igf genes but also reveal a novel role of IGF signaling in midline formation and notochord development in a vertebrate model

Duplication of the IGFBP-2 gene in teleost fish: protein structure and functionality conservation and gene expression divergence.

BACKGROUND: Insulin-like growth factor binding protein-2 (IGFBP-2) is a secreted protein that binds and regulates IGF actions in controlling growth, development, reproduction, and aging. Elevated expression of IGFBP-2 is often associated with progression of many types of cancers. METHODOLOGY/PRINCIPAL FINDINGS: We report the identification and characterization of two IGFBP-2 genes in zebrafish and four other teleost fish. Comparative genomics and structural analyses suggest that they are co-orthologs of the human IGFBP-2 gene. Biochemical assays show that both zebrafish igfbp-2a and -2b encode secreted proteins that bind IGFs. These two genes exhibit distinct spatiotemporal expression patterns. During embryogenesis, IGFBP-2a mRNA is initially detected in the lens, then in the brain boundary vasculature, and subsequently becomes highly expressed in the liver. In the adult stage, liver has the highest levels of IGFBP-2a mRNA, followed by the brain. Low levels of IGFBP-2a mRNA were detected in muscle and in the gonad in male adults only. IGFBP-2b mRNA is detected initially in all tissues at low levels, but later becomes abundant in the liver. In adult males, IGFBP-2b mRNA is only detected in the liver. In adult females, it is also found in the gut, kidney, ovary, and muscle. To gain insights into how the IGFBP-2 genes may have evolved through partitioning of ancestral functions, functional and mechanistic studies were carried out. Expression of zebrafish IGFBP-2a and -2b caused significant decreases in the growth and developmental rates and their effects are comparable to that of human IGFBP-2. IGFBP-2 mutants with altered IGF binding-, RGD-, and heparin-binding sites were generated and their actions examined. While mutating the RGD and heparin binding sites had little effect, altering the IGF binding site abolished its biological activity. CONCLUSIONS/SIGNIFICANCE: These results suggest that IGFBP-2 is a conserved regulatory protein and it inhibits growth and development primarily by binding to and inhibiting IGF actions in vivo. The duplicated IGFBP-2 genes may provide additional flexibility in the regulation of IGF activities