doi: 10.1161/01.RES.0000217601.30361.b1
© 2006 American Heart Association, Inc.
Editorials |
Fishing for Function in Zebrafish
From the Cardiovascular Research Institute and Department of Medicine, University of Rochester School of Medicine, Rochester, NY.
Correspondence to Joseph M. Miano, University of Rochester School of Medicine, 601 Elmwood Ave, Rochester, New York 14642. E-mail j.m.miano{at}rochester.edu
See related article, pages 846–855
Key Words: smooth muscle • zebrafish • morpholino • acetyltransferase • rapamycin
Zebrafish have emerged as a powerful and inexpensive vertebrate model system to study the regulation and function of genes. Though this model has been under investigation for more than half a century,1 97% of the published work relating to zebrafish has accumulated in only the last 15 years (Figure). Two important developments have facilitated the rapid ascent of zebrafish as a model system, particular for vascular biology. First, forward genetic screens in zebrafish identified a number of fascinating vascular phenotypes such as gridlock (aortic coarctation) and cloche (loss of endothelial cells [ECs]).2,3 Second, microangiography, coupled to transgenic lines of fish carrying an endothelial cell promoter–driven reporter (eg, green fluorescent protein, GFP), has allowed for the mapping of the zebrafish vasculature in stunning detail with never-before-seen vascular processes amenable to real time visualization.4 These technologies, and evolving tools in reverse genetics, have led to the identification of numerous genes linked to vascular pathology, some of which phenocopy diseases seen in humans (Table). As the Table indicates, essentially all of the zebrafish vascular phenotypes involve perturbations in endothelial cell differentiation, migration, and vascular tube formation with little insight into the role of supportive smooth muscle cells (SMCs). Difficulty in assessing vascular SMC biology in zebrafish stems from our incomplete understanding of the timing of SMC recruitment to the vessel wall, the extent of SMC investment, and the notable lack of SMC markers. Thus, to realize the full potential of zebrafish as a model for vascular biology, developments in these areas of research are necessary, particularly given the importance of SMC in mammalian vascular pathobiology.
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Normal adult vascular SMCs exhibit a quiescent sessile phenotype to carry out their primary function of contraction. The contractile phenotype is compromised in diseases of the vessel wall where SMCs are seen to migrate, proliferate, and elaborate an extensive extracellular matrix within an evolving new layer of the vessel wall—the neointima. This process of SMC phenotypic modulation appears to involve the reinstatement of an embryonic program of gene expression with at least a temporary suspension of the adult contractile gene program.5 In support of this concept are seminal studies from the laboratory of the late Dr Richard Majack demonstrating that embryonic and neointimal SMCs exhibit autonomous growth as compared with late fetal-adult SMCs which show nonautonomous growth potential.6,7 This implies the existence of distinct programs of gene expression at the embryonic-fetal interface as rapidly growing SMC accumulate more contractile proteins and begin entering a state of quiescence. What might the genes be that reduce SMC growth while maintaining a contractile phenotype at this interface? One candidate could be myocardin, which exhibits high expression in mature SMCs, activates contractile gene expression, and suppresses growth, only to be attenuated when SMCs lose their contractile phenotype.8,9 In this model, reduced myocardin expression might also allow for the emergence of an autonomous growth phenotype, perhaps by allowing the factor it binds (serum response factor) to associate with other proteins that direct growth as opposed to quiescence.10
And what might those genes be that become activated in the transition from nonautonomous growth to autonomous (disease-associated) growth? Several screening studies have sought to address this question with work from one of Dr Majack’s protégés of special note in present context. Using a subtractive hybridization approach, Weiser-Evans et al cloned out several novel genes that were preferentially expressed in SMCs with an autonomous-replicating phenotype.11 One of the novel genes, called embryonic growth-associated protein (EGAP), showed expression that paralleled the embryonic-fetal growth curve with reactivated expression after vascular injury to the adult vessel wall. Antisense oligonucleotides to EGAP attenuated the autonomous growth phenotype in both embryonic and neointimal SMCs, suggesting the protein functioned in some capacity to effectuate growth.11 Now, in this issue of Circulation Research,12 the same laboratory has provided further insight into the identity of EGAP and its potential function in vivo using zebrafish as a model system.
Amino acid homology studies of EGAP revealed striking conservation (21%) with a yeast protein (Mak10p) encoding a noncatalytic subunit of a multiprotein complex involved in cotranslational acetylation of N-terminal amino acids in an estimated 85% of eukaryotic proteins.13 There are three N-terminal acetyltransferases (NatA, NatB, and NatC), each having a preferential bias for N-terminal amino acid acetylation.14 EGAP (hereafter referred to by its official HUGO Nomenclature Committee assigned gene symbol, MAK10) and its two associated subunits (MAK3 and MAK31) comprise the NatC complex.14 Authors cloned MAK3 and MAK10 and performed fluorescence confocal microscopy and coimmunoprecipitation studies to show physical and functional association among all three subunits. Consistent with the earlier antisense knockdown studies of MAK10,11 growth potential could only be realized if all three subunits were present in a cell. To begin exploring the role of MAK10 in vivo, authors turned to zebrafish and showed first that expression of zMAK10 was expressed in blood vessels at a time when circulation commences (
24 hours after fertilization); for reasons discussed above, it was not possible to distinguish endothelial cell from SMC expression of zMAK10. The authors then designed morpholino oligonucleotides (MO) to splice donor sites in zMAK10 and zMAK3 to effectively knockdown endogenous expression of each respective subunit. Injected embryos were then examined for signs of developmental defects. Both morphants showed evidence of developmental delay as revealed by reduced somite number, body length, and DNA synthesis. To show the developmental delay was specific to the gene knocked down and not secondary to some other effect, rescue experiments were performed successfully with wild-type zMAK10. Thus, for the first time, authors have shown the requirement for a NAT complex in normal vertebrate development.
To evaluate the role of zMAK10 in zebrafish vascular development, the authors knocked down zMAK10 in a transgenic line carrying an endothelial-restricted reporter (Fli1-EGFP) and then followed vascular development with fluorescence microscopy. Compared with controls, zMAK10 knockdown resulted in disorganized vessels with abnormal vascular lumen formation and truncated intersomitic vessels, phenotypes that could be rescued with wild-type zMAK10. These vascular perturbations likely stem from defects in endothelial cell migration/differentiation though a role in SMCs cannot formally be ruled out. Based on previous work from the Weiser-Evans laboratory showing target of rapamycin (TOR) as an essential mediator of SMC growth,15 as well as its N-terminal amino acid sequence (M-L) matching the preferred sequence for NatC-mediated acetylation,14 authors next evaluated the expression of zTOR in wild-type versus zMAK10 knockdown animals. Results from both whole mount immunohistochemistry and Western blotting revealed a profound decrease in expression of zTOR with zMAK10 knockdown. The mechanism for reduced TOR expression is unclear at this time, but it is intriguing to consider the possibility that TOR is a direct substrate for NatC and that acetylation of TOR is necessary for its stabilization. In a final series of elegant experiments, authors used rapamycin (inhibitor of TOR) to show similar vascular defects seen with the zMAK10 knockdown and then rescued the zMAK10 phenotype with a constitutively active mTOR, suggesting that TOR indeed lies within the zMAK10 pathway either as a direct substrate or a downstream target of another substrate. Collectively, these novel findings offer a model for cellular growth in vivo wherein NatC acetylates TOR to mediate autonomous growth potential. It will be important to further evaluate this model and assess its activity in vascular SMCs.
The genomics revolution has taught us much about the nature of life. For one, there seems to be little association between genome size and complexity of an organism; our genome (3.1 Gb) is only 20% of that in wheat (16 Gb), and both overshadow that of pufferfish (0.4 Gb). In addition, the number of genes in a genome is often disproportionate to the complexity of a species; the nematode, Caenorhabditis elegans, for example, has nearly as many genes (19 000) as humans (23 000). Therefore, to achieve the requisite integrative systems biology present in complex species such as our own, a large (100 000) proteome exists. Superimposed on this level of protein diversity are the hundreds of (co)posttranslational chemical modifications, including N-terminal acetylation, that impart specific instructions dictating the function (or fate) of a protein in any given cellular context. The report by Wenzlau et al12 documents, for the first time, the essential requirement of one specific subunit (MAK10) of an N-terminal acetyltransferase (NatC) in a vertebrate system and hints at a potential substrate (TOR) known to be of critical importance in growth processes. The challenges ahead will be to define the substrates of NatC and delineate the functional consequences of this cotranslational modification, particularly as they may relate to vascular SMC growth during development and in disease. Zebrafish will continue to serve as an attractive model system for vascular biology both in terms of elucidating (co)posttranslational modifications of proteins and their functions and in the high throughput screening16 that surely lies ahead as we continue to define drugable targets in the vessel wall for therapeutic means.
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Acknowledgments |
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The author is supported by grants from the National Institutes of Health (HL-62572 and HL-70077) and is an Established Investigator of the American Heart Association (0340075N). He dedicates his work to A.M.M. and E.A.M.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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Related Article:
Circ. Res. 2006 98: 846-855.
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