Mutations in gfpt1 and skiv2l2 cause distinct stage-specific defects in larval melanocyte regeneration in zebrafish.
Mutations in gfpt1 and skiv2l2 cause distinct stage-specific defects in larval melanocyte regeneration in zebrafish.
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DOI：
10.1371/journal.pgen.0030088
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发表时间：
2007-06
期刊：
影响因子：
4.5
通讯作者：
Johnson, Stephen L.
中科院分区：
文献类型：
--
作者：
Yang, Chao-Tsung;Hindes, Anna E.;Hultman, Keith A.;Johnson, Stephen L.
在斑马鱼中建立单细胞再生模式为研究再生过程的遗传机制提供了机会。我们之前已经证明，在小分子MoTP消融幼体黑素细胞后，再生黑素细胞产生于静止的黑素细胞前体的细胞分裂。MoTP易于消融黑素细胞，这使我们能够对这种前体或干细胞再生的特定机制进行正向遗传筛选。在这里，我们报道了两个突变体的鉴定，eartha j23e1和julie j24e1从黑素细胞消融筛选。这两种突变体都发育成正常的幼虫黑素细胞，但在黑素细胞消融后，每种突变都会导致黑素细胞再生中明显的阶段特异性缺陷。定位克隆表明，地球j23e1突变为谷氨酰胺-6-磷酸生物合成限速酶gfpt1（谷氨酰胺：果糖-6-磷酸转氨酶1）的无义突变。我们的分析表明，gfpt1突变在再生后期特异性影响黑素细胞分化（以黑色素产生为标志），并且gfpt1在黑素细胞中细胞自主作用，促进黑色素细胞的个体发生变暗。我们发现julie j24e1突变是skiv2l2（超级杀手病毒杀活性2-like 2）的剪接位点突变，skiv2l2是一种预测的DEAD-box RNA解旋酶。我们的原位分析显示，skiv2l2的突变导致细胞增殖缺陷，这表明skiv2l2在黑素细胞再生的早期阶段调节成黑素细胞增殖起作用。这一发现与先前描述的在幼虫黑素细胞再生过程中细胞分裂的作用一致。对这些突变体的分析揭示了它们在黑素细胞再生中的阶段特异性作用。有趣的是，这些突变体不仅在再生过程的早期阶段识别再生特异性功能，而且在再生黑素细胞分化的后期也识别再生特异性功能。我们认为，在这个突变体筛选中发现的再生机制可能揭示了胚胎发生过程中建立分化细胞的机制与幼虫或成体生长过程中建立分化细胞的机制之间的根本差异。个体发育和再生的程序有许多共同的组成部分。再生和发育之间的遗传需求差异可能确定干细胞在胚胎后阶段维持细胞群的特定机制，或确定其他再生特异性功能。在这里，我们利用一种前向遗传方法，利用单细胞类型消融和再生来分离斑马鱼黑素细胞再生的特定机制。在化学消融黑素细胞后，斑马鱼幼虫从未分化的前体或干细胞中重建其幼虫的色素模式。我们分离了两种斑马鱼突变体，它们正常发育胚胎黑素细胞，但在消融后不能再生黑素细胞。这种表型表明突变基因的再生特异性作用。我们进一步鉴定了gfpt1和skiv2l2的突变，并显示了它们在黑素细胞再生中的阶段特异性作用。有趣的是，这些突变体不仅在再生过程的早期阶段（skiv2l2）识别再生特异性功能，而且在再生黑素细胞分化的后期阶段（gfpt1）也识别再生特异性功能。我们认为，在这个突变体筛选中确定的再生机制可能揭示了胚胎发生过程中建立分化细胞的机制与幼虫或成体生长过程中建立分化细胞的机制之间的根本差异。
The establishment of a single cell type regeneration paradigm in the zebrafish provides an opportunity to investigate the genetic mechanisms specific to regeneration processes. We previously demonstrated that regeneration melanocytes arise from cell division of the otherwise quiescent melanocyte precursors following larval melanocyte ablation with a small molecule, MoTP. The ease of ablating melanocytes by MoTP allows us to conduct a forward genetic screen for mechanisms specific to regeneration from such precursors or stem cells. Here, we reported the identification of two mutants, eartha j23e1 and julie j24e1 from a melanocyte ablation screen. Both mutants develop normal larval melanocytes, but upon melanocyte ablation, each mutation results in a distinct stage-specific defect in melanocyte regeneration. Positional cloning reveals that the eartha j23e1 mutation is a nonsense mutation in gfpt1 (glutamine:fructose-6-phosphate aminotransferase 1), the rate-limiting enzyme in glucosamine-6-phosphate biosynthesis. Our analyses reveal that a mutation in gfpt1 specifically affects melanocyte differentiation (marked by melanin production) at a late stage during regeneration and that gfpt1 acts cell autonomously in melanocytes to promote ontogenetic melanocyte darkening. We identified that the julie j24e1 mutation is a splice-site mutation in skiv2l2 (superkiller viralicidic activity 2-like 2), a predicted DEAD-box RNA helicase. Our in situ analysis reveals that the mutation in skiv2l2 causes defects in cell proliferation, suggesting that skiv2l2 plays a role in regulating melanoblast proliferation during early stages of melanocyte regeneration. This finding is consistent with previously described role for cell division during larval melanocyte regeneration. The analyses of these mutants reveal their stage-specific roles in melanocyte regeneration. Interestingly, these mutants identify regeneration-specific functions not only in early stages of the regeneration process, but also in late stages of differentiation of the regenerating melanocyte. We suggest that mechanisms of regeneration identified in this mutant screen may reveal fundamental differences between the mechanisms that establish differentiated cells during embryogenesis, and those involved in larval or adult growth. Programs of ontogenetic development and regeneration share many components. Differences in genetic requirements between regeneration and development may identify mechanisms specific to the stem cells that maintain cell populations in postembryonic stages, or identify other regeneration-specific functions. Here, we utilize a forward genetic approach that takes advantage of single cell type ablation and regeneration to isolate mechanisms specific to regeneration of the zebrafish melanocyte. Upon chemical ablation of melanocytes, zebrafish larvae reconstitute their larval pigment pattern from undifferentiated precursors or stem cells. We isolated two zebrafish mutants that develop embryonic melanocytes normally but fail to regenerate their melanocytes upon ablation. This phenotype suggests the regeneration-specific roles of the mutated genes. We further identified the mutations in gfpt1 and skiv2l2 and show their stage-specific roles in melanocyte regeneration. Interestingly, these mutants identify regeneration-specific functions not only in early stages of the regeneration process (skiv2l2), but also in late stages of differentiation of the regenerating melanocyte (gfpt1). We suggest that mechanisms of regeneration identified in this mutant screen may reveal fundamental differences between the mechanisms that establish differentiated cells during embryogenesis and those involved in larval or adult growth.
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DOI：
10.1007/s10162-002-3022-x
发表时间：
2003-06-01
影响因子：
2.4
作者：
Harris, JA;Cheng, AG;Rubel, EW
通讯作者：
Rubel, EW
影响因子：
2.7
作者：
Chiang, EFL;Pai, CI;Chung, BC
通讯作者：
Chung, BC
影响因子：
56.9
作者：
Giraldez, AJ;Mishima, Y;Schier, AF
通讯作者：
Schier, AF
影响因子：
20.3
作者：
Blair, A;Hogge, DE;Sutherland, HJ
通讯作者：
Sutherland, HJ
影响因子：
64.8
作者：
HO, RK;KANE, DA
通讯作者：
KANE, DA