Mackereth Melinda D, Kwak Su-Jin, Fritz Andreas, Riley Bruce B
Department of Biology, Emory University, Atlanta, GA 30322, USA.
Development. 2005 Jan;132(2):371-82. doi: 10.1242/dev.01587. Epub 2004 Dec 16.
Vertebrate Pax2 and Pax8 proteins are closely related transcription factors hypothesized to regulate early aspects of inner ear development. In zebrafish and mouse, Pax8 expression is the earliest known marker of otic induction, and Pax2 homologs are expressed at slightly later stages of placodal development. Analysis of compound mutants has not been reported. To facilitate analysis of zebrafish pax8, we completed sequencing of the entire gene, including the 5' and 3' UTRs. pax8 transcripts undergo complex alternative splicing to generate at least ten distinct isoforms. Two different subclasses of pax8 splice isoforms encode different translation initiation sites. Antisense morpholinos (MOs) were designed to block translation from both start sites, and four additional MOs were designed to target different exon-intron boundaries to block splicing. Injection of MOs, individually and in various combinations, generated similar phenotypes. Otic induction was impaired, and otic vesicles were small. Regional ear markers were expressed correctly, but hair cell production was significantly reduced. This phenotype was strongly enhanced by simultaneously disrupting either of the co-inducers fgf3 or fgf8, or another early regulator, dlx3b, which is thought to act in a parallel pathway. In contrast, the phenotype caused by disrupting foxi1, which is required for pax8 expression, was not enhanced by simultaneously disrupting pax8. Disrupting pax8, pax2a and pax2b did not further impair otic induction relative to loss of pax8 alone. However, the amount of otic tissue gradually decreased in pax8-pax2a-pax2b-deficient embryos such that no otic tissue was detectable by 24 hours post-fertilization. Loss of otic tissue did not correlate with increased cell death, suggesting that otic cells dedifferentiate or redifferentiate as other cell type(s). These data show that pax8 is initially required for normal otic induction, and subsequently pax8, pax2a and pax2b act redundantly to maintain otic fate.
脊椎动物的Pax2和Pax8蛋白是密切相关的转录因子，据推测它们可调节内耳发育的早期阶段。在斑马鱼和小鼠中，Pax8的表达是已知最早的耳诱导标志物，而Pax2同源物在基板发育的稍晚阶段表达。尚未见关于复合突变体的分析报道。为便于对斑马鱼pax8进行分析，我们完成了整个基因的测序，包括5'和3'非翻译区。pax8转录本经历复杂的可变剪接，产生至少十种不同的异构体。pax8剪接异构体的两个不同亚类编码不同的翻译起始位点。设计反义吗啉代寡核苷酸（MO）以阻断两个起始位点的翻译，并设计另外四种MO靶向不同的外显子-内含子边界以阻断剪接。单独或组合注射MO产生了相似的表型。耳诱导受损，耳泡较小。耳部区域标志物表达正确，但毛细胞产生显著减少。同时破坏共同诱导因子fgf3或fgf8，或另一个早期调节因子dlx3b（被认为在平行途径中起作用）会强烈增强这种表型。相比之下，破坏pax8表达所需的foxi1所导致的表型，不会因同时破坏pax8而增强。相对于单独缺失pax8，破坏pax8、pax2a和pax2b不会进一步损害耳诱导。然而，在pax8-pax2a-pax2b缺陷的胚胎中，耳组织的量逐渐减少，以至于在受精后24小时无法检测到耳组织。耳组织的丧失与细胞死亡增加无关，这表明耳细胞去分化或重新分化为其他细胞类型。这些数据表明，pax8最初是正常耳诱导所必需的，随后pax8、pax2a和pax2b起冗余作用以维持耳命运。
