BACKGROUND Previous work aimed at understanding the gene regulatory networks (GRNs) governing caudal hindbrain formation identified morphogens such as Retinoic Acid (RA) and Fibroblast growth factors (FGFs), as well as transcription factors like hoxb1b, hoxb1a, hnf1ba, and valentino as being required for rhombomere (r) r4-r6 formation in zebrafish. Considering that the caudal hindbrain is relatively complex - for instance, unique sets of neurons are formed in each rhombomere segment - it is likely that additional essential genes remain to be identified and integrated into the caudal hindbrain GRN. METHODS By taking advantage of gene expression data available in the Zebrafish Information Network (ZFIN), we identified 84 uncharacterized genes that are expressed in r4-r6. We selected a representative set of 22 genes and assayed their expression patterns in hoxb1b, hoxb1a, hnf1b, and valentino mutants with the goal of positioning them in the caudal hindbrain GRN. We also investigated the effects of RA and FGF on the expression of this gene set. To examine whether these genes are necessary for r4-r6 development, we analyzed germline mutants for six of the genes (gas6, gbx1, sall4, eglf6, celf2, and greb1l) for defects in hindbrain development. RESULTS Our results reveal that r4 gene expression is unaffected by the individual loss of hoxb1b, hoxb1a or RA, but is under the combinatorial regulation of RA together with hoxb1b. In contrast, r5/r6 gene expression is dependent on RA, FGF, hnf1ba and valentino - as individual loss of these factors abolishes r5/r6 gene expression. Our analysis of six mutant lines did not reveal rhombomere or neuronal defects, but transcriptome analysis of one line (gas6 mutant) identified expression changes for genes involved in several developmental processes - suggesting that these genes may have subtle roles in hindbrain development. CONCLUSION We conclude that r4-r6 formation is relatively robust, such that very few genes are absolutely required for this process. However, there are mechanistic differences in r4 versus r5/r6, such that no single factor is required for r4 development while several genes are individually required for r5/r6 formation.

中文翻译：

---

新的尾后脑基因的分析揭示了在胚胎斑马鱼中菱形4和5/6中基因表达的不同调控逻辑。

背景技术先前旨在了解控制尾后脑形成的基因调控网络（GRN）的工作已确定了诸如视黄酸（RA）和成纤维细胞生长因子（FGF）等形态发生子，以及诸如hoxb1b，hoxb1a，hnf1ba和valentino之类的转录因子。斑马鱼中菱形（r）r4-r6形成所必需的。考虑到尾后脑相对复杂-例如，在每个菱形节段中形成了独特的神经元集-很可能还有其他重要基因有待鉴定并整合到尾后脑GRN中。方法利用斑马鱼信息网络（ZFIN）中可用的基因表达数据，我们鉴定了8​​4个在r4-r6中表达的未表征基因。我们选择了代表的22个基因组，并分析了它们在hoxb1b，hoxb1a，hnf1b和valentino突变体中的表达模式，目的是将它们定位在尾后脑GRN中。我们还研究了RA和FGF对该基因集表达的影响。为了检查这些基因对于r4-r6发育是否必需，我们分析了六个基因（gas6，gbx1，sall4，eglf6，celf2和greb11l）中后代发育缺陷的种系突变体。结果我们的结果表明，r4基因表达不受hoxb1b，hoxb1a或RA个体丢失的影响，但受RA和hoxb1b共同调控。相反，r5 / r6基因表达依赖于RA，FGF，hnf1ba和valentino-因为这些因素的个体丧失消除了r5 / r6基因表达。我们对6个突变体系的分析未发现菱形或神经元缺陷，但对1个系（gas6突变体）的转录组分析确定了参与多个发育过程的基因的表达变化-暗示这些基因在后脑发育中可能具有微妙的作用。结论我们得出结论，r4-r6的形成相对稳定，因此该过程绝对需要很少的基因。但是，r4与r5 / r6的机理不同，因此r4的发育不需要单个因素，而r5 / r6的形成则需要几个基因。但是对一个品系（gas6突变体）的转录组分析确定了参与几个发育过程的基因的表达变化-暗示这些基因在后脑发育中可能具有微妙的作用。结论我们得出结论，r4-r6的形成相对稳定，因此该过程绝对需要很少的基因。但是，r4与r5 / r6的机理不同，因此r4的形成不需要单个因素，而r5 / r6的形成则需要几个基因。但是对一个品系（gas6突变体）的转录组分析确定了参与几个发育过程的基因的表达变化-暗示这些基因在后脑发育中可能具有微妙的作用。结论我们得出结论，r4-r6的形成相对稳定，因此该过程绝对需要很少的基因。但是，r4与r5 / r6的机理不同，因此r4的发育不需要单个因素，而r5 / r6的形成则需要几个基因。