Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St/LRB815, Worcester, MA, USA.
Neural Dev. 2018 Jun 26;13(1):13. doi: 10.1186/s13064-018-0112-y.
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.
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.
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.
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.
先前旨在理解调控后脑尾段形成的基因调控网络（GRNs）的工作，鉴定了形态发生素如视黄酸（RA）和纤维母细胞生长因子（FGFs），以及转录因子如 hoxb1b、hoxb1a、hnf1ba 和 valentino 等，是斑马鱼后脑尾段（r）r4-r6 形成所必需的。鉴于后脑尾段相对复杂 - 例如，每个 r 节段形成独特的神经元集合 - 很可能还有其他必需基因有待鉴定并整合到后脑尾段 GRN 中。
我们利用 Zebrafish Information Network（ZFIN）中提供的基因表达数据，鉴定了 84 个在 r4-r6 中表达的未被表征的基因。我们选择了一组具有代表性的 22 个基因，并在 hoxb1b、hoxb1a、hnf1b 和 valentino 突变体中检测它们的表达模式，目的是将它们定位到后脑尾段 GRN 中。我们还研究了 RA 和 FGF 对这个基因集表达的影响。为了研究这些基因是否对 r4-r6 的发育是必需的，我们分析了 6 个基因（gas6、gbx1、sall4、eglf6、celf2 和 greb1l）的种系突变体在后脑发育方面是否存在缺陷。
我们的结果表明，单个 hoxb1b、hoxb1a 或 RA 的缺失并不影响 r4 基因的表达，但 RA 与 hoxb1b 的组合调控 r4 基因的表达。相比之下，r5/r6 基因的表达依赖于 RA、FGF、hnf1ba 和 valentino - 因为这些因子的单个缺失会使 r5/r6 基因的表达消失。我们对六个突变系的分析未显示 r 节段或神经元缺陷，但对其中一个系（gas6 突变体）的转录组分析鉴定了涉及几个发育过程的基因的表达变化 - 表明这些基因可能在后脑发育中有微妙的作用。
我们得出结论，r4-r6 的形成相对稳健，因此这个过程只需要很少的基因。然而，r4 与 r5/r6 之间存在机制上的差异，即单个因子不是 r4 发育所必需的，而几个基因是 r5/r6 形成所必需的。
