Djiotsa Joachim, Verbruggen Vincianne, Giacomotto Jean, Ishibashi Minaka, Manning Elisabeth, Rinkwitz Silke, Manfroid Isabelle, Voz Marianne L, Peers Bernard
Molecular Biology and Genetic Engineering, Giga-Research, University of Liège, 1 avenue de l'Hôpital B34, Sart-Tilman B-4000, Belgium.
BMC Dev Biol. 2012 Dec 17;12:37. doi: 10.1186/1471-213X-12-37.
Genetic studies in mouse have demonstrated the crucial function of PAX4 in pancreatic cell differentiation. This transcription factor specifies β- and δ-cell fate at the expense of α-cell identity by repressing Arx gene expression and ectopic expression of PAX4 in α-cells is sufficient to convert them into β-cells. Surprisingly, no Pax4 orthologous gene can be found in chicken and Xenopus tropicalis raising the question of the function of pax4 gene in lower vertebrates such as in fish. In the present study, we have analyzed the expression and the function of the orthologous pax4 gene in zebrafish.
pax4 gene is transiently expressed in the pancreas of zebrafish embryos and is mostly restricted to endocrine precursors as well as to some differentiating δ- and ε-cells but was not detected in differentiating β-cells. pax4 knock-down in zebrafish embryos caused a significant increase in α-cells number while having no apparent effect on β- and δ-cell differentiation. This rise of α-cells is due to an up-regulation of the Arx transcription factor. Conversely, knock-down of arx caused to a complete loss of α-cells and a concomitant increase of pax4 expression but had no effect on the number of β- and δ-cells. In addition to the mutual repression between Arx and Pax4, these two transcription factors negatively regulate the transcription of their own gene. Interestingly, disruption of pax4 RNA splicing or of arx RNA splicing by morpholinos targeting exon-intron junction sites caused a blockage of the altered transcripts in cell nuclei allowing an easy characterization of the arx- and pax4-deficient cells. Such analyses demonstrated that arx knock-down in zebrafish does not lead to a switch of cell fate, as reported in mouse, but rather blocks the cells in their differentiation process towards α-cells.
In zebrafish, pax4 is not required for the generation of the first β- and δ-cells deriving from the dorsal pancreatic bud, unlike its crucial role in the differentiation of these cell types in mouse. On the other hand, the mutual repression between Arx and Pax4 is observed in both mouse and zebrafish. These data suggests that the main original function of Pax4 during vertebrate evolution was to modulate the number of pancreatic α-cells and its role in β-cells differentiation appeared later in vertebrate evolution.
小鼠遗传学研究已证明PAX4在胰腺细胞分化中具有关键作用。该转录因子通过抑制Arx基因表达来确定β细胞和δ细胞的命运，而牺牲α细胞特性，并且PAX4在α细胞中的异位表达足以将它们转化为β细胞。令人惊讶的是，在鸡和热带爪蟾中未发现Pax4直系同源基因，这就提出了pax4基因在鱼类等低等脊椎动物中的功能问题。在本研究中，我们分析了斑马鱼中直系同源pax4基因的表达和功能。
pax4基因在斑马鱼胚胎胰腺中短暂表达，主要局限于内分泌前体以及一些正在分化的δ细胞和ε细胞，但在正在分化的β细胞中未检测到。斑马鱼胚胎中pax4基因敲低导致α细胞数量显著增加，而对β细胞和δ细胞分化没有明显影响。α细胞的这种增加是由于Arx转录因子的上调。相反，敲低arx导致α细胞完全缺失，同时pax4表达增加，但对β细胞和δ细胞数量没有影响。除了Arx和Pax4之间的相互抑制外，这两种转录因子还对自身基因的转录起负调节作用。有趣的是，通过靶向外显子 - 内含子连接位点的吗啉代寡核苷酸破坏pax4 RNA剪接或arx RNA剪接会导致改变的转录本在细胞核中受阻，从而便于对arx和pax4缺陷细胞进行表征。此类分析表明，斑马鱼中敲低arx不会像在小鼠中报道的那样导致细胞命运转变，而是将细胞阻断在其向α细胞的分化过程中。
与Pax4在小鼠中对这些细胞类型分化的关键作用不同，在斑马鱼中，源自背侧胰腺芽的第一批β细胞和δ细胞的产生不需要pax4。另一方面，在小鼠和斑马鱼中均观察到Arx和Pax4之间的相互抑制。这些数据表明，在脊椎动物进化过程中，Pax4的主要原始功能是调节胰腺α细胞的数量，其在β细胞分化中的作用在脊椎动物进化后期才出现。
