Mintzer K A, Lee M A, Runke G, Trout J, Whitman M, Mullins M C
University of Pennsylvania School of Medicine, Department of Cell and Developmental Biology, Philadelphia, PA 19104-6058, USA.
Development. 2001 Mar;128(6):859-69. doi: 10.1242/dev.128.6.859.
TGFbeta signaling pathways of the bone morphogenetic protein (BMP) subclass are essential for dorsoventral pattern formation of both vertebrate and invertebrate embryos. Here we determine by chromosomal mapping, linkage analysis, cDNA sequencing and mRNA rescue that the dorsalized zebrafish mutant lost-a-fin (laf) is defective in the gene activin receptor-like kinase 8 (alk8), which encodes a novel type I TGFbeta receptor. The alk8 mRNA is expressed both maternally and zygotically. Embyros that lack zygotic, but retain maternal Laf/Alk8 activity, display a weak dorsalization restricted to the tail and die by 3 days postfertilization. We rescued the laf dorsalized mutant phenotype by alk8 mRNA injection and generated homozygous laf/alk8 mothers to investigate the maternal role of Laf/Alk8 activity. Adult fish lacking Laf/Alk8 activity are fertile, exhibit a growth defect and are significantly smaller than their siblings. Embryos derived from homozygous females, which lack both maternal and zygotic Laf/Alk8 activity, display a strongly dorsalized mutant phenotype, no longer limited to the tail. These mutant embryos lack almost all gastrula ventral cell fates, with a concomitant expansion of dorsal cell types. During later stages, most of the somitic mesoderm and neural tissue circumscribe the dorsoventral axis of the embryo. Zygotic laf/alk8 mutants can be rescued by overexpression of the BMP signal transducer Smad5, but not the Bmp2b or Bmp7 ligands, consistent with the Laf/Alk8 receptor acting within a BMP signaling pathway, downstream of a Bmp2b/Bmp7 signal. Antibodies specific for the phosphorylated, activated form of Smad1/5, show that BMP signaling is nearly absent in gastrula lacking both maternal and zygotic Laf/Alk8 activity, providing further evidence that Laf/Alk8 transduces a BMP signal. In total, our work strongly supports the role of Laf/Alk8 as a type I BMP receptor required for the specification of ventral cell fates.
骨形态发生蛋白（BMP）亚类的转化生长因子β（TGFβ）信号通路对于脊椎动物和无脊椎动物胚胎的背腹模式形成至关重要。在此，我们通过染色体定位、连锁分析、cDNA测序和mRNA拯救确定，背化的斑马鱼突变体失鳍（laf）在激活素受体样激酶8（alk8）基因中存在缺陷，该基因编码一种新型的I型TGFβ受体。alk8 mRNA在母源和合子阶段均有表达。缺乏合子但保留母源Laf/Alk8活性的胚胎，表现出局限于尾部的轻微背化，并在受精后3天死亡。我们通过注射alk8 mRNA拯救了laf背化突变体表型，并产生了纯合的laf/alk8母本，以研究Laf/Alk8活性的母源作用。缺乏Laf/Alk8活性的成年鱼可育，但表现出生长缺陷，且明显小于其同胞。来自纯合雌鱼的胚胎，既缺乏母源也缺乏合子Laf/Alk8活性，表现出强烈的背化突变体表型，不再局限于尾部。这些突变胚胎几乎缺乏所有原肠胚期的腹侧细胞命运，同时背侧细胞类型相应扩展。在后期阶段，大多数体节中胚层和神经组织环绕着胚胎的背腹轴。合子laf/alk8突变体可通过过表达BMP信号转导分子Smad5而得到拯救，但不能通过过表达Bmp2b或Bmp7配体得到拯救，这与Laf/Alk8受体在Bmp2b/Bmp7信号下游的BMP信号通路中起作用一致。针对磷酸化、活化形式的Smad1/5的特异性抗体表明，在既缺乏母源也缺乏合子Laf/Alk8活性的原肠胚中，BMP信号几乎不存在，这进一步证明Laf/Alk8可转导BMP信号。总之，我们的工作有力地支持了Laf/Alk8作为确定腹侧细胞命运所需的I型BMP受体的作用。
