The Role of Fgf Signaling in Vertebrate Development

这个项目的长期目标是了解一个重要的信号配体家族，即成纤维细胞生长因子(FGFs)是如何控制广泛的细胞生物学行为的，如增殖、细胞死亡、迁移、干细胞维持和基因表达。特别是，我们使用复杂的小鼠遗传学来了解成纤维细胞生长因子信号在中胚层谱系中的作用，特别强调体轴的延伸和体节的形成(节段性中胚层片段是脊椎动物肌肉、真皮和椎体的基石)。我们的工作表明，遗传冗余是这一生物学的一个重要方面；因此，这个项目的所有工作都是为了全面表征中胚层谱系中成纤维细胞生长因子信号的遗传冗余。这项工作与许多癌症病例有关，在这些病例中，可能有不止一个成纤维细胞生长因子基因受损。为了实现这一点，我们创造了重要的Cre小鼠品系并对其进行了表征，这些品系是允许控制早期胚胎中基因表达的工具。其中包括TCre(在早期初生的中胚层中表达；见发育。132：3859-71。)，TCreERT2(在所有胚胎阶段活跃于新生的中胚层；见PLoS one。8：E62479)和Tbx4-Cre(在包括尿囊、后肢和外生殖器的后中胚层区域表达；见Dev dyn。240：2290-300。DOI：10.1002/dvdy.22731)。特别是，TCRE在该领域产生了重大影响，在50多份出版物中发挥了重要作用。例如，TCre和TCreERT2在证明Wnt5a/Ror2信号通过控制中间中胚层延伸(Hum Mol Genet)来调节肾脏形态发生的合作努力中都具有重要意义。2014年7月31日PII：ddu397)。除了为小鼠遗传学社区提供有价值的小鼠品系外，该项目还发表了一些论文，记录了我们对早期胚胎中成纤维细胞生长因子信号的主要见解。我们发表了Fgf8在体细胞发生中不是必需的，尽管一系列高调的工作已经将它放在当前模型的中心位置。然而，在与NCI的同事Alan Perantoni的合作中，我们证明了Fgf8对于肾脏和男性生殖道的发育是必不可少的。132：3859-71，发展。138：5369-78)。这些突变体的一个特征是由于Fgf8信号的丢失而导致细胞的异常死亡。在过去的一年里，我们发表了一项研究，表明我们可以通过移除促凋亡基因bax和bak来恢复这些突变体的细胞存活。这种基因操作恢复了肾脏发育的一些措施，但肾脏发生不正常，揭示了Fgf8在这一过程中的功能(分化，2023,130：7-15)。我们发现，FGF8和FGF4一起是体细胞发生所必需的：振荡基因结构域、WNT途径基因和未分化的有丝分裂前中胚层的标记的表达(《学报》，S，A。108：4018-23)。通过研究我们通过基因恢复WNT信号的成纤维细胞生长因子突变体，我们证明了在这个过程中，成纤维细胞生长因子信号独立于WNT信号起作用。我们发现的功能冗余与癌症有关，因为这两种FGFs在睾丸肿瘤中都被发现异常活跃。在最近的研究中，我们更深入地研究了这项工作，并表明Fgf4突变体(但不是Fgf8突变体)显示了一系列脊柱缺陷，这些缺陷模拟了由有缺陷的凹槽振荡引起的人类椎骨节段性缺陷的频谱。这项工作的一个关键创新是我们采用了计算建模来生成胚胎的体积子集。然后，我们量化这些体积内受Fgf4信号丢失影响的关键目标基因的mRNA水平(eLife 2020；9：e55608)。DOI：https://doi.org/10.7554/eLife.55608).我们将这项技术创新应用于与约翰·霍普金斯医学院权氏实验室就WNT信号在心脏发育中的作用发表的一项合作研究中(美国学报，2023,120(4)：e2217687120)。在最近的另一篇文章中，我们证明了Fgf8亚家族(Fgf8、Fgf17和Fgf18)是脊椎动物胚胎关闭腹侧体壁所必需的。这一过程中的缺陷是人类出生缺陷的一大类，是一个重大的健康负担(Development(2020)147，dev189506)。DOI：10.1242/dev.189506)。我们正在继续研究成纤维细胞生长因子信号中的遗传冗余在胚胎发育的几个方面。我们目前专注于两个方面的发育：产生激发条纹，产生所有的胚胎中胚层，以及体节分化成其派生的谱系(肌肉和骨骼)。

The long term goal of this project is to understand how an important family of signaling ligands, called Fibroblast Growth Factors (FGFs), control a wide spectrum of cell biological behaviors such as proliferation, cell death, migration, stem cell maintenance and gene expression. In particular, we use complex mouse genetics to understand the role of FGF signaling in mesodermal lineages with a special emphasis on extension of the body axis and formation of somites (segmented mesodermal segments that are the building blocks of vertebrate muscle, dermis and vertebral bodies). Our work has made clear that genetic redundancy is an important aspect of this biology; therefore all work in this project emerges from an effort to comprehensively characterize the genetic redundancy of FGF signaling in the mesodermal lineage. Such work is relevant to many cases of cancer where more than one FGF gene may be damaged. To achieve this, we have generated and characterized important Cre mouse lines, which are tools that allow the control of gene expression in the early embryo. These include TCre (expressed in the early emerging nascent mesoderm; see Development. 132: 3859-71. ), TCreERT2 (active in emerging nascent mesoderm at all embryonic stages; see PLoS ONE. 8: e62479) and Tbx4-Cre (expressed in a posterior mesodermal domain that includes the allantois, hindlimb, and external genitalia; see Dev Dyn. 240: 2290-300. doi: 10.1002/dvdy.22731). TCre, in particular, has had a major impact on the field, being essential in over 50 publications. For example, both TCre and TCreERT2 have important in a collaborative effort to demonstrate that Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension (Hum Mol Genet. 2014 Jul 31. pii: ddu397). Besides providing the mouse genetics community with valuable mouse lines, this project has yielded papers that document our major insights regarding FGF signaling in the early embryo. We published that Fgf8 not required for somitogenesis, although a body of high-profile work had placed it in a central position in current models. However, in collaboration with NCI colleague, Alan Perantoni, we demonstrated that Fgf8 was essential for development of the kidney and male reproductive tract (Development. 132: 3859-71, Development. 138: 5369-78). A feature of these mutants was aberrant cell death due to loss of Fgf8 signaling. This past year, we published a study, showing we can restore cell survival to such mutants, by removing the pro-apoptotic genes, Bax and Bak. This genetic manipulation restores some measure of kidney development, but nephrogenesis is not normal, revealing new insights into Fgf8 function in this process (Differentiation, 2023, 130:7-15). We showed that Fgf8, together with Fgf4, are required for essential aspects of somitogenesis: expression of oscillating gene domains, WNT pathway genes and markers of undifferentiated presomitic mesoderm (Proc Natl Acad Sci U S A. 108: 4018-23). By examining FGF mutants in which we genetically restored WNT signaling, we demonstrated that FGF signaling operates independently of WNT signaling in this process. The functional redundancy that we uncovered has implications for cancer as both FGFs have been found to be aberrantly active in testicular tumors. In recent studies we have delved deeper into this work and have shown that Fgf4 mutants (but not Fgf8 mutants) display a range of vertebral defects that model a spectrum of human Segmentation Defects of the Vertebrae caused by defective Notch oscillations. A key innovation in this work is our adaption of computational modeling to generate embryonic volumetric subsets of the embryo. We then quantify mRNA levels of key target genes, affected by the loss of Fgf4 signaling, within these volumes (eLife 2020;9:e55608. DOI: https://doi.org/10.7554/eLife.55608). We applied this technical innovation in a collaborative study published with the Kwon lab at Johns Hopkins Medical School on the role of WNT signaling in heart development (Proc Natl Acad Sci U S A., 2023, 120(4):e2217687120). In another recent publication, we demonstrated that the Fgf8 subfamily (Fgf8, Fgf17 and Fgf18) are required for closing the ventral body wall in the vertebrate embryo. Defects in this process are a major class of human birth defect and a significant health burden (Development (2020) 147, dev189506. doi:10.1242/dev.189506). We are continuing to study genetic redundancy in FGF signaling in several aspects of embryonic development. We are currently focusing on two aspects of development: generation of the promitive streak, which generates all embryonic mesoderm and differentiation of the somite into its derivative lineages (muscle and bone).