The Role of Fgf Signaling in Vertebrate Development

该项目的长期目标是了解一个重要的信号配体家族，称为成纤维细胞生长因子（FGFs），如何控制广泛的细胞生物学行为，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理解FGF信号在中胚层谱系中的作用，特别强调了体轴的延伸和体节的形成（分割的中胚层节段是脊椎动物肌肉、真皮和椎体的基石）。我们的工作已经清楚地表明，基因冗余是这种生物学的一个重要方面；因此，本项目的所有工作都是为了全面表征中胚层谱系中FGF信号的遗传冗余。这项工作与许多可能有一个以上FGF基因受损的癌症病例有关。为了实现这一目标，我们已经产生并表征了重要的Cre小鼠系，这是允许控制早期胚胎基因表达的工具。其中包括TCre（在早期新生中胚层中表达；见发育，132:3859-71）。TCreERT2(活跃于所有胚胎阶段的新生中胚层；见PLoS ONE。8: e62479)和Tbx4-Cre(表达于包括尿囊、后肢和外生殖器在内的后中胚层域；见《动物医学杂志》240:2290-300。doi: 10.1002 / dvdy.22731)。特别是tcree对该领域产生了重大影响，在50多种出版物中发挥了重要作用。例如，TCreERT2和TCreERT2在合作证明Wnt5a/Ror2信号通过控制中胚层延伸调节肾脏形态发生方面发挥了重要作用（Hum Mol Genet. 2014 july 31）。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FGF信号传导的主要见解。我们发表了Fgf8不是体细胞发生所必需的，尽管一系列备受瞩目的工作已经将其置于当前模型的中心位置。然而，在与NCI同事Alan Perantoni的合作中，我们证明了Fgf8对肾脏和男性生殖道的发育至关重要（development . 132: 3859-71, development . 138: 5369-78）。这些突变体的一个特征是由于Fgf8信号的缺失导致细胞异常死亡。去年，我们发表了一项研究，表明我们可以通过去除促凋亡基因Bax和Bak来恢复这些突变体的细胞存活。这种基因操作在一定程度上恢复了肾脏的发育，但肾脏形成并不正常，这揭示了Fgf8在这一过程中的功能（分化，2023,130:7-15）。我们发现Fgf8和Fgf4在体细胞发生的基本方面是必需的：振荡基因域、WNT通路基因和未分化的体前中胚层标志物的表达（Proc Natl Acad science，美国，108:4018-23）。通过检测基因恢复WNT信号的FGF突变体，我们证明了FGF信号在这一过程中独立于WNT信号传导。我们发现的功能冗余对癌症有影响，因为两种fgf在睾丸肿瘤中都异常活跃。在最近的研究中，我们对这项工作进行了更深入的研究，并表明Fgf4突变体（而不是Fgf8突变体）显示出一系列椎体缺陷，这些缺陷模拟了由缺陷Notch振荡引起的人类椎体分割缺陷的频谱。这项工作的一个关键创新是我们采用计算建模来生成胚胎的胚胎体积子集。然后，我们量化了这些体积内受Fgf4信号缺失影响的关键靶基因的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Kwon实验室发表的一项关于WNT信号在心脏发育中的作用的合作研究中（Proc Natl Acad Sci us, 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）是关闭脊椎动物胚胎腹壁所必需的。这一过程中的缺陷是一类主要的人类出生缺陷和重大的健康负担(发展（2020）147,dev189506。doi: 10.1242 / dev.189506)。我们正在继续研究胚胎发育过程中FGF信号的遗传冗余。我们目前专注于发育的两个方面：产生所有胚胎中胚层的前体细胞条纹的产生和体体向其衍生谱系（肌肉和骨）的分化。

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).