22.8.5的PNAS
1. Fibroblasts and Macrophages Employ Different Modes of Compartment Size Control
Fig 1A：Factors that control compartment size, and ultimately tissue and organ size, generally fall into two categories.
Fig 1B, C：在此前的研究中1，作者证实成纤维细胞的增殖受到space availability的限制。尽管生长因子信号存在的条件下，space constraints set the carrying capacity of fibroblasts in vitro. 因此作者推测成纤维细胞和巨噬细胞控制细胞数量使用的是不同的策略。因此作者使用Edu检测了不同细胞密度下FB和MP的增殖情况(2h)，发现和MP相比，FB的密度依赖性更高。
Fig 1D, E：随后作者探究了FB和MP生长时对生长因子的依赖性，发现FB对PDGF-BB的依赖比较小，曲线很快变水平。而MP的生长则明显呈CSF-1依赖。
Interestingly, even though proliferation of FB requires growth factors, in all conditions we have examined, space constraints regulate proliferation of FC almost 5 times more than the addition of growth factors; in contrast, growth factors influence proliferation of MP about 5 times more than changes in cell density. 很有意思哦，是不是结构细胞的生长主要受到space的限制，而免疫细胞主要受到生长激素的限制？
2. Fibroblasts Display Density-Dependent Gene-Expression Programs
Fig 2A：为了验证前面的假设，作者对4个密度的FB进行了RNAseq。（The highest cell density is close to the theoretical carrying capacity estimated previously (highly confluent), and the lowest cell density is close to the threshold at which fibroblasts cannot survive in monoculture (sparse).）作者鉴定出了1826个显著高表达(transcripts per million [TPM] > 2)且受到细胞密度调控的基因。3个cluster(L1-L3)在低细胞密度中高表达，5个cluster(H1-H5)在低细胞密度中高表达。
这个图，可以两组两组比差异基因，再把持续上调/持续下调的基因挑出来画热图就行。基因分cluster可以用hclust来做。
Fig 2B：随后作者对LD和HD的基因做了富集分析（主图展示的是LD和HD的所有基因做的富集，分cluster的富集在附件里）。LD的基因主要与ribosome biogenesis, cell cycle, DNA replication, and purine and pyrimidine metabolism相关，与LD的细胞正处于合成代谢和细胞增殖的状态相一致。HD的基因主要与lysosomal function, ECM, and protein digestion and absorption有关。而在信号通路上，LD主要富集到MAPK, Hippo-YAP, TGF-b, Ras, VEGF, TNF 和 IL-17 信号通路。HD主要富集到Wnt信号通路（KEGG做的）。为了验证前面这个结果，作者在Molecular Signature database数据库（也就是GSEA那个数据库）中针对性的对前面那几个pathway重新做了富集，结果提示Hippo-YAP, TGF-b, and Wnt activation demonstrate the strongest enrichment for genes induced at low cell density, and genes repressed by TGF-b show the strongest enrichment at high cell density.
这个思路可以参考，先GO-KEGG再GSEA
Overall, functional enrichment analysis revealed that Hippo-YAP and TGF-b signaling pathways are most highly correlated with density-dependent gene expression.
Fig 2C：由于前面的结果是测的in vitro培养的FB，体内体外环境还是有差异的。Inspired by previous work that reconstructed the spatial environ- ment of single cells based on gradient expression2,3, we developed a computational algorithm to estimate “cell density” for individual cells based on their relative expression of density- dependent genes.
Fig 2D：由于Fibrosis is characterized by excessive fibroblast proliferation，使用上面的策略，作者比较了已发表的单细胞数据中HC和idiopathic pulmonary fibrosis (IPF) 或 chronic obstructive pulmonary disease (COPD)的成纤维密度，发现fibroblasts isolated from IPF patients display an increased tendency for high cell density，与这种疾病存在invasive expansion of myofibroblasts相一致。COPD患者的成纤维细胞则没有这种现象。
这里其实看起来好像就是用前面鉴定出的density相关基因在HC和IPF患者的成纤维细胞中算了一下density评分，发现增高。
These results suggest that the density-dependent expression programs identified in vitro may represent changes in fibroblasts that occur during fibrotic disease.
Fig 2
分cluster的富集
3. Space Availability Modulates the Hippo-YAP and TGF-β Signaling Pathways
关于Hippo-YAP 和 TGF-β 通路的背景知识：
简言之，Hippo-YAP 抑制条件下，YAP/TAZ这个转录共激活蛋白会激活结合并激活 转录因子TEAF。而在Hippo活化情况下，被磷酸化的Lats激酶会磷酸化下游YAP/TAZ，引起YAP和TAZ的泛素化降解；TGF-β信号通路的激活会活化SMAD，启动入核调控。
Fig 3A：作者发现TEAD和SMAD的binding sequence motif都在LD上调基因的promoter区被显著富集。而在HD上调基因的promoter区只显著富集到SMAD。SMAD proteins can act as both transcriptional activators and repressor. Distinct SMAD motifs enriched among the genes induced at either high or low densities suggest that cooperation with other transcriptional coactivators or corepressors may determine density-dependent activation or repression in addition to TGF-b signaling.
Fig 3B, C：随后作者验证了Hippo-YAP 和 TGF-β通路的活化情况。B图中，随着细胞密度的下降，p-SMAD2/3(TGF-β通路活化指标)的表达增加。C图中，低密度时YAP1和细胞核共定位。随着细胞密度的增加，YAP1逐渐excluded from the nucleus.
Collectively, these data demonstrate that the activity of both TGF-β signaling and Hippo-YAP signaling are regulated in fibroblasts in a cell density-dependent manner, in response to space limitation.
Fig 3D, E：此外，一些Hippo-YAP靶基因如 Nppb, Akred1, Bdnf, Ctgf, and Cyr61以及 TGF-β 靶基因如 Serpine1, Acta1, Col2a1, Hbegf, and Ngf 都呈密度依赖性表达。
4. YAP1 and TGF-β Signaling Control Expression of Different Growth Factors in Response to Space Limitation
在3E的结果中，我们看到很多生长因子也呈密度依赖性表达。比如neurotrophic growth factors (Bdnf, Ngf, Nif3, Ptn), epidermal growth factors (Ereg, Hbegf), hepatocyte growth factor Hgf, and myeloid growth factor Csf1. 此外一些趋化因子如Ccl8, Cxcl14, Cxcl15, 细胞因子如Il33也呈时间依赖性表达(附件)。In particular, the expression of Csf1 was inversely related to fibroblast density, suggesting that density sensing by fibroblasts is coupled with Csf1 production for macrophages.
Fig 4A：为了探究哪个通路调控了FB表达Csf1，作者探究了活化YAP1, TGF-β 或 Wnt信号通路是否可以调控Csf1的表达。重组的TGF-β 或 WNT3A 在体外没有诱导出Csf1的表达，但是诱导了Hbegf 和 Ctgf 的表达(附件)。而在YAP1持续活化的小鼠，其成纤维细胞的Csf1表达增加。 提示成纤维Csf1的表达被YAP1, 而不是 TGF-β 或 Wnt信号通路激活。
Fig 4B：随后作者使用siRNA干预小鼠胚胎成纤维细胞，发现knocking down Yap1 降低了 Csf1 的表达接近一倍，similar to the difference between low and high cell densities。而 knockdown Smad4 (the central transcription factor in TGF-b signaling)则没有显著影响。
Fig 4C：为了进一步探究TGF-β信号通路对密度依赖性生长因子表达的作用，作者使用了genetic and pharmacological targeting策略。作者首先从Tgfbr2fl/fl鼠分离了成纤维细胞，分别转染了GFP 或Cre-GFP viral vectors，并分离了GFP阳性的也就是转染成功的细胞。发现低密度培养的细胞，是否敲除Tgfb2对Csf1r表达影响不大，而Hbegf, Ctgf, 和 Serpine1 (genes regulated by cell density and TGF-b signaling) were significantly reduced or entirely abolished. 提示TGFBR2参与一系列density-dependent 基因如Hbegf 和 Ctgf 的调控，而Csf1则是受到YAP1的调控，独立于TGF-β信号通路。
Fig 4
5. YAP1 Regulates the Expression of Csf1 via a Conserved Distal Enhancer
随后作者想要去探究Hippo pathway是怎样调控Csf1的表达的。Csf1 并不是一个已知的 YAP1 靶基因而且它的promoter区缺乏TEAD transcription factors的结合序列。因此作者推测 YAP1可能通过distal regulatory elements调节Csf1的表达。
Fig 4D：为了验证上面的猜想，作者对成纤维细胞的内源性YAP1蛋白进行了ChIP-seq。并在Csf1转录起始位点上游33 和 36 kb 发现了2个distinct binding sites。
对组蛋白修饰的分析结果提示两个YAP1的binding sites are within regions that are enriched for H3K27ac but lack H3K4me1 and H3K4me3 marks, suggesting that YAP1 physically occupies two active distal enhancers of Csf1。
Fig 4E：在global水平，作者观察到approximately three times more YAP1 binding events at low density compared to YAP1 binding events at high density. Both TEAD4 and AP-1 motifs were identified as highly enriched in YAP1 peaks, yet this enrichment was more robust at low density.
Fig 4
These data suggest that Csf1 expression is regulated by cell type-specific enhancers, and that density-dependent control of Csf1 by YAP1 is a feature of fibroblasts.
6. Fibroblasts Produce CSF1 to Support Macrophage Populations
为了探究成纤维细胞中YAP调控的Csf1表达可以直接调控巨噬细胞数量，作者使用了前面第4部分中的YAP1持续活化的小鼠，分离了YAP1CA FB，与巨噬细胞共培养。
Fig 4F：YAP1的持续激活引起了成纤维数量的增加，consistent with a role for Hippo-YAP signaling in autonomous control of cell proliferation. 此外，巨噬细胞数量、巨噬细胞/FB的比例在YAP活化时都增加。
Fig 4G：With elevated expression of Csf1, macrophages and fibroblasts still exhibited a stable ratio regardless of starting conditions.
Fig 4
7. Space Limitation Regulates Gene Expression through Actin-Dependent Mechanisms
Fig 5A：此外，作者观察到在更高密度下细胞的体积更小，细胞核的长度和宽度更小，厚度则增加。
Fig 5B：细胞密度越大，细胞越接近球状，indicating that nuclei at low and high cell densities experience different mechanical pressure.
Previously, it was reported that stiff matrices promote nucleation of actin fibers that apply pressure to the nucleus, leading to YAP1 nuclear translocation.
Fig 5C, D：在研究中，作者发现在低细胞密度下actin常常更离散，更容易被观察到，而且出现更强的荧光强度。因此作者假设actin fiber的形成直接调控密度依赖性生长因子的表达。
Fig 5E, F：随后作者使用CN03刺激了成纤维细胞2h。结果显示actin filaments formed across the length of cells, and that YAP1 was found almost exclusively in the nucleus even at high cell density. In addition to YAP1-regulated growth factors, two growth factors that are strongly dependent on cell density and TGF-à signaling, Hbegf and Ctgf, are also activated by RhoA, but independent of YAP1.
CN03：RhoA激活因子(CN03)，可防止RhoA- gtp水解并将RhoA锁在活性状态，RhoA主要与细胞骨架调控有关，主要参与肌动蛋白应激纤维的形成和肌动蛋白的收缩性。
Fig 5
Given the distinct change of nuclear shape at different cell densities, these data suggest that sensing space availability may be dependent on RhoA signaling, and that density-dependent expression programs may be controlled through mechanical forces acting on the nucleus.
8. Environmental Conditions Regulate Growth Factor Expression
由于巨噬细胞的生长主要受Csf1调控而不是space大小，作者随后想要去探究其他的外界环境因素如营养和氧气等是否影响细胞群的大小。
Fig 6A：作者探究了amino acid deprivation, glucose deprivation 和 hypoxia条件下成纤维细胞生长因子的表达。不同条件下都存在着生长因子的上调和下调。
Fig 6B：此外作者又纳入了oxidative, osmotic, and endoplasmic reticulum (ER) stress条件下生长因子的表达，做了相关性分析，发现the overall cellular responses to these stress conditions and nutrient limitations showed poor correlation.
Fig 6C：展示了不同条件下生长因子的差异性表达
Fig 6
Discussion
The population sizes of different cell types within a tissue compartment must be tightly regulated to ensure proper tissue functioning, prevent overgrowth, and allow for regeneration and repair (Fig 6D). Two modes of cell number control have been described previously: one is based on space availability, which can be mediated through either cell–cell or cell–ECM contact; the other is based on growth factor availability (2, 7, 15). However, whether these control strategies function independently from each other is not known. Here we demonstrated that fibroblasts and macrophages, two universal cell types within tissues, each use a different strategy to control their numbers. Fibroblast proliferation is more sensitive to the availability of space, while macrophage expansion is highly sensitive to the availability of a growth factor. Moreover, we found that production of the macrophage- specific growth factor CSF1 by fibroblasts was directly regulated by Hippo-YAP signaling in response to space limitation. This coupling between space availability with growth factor production provides a simple link between the two different modes of cell number control (Fig. 6D).
参考文献：
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