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 1D, E：隨后作者探究了FB和MP生長時對生長因子的依賴性铲球，發(fā)現(xiàn)FB對PDGF-BB的依賴比較小彻况，曲線很快變水平谁尸。而MP的生長則明顯呈CSF-1依賴。
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. 很有意思哦纽甘，是不是結(jié)構(gòu)細(xì)胞的生長主要受到space的限制良蛮，而免疫細(xì)胞主要受到生長激素的限制？

2. Fibroblasts Display Density-Dependent Gene-Expression Programs

Fig 2A：為了驗證前面的假設(shè)悍赢，作者對4個密度的FB進(jìn)行了RNAseq决瞳。（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).）作者鑒定出了1826個顯著高表達(dá)(transcripts per million [TPM] > 2)且受到細(xì)胞密度調(diào)控的基因。3個cluster(L1-L3)在低細(xì)胞密度中高表達(dá)左权，5個cluster(H1-H5)在低細(xì)胞密度中高表達(dá)皮胡。

這個圖，可以兩組兩組比差異基因赏迟，再把持續(xù)上調(diào)/持續(xù)下調(diào)的基因挑出來畫熱圖就行屡贺。基因分cluster可以用hclust來做。

Fig 2B：隨后作者對LD和HD的基因做了富集分析（主圖展示的是LD和HD的所有基因做的富集甩栈，分cluster的富集在附件里）泻仙。LD的基因主要與ribosome biogenesis, cell cycle, DNA replication, and purine and pyrimidine metabolism相關(guān)，與LD的細(xì)胞正處于合成代謝和細(xì)胞增殖的狀態(tài)相一致量没。HD的基因主要與lysosomal function, ECM, and protein digestion and absorption有關(guān)玉转。而在信號通路上，LD主要富集到MAPK, Hippo-YAP, TGF-b, Ras, VEGF, TNF 和 IL-17 信號通路允蜈。HD主要富集到Wnt信號通路（KEGG做的）冤吨。為了驗證前面這個結(jié)果，作者在Molecular Signature database數(shù)據(jù)庫（也就是GSEA那個數(shù)據(jù)庫）中針對性的對前面那幾個pathway重新做了富集饶套，結(jié)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.

這個思路可以參考漩蟆，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：由于前面的結(jié)果是測的in vitro培養(yǎng)的FB，體內(nèi)體外環(huán)境還是有差異的妓蛮。Inspired by previous work that reconstructed the spatial environ- ment of single cells based on gradient expression^2,3, 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怠李，使用上面的策略，作者比較了已發(fā)表的單細(xì)胞數(shù)據(jù)中HC和idiopathic pulmonary fibrosis (IPF) 或 chronic obstructive pulmonary disease (COPD)的成纖維密度蛤克，發(fā)現(xiàn)fibroblasts isolated from IPF patients display an increased tendency for high cell density捺癞，與這種疾病存在invasive expansion of myofibroblasts相一致。COPD患者的成纖維細(xì)胞則沒有這種現(xiàn)象构挤。

這里其實看起來好像就是用前面鑒定出的density相關(guān)基因在HC和IPF患者的成纖維細(xì)胞中算了一下density評分髓介，發(fā)現(xiàn)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

關(guān)于Hippo-YAP 和 TGF-β 通路的背景知識：

簡言之筋现，Hippo-YAP 抑制條件下唐础，YAP/TAZ這個轉(zhuǎn)錄共激活蛋白會激活結(jié)合并激活 轉(zhuǎn)錄因子TEAF。而在Hippo活化情況下矾飞，被磷酸化的Lats激酶會磷酸化下游YAP/TAZ一膨，引起YAP和TAZ的泛素化降解；TGF-β信號通路的激活會活化SMAD洒沦，啟動入核調(diào)控豹绪。

Fig 3A：作者發(fā)現(xiàn)TEAD和SMAD的binding sequence motif都在LD上調(diào)基因的promoter區(qū)被顯著富集。而在HD上調(diào)基因的promoter區(qū)只顯著富集到SMAD申眼。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：隨后作者驗證了Hippo-YAP 和 TGF-β通路的活化情況瞒津。B圖中，隨著細(xì)胞密度的下降括尸，p-SMAD2/3(TGF-β通路活化指標(biāo))的表達(dá)增加仲智。C圖中，低密度時YAP1和細(xì)胞核共定位姻氨。隨著細(xì)胞密度的增加钓辆，YAP1逐漸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 都呈密度依賴性表達(dá)。

4. YAP1 and TGF-β Signaling Control Expression of Different Growth Factors in Response to Space Limitation

在3E的結(jié)果中前联，我們看到很多生長因子也呈密度依賴性表達(dá)功戚。比如neurotrophic growth factors (Bdnf, Ngf, Nif3, Ptn), epidermal growth factors (Ereg, Hbegf), hepatocyte growth factor Hgf, and myeloid growth factor Csf1. 此外一些趨化因子如Ccl8, Cxcl14, Cxcl15, 細(xì)胞因子如Il33也呈時間依賴性表達(dá)(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：為了探究哪個通路調(diào)控了FB表達(dá)Csf1似嗤，作者探究了活化YAP1, TGF-β 或 Wnt信號通路是否可以調(diào)控Csf1的表達(dá)啸臀。重組的TGF-β 或 WNT3A 在體外沒有誘導(dǎo)出Csf1的表達(dá)，但是誘導(dǎo)了Hbegf 和 Ctgf 的表達(dá)(附件)烁落。而在YAP1持續(xù)活化的小鼠乘粒，其成纖維細(xì)胞的Csf1表達(dá)增加。 提示成纖維Csf1的表達(dá)被YAP1, 而不是 TGF-β 或 Wnt信號通路激活伤塌。
Fig 4B：隨后作者使用siRNA干預(yù)小鼠胚胎成纖維細(xì)胞灯萍，發(fā)現(xiàn)knocking down Yap1 降低了 Csf1 的表達(dá)接近一倍，similar to the difference between low and high cell densities每聪。而 knockdown Smad4 (the central transcription factor in TGF-b signaling)則沒有顯著影響旦棉。
Fig 4C：為了進(jìn)一步探究TGF-β信號通路對密度依賴性生長因子表達(dá)的作用，作者使用了genetic and pharmacological targeting策略药薯。作者首先從Tgfbr2^fl/fl鼠分離了成纖維細(xì)胞绑洛，分別轉(zhuǎn)染了GFP 或Cre-GFP viral vectors，并分離了GFP陽性的也就是轉(zhuǎn)染成功的細(xì)胞童本。發(fā)現(xiàn)低密度培養(yǎng)的細(xì)胞真屯，是否敲除Tgfb2對Csf1r表達(dá)影響不大，而Hbegf, Ctgf, 和 Serpine1 (genes regulated by cell density and TGF-b signaling) were significantly reduced or entirely abolished. 提示TGFBR2參與一系列density-dependent 基因如Hbegf 和 Ctgf 的調(diào)控穷娱，而Csf1則是受到Y(jié)AP1的調(diào)控绑蔫，獨立于TGF-β信號通路。

Fig 4

5. YAP1 Regulates the Expression of Csf1 via a Conserved Distal Enhancer

隨后作者想要去探究Hippo pathway是怎樣調(diào)控Csf1的表達(dá)的鄙煤。Csf1 并不是一個已知的 YAP1 靶基因而且它的promoter區(qū)缺乏TEAD transcription factors的結(jié)合序列。因此作者推測 YAP1可能通過distal regulatory elements調(diào)節(jié)Csf1的表達(dá)茶袒。

Fig 4D：為了驗證上面的猜想梯刚，作者對成纖維細(xì)胞的內(nèi)源性YAP1蛋白進(jìn)行了ChIP-seq。并在Csf1轉(zhuǎn)錄起始位點上游33 和 36 kb 發(fā)現(xiàn)了2個distinct binding sites薪寓。
對組蛋白修飾的分析結(jié)果提示兩個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亡资。
Fig 4E：在global水平，作者觀察到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

為了探究成纖維細(xì)胞中YAP調(diào)控的Csf1表達(dá)可以直接調(diào)控巨噬細(xì)胞數(shù)量向叉，作者使用了前面第4部分中的YAP1持續(xù)活化的小鼠锥腻，分離了YAP1^CA FB，與巨噬細(xì)胞共培養(yǎng)母谎。
Fig 4F：YAP1的持續(xù)激活引起了成纖維數(shù)量的增加瘦黑，consistent with a role for Hippo-YAP signaling in autonomous control of cell proliferation. 此外，巨噬細(xì)胞數(shù)量、巨噬細(xì)胞/FB的比例在YAP活化時都增加幸斥。
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：此外匹摇，作者觀察到在更高密度下細(xì)胞的體積更小，細(xì)胞核的長度和寬度更小甲葬，厚度則增加廊勃。
Fig 5B：細(xì)胞密度越大，細(xì)胞越接近球狀经窖，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：在研究中坡垫，作者發(fā)現(xiàn)在低細(xì)胞密度下actin常常更離散，更容易被觀察到画侣，而且出現(xiàn)更強(qiáng)的熒光強(qiáng)度冰悠。因此作者假設(shè)actin fiber的形成直接調(diào)控密度依賴性生長因子的表達(dá)。
Fig 5E, F：隨后作者使用CN03刺激了成纖維細(xì)胞2h棉钧。結(jié)果顯示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)屿脐，可防止RhoA- gtp水解并將RhoA鎖在活性狀態(tài)，RhoA主要與細(xì)胞骨架調(diào)控有關(guān)宪卿，主要參與肌動蛋白應(yīng)激纖維的形成和肌動蛋白的收縮性的诵。

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

由于巨噬細(xì)胞的生長主要受Csf1調(diào)控而不是space大小，作者隨后想要去探究其他的外界環(huán)境因素如營養(yǎng)和氧氣等是否影響細(xì)胞群的大小佑钾。
Fig 6A：作者探究了amino acid deprivation, glucose deprivation 和 hypoxia條件下成纖維細(xì)胞生長因子的表達(dá)西疤。不同條件下都存在著生長因子的上調(diào)和下調(diào)。
Fig 6B：此外作者又納入了oxidative, osmotic, and endoplasmic reticulum (ER) stress條件下生長因子的表達(dá)休溶，做了相關(guān)性分析代赁，發(fā)現(xiàn)the overall cellular responses to these stress conditions and nutrient limitations showed poor correlation.
Fig 6C：展示了不同條件下生長因子的差異性表達(dá)

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).

參考文獻(xiàn)：

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