Granulosal and thecal expression of bone morphogenetic protein- and activin-binding protein mRNA transcripts during bovine follicle development and factors modulating their expression in vitro

Evidence supports local roles for transforming growth factor β superfamily members including activins and bone morphogenetic proteins (BMP) in follicle development. Access of these ligands to signalling receptors is likely modulated by extracellular binding proteins (BP). In this study, we comparedex vivoexpression of four BPs (chordin, gremlin, noggin and follistatin) in granulosal (GC) and theca interna (TC) compartments of developing bovine antral follicles (1–18 mm). Effects of FSH and IGF on BMP and BP expression by cultured GC, and effects of LH and BMPs on BP expression by cultured TC were also examined. Follicular expression of all four BP transcripts was higher in GC than TC compartments (P<0.001) a finding confirmed by immunohistochemistry. Follicle category affected (P<0.01) gremlin and follistatin mRNA abundance, with a significant cell-type×follicle category interaction for chordin, follistatin and noggin. Noggin transcript abundance was lower (P<0.05) in GC of large ‘E-active’ than ‘E-inactive’ follicles while follistatin mRNA level was higher (P<0.01). FSH enhanced CYP19, FSHR, INHBA and follistatin by GC without affecting BMP or BMP–BP expression. IGF increased CYP19 and follistatin, reduced BMP4, noggin and gremlin but did not affect chordin orFSHRmRNA levels. LH increased TC androgen secretion but had no effect on BMP or BP expression. BMPs uniformly suppressed TC androgen production whilst increasing chordin, noggin and gremlin mRNA levels up to 20-fold (P<0.01). These findings support the hypothesis that extracellular BP, mostly from GC, contribute to the regulation of intrafollicular BMP/activin signalling. Enhancement of thecal BP expression by BMP implies an autoregulatory feedback role to prevent excessive signalling.

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Proteoglycan-4 is correlated with longer survival in HCC patients and enhances sorafenib and regorafenib effectiveness via CD44 in vitro

Cell Death and Disease ◽

10.1038/s41419-020-03180-8 ◽

2020 ◽

Vol 11 (11) ◽

Author(s):

Francesco Dituri ◽

Rosanna Scialpi ◽

Tannin A. Schmidt ◽

Martina Frusciante ◽

Serena Mancarella ◽

...

Keyword(s):

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Ex Vivo ◽

Mrna Levels ◽

Drug Effectiveness ◽

Proteoglycan 4 ◽

Tumor Expression ◽

A Prospective Study ◽

Hcc Cells

AbstractSorafenib and regorafenib administration is among the preferential approaches to treat hepatocellular carcinoma (HCC), but does not provide satisfactory benefits. Intensive crosstalk occurring between cancer cells and other multiple non-cancerous cell subsets present in the surrounding microenvironment is assumed to affect tumor progression. This interplay is mediated by a number of soluble and structural extracellular matrix (ECM) proteins enriching the stromal milieu. Here we assess the HCC tumor expression of the ECM protein proteoglycan 4 (PRG4) and its potential pharmacologic activity either alone, or in combination with sorafenib and regorafenib. PRG4 mRNA levels resulted strongly correlated with increased survival rate of HCC patients (p = 0.000) in a prospective study involving 78 HCC subjects. We next showed that transforming growth factor beta stimulates PRG4 expression and secretion by primary human HCC cancer-associated fibroblasts, non-invasive HCC cell lines, and ex vivo specimens. By functional tests we found that recombinant human PRG4 (rhPRG4) impairs HCC cell migration. More importantly, the treatment of HCC cells expressing CD44 (the main PRG4 receptor) with rhPRG4 dramatically enhances the growth-limiting capacity of sorafenib and regorafenib, whereas not significantly affecting cell proliferation per se. Conversely, rhPRG4 only poorly potentiates drug effectiveness on low CD44-expressing or stably CD44-silenced HCC cells. Overall, these data suggest that the physiologically-produced compound PRG4 may function as a novel tumor-suppressive agent by strengthening sorafenib and regorafenib effects in the treatment of HCC.

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Antagonistic Effects of Insulin and TGF-β3 during Chondrogenic Differentiation of Human BMSCs under a Minimal Amount of Factors

Cells Tissues Organs ◽

10.1159/000442411 ◽

2016 ◽

Vol 201 (2) ◽

pp. 88-96 ◽

Cited By ~ 6

Author(s):

Emilio Satoshi Hara ◽

Mitsuaki Ono ◽

Yuya Yoshioka ◽

Junji Ueda ◽

Yuri Hazehara ◽

...

Keyword(s):

Chondrogenic Differentiation ◽

Transforming Growth Factor ◽

Direct Interaction ◽

Transforming Growth Factor Β ◽

Treated Group ◽

Minimal Amount ◽

Mrna Levels ◽

Dependent Manner ◽

Antagonistic Effects

Growth factors are crucial regulators of cell differentiation towards tissue and organ development. Insulin and transforming growth factor-β (TGF-β) have been used as the major factors for chondrogenesis in vitro, by activating the AKT and Smad signaling pathways. Previous reports demonstrated that AKT and Smad3 have a direct interaction that results in the inhibition of TGF-β-mediated cellular responses. However, the result of this interaction between AKT and Smad3 during the chondrogenesis of human bone marrow-derived stem/progenitor cells (hBMSCs) is unknown. In this study, we performed functional analyses by inducing hBMSCs into chondrogenesis with insulin, TGF-β3 or in combination, and found that TGF-β3, when applied concomitantly with insulin, significantly decreases an insulin-induced increase in mRNA levels of the master regulator of chondrogenesis, SOX9, as well as the regulators of the 2 major chondrocyte markers, ACAN and COL2A1. Similarly, the insulin/TGF-β3-treated group presented a significant decrease in the deposition of cartilage matrix as detected by safranin O staining of histological sections of hBMSC micromass cultures when compared to the group stimulated with insulin alone. Intracellular analysis revealed that insulin-induced activation of AKT suppressed Smad3 activation in a dose-dependent manner. Accordingly, insulin/TGF-β3 significantly decreased the TGF-β3-induced increase in mRNA levels of the direct downstream factor of TGF-β/Smad3, CCN2/CGTF, compared to the group stimulated with TGF-β3 alone. On the other hand, insulin/TGF-β3 stimulation did not suppress insulin-induced expression of the downstream targets TSC2 and DDIT4/REDD1. In summary, insulin and TGF-β3 have antagonistic effects when applied concomitantly, with a minimal number of factors. The application of an insulin/TGF-β3 combination without further supplementation should be used with caution in the chondrogenic differentiation of hBMSCs.

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Intercellular coupling between peripheral circadian oscillators by TGF-β signaling

Science Advances ◽

10.1126/sciadv.abg5174 ◽

2021 ◽

Vol 7 (30) ◽

pp. eabg5174

Author(s):

Anna-Marie Finger ◽

Sebastian Jäschke ◽

Marta del Olmo ◽

Robert Hurwitz ◽

Adrián E. Granada ◽

...

Keyword(s):

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Clock Genes ◽

Ex Vivo ◽

Three Dimensional ◽

Transforming Growth Factor Β ◽

Molecular Clocks ◽

Peripheral Oscillators ◽

Circadian Oscillators

Coupling between cell-autonomous circadian oscillators is crucial to prevent desynchronization of cellular networks and disruption of circadian tissue functions. While neuronal oscillators within the mammalian central clock, the suprachiasmatic nucleus, couple intercellularly, coupling among peripheral oscillators is controversial and the molecular mechanisms are unknown. Using two- and three-dimensional mammalian culture models in vitro (mainly human U-2 OS cells) and ex vivo, we show that peripheral oscillators couple via paracrine pathways. We identify transforming growth factor–β (TGF-β) as peripheral coupling factor that mediates paracrine phase adjustment of molecular clocks through transcriptional regulation of core-clock genes. Disruption of TGF-β signaling causes desynchronization of oscillator networks resulting in reduced amplitude and increased sensitivity toward external zeitgebers. Our findings reveal an unknown mechanism for peripheral clock synchrony with implications for rhythmic organ functions and circadian health.

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Gene expression in mouse ovarian follicle development in vivo versus an ex vivo alginate culture system

Reproduction ◽

10.1530/rep-10-0481 ◽

2011 ◽

Vol 142 (2) ◽

pp. 309-318 ◽

Cited By ~ 26

Author(s):

Elizabeth M Parrish ◽

Anaar Siletz ◽

Min Xu ◽

Teresa K Woodruff ◽

Lonnie D Shea

Keyword(s):

Gene Expression ◽

Transforming Growth Factor ◽

Ex Vivo ◽

Expression Profiles ◽

Ovarian Follicle ◽

Gene Expression Profiles ◽

Follicle Development ◽

Α Subunit

Ovarian follicle maturation results from a complex interplay of endocrine, paracrine, and direct cell–cell interactions. This study compared the dynamic expression of key developmental genes during folliculogenesis in vivo and during in vitro culture in a 3D alginate hydrogel system. Candidate gene expression profiles were measured within mouse two-layered secondary follicles, multi-layered secondary follicles, and cumulus–oocyte complexes (COCs). The expression of 20 genes involved in endocrine communication, growth signaling, and oocyte development was investigated by real-time PCR. Gene product levels were compared between i) follicles of similar stage and ii) COCs derived either in vivo or by in vitro culture. For follicles cultured for 4 days, the expression pattern and the expression level of 12 genes were the same in vivo and in vitro. Some endocrine (cytochrome P450, family 19, subfamily A, polypeptide 1 (Cyp19a1) and inhibin βA subunit (Inhba)) and growth-related genes (bone morphogenetic protein 15 (Bmp15), kit ligand (Kitl), and transforming growth factor β receptor 2 (Tgfbr2)) were downregulated relative to in vivo follicles. For COCs obtained from cultured follicles, endocrine-related genes (inhibin α-subunit (Inha) and Inhba) had increased expression relative to in vivo counterparts, whereas growth-related genes (Bmp15, growth differentiation factor 9, and kit oncogene (Kit)) and zona pellucida genes were decreased. However, most of the oocyte-specific genes (e.g. factor in the germline α (Figla), jagged 1 (Jag1), and Nlrp5 (Mater)) were expressed in vitro at the same level and with the same pattern as in vivo-derived follicles. These studies establish the similarities and differences between in vivo and in vitro cultured follicles, guiding the creation of environments that maximize follicle development and oocyte quality.

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Evidence for an inhibitory role of bone morphogenetic protein(s) in the follicular–luteal transition in cattle

Reproduction ◽

10.1530/rep-08-0198 ◽

2009 ◽

Vol 137 (1) ◽

pp. 67-78 ◽

Cited By ~ 28

Author(s):

Amjad R Kayani ◽

Claire Glister ◽

Philip G Knight

Keyword(s):

Bone Morphogenetic Proteins ◽

Mrna Level ◽

Protein S ◽

Transcript Abundance ◽

Cell Types ◽

Suppressive Effect ◽

Cell Number ◽

Activin A ◽

Activin Receptors

Bone morphogenetic proteins (BMPs) and their receptors are expressed in ovarian theca cells (TC) and granulosa cells (GC) and BMPs have been implicated in the regulation of several aspects of follicle development including thecal androgen production and granulosal oestrogen production. Their potential involvement in luteal function has received less attention. In this study, we first compared relative abundance of mRNA transcripts for BMPs, activin-βA and BMP/activin receptors in bovine corpus luteum (CL) and follicular theca and granulosa layers before undertaking functional in vitro experiments to test the effect of selected ligands (BMP6 and activin A) on luteinizing bovine TC and GC. Relative to β-actin transcript abundance, CL tissue contained more BMP4 and -6 mRNA than granulosa, more BMP2 mRNA than theca but much less activin-βA mRNA than both granulosa and theca. Transcripts for all seven BMP/activin receptors were readily detected in each tissue and two transcripts (BMPRII, ActRIIA) were more abundant in CL than either theca or granulosa, consistent with tissue responsiveness. In vitro luteinization of TC and GC from antral follicles (4–6 mm) was achieved by culturing with 5% serum for 6 days. Treatment with BMP6 (0, 2, 10, and 50 ng/ml) and activin A (0, 2, 10 and 50 ng/ml) under these conditions dose-dependently suppressed forskolin-induced progesterone (P4) secretion from both cell types without affecting cell number. BMP6 reduced forskolin-stimulated upregulation of STAR mRNA and raised ‘basal’ CYP17A1 mRNA level in theca-lutein cells without affecting expression of CYP11A1 or hydroxy-Δ-5-steroid dehydrogenase, 3 β- and steroid Δ-isomerase 1 (HSD3B1). In granulosa-lutein cells, STAR transcript abundance was not affected by BMP6, whereas forskolin-induced expression of CYP11A1, HSD3B1, CYP19A1 and oxytocin transcripts was reduced. In both cell types, follistatin attenuated the suppressive effect of activin A and BMP6 on forskolin-induced P4 secretion but had no effect alone. Granulosa-lutein cells secreted low levels of endogenous activin A (∼1 ng/ml); BMP6 reduced this, while raising follistatin secretion thus decreasing activin A:follistatin ratio. Collectively, these findings support inhibitory roles for BMP/activin signalling in luteinization and steroidogenesis in both TC and GC.

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Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production

Reproduction ◽

10.1530/rep-10-0216 ◽

2010 ◽

Vol 140 (5) ◽

pp. 699-712 ◽

Cited By ~ 40

Author(s):

Claire Glister ◽

Leanne Satchell ◽

Philip G Knight

Keyword(s):

Bone Morphogenetic Proteins ◽

Physiological Role ◽

Antral Follicle ◽

Suppressive Effect ◽

Type I ◽

Follicle Development ◽

Androgen Synthesis ◽

Theca Interna ◽

Bmp Signalling

We reported previously that bone morphogenetic proteins (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC)in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1–18 mm). Abundance of mRNA transcripts for four BMPs (BMP2,BMP4,BMP6, andBMP7) and associated type I (BMPR1A,BMPR1B,ACVR1andACVR1B) and type II (BMPR2,ACVR2AandACVR2B) receptors showed relatively modest, though significant, changes during follicle development.BMP2was selectively expressed in GC, whileBMP6,BMP7and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold;P<0.001) as follicles grew from 1–2 to 9–10 mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TCin vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressedCYP17mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

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The effect of TGFβ1 on thermogenic markers is dependent on the degree of adipocyte differentiation

Bioscience Reports ◽

10.1042/bsr20194262 ◽

2020 ◽

Vol 40 (5) ◽

Author(s):

Babu R. Maharjan ◽

Susan V. McLennan ◽

Stephen M. Twigg ◽

Paul F. Williams

Keyword(s):

Adipocyte Differentiation ◽

Transforming Growth Factor ◽

Cell Metabolism ◽

Transforming Growth Factor Β ◽

Mrna Levels ◽

Mature Adipocyte ◽

Fat Cell ◽

Variable Expression ◽

Ucp2 Mrna

Abstract Transforming growth factor β (TGFβ) a multifunctional cytokine is known to regulate cell proliferation, differentiation, migration and survival. Although there is variable expression of modulators of TGFβ action during differentiation, a differential effect on fat cell metabolism at the different stages of adipocyte differentiation was unclear. In the present study, 3T3L1 cells were used as an in vitro model to study the effect of TGFβ on adipogenic and thermogenic markers at various stages of preadipocyte to mature adipocyte differentiation. As in our earlier studies on the effect of TGFβ on CEBP’s, we used a standard differentiation mix, and one with the addition of rosiglitazone. RhTGFβ1 was added to undifferentiated adipocytes (preadipocytes) and to adipocytes at day 0 (commitment stage) as well as day 10 (terminal differentiation). Cellular responses in terms of Pref1, PPARγ, TLE3, PGC1α, PRDM16, UCP1 and UCP2 mRNA levels and selected protein products, were determined. Increases in PPARγ, PRDM16, UCP1 and UCP2 mRNA and decreases in Pref1 are good indicators of successful differentiation. The early addition of rhTGFβ1 during commitment stage decreased PPARγ, PRDM16, TLE3, UCP1 and UCP2 mRNA and decreased PRDM16 protein consistent with our earlier report on the inhibition of CEBP’s by TGFβ and CCN2. The addition of rhTGFβ1 to mature adipocyte at day 10 increased UCP1 mRNA and increased PRDM16 and UCP1 proteins. In the present study, our results suggest that TGFβ1 added late enhances the thermogenic potential of mature cells and causes 3T3L1 cells to differentiate to resemble brown or beige rather than white adipose tissue.

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TGF-β superfamily expression and actions in the endometrium and placenta

Reproduction ◽

10.1530/rep.1.01076 ◽

2006 ◽

Vol 132 (2) ◽

pp. 217-232 ◽

Cited By ~ 246

Author(s):

Rebecca L Jones ◽

Chelsea Stoikos ◽

Jock K Findlay ◽

Lois A Salamonsen

Keyword(s):

Embryo Development ◽

Bone Morphogenetic Proteins ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Maternal Serum ◽

Preimplantation Embryos ◽

Tissue Remodelling ◽

Hormone Production ◽

Tgfβ Superfamily

Transforming growth factor β (TGFβ) superfamily members are closely associated with tissue remodelling events and reproductive processes. This review summarises the current state of knowledge regarding the expression and actions of TGFβ superfamily members in the uterus, during the menstrual cycle and establishment of pregnancy. TGFβs and activin β subunits are abundantly expressed in the endometrium, where roles in preparation events for implantation have been delineated, particularly in promoting decidualisation of endometrial stroma. These growth factors are also expressed by epithelial glands and secreted into uterine fluid, where interactions with preimplantation embryos are anticipated. Knockout models and embryo culture experiments implicate activins, TGFβs, nodal and bone morphogenetic proteins (BMPs) in promoting pre- and post-implantation embryo development. TGFβ superfamily members may therefore be important in the maternal support of embryo development. Following implantation, invasion of the decidua by fetal trophoblasts is tightly modulated. Activin promotes, whilst TGFβ and macrophage inhibitory cytokine-1 (MIC-1) inhibit, trophoblast migration in vitro, suggesting the relative balance of TGFβ superfamily members participate in modulating the extent of decidual invasion. Activins and TGFβs have similar opposing actions in regulating placental hormone production. Inhibins and activins are produced by the placenta throughout pregnancy, and have explored as a potential markers in maternal serum for pregnancy and placental pathologies, including miscarriage, Down’s syndrome and pre-eclampsia. Finally, additional roles in immunomodulation at the materno-fetal interface, and in endometrial inflammatory events associated with menstruation and repair, are discussed.

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Host and Pathogen Glycosaminoglycan-Binding Proteins Modulate Antimicrobial Peptide Responses inDrosophila melanogaster

Infection and Immunity ◽

10.1128/iai.00254-10 ◽

2010 ◽

Vol 79 (2) ◽

pp. 606-616 ◽

Cited By ~ 8

Author(s):

Zhipeng Wang ◽

Lindsay A. Flax ◽

Melissa M. Kemp ◽

Robert J. Linhardt ◽

Miriam J. Baron

Keyword(s):

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Bone Morphogenetic Proteins ◽

Transforming Growth Factor ◽

Streptococcal Infection ◽

Transforming Growth Factor Β ◽

Sulfated Polysaccharide ◽

Host Cells ◽

Bacterial Surface

ABSTRACTDuring group B streptococcal infection, the alpha C protein (ACP) on the bacterial surface binds to host cell surface heparan sulfate proteoglycans (HSPGs) and facilitates entry of bacteria into human epithelial cells. Previous studies in aDrosophila melanogastermodel showed that binding of ACP to the sulfated polysaccharide chains (glycosaminoglycans) of HSPGs promotes host death and is associated with higher bacterial burdens. We hypothesized that ACP-glycosaminoglycan binding might determine infection outcome by altering host responses to infection, such as expression of antimicrobial peptides. As glycosaminoglycans/HSPGs also interact with a number of endogenous secreted signaling molecules inDrosophila, we examined the effects of host and pathogen glycosaminoglycan/HSPG-binding structures in host survival of infection and antimicrobial peptide expression. Strikingly, host survival after infection with wild-type streptococci was enhanced among flies overexpressing the endogenous glycosaminoglycan/HSPG-binding morphogen Decapentaplegic—a transforming growth factor β-likeDrosophilahomolog of mammalian bone morphogenetic proteins—but not by flies overexpressing a mutant, non-glycosaminoglycan-binding Decapentaplegic, or the other endogenous glycosaminoglycan/HSPG-binding morphogens, Hedgehog and Wingless. While ACP-glycosaminoglycan binding was associated with enhanced transcription of peptidoglycan recognition proteins and antimicrobial peptides, Decapentaplegic overexpression suppressed transcription of these genes during streptococcal infection. Further, the glycosaminoglycan-binding domain of ACP competed with Decapentaplegic for binding to the soluble glycosaminoglycan heparin in anin vitroassay. These data suggest that, in addition to promoting bacterial entry into host cells, ACP competes with Decapentaplegic for binding to glycosaminoglycans/HSPGs during infection and that these bacterial and endogenous glycosaminoglycan-binding structures determine host survival and regulate antimicrobial peptide transcription.

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Optogenetic control of the Bone Morphogenetic Protein signalling pathway through engineered blue light-sensitive receptors

10.1101/2020.04.27.063073 ◽

2020 ◽

Author(s):

Paul A. Humphreys ◽

Steven Woods ◽

Christopher A. Smith ◽

Stuart A. Cain ◽

Robert Lucas ◽

...

Keyword(s):

Blue Light ◽

Bone Morphogenetic Proteins ◽

Target Genes ◽

Transforming Growth Factor ◽

Nuclear Translocation ◽

Transforming Growth Factor Β ◽

Signalling Pathway ◽

Bmp Signalling ◽

Optogenetic Control

AbstractBone Morphogenetic Proteins (BMPs) are members of the Transforming Growth Factor β (TGFβ) superfamily and have crucial roles during development; including mesodermal patterning and specification of renal, hepatic and skeletal tissues. In vitro developmental models currently rely upon costly and unreliable recombinant BMP proteins that do not enable dynamic or precise perturbation of the BMP signalling pathway. Here, we develop a novel optogenetic BMP signalling system (optoBMP) that enables rapid induction of the canonical BMP signalling pathway through illumination with blue light. We demonstrate the utility of the optoBMP system in multiple human cell lines to initiate signal transduction through phosphorylation and nuclear translocation of SMAD1/5, leading to upregulation of BMP target genes including Inhibitors of DNA binding ID2 and ID4. Furthermore, we demonstrate how the optoBMP system can be used to fine-tune activation of the BMP signalling pathway through variable light stimulation. Optogenetic control of BMP signalling will enable dynamic and high-throughput intervention across a variety of applications in cellular and developmental systems.

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