Conserved regulation of Nodal-mediated left-right patterning in zebrafish and mouse.

Nodal is the major effector of left-right axis development. In mice, Nodal forms heterodimers with Gdf1 and is inhibited by Cerl2/Dand5 at the node, and by Lefty1 in the lateral plate mesoderm (LPM). Studies in zebrafish have suggested some parallels, but also differences, between left-right patterning in mouse and zebrafish. To address these discrepancies, we generated single and double zebrafish mutants for southpaw (spaw, the Nodal ortholog), dand5 and lefty1, and performed biochemical and activity assays with Spaw and Vg1/Gdf3 (the Gdf1 ortholog). Contrary to previous findings, spaw mutants failed to initiate spaw expression in the LPM, and asymmetric heart looping was absent, similar to mouse Nodal mutants. In blastoderm assays, Vg1 and Spaw were interdependent for target gene induction, and contrary to previous results, formed heterodimers. Loss of Dand5 or Lefty1 caused bilateral spaw expression, similar to mouse mutants, and Lefty1 was replaceable with a uniform Nodal signaling inhibitor. Collectively, these results indicate that Dand5 activity biases Spaw-Vg1 heterodimer activity to the left, Spaw around Kupffer's vesicle induces the expression of spaw in the LPM and global Nodal inhibition maintains the left bias of Spaw activity, demonstrating conservation between zebrafish and mouse mechanisms of left-right patterning.

Nr5a homologues in the ricefield eel Monopterus albus: Alternative splicing, tissue-specific expression, and differential roles on the activation of cyp19a1a promoter in vitro.

Nr5a (Fushi tarazu factor 1, Ftz-F1) homologues belong to the nuclear receptor superfamily, and are involved in the regulation of reproduction in vertebrates. Four genes encoding Nr5a homologues were present in the genome of ricefield eel, which are designated as nr5a1a, nr5a1b, nr5a2, and nr5a5 in the present study. Alternatively spliced transcripts were identified for nr5a1a and nr5a1b genes. Sequence analysis indicated that nr5a5 is possibly a paralog of nr5a2, and nr5a1b is lost during evolution in some teleosts including tilapia and medaka. Ricefield eel nr5a genes exhibit tissue-specific expression patterns, with nr5a1a and nr5a1b resembling that of the SF-1/Ad4BP (NR5A1) subfamily, and nr5a2 and nr5a5 resembling that of the NR5A2/LRH/FTF subfamily. Transcriptomic analysis revealed parallel expression profiles of nr5a1a, foxl2, and cyp19a1a in ovarian follicles during vitellogenesis, with peak values at the late vitellogenic stage. Real-time PCR indicated that the expression levels of nr5a1a and foxl2 in gonads were decreased significantly during the sexual transition from female to the late intersexual stage. In vitro transient transfection assay showed that Nr5a1a up-regulated ricefield eel cyp19a1a promoter activities synergistically with Foxl2. However, Nr5a1b, Nr5a2, and Nr5a5 could neither activate ricefield eel cyp19a1a promoter alone nor enhance the stimulatory effects of Foxl2 on cyp19a1a promoter activities. Collectively, the above data suggest that Nr5a homologues may have diverse and differential roles in the tissues of ricefield eels. The up-regulation of gonadal nr5a1a and foxl2 during vitellogenesis may be important for the ovarian development whereas their down-regulation during the sexual transition period may be important for the sex change process of ricefield eels, possibly through the regulation of cyp19a1a gene expression.

Nodal and BMP dispersal during early zebrafish development.

The secreted TGF-ß superfamily signals Nodal and BMP coordinate the patterning of vertebrate embryos. Nodal specifies endoderm and mesoderm during germ layer formation, and BMP specifies ventral fates and patterns the dorsal/ventral axis. Five major models have been proposed to explain how the correct distributions of Nodal and BMP are achieved within tissues to orchestrate embryogenesis: source/sink, transcriptional determination, relay, self-regulation, and shuttling. Here, we discuss recent experiments probing these signal dispersal models, focusing on early zebrafish development.

p38 MAPK as an essential regulator of dorsal-ventral axis specification and skeletogenesis during sea urchin development: a re-evaluation.

Dorsal-ventral axis formation in the sea urchin embryo relies on the asymmetrical expression of the TGFß Nodal. The p38-MAPK pathway has been proposed to be essential for dorsal-ventral axis formation by acting upstream of nodal expression. Here, we report that, in contrast to previous studies that used pharmacological inhibitors of p38, manipulating the activity of p38 by genetic means has no obvious impact on morphogenesis. Instead, we discovered that p38 inhibitors strongly disrupt specification of all germ layers by blocking signalling from the Nodal receptor and by interfering with the ERK pathway. Strikingly, while expression of a mutant p38 that is resistant to SB203580 did not rescue dorsal-ventral axis formation or skeletogenesis in embryos treated with this inhibitor, expression of mutant Nodal receptors that are resistant to SB203580 fully restored nodal expression in SB203580-treated embryos. Taken together, these results establish that p38 activity is not required for dorsal-ventral axis formation through nodal expression nor for skeletogenesis. Our results prompt a re-evaluation of the conclusions of several recent studies that linked p38 activity to dorsal-ventral axis formation and to patterning of the skeleton.

Translational co-regulation of a ligand and inhibitor by a conserved RNA element.

In many organisms, transcriptional and post-transcriptional regulation of components of pathways or processes has been reported. However, to date, there are few reports of translational co-regulation of multiple components of a developmental signaling pathway. Here, we show that an RNA element which we previously identified as a dorsal localization element (DLE) in the 3'UTR of zebrafish nodal-related1/squint (ndr1/sqt) ligand mRNA, is shared by the related ligand nodal-related2/cyclops (ndr2/cyc) and the nodal inhibitors, lefty1 (lft1) and lefty2 mRNAs. We investigated the activity of the DLEs through functional assays in live zebrafish embryos. The lft1 DLE localizes fluorescently labeled RNA similarly to the ndr1/sqt DLE. Similar to the ndr1/sqt 3'UTR, the lft1 and lft2 3'UTRs are bound by the RNA-binding protein (RBP) and translational repressor, Y-box binding protein 1 (Ybx1), whereas deletions in the DLE abolish binding to Ybx1. Analysis of zebrafish ybx1 mutants shows that Ybx1 represses lefty1 translation in embryos. CRISPR/Cas9-mediated inactivation of human YBX1 also results in human NODAL translational de-repression, suggesting broader conservation of the DLE RNA element/Ybx1 RBP module in regulation of Nodal signaling. Our findings demonstrate translational co-regulation of components of a signaling pathway by an RNA element conserved in both sequence and structure and an RBP, revealing a 'translational regulon'.

A novel TGFß modulator that uncouples R-Smad/I-Smad-mediated negative feedback from R-Smad/ligand-driven positive feedback.

As some of the most widely utilised intercellular signalling molecules, transforming growth factor ß (TGFß) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFß signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFß signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFß signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFß signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFß signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFß family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFß signalling and thereby embryonic patterning.

Xenopus pitx3 target genes lhx1 and xnr5 are identified using a novel three-fluor flow cytometry-based analysis of promoter activation and repression.

BACKGROUND: Pitx3 plays a well understood role in directing development of lens, muscle fiber, and dopaminergic neurons; however, in Xenopus laevis, it may also play a role in early gastrulation and somitogenesis. Potential downstream targets of pitx3 possess multiple binding motifs that would not be readily accessible by conventional promoter analysis. RESULTS: We isolated and characterized pitx3 target genes lhx1 and xnr5 using a novel three-fluor flow cytometry tool that was designed to dissect promoters with multiple binding sites for the same transcription factor. This approach was calibrated using a known pitx3 target gene, Tyrosine hydroxylase. CONCLUSIONS: We demonstrate how flow cytometry can be used to detect gene regulatory changes with exquisite precision on a cell-by-cell basis, and establish that in HEK293 cells, pitx3 directly activates lhx1 and represses xnr5. Developmental Dynamics 246:657-669, 2017. © 2017 Wiley Periodicals, Inc.

The transcription factor Vox represses endoderm development by interacting with Casanova and Pou2.

Endoderm and mesoderm are both formed upon activation of Nodal signaling but how endoderm differentiates from mesoderm is still poorly explored. The sox-related gene casanova (sox32) acts downstream of the Nodal signal, is essential for endoderm development and requires the co-factor Pou2 (Pou5f1, Oct3, Oct4) in this process. Conversely, BMP signals have been shown to inhibit endoderm development by an as yet unexplained mechanism. In a search for Casanova regulators in zebrafish, we identified two of its binding partners as the transcription factors Pou2 and Vox, a member of the Vent group of proteins also involved in the patterning of the gastrula. In overexpression studies we show that vox and/or Vent group genes inhibit the capacity of Casanova to induce endoderm, even in the presence of its co-factor Pou2, and that Vox acts as a repressor in this process. We further show that vox, but not other members of the Vent group, is essential for defining the proper endodermal domain size at gastrulation. In this process, vox acts downstream of BMPs. Cell fate analysis further shows that Vox plays a key role downstream of BMP signals in regulating the capacity of Nodal to induce endoderm versus mesoderm by modulating the activity of the Casanova/Pou2 regulatory system.

Multicellular Mathematical Modelling of Mesendoderm Formation in Amphibians.

The earliest cell fate decisions in a developing embryo are those associated with establishing the germ layers. The specification of the mesoderm and endoderm is of particular interest as the mesoderm is induced from the endoderm, potentially from an underlying bipotential group of cells, the mesendoderm. Mesendoderm formation has been well studied in an amphibian model frog, Xenopus laevis, and its formation is driven by a gene regulatory network (GRN) induced by maternal factors deposited in the egg. We have recently demonstrated that the axolotl, a urodele amphibian, utilises a different topology in its GRN to specify the mesendoderm. In this paper, we develop spatially structured mathematical models of the GRNs governing mesendoderm formation in a line of cells. We explore several versions of the model of mesendoderm formation in both Xenopus and the axolotl, incorporating the key differences between these two systems. Model simulations are able to reproduce known experimental data, such as Nodal expression domains in Xenopus, and also make predictions about how the positional information derived from maternal factors may be interpreted to drive cell fate decisions. We find that whilst cell-cell signalling plays a minor role in Xenopus, it is crucial for correct patterning domains in axolotl.

Integration of nodal and BMP signals in the heart requires FoxH1 to create left-right differences in cell migration rates that direct cardiac asymmetry.

Failure to properly establish the left-right (L/R) axis is a major cause of congenital heart defects in humans, but how L/R patterning of the embryo leads to asymmetric cardiac morphogenesis is still unclear. We find that asymmetric Nodal signaling on the left and Bmp signaling act in parallel to establish zebrafish cardiac laterality by modulating cell migration velocities across the L/R axis. Moreover, we demonstrate that Nodal plays the crucial role in generating asymmetry in the heart and that Bmp signaling via Bmp4 is dispensable in the presence of asymmetric Nodal signaling. In addition, we identify a previously unappreciated role for the Nodal-transcription factor FoxH1 in mediating cell responsiveness to Bmp, further linking the control of these two pathways in the heart. The interplay between these TGFß pathways is complex, with Nodal signaling potentially acting to limit the response to Bmp pathway activation and the dosage of Bmp signals being critical to limit migration rates. These findings have implications for understanding the complex genetic interactions that lead to congenital heart disease in humans.

Zebrafish Rnf111 is encoded by multiple transcripts and is required for epiboly progression and prechordal plate development.

Arkadia (also known as RING finger 111) encodes a nuclear E3 ubiquitin ligase that targets intracellular effectors and modulators of TGFß/Nodal-related signaling for polyubiquitination and proteasome-dependent degradation. In the mouse, loss of Arkadia results in early embryonic lethality, with defects attributed to compromised Nodal signaling. Here, we report the isolation of zebrafish arkadia/rnf111, which is represented by 5 transcript variants. arkadia/rnf111 is broadly expressed during the blastula and gastrula stages, with eventual enrichment in the anterior mesendoderm, including the prechordal plate. Morpholino knockdown experiments reveal an unexpected role for Arkadia/Rnf111 in both early blastula organization and epiboly progression. Using a splice junction morpholino, we present additional evidence that arkadia/rnf111 transcript variants containing a 3' alternative exon are specifically required for epiboly progression in the late gastrula. This result suggests that arkadia/rnf111 transcript variants encode functionally relevant protein isoforms that provide additional intracellular flexibility and regulation to the Nodal signaling pathway.

Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb.

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4-6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4-6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4-5-6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity.

Dorsal activity of maternal squint is mediated by a non-coding function of the RNA.

Despite extensive study, the earliest steps of vertebrate axis formation are only beginning to be elucidated. We previously showed that asymmetric localization of maternal transcripts of the conserved zebrafish TGFß factor Squint (Sqt) in 4-cell stage embryos predicts dorsal, preceding nuclear accumulation of ß-catenin. Cell ablations and antisense oligonucleotides that deplete Sqt lead to dorsal deficiencies, suggesting that localized maternal sqt functions in dorsal specification. However, based upon analysis of sqt and Nodal signaling mutants, the function and mechanism of maternal sqt was debated. Here, we show that sqt RNA may function independently of Sqt protein in dorsal specification. sqt insertion mutants express localized maternal sqt RNA. Overexpression of mutant/non-coding sqt RNA and, particularly, the sqt 3'UTR, leads to ectopic nuclear ß-catenin accumulation and expands dorsal gene expression. Dorsal activity of sqt RNA requires Wnt/ß-catenin but not Oep-dependent Nodal signaling. Unexpectedly, sqt ATG morpholinos block both sqt RNA localization and translation and abolish nuclear ß-catenin, providing a mechanism for the loss of dorsal identity in sqt morphants and placing maternal sqt RNA upstream of ß-catenin. The loss of early dorsal gene expression can be rescued by the sqt 3'UTR. Our findings identify new non-coding functions for the Nodal genes and support a model wherein sqt RNA acts as a scaffold to bind and deliver/sequester maternal factors to future embryonic dorsal.

Embryonic mesoderm and endoderm induction requires the actions of non-embryonic Nodal-related ligands and Mxtx2.

Vertebrate mesoderm and endoderm formation requires signaling by Nodal-related ligands from the TGFß superfamily. The factors that initiate Nodal-related gene transcription are unknown in most species and the relative contributions of Nodal-related ligands from embryonic, extraembryonic and maternal sources remain uncertain. In zebrafish, signals from the yolk syncytial layer (YSL), an extraembryonic domain, are required for mesoderm and endoderm induction, and YSL expression of nodal-related 1 (ndr1) and ndr2 accounts for a portion of this activity. A variable requirement of maternally derived Ndr1 for dorsal and anterior axis formation has also been documented. Here we show that Mxtx2 directly activates expression of ndr2 via binding to its first intron and is required for ndr2 expression in the YSL. Mxtx2 is also required for the Nodal signaling-independent expression component of the no tail a (ntla) gene, which is required for posterior (tail) mesoderm formation. Therefore, Mxtx2 defines a new pathway upstream of Nodal signaling and posterior mesoderm formation. We further show that the co-disruption of extraembryonic Ndr2, extraembryonic Ndr1 and maternal Ndr1 eliminates endoderm and anterior (head and trunk) mesoderm, recapitulating the loss of Nodal signaling phenotype. Therefore, non-embryonic sources of Nodal-related ligands account for the complete spectrum of early Nodal signaling requirements. In summary, the induction of mesoderm and endoderm depends upon the combined actions of Mxtx2 and Nodal-related ligands from non-embryonic sources.

Direct and indirect control of oral ectoderm regulatory gene expression by Nodal signaling in the sea urchin embryo.

The Nodal signaling pathway is known from earlier work to be an essential mediator of oral ectoderm specification in the sea urchin embryo, and indirectly, of aboral ectoderm specification as well. Following expression of the Nodal ligand in the future oral ectoderm during cleavage, a sequence of regulatory gene activations occur within this territory which depend directly or indirectly on nodal gene expression. Here we describe additional regulatory genes that contribute to the oral ectoderm regulatory state during specification in Strongylocentrotus purpuratus, and show how their spatial expression changes dynamically during development. By means of system wide perturbation analyses we have significantly improved current knowledge of the epistatic relations among the regulatory genes of the oral ectoderm. From these studies there emerge diverse circuitries relating downstream regulatory genes directly and indirectly to Nodal signaling. A key intermediary regulator, the role of which had not previously been discerned, is the not gene. In addition to activating several genes earlier described as targets of Nodal signaling, the not gene product acts to repress other oral ectoderm genes, contributing crucially to the bilateral spatial organization of the embryonic oral ectoderm.

Conservation defines functional motifs in the squint/nodal-related 1 RNA dorsal localization element.

RNA localization is emerging as a general principle of sub-cellular protein localization and cellular organization. However, the sequence and structural requirements in many RNA localization elements remain poorly understood. Whereas transcription factor-binding sites in DNA can be recognized as short degenerate motifs, and consensus binding sites readily inferred, protein-binding sites in RNA often contain structural features, and can be difficult to infer. We previously showed that zebrafish squint/nodal-related 1 (sqt/ndr1) RNA localizes to the future dorsal side of the embryo. Interestingly, mammalian nodal RNA can also localize to dorsal when injected into zebrafish embryos, suggesting that the sequence motif(s) may be conserved, even though the fish and mammal UTRs cannot be aligned. To define potential sequence and structural features, we obtained ndr1 3'-UTR sequences from approximately 50 fishes that are closely, or distantly, related to zebrafish, for high-resolution phylogenetic footprinting. We identify conserved sequence and structural motifs within the zebrafish/carp family and catfish. We find that two novel motifs, a single-stranded AGCAC motif and a small stem-loop, are required for efficient sqt RNA localization. These findings show that comparative sequencing in the zebrafish/carp family is an efficient approach for identifying weak consensus binding sites for RNA regulatory proteins.

Subnuclear development of the zebrafish habenular nuclei requires ER translocon function.

The dorsal habenular nuclei (Dh) of the zebrafish are characterized by significant left-right differences in gene expression, anatomy, and connectivity. Notably, the lateral subnucleus of the Dh (LsDh) is larger on the left side of the brain than on the right, while the medial subnucleus (MsDh) is larger on the right compared to the left. A screen for mutations that affect habenular laterality led to the identification of the sec61a-like 1(sec61al1) gene. In sec61al1(c163) mutants, more neurons in the LsDh and fewer in the MsDh develop on both sides of the brain. Generation of neurons in the LsDh occurs more rapidly and continues for a longer time period in mutants than in WT. Expression of Nodal pathway genes on the left side of the embryos is unaffected in mutants, as is the left sided placement of the parapineal organ, which promotes neurogenesis in the LsDh of WT embryos. Ultrastructural analysis of the epithalamus indicates that ventricular precursor cells, which form an epithelium in WT embryos, lose apical-basal polarity in sec61al1(c163) mutants. Our results show that in the absence of sec61al1, an excess of precursor cells for the LsDh exit the ventricular region and differentiate, resulting in formation of bilaterally symmetric habenular nuclei.

The zebrafish maternal-effect gene mission impossible encodes the DEAH-box helicase Dhx16 and is essential for the expression of downstream endodermal genes.

Early animal embryonic development requires maternal products that drive developmental processes prior to the activation of the zygotic genome at the mid-blastula transition. During and after this transition, maternal products may continue to act within incipient zygotic developmental programs. Mechanisms that control maternally-inherited products to spatially and temporally restrict developmental responses remain poorly understood, but necessarily depend on posttranscriptional regulation. We report the functional analysis and molecular identification of the zebrafish maternal-effect gene mission impossible (mis). Our studies suggest requirements for maternally-derived mis function in events that occur during gastrulation, including cell movement and the activation of some endodermal target genes. Cell transplantation experiments show that the cell movement defect is cell autonomous. Within the endoderm induction pathway, mis is not required for the activation of early zygotic genes, but is essential to implement nodal activity downstream of casanova/sox 32 but upstream of sox17 expression. Activation of nodal signaling in blastoderm explants shows that the requirement for mis function in endoderm gene induction is independent of the underlying yolk cell. Positional cloning of mis, including genetic rescue and complementation analysis, shows that it encodes the DEAH-box RNA helicase Dhx16, shown in other systems to act in RNA regulatory processes such as splicing and translational control. Analysis of a previously identified insertional dhx16 mutation shows that the zygotic component of this gene is also essential for embryonic viability. Our studies provide a striking example of the interweaving of maternal and zygotic genetic functions during the egg-to-embryo transition. Maternal RNA helicases have long been known to be involved in the development of the animal germ line, but our findings add to growing evidence that these factors may also control specific gene expression programs in somatic tissues.

Xrel3/XrelA attenuates ß-catenin-mediated transcription during mesoderm formation in Xenopus embryos.

In Xenopus laevis embryonic development, activation of the Wnt/ß-catenin pathway promotes mesoderm cell fate determination via Xnr (Xenopus nodal-related) expression. We have demonstrated previously that Rel/NF-κB (nuclear factor κB) proteins expressed in presumptive ectoderm limit the activity of Xnrs to the marginal zone of embryos during mesoderm induction, which assists to distinguish mesoderm from ectoderm. The mechanism of this regulation, however, is unknown. In the present study, we investigated whether Rel/NF-κB proteins are able to modulate mesoderm formation by mediating Wnt/ß-catenin signalling. We determined that ectopic expression of XrelA or Xrel3 in the dorsal marginal zone perturbed dorsal mesoderm formation by down-regulating multiple Wnt/ß-catenin target genes including Xnr3, Xnr5 and Xnr6. Ventral co-expression of XrelA or Xrel3 with either wild-type ß-catenin or constitutively active ß-cateninS37A abrogated ß-catenin-induced axis duplication and attenuated ß-catenin-stimulated reporter transcription. Lastly, we provide evidence that Xrel3, but not XrelA, can interact with ß-catenin without affecting the association of ß-catenin with other transcriptional co-activators in vitro. Both Xrel3 and XrelA, however, prevented the accumulation, in nuclei, of exogenously expressed and endogenous ß-catenin in vivo. These results suggest that Rel proteins are able to bind ß-catenin and attenuate ß-catenin-mediated transcription by nuclear exclusion.

The fibroblast integrin alpha11beta1 is induced in a mechanosensitive manner involving activin A and regulates myofibroblast differentiation.

Fibrotic tissue is characterized by an overabundance of myofibroblasts. Thus, understanding the factors that induce myofibroblast differentiation is paramount to preventing fibrotic healing. Previous studies have shown that mechanical stress derived from the integrin-mediated interaction between extracellular matrix and the cytoskeleton promotes myofibroblast differentiation. Integrin alpha11beta1 is a collagen receptor on fibroblasts. To determine whether alpha11beta1 can act as a mechanosensor to promote the myofibroblast phenotype, mouse embryonic fibroblasts and human corneal fibroblasts were utilized. We found that alpha11 mRNA and protein levels were up-regulated in mouse embryonic fibroblasts grown in attached three-dimensional collagen gels and conversely down-regulated in cells grown in floating gels. alpha11 up-regulation could be prevented by manually detaching the collagen gels or by cytochalasin D treatment. Furthermore, SB-431542, an inhibitor of signaling via ALK4, ALK5, and ALK7, prevented the up-regulation of alpha11 and the concomitant phosphorylation of Smad3 under attached conditions. In attached gels, TGF-beta1 was secreted in its inactive form but surprisingly not further activated, thus not influencing alpha11 regulation. However, inhibition of activin A attenuated the up-regulation of alpha11. To determine the role of alpha11 in myofibroblast differentiation, human corneal fibroblasts were transfected with small interfering RNA to alpha11, which decreased alpha-smooth muscle actin expression and myofibroblast differentiation. Our data suggest that alpha11beta1 is regulated by cell/matrix stress involving activin A and Smad3 and that alpha11beta1 regulates myofibroblast differentiation.