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TGF-b Family Signaling in Epithelial Differentiation and Epithelial–Mesenchymal Transition

Kaoru Kahata,1 Mahsa Shahidi Dadras,1,2 and Aristidis Moustakas1,2

1Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, SE–751 24 Uppsala, Sweden 2Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, SE–751 23 Uppsala, Sweden Correspondence: [email protected]

Epithelia exist in the animal body since the onset of embryonic development; they generate tissue barriers and specify organs and glands. Through epithelial–mesenchymal transitions (EMTs), epithelia generate mesenchymal cells that form new tissues and promote healing or disease manifestation when epithelial homeostasis is challenged physiologically or patho- logically. Transforming growth factor-bs (TGF-bs), activins, bone morphogenetic proteins (BMPs), and growth and differentiation factors (GDFs) have been implicated in the regulation of epithelial differentiation. These TGF-b family ligands are expressed and secreted at sites where the epithelium interacts with the mesenchyme and provide paracrine queues from the mesenchyme to the neighboring epithelium, helping the specification of differentiated ep- ithelial cell types within an organ. TGF-b ligands signal via Smads and cooperating kinase pathways and control the expression or activities of key transcription factors that promote either epithelial differentiation or mesenchymal transitions. In this review, we discuss evi- dence that illustrates how TGF-b family ligands contribute to epithelial differentiation and induce mesenchymal transitions, by focusing on the embryonic ectoderm and tissues that form the external mammalian body lining.

TGF-b FAMILY MEMBERS IN EPITHELIAL activity and activate intracellular effectors such DIFFERENTIATION as Smad proteins and various signaling branch- es of protein kinases, lipid kinases, and small he transforming growth factor-b (TGF-b) GTPases (Hata and Chen 2016; Zhang 2017). Tfamily consists of secreted polypeptides The Smad branch of signaling mediators in- that include TGF-b1, TGF-b2, and TGF-b3, cludes receptor-activated Smads (R-Smads), activins, bone morphogenetic proteins (BMPs), a common mediator Smad (co-Smad), and and growth and differentiation factors (GDFs). inhibitory Smads (I-Smads). Five different R- These ligands signal after binding to type II Smads (Smad1, Smad2, Smad3, Smad5, and and type I receptors that show protein kinase Smad8) are directly phosphorylated by the

Editors: Rik Derynck and Kohei Miyazono Additional Perspectives on The Biology of the TGF-b Family available at www.cshperspectives.org Copyright # 2018 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a022194 Cite this article as Cold Spring Harb Perspect Biol 2018;10:a022194

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type I receptors. Phosphorylated R-Smads form enchymal cells. In addition to the mesenchymal heteromeric complexes with Smad4, the only inputs, epithelial cells are also capable of trans- mammalian co-Smad. Smad6 and Smad7 are differentiating to other cell types, through I-Smads, whose genes are transcriptionally in- processes that are collectively termed epithelial duced by the TGF-b, activin, and BMP path- plasticity. When the transdifferentiation gener- ways and limit the activities of these pathways ates mesenchymal cells, the process is best (Miyazawa and Miyazono 2016). The TGF-b known as epithelial–mesenchymal transition family is implicated in multiple stages of early (EMT) (Hay 1995; Lim and Thiery 2012). embryonic development; a prominent example EMT can be reversible and then leads to the is nodal, which signals the generation of prox- transdifferentiation from mesenchymal to epi- imodistal polarity in the early embryo (Schier thelial cells, known as mesenchymal–epithelial 2003; Robertson 2014). As embryonic morpho- transition (MET) (Nieto 2013). However, cases genesis proceeds, nodal also specifies endoder- of MET in which the starting cell source is a mal tissue differentiation, and controls the differentiated mesenchymal cell (e.g., a fibro- anteroposterior pattern of the embryo (Schier blast) have been reported mainly in the field 2003; Robertson 2014). In addition, left–right of induced pluripotent stem (iPS) cell technol- body asymmetry is regulated by nodal and ogy (Sanchez-Alvarado and Yamanaka 2014). BMPs (Shiratori and Hamada 2014). On the EMTand MET play critical roles in disease path- other hand, dorsoventral embryonic polarity is ogenesis (Kalluri and Weinberg 2009), such as controlled by BMP-specific extracellular antag- in cancer, in which TGF-b-induced EMT em- onists, such as chordin and noggin, which limit powers prometastatic potential, and in tissue the binding of BMPs to their signaling receptors fibrosis, in which BMP-induced MET counter- (Bierand De Robertis 2015). Thus, BMPs secret- acts fibrosis. In this review, we focus on roles of ed by dorsal embryonic tissue (i.e., the Spemann EMT and MET during normal development of organizer) repress gene expression, and BMPs epithelial organs. secreted by ventral tissue activate gene expres- Hallmarks of EMT are the remodeling of sion, generating a dorsoventral polar pattern of tight, adherens, and desmosomal junctions at differentiation in the emerging embryonic tis- the plasma membrane and remodeling of the sue (Bier and De Robertis 2015). Nodal and cytoskeleton, including actin microfilaments, BMPs and their extracellular antagonists, acting microtubules, and keratin or vimentin interme- in concentration-dependent gradients across diate filaments; these processes are driven by the early embryonic tissue, enable progenitor remodeling of polarity complexes in epithelial cells to generate the three embryonic line- cells (Wheelock et al. 2008; Nelson 2009; Huang ages—ectoderm, endoderm and mesoderm. et al. 2012). The TGF-b family plays prominent These three lineages subsequently generate di- roles in directing EMT and MET, and much is rectly or indirectly (i.e., through epithelial– already understood in relation to the signaling mesenchymal interactions) differentiated epi- pathways that mediate this epithelial response thelial cell types, which populate the various and the gene programs that control transdiffer- tissues discussed in this review. entiation (Lamouille et al. 2014). Historically, evidence that TGF-b members induce EMT was gathered in studies of heart EPITHELIAL–MESENCHYMAL AND valve formation; TGF-b2 transforms endothe- MESENCHYMAL–EPITHELIAL TRANSITIONS lial cells to mesenchymal cells that generate the IN EPITHELIAL ORGANOGENESIS cushions that line the septa in the heart valves Epithelial morphogenesis proceeds through (Mercado-Pimentel and Runyan 2007). This successive cycles of induction of epithelial pro- example expands the concept of EMT beyond liferation by the adjacent mesenchymal layer, epithelial cells and informs us that plasticity in followed by differentiation cycles, which are differentiation also takes place in the endothe- also positively or negatively controlled by mes- lium, thus named endothelial–mesenchymal

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TGF-b Family in Epithelial Differentiation

transition (EndMT). Another critical observa- ing the other two layers (Robertson 2014). How- tion of EMT induced by TGF-b1 involved ever, gastrulation additionally encompasses mouse mammary epithelial NMuMG cells substantial epithelial cell invagination and (Miettinen et al. 1994). Subsequent research movement without EMT. defined molecular pathways and gene-based The epiblast EMT involves embryonic TGF- mechanisms that mediate the EMT program in b family ligands such as mouse nodal and NMuMG cells (Valcourt et al. 2005). In addi- chicken Vg1, or the mammalian Vg1 orthologs tion, TGF-b members drive EMT while arrest- GDF-1 and GDF-3 (Shah et al. 1997; Chea et al. ing the cell cycle of NMuMG cells (Gal et al. 2005; Andersson et al. 2007). Classical studies 2008). It is worth noting that epithelial cell cycle established that loss-of-function Nodal muta- arrest by TGF-bs, activins or BMPs is a hallmark tions in mice lead to defective gastrulation response that characterizes this family of cyto- (Robertson 2014). Moreover, transplantation kines (Massague´ 2012). It was also shown that experiments, aimed at rescuing the mutant BMPs, in a concentration-dependent manner, phenotype, showed that only very low doses of can overcome the positive effects of TGF-b in nodal protein, expressed by few cells, are suffi- driving EMT, thus promoting the epithelial cient and required to mediate the epiblast EMT phenotype (Kowanetz et al. 2004), which is rem- (Varlet et al. 1997). The binding of nodal to its iniscent of a positive effect of BMPs toward receptors is controlled by the extracellular in- MET (Zeisberg et al. 2003). However, it should hibitors lefty and cerberus-like, so that only cells be kept in mind that BMP signaling can also positioned at the correct place during tissue promote EMT in cancer (Davis et al. 2016) or ingression undergo EMT (Perea-Gomez et al. during neural crest delamination, as will be de- 2002; Bertocchini et al. 2004). Cerberus-like an- scribed later. Finally, studies in the cancer field tagonizes not only nodal but also BMPs and revealed a role of EMT in the generation of Wnts, which is important as explained below. epithelial stemness, and this concept has been Similar to nodal, gain-of-function overexpres- extended to differentiation of normal mamma- sion of Vg1 promotes epiblast EMT and causes ry epithelium, and embryonic stem cell biology, partial duplication of the chicken primitive including iPS cell technology (Watabe and streak (Shah et al. 1997; Chea et al. 2005), Miyazono 2009; Samavarchi-Tehrani et al. whereas loss-of-function mutations in mouse 2010; Tam and Weinberg 2013). Gdf1 and Gdf3 suppress epiblast EMT (Anders- son et al. 2007). The embryonic epithelial cells need to be sensitized by Wnt signaling, which EPIBLAST EPITHELIAL TO MESODERM ensures that the cells express TGF-b family re- DIFFERENTIATION ceptors (Skromne and Stern 2001). Thus, Wnt Gastrulation generates three layers of embryonic signaling acts upstream of nodal, Vg1, or GDF- tissue—ectoderm, mesoderm, and endoderm. 1/3; therefore, altering Wnt levels in developing Each of these layers has distinct developmental gastrulae mimics experimental perturbations fates as they generate the adult body tissues, with of nodal, Vg1, or GDF-1/3 (Skromne and Stern nerve and skin being derived from ectoderm, 2001). In addition, the epiblast EMT must muscle from mesoderm, and gut from endo- be regulated temporally, and this is partially derm (Robertson 2014). During gastrulation, achieved by activation of fibroblast growth the epithelial cells of the epiblast generate, factor (FGF) signaling in mesenchymal cells through EMT, motile mesenchymal cells that (Mathieu et al. 2004). FGF and nodal cooperate migrate into the blastocyst cavity to populate to sustain the expression of key transcription the space and give rise to the germ layers. In factors that drive the EMT during gastrulation, this manner, EMT gives rise to two embryonic including Snail family proteins (Mathieu et al. tissue layers, mesoderm and endoderm, local- 2004). Despite the defective gastrulation in em- ized toward the center of the body; ectoderm bryos lacking the Snail1 gene, Snail transcrip- lies toward the surface of the embryo surround- tional activity has not yet been firmly linked to

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K. Kahata et al.

induction of the mesodermal differentiation Wnt, FGF,and BMP signaling pathways reg- program (Acloque et al. 2009). Instead, the ulate gene expression during neural crest devel- transcription factor eomesodermin (Eomes) opment (Sauka-Spengler and Bronner-Fraser may regulate the specification of mesoderm by 2008). Canonical and noncanonical Wnt sig- EMT in the gastrulating embryo (Arnold et al. naling regulate the proliferation of neural crest 2008). This has been shown by inactivation of precursors and the migration of delaminating Eomes, which does not perturb Snail expression cells (Garcia-Castro et al. 2002; Carmona-Fon- in the embryonic cells, despite the loss of EMT taine et al. 2008). In avian embryos, BMP-4 is and mesodermal differentiation in the mutant expressed in the dorsal neural tube that triggers embryos (Arnold et al. 2008). More studies on the expression of the transcription factor the molecular cooperation between Snail and c-Myb, and converts epithelial neural plate cells Eomes and the identification of distinct and into mesenchymal neural crest cells (Sela-Do- common target genes during gastrulation are nenfeld and Kalcheim 1999; Karafiat et al. warranted. One such target gene is the polarity 2005). The BMP inhibitor noggin, on the other gene Crumbs3, whose expression is repressed by hand, blocks the migration of neural crest cells Snail, resulting in disassembly of tight junctions until noggin levels are eventually down-regulat- in embryonic epithelial cells (Whiteman et al. ed by signals from the somites (Sela-Donenfeld 2008). Furthermore, TGF-b family members and Kalcheim 1999, 2000; Karafiat et al. 2005). induce Snail expression during the EMT and The cross talk between BMP and Wnt signaling activate the p38 mitogen activated protein during delamination of the neural crest cells kinase (MAPK) through its binding protein, involves release of BMP signaling from noggin, p38-interacting protein (p38IP) (Zohn et al. leading to increases in Wnt1 expression, which 2006). The function of p38IP relates to E-cad- eventually promotes cell cycle entry and delami- herin degradation during EMT (Zohn et al. nation (Burstyn-Cohen et al. 2004). 2006). However, similar to Snail, the exact roles At the transcription factor level, the Sox9, of p38IP and the p38 MAPK downstream from Foxd3, and Snail1 genes are necessary for the nodal or GDF-1/3 remain to be clarified. regulation of delamination during neural crest EMT during primitive streak formation EMT (Dottori et al. 2001; Cheung et al. 2005). shows mechanistic parallels with the changes Sox9 is required for EMT and survival of the observed in cell culture and in studies of EMT neural crest cells in the trunk of the crest during tissue fibrosis or cancer progression. (Cheung et al. 2005). Snail and Slug (encoded Deciphering molecular mechanisms of embry- by the Snail2 gene), together with Sox9, induce onic EMT is of direct relevance to the patho- neural crest delamination and EMT in neural physiology of adult diseases. epithelial cells, whereas FoxD3 regulates the neural crest cell migration by repressing the ex- pression of cell–cell adhesion molecules and NEUROECTODERMAL DELAMINATION promoting the expression of migratory mark- AND NEURAL CREST EMT ers, including human natural killer antigen-1 The neural crest represents an embryonic stem (HNK-1) and cadherin-7 (Dottori et al. 2001; cell population, unique to vertebrates, that is Cheung et al. 2005). In addition, BMP signaling located at the border of the neural plate in the generates complexes of Smad1 and/or Smad5 early embryo and forms the central nervous with the transcription factor Sox5, which system. After the neural tube closure, neural activate the expression of several genes before crest cells reside in the dorsal neural tube and the delamination of the neuroectodermal cells undergo EMT to generate numerous deriva- (Nordin and LaBonne 2014). The activity of tives, including peripheral neurons, glial and these complexes is also important during other Schwann cells, pigment cells, and facial skeleton ectodermal tissue movements, in the blastula or cells (Bronner-Fraser and Fraser 1988; Bronner during neural plate formation (Nordin and 2012; Rogers et al. 2013). LaBonne 2014).

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TGF-b Family in Epithelial Differentiation

Similar to other EMTs, a positive forward the EMT induced by BMP, whereas cadherin- regulatory loop has been revealed in the neural 6B contributes to the delamination process crest. Accordingly, BMP signaling induces the (Park and Gumbiner 2010). The mechanism avian Snail2 gene using Sox9 as a transcriptional of BMP receptor activation by cadherin-6B cofactor (Sakai et al. 2006). Sox9 and Slug also may not involve BMP ligands, but, rather, in- form complexes, and together with signaling teraction of cadherin-6B with the BMP type II input by protein kinase A, they amplify the receptor (BMPRII), which recruits the LIM expression of Snail2, resulting in sustained domain kinase LIMK1 to its carboxy-terminal Slug protein activity throughout the neural tail, providing signals toward cofilin that me- crest delamination process (Sakai et al. 2006). diate a reorganization of the actin cytoskeleton Another transcription factor that promotes (Park and Gumbiner 2012). Smad signaling EMT, zinc-finger E box–binding homeobox 2 mediated by BMPRII and BMPRI is not re- (ZEB2, also known as SIP1), contributes to the quired for this mechanism, and activation of neural crest mesenchymal transition (Rogers actin reorganization is sufficient to cooperate et al. 2013). Reduction of ZEB2 expression by with Sox9 for effective neural crest delamina- morpholino oligonucleotides revealed that its tion (Park and Gumbiner 2012). Furthermore, activity is required for the proper expression loss of N-cadherin and gain of cadherin-6 by of FoxD3 and subsequent transcriptional in- neural crest mesenchymal cells, results in ac- duction of Sox10 in the delaminating neural cumulation of F-actin in specialized apical crest cells that engage into migration (Rogers neural crest cell tails (Clay and Halloran et al. 2013). Overall, Snail and ZEB family 2014). This mechanism controls the distribu- transcription factors regulate the expression of tion of active Rho GTPase at the apical mem- cadherins and other junctional components brane during cell detachment and thus pro- (Cano et al. 2000; Ikenouchi et al. 2003; Peinado motes cell movement (Clay and Halloran et al. 2007). These EMT transcription factors 2014). Thus, neural crest EMT resembles the bind to conserved E-box sequences in regulato- EMTs of epithelial tissues, but also shows dis- ry promoter sequences of their target genes, tinct characteristics that require further under- such as the genes encoding E-cadherin, claudin, standing. In particular, the coordination of or occludin, and operate downstream from BMP signaling with other pathways remains TGF-b signaling in cooperation with the acti- to be fully characterized in the delaminating vated Smads (Vincent et al. 2009). neural crest cells. ZEB2 mediates the switch from E-cadherin to N-cadherin expression (Rogers et al. 2013). THE TGF-b FAMILY IN THE DEVELOPMENT In addition, several other cadherins mediate dif- OF EXTERNAL EPITHELIALTISSUES AND EMT ferent functions in premigratory and migratory neural crest cells. Accordingly, cadherin-6B is Skin and Hair Follicle Development important at the premigratory stage, and cad- Defining Ectoderm herin-7 and cadherin-11 facilitate cell migration during the mesenchymal transition (Nakagawa The epidermis and its appendages, such as and Takeichi 1998; Vallin et al. 1998). Interest- hair follicles, sebaceous glands, and sweat ingly, cadherin-6B plays a dual regulatory role glands, are derived from ectoderm. Ectoderm through cross talk with BMP signaling in the develops into epidermis and nervous system, neural crest (Park and Gumbiner 2010, 2012). and BMPs specify ectodermal identity (Fig. Cadherin-6B activates BMP signaling, which 1A). Based on a classical “default model” of promotes the EMT, whereas BMP signaling neural induction, BMP, in particular BMP-4, induces cadherin-6B expression and represses is an epidermal inducer, and chordin, noggin, N-cadherin expression in the premigratory and follistatin are extracellular antagonists se- neural crest (Park and Gumbiner 2010). In creted from cells in the Spemann organizer, this embryonic tissue, N-cadherin antagonizes which block BMP signaling and induce neural

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Figure 1. Roles of bone morphogenetic protein (BMP) signaling in epidermal development. (A) Ectoderm definition. Ectodermal cells (pink) are separated from the neural plate cells (yellow) by small patches of border cells (gray). (Legend continues on following page.) Downloaded from http://cshperspectives.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press

TGF-b Family in Epithelial Differentiation

development (reviewed in Stern 2005). Various 2016). The adjacent epidermal cells provide studies have shown that activation followed by both Hh and TGF-b2, which promote prolifer- repression of BMP signaling is not sufficient to ation and extracellular matrix synthesis in the specify the fate of ectoderm, and that neural two fibroblast types. Although the two fibroblast induction depends on FGF signaling in addi- lineages develop independently from each other, tion to the BMP signaling shutoff (Delaune they also maintain their responsiveness to the et al. 2005; Stern 2005). Furthermore, Wnt-in- original Wnt signaling input (Lichtenberger duced b-catenin signaling governs ectodermal et al. 2016). Later in skin development, basal patterning, by blocking ectodermal cells from progenitor cells divide and give rise to interme- responding to FGFs and preserving their re- diate cells that migrate a short distance to enter sponse to BMPs (Stern 2005; Patthey and the final differentiation zone of the dermal and Gunhaga 2014). Appropriate formation of the epidermal layers (Zhu et al. 2014). This later border region between epidermis and nervous phase is also driven by Wnt and b-catenin sig- system, which develops into neural crest and naling, which induces expression of BMP-2, peripheral nervous system, requires temporal BMP-4, or BMP-7 in the dermal cells (Zhu activation of BMP signaling under the control et al. 2014). BMP–Smad signaling then induces of Wnt-induced b-catenin (reviewed in Patthey expression of FGF7 and FGF10, which directly and Gunhaga 2014). Ectopic BMP narrows the promote epidermal differentiation in the adja- border region, resulting in a wider epidermal cent cell layer (Zhu et al. 2014). FGF signaling, in region, and overexpressed BMP antagonists en- cooperation with the transcription factor p63, large the neural plate arising from the border generates differentiated epidermal cells, which region (Stern 2005), which suggests that BMP move away from the mitotic layer of the basal controls ectodermal expression by defining the epidermal region, generating the stratified epi- position of the border. At this developmental dermal epithelium (Zhu et al. 2014). stage, the interaction of Sox5 with BMP-specific Smads plays important roles (Nordin and La- Overview of Epidermal Development Bonne 2014). In addition, Wnt-induced b-cat- enin activity in the dermis controls the genera- Once epidermis is specified, hair follicle forma- tion of two types of dermal fibroblasts; the tion begins with local thickening of the epitheli- papillary fibroblast lineage responds to Hedge- um (hair follicle placode), which is accompanied hog (Hh) signals and the reticular fibroblast lin- by condensation of the underlying mesenchyme eage responds to TGF-b2 (Lichtenberger et al. (dermal condensation) that becomes the der-

Figure 1. (Continued) These embryonic cell types are defined anatomically and based on the pathways that control their developmental fate. In ectodermal cells, Wnt-induced b-catenin signals are active, and BMP signaling is preserved and induces epidermis, whereas fibroblast growth factor (FGF) signaling promotes neural commitment in which Wnt–b-catenin signaling is silenced. Intermediate intensity of Wnt and BMP signals in the border region defines the limits of these embryonic territories. (B–E) Hair follicle development and hair cycling. (B) Wnt–b-catenin (þ) and FGF (þ) signaling induce placode formation accompanied by dermal condensation underneath. BMP (þ) signaling on either side of the hair follicle placode inhibits its formation. (C) Hair follicle placode “downgrowth” away from the surface of the skin and engulfment of dermal conden- sations at the tip of the hair germ that grows downward. (D) Proliferation and differentiation of bulge cells toward outer root sheath (ORS) cells are blocked by BMP signals, whereas BMP signals are required for terminal differentiation of matrix cells to inner root sheath (IRS) cells that generate the hair shaft. Hair follicle stem cells (HFSCs) are accommodated in the bulge and hair germ. Bulge cells remain in a quiescent stage of their cell cycle and BMP signaling contributes to their lengthy life span. Hair germ cells are more prone to be activated by dermal signals and contribute postnatally to hair follicle regeneration. The three phases of the postnatal hair cycle are also shown. (E) At the end of the telogen phase, the dermal papilla secretes noggin, which inhibits BMP signaling; BMPs inhibit bulge cell differentiation to hair germ (HG) cells and matrix cells as explained in D.

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mal papilla (Fig. 1B). The hair follicle placode cells need epidermal signals to form dermal pa- grows downward, away from the skin surface, a pillae (Davidson and Hardy 1952; Kollar 1970; process known as “follicular downgrowth,” and Hardy 1992). Instructive signals consist of acti- penetrates into the dermis, whereas the leading vators (ligands) and inhibitors (antagonists) for edge engulfs the underlying dermal condensa- certain signaling pathways, and their balance de- tion at the hair germ stage (Fig. 1C). The out- termines the strength of signals and their effects. ermost cells give rise to the outer root sheath In epidermal development, BMP signaling is a (ORS), which surrounds the hair follicles and is pivotal instructive pathway that operates during continuous with the basal layer of the interfol- mesenchymal and epithelial interactions. licular epidermis. The ORS is supposed to con- tain less differentiated cells than cells found in The BMPs in Hair Follicle Development other compartments of the hair follicle. Rapidly proliferating transit-amplifying cells located in Hair follicle formation is initiated by Wnt sig- the matrix (root sheath) of the hair follicle naling through b-catenin (Fig. 1B) (Zhou et al. emerge from the innermost cells, and differen- 1995; Gat et al. 1998; Andl et al. 2002). Wnt tiate into the supportive inner root sheath (IRS) induces the expression of Wnt inhibitors, such and into the hair shaft forming seven concentric as dickkopf (Dkk), whose concentration deter- layers (Fig. 1D). The epidermis and its ap- mines hair follicle position and patterning, pendages consistently renew after birth as they perhaps through a reaction–diffusion model derive from the multipotent epidermal stem (Jiang et al. 2004; Sick et al. 2006; Zhang et al. cells (Moore and Lemischka 2006; Blanpain 2008). Epidermal cells without Wnt or b-cate- and Fuchs 2014). The interfollicular epidermis nin signaling proliferate to form stratified inter- undergoes frequent turnover and is replenished follicular epithelium, and Wnt-induced b-cat- by epidermal stem cells that are located in the enin signaling correlates with the formation innermost (basal) layer of stratified epidermis of the hair follicle placode. Epithelial signals (Moore and Lemischka 2006; Blanpain and including FGF enable the formation of a Fuchs 2014). In contrast, hair follicle stem dermal condensation underneath the placode cells (HFSCs) only periodically proliferate and (Huh et al. 2013). Mice with an inactivated regenerate through the hair cycle that consists gene encoding the BMP antagonist noggin of three phases—catagen (apoptosis-driven show, among other phenotypes, delayed hair regression), telogen (quiescence), and anagen follicle induction (Botchkarev et al. 1999; Mou (growth and hair shaft formation) (Doma et al. 2006). Conversely, addition of exogenous et al. 2013). Hair follicle development during noggin on explanted embryonic skin cultures embryogenesis and the postnatal anagen phase promotes hair follicle formation (Botchkarev are thought to be regulated by similar mecha- et al. 1999; Mou et al. 2006). Accordingly, loss nisms that generate the hair shaft–producing of the negative control of BMP-4 activity results bulb (Fig. 1D,E; details about epidermal stem in disturbed hair follicle formation and leads to cells are presented below). progressive hair loss (Botchkarev et al. 1999; Mou et al. 2006). Furthermore, nuclear phos- pho-Smad1 cannot be detected in the follicular Mesenchymal and Epithelial Interaction placode region where Wnt signaling through Epidermal development and postnatal hair cy- b-catenin is activated (Mou et al. 2006). These cling are orchestrated by reciprocal instructive observations support an inhibitory role of BMP interactions between epithelium (epidermis) signaling in hair follicle formation. BMPs can and mesenchyme (dermis), as shown in cocul- attenuate the proliferation and migration and ture experiments of epidermal and dermal com- induce apoptosis of hair follicle epithelium ponents from embryonic skin (Sengel 1990). (Blessing et al. 1993; Botchkarev et al. 1999). Epidermal cells receive instructive signals from BMP signaling activity also correlates with the underlying dermis, and the mesenchymal the loss of expression of essential factors that

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TGF-b Family in Epithelial Differentiation

contribute to hair follicle development, such as Hoxc13, are expressed in the differentiating Sonic Hedgehog (Shh), the tumor necrosis fac- IRS and hair shaft (Godwin and Capecchi tor (TNF) family member ectodysplasin A 1998; Meier et al. 1999; Kulessa et al. 2000; (EDA), and its receptor EDAR (Kobielak et al. Tkatchenko et al. 2001; Ma et al. 2003; Johns 2003; Andl et al. 2004). Shh is a key regulator of et al. 2005). GATA-binding protein 3 (GATA-3) hair follicle morphogenesis during embryonic expression is also induced by BMP-4 and main- development and after birth during the anagen tains BMP-4 levels through a positive feedback stage (Jung et al. 1998; St-Jacques et al. 1998; loop, which is necessary for the differentiation Chiang et al. 1999; Wang et al. 2000). BMP-4 of the IRS (Kobielak et al. 2003). signaling mediates repression of Shh expression, whereas noggin activates Shh expression (Botch- Expression of BMPs and Antagonists in karev et al. 2001; Blanpain and Fuchs 2006; Hair Follicles Gao et al. 2008). EDA and its receptor EDAR cooperate with Wnt–b-catenin signaling to BMP-2 expression is only observed in mouse give rise to the specialized mechanosensory whisker follicles (Lyons et al. 1990; Bitgood guard hair follicles. EDA signaling through and McMahon 1995), and BMP-4 expression EDAR also increases the expression of connec- is more dominant in dermal condensations tive tissue growth factor (CTGF) and moderately and papillae, although it is expressed in the increases the expression of the BMP and activin IRS and precortical cells at later developmental antagonist follistatin (Pummila et al. 2007). Fol- stages (Fig. 1D) (Bitgood and McMahon 1995). listatin, acting as a BMP antagonist, can then BMP-6 is expressed in both the epithelial tran- block BMP signaling, and BMPs, in turn, sit-amplifying matrix cells and the mesenchy- repress epidermal EDAR expression (Mou et al. mal dermal papillae, which regulate each other 2006; Pummila et al. 2007). CTGF has been through mesenchymal–epithelial interaction shown to act as either a weak BMP antagonist (Rendl et al. 2008). CTGF antagonizes the ac- or a factor that potentiates TGF-b signaling and tivity of BMP during the development of pri- promotes embryonic fibroblast condensation. mary hair follicles (Mou et al. 2006), whereas Thus, CTGF can possibly act indirectly against other types of hair follicles are more dependent the BMPs, through other BMP antagonists that on noggin as BMP antagonist (van Genderen are secreted by fibroblasts (Pummila et al. et al. 1994). Noggin is expressed in the dermal 2007), a mechanism of action that remains to condensations and papillae (mesenchyme) be clarified. throughout hair morphogenesis (Botchkarev By inactivating BMP signaling, noggin in- et al. 1999; Jamora et al. 2005), and its expres- duces the expression of lymphoid enhancer- sion is guided by epithelial signals, such as those binding factor 1 (LEF1), a transcription factor induced by Shh, laminin-511, platelet-derived downstream from the Wnt–b-catenin pathway growth factor (PDGF), and dermal signals in- (Botchkarev et al. 1999; Jamora et al. 2003). cluding those by integrin b1 and PDGF receptor Lack of noggin attenuates the activity of Wnt- a (PDGFRa) (Rishikaysh et al. 2014). The tran- induced b-catenin signaling, and inactivation scription factor Sox2 is expressed in dermal pa- of the gene encoding the BMP receptor pillae and activates the expression of Sostdc1 BMPRIA/ALK-3 results in sustained LEF1 ex- (sclerostin domain-containing 1, also known pression (Kobielak et al. 2003; Andl et al. 2004), as ectodin or USAG-1), a potent BMP inhibitor, which confirms that blocking BMP signaling leading to repression of BMP-6 expression in enhances and preserves the activity of the matrix cells (Clavel et al. 2012). Ablation of Wnt–b-catenin-LEF1 pathway (Fuchs 2007). Sox2 in dermal papillae decreases the expression Additionally, BMP signaling is essential for the of Sostdc1, which leads to activation of BMP-6 terminal differentiation of the hair shaft and the expression in matrix cells, and thus inhibits IRS from the transit-amplifying matrix cells; their migration toward the hair shaft compart- BMP target genes, such as Msx2, Foxn1, and ments, resulting in delay of hair growth.

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K. Kahata et al.

Epidermal Development and Smads (Fig. 1E) (Greco et al. 2009). The transition of HFSCs from the upper bulge toward the HG via R-Smads are highly expressed in the epidermis the lower bulge is observed at the end of the and hair follicles (He et al. 2001) and may have telogen phase, and HG cells directly contribute redundant roles. In contrast to inactivation of to the regeneration of hair follicles during the the expression of individual R-Smads, hair fol- anagen phase. licle development is severely abrogated when The transition of the HFSCs is regulated by Smad4 expression is deleted in keratinocytes dermal signals consisting of Wnts and noggin (Yang et al. 2005; Qiao et al. 2006). The pheno- (Greco et al. 2009; Rompolas et al. 2013). Inter- types of hair follicles in Smad42/2 mice are estingly, the Wnt and BMP signaling pathways very similar to those of Bmpr1a2/2 mice, are cyclically activated out of phase during the which suggests that hair follicle development hair regeneration cycle; this mechanism is main- is controlled by BMP–Smad4 signaling (Kobie- tained by the periodic regulation of expression of lak et al. 2003; Andl et al. 2004; Ming Kwan BMP-2 and BMP-4 by dermal cells and the out- et al. 2004; Yuhki et al. 2004). Loss of Smad4 of-phase, periodic Wnt-induced b-catenin sig- results in the depletion of HFSCs because of naling in the stem cells (Plikus et al. 2008). Ac- increased activity by b-catenin; increased b- tivated Wnt–b-catenin signals induce HG cells catenin activity transcriptionally induces the to proliferate and differentiate into matrix cells expression of c-Myc, which causes rapid cell leading to induction of anagen (Korinek et al. cycles and eventually exhausts the proliferative 1998; Pinto et al. 2003; Kuhnert et al. 2004; Blan- potential of the HFSCs, achieving their deple- pain and Fuchs 2006). Simultaneously, HG cells tion (Yang et al. 2009). Smad7 expression is express Shh that promotes the differentiation of very low in normal keratinocytes (He et al. lower bulge cells into ORS and induces dermal 2001) but is often increased under pathological papillae to express noggin (Ito et al. 2005; Jiang conditions, such as in aged and photoaged hu- and Hui 2008; Rompolas et al. 2013; Hsu et al. man skin (He et al. 2002; Quan et al. 2002) and 2014). In turn, noggin helps HG cells to express in skin cancer (He et al. 2001). Mice overex- Shh (Botchkarev et al. 2001, 2002). pressing Smad7 under the control of the kera- Inactivation of Bmpr1a, which encodes the tin-5 (Krt5) promoter show multiple develop- only known BMP type I receptor expressed in mental defects in their stratified epithelia, hair follicles (Botchkarev et al. 1999), induces including impairment of hair follicle develop- quiescent stem cells to enter the cell cycle lead- ment (He et al. 2002). ing to failure of hair follicle maintenance (Ko- bielak et al. 2003; Andl et al. 2004; Yuhki et al. Epidermal Stem Cells and Hair Cycling 2004; Zhang et al. 2006). This result suggests Accumulating evidence has revealed that stem that BMPs are essential for maintaining quies- cell niches are heterogeneous and compartmen- cence of HFSCs, which is in part explained by talized. Stem cells residing in different epider- the transcriptional activation of the gene encod- mal niches show different levels of quiescence ing the cell cycle inhibitor p27Kip1 by BMP sig- and fate (reviewed in Blanpain and Fuchs 2014). naling (Sharov et al. 2006). Supporting this no- Among HFSCs, most quiescent cells reside in tion, BMP-6 is expressed and Smad1 is activated the upper half of the bulge region underneath in HFSCs in the bulge (Andl et al. 2002, 2004; the sebaceous glands, and the cells in the lower Blanpain et al. 2004), and the expression of oth- half of the bulge are more likely to proliferate er Smads and their target genes is enriched in (Morris et al. 2004; Tumbar et al. 2004; Levy the bulge region (He et al. 2001; Morris 2004; et al. 2005; Zhang et al. 2009; Hsu et al. 2011; Tumbar et al. 2004). Acting upstream of BMPs Rompolas et al. 2013). The hair germ (HG) just in the HFSCs, the transcription factor FoxC1 under the bulge and in close proximity to the contributes to the self-renewal of the HFSCs dermal papilla represents another niche, where by inducing the expression of the nuclear factor HFSCs are prone to be activated and proliferate of activated T cells 1 (NFATc1)and BMP ligands

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TGF-b Family in Epithelial Differentiation

(Wang et al. 2016). In this manner, FoxC1 pro- sufficient to mediate the biological effects of motes the quiescent state of the stem cells, and BMPs, causing a prolonged anagen phase in genetic loss of the Foxc1 gene in activated stem the follicles in the absence of Smad1 and cells correlates with failure of the HFSCs to en- Smad5. Additionally, BMP signaling is respon- ter quiescence, whereas later ablation of Foxc1 in sible for suppressing histone 3 methylation lev- quiescent hair follicle cells residing in the stem els across the genome in HFSCs that enter qui- cell niche does not impair the ability of younger escence and differentiate into hair follicle cells HFSCs to enter the quiescent stage that relates (Lee et al. 2016). BMPs induce the expression of to differentiation. specific histone demethylases and repress ex- BMPs also repress Wnt-induced b-catenin pression of some histone methyltransferases signaling in the hair follicles (Jamora et al. 2003; (Lee et al. 2016). An important question that Kobielak et al. 2003; Blanpain et al. 2004). It has remains is whether these epigenetic changes ob- been suggested that paracrine BMP-4 and auto- served during the hair follicle cycle correlate with crine BMP-6 signaling maintain the quiescence the expression of specific genes and/or may re- in the niche (Blanpain et al. 2004). At the end of flect a conformational state of the chromatin the anagen phase, regeneration of the hair shaft that adapts to each stage of differentiation. and IRS from matrix cells is completed, con- In summary, BMPs maintain quiescent stem comitantly with the terminal differentiation of cells in the niche, and BMP signaling needs to be the hair follicles. As mentioned, BMPs are re- blocked in HG at the onset of anagen to allow quired for terminal differentiation of the hair HFSCs to proliferate and differentiate into ma- shaft. In the same way, the completion of regen- trix cells. Conversely, however, matrix cells and eration requires active BMP signals. Targeted hair shaft progenitors require BMPs to complete inactivation of Bmpr1a at this stage results in the generation of new HFSCs. Similar to embry- accumulation of highly proliferative and undif- onic hair follicle development, spatiotemporal ferentiated matrix cells and impaired differenti- activation of BMP signals is important for post- ation of the IRS and hair shaft (Ming Kwan et al. natal hair cycling and maintenance of HFSCs. 2004). Transgenic mice that express noggin in hair matrix cells and hair shaft precursor cells Roles of TGF-bs and Activins in Epidermal show accumulation of undifferentiated progen- Development itor cells, leading to severely impaired hair shaft differentiation (Kulessa et al. 2000). Consistent- TGF-b plays an important role in skin regener- ly, BMP-2 and BMP-4 are expressed during late ation (e.g., during wound healing) and is in- anagen and early telogen, and are absent during volved in normal epidermal development and late telogen and early anagen (Callahan and Oro maintenance of homeostasis, acting as a potent 2001). epithelial growth inhibitor. Transgenic mice The expression of Smads and their target that express Smad2 specifically in keratinocytes genes is diminished in HG, developing ORS, show severe thickening of their stratified epithe- and proliferating matrix cells. The strongest lium and decreased number of hair follicles (Ito BMP activity is observed in the differentiating et al. 2001), suggesting that Smad2 activation in IRS and hair shaft (Kobielak et al. 2003; Gen- response to TGF-b or activin signaling pro- ander et al. 2014). Elimination of Smad1 and motes tissue proliferation. TGF-b signaling is Smad5 expression during hair follicle morpho- also activated in HFSCs (Blanpain et al. 2004; genesis by conditional double gene inactivation Morris et al. 2004; Tumbar et al. 2004). At early in mice results in lack of differentiated whiskers stages of hair follicle development, neither the and associated death of the pups after birth epithelium nor the mesenchyme expresses (Kandyba et al. 2014). This experiment shows TGF-b at detectable levels (Lyons et al. 1990). that BMPs maintain HFSC quiescence through As the follicles grow downward away from the Smad1 and Smad5 signaling, and that the pres- skin surface, a process usually described as fol- ence of activated Smad8 in the HFSCs is not licular “downgrowth,” TGF-b1 starts to be ex-

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K. Kahata et al.

pressed in the placode epithelium and, at a later Inactivation of the Inhba gene, encoding in- stage, the IRS expresses TGF-b1, and the ORS hibin bA (also known as activin bA), in mice expresses TGF-b2 and TGF-b3 (Lyons et al. leads to loss of activin A and inhibin A, causing 1990). The expression of all three TGF-bsis lack of whiskers and their follicles (Matzuk gradually diminished after maturation (Lyons 1995). Conversely, Fst2/2 mice that lack activin et al. 1990). TGF-b2 induces this “downgrowth” inhibition by follistatin show a hyperkeratotic of hair follicles, which requires the Ras-extracel- epidermis and abnormally shaped whiskers lular signal-regulated kinase (Erk) MAPK path- (Matzuk et al. 1995), which suggests that activin way leading to induction of Snail expression promotes normal hair follicle development. (Foitzik et al. 1999; Jamora et al. 2005). In ad- This conclusion is supported by analysis of the dition, follicular “downgrowth” or regression back skin of E18.5 mouse embryos carrying a induced by TGF-b involves extensive apoptosis Fst2/2 germline mutation or expressing an that appears as a gradient of cells with increas- Inhba transgene under the Krt14 promoter, the ing death rates, so that a large number of follic- latter causing activin A overexpression in the ular epithelial cells disappear (Mesa et al. 2015). prenatal mouse skin (Nakamura et al. 2003). The surviving basal epithelial cells phagocytose Both mouse models show a delay in hair follicle the dying layer of cells so that the follicle is ready morphogenesis (Nakamura et al. 2003). Ex vivo for regeneration during the next cycle of growth treatment of mouse hair follicle cultures with and differentiation. activin B (inhibin bB-bB) induces potent entry TGF-b1 is necessary for entry into the cata- into the anagen stage (Jia et al. 2013). Keratino- gen phase (Foitzik et al. 2000), and its activity is cytes and stromal cells in the developing skin controlled by the small leucine-rich repeat pro- respond to activins, as examined in transgenic teoglycan Tsukushi, which binds directly to mice expressing a dominant-negative mutant TGF-b1 and mediates activation of its signaling ActRIB/ALK-4 receptor in their keratinocytes pathway in the hair follicles (Niimori et al. (Bamberger et al. 2005). These mice show per- 2012). Consequently, mice lacking Tsukushi ex- turbed hair follicle morphogenesis; overexpress- pression caused by Tsku inactivation have a de- ing inhibin bA in the hair follicles can at least layed hair cycle similar to the Tgfb12/2 mice partially rescue the effect of the dominant-neg- (Foitzik et al. 2000; Niimori et al. 2012). TGF- ative ActRIB/ALK-4 (Nakamura et al. 2003; b2 controls HFSC regeneration; it is secreted by Bamberger et al. 2005), suggesting that activin the dermal papillae and activates the TGF-b re- signals in the epidermis through this receptor. ceptors and Smad2 and Smad3 signaling in Transgenic mice expressing individual TGF- HFSCs, and induces the expression of the trans- b family ligands have revealed roles in overall membrane protein TMEFF1 (transmembrane epidermal differentiation beyond effects on hair protein with EGF-like and two follistatin-like follicle morphogenesis. Analyses of mice over- domains 1, also known as tomoregulin-1) (Osh- expressing TGF-b1 support the growth inhibi- imori and Fuchs 2012). TMEFF1 can associate tory action of this cytokine in the epidermis, as with BMPs through its follistatin-homology do- it potently arrests the keratinocyte cell cycle, mains, and inactivates BMP signaling, thus sup- consistent with cell cycle studies of keratino- pressing HFSC generation (Oshimori and Fuchs cytes in culture (reviewed in Massague´ 2012). 2012), as mentioned earlier. Whether the activ- Hyperkeratosis and a sparse distribution of hair ity of TGF-b2 is controlled extracellularly by follicles are seen in mice overexpressing activat- Tsukushi, as for TGF-b1, remains to be exam- ed TGF-b1 from the Krt1 promoter that drives ined (Niimori et al. 2012). Kindlin-1, which keratin-1 expression, leading to a shiny and rig- upon mutational inactivation of its gene causes id skin that prohibits proper movement and the Kindler syndrome in humans, alters the ho- breathing, and causes rapid postnatal death meostasis of stem cells by promoting integrin- (Sellheyer et al. 1993). These mice show low mediated TGF-b activation and inhibiting the numbers of dividing keratinocytes and few pro- canonical Wnt pathway (Rognoni et al. 2014). liferating hair follicle cells in their skin (Sell-

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TGF-b Family in Epithelial Differentiation

heyer et al. 1993). In contrast, expression of ac- genic BMP-6 in small epidermal patches with tive TGF-b1 from the Krt10 promoter results in low expression level show increased prolifera- mitotic activity of epidermal cells, indicative of tion and hyperkeratosis, whereas mice express- hyperproliferation (Cui et al. 1995). However, ing high and uniform levels of BMP-6 show when homeostasis is perturbed after chemical potent growth inhibition during epidermal challenge that leads to skin hyperplasia, the proliferation before or during birth (Blessing transgenic TGF-b1 acts as a growth inhibitor et al. 1996). Noggin expression from the Krt5 requiring the function of the TGF-b type II re- promoter in mouse epidermis blocks eyelid ceptor (TbRII) (Cui et al. 1995). Parallel ap- opening in the affected pups because of ineffi- proaches using inducible expression of active cient apoptosis in the eyelid skin and defective TGF-b1 in the epidermis confirm that TGF- epidermal differentiation (Sharov et al. 2006). b1 limits the proliferation of keratinocytes in Thus, similar to the patterning of the skin and vivo (Wang et al. 1999; Liu et al. 2001). Active connective tissue around the digits of the arms TGF-b1 signaling in the epidermis can even and feet, BMPs control eyelid skin differentia- resist to oncogenic stimuli that initiate neoplas- tion by coordinating epithelial apoptosis and tic transformation (Wang et al. 1999). On the differentiation. other hand, increased epidermal proliferation An additional specification in skin differen- due to TGF-b1 overexpression is associated tiation is the generation of ciliated epithelial with a strong negative feedback response that cells. Cilia have diverse physiological functions, includes Smad7 up-regulation (Liu et al. by directing local fluid or air flow and serving as 2001). In a similar manner, expression of a sensory appendages. TGF-b signaling through dominant-negative TbRII mutant that blocks Smad2 and Smad3 controls the length of the endogenous TGF-b signaling under the control cilium during embryogenesis, including in epi- of the Lor promoter (of the gene encoding lor- dermal development (To¨zser et al. 2015). In the icrin) in the basal and suprabasal epidermal lay- epidermis, TGF-b signaling regulates the ex- ers, leads to a strong hyperkeratotic phenotype pression of the ciliary organizer, Meckel syn- (Wang et al. 1997). Expression of the same drome 1–related protein (MKSR), also known dominant-negative TbRII in the basal compart- as B9 domain-containing 1 (B9D1) protein ment of the skin and in hair follicle cells leads to (To¨zser et al. 2015). B9D1/MKSR is part of a relatively normal skin differentiation, but con- multiprotein complex localized at the transition tributes synergisitcally to skin carcinogenesis zone between the intracellular basal body and when the mice are treated with radiation or the membrane-protruding axoneme of the cili- chemical carcinogens (Amendt et al. 1998). um, and regulates the assembly, growth, and Overexpression of activin bA in mouse epi- length of the cilium. This mechanism of cilio- dermis from the Krt14 promoter results in a genesis is relevant in other developmental con- dramatically enlarged epidermis because of in- texts ranging from gastrulation and neurulation creased proliferation of keratinocytes, and pro- to spermatid differentiation (To¨zser et al. 2015). nounced fibrosis, in part supported by an adi- BMP signaling also controls differentiation of pocyte to mesenchymal transdifferentiation ciliary epithelia in embryos, through coopera- (Munz et al. 1999). Mice expressing BMP-6 tion with Notch, specifying the localization of from the Krt10 promoter also show severe skin multiciliated and secretory cells in the outer lay- malformations leading to psoriasis, supporting er of the embryonic epidermis (Cibois et al. the link between BMP-6 function and Wnt 2015). Whereas BMPs drive differentiation, pathway activation in keratinocyte proliferation TGF-bs may control ciliary assembly and length. (Blessing et al. 1996), as discussed earlier. This study, similar to the TGF-b1 studies, revealed Feather Development that the pattern of transgene expression within the tissue defines the response during epidermal Feathers, which enable birds to fly, are highly differentiation. Thus, mice expressing the trans- organized epidermal appendages with a hierar-

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Proliferation Differentiation 2018;10:a022194 Figure 2. Branching and evolution of feathers. (A,B) TGF-b2 localizes in the border where epithelium and mesenchyme interact. The multilayered epidermis can be divided into epidermal ridges (dark blue cells) by the ridge-forming activator noggin and the ridge-forming inhibitor BMP-4. Noggin and BMP-4 are distributed in the mesenchyme (not shown) and their balanced action is crucial for normal specification of these primordial ridge cells. (B,C) High BMP-4 levels at the proliferative zone lead to the formation of a cylindrical structure by the epithelial cells. (Legend continues on following page.) Downloaded from http://cshperspectives.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press

TGF-b Family in Epithelial Differentiation

chical branched pattern, presenting some simi- the feather with evenly spaced barbs (Fig. 2) larities to scales in fish and hairs in mammals (Noramly and Morgan 1998; Yu et al. 2002). (Fig. 2) (Yu et al. 2002). In situ RNA hybridiza- BMPs exert a lateral inhibitory mechanism tion and immunohistochemistry revealed that that leads to segmental patterning of the feather TGF-b2 and TGF-b3 are expressed in the pri- tract, guaranteeing that feather buds do not mordial buds of the feathers during late stages grow in a dense and disorganized pattern, and of chicken embryogenesis (Jakowlew et al. are surrounded by space for future growth of 1994). TGF-b2 is expressed in the junctional differentiating tissue (Noramly and Morgan space where epithelium and mesenchyme inter- 1998; Yu et al. 2002). act (Fig. 2). When applied on chicken skin In the feather bud cells, BMPs signal through explants, TGF-b2 induces differentiation of BMPRII, BMPRIA/ALK-3, and BMPRIB/ALK- feather buds, whereas TGF-b1 cannot show 6 (Ashique et al. 2002), and regulate the expres- this response (Ting-Berreth and Chuong 1996). sion of the FGF receptor FGFR2, which is Opposing the activities of TGF-b2, BMPs expressed in cells aligned along the area where induce responses that reflect antagonism to the epithelium contacts the mesenchyme. Con- TGF-bs; BMP-2 and BMP-4 suppress feather versely, FGF signaling regulates the expression primordium growth and differentiation in sim- and secretion of BMPs along the patterned ilar explant assays as those that were used to bud (Jung et al. 1998; Noramly and Morgan show induction of feather bud differentiation 1998). In addition to FGFR2 signaling, BMP (Jung et al. 1998). Shh signaling induces the signaling induces expression of transcription expression of BMP-2 and BMP-4, and BMP factors, such as Msx1 and Twist2 (also known signaling suppresses Shh expression in a feed- as cDermo-1 in chicken), that drive prolifera- back mechanism (Fig. 2) (Harris et al. 2005). tion of the whole feather bud (Scaal et al. 2002). Additionally, microRNAs (miRNAs) control Although BMP inhibits bud outgrowth in the both Shh and BMP-2 expression in the devel- neighboring space, the proliferation and differ- oping feather bud (Zhang et al. 2013). Besides entiation of the primordial bud cells that secrete explant assays using TGF-b- or BMP-impreg- these BMPs is not affected at all. This lack of nated beads, retroviral delivery of BMP-2, BMP- growth inhibition is maintained by bud cells, 4, or noggin to developing feather bud ecto- which locally secrete follistatin, thus protecting derm confirmed the role of BMPs as suppres- them from autocrine BMP actions (Patel et al. sors of feather differentiation (Noramly and 1999). In fact, follistatin and another BMP in- Morgan 1998). Feather buds are arranged in a hibitor, gremlin, are secreted by interbud der- periodic pattern along the central morphoge- mal cells in response to BMP through a feedback netic stalk that will develop into the rachis of mechanism (Fig. 2) (Ohyama et al. 2001; Bardot

Figure 2. (Continued) Within the epidermis, when the level of noggin is high, the epithelia gradually start to form multiple barb ridges (blue cells). The role and localization of the upstream Sonic Hedgehog (Shh) is also indicated. (D) Expression of BMP-2 and BMP-4 is regulated by Shh signaling. Bone morphogenetic proteins (BMPs) mediate a lateral inhibitory mechanism that leads to arrangement of feather buds in a periodic pattern. BMP signaling in the feather bud cells (brown) regulates the expression of FGFR2. Moreover, BMP induces the expression of transcriptional regulators Msx1 and Twist2 that drive feather bud proliferation. Follistatin and gremlin, secreted by feather interbud dermal cells moderate the autocrine action of BMP signaling via a feedback mechanism. TSC-22 expression is repressed by BMP signaling and induced by FGF signaling, and is required for feather cell differentiation. Marginal plate cells that express Shh and BMP-2 undergo apoptosis, and Shh- negative barb ridge cells continue proliferating. (E,F) Barb ridge cells express BMP-2 and BMP-4 and form the more differentiated structures known as barbules. Barbule plate cells form cilia and hooklets. Noggin activity toward the end of feather formation is reduced and leads to formation of the calamus without branches at the proximal end of the feather shaft. The site with higher BMP activity forms the bilateral symmetric feather. (The image has been inspired by the article of Yu et al. 2002.)

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K. Kahata et al.

et al. 2004). This patterning mechanism gener- enchymal cells that cannot progress to terminal ates gradients of spatially organized BMP sig- differentiation, and thus nail development is naling that is interpreted by the transcription severely defective (Cai and Ma 2011). The com- factor named transforming growth factor-b- mon processes that shape differentiation into stimulated clone-22 (TSC-22), whose expres- hairs and nails are also highlighted by studies sion is repressed by BMP signaling and activated of the transcription factor FoxI3, whose expres- by FGF signaling, and which is critical for feath- sion is induced by activin A in epithelial cells er cell differentiation (Dohrmann et al. 2002). (Shirokova et al. 2013), and is required for hair, The studies of feather morphogenesis pro- tooth, and nail development (Jussila et al. 2015; vide beautiful examples of feedback regulation Shirokova et al. 2016). and reciprocal cross talk between epithelial cells Embryonic morphogenesis of digits and and adjacent mesenchyme that leads to the for- nails is also recapitulated, at least in part, in mation of highly patterned appendages. young children and in rodents following ampu- tation of the distal digit (Han et al. 2008). Similar, but not identical, to embryonic mor- Nail Morphogenesis phogenesis, amputated digits regenerate based The development of vertebrate nails is intimate- on the proliferative and differentiation activity ly coordinated with the development of the of a blastema that secretes BMP-4 to pattern extremities of limbs (fingers/toes-claws, etc.), chondrogenesis and osteogenesis, so that the a morphogenetic program that is regulated by amputated digit will form again, albeit only to BMPs. This has been shown by tissue-specific a partial size relative to the original size (Han and developmental stage-specific overexpres- et al. 2008). This regenerative mechanism takes sion of noggin from the Msx2 promoter, which place after birth and can last up to sexual ma- targets expression to the apical ectodermal ridge turity, which is the period of physiological that governs limb mesenchyme growth and growth and development of vertebrate digits patterning (Wang et al. 2004). As a result, en- (Han et al. 2008). dogenous BMPs acting during limb bud differ- entiation are inactivated and the embryo devel- Palate Differentiation and Fusion ops digital fusions or polydactyly, whereas the ventral footpads grow many nails (Wang et al. In situ RNA hybridization analysis during pal- 2004). The apoptotic mechanisms that pattern ate development and fusion revealed the expres- the growing digits of these animals in response sion of all three TGF-b isoforms in a cell type- to BMP are inactivated because of the presence specific manner (Pelton et al. 1990). These early of noggin, and thus mesodermal differentiation studies suggested that TGF-b3 may mediate the is prolonged and unrestricted, causing these transdifferentiation of palate epithelial cells to abnormalities (Wang et al. 2004). mesenchymal tissue, whereas TGF-b1 might Two key transcription factors drive mesen- regulate mesenchymal cell proliferation and chymal stem cell differentiation into nail cells— extracellular matrix deposition (Pelton et al. Msx2 and FoxN1. Primordial epithelial cells 1990). The critical role of TGF-b3 is shown by secrete BMPs, which signal via their receptors the severe defects in palate development and on the adjacent mesenchymal stem cells and closure observed in the Tgfb32/2 mice causing induce expression of Msx2 and FoxN1, promot- a cleft in the secondary palate (Fig. 3) (Kaarti- ing mesenchymal differentiation (Cai and Ma nen et al. 1995) that is not seen in mice lacking 2011). Although Msx2 promotes terminal nail either of the other two TGF-b isoforms. Genetic cell differentiation, FoxN1 acts in a pathway that replacement of TGF-b3 by TGF-b1 expression, shares mechanistic aspects with hair follicle dif- by introducing a TGF-b1 cDNA into the delet- ferentiation (Cai and Ma 2011). Accordingly, ed Tgfb3 exon, results in incomplete rescue of inactivation of Msx2 and Foxn1 in the germline the cleft palate phenotype (Yang and Kaartinen causes extensive proliferation of primitive mes- 2007). TGF-b1 can initiate fusion of the two

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TGF-b Family in Epithelial Differentiation

Cell cycle arrest Migration Apoptosis Palatal epithelium ? Follistatin TGF-β3

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? Twist1/LEF1

Oral cavity E-cadherin Vimentin

EMT

Tongue

Figure 3. Molecular control of palatal development. TGF-b3 plays a crucial role in palatal development and closure. TGF-b3 signals through TbRII and TbRI receptors to stimulate the formation of Smad2–Smad3– Smad4 complexes. The ubiquitin ligase TRIM33 also participates in complexes with Smad2 and/or Smad3; cooperative actions between Smad, TRIM33, and TAK1 signaling may activate expression of the epithelial– mesenchymal transition (EMT) transcription factors Twist1 and LEF1 (note the “question mark,” as the latter mechanism is not firmly proven), which directly activate the expression of mesenchymal genes, such as the gene encoding vimentin, and suppress the expression of epithelial genes including the Cdh1 gene encoding E- cadherin during the palatal EMT. Smad complexes may also interact with IRF6 to regulate additional target genes during EMT. The possible involvement of follistatin, which may (note the “question mark”) indirectly limit TGF-b3 activity, and ActRI/ALK-2 as an alternative type I receptor, are also indicated to reflect the discussion in the main text. The sequence of events induced by TGF-b is epithelial cell cycle arrest, followed by cell migration and eventual apoptosis at the seam (top of the figure). (The image has been inspired by Bush and Jiang 2012.)

apposed epithelial sheets, but fails to complete et al. 2007). Such ex vivo effects of TGF-b3 the fusion process (Yang and Kaartinen 2007). would be expected to be recapitulated by treat- Thus, TGF-b1 and TGF-b3 play roles in palatal ment with exogenous TGF-b1 or TGF-b2, differentiation, but TGF-b3 may preferentially based on the redundant signaling pathways in- induce the local apoptosis that is required for duced by these three isoforms, which remains to palate fusion at the seam, where the two sides of be studied. Furthermore, compound transgenic the tissue normally fuse (Murillo et al. 2009). mice show that TGF-b3 signals via TbRI in the Accordingly, treatment of primary epithelial palate (Fig. 3), because an activated TbRI recep- cells from the palatal medial edge seam with tor can rescue the cleft palate of Tgfb32/2 mice, TGF-b3 induces a program that starts with and targeted inactivation of TbRI expression in cell cycle arrest, followed by cell migration and the palatal epithelium prevents completion of local apoptosis, suggesting an in vitro recon- palatal differentiation and fusion (Dudas et al. struction of the palatal fusion process (Ahmed 2004). Palatal fusion requires the coordinate

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signaling by Smads and TGF-b-activated kinase of the craniofacial defects of these constitutively 1 (TAK1) downstream from TGF-b3 in vivo active Bmpr1a transgenic mice is achieved fol- (Lane et al. 2015). Silencing Smad4 or Map3k7 lowing pifithrin-a treatment (Hayano et al. (encoding TAK1) expression individually, leads 2015). Thus, limited BMP signaling activity to weak palatal phenotypes; combined inactiva- may be associated with normal completion of tion of Smad4 and Map3k7 expression leads to the palatal fusion to the overlaying nasal sep- the severe palatal phenotype seen in Tgfb32/2 tum, whereas TGF-b promotes the overall pro- mice (Lane et al. 2015). The chromatin regula- cess of secondary palate formation. tor and ubiquitin ligase tripartite motif con- In addition, conditional inactivation of taining 33 (TRIM33, also known as TIF1g or TbRII expression in the palatal epithelium ectodermin) forms complexes with Smad2 and also gives rise to cleft palate, with a concomitant Smad3 and regulates expression of specific reduction in muscle mass, and inactivation of genes. Maximal cleft palate defects are scored Wnt signaling, through increased expression when Trimm33 expression is silenced in combi- of the Wnt antagonists Dkk1 and Dkk4 that nation with inactivation of Smad4 and Map3k7 normally support palatal muscle growth (Iwata expression (Lane et al. 2015), attesting to the et al. 2014). A cleft palate phenotype has been physiological cooperation between three signal- observed in the mouse following inactivation of ing branches, Smad2/3–TRIM33, Smad2/3– the Irf6 gene, which encodes the interferon Smad4, and TAK1 downstream from TGF-b3 regulatory factor 6 (IRF6) transcription factor (Fig. 3). (Iwata et al. 2013). This cleft palate phenotype The type I receptor ActRI/ALK-2 has also can be rescued by concomitant deletion of the been proposed to mediate TGF-b3 signals in the Smad4 gene, suggesting that Smads cooperate palate epithelium, possibly acting in a less po- with IRF6 in the regulation of target genes tent manner (Fig. 3) (Noda et al. 2015). How- (Iwata et al. 2013). ever, ActRI acts during palatal fusion as a BMP TGF-b3 induces expression of Twist1 in pal- signaling receptor, which activates BMP-specif- atal epithelial cells (Fig. 3) during a time win- ic Smad proteins (Noda et al. 2015). An activat- dow before the fusion of the epithelial sheets ed mutant of ActRI induces BMP Smad signal- (Yu et al. 2008). However, Twist1 expression ing and causes cleft palate because the epithelial alone is not sufficient to drive palatal fusion, layer in the medial edge fails to undergo apo- as shown using siRNA-mediated silencing (Yu ptosis and has increased proliferation, and the et al. 2008). In parallel, Smad-induced associa- mesenchymal and muscle layers fail to fuse tion of the transcription factor LEF1 to the (Noda et al. 2015). In a related but seemingly promoter of the Cdh1 gene, which encodes E- opposite scenario, mice with a gain-of-function cadherin, may enforce repression of E-cadherin mutation in the BMPRIA receptor show a vari- expression and promote the onset of palatal ety of defects, mostly associated with craniofa- EMT (Fig. 3) (Nawshad et al. 2007). The cial development, because of malformations in Smad–LEF1 complex may also directly regulate the neural crest (Hayano et al. 2015). A number the expression of mesenchymal genes during of these mice also show defective closure of their palatal EMT, such as the gene encoding vimen- secondary palate, suggesting that enhanced tin (Nawshad et al. 2007). Finally, uncontrolled BMP–Smad signaling causes epithelial cell EMT in the developing palate may be inhibited apoptosis (Hayano et al. 2015). Such palatal by the extracellular antagonist follistatin, which apoptosis depends on p53 activity, and those is coexpressed in the palatal epithelium with constitutively active Bmpr1a transgenic mice TGF-b3 (Nogai et al. 2008). However, the pro- that show defective closure of their secondary posed inactivation of TGF-b3 by follistatin palate can form relatively normal palatal fusion during palate development, based on a direct to the nasal septum when pharmacologically physical complex between TGF-b3 and follista- treated with pifithrin-a, which inhibits p53 ac- tin, requires further investigation in vitro and in tivity. In contrast, only weak and partial rescue developing embryos (Nogai et al. 2008). Thus,

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TGF-b Family in Epithelial Differentiation

EMT is an integral developmental program that tal epithelial cells show a block in early stage mediates palatal fusion. tooth development (Wang et al. 2012). BMP signaling activates the expression of the tran- scription factor Pitx2, which contributes to Tooth Development differentiation of dental epithelial cells in the Consistent with the inhibition of the activin and growing dental bud (Wang et al. 2012). Pitx2 BMP functions by follistatin, Fst2/2mice show activates expression of the miR-200c microRNA, general growth retardation, and, among other which, among other targets, represses noggin affected organs, perturbed tooth development expression (Cao et al. 2013). Thus, silencing (Matzuk et al. 1995). Multiple TGF-b family miR-200c expression results in defective termi- members control tooth cell differentiation. Ac- nal tooth differentiation that correlates with en- tivin bA is required for normal tooth develop- hanced noggin levels in the developing dental ment, and several but not all teeth of Inhba2/2 epithelium (Cao et al. 2013). However, noggin mice fail to progress into terminal development interacts with and inhibits not only BMPs but and remain arrested as undifferentiated primor- also extracellular Wnts, thus blocking Wnt dial buds (Ferguson et al. 1998). The tooth phe- signaling in the dental epithelium (Yuan et al. notype of Inhba2/2 mice is rescued by activin A 2015). Although interaction of noggin with released from beads that are implanted into the several BMPs has been studied down to the level developing mandibles (Ferguson et al. 1998). of crystallographic detail, the interaction be- Conditional overexpression of the inhibito- tween noggin and Wnts has only been analyzed ry Smad7 in stratified epithelia of mice also re- using antibody-based techniques, such as prox- sults in defective tooth development, in addi- imity ligation assays and colocalization studies tion to other skin-related phenotypes (Klopcic using immunohistochemistry (Yuan et al. et al. 2007). This study shows that TGF-b family 2015); thus, more rigorous biochemical analysis signaling controls the terminal differentiation is awaited to further establish the noggin–Wnt of ameloblasts and deposition of enamel layers interactions. Therefore, the phenotype resulting in developing teeth (Klopcic et al. 2007). Addi- from transgenic noggin expression supports the tionally, conditional inactivation of the Tgfbr2 interdependence of Wnt and BMP signaling, gene, encoding the TbRII receptor, in cells both controlling the expression of the Pitx2 derived from the cranial neural crest prevents gene, which is essential for odontogenesis odontoblast differentiation and results in loss (Yuan et al. 2015). of dentin deposition that supports terminal Further support on the role of BMP signal- tooth morphogenesis (Oka et al. 2007). Matu- ing in promoting tooth development stems ration of odontoblasts can be rescued by addi- from studies of conditional inactivation of the tion of TGF-b2 to tooth explants, confirming Bmpr1a gene (Yang et al. 2013). Doxycycline- that TGF-b signaling controls terminal tooth inducible expression of the Cre recombinase development (Oka et al. 2007). from a hybrid Krt5-rtTA promoter and doxycy- In this differentiation program, BMPs cline treatment of embryos one day before the stored within the enamel matrix promote sig- onset of dental epithelial differentiation, results naling in cells of the dental follicle (Ke´moun in repression of BMP signaling from dental et al. 2007). Dental follicle cells, cultured in epithelial cells, including the ameloblasts, and the presence of exogenous BMP-2 and BMP-7, causes a switch in differentiation (Yang et al. induce the expression of proteins that define 2013). Thus, epithelial cells that normally gen- cementoblast differentiation (Ke´moun et al. erate the crown of the tooth give rise to cells of 2007). Conversely, addition of noggin represses the root of the tooth, resulting in abnormal and this induction, depletes the BMPs in the micro- excessive root growth (Yang et al. 2013). At this environment, and blocks cementoblast protein stage of tooth development, BMP signaling nor- expression (Ke´moun et al. 2007). Additionally, mally represses paracrine Wnt signaling, which transgenic mice expressing noggin in their den- is hyperactivated in Bmpr1a2/2 odontoblasts

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(Yang et al. 2013). Furthermore, conditional Downstream from BMP signaling, the Shh Smad4 inactivation in odontoblasts blocks cell pathway contributes to the generation of dental differentiation and extracellular matrix deposi- epithelial stem cells that slowly self-renew and tion, leading to rudimentary tooth develop- contribute to the continuous growth of the ment (Gao et al. 2009). Loss of Smad4 also mouse incisors (Li et al. 2015b). Shh signaling results in reduced paracrine signaling by BMPs via transcriptional elevation of Gli levels pro- that are secreted by odontoblasts and act on motes the expression of Sox2, a transcription neighboring epithelial cells of the developing factor that defines stemness in many cell line- tooth root. This perturbed signaling circuit re- ages (Li et al. 2015b). BMP-induced Smad sults in keratocystic odontogenic tumors that signaling suppresses expression of Shh, thus may result from insufficient suppression of the limiting the self-renewing potential of the den- proliferation of these epithelial cells caused tal stem cells (Li et al. 2015b). Accordingly, cell by reduced BMP signaling (Gao et al. 2009). type-specific ablation of Smad4 promotes Shh Following its conditional inactivation at distinct signaling through Gli proteins, and enhances stages of odontogenesis, Smad4 loss does not the stem cell population, causing continuous affect early differentiation but disrupts the late growth of incisors, and aberrant growth of mo- stages of odontoblast differentiation (Kim et al. lar teeth, whose growth is normally suppressed 2015). Thus, BMP signaling with Smad4 as co- by BMP signaling (Li et al. 2015b). activator may instruct odontoblasts to express The contribution of Wnt signaling in con- collagens and deposit the secondary dentin lay- trolling tooth development, which often results er that completes odontogenesis (Kim et al. in effects that are opposite to those of BMPs, has 2015). been analyzed at the genome-wide level and GDF-5, also known as cartilage-derived through mathematical modeling (O’Connell morphogenetic protein-1 or BMP-14, is post- et al. 2012). This study defines reciprocal Wnt natally expressed in cells of the periodontal and BMP pathway cross talk that regulates large ligament, especially after initiation of enamel numbers of genes in the two juxtaposed tissues, synthesis (Liu et al. 2016). In this context, the dental epithelium and the adjacent mesen- GDF-5 may act upstream of BMP-2, which chyme, during progression of tooth develop- then promotes ameloblast differentiation (Liu ment (O’Connell et al. 2012). et al. 2016), although the exact role of GDF-5 as TGF-b1-induced Smad signaling has been an upstream “weak” activator of BMP-2 activity analyzed in odontoblast culture differentiation remains to be clarified. The importance of BMP experiments (Lee et al. 2011). TGF-b activates, signaling in tooth development is also under- and recruits, ubiquitin ligases to the transcrip- scored by tooth phenotype resulting from ge- tional regulator, nuclear factor I-C (NFI-C), netic mutations in the human GREM2 gene, whose activity is important for differentiation which encodes “protein related to DAN and of the dental root (Lee et al. 2011). Thus, TGF- cerberus” (PRDC), and the corresponding phe- b1 signaling represses the expression of NFI-C, notype of Grem22/2 mice with characteristic which in turn represses TGF-b signaling by re- dental defects, including small and malformed ducing the phosphorylated Smad2 and Smad3 incisors (Kantaputra et al. 2015). Multiple hu- levels, through an undefined mechanism (Lee man tooth defects have been associated with et al. 2011). This may generate a double-negative mutations in the GREM2 gene and can be dis- regulatory network that is important for odon- cerned from contributions by other develop- tobast differentiation. mental regulators such as Wnt family members Teeth grow continuously during adult (Kantaputra et al. 2015). The autosomal-dom- mouse life, a process promoted by adult dental inant character of these GREM2 mutations also stem cells and cytokines. TGF-b signaling underscores the importance of dose-depen- through TbRI regulates the expression and sig- dence in the regulation of BMP signaling during naling by FGF family members, which are re- dental development (Kantaputra et al. 2015). quired for self-renewal of the dental stem cells

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TGF-b Family in Epithelial Differentiation

(Zhao et al. 2011). Conditional Tgfbr1 inactiva- epithelium, and excessive macrophage recruit- tion in the dental mesenchyme, which is derived ment to the Tgfb22/2 corneal epithelium from the embryonic neural crest, drastically re- suggests that the mutant phenotype results duces the expression of FGFs and the popula- from abnormal macrophage function (Saika tion of dental stem cells, whereas addition of et al. 2001). Furthermore, overexpression of FGF10 to explanted developing teeth rescues dominant-negative mutants of TbRI or TbRII the defect caused by TbRI loss (Zhao et al. in the lens epithelium results in a strong block 2011). in lens epithelial differentiation, which causes GDF-11/BMP-11, a distant member of the extensive cell death and leads to cataract forma- TGF-b family, has been studied for its role in tion (de Iongh et al. 2001). During lens craniofacial development (Lee and Lee 2015). differentiation, the dominant-negative TGF-b The binding of GDF-11 to its receptors is inhib- receptors disrupt the expression and structural ited by the extracellular GDF-associated serum organization of critical filamentous proteins protein 1 (GASP1) (Lee and Lee 2013). Tooth in the mature lens fibers, because of lack of buds start expressing GASP1 before embryonic architectural coordination between the actin age of E17.5, and mice with loss-of-function cytoskeleton and the assembly of fibrous pro- mutations in both the Gdf11 and Gasp1 genes teins (de Iongh et al. 2001). On the other hand, show enhanced cleft palate and abnormal inci- lens-specific Tgfbr2 ablation results in normal sors in the lower mandible (Lee and Lee 2015). lens development (Beebe et al. 2004), suggest- The incisor defect is due to lack of proliferation ing that TGF-b family members other than the and differentiation in the tooth buds for these TGF-b isoforms (e.g., activins) might control specific incisors only. Loss of Gdf11 only does the differentiation program that generates the not result in this phenotype, suggesting that lens fibers. Perhaps, dominant-negative TGF- individual tooth buds selectively control their b receptor expression might interfere with development by the coordinate activity of signaling components downstream from such GASP1 and GDF-11 (Lee and Lee 2015). alternative ligands, whose identity remains to be firmly established. The BMPs play instructive roles in the dif- THE TGF-b FAMILY IN EPITHELIAL ferentiation of the lens epithelium. In develop- DIFFERENTIATION AND EMT IN THE EYE ing chick eyes, BMP-induced Smad1 activation TGF-b family members also regulate epithelial is seen during elongation and differentiation differentiation in eye development. This is illus- of the lens fiber cells (Belecky-Adams et al. trated by their activities in lens and retinal epi- 2002). Overexpression of noggin in the chick thelial cell differentiation. retina perturbs lens cell differentiation and in- duces apoptosis, causing tissue degradation (Belecky-Adams et al. 2002). The impaired Lens Differentiation differentiation of the lens epithelium can be Although BMPs control embryonic lens differ- rescued ex vivo by BMP-2, BMP-4 or BMP-7, entiation, the TGF-bs regulate late lens devel- which overcome the defect caused by noggin opment and have been linked to pathogenetic (Belecky-Adams et al. 2002). Supporting the processes in the lens (de Iongh et al. 2001; Saika role of BMPs during lens epithelial differentia- et al. 2001). Genetic ablation of the individual tion, mouse lens epithelium-specific Bmpr1a TGF-b genes in the mouse corneal epithelium inactivation results in small lenses with thin showed that Tgfb2 inactivation, but not inacti- cell layers (Beebe et al. 2004). A similar mouse vation of Tgfb1 or Tgfb3, results in abnormal lens-specific Actr1 inactivation also results in differentiation and defective fusion of the cor- small lenses, because of enhanced apoptosis nea to the neighboring lens (Saika et al. 2001). (Rajagopal et al. 2008). In this context, ActRI/ TGF-b2 regulates extracellular matrix deposi- ALK-2 activation promotes cell proliferation at tion in the developing stroma of the corneal early stages of lens development, and cell cycle

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arrest during late stages of lens development, apparent phenotype, high level TGF-b1 expres- which is required for lens fiber differentiation sion results in the development of small eyes (Rajagopal et al. 2008). with enhanced mesenchymal cell numbers, The molecular programs regulated by BMP excessive extracellular matrix deposition, and signaling in lens epithelial cells are not well dramatic apoptosis of lens epithelial cells, sug- characterized; however, in chick lens explant cul- gesting a role of TGF-b1 in promoting fibrotic tures, BMP signaling cooperates with FGF sig- EMT (Flu¨gel-Koch et al. 2002). Studies of lens naling to induce the expression of CCDC80/ epithelial cells in culture highlight a role of equarin, a cell-cycle and differentiation regula- Smad3 as mediator of the TGF-b-induced tor (Jarrin et al. 2012). BMP signaling also EMT response in tissue injury and fibrosis regulates the expression of FGF receptors so (Saika et al. 2004). In addition to Smad3, the that lens epithelial cells gain responsiveness to phosphatidylinositol 30 kinase and Erk MAPK FGFs (Boswell and Musil 2015). Subsequently, pathways contribute to the regulation of target FGF receptor signaling induces BMP4 expres- genes, such as those encoding Snail, Slug or sion, thus forming a positive feedback loop, a-smooth muscle actin, by TGF-b during lens which provides sustained BMP responses during EMT (Cho et al. 2007; Choi et al. 2007; Chen lens formation (Boswell and Musil 2015). et al. 2014). BMP-7 signaling antagonizes TGF- Similar and conserved mechanisms may b-induced EMT in in lens epithelial cell operate in mouse and chick lens development. cultures, and protects epithelial differentiation Accordingly, inactivation of the expression of by inducing the expression of the transcription BMPRIA or ActRI in ectodermal tissue that factors Id2 and Id3 (Kowanetz et al. 2004). generates the lens progenitor cells prevents lens Adenoviral BMP-7 delivery directly to the placode formation and subsequent lens cell in- adult mouse lens induces Id2 and Id3 expres- vagination, highlighting the requirement of sion in the lens epithelium, whereas adenoviral these two BMP receptors (Huang et al. 2015). Id2 or Id3 expression promotes endogenous The embryonic ectodermal tissue attempts to BMP-7 expression (Saika et al. 2006). These compensate the loss of receptor signaling by se- three interventions protect the lens epithelium creting high amounts of BMPs, such as BMP-7, from injury-mediated EMT and resulting leading to perturbed BMP morphogenetic fibrosis. gradients, aberrant regulation of target genes, especially those of the apoptotic program, and Retinal Epithelium Formation abnormalities in the fission of the optic cavity that surrounds the lens (Huang et al. 2015). The Early studies using whole rat embryo cultures at physiological requirement of BMP signaling several developmental stages and treatment of during lens development is also supported by the embryos with antibodies against BMP-7 studies of myopic eye development in guinea gave evidence for an overt defect in eye devel- pigs, caused by lens injury (Li et al. 2015a). Dur- opment (Solursh et al. 1996). BMP-7 is ex- ing progression of myopia, BMP-2 expression pressed in the rat retinal epithelium, lens, and and phospho-Smad1/5 levels decrease (Li et al. cornea, as shown by in situ hybridization and 2015a). The requirement of BMP-2 signaling is immunohistochemistry (Solursh et al. 1996). thought to depend on the regulation of extracel- Human embryonic stem cells have been dif- lular matrix biosynthesis (e.g., of collagens or ferentiated into retinal epithelial cells using a glycosaminoglycans) and the induction of chon- mixture of growth factors, including TGF-b drogenic genes (Li et al. 2015a). Thus, reduced family proteins, showing their need in retinal BMP signaling and low chondrogenic gene ex- epithelial differentiation (Idelson et al. 2009). pression prevent terminal differentiation during TGF-b can induce EMT in retinal epithelial cells lens injury progression and cause myopia. at least in part via transcriptional induction of Although low level transgenic expression of Snail1 (Li et al. 2011). Smad signaling induces TGF-b1 in the mouse lens and cornea has no expression of the guanine exchange factor, GEF-

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TGF-b Family in Epithelial Differentiation

H1 that activates the small GTPase RhoA and studied in culture, has been linked to patholog- promotes actin cytoskeletal remodeling that ical changes. couples retinal EMT to cell migration (Tsapara et al. 2010). Furthermore, retinal epithelial cells CONCLUSIONS AND PERSPECTIVES undergo cell cycle arrest in response to TGF-b; however, the concomitant transcriptional in- This review illustrates roles of TGF-b family duction of survivin expression by TGF-b over- members in an array of, but not all, epithelial comes cytostasis and promotes cell survival tissues. Unfortunately, it is no longer possible during EMT (Lee et al. 2013). to comprehensively review the overwhelming The retinal epithelium receives important literature on the topic. Mouse models with cues for its differentiation from the juxtaposed cell-specific gene inactivation or overexpression lens (Heermann et al. 2015). The optic cup have revealed the sequential action of multiple develops around the differentiating lens, and TGF-b family members and their cross talk with this involves cell proliferation in three dimen- other pathways, primarily Wnt, FGF, Hh, or sions that gradually decreases (Heermann et al. MAPK signaling modules. Many details on sig- 2015). In zebrafish eyes, epithelial cells migrate nal transduction mechanisms are derived from from the apices of the lens and form streams of cell culture studies. However, their translation tissue remodeling along the rims of the optic into the in vivo situation increases steadily cup (Heermann et al. 2015). BMP signaling is and presents a firm requirement for future essential for the movement of these epithelial research. In addition, processes characterized cells, which comprise retinal stem cells, and in- using small rodent models do not always repre- hibition of BMP signaling by antagonists, such sent human physiology, especially in relation to as follistatin and the decoy receptor, BMP and adult tissue homeostasis and pathogenesis. activin membrane-bound inhibitor (BAMBI), Thus, it is important to further define the disrupts their morphogenetic fate (Heermann TGF-b family biology in a human context, pos- et al. 2015). Completion of this differentiation sibly with high affinity reagents that allow sen- program in the developing eye, which ensures sitive detection of proteins and their complexes physiological fission between the lens and the in tissues and biological fluids. optic cup cells, therefore relies on controlled We also touched on the process of EMT, BMP activity (Heermann et al. 2015). Chick without covering its importance in cancer pro- eye studies have clarified that, at early stages of gression or fibrosis. It is evident that EMT con- eye development, lens-derived BMP activity is tributes to organ development, and it is even required for the specification of the retinal ep- clearer that TGF-b family members are potent ithelial layer and the generation of the neural regulators of various transdifferentiations dur- retina that contacts the optic nerve cells in the ing organogenesis. Distinct from the EMTs, but brain (Pandit et al. 2015). Lens-derived BMPs equally important, are the abundant epithelial– signal in cross talk with Wnts, and specify reti- mesenchymal interactions that specify the pro- nal epithelial cell differentiation (Pandit et al. liferation and differentiation of new cell types. 2015). In addition, this signaling cross talk lim- This area of biology will continue to shed light its the expansion of telencephalic neuronal cells, on fundamental processes that depend on the a step required for the development of the neu- plastic interaction between neighboring cell ral retina and its contacts with eye and brain types. The time approaches when the develop- cells (Pandit et al. 2015). mental processes that generate new epithelia, Thus, the control of epithelial cell differ- and the mechanisms by which mesenchyme entiation in the eye by TGF-b family mem- specifies epithelial differentiation, will be com- bers is best documented in the case of the pleted. Not all TGF-b family members have yet lens and cornea. Retinal epithelial cells also re- been studied for their roles in epithelial biology. ceive signals from these growth factors in vivo, Future studies are awaited to enlighten this ex- and EMT in retinal tissue, which has been citing front in developmental and cell biology.

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ACKNOWLEDGMENTS chondrogenesis and feather formation in the chicken em- bryo. Int J Dev Biol 46: 243–253. We thank all past and present members of the Bamberger C, Scharer A, Antsiferova M, Tychsen B, Pankow TGF-b signaling and STEP groups for their S, Muller M, Rulicke T, Paus R, Werner S. 2005. Activin contributions to the scientific work emanating controls skin morphogenesis and wound repair predom- inantly via stromal cells and in a concentration-depen- from our laboratory. We thank Carl-Henrik dent manner via keratinocytes. Am J Pathol 167: 733– Heldin for his support, guidance, and a thor- 747. ough reading of the manuscript during prepa- Bardot B, Lecoin L, Fliniaux I, Huillard E, Marx M, Viallet JP. 2004. Drm/Gremlin, a BMP antagonist, defines the ration. This review summarizes work from a interbud region during feather development. Int J Dev large number of published papers; however, be- Biol 48: 149–156. cause of space limitations we have been unable Beebe D, Garcia C, Wang X, Rajagopal R, Feldmeier M, Kim to include all relevant publications in our dis- JY, Chytil A, Moses H, Ashery-Padan R, Rauchman M. 2004. Contributions by members of the TGFb superfam- cussion and thus cite other review articles. We ily to lens development. Int J Dev Biol 48: 845–856. apologize to those authors whose relevant work Belecky-Adams TL, Adler R, Beebe DC. 2002. Bone mor- has not been included in this review. We ac- phogenetic protein signaling and the initiation of lens knowledge funding by the Ludwig Institute for fiber cell differentiation. Development 129: 3795–3802. Cancer Research, the Swedish Cancer Society, Bertocchini F, Skromne I, Wolpert L, Stern CD. 2004. 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TGF-β Family Signaling in Epithelial Differentiation and Epithelial − Mesenchymal Transition

Kaoru Kahata, Mahsa Shahidi Dadras and Aristidis Moustakas

Cold Spring Harb Perspect Biol 2018; doi: 10.1101/cshperspect.a022194 originally published online February 28, 2017

Subject Collection The Biology of the TGF-β Family

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TGF-β Family Signaling in Neural and Neuronal Signaling Cross Talk between TGF-β/Smad and Differentiation, Development, and Function Other Signaling Pathways Emily A. Meyers and John A. Kessler Kunxin Luo

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Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved