Apical ectodermal ridge regulates three principal axes of the developing limb

Apical ectodermal ridge regulates three principal axes of the developing limb 顶端外胚层嵴对肢体发育的立体调控 1 2 first-author 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhanglan@sdpei.edu.cn †E-mail:zhanglan@sdpei.edu.cn zhanglan@sdpei.edu.cn Centre for Anatomy and Human Identification, University of Dundee, Dundee DD1 5EH, UK Centre for Anatomy and Human Identification, University of Dundee, Dundee DD1 5EH, UK Collaborative Innovation Center for Sports Health Promotion, Shandong Sport University, Jinan 250102, China Collaborative Innovation Center for Sports Health Promotion, Shandong Sport University, Jinan 250102, China Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog ( Shh ) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 ( En1 ) expression, and thus represses Wnt family member 7a ( Wnt7a ) expression in the ventral ectoderm by the expression of Fgfs , Sp6/8 , and bone morphogenetic protein ( Bmp ) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously. 理解胚胎肢体发育有助于更加全面地了解肢体的发生和分化,同时对于相关基因疗法和肢体再生具有临床指导意义.本文综述了顶端外胚层嵴(AER)参与胚胎肢体发育调控的研究进展及机理,重点总结了AER对肢体发育三个轴向的立体调控,包括基因表达调控、信号通路及相关调控中心,并进一步讨论了各信号中心之间的交互作用及现有研究待解决问题,从而加深对肢体发育异常、肢端异常等疾病的理解. Apical ectodermal ridge (AER) Limb development Fibroblast growth factor (FGF) Zone of polarizing activity (ZPA) 顶端外胚层嵴(AER) 肢体发育 成纤维细胞生长因子(FGF) 极化活性区(APZ) Introduction 1 Studies on vertebrate limb development are of great significance for the understanding of pattern formation and morphogenesis in embryology (Casanova et al., 2011 2011 ; Rodriguez-Leon et al., 2013 2013 ). A good conception of limb development both includes the outgrowth and differentiation of the limb, and provides information of clinical relevance, such as reasons for congenital limb deficiencies, or clues about regeneration. Research to date has revealed that limb development begins with the formation of two paired limb buds (the forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER) (Fig. 1 1 ) (Irvine and Rauskolb, 2001 2001 ; Towers and Tickle, 2009 2009 ). It is believed that the AER is a signalling centre with a key role in regulating limb growth and development (Casanova et al., 2011 2011 ; Mallick, 2013 2013 ). The green layer indicates the apical ectodermal ridge. AER: apical ectodermal ridge. Reprinted from Zeller et al. ( 2009 2009 ), Copyright 2009, with permission from Springer Nature The AER is a transitional embryonic structure that regulates gene expression involved with limb development in four stages through the secretion of signalling molecules (Mallick, 2013 2013 ; Rodriguez-Leon et al., 2013 2013 ). Thus, an understanding of AER development is essential for further research on limb growth. For the initiation of AER function, fibroblast growth factor 10 (FGF10) (encoded by Fgf10 gene) expressed from mesenchymal cells binds the epithelial FGF receptor 2b (FGFR2b) with heparin sulfate to activate Fgf8 in the AER precursor cells (Barrow et al., 2003 2003 ; Itoh and Ohta, 2014 2014 ). During maturation, Engrailed-1 ( En1 ) is required to repress the expression of Wnt family member 7a ( Wnt7a ) to the dorsal ectoderm in order to form the dorsal–ventral boundary (Logan et al., 1997 1997 ), while the upstream of En1 is regulated by bone morphogenetic protein (BMP) (Pizette et al., 2001 2001 ). Furthermore, Casanova et al. ( 2011 2011 ) discovered that AT-rich interaction domain 3b ( Arid3b ) participated in AER maturation. The maintenance stage is highly relevant to limb outgrowth and pattern formation, where several signalling pathways function within the AER through a positive feedback loop termed sonic hedgehog (SHH)-Gremlin 1 (GREM1)-FGF regulatory loop (Bouldin et al., 2010 2010 ; Rodriguez-Leon et al., 2013 2013 ). Following the disconnection of the SHH-GREM1-FGF regulatory loop, the regression stage of the AER occurs for size regulation (Scherz et al., 2004 2004 ). Vertebral limb growth and development proceeds in three main axes (Fig. 2 2 ), with the AER acting throughout this process. Based on classical manipulation experiments (Saunders, 1948 1948 ), this structure was used to be considered to control proximodistal patterning. However, the ridge also influences the regulation of anteroposterior patterning, dorsoventral patterning, and the shaping of the limbs (Choi et al., 2012 2012 ; Delgado and Torres, 2017 2017 ). This review aims to summarise the specific roles of the AER in three main axes and in shaping the developing limbs, by conducting a comprehensive search in the database sources up to 2019: MEDLINE, Embase, Scopus, Google Scholar, Cochrane Library, and Web of Science databases. The search terms used were “apical ectodermal ridge,” “developing limb/limb development,” and “limb bud” for searching valuable information in the title, keywords, and abstract. No restrictions were placed on the type of articles, study design, language, or publication year. The searching results were manually reviewed to exclude irrelevant articles. Proximodistal axis: proximal to distal (stylopod to autopod); Anteroposterior axis: anterior to posterior (thumb (digit 1) to little finger (digit 5)); Dorsoventral axis: dorsal to ventral (back of the hand to the palm). Reprinted from Duboc and Logan ( 2009 2009 ), Copyright 2009, with permission from Elsevier Role of AER in proximodistal patterning 1 The primary function of the AER is a regulation of proximodistal patterning. A truncation of the limb skeleton may occur in any stage of limb development when the AER is removed in experiments (Saunders, 1948 1948 ; Summerbell et al., 1973 1973 ). The removal of the AER results in arrested limb development, with the achieved degree of development determined by the stage of development at which the AER is removed (Saunders, 1948 1948 ; Tickle, 2003 2003 ). These findings help us to better understand the mechanism of limb hypoplasia and truncation along the proximodistal axis in humans. Current models for specifying positional information in proximodistal patterning 2 Several models have been proposed to explain proximodistal patterning, such as the progress zone (PZ) model (Summerbell et al., 1973 1973 ), the early specification model (Dudley et al., 2002 2002 ), and the two-signal gradient model (Tabin and Wolpert, 2007 2007 ; Cooper et al., 2011 2011 ). In the PZ model, positional information of the developing limb is from the PZ immediately beneath the AER (Summerbell et al., 1973 1973 ; Wolpert, 2002 2002 ). The PZ acts as a developmental clock suggesting that the length of time undifferentiated mesenchymal cells spend in the PZ specifies their positional value along the proximodistal axis (Wolpert, 2002 2002 ; Tickle, 2003 2003 ; Towers and Tickle, 2009 2009 ). That is, positional values in the PZ model are produced when cells leave the zone (Tickle, 2003 2003 ). However, the early specification model indicates that specific positional information is assigned upon the formation of the limb bud (Dudley et al., 2002 2002 ; Tickle and Wolpert, 2002 2002 ). The difference in positional values between these two models is illustrated in Fig. 3 3 . As for the two-signal gradient model, it involves retinoic acid (RA) signalling as a proximal signal and FGFs as distal signals to regulate proximodistal patterning simultaneously (Mariani et al., 2008 2008 ). Left: progress zone (PZ) model; positional values along the proximodistal axis. The shaded region is the “PZ.” Right: early specification model; positional values are specified at the formation of the limb bud. Reprinted from Tickle ( 2003 2003 ), Copyright 2003, with permission from Elsevier Therefore, the PZ model and early specification model contradict each other. Moreover, studies in recent years have supplied evidence for the two-signal gradient model (Roselló-Díez et al., 2014 2014 ). In fact, there is still no consensus about models among researchers, but each of these models contributes to the understanding of limb development and roles of the AER, in particular, the signals expressed in the AER. Thus, substantial evidence is still required to propose or support a valid model that credibly explains the mechanism of proximodistal patterning. Molecular and genetic bases of proximodistal patterning 2 FGF signalling is instructive in proximodistal patterning, and has been considered as the primary component of the AER signalling (Mallick, 2013 2013 ). Evidence for the function of FGFs in proximodistal patterning was supplied by an FGF-soaked bead experiment in chick wing buds. During the experiment, the AER was replaced by an FGF-soaked bead compared with one no-treatment group and one ridge-removal group without the FGF bead. Results showed that the FGF-soaked bead rescued the outgrowth and proximodistal patterning even with the AER removed (Niswander et al., 1993 1993 ). Using mouse embryos, Moon and Capecchi ( 2000 2000 ) tested the function of FGF signalling by the direct inactivation of Fgf genes ( Fgf8 ) in the AER. As a result, bud outgrowth was reduced and truncation appeared. A similar study by Lewandoski et al. ( 2000 2000 ) also supported these results. However, the inactivation of other Fgf genes does not affect limb development, except by the simultaneous inactivation of Fgf4 and Fgf8 (Martin, 1998 1998 ; Moon et al., 2000 2000 ; Sun et al., 2000 2000 ; Towers and Tickle, 2009 2009 ). The simultaneous absence of Fgf4 and Fgf8 in the AER at different stages of limb development eventually results in a lack of induction of limb development (Sun et al., 2002 2002 ). Besides, both of these genes in the AER are involved in the maintenance of the undifferentiated zone adjacent to the AER. The AER produces a large number of FGF proteins encoded by Fgf4 , Fgf8 , Fgf9 , and Fgf17 (in a mouse embryo), or Fgf19 (in a chick embryo), which function at different stages of limb development (Fig. 4 4 ) (Tickle, 2003 2003 ; Rodriguez-Leon et al., 2013 2013 ). The gene Fgf8 participates in regulating the outgrowth and development throughout proximodistal patterning, while other Fgf genes are activated at specific stages. The expression of Fgf4 and Fgf17 lasts until the beginning of digit patterning (Niswander and Martin, 1992 1992 ), while the expression of Fgf9 still persists in the later stage of digit patterning (Hajihosseini and Heath, 2002 2002 ). P-D: proximal-distal; LPM: lateral plate mesoderm; AER: apical ectodermal ridge; FGF: fibroblast growth factor. Reprinted from Tickle ( 2003 2003 ), Copyright 2003, with permission from Elsevier There are also other molecules and genes regulating proximodistal patterning, such as RA signalling (Yashiro et al., 2004 2004 ), T-box transcription factor 5 ( Tbx5 ) and Tbx4 (Nishimoto et al., 2015 2015 ), myeloid ecotropic viral integration site ( Meis ) (Mercader et al., 1999 1999 , 2000 2000 ), Homeobox A11 ( Hoxa11 ) and Hoxa13 (Nelson et al., 1996 1996 ; Mercader et al., 2009 2009 ), and Shh (Laufer et al., 1994 1994 ; Niswander et al., 1994 1994 ). The disruption of RA signalling through the deletion of retinaldehyde dehydrogenase 2 ( Raldh2 ) leads to forelimb abnormalities (Niederreither et al., 2002 2002 ). Moreover, RA signalling controls the expression of Tbx5 serving the forelimb and Tbx4 serving the hindlimb with the expression of Hox genes and β-catenin, and later through an Fgf10 -AER- Fgf8 feed-forward mechanism to regulate limb growth and development (Fig. 5 5 ) (Nishimoto et al., 2015 2015 ). Through the feed-forward loop, Fgf10 is regulated by the RA both directly and indirectly through Tbx genes. The corresponding genes encode MEIS1/2, HOXA11, and HOXA13 proteins as proximodistal markers in the stylopod, zeugopod, and autopod, respectively (Fig. 6 6 ) (Roselló-Díez et al., 2014 2014 ). The Shh gene, expressed in the zone of polarizing activity (ZPA), primarily regulates anteroposterior patterning. However, the expression of Fgf4 requires SHH signalling through the SHH-FGH feedback loop to regulate proximodistal patterning (Fig. 7 7 ) (Pownall and Isaacs, 2010 2010 ). The factors retinoic acid (RA)/β-catenin/Homeobox ( Hox ) cooperatively activate T-box transcription factor 5 ( Tbx5 ) in the forelimb and Tbx4 in the hindlimb. Genes fibroblast growth factor 10 ( Fgf10 ) and Fgf8 are regulated by RA both directly, and indirectly with Tbx . PZ: progress zone; AER: apical ectodermal ridge PD: programmed death; Meis : myeloid ecotropic viral integration site; Hoxa : Homeobox A. Reprinted from Roselló-Díez et al. ( 2014 2014 ), Copyright 2014, with permission from the Company of Biologists Ltd. ZPA: zone of polarizing activity; SHH: sonic hedgehog; AER: apical ectodermal ridge; FGF: fibroblast growth factor Roles of FGF10 signalling in pre-AER and post-AER induction 2 The factor FGF10, as a paracrine FGF, plays different roles in pre-and post-AER phases along with limb development. During pre-AER induction, Fgf10 functions in the lateral plate mesoderm to initiate the formation of AER through Fgf10 /AER- Fgfr2b signalling, and then establishes the Fgf10 -AER- Fgf8 feed-forward loop (Sekine et al., 1999 1999 ; Danopoulos et al., 2013 2013 ). Moreover, a new function of FGF10 found in a study on FGF10 null embryos is that FGF10 involves the regulation of epithelial to mesenchymal transition, thus allowing the formation of the progenitors of the limb (Gros and Tabin, 2014 2014 ; Jin et al., 2019 2019 ). It remains unclear, however, whether epithelial to mesenchymal transition is the direct consequence of Fgf10 expression. During post-AER induction, Fgf10 is responsible for maintaining the integrity of AER and FGFR2b signalling by promoting Fgf8 and Wnt3a expression in the AER, which in turn maintain Fgf10 expression in the PZ to initiate the amplification of the progenitors of the limb (Sekine et al., 1999 1999 ; Mariani et al., 2008 2008 ; Jin et al., 2019 2019 ). Collectively, the FGF family emanating from the AER regulates proximodistal patterning directly with different functions in corresponding stages of limb outgrowth and development. Meanwhile, newer “players” are being found that participate in the proximodistal patterning via specific feedback loops. Role of AER in anteroposterior patterning 1 The main pathway where the AER participates in regulating anteroposterior patterning is the SHH-GREM1-FGF regulatory loop. As mentioned in the Introduction section, this feedback loop is essential for the maintenance stage of the AER. The gene Shh mediates anteroposterior patterning, and only exists in the posterior area of the limb bud, which is the so-called PA (Duboc and Logan, 2009 2009 ). Within the SHH-GREM1-FGF regulatory loop (Fig. 8 8 ), the expression of Fgf genes, including Fgf4 , Fgf8 , Fgf9 , and Fgf17 , is required for the maintenance of SHH signalling. This loop is positively mediated by Grem1 as an antagonist of Bmp4 , which is responsible for a negative regulation of Fgf expression in the AER (Duboc and Logan, 2009 2009 ). SHH: sonic hedgehog; GREM1: Gremlin 1; FGF: fibroblast growth factor; BMP: bone morphogenetic protein. Reprinted from Duboc and Logan ( 2009 2009 ), Copyright 2009, with permission from Elsevier Additionally, FGFs regulate E26 transformation-specific (ETS) variant 4 ( Etv4 ) and Etv5 of the ETS transcription factors, which are involved with restricting the expression of Shh in the anterior mesenchyme (Zhang et al., 2009 2009 ). Authors of the same study found that the inactivation of Fgf genes in the AER or FGFRs leads to reduction of Etv4 and Etv5 expression. Furthermore, the ectopic expression of Shh gene occurs in the anterior mesenchyme and extra digits appear as Etv4 and Etv5 are inactivated (Fig. 9 9 ). These results are also supported by Lettice et al. ( 2012 2012 ). The asterisk indicates the extra digit. Tcre : transcervical resection of endometrium; Etv : E26 transformation-specific (ETS) variant; mt: metatarsal. Reprinted from Zhang et al. ( 2009 2009 ), Copyright 2009, with permission from Elsevier Thus, the AER not only promotes Shh expression in ZPA for proliferation, but also inhibits Shh expression in the anterior mesenchyme. Anteroposterior patterning is regulated through the dual-direction regulatory loop established between signals from the ZPA (SHH) and AER (FGFs). Role of AER in dorsoventral patterning 1 Dorsoventral patterning is regulated by the interaction between the AER and the ectoderm (dorsal and ventral regions). During normal limb development, the Wnt7a gene is expressed in the dorsal ectoderm, while En1 expression is only detected in the ventral ectoderm (Delgado and Torres, 2017 2017 ). Findings increasingly support that the Fgf8 , Sp6 / 8 , and Bmp2 / 4 / 7 expressed in the AER have crucial roles in dorsoventral patterning (Fig. 10 10 ) (Fernandez-Teran and Ros, 2008 2008 ; Haro et al., 2014 2014 ). The genes Fgfs , Sp6 / 8 , and Bmps activate the expression of En1 , which in turn acts by restricting the expression of Wnt7a in the ventral ectoderm (Loomis et al., 1996 1996 ). Conversely, Wnt7a expression in the dorsal ectoderm induces LIM homeodomain factor 1b ( Lmx1b ), and thus it triggers dorsal differentiation. AER: apical ectodermal ridge; Shh : sonic hedgehog; Wnt7a : Wnt family member 7a; En1 : Engrailed-1; Lmx1b : LIM homeodomain factor 1b; Bmp : bone morphogenetic protein; Fgf : fibroblast growth factor. Reprinted from Delgado and Torres ( 2017 2017 ), Copyright 2017, with permission from Elsevier Furthermore, Bmps , Wnt7a , and En1 give feedback to the AER through the SHH-FGF loop, that is, these factors are also crucial to the initiation, maturation, and maintenance of the AER as mentioned. In this way, the location of the AER at the boundary between the ventral and dorsal ectoderms can be partly explained. Role of AER in shaping the developing limb 1 Levels of the FGF4 and FGF8 proteins expressed in the AER affect the patterning of individual digits, and are regulated by Bmps in the AER (Fig. 11 11 ). (a) During normal development, bone morphogenetic protein (BMP) receptor 1a (BMPR1a) downregulates fibroblast growth factor 8 ( Fgf8 ) expression to activate apoptosis in the interdigit zone. (b) After removal of Bmpr1a , the expansion of Fgf4/8 expression causes abnormal cell survival, and thus webbed digits appear. Reprinted from Pajni-Underwood et al. ( 2007 2007 ), Copyright 2007, with permission from the Company of Biologists Ltd. When the gene BMP receptor 1a ( Bmpr1a ) is inactivated, expression of Fgf4 and Fgf8 in the AER is upregulated, causing webbed digits and less cell death in the interdigit zone (Pajni-Underwood et al., 2007 2007 ). This suggests that Fgf4 and Fgf8 can prevent apoptosis in the interdigital region. Additionally, the removal of Bmp2/4 from the AER also results in increased expression of Fgf4 and Fgf8 with the same results. Furthermore, a study by Choi et al. ( 2012 2012 ) including the removal of BMP ligands ( Bmp2/4/7 ) from the AER reported polydactyly and webbed digits. Hence, Fgf4/8 and Bmps from the AER are responsible for shaping developing digits. Signalling interactions across different signalling centres during limb development 1 As explained previously, the AER, PZ, and ZPA, seen as three signalling centres besides the mesenchyme, regulate almost all phases of limb development simultaneously. Meanwhile, four AER compartments interact with each other by respective signalling pathways during the process. Jin et al. ( 2019 2019 ) linked signalling to these four compartments with effects of Fgf10 (Fig. 12 12 ), clearly showing pathways of RA/ Hox /β-catenin signalling to Tbx4 / Tbx5 , Fgf10- AER- Fgf8 feedback loop, SHH-GREM1-FGF regulatory loop, and repression of FGFs on Shh expression, as mentioned above. Additionally, Fgf10 express is stabilized by Wnt2b (forelimb) and Wnt8c (hindlimb), and Fgf8 expression is stabilized by Wnt3a (Kawakami et al., 2001 2001 ). Furthermore, BMP signalling also upregulates Tbx expression, except for initiating Grem1 expression (Rodriguez-Esteban et al., 1999 1999 ; Sheeba and Logan, 2017 2017 ). Reciprocal interactions form a basic loop to implement a regulation by signalling centres for the outgrowth and patterning of the limb. The limb bud consists of mesenchyme, progress zone (PZ), zone of polarizing activity (ZPA), and apical ectodermal ridge (AER) from proximal to distal. The closed loop regulating the outgrowth and development of the limb bud includes retinoic acid (RA)/homeobox ( Hox )/β-catenin signalling, a fibroblast growth factor 10 ( Fgf10 )-AER- Fgf8 feedback loop, and a sonic hedgehog (SHH) - Gremlin 1 (GREM1)-FGF regulatory loop. Tbx : T-box transcription factor; Fgfr2b : FGF receptor 2b; Etv4 : E26 transformation-specific (ETS) variant 4; Bmp : bone morphogenetic protein Conclusions 1 The AER contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cellular activities. The principal function of the AER is the regulation of proximodistal patterning. This is achieved directly through FGFs, while the specific model for explaining proximodistal patterning remains unclear. During anteroposterior patterning, the AER acts as a dual-direction regulator in which it promotes Shh expression in ZPA for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates En1 expression, and thus it suppresses Wnt7a expression in the ventral ectoderm by the expression of Fgfs , Sp6 / 8 , and Bmps . It also plays a vital role in shaping the individual digits, as the levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. Consequently, it is important to understand the crosstalk among the three main axes of AER to accurately describe limb growth and pattern formation, as different processes occur simultaneously in limb development. References JR Barrow , KR Thomas , O Boussadia-Zahui , 等 . Ectodermal Wnt3/β-catenin signaling is required for the establishment and maintenance of the apical ectodermal ridge . 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FGF-regulated Etv genes are essential for repressing Shh expression in mouse limb buds Compliance with ethics guidelines Guo-hao LIN and Lan ZHANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by either of the authors. 10.1631/jzus.B2000285 Project supported by the Key Research and Development Project of Shandong Province (No. 2017G006043), China 2020-05-31 2020-08-06 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 21