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Segment Identity and Cell Segregation in the Vertebrate Hindbrain
CHAPTER THIRTY-FOUR
Segment Identity and Cell Segregation in the Vertebrate Hindbrain Megan Addison, David G. Wilkinson1 The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Vertebrate Hindbrain Segmentation 3. Segment Identity 3.1 Induction of Segmental Identity in the Vertebrate Hindbrain 3.2 Establishment of Mutually Exclusive Segmental Identity 3.3 Plasticity in Cell Identity 4. Cell Segregation 4.1 Restriction of Cell Intermingling Across Segment Borders 4.2 Coupling Between Cell Identity and Cell Segregation 5. Summary and Future Perspectives Acknowledgments References
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Abstract The subdivision of tissues into sharply demarcated regions with distinct and homogenous identity is an essential aspect of embryonic development. Along the anteroposterior axis of the vertebrate nervous system, this involves signaling which induces spatially restricted expression of transcription factors that specify regional identity. The spatial expression of such transcription factors is initially imprecise, with overlapping expression of genes that specify distinct identities, and a ragged border at the interface of adjacent regions. This pattern becomes sharpened by establishment of mutually exclusive expression of transcription factors, and by cell segregation that underlies formation of a straight border. In this review, we discuss studies of the vertebrate hindbrain which have revealed how discrete regional identity is established, the roles of Eph ephrin signaling in cell segregation and border sharpening, and how cell identity and cell segregation are coupled.
Current Topics in Developmental Biology, Volume 117 ISSN 0070-2153 http://dx.doi.org/10.1016/bs.ctdb.2015.10.019
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2016 Elsevier Inc. All rights reserved.
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1. INTRODUCTION In many tissues, the generation of a complex organization of cell types involves an initial subdivision into domains along one or several body axes, each with a distinct identity. At the molecular level, regional identity is determined by specific transcription factors which have spatially restricted expression within the tissue induced by intercellular signaling. An extensively studied example is the patterning along the anteroposterior axis of the vertebrate neural epithelium, in which graded signals induce transcription factors that define distinct regions of the brain and spinal cord. At early stages, the borders of adjacent regions are imprecise, and then are sharpened, reflecting that correct tissue organization requires the formation of discrete territories (Batlle & Wilkinson, 2012; Dahmann, Oates, & Brand, 2011). The sharpening of borders involves two processes (Kiecker & Lumsden, 2005; Fig. 1). First, cells at the prospective border of adjacent regions may initially coexpress transcription factors that specify distinct identities, likely reflecting an intrinsic imprecision in the interpretation of graded signals. Such overlaps can be resolved through cross-repression that establishes mutually exclusive expression of transcription factors. Second, once distinct identities are established, the border of adjacent regions is initially ragged due to the spatial imprecision in cell specification, as well as cell intermingling that occurs during tissue growth and morphogenetic movements. The ragged interface is then straightened by segregation of the cells with distinct identity. Direct evidence for an imprecise pattern generated by noisy inductive signaling being sharpened by cell segregation has been obtained by in vivo imaging studies of the ventral neural tube in zebrafish (Xiong et al., 2013). This implies that mechanisms that underlie cell segregation are upregulated in concert with, or downstream of, regional specification. Understanding of how sharp regional domains are formed thus requires elucidation of interconnected processes: the establishment of mutually exclusive expression of transcription factors that specify regional identity, cell segregation and border sharpening, and the coupling between regional identity and cell segregation. In this review, we discuss current understanding of these mechanisms in the segmentation of the vertebrate hindbrain.
2. VERTEBRATE HINDBRAIN SEGMENTATION During development of the vertebrate hindbrain, the neuroepithelium becomes subdivided into seven morphological units, known as rhombomeres
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1 Induction of segmental identity Anterior
Posterior
[RA]B
[RA]A
pre-r3
pre-r4 [RA]A
hoxb1
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[RA]
hoxb1 + krox20
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2 Establishment of mutually exclusive expression Dual-expressing cell; intermediate identity
hoxb1
krox20
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3 Cell segregation and border sharpening
r4 identity
EphA4
r3 identity
Figure 1 Border formation during hindbrain segmentation. The left-hand side depicts the progressive sharpening of segment borders during hindbrain development, illustrated for the r3/r4 border. During induction of segmental identity, graded retinoic acid (RA) upregulates expression of hoxb1 and krox20, with some cells at the prospective border expressing both genes (top right). hoxb1 and krox20 expression becomes mutually exclusive through cross-repression, thus resolving the identity of those cells which initially express both genes (bottom right). Autoregulatory loops maintain the expression of hoxb1 and krox20. Krox20 upregulates EphA4 in r3, while unknown factors (perhaps directly or indirectly downstream of hoxb1) upregulate ephrinB3 in r4. Signaling of EphA4 and ephrinB3, and of other Eph–ephrin pairs, underlies cell segregation that sharpens the segment borders.
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(r1–r7). The subdivision of the hindbrain underlies the segmental patterning of neuronal organization (Clarke & Lumsden, 1993; Hanneman, Trevarrow, Metcalfe, Kimmel, & Westerfield, 1988; Lumsden & Keynes, 1989), as well as specification of branchial neural crest cells and establishment of their migratory pathways (Birgbauer, Sechrist, Bronner-Fraser, & Fraser, 1995; Lumsden, Sprawson, & Graham, 1991). At the molecular level, anteroposterior identity is determined by segmentally expressed Hox genes which have overlapping and nested expression patterns in the hindbrain (Tumpel, Wiedemann, & Krumlauf, 2009). For example, Hoxb1 (hoxb1a in zebrafish) confers regional identity to r4 (Bell, Wingate, & Lumsden, 1999; McClintock, Kheirbek, & Prince, 2002; Rohrschneider, Elsen, & Prince, 2007; Studer, Lumsden, Ariza-McNaughton, Bradley, & Krumlauf, 1996). Specific Hox genes together with other transcription factors are required for segmentation of the hindbrain. For example, Krox20 is essential for the formation of r3 and r5 (SchneiderMaunoury, Seitanidou, Charnay, & Lumsden, 1997; Schneider-Maunoury et al., 1993; Swiatek & Gridley, 1993; Voiculescu et al., 2001), while MafB is required for formation of r5 in mouse (Manzanares, Trainor, et al., 1999), and for segmentation to form r5 and r6 in zebrafish (Moens, Yan, Appel, Force, & Kimmel, 1996). As discussed in detail later, sharp borders form at the interface of adjacent rhombomeres, across which cell intermingling is prevented through signaling by Eph receptors and ephrins. In parallel, specialized cells with distinct cellular and molecular properties are induced to form at the interfaces of rhombomeres (Guthrie & Lumsden, 1991; Heyman, Faissner, & Lumsden, 1995; Lumsden & Keynes, 1989). There is a lower proliferation of boundary cells and accumulation of extracellular matrix which could in principle contribute to maintenance of sharp borders, but it has been found that cell intermingling is still restricted after blocking of boundary cell formation in chick (Nittenberg et al., 1997). There is increasing evidence that hindbrain boundary cells function as signaling centers that regulate segmental gene expression in chick (Sela-Donenfeld, Kayam, & Wilkinson, 2009), and the patterning of cell differentiation within rhombomeres in zebrafish (Gonzalez-Quevedo, Lee, Poss, & Wilkinson, 2010; Terriente, Gerety, Watanabe-Asaka, Gonzalez-Quevedo, & Wilkinson, 2012).
3. SEGMENT IDENTITY 3.1 Induction of Segmental Identity in the Vertebrate Hindbrain The induction of different transcription factors along the anteroposterior axis of the hindbrain involves a combination of transient posteriorizing
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signals, including graded retinoic acid, and Wnt and Fgf proteins (Hernandez, Putzke, Myers, Margaretha, & Moens, 2007; Kudoh, Wilson, & Dawid, 2002; White, Nie, Lander, & Schilling, 2007). Retinoic acid is synthesized in the paraxial mesoderm posterior to the hindbrain and is necessary for the specification of hindbrain segments posterior to the r3/4 border. Domains of Hox expression here are patterned by differential responses to graded levels of retinoic acid along the anteroposterior axis. Retinoic acid can induce the expression of certain Hox genes directly by activating RAR/RXR heterodimers at retinoic acid response elements (RAREs) in regulatory regions, for example, of members of the Hox1 and Hox4 paralogous groups (Marshall et al., 1994; Nolte, Amores, Nagy Kovacs, Postlethwait, & Featherstone, 2003). The expression of other posterior retinoic acid-responsive hox genes, including Hox3 genes, is regulated indirectly by retinoic acid; for example, Hoxa3 expression in r5 and r6 is regulated by MafB, which is itself regulated in part by retinoic acid (via Vhnf1) (Hernandez, Rikhof, Bachmann, & Moens, 2004; Manzanares, Cordes, et al., 1999). More posterior retinoic acid-responsive Hox genes require higher levels of retinoic acid for their induction and are expressed later than more anterior retinoic acid-responsive Hox genes, which are induced at lower levels of retinoic acid (Dupe & Lumsden, 2001; Maves & Kimmel, 2005). However, the spatial patterning of Hox gene expression along the anteroposterior axis by retinoic acid is not purely achieved by differential sensitivities to retinoic acid, as the RAREs of different Hox genes have been found to have comparable sensitivities (Nolte et al., 2003). It has also been shown that Hox genes expressed in more posterior hindbrain domains do not require longer exposures to retinoic acid for their induction (Hernandez et al., 2007; Maves & Kimmel, 2005). The mechanisms by which the graded retinoic acid activity required for hindbrain segmentation is generated and maintained are not completely understood. The concentration of retinoic acid along the anteroposterior axis of the hindbrain is influenced both spatially and temporally by members of the Cyp26 family of cytochrome p450 enzymes—Cyp26a1, b1 and c1— which degrade retinoic acid. In the absence of Cyp26 enzymes, the anteroposterior patterning of the hindbrain is disrupted (Hernandez et al., 2007). Furthermore, exogenous retinoic acid applied over a 20-fold concentration range and at a variety of developmental stages is sufficient to correctly pattern the hindbrain of embryos that are depleted of endogenous retinoic acid, and Cyp26 enzymes are required for this rescue (Dupe & Lumsden, 2001; Hernandez et al., 2007; Maves & Kimmel, 2005). Cyp26a1 is induced by retinoic acid itself and has an important role in the negative feedback of
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retinoic acid signaling (White et al., 2007). This self-enhanced degradation allows retinoic acid-induced patterning to be robust despite variations in retinoic acid levels. Cyp26b1 and cyp26c1 are segmentally and dynamically expressed during hindbrain segmentation and refine retinoic acid responsiveness within the hindbrain through localized retinoic acid degradation (White & Schilling, 2008). It is not clear whether segmentally expressed Cyp26 enzymes generate a step-wise gradient of retinoic acid. In mouse, use of a RARE–lacZ reporter has revealed distinct boundaries of retinoic acid responsiveness, which move during hindbrain segmentation (Rossant, Zirngibl, Cado, Shago, & Goguere, 1991; Sirbu, Gresh, Barra, & Duester, 2005). However, a RARE-eYFP transgenic zebrafish line has revealed a smooth gradient of retinoic acid responsiveness, though only at later stages of hindbrain segmentation (White et al., 2007). More recently, a smooth gradient of retinoic acid in the zebrafish hindbrain has been detected by visualization of unbound intracellular retinoic acid using FRET-based genetically encoded retinoic acid probes (Shimozono, Iimura, Kitaguchi, Higashijima, & Miyawaki, 2013). Fgfs have important roles in hindbrain patterning, in part by inhibiting retinoic acid-mediated cyp26a1 upregulation (White et al., 2007). This enables Fgfs to influence the retinoic acid gradient within the hindbrain, which has been suggested to help couple growth of the hindbrain with a corresponding expansion of the retinoic acid gradient (Schilling, Nie, & Lander, 2012; White et al., 2007). In combination with retinoic acid, Fgfs and Wnts also initiate the expression of certain posterior genes, including hoxb1b (posteriorly from pre-r4) and vhnf1, (in pre-r5–r6) (Kudoh et al., 2002). Later in hindbrain segmentation, fgf3 and fgf8 are upregulated by Hox1 proteins in r4, and are involved in the patterning of surrounding rhombomeres (Hernandez et al., 2004; Marin & Charnay, 2000; Maves, Jackman, & Kimmel, 2002; Walshe, Maroon, McGonnell, Dickson, & Mason, 2002; Waskiewicz, Rikhof, & Moens, 2002; Wiellette & Sive, 2003). For example, Fgf signals from r4 cooperate with vhnf1 and retinoic acid to drive initiation of mafB expression in r5 and r6 (Hernandez et al., 2004), and Fgfs also contribute to induction of krox20 expression (Labalette et al., 2015).
3.2 Establishment of Mutually Exclusive Segmental Identity The expression domains of segmentally expressed transcription factors initially have diffuse borders, in which some cells coexpress factors that confer
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conflicting identities and thus have an intermediate identity. For example in zebrafish, some cells at the r3/r4 and r4/r5 interfaces express both krox20 and hoxb1a, which confer opposing identities; likewise, ectopic krox20-expressing cells within r4 can also contain hoxb1a transcripts (Zhang et al., 2012). This raises the question of how one or the other gene is downregulated in order to establish and maintain a single identity. There have been detailed investigations of gene regulatory interactions in the specification of r3, r4, and r5 in the hindbrain. Following initiation of krox20 expression, an autoregulatory loop amplifies and maintains krox20 expression in r3 and r5 (Bouchoucha et al., 2013; Chomette, Frain, Cereghini, Charnay, & Ghislain, 2006; Giudicelli, Taillebourg, Charnay, & Gilardi-Hebenstreit, 2001). Likewise, Hoxb1 upregulates its own expression in r4 via an autoregulatory loop (P€ opperl et al., 1995). Mutually exclusive expression is established since Krox20 and Hoxb1 repress each other: ectopic overexpression of krox20 in r4 causes downregulation of hoxb1 expression (Giudicelli et al., 2001), while hoxb1 represses krox20 expression indirectly via activation of Nlz factors (Labalette et al., 2015). These interactions create a bistable switch, which enables cells to adopt an exclusive identity and commit to a particular fate following transient inputs to identity specification by morphogens (Fig. 1). This generates sharp spatial transitions in identity despite shallow and noisy morphogen gradients (Bouchoucha et al., 2013). Computational modeling has demonstrated that such a bistable switch regulating krox20 and hoxb1 expression, in combination with noise in the retinoic acid gradient, can contribute to border sharpening in the hindbrain by driving the refinement of gene expression domains via noise-induced switching of cell identity (Schilling et al., 2012; Zhang et al., 2012).
3.3 Plasticity in Cell Identity Intermingling of cells occurs during hindbrain development, driven by cell division and morphogenetic rearrangements such as convergent extension. This is revealed by the dispersal of the progeny of individual cells in the chick hindbrain, which when labeled at early stages can contribute to adjacent rhombomeres, but when labeled after morphological boundary formation are restricted to single segments (Fraser, Keynes, & Lumsden, 1990). This raises the question of how sharp gene expression boundaries are established despite some cell movement between segments at early stages. There is evidence that cells are capable of switching their identity due to plasticity in Hox
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expression status. In mouse embryos, transpositions of small numbers of cells between rhombomeres have found that cells will change Hox expression (Trainor & Krumlauf, 2000). Likewise, in zebrafish, cells can alter their hox expression following transplantation between rhombomeres at early stages, when border sharpening is occurring (Kemp, Cooke, & Moens, 2009; Schilling, Prince, & Ingham, 2001). Transplantations at later stages, when segmental gene expression domains are sharp, revealed a progressive loss of plasticity in cell identity (Schilling et al., 2001); by these stages, there is no longer cell mixing between segments (Calzolari, Terriente, & Pujades, 2014), and thus switching is not required during normal development. Cells transplanted to more posterior regions of the hindbrain have been found to exhibit increased plasticity compared to posterior-to-anterior transplantations (Grapin-Botton, Bonnin, McNaughton, Krumlauf, & Le Douarin, 1995; Itasaki, Sharpe, Morrison, & Krumlauf, 1996; Schilling et al., 2001), which may reflect the importance of posteriorizing signals in regulating Hox expression. Intriguingly, cell plasticity depends on the size of the group of transplanted cells, with larger groups maintaining their original Hox expression at a different position along the anteroposterior axis (Schilling et al., 2001; Trainor & Krumlauf, 2000). A failure to switch identity can account for the groups of krox20-expressing cells present in evennumbered segments following increased intermingling due to dominant negative blocking of Eph receptors (Xu, Alldus, Holder, & Wilkinson, 1995). These observations suggest that community effects have a role in the maintenance of segmental gene expression. The finding that Krox20 nonautonomously induces its own expression in adjacent cells (Giudicelli et al., 2001) suggests a model in which it upregulates a signal which establishes and maintains homogenous identity within groups of cells. It is not clear from these experiments the extent to which cell plasticity contributes to the establishment and maintenance of distinct segmental identity. Lineage analysis in chick reveals cell intermingling at early stages in hindbrain segmentation (Fraser et al., 1990), but the extent to which this occurs has not been quantitated. Use of Cre-mediated recombination in mouse to permanently label cells of the hindbrain that have expressed the r3/r5 marker, Krox20, has found some labeled cells in even-numbered rhombomeres (Voiculescu et al., 2001). This is consistent with cells dynamically expressing Krox20, and suggests that switching of cell identity occurs. However, such labeling could reflect resolution of initial overlapping expression, for example of Krox20 and Hoxb1, rather than intermingling of cells that have established an r3/r5 identity. In zebrafish, cells expressing
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stable fluorescent reporters of r3 and r5 cell identity have not been detected in adjacent segments once sharpening of gene expression domains is complete (Calzolari et al., 2014). This suggests that in zebrafish there is little, if any, intermingling between segments and cell identity switching in normal development. However, it is possible that early intermingling is not detected since insufficient levels of the transgenic fluorescent reporter have accumulated in cells with transient r3/r5 identity.
4. CELL SEGREGATION 4.1 Restriction of Cell Intermingling Across Segment Borders During early hindbrain development, the initially ragged expression limits of molecular determinants of rhombomere identity become straight (Irving, Nieto, DasGupta, Charnay, & Wilkinson, 1996; Oxtoby & Jowett, 1993). This sharpening is presumably driven by cell segregation mechanisms that underlie the restriction of intermingling between hindbrain segments (Calzolari et al., 2014; Fraser et al., 1990; Jimenez-Guri et al., 2010; Mathis, Sieur, Voiculescu, Charnay, & Nicolas, 1999). Transplantation experiments in chick showed that the inhibition of cell mixing involves cell properties that differ most prominently between odd versus even-numbered rhombomeres (Guthrie, Prince, & Lumsden, 1993). There is much evidence that interactions between Eph receptor tyrosine kinases and ephrins have a major role in this restriction of cell mixing. Eph receptors and ephrins comprise a cell contact-dependent signaling system, which upon binding and clustering transduce signals bidirectionally (Klein, 2012). In mouse, several Eph receptors are expressed in r3 and r5, and ephrinB proteins that they bind are expressed in even-numbered segments (Xu, Mellitzer, & Wilkinson, 2000). There is a different situation in zebrafish (Chan et al., 2001; Cooke et al., 2001; Xu et al., 1995), in which Eph–ephrin pairs that have high affinity have complementary expression, but not simply in odd versus even-numbered segments: expression of EphA4 and ephrinB3 occurs in r3/r5 and r2/r4/r6, respectively; expression of EphB4a and ephrinb2a in r5/r6 and r2/r4/r7, respectively. The effects of blocking or knockdown of EphA4a and ephrinB2a reveal that Eph–ephrin signaling is required to inhibit cell intermingling and sharpen segment borders in zebrafish (Cooke, Kemp, & Moens, 2005; Kemp et al., 2009; Xu et al., 1995). The restriction of cell mixing is mediated, at least in part, by Eph–ephrin pairs with complementary expression
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that are interacting at the border (Mellitzer, Xu, & Wilkinson, 1999; Xu, Mellitzer, Robinson, & Wilkinson, 1999). Eph–ephrin signaling could act through one or several mechanisms that have been shown to drive cell segregation in other tissues: differential cell–cell adhesion, cortical tension, and cell repulsion (Batlle & Wilkinson, 2012; Cayuso, Xu, & Wilkinson, 2015; Fagotto, 2014; Fagotto, Winklbauer, & Rohani, 2014). A recent study has shown that there is an Eph–ephrin-dependent accumulation of actomyosin cables at segment borders, which can potentially generate increased cortical tension that restricts cell intermingling (Calzolari et al., 2014). Since the formation of actomyosin cables occurs several hours after border sharpening, cortical tension may play a late role in boundary stabilization. Intriguingly, mosaic knockdown of EphA4 or of ephrinB2a in zebrafish was found to lead to segregation of the knockdown cells to the borders of the segments expressing the corresponding Eph receptor or ephrin (Cooke et al., 2005; Kemp et al., 2009). The mechanism(s) that drives segregation of cells with decreased Eph or ephrin expression within segments is not known, but in principle could involve differential adhesion, tension, or repulsion. This suggests that EphA4 and ephrinB2 activity within segments contributes to cell segregation, presumably acting in parallel with bidirectional signaling at the segment borders to drive sharpening.
4.2 Coupling Between Cell Identity and Cell Segregation The finding that border sharpening requires complementary expression of interacting Eph receptors and ephrins raises the question of how their segmental expression in the hindbrain is regulated. One possibility is that this is mediated by the transcription factors that underlie segmentation and anteroposterior identity in the hindbrain. Indeed, mafB is required to upregulate EphB4a and suppress ephrinB2a expression in r5 and r6, such that this Eph–ephrin pair is in complementary domains (Cooke et al., 2001; Hernandez et al., 2004). Likewise, Hoxa2 is required for EphA7 expression in r3 (Taneja et al., 1996). However, due to the cross-regulation that occurs between mafB, Krox20, and Hox genes, it is not known from these studies which of the transcription factors are the direct regulator of Eph receptor or ephrin expression. Evidence has been obtained by analysis of enhancer elements of the EphA4 gene showing that Krox20 directly regulates EphA4 expression in r3 and r5 (Theil et al., 1998). Since Krox20 also directly regulates expression of specific Hox genes (Tumpel et al., 2009), there is thus a coupling of anteroposterior identity and cell segregation. Direct regulation
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has also been revealed in studies showing that EphA2 expression in r4 is upregulated by Hoxa1 and Hoxb1 (Studer et al., 1998) through binding to Hox/Pbx binding sites in an EphA2 enhancer (Chen & Ruley, 1998). However, the roles of EphA2 in the hindbrain are not known. A recent study has given further insights into the relationships between Hox genes and cell segregation in the hindbrain (Prin, Serpente, Itasaki, & Gould, 2014). The anterior limit of Hoxb4 and Hoxd4 expression is at the r6/r7 boundary, which is absent in double Hoxb4/Hoxd4 mutants, as seen by loss of molecular markers of boundary cells and of the morphological constriction. Furthermore, boundary formation in more anterior regions of the hindbrain is disrupted when Hox4 genes are ectopically expressed. Importantly, it was found that mosaic ectopic expression of Hox4 genes leads to segregation of Hox4+ and Hox4– cells. This segregation is accompanied by formation of an apical constriction in whichever of the two populations is in the minority, and alterations in the apical surface area of cells 3–4 diameters away from the Hox4+/Hox4– border. These observations are consistent with a tension-based mechanism, which may involve Eph–ephrin signaling since ectopic expression of ephrinB2 creates similar constrictions. Furthermore, Hox4 genes underlie the posterior limit of EphA7 expression at the r6/r7 border by downregulating its expression in r7, albeit it is not known whether EphA7 is involved in boundary formation. Taken together, these findings reveal that Hox4 genes underlie formation of the r6/r7 boundary and regulate mechanisms that drive cell segregation. Since cell segregation also occurs following ectopic expression of Hoxa2 and Hoxa3, this role may be a general feature of Hox genes (Prin et al., 2014). Evidence that Hox genes act through regulation of Eph–ephrin expression is currently circumstantial, and it is possible that cell segregation also involves other Hox targets (Prin et al., 2014). The results of overexpression experiments show that krox20 has a dominant role in cell segregation in r3 and r5 through regulation of EphA4, which overrides the control of cell segregation by Hox genes in these segments. Hox genes may thus act in the other rhombomeres to promote border formation.
5. SUMMARY AND FUTURE PERSPECTIVES Major progress has been made in elucidating the signaling and gene regulatory networks that underlie segmental expression of key transcription factors in hindbrain patterning, including Hox genes, krox20, and mafB. These involve positive feedback and mutual repression that establishes
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complementary segmental expression. Recent studies have focussed on cross-repression of hoxb1 and krox20 at the borders of r4, and it is important to elucidate the regulatory relationships that establish discrete cell identities across the other boundaries in the hindbrain. Another important question is whether such networks account for cell identity switching following transplantation between segments, and which may also occur during cell intermingling in normal development. Presumably, the ectopic cells are reading a different level of graded retinoic acid and other signals at the new anteroposterior position. Another possibility is that ectopic cells come under the influence of signaling from their new neighbors, as suggested by the lack of identity switching when groups of cells are transplanted. How does the gene regulatory network respond to such changes in the signaling environment? In particular, how does altered signaling overcome positive feedback loops that maintain krox20 and hoxb1 gene expression? Do such loops and/ or other mechanisms underlie the decreasing ability of cells to switch identity at later stages of hindbrain development? There has been less progress in understanding the coupling between segment identity and cell segregation. It is clear that Hox genes, krox20, and mafB are important regulators which lie upstream of Eph receptor and ephrin expression in the hindbrain. However, for the Eph receptors and ephrins known to underlie cell segregation in the hindbrain, the only direct link shown thus far is the upregulation of EphA4 by krox20. It will, in particular, be interesting to uncover whether Hox genes are direct regulators of the relevant Eph receptors and ephrins, and how the gene regulatory network in the hindbrain establishes the complementary expression of Eph–ephrin pairs that is required for cell segregation.
ACKNOWLEDGMENTS We are grateful to Alex Gould, Fabrice Prin, Jordi Cayuso, and Qiling Xu for their valuable comments on this chapter. Work in the author’s lab is funded by the Francis Crick Institute.
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Segment Identity and Cell Segregation in the Vertebrate Hindbrain Megan Addison, David G. Wilkinson1 The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Vertebrate Hindbrain Segmentation 3. Segment Identity 3.1 Induction of Segmental Identity in the Vertebrate Hindbrain 3.2 Establishment of Mutually Exclusive Segmental Identity 3.3 Plasticity in Cell Identity 4. Cell Segregation 4.1 Restriction of Cell Intermingling Across Segment Borders 4.2 Coupling Between Cell Identity and Cell Segregation 5. Summary and Future Perspectives Acknowledgments References
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Abstract The subdivision of tissues into sharply demarcated regions with distinct and homogenous identity is an essential aspect of embryonic development. Along the anteroposterior axis of the vertebrate nervous system, this involves signaling which induces spatially restricted expression of transcription factors that specify regional identity. The spatial expression of such transcription factors is initially imprecise, with overlapping expression of genes that specify distinct identities, and a ragged border at the interface of adjacent regions. This pattern becomes sharpened by establishment of mutually exclusive expression of transcription factors, and by cell segregation that underlies formation of a straight border. In this review, we discuss studies of the vertebrate hindbrain which have revealed how discrete regional identity is established, the roles of Eph ephrin signaling in cell segregation and border sharpening, and how cell identity and cell segregation are coupled.
Current Topics in Developmental Biology, Volume 117 ISSN 0070-2153 http://dx.doi.org/10.1016/bs.ctdb.2015.10.019
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1. INTRODUCTION In many tissues, the generation of a complex organization of cell types involves an initial subdivision into domains along one or several body axes, each with a distinct identity. At the molecular level, regional identity is determined by specific transcription factors which have spatially restricted expression within the tissue induced by intercellular signaling. An extensively studied example is the patterning along the anteroposterior axis of the vertebrate neural epithelium, in which graded signals induce transcription factors that define distinct regions of the brain and spinal cord. At early stages, the borders of adjacent regions are imprecise, and then are sharpened, reflecting that correct tissue organization requires the formation of discrete territories (Batlle & Wilkinson, 2012; Dahmann, Oates, & Brand, 2011). The sharpening of borders involves two processes (Kiecker & Lumsden, 2005; Fig. 1). First, cells at the prospective border of adjacent regions may initially coexpress transcription factors that specify distinct identities, likely reflecting an intrinsic imprecision in the interpretation of graded signals. Such overlaps can be resolved through cross-repression that establishes mutually exclusive expression of transcription factors. Second, once distinct identities are established, the border of adjacent regions is initially ragged due to the spatial imprecision in cell specification, as well as cell intermingling that occurs during tissue growth and morphogenetic movements. The ragged interface is then straightened by segregation of the cells with distinct identity. Direct evidence for an imprecise pattern generated by noisy inductive signaling being sharpened by cell segregation has been obtained by in vivo imaging studies of the ventral neural tube in zebrafish (Xiong et al., 2013). This implies that mechanisms that underlie cell segregation are upregulated in concert with, or downstream of, regional specification. Understanding of how sharp regional domains are formed thus requires elucidation of interconnected processes: the establishment of mutually exclusive expression of transcription factors that specify regional identity, cell segregation and border sharpening, and the coupling between regional identity and cell segregation. In this review, we discuss current understanding of these mechanisms in the segmentation of the vertebrate hindbrain.
2. VERTEBRATE HINDBRAIN SEGMENTATION During development of the vertebrate hindbrain, the neuroepithelium becomes subdivided into seven morphological units, known as rhombomeres
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1 Induction of segmental identity Anterior
Posterior
[RA]B
[RA]A
pre-r3
pre-r4 [RA]A
hoxb1
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krox20
r3 identity
[RA]
hoxb1 + krox20
Intermediate identity
2 Establishment of mutually exclusive expression Dual-expressing cell; intermediate identity
hoxb1
krox20
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3 Cell segregation and border sharpening
r4 identity
EphA4
r3 identity
Figure 1 Border formation during hindbrain segmentation. The left-hand side depicts the progressive sharpening of segment borders during hindbrain development, illustrated for the r3/r4 border. During induction of segmental identity, graded retinoic acid (RA) upregulates expression of hoxb1 and krox20, with some cells at the prospective border expressing both genes (top right). hoxb1 and krox20 expression becomes mutually exclusive through cross-repression, thus resolving the identity of those cells which initially express both genes (bottom right). Autoregulatory loops maintain the expression of hoxb1 and krox20. Krox20 upregulates EphA4 in r3, while unknown factors (perhaps directly or indirectly downstream of hoxb1) upregulate ephrinB3 in r4. Signaling of EphA4 and ephrinB3, and of other Eph–ephrin pairs, underlies cell segregation that sharpens the segment borders.
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(r1–r7). The subdivision of the hindbrain underlies the segmental patterning of neuronal organization (Clarke & Lumsden, 1993; Hanneman, Trevarrow, Metcalfe, Kimmel, & Westerfield, 1988; Lumsden & Keynes, 1989), as well as specification of branchial neural crest cells and establishment of their migratory pathways (Birgbauer, Sechrist, Bronner-Fraser, & Fraser, 1995; Lumsden, Sprawson, & Graham, 1991). At the molecular level, anteroposterior identity is determined by segmentally expressed Hox genes which have overlapping and nested expression patterns in the hindbrain (Tumpel, Wiedemann, & Krumlauf, 2009). For example, Hoxb1 (hoxb1a in zebrafish) confers regional identity to r4 (Bell, Wingate, & Lumsden, 1999; McClintock, Kheirbek, & Prince, 2002; Rohrschneider, Elsen, & Prince, 2007; Studer, Lumsden, Ariza-McNaughton, Bradley, & Krumlauf, 1996). Specific Hox genes together with other transcription factors are required for segmentation of the hindbrain. For example, Krox20 is essential for the formation of r3 and r5 (SchneiderMaunoury, Seitanidou, Charnay, & Lumsden, 1997; Schneider-Maunoury et al., 1993; Swiatek & Gridley, 1993; Voiculescu et al., 2001), while MafB is required for formation of r5 in mouse (Manzanares, Trainor, et al., 1999), and for segmentation to form r5 and r6 in zebrafish (Moens, Yan, Appel, Force, & Kimmel, 1996). As discussed in detail later, sharp borders form at the interface of adjacent rhombomeres, across which cell intermingling is prevented through signaling by Eph receptors and ephrins. In parallel, specialized cells with distinct cellular and molecular properties are induced to form at the interfaces of rhombomeres (Guthrie & Lumsden, 1991; Heyman, Faissner, & Lumsden, 1995; Lumsden & Keynes, 1989). There is a lower proliferation of boundary cells and accumulation of extracellular matrix which could in principle contribute to maintenance of sharp borders, but it has been found that cell intermingling is still restricted after blocking of boundary cell formation in chick (Nittenberg et al., 1997). There is increasing evidence that hindbrain boundary cells function as signaling centers that regulate segmental gene expression in chick (Sela-Donenfeld, Kayam, & Wilkinson, 2009), and the patterning of cell differentiation within rhombomeres in zebrafish (Gonzalez-Quevedo, Lee, Poss, & Wilkinson, 2010; Terriente, Gerety, Watanabe-Asaka, Gonzalez-Quevedo, & Wilkinson, 2012).
3. SEGMENT IDENTITY 3.1 Induction of Segmental Identity in the Vertebrate Hindbrain The induction of different transcription factors along the anteroposterior axis of the hindbrain involves a combination of transient posteriorizing
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signals, including graded retinoic acid, and Wnt and Fgf proteins (Hernandez, Putzke, Myers, Margaretha, & Moens, 2007; Kudoh, Wilson, & Dawid, 2002; White, Nie, Lander, & Schilling, 2007). Retinoic acid is synthesized in the paraxial mesoderm posterior to the hindbrain and is necessary for the specification of hindbrain segments posterior to the r3/4 border. Domains of Hox expression here are patterned by differential responses to graded levels of retinoic acid along the anteroposterior axis. Retinoic acid can induce the expression of certain Hox genes directly by activating RAR/RXR heterodimers at retinoic acid response elements (RAREs) in regulatory regions, for example, of members of the Hox1 and Hox4 paralogous groups (Marshall et al., 1994; Nolte, Amores, Nagy Kovacs, Postlethwait, & Featherstone, 2003). The expression of other posterior retinoic acid-responsive hox genes, including Hox3 genes, is regulated indirectly by retinoic acid; for example, Hoxa3 expression in r5 and r6 is regulated by MafB, which is itself regulated in part by retinoic acid (via Vhnf1) (Hernandez, Rikhof, Bachmann, & Moens, 2004; Manzanares, Cordes, et al., 1999). More posterior retinoic acid-responsive Hox genes require higher levels of retinoic acid for their induction and are expressed later than more anterior retinoic acid-responsive Hox genes, which are induced at lower levels of retinoic acid (Dupe & Lumsden, 2001; Maves & Kimmel, 2005). However, the spatial patterning of Hox gene expression along the anteroposterior axis by retinoic acid is not purely achieved by differential sensitivities to retinoic acid, as the RAREs of different Hox genes have been found to have comparable sensitivities (Nolte et al., 2003). It has also been shown that Hox genes expressed in more posterior hindbrain domains do not require longer exposures to retinoic acid for their induction (Hernandez et al., 2007; Maves & Kimmel, 2005). The mechanisms by which the graded retinoic acid activity required for hindbrain segmentation is generated and maintained are not completely understood. The concentration of retinoic acid along the anteroposterior axis of the hindbrain is influenced both spatially and temporally by members of the Cyp26 family of cytochrome p450 enzymes—Cyp26a1, b1 and c1— which degrade retinoic acid. In the absence of Cyp26 enzymes, the anteroposterior patterning of the hindbrain is disrupted (Hernandez et al., 2007). Furthermore, exogenous retinoic acid applied over a 20-fold concentration range and at a variety of developmental stages is sufficient to correctly pattern the hindbrain of embryos that are depleted of endogenous retinoic acid, and Cyp26 enzymes are required for this rescue (Dupe & Lumsden, 2001; Hernandez et al., 2007; Maves & Kimmel, 2005). Cyp26a1 is induced by retinoic acid itself and has an important role in the negative feedback of
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retinoic acid signaling (White et al., 2007). This self-enhanced degradation allows retinoic acid-induced patterning to be robust despite variations in retinoic acid levels. Cyp26b1 and cyp26c1 are segmentally and dynamically expressed during hindbrain segmentation and refine retinoic acid responsiveness within the hindbrain through localized retinoic acid degradation (White & Schilling, 2008). It is not clear whether segmentally expressed Cyp26 enzymes generate a step-wise gradient of retinoic acid. In mouse, use of a RARE–lacZ reporter has revealed distinct boundaries of retinoic acid responsiveness, which move during hindbrain segmentation (Rossant, Zirngibl, Cado, Shago, & Goguere, 1991; Sirbu, Gresh, Barra, & Duester, 2005). However, a RARE-eYFP transgenic zebrafish line has revealed a smooth gradient of retinoic acid responsiveness, though only at later stages of hindbrain segmentation (White et al., 2007). More recently, a smooth gradient of retinoic acid in the zebrafish hindbrain has been detected by visualization of unbound intracellular retinoic acid using FRET-based genetically encoded retinoic acid probes (Shimozono, Iimura, Kitaguchi, Higashijima, & Miyawaki, 2013). Fgfs have important roles in hindbrain patterning, in part by inhibiting retinoic acid-mediated cyp26a1 upregulation (White et al., 2007). This enables Fgfs to influence the retinoic acid gradient within the hindbrain, which has been suggested to help couple growth of the hindbrain with a corresponding expansion of the retinoic acid gradient (Schilling, Nie, & Lander, 2012; White et al., 2007). In combination with retinoic acid, Fgfs and Wnts also initiate the expression of certain posterior genes, including hoxb1b (posteriorly from pre-r4) and vhnf1, (in pre-r5–r6) (Kudoh et al., 2002). Later in hindbrain segmentation, fgf3 and fgf8 are upregulated by Hox1 proteins in r4, and are involved in the patterning of surrounding rhombomeres (Hernandez et al., 2004; Marin & Charnay, 2000; Maves, Jackman, & Kimmel, 2002; Walshe, Maroon, McGonnell, Dickson, & Mason, 2002; Waskiewicz, Rikhof, & Moens, 2002; Wiellette & Sive, 2003). For example, Fgf signals from r4 cooperate with vhnf1 and retinoic acid to drive initiation of mafB expression in r5 and r6 (Hernandez et al., 2004), and Fgfs also contribute to induction of krox20 expression (Labalette et al., 2015).
3.2 Establishment of Mutually Exclusive Segmental Identity The expression domains of segmentally expressed transcription factors initially have diffuse borders, in which some cells coexpress factors that confer
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conflicting identities and thus have an intermediate identity. For example in zebrafish, some cells at the r3/r4 and r4/r5 interfaces express both krox20 and hoxb1a, which confer opposing identities; likewise, ectopic krox20-expressing cells within r4 can also contain hoxb1a transcripts (Zhang et al., 2012). This raises the question of how one or the other gene is downregulated in order to establish and maintain a single identity. There have been detailed investigations of gene regulatory interactions in the specification of r3, r4, and r5 in the hindbrain. Following initiation of krox20 expression, an autoregulatory loop amplifies and maintains krox20 expression in r3 and r5 (Bouchoucha et al., 2013; Chomette, Frain, Cereghini, Charnay, & Ghislain, 2006; Giudicelli, Taillebourg, Charnay, & Gilardi-Hebenstreit, 2001). Likewise, Hoxb1 upregulates its own expression in r4 via an autoregulatory loop (P€ opperl et al., 1995). Mutually exclusive expression is established since Krox20 and Hoxb1 repress each other: ectopic overexpression of krox20 in r4 causes downregulation of hoxb1 expression (Giudicelli et al., 2001), while hoxb1 represses krox20 expression indirectly via activation of Nlz factors (Labalette et al., 2015). These interactions create a bistable switch, which enables cells to adopt an exclusive identity and commit to a particular fate following transient inputs to identity specification by morphogens (Fig. 1). This generates sharp spatial transitions in identity despite shallow and noisy morphogen gradients (Bouchoucha et al., 2013). Computational modeling has demonstrated that such a bistable switch regulating krox20 and hoxb1 expression, in combination with noise in the retinoic acid gradient, can contribute to border sharpening in the hindbrain by driving the refinement of gene expression domains via noise-induced switching of cell identity (Schilling et al., 2012; Zhang et al., 2012).
3.3 Plasticity in Cell Identity Intermingling of cells occurs during hindbrain development, driven by cell division and morphogenetic rearrangements such as convergent extension. This is revealed by the dispersal of the progeny of individual cells in the chick hindbrain, which when labeled at early stages can contribute to adjacent rhombomeres, but when labeled after morphological boundary formation are restricted to single segments (Fraser, Keynes, & Lumsden, 1990). This raises the question of how sharp gene expression boundaries are established despite some cell movement between segments at early stages. There is evidence that cells are capable of switching their identity due to plasticity in Hox
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expression status. In mouse embryos, transpositions of small numbers of cells between rhombomeres have found that cells will change Hox expression (Trainor & Krumlauf, 2000). Likewise, in zebrafish, cells can alter their hox expression following transplantation between rhombomeres at early stages, when border sharpening is occurring (Kemp, Cooke, & Moens, 2009; Schilling, Prince, & Ingham, 2001). Transplantations at later stages, when segmental gene expression domains are sharp, revealed a progressive loss of plasticity in cell identity (Schilling et al., 2001); by these stages, there is no longer cell mixing between segments (Calzolari, Terriente, & Pujades, 2014), and thus switching is not required during normal development. Cells transplanted to more posterior regions of the hindbrain have been found to exhibit increased plasticity compared to posterior-to-anterior transplantations (Grapin-Botton, Bonnin, McNaughton, Krumlauf, & Le Douarin, 1995; Itasaki, Sharpe, Morrison, & Krumlauf, 1996; Schilling et al., 2001), which may reflect the importance of posteriorizing signals in regulating Hox expression. Intriguingly, cell plasticity depends on the size of the group of transplanted cells, with larger groups maintaining their original Hox expression at a different position along the anteroposterior axis (Schilling et al., 2001; Trainor & Krumlauf, 2000). A failure to switch identity can account for the groups of krox20-expressing cells present in evennumbered segments following increased intermingling due to dominant negative blocking of Eph receptors (Xu, Alldus, Holder, & Wilkinson, 1995). These observations suggest that community effects have a role in the maintenance of segmental gene expression. The finding that Krox20 nonautonomously induces its own expression in adjacent cells (Giudicelli et al., 2001) suggests a model in which it upregulates a signal which establishes and maintains homogenous identity within groups of cells. It is not clear from these experiments the extent to which cell plasticity contributes to the establishment and maintenance of distinct segmental identity. Lineage analysis in chick reveals cell intermingling at early stages in hindbrain segmentation (Fraser et al., 1990), but the extent to which this occurs has not been quantitated. Use of Cre-mediated recombination in mouse to permanently label cells of the hindbrain that have expressed the r3/r5 marker, Krox20, has found some labeled cells in even-numbered rhombomeres (Voiculescu et al., 2001). This is consistent with cells dynamically expressing Krox20, and suggests that switching of cell identity occurs. However, such labeling could reflect resolution of initial overlapping expression, for example of Krox20 and Hoxb1, rather than intermingling of cells that have established an r3/r5 identity. In zebrafish, cells expressing
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stable fluorescent reporters of r3 and r5 cell identity have not been detected in adjacent segments once sharpening of gene expression domains is complete (Calzolari et al., 2014). This suggests that in zebrafish there is little, if any, intermingling between segments and cell identity switching in normal development. However, it is possible that early intermingling is not detected since insufficient levels of the transgenic fluorescent reporter have accumulated in cells with transient r3/r5 identity.
4. CELL SEGREGATION 4.1 Restriction of Cell Intermingling Across Segment Borders During early hindbrain development, the initially ragged expression limits of molecular determinants of rhombomere identity become straight (Irving, Nieto, DasGupta, Charnay, & Wilkinson, 1996; Oxtoby & Jowett, 1993). This sharpening is presumably driven by cell segregation mechanisms that underlie the restriction of intermingling between hindbrain segments (Calzolari et al., 2014; Fraser et al., 1990; Jimenez-Guri et al., 2010; Mathis, Sieur, Voiculescu, Charnay, & Nicolas, 1999). Transplantation experiments in chick showed that the inhibition of cell mixing involves cell properties that differ most prominently between odd versus even-numbered rhombomeres (Guthrie, Prince, & Lumsden, 1993). There is much evidence that interactions between Eph receptor tyrosine kinases and ephrins have a major role in this restriction of cell mixing. Eph receptors and ephrins comprise a cell contact-dependent signaling system, which upon binding and clustering transduce signals bidirectionally (Klein, 2012). In mouse, several Eph receptors are expressed in r3 and r5, and ephrinB proteins that they bind are expressed in even-numbered segments (Xu, Mellitzer, & Wilkinson, 2000). There is a different situation in zebrafish (Chan et al., 2001; Cooke et al., 2001; Xu et al., 1995), in which Eph–ephrin pairs that have high affinity have complementary expression, but not simply in odd versus even-numbered segments: expression of EphA4 and ephrinB3 occurs in r3/r5 and r2/r4/r6, respectively; expression of EphB4a and ephrinb2a in r5/r6 and r2/r4/r7, respectively. The effects of blocking or knockdown of EphA4a and ephrinB2a reveal that Eph–ephrin signaling is required to inhibit cell intermingling and sharpen segment borders in zebrafish (Cooke, Kemp, & Moens, 2005; Kemp et al., 2009; Xu et al., 1995). The restriction of cell mixing is mediated, at least in part, by Eph–ephrin pairs with complementary expression
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that are interacting at the border (Mellitzer, Xu, & Wilkinson, 1999; Xu, Mellitzer, Robinson, & Wilkinson, 1999). Eph–ephrin signaling could act through one or several mechanisms that have been shown to drive cell segregation in other tissues: differential cell–cell adhesion, cortical tension, and cell repulsion (Batlle & Wilkinson, 2012; Cayuso, Xu, & Wilkinson, 2015; Fagotto, 2014; Fagotto, Winklbauer, & Rohani, 2014). A recent study has shown that there is an Eph–ephrin-dependent accumulation of actomyosin cables at segment borders, which can potentially generate increased cortical tension that restricts cell intermingling (Calzolari et al., 2014). Since the formation of actomyosin cables occurs several hours after border sharpening, cortical tension may play a late role in boundary stabilization. Intriguingly, mosaic knockdown of EphA4 or of ephrinB2a in zebrafish was found to lead to segregation of the knockdown cells to the borders of the segments expressing the corresponding Eph receptor or ephrin (Cooke et al., 2005; Kemp et al., 2009). The mechanism(s) that drives segregation of cells with decreased Eph or ephrin expression within segments is not known, but in principle could involve differential adhesion, tension, or repulsion. This suggests that EphA4 and ephrinB2 activity within segments contributes to cell segregation, presumably acting in parallel with bidirectional signaling at the segment borders to drive sharpening.
4.2 Coupling Between Cell Identity and Cell Segregation The finding that border sharpening requires complementary expression of interacting Eph receptors and ephrins raises the question of how their segmental expression in the hindbrain is regulated. One possibility is that this is mediated by the transcription factors that underlie segmentation and anteroposterior identity in the hindbrain. Indeed, mafB is required to upregulate EphB4a and suppress ephrinB2a expression in r5 and r6, such that this Eph–ephrin pair is in complementary domains (Cooke et al., 2001; Hernandez et al., 2004). Likewise, Hoxa2 is required for EphA7 expression in r3 (Taneja et al., 1996). However, due to the cross-regulation that occurs between mafB, Krox20, and Hox genes, it is not known from these studies which of the transcription factors are the direct regulator of Eph receptor or ephrin expression. Evidence has been obtained by analysis of enhancer elements of the EphA4 gene showing that Krox20 directly regulates EphA4 expression in r3 and r5 (Theil et al., 1998). Since Krox20 also directly regulates expression of specific Hox genes (Tumpel et al., 2009), there is thus a coupling of anteroposterior identity and cell segregation. Direct regulation
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has also been revealed in studies showing that EphA2 expression in r4 is upregulated by Hoxa1 and Hoxb1 (Studer et al., 1998) through binding to Hox/Pbx binding sites in an EphA2 enhancer (Chen & Ruley, 1998). However, the roles of EphA2 in the hindbrain are not known. A recent study has given further insights into the relationships between Hox genes and cell segregation in the hindbrain (Prin, Serpente, Itasaki, & Gould, 2014). The anterior limit of Hoxb4 and Hoxd4 expression is at the r6/r7 boundary, which is absent in double Hoxb4/Hoxd4 mutants, as seen by loss of molecular markers of boundary cells and of the morphological constriction. Furthermore, boundary formation in more anterior regions of the hindbrain is disrupted when Hox4 genes are ectopically expressed. Importantly, it was found that mosaic ectopic expression of Hox4 genes leads to segregation of Hox4+ and Hox4– cells. This segregation is accompanied by formation of an apical constriction in whichever of the two populations is in the minority, and alterations in the apical surface area of cells 3–4 diameters away from the Hox4+/Hox4– border. These observations are consistent with a tension-based mechanism, which may involve Eph–ephrin signaling since ectopic expression of ephrinB2 creates similar constrictions. Furthermore, Hox4 genes underlie the posterior limit of EphA7 expression at the r6/r7 border by downregulating its expression in r7, albeit it is not known whether EphA7 is involved in boundary formation. Taken together, these findings reveal that Hox4 genes underlie formation of the r6/r7 boundary and regulate mechanisms that drive cell segregation. Since cell segregation also occurs following ectopic expression of Hoxa2 and Hoxa3, this role may be a general feature of Hox genes (Prin et al., 2014). Evidence that Hox genes act through regulation of Eph–ephrin expression is currently circumstantial, and it is possible that cell segregation also involves other Hox targets (Prin et al., 2014). The results of overexpression experiments show that krox20 has a dominant role in cell segregation in r3 and r5 through regulation of EphA4, which overrides the control of cell segregation by Hox genes in these segments. Hox genes may thus act in the other rhombomeres to promote border formation.
5. SUMMARY AND FUTURE PERSPECTIVES Major progress has been made in elucidating the signaling and gene regulatory networks that underlie segmental expression of key transcription factors in hindbrain patterning, including Hox genes, krox20, and mafB. These involve positive feedback and mutual repression that establishes
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complementary segmental expression. Recent studies have focussed on cross-repression of hoxb1 and krox20 at the borders of r4, and it is important to elucidate the regulatory relationships that establish discrete cell identities across the other boundaries in the hindbrain. Another important question is whether such networks account for cell identity switching following transplantation between segments, and which may also occur during cell intermingling in normal development. Presumably, the ectopic cells are reading a different level of graded retinoic acid and other signals at the new anteroposterior position. Another possibility is that ectopic cells come under the influence of signaling from their new neighbors, as suggested by the lack of identity switching when groups of cells are transplanted. How does the gene regulatory network respond to such changes in the signaling environment? In particular, how does altered signaling overcome positive feedback loops that maintain krox20 and hoxb1 gene expression? Do such loops and/ or other mechanisms underlie the decreasing ability of cells to switch identity at later stages of hindbrain development? There has been less progress in understanding the coupling between segment identity and cell segregation. It is clear that Hox genes, krox20, and mafB are important regulators which lie upstream of Eph receptor and ephrin expression in the hindbrain. However, for the Eph receptors and ephrins known to underlie cell segregation in the hindbrain, the only direct link shown thus far is the upregulation of EphA4 by krox20. It will, in particular, be interesting to uncover whether Hox genes are direct regulators of the relevant Eph receptors and ephrins, and how the gene regulatory network in the hindbrain establishes the complementary expression of Eph–ephrin pairs that is required for cell segregation.
ACKNOWLEDGMENTS We are grateful to Alex Gould, Fabrice Prin, Jordi Cayuso, and Qiling Xu for their valuable comments on this chapter. Work in the author’s lab is funded by the Francis Crick Institute.
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