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Hox Genes and the Hindbrain
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Hox Genes and the Hindbrain: A Study in Segments Robb Krumlauf1 Stowers Institute for Medical Research, Kansas City, Missouri, USA Department of Anatomy & Cell Biology, Kansas University Medical School, Kansas City, Kansas, USA 1 Corresponding author: e-mail address: [email protected]
Abstract The hindbrain develops through a process of segmentation which is coupled with the ordered expression of Hox genes to generate regional diversity of key neural and craniofacial derivatives during head development. This is a fundamental feature governed by a gene regulatory network conserved to the base of vertebrate evolution.
The vertebrate hindbrain and its relationship to head development are a good model system for understanding fundamental mechanisms of patterning and morphogenesis during development. The vertebrate central nervous system (CNS) has long been postulated to be organized into segments, referred to as neuromeres, based on morphological features. For over a century and a half wide, variation in observations between animals and stages of development led to considerable debate on the validity of this hypothesis. However, a little over 25 years ago, several studies on properties of the hindbrain in chick and mouse embryos reawakened interest in the idea of metameric segments as basic units of organization for some regions of the CNS. The hindbrain is a highly conserved complex coordination center in the CNS. Neurofilament immunostaining and DiI tracing of cranial nerve roots in the chick embryo revealed a segmental organization of the branchiomotor nerves and that nerve roots develop in a two-segment periodicity (Lumsden & Keynes, 1989). Gene expression studies in the mouse revealed that the zinc-finger transcription factor Krox20 is expressed in two alternate segments and that members of the Hox homeobox gene clusters are
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coordinately expressed in nested domains of the hindbrain, in a manner that correlates with specific segments (Hunt et al., 1991; Murphy, Davidson, & Hill, 1989; Wilkinson, Bhatt, Chavrier, Bravo, & Charnay, 1989; Wilkinson, Bhatt, Cook, Boncinelli, & Krumlauf, 1989). This opened the flood gates for a large number of cellular and molecular studies in different vertebrate model systems that have convincingly demonstrated that the formation of regional diversity in the hindbrain is achieved through a process of segmentation, which ultimately gives rise to well-defined regions of the adult brain. This segmental organization is critical for patterning of the cranial neural crest which generates most of the bone and connective tissues of head and facial structures. The Hox family of transcription factors is coupled to this process and provides a molecular framework for specifying the unique identities of hindbrain segments (rhombomeres) and facial structures. This body of research has collectively provided mechanistic insight into how the hindbrain arises through a process of segmentation to generate and pattern a series of distinct neural and craniofacial structures (as reviewed in Alexander, Nolte, & Krumlauf, 2009; Lumsden, 2004; Lumsden & Krumlauf, 1996; Moens & Prince, 2002; Santagati & Rijli, 2003; Trainor & Krumlauf, 2000; Tumpel, Wiedemann, & Krumlauf, 2009). The dynamic process of hindbrain segmentation divides the developing neural plate in this region into eight lineage-restricted cellular compartments, termed rhombomeres (r). Cells freely mix along the anterioposterior (AP) axis of the neural epithelium during early stages (Fraser, Keynes, & Lumsden, 1990), but changes in cell adhesive properties (Guthrie, Prince, & Lumsden, 1993), Eph/ephrin signaling (Cooke & Moens, 2002; Mellitzer, Xu, & Wilkinson, 1999; Sela-Donenfeld & Wilkinson, 2005; Xu, Mellitzer, Robinson, & Wilkinson, 1999), and the formation of distinct boundary cell populations with signaling properties (Cheng et al., 2004; Gonzalez-Quevedo, Lee, Poss, & Wilkinson, 2010) progressively limit intermingling between rhombomeres. During this process, Hox gene expression becomes tightly coupled to specific segments and provides a combinatorial code which modulates differentiation programs and leads to the generation of unique identities in each rhombomere (Hunt et al., 1991; Wilkinson, Bhatt, Cook, et al., 1989). This segmentation has a major impact on patterning craniofacial development through its effects on formation, migration, and patterning of cranial neural crest cells, whose derivatives form most of the bone and connective tissue of the vertebrate head (Birgbauer, Sechrist, Bronner-Fraser, & Fraser, 1995; Nieto, Sechrist, Wilkinson, & Bronner-Fraser, 1995; Santagati & Rijli, 2003;
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Simoes-Costa & Bronner, 2015; Trainor & Krumlauf, 2000; Trainor, Melton, & Manzanares, 2003). Studies employing gain and loss of function analyses in mouse, zebra fish, and other vertebrate models have validated the diverse roles of Hox genes in regulating multiple aspects of segmentation, segmental identity, and neural crest patterning (reviewed in Alexander et al., 2009; Maconochie, Nonchev, Morrison, & Krumlauf, 1996; Moens & Prince, 2002; Santagati & Rijli, 2003). A major focus of my group’s research over many years has been to develop a deep level of understanding of the cis-regulatory components of Hox clusters, their cognate upstream factors, and the signaling inputs which govern the segment-restricted expression and function of Hox genes in hindbrain development. Because the segmental processes of head development are highly conserved among jawed vertebrates, comparative studies between different species have greatly facilitated the characterization of gene regulatory cascades that control early hindbrain development. Investigation of the mechanistic basis for regulation of expression of the nine Hox genes implicated in hindbrain segmentation, using mouse, chicken, and zebra fish experimental models, has enabled the identification and functional characterization of the majority of cis-regulatory modules which direct restricted expression of these Hox genes in individual rhombomeres (reviewed in Alexander et al., 2009; Parrish, Nolte, & Krumlauf, 2009; Tu¨mpel, Maconochie, Wiedemann, & Krumlauf, 2002; Tumpel, Wiedemann, & Krumlauf, 2009). The organization and properties of this cis-circuitry have helped to build a picture of the Hox gene regulatory network (GRN) for the hindbrain and several general principles emerged from these analyses (Tu¨mpel et al., 2009). The overall pattern of segmental expression for any given gene is the result of the combined activity of multiple independent cis-modules which control specific subsets of the expression domain. Each module is subject to its own distinct regulatory inputs and timing, resulting in unique regulatory circuits in each segment that confer the differential regulation of Hox genes. Retinoid and Fgf signaling are involved in transiently inducing early nested domains of Hox expression in the CNS (Bel-Vialar, Itasaki, & Krumlauf, 2002; Deschamps & van Nes, 2005; Hernandez, Rikhof, Bachmann, & Moens, 2004), which later become segmentally restricted through the combined action of transcription factors (e.g., Kreisler, Krox20, vhnf1, and Hox) in multiple modules. Direct auto- and crossregulatory interactions between the Hox genes themselves play a significant role in maintaining the rhombomere-restricted domains of expression
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(Gould, Morrison, Sproat, White, & Krumlauf, 1997; Manzanares et al., 2001; Tu¨mpel et al., 2007). This modular organization of regulatory elements helps to explain variations in the levels of expression within specific rhombomeres and the complex temporal dynamics associated with establishing Hox expression. It also provides insight into how Hox genes integrate cues from the key signaling pathways involved in control of axial elongation and patterning. The dynamic process through which Hoxb1 expression becomes segmentally restricted to r4 in vertebrates (Frohman, Boyle, & Martin, 1990; Murphy et al., 1989; Sundin & Eichele, 1990; Wilkinson, Bhatt, Cook, et al., 1989) is illustrated in Fig. 1A and serves as a good example to illustrate some of the general regulatory principles of the Hox GRN. Figure 1B represents a schematic of the Hoxb1 locus marking some of the enhancers, binding sites, and upstream factors involved in generating the r4-restricted domain of expression. These coordinate the temporal series of steps leading to the r4-restricted domain, as illustrated at the bottom of Fig. 1B. The process is triggered, by the newly forming somites adjacent to the caudal hindbrain. They express the enzyme Raldh2, which synthesizes retinoids that spread into the hindbrain to activate transcription (Begemann, Schilling, Rauch, Geisler, & Ingham, 2001; Molotkova, Molotkov, Sirbu, & Duester, 2005; Niederreither et al., 2003). A neural enhancer 30 of Hoxb1 contains a retinoic acid response element (RARE) (Marshall et al., 1994) which is directly stimulated by the somite-derived retinoids to activate expression of Hoxb1 which extends anteriorly toward the midbrain (Mb). The Hoxa1 gene also contains an analogous 30 RARE and is activated in the same manner as Hoxb1 but slightly earlier (Dupe´ et al., 1997). During this stage, a signaling center in the Mb involving the Fgf pathway induces the expression of Cyp26a, which encodes an enzyme that degrades retinoids (Duester, 2008; Hernandez, Putzke, Myers, Margaretha, & Moens, 2007; Sirbu, Gresh, Barra, & Duester, 2005). These opposing influences limit the initial anterior expansion of Hoxb1 and Hoxa1 expression to the r2/r3 boundary (Sirbu et al., 2005). In the next phase, Krox20 is induced specifically in future r3 and r5 (Giudicelli, Taillebourg, Charnay, & GilardiHebenstreit, 2001; Schneider-Maunoury et al., 1993; Wilkinson, Bhatt, Chavrier, et al., 1989) and binds to an r3/r5 repressor element upstream of Hoxb1 (Fig. 1B). This repressor also contains an RARE, and together with Krox20, it inhibits expression specifically in r3 and r5 (Studer, Popperl, Marshall, Kuroiwa, & Krumlauf, 1994). Concurrently, levels of Hoxb1 expression in r4 are also stimulated by Hoxa1 via a Hox response
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Figure 1 Temporal dynamics in the segmentally restricted expression and regulation of Hoxb1 in r4. (A) Sketches of in situ expression profiles of Hoxb1 in mouse embryos showing changes over time. (B) At the top is a diagram of the Hoxb1 gene with enhancer regions, binding sites, and regulatory inputs from transcription factors. At the bottom is a drawing of how the initial broad domain of Hoxb1 becomes progressively restricted to r4 through the input of repressive and activation inputs.
element upstream of the gene (P€ opperl et al., 1995; Studer et al., 1998b; Tvrdik & Capecchi, 2006). In the next step, Hoxb1 autoregulates its own expression in conjunction with the cofactors Pbx and Meis through its highly conserved Hox response element (Gavalas et al., 1998; Moens & Selleri, 2006; P€ opperl et al., 1995; Studer et al., 1998b; Vitobello et al., 2011; Waskiewicz, Rikhof,
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Hernandez, & Moens, 2001). Hoxb1 and Hoxa1 repress the expression of Krox20 in r4 (Helmbacher et al., 1998). In the r6 region, expression of Hoxb3 and Hoxa3 is activated and they bind to the same Hox response element and repress Hoxb1 in r6 (Gaufo, Thomas, & Capecchi, 2003; Wong et al., 2011). In the final step, as somites differentiate they cease production of Raldh2; hence, there is no longer broad stimulation of Hoxb1 by retinoids in the hindbrain region. The Hoxa1 gene does not possess an autoregulatory element and its expression rapidly declines (Dupe´ et al., 1997; Studer et al., 1998b; Tvrdik & Capecchi, 2006). Through the repressive and auto- and cross-regulatory feedback loops, Hoxb1 maintains its own expression in r4, and also begins to receive positive inputs from Hoxb2 and Hoxa2, which it activates in r4 (Davenne et al., 1999; Gavalas, Ruhrberg, Livet, Henderson, & Krumlauf, 2003; Maconochie et al., 1997; Pattyn et al., 2003; Tu¨mpel et al., 2007). This illustrates how rhombomere-restricted expression is achieved through the integration of activation in a broad domains followed by successive sculpting by repression and activation. These studies have underscored a key role for retinoic acid (RA) signaling in transient induction of the early ordered and nested domains of Hox expression in the CNS. RA signaling is implicated in early positioning of the anterior boundaries of 30 HoxB genes (paralog groups 1–5) (Bel-Vialar et al., 2002; Marshall et al., 1994; Sirbu et al., 2005; Studer et al., 1998a) and later in the rostral expansion of the expression domains of 50 genes in the cluster (Ahn, Mullan, & Krumlauf, 2014). Even though no RAREs have been identified in the 50 region of the HoxB cluster, several RAREs are present around Hoxb4 and Hoxb5 (Gould, Itasaki, & Krumlauf, 1998; Oosterveen et al., 2003; Sharpe, Nonchev, Gould, Whiting, & Krumlauf, 1998). Recent experiments have demonstrated that RAREs embedded within and adjacent to the HoxB cluster act on several genes through enhancer sharing and long-range regulation (Ahn et al., 2014; Nolte, Jinks, Wang, Martinez Pastor, & Krumlauf, 2013). These studies identify the positions of highly conserved cis-elements that mediate the responses of Hox clusters to retinoids and suggest that they may be a contributing factor in maintaining the clustered organization and coupling of Hox genes to axial patterning in the CNS. The knowledge gained from this GRN framework (Alexander et al., 2009; Tu¨mpel et al., 2009) enables exploration of the degree to which it is conserved during evolution of vertebrates (Nolte, Ahn, & Krumlauf, 2012). By integrating the information on Hox enhancers, regulatory networks, and inputs from signaling pathways (RA, Fgfs, and Wnts) in
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vertebrates, it is possible to investigate their conservation or divergence during chordate evolution. For example, amphioxus, a cephalochordate, contains a single Hox cluster, and a screen for regulatory elements in the amphioxus Hox genes that function in transgenic mice or chicken embryos revealed no cis-elements capable of mediating segmental expression in the vertebrate hindbrain (Manzanares et al., 2000). However, several neural enhancers containing RAREs were found in the amphiHox cluster and these displayed a dependence upon retinoid signaling (Manzanares et al., 2000; Wada, Escriva, Zhang, & Laudet, 2006). It is intriguing that they are located in the same relative positions that RARE-dependent neural enhancers are found in several vertebrate Hox clusters (Ahn et al., 2014). This suggests that these RARE-based control modules might represent conserved regulatory features of an ancient cluster present before the emergence of vertebrates. In accord with this idea, studies by Chris Lowe and colleagues on the acorn worm Saccoglossus kowalevskii, a hemichordate, have shown that the A/P domain map of transcription factors (including Hox) and signaling ligands can surprisingly be used to align the bodies of hemichordates and chordates (Gerhart, Lowe, & Kirschner, 2005; Lowe et al., 2003; Pani et al., 2012). These findings imply that the axial signaling centers must have evolved long ago in a common chordate ancestor. Hence, the conserved positions of RAREs in amphiHox and vertebrate clusters might reflect an ancient cis-signature of retinoid signaling involved in generating ordered domains of Hox neural expression. It will be interesting to determine whether such RAREs exist in the hemichordate Hox cluster (Freeman et al., 2012). While analyses in amphioxus and Ciona have found that none of the cis-elements responsible for the segmental expression of Hox genes in the vertebrate hindbrain appear to be present in these chordates (Manzanares et al., 2000; Natale et al., 2011), comparative studies in lamprey, zebra fish, and mice have begun to provide insight on the question of when ordered domains of Hox expression became coupled to hindbrain segmentation in the origin of chordates. As detailed above, all jawed vertebrates pass through an embryonic stage in which the hindbrain is divided into rhombomeric compartments and the hindbrain “Hox code” and GRN shows remarkable conservation from zebra fish to mammals. Due to its position at the base of the vertebrate family tree, the sea lamprey, a jawless fish (cyclostome), can give unique insights into the ancestry of vertebrates (Shimeld & Donoghue, 2012). Lampreys have neural crest cells with a conserved neural crest GRN (Sauka-Spengler, Meulemans, Jones, & Bronner-Fraser, 2007).
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However, it has been unclear whether lamprey embryos develop a segmented hindbrain associated with segmental Hox expression (Cohn, 2002; Murakami et al., 2004; Murakami, Uchida, Rijli, & Kuratani, 2005; Pascual-Anaya et al., 2012; Takio et al., 2004, 2007). However, a recent study showing that enhancers from the Hox clusters of jawed vertebrates mediated rhombomere-restricted domains of reporter expression in the lamprey hindbrain indicates that the lamprey must have the upstream regulatory factors of the hindbrain GRN capable of directing segmental expression (Parker, Bronner, & Krumlauf, 2014). This work also uncovered ordered segmental expression of endogenous lamprey Hox genes and identified an enhancer that mediates the rhombomeric expression of a group 2 Hox gene in r2 and r4 (Parker et al., 2014). This enhancer is located in the same relative position as the mouse Hoxa2 r2/r4 segmental enhancer (Tu¨mpel et al., 2007; Tu¨mpel, Cambronero, Sims, Krumlauf, & Wiedemann, 2008; Tu¨mpel, Cambronero, Wiedemann, & Krumlauf, 2006). This insight from regulatory analysis suggests that key aspects of the hindbrain GRN, including Hox genes, were already present in the common ancestor of lamprey and jawed vertebrates. Therefore, during the evolution of the vertebrate head, segmental Hox expression in the hindbrain appears to be a fundamental innovation wired into the GRNs of the developmental program at the base of vertebrate tree. Consistent with this idea, analysis of conserved noncoding regions in the lamprey genome has shown that many of these are associated with cis-elements that direct Hox-dependent gene regulatory activities that may pattern head development (Parker, Piccinelli, Sauka-Spengler, Bronner, & Elgar, 2011). A major challenge for the future is that though the function of Hox proteins is critical for specification of regional properties of hindbrain segments and other tissues, very little is known about the downstream target loci where they are recruited as protein complexes to exert their functional activities. The 39 mammalian Hox proteins have very similar structures and in vitro binding specificities (Slattery et al., 2011), so their individual specificity is likely to be modulated by subtle differences in cofactors, interacting proteins, target sites, or other unknown processes (Mann, Lelli, & Joshi, 2009; Merabet, Hudry, Saadaoui, & Graba, 2009; Saadaoui et al., 2011). It seems likely that differences in the downstream targets of Hox genes between species may be a central aspect of how they regulate common processes in a distinct manner. It will be essential to identify in vivo relevant Hox response elements to gain insight into the sites, binding partners, and mechanisms of transcriptional regulation essential for their function in hindbrain
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and other tissues and to link these loci to the pathways and cellular processes they modulate. While we understand a great deal about the ground plan of segmentation and how distinct segmental identities are established, there is a major gap in our knowledge on how this modulates the programs that control the formation and identity of distinct neuronal populations within the segments. Most of the focus on expression of Hox genes in the CNS has been on their AP domains and boundaries. However, each of the four mouse Hox clusters displays distinct dorsoventral (DV) patterns of expression that correlate with the birth of different classes of neurons (Graham, Maden, & Krumlauf, 1991). This is illustrated in Fig. 2A. All of the HoxB genes are initially expressed uniformly throughout the DV axis of the neural tube, but over time they become progressively restricted with the highest levels in dorsal domains. Figure 2C illustrates how the r4-restricted domain of Hoxb1 is further limited in later stages along the DV axis to specific populations of differentiating neurons in r4. These stripes of neuronal expression are significant, as in Hox mutants these cell populations fail to develop properly (Gavalas et al., 2003; Pattyn et al., 2003). Different patterns of DV expression are observed for the other Hox clusters (Fig. 2B). Elegant work from the groups of Jessell and Dasen have detailed the roles of Hox genes in regulating diversity and identities of motor neuron pools in the spinal cord (Dasen, De Camilli, Wang, Tucker, & Jessell, 2008; Dasen & Jessell, 2009; Dasen, Liu, & Jessell, 2003; Dasen, Tice, Brenner-Morton, & Jessell, 2005; Jung et al., 2010; Philippidou, Walsh, Aubin, Jeannotte, & Dasen, 2012). This clearly demonstrates that the DV restrictions to expression in later stages are functionally meaningful in the spinal cord; however, little is known about whether and how these dynamic DV patterns of expression translate to regulation of neuronal differentiation programs in the hindbrain. Dissecting these cellular programs during the establishment and differentiation of neuronal populations will be critical for understanding how the hindbrain generates the components and circuits associated with its role as a complex coordination center of the CNS. Finally, another important direction for the future is to understand how hindbrain segmentation and Hox genes play roles in later stages of elaboration of the CNS to generate adult structures and circuits. Insight is arising from conditional mouse mutants that reveals roles in somatosensory circuits, respiratory circuits, and generation of neural progenitor pools (Briscoe & Wilkinson, 2004; Chatonnet et al., 2003; Davenne et al., 1999; del Toro et al., 2001; Di Bonito, Glover, & Studer, 2013; Di Bonito, Narita,
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Figure 2 Dorsoventral changes in Hox expression in the neural tube. (A) Cross-sectional diagrams of the developing neural tube showing how HoxB genes become enriched in dorsal regions. (B) Comparison of the different dorsoventral distributions of genes from separate Hox clusters. (C) Sketch showing that the r4 expression of Hoxb1 becomes restricted to small longitudinal stripes of neurons in r4.
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et al., 2013; Oury et al., 2006; Pasqualetti, Diaz, Renaud, Rijli, & Glover, 2007; Pattyn et al., 2003). With the development of new tools and technologies, there is hope that efforts will be successful in understanding the full implications of how hindbrain segmentation eventually leads to the full elaboration of this region of the CNS and assess how variations in the process impact development, disease, and evolution.
ACKNOWLEDGMENTS I would like to thank Mark Miller for the illustrations and the many friends, colleagues, and lab members who over the years have participated in the research and exchange of ideas that have made this such a wonderful area to study.
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Hox Genes and the Hindbrain: A Study in Segments Robb Krumlauf1 Stowers Institute for Medical Research, Kansas City, Missouri, USA Department of Anatomy & Cell Biology, Kansas University Medical School, Kansas City, Kansas, USA 1 Corresponding author: e-mail address: [email protected]
Abstract The hindbrain develops through a process of segmentation which is coupled with the ordered expression of Hox genes to generate regional diversity of key neural and craniofacial derivatives during head development. This is a fundamental feature governed by a gene regulatory network conserved to the base of vertebrate evolution.
The vertebrate hindbrain and its relationship to head development are a good model system for understanding fundamental mechanisms of patterning and morphogenesis during development. The vertebrate central nervous system (CNS) has long been postulated to be organized into segments, referred to as neuromeres, based on morphological features. For over a century and a half wide, variation in observations between animals and stages of development led to considerable debate on the validity of this hypothesis. However, a little over 25 years ago, several studies on properties of the hindbrain in chick and mouse embryos reawakened interest in the idea of metameric segments as basic units of organization for some regions of the CNS. The hindbrain is a highly conserved complex coordination center in the CNS. Neurofilament immunostaining and DiI tracing of cranial nerve roots in the chick embryo revealed a segmental organization of the branchiomotor nerves and that nerve roots develop in a two-segment periodicity (Lumsden & Keynes, 1989). Gene expression studies in the mouse revealed that the zinc-finger transcription factor Krox20 is expressed in two alternate segments and that members of the Hox homeobox gene clusters are
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coordinately expressed in nested domains of the hindbrain, in a manner that correlates with specific segments (Hunt et al., 1991; Murphy, Davidson, & Hill, 1989; Wilkinson, Bhatt, Chavrier, Bravo, & Charnay, 1989; Wilkinson, Bhatt, Cook, Boncinelli, & Krumlauf, 1989). This opened the flood gates for a large number of cellular and molecular studies in different vertebrate model systems that have convincingly demonstrated that the formation of regional diversity in the hindbrain is achieved through a process of segmentation, which ultimately gives rise to well-defined regions of the adult brain. This segmental organization is critical for patterning of the cranial neural crest which generates most of the bone and connective tissues of head and facial structures. The Hox family of transcription factors is coupled to this process and provides a molecular framework for specifying the unique identities of hindbrain segments (rhombomeres) and facial structures. This body of research has collectively provided mechanistic insight into how the hindbrain arises through a process of segmentation to generate and pattern a series of distinct neural and craniofacial structures (as reviewed in Alexander, Nolte, & Krumlauf, 2009; Lumsden, 2004; Lumsden & Krumlauf, 1996; Moens & Prince, 2002; Santagati & Rijli, 2003; Trainor & Krumlauf, 2000; Tumpel, Wiedemann, & Krumlauf, 2009). The dynamic process of hindbrain segmentation divides the developing neural plate in this region into eight lineage-restricted cellular compartments, termed rhombomeres (r). Cells freely mix along the anterioposterior (AP) axis of the neural epithelium during early stages (Fraser, Keynes, & Lumsden, 1990), but changes in cell adhesive properties (Guthrie, Prince, & Lumsden, 1993), Eph/ephrin signaling (Cooke & Moens, 2002; Mellitzer, Xu, & Wilkinson, 1999; Sela-Donenfeld & Wilkinson, 2005; Xu, Mellitzer, Robinson, & Wilkinson, 1999), and the formation of distinct boundary cell populations with signaling properties (Cheng et al., 2004; Gonzalez-Quevedo, Lee, Poss, & Wilkinson, 2010) progressively limit intermingling between rhombomeres. During this process, Hox gene expression becomes tightly coupled to specific segments and provides a combinatorial code which modulates differentiation programs and leads to the generation of unique identities in each rhombomere (Hunt et al., 1991; Wilkinson, Bhatt, Cook, et al., 1989). This segmentation has a major impact on patterning craniofacial development through its effects on formation, migration, and patterning of cranial neural crest cells, whose derivatives form most of the bone and connective tissue of the vertebrate head (Birgbauer, Sechrist, Bronner-Fraser, & Fraser, 1995; Nieto, Sechrist, Wilkinson, & Bronner-Fraser, 1995; Santagati & Rijli, 2003;
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Simoes-Costa & Bronner, 2015; Trainor & Krumlauf, 2000; Trainor, Melton, & Manzanares, 2003). Studies employing gain and loss of function analyses in mouse, zebra fish, and other vertebrate models have validated the diverse roles of Hox genes in regulating multiple aspects of segmentation, segmental identity, and neural crest patterning (reviewed in Alexander et al., 2009; Maconochie, Nonchev, Morrison, & Krumlauf, 1996; Moens & Prince, 2002; Santagati & Rijli, 2003). A major focus of my group’s research over many years has been to develop a deep level of understanding of the cis-regulatory components of Hox clusters, their cognate upstream factors, and the signaling inputs which govern the segment-restricted expression and function of Hox genes in hindbrain development. Because the segmental processes of head development are highly conserved among jawed vertebrates, comparative studies between different species have greatly facilitated the characterization of gene regulatory cascades that control early hindbrain development. Investigation of the mechanistic basis for regulation of expression of the nine Hox genes implicated in hindbrain segmentation, using mouse, chicken, and zebra fish experimental models, has enabled the identification and functional characterization of the majority of cis-regulatory modules which direct restricted expression of these Hox genes in individual rhombomeres (reviewed in Alexander et al., 2009; Parrish, Nolte, & Krumlauf, 2009; Tu¨mpel, Maconochie, Wiedemann, & Krumlauf, 2002; Tumpel, Wiedemann, & Krumlauf, 2009). The organization and properties of this cis-circuitry have helped to build a picture of the Hox gene regulatory network (GRN) for the hindbrain and several general principles emerged from these analyses (Tu¨mpel et al., 2009). The overall pattern of segmental expression for any given gene is the result of the combined activity of multiple independent cis-modules which control specific subsets of the expression domain. Each module is subject to its own distinct regulatory inputs and timing, resulting in unique regulatory circuits in each segment that confer the differential regulation of Hox genes. Retinoid and Fgf signaling are involved in transiently inducing early nested domains of Hox expression in the CNS (Bel-Vialar, Itasaki, & Krumlauf, 2002; Deschamps & van Nes, 2005; Hernandez, Rikhof, Bachmann, & Moens, 2004), which later become segmentally restricted through the combined action of transcription factors (e.g., Kreisler, Krox20, vhnf1, and Hox) in multiple modules. Direct auto- and crossregulatory interactions between the Hox genes themselves play a significant role in maintaining the rhombomere-restricted domains of expression
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(Gould, Morrison, Sproat, White, & Krumlauf, 1997; Manzanares et al., 2001; Tu¨mpel et al., 2007). This modular organization of regulatory elements helps to explain variations in the levels of expression within specific rhombomeres and the complex temporal dynamics associated with establishing Hox expression. It also provides insight into how Hox genes integrate cues from the key signaling pathways involved in control of axial elongation and patterning. The dynamic process through which Hoxb1 expression becomes segmentally restricted to r4 in vertebrates (Frohman, Boyle, & Martin, 1990; Murphy et al., 1989; Sundin & Eichele, 1990; Wilkinson, Bhatt, Cook, et al., 1989) is illustrated in Fig. 1A and serves as a good example to illustrate some of the general regulatory principles of the Hox GRN. Figure 1B represents a schematic of the Hoxb1 locus marking some of the enhancers, binding sites, and upstream factors involved in generating the r4-restricted domain of expression. These coordinate the temporal series of steps leading to the r4-restricted domain, as illustrated at the bottom of Fig. 1B. The process is triggered, by the newly forming somites adjacent to the caudal hindbrain. They express the enzyme Raldh2, which synthesizes retinoids that spread into the hindbrain to activate transcription (Begemann, Schilling, Rauch, Geisler, & Ingham, 2001; Molotkova, Molotkov, Sirbu, & Duester, 2005; Niederreither et al., 2003). A neural enhancer 30 of Hoxb1 contains a retinoic acid response element (RARE) (Marshall et al., 1994) which is directly stimulated by the somite-derived retinoids to activate expression of Hoxb1 which extends anteriorly toward the midbrain (Mb). The Hoxa1 gene also contains an analogous 30 RARE and is activated in the same manner as Hoxb1 but slightly earlier (Dupe´ et al., 1997). During this stage, a signaling center in the Mb involving the Fgf pathway induces the expression of Cyp26a, which encodes an enzyme that degrades retinoids (Duester, 2008; Hernandez, Putzke, Myers, Margaretha, & Moens, 2007; Sirbu, Gresh, Barra, & Duester, 2005). These opposing influences limit the initial anterior expansion of Hoxb1 and Hoxa1 expression to the r2/r3 boundary (Sirbu et al., 2005). In the next phase, Krox20 is induced specifically in future r3 and r5 (Giudicelli, Taillebourg, Charnay, & GilardiHebenstreit, 2001; Schneider-Maunoury et al., 1993; Wilkinson, Bhatt, Chavrier, et al., 1989) and binds to an r3/r5 repressor element upstream of Hoxb1 (Fig. 1B). This repressor also contains an RARE, and together with Krox20, it inhibits expression specifically in r3 and r5 (Studer, Popperl, Marshall, Kuroiwa, & Krumlauf, 1994). Concurrently, levels of Hoxb1 expression in r4 are also stimulated by Hoxa1 via a Hox response
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Figure 1 Temporal dynamics in the segmentally restricted expression and regulation of Hoxb1 in r4. (A) Sketches of in situ expression profiles of Hoxb1 in mouse embryos showing changes over time. (B) At the top is a diagram of the Hoxb1 gene with enhancer regions, binding sites, and regulatory inputs from transcription factors. At the bottom is a drawing of how the initial broad domain of Hoxb1 becomes progressively restricted to r4 through the input of repressive and activation inputs.
element upstream of the gene (P€ opperl et al., 1995; Studer et al., 1998b; Tvrdik & Capecchi, 2006). In the next step, Hoxb1 autoregulates its own expression in conjunction with the cofactors Pbx and Meis through its highly conserved Hox response element (Gavalas et al., 1998; Moens & Selleri, 2006; P€ opperl et al., 1995; Studer et al., 1998b; Vitobello et al., 2011; Waskiewicz, Rikhof,
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Hernandez, & Moens, 2001). Hoxb1 and Hoxa1 repress the expression of Krox20 in r4 (Helmbacher et al., 1998). In the r6 region, expression of Hoxb3 and Hoxa3 is activated and they bind to the same Hox response element and repress Hoxb1 in r6 (Gaufo, Thomas, & Capecchi, 2003; Wong et al., 2011). In the final step, as somites differentiate they cease production of Raldh2; hence, there is no longer broad stimulation of Hoxb1 by retinoids in the hindbrain region. The Hoxa1 gene does not possess an autoregulatory element and its expression rapidly declines (Dupe´ et al., 1997; Studer et al., 1998b; Tvrdik & Capecchi, 2006). Through the repressive and auto- and cross-regulatory feedback loops, Hoxb1 maintains its own expression in r4, and also begins to receive positive inputs from Hoxb2 and Hoxa2, which it activates in r4 (Davenne et al., 1999; Gavalas, Ruhrberg, Livet, Henderson, & Krumlauf, 2003; Maconochie et al., 1997; Pattyn et al., 2003; Tu¨mpel et al., 2007). This illustrates how rhombomere-restricted expression is achieved through the integration of activation in a broad domains followed by successive sculpting by repression and activation. These studies have underscored a key role for retinoic acid (RA) signaling in transient induction of the early ordered and nested domains of Hox expression in the CNS. RA signaling is implicated in early positioning of the anterior boundaries of 30 HoxB genes (paralog groups 1–5) (Bel-Vialar et al., 2002; Marshall et al., 1994; Sirbu et al., 2005; Studer et al., 1998a) and later in the rostral expansion of the expression domains of 50 genes in the cluster (Ahn, Mullan, & Krumlauf, 2014). Even though no RAREs have been identified in the 50 region of the HoxB cluster, several RAREs are present around Hoxb4 and Hoxb5 (Gould, Itasaki, & Krumlauf, 1998; Oosterveen et al., 2003; Sharpe, Nonchev, Gould, Whiting, & Krumlauf, 1998). Recent experiments have demonstrated that RAREs embedded within and adjacent to the HoxB cluster act on several genes through enhancer sharing and long-range regulation (Ahn et al., 2014; Nolte, Jinks, Wang, Martinez Pastor, & Krumlauf, 2013). These studies identify the positions of highly conserved cis-elements that mediate the responses of Hox clusters to retinoids and suggest that they may be a contributing factor in maintaining the clustered organization and coupling of Hox genes to axial patterning in the CNS. The knowledge gained from this GRN framework (Alexander et al., 2009; Tu¨mpel et al., 2009) enables exploration of the degree to which it is conserved during evolution of vertebrates (Nolte, Ahn, & Krumlauf, 2012). By integrating the information on Hox enhancers, regulatory networks, and inputs from signaling pathways (RA, Fgfs, and Wnts) in
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vertebrates, it is possible to investigate their conservation or divergence during chordate evolution. For example, amphioxus, a cephalochordate, contains a single Hox cluster, and a screen for regulatory elements in the amphioxus Hox genes that function in transgenic mice or chicken embryos revealed no cis-elements capable of mediating segmental expression in the vertebrate hindbrain (Manzanares et al., 2000). However, several neural enhancers containing RAREs were found in the amphiHox cluster and these displayed a dependence upon retinoid signaling (Manzanares et al., 2000; Wada, Escriva, Zhang, & Laudet, 2006). It is intriguing that they are located in the same relative positions that RARE-dependent neural enhancers are found in several vertebrate Hox clusters (Ahn et al., 2014). This suggests that these RARE-based control modules might represent conserved regulatory features of an ancient cluster present before the emergence of vertebrates. In accord with this idea, studies by Chris Lowe and colleagues on the acorn worm Saccoglossus kowalevskii, a hemichordate, have shown that the A/P domain map of transcription factors (including Hox) and signaling ligands can surprisingly be used to align the bodies of hemichordates and chordates (Gerhart, Lowe, & Kirschner, 2005; Lowe et al., 2003; Pani et al., 2012). These findings imply that the axial signaling centers must have evolved long ago in a common chordate ancestor. Hence, the conserved positions of RAREs in amphiHox and vertebrate clusters might reflect an ancient cis-signature of retinoid signaling involved in generating ordered domains of Hox neural expression. It will be interesting to determine whether such RAREs exist in the hemichordate Hox cluster (Freeman et al., 2012). While analyses in amphioxus and Ciona have found that none of the cis-elements responsible for the segmental expression of Hox genes in the vertebrate hindbrain appear to be present in these chordates (Manzanares et al., 2000; Natale et al., 2011), comparative studies in lamprey, zebra fish, and mice have begun to provide insight on the question of when ordered domains of Hox expression became coupled to hindbrain segmentation in the origin of chordates. As detailed above, all jawed vertebrates pass through an embryonic stage in which the hindbrain is divided into rhombomeric compartments and the hindbrain “Hox code” and GRN shows remarkable conservation from zebra fish to mammals. Due to its position at the base of the vertebrate family tree, the sea lamprey, a jawless fish (cyclostome), can give unique insights into the ancestry of vertebrates (Shimeld & Donoghue, 2012). Lampreys have neural crest cells with a conserved neural crest GRN (Sauka-Spengler, Meulemans, Jones, & Bronner-Fraser, 2007).
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However, it has been unclear whether lamprey embryos develop a segmented hindbrain associated with segmental Hox expression (Cohn, 2002; Murakami et al., 2004; Murakami, Uchida, Rijli, & Kuratani, 2005; Pascual-Anaya et al., 2012; Takio et al., 2004, 2007). However, a recent study showing that enhancers from the Hox clusters of jawed vertebrates mediated rhombomere-restricted domains of reporter expression in the lamprey hindbrain indicates that the lamprey must have the upstream regulatory factors of the hindbrain GRN capable of directing segmental expression (Parker, Bronner, & Krumlauf, 2014). This work also uncovered ordered segmental expression of endogenous lamprey Hox genes and identified an enhancer that mediates the rhombomeric expression of a group 2 Hox gene in r2 and r4 (Parker et al., 2014). This enhancer is located in the same relative position as the mouse Hoxa2 r2/r4 segmental enhancer (Tu¨mpel et al., 2007; Tu¨mpel, Cambronero, Sims, Krumlauf, & Wiedemann, 2008; Tu¨mpel, Cambronero, Wiedemann, & Krumlauf, 2006). This insight from regulatory analysis suggests that key aspects of the hindbrain GRN, including Hox genes, were already present in the common ancestor of lamprey and jawed vertebrates. Therefore, during the evolution of the vertebrate head, segmental Hox expression in the hindbrain appears to be a fundamental innovation wired into the GRNs of the developmental program at the base of vertebrate tree. Consistent with this idea, analysis of conserved noncoding regions in the lamprey genome has shown that many of these are associated with cis-elements that direct Hox-dependent gene regulatory activities that may pattern head development (Parker, Piccinelli, Sauka-Spengler, Bronner, & Elgar, 2011). A major challenge for the future is that though the function of Hox proteins is critical for specification of regional properties of hindbrain segments and other tissues, very little is known about the downstream target loci where they are recruited as protein complexes to exert their functional activities. The 39 mammalian Hox proteins have very similar structures and in vitro binding specificities (Slattery et al., 2011), so their individual specificity is likely to be modulated by subtle differences in cofactors, interacting proteins, target sites, or other unknown processes (Mann, Lelli, & Joshi, 2009; Merabet, Hudry, Saadaoui, & Graba, 2009; Saadaoui et al., 2011). It seems likely that differences in the downstream targets of Hox genes between species may be a central aspect of how they regulate common processes in a distinct manner. It will be essential to identify in vivo relevant Hox response elements to gain insight into the sites, binding partners, and mechanisms of transcriptional regulation essential for their function in hindbrain
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and other tissues and to link these loci to the pathways and cellular processes they modulate. While we understand a great deal about the ground plan of segmentation and how distinct segmental identities are established, there is a major gap in our knowledge on how this modulates the programs that control the formation and identity of distinct neuronal populations within the segments. Most of the focus on expression of Hox genes in the CNS has been on their AP domains and boundaries. However, each of the four mouse Hox clusters displays distinct dorsoventral (DV) patterns of expression that correlate with the birth of different classes of neurons (Graham, Maden, & Krumlauf, 1991). This is illustrated in Fig. 2A. All of the HoxB genes are initially expressed uniformly throughout the DV axis of the neural tube, but over time they become progressively restricted with the highest levels in dorsal domains. Figure 2C illustrates how the r4-restricted domain of Hoxb1 is further limited in later stages along the DV axis to specific populations of differentiating neurons in r4. These stripes of neuronal expression are significant, as in Hox mutants these cell populations fail to develop properly (Gavalas et al., 2003; Pattyn et al., 2003). Different patterns of DV expression are observed for the other Hox clusters (Fig. 2B). Elegant work from the groups of Jessell and Dasen have detailed the roles of Hox genes in regulating diversity and identities of motor neuron pools in the spinal cord (Dasen, De Camilli, Wang, Tucker, & Jessell, 2008; Dasen & Jessell, 2009; Dasen, Liu, & Jessell, 2003; Dasen, Tice, Brenner-Morton, & Jessell, 2005; Jung et al., 2010; Philippidou, Walsh, Aubin, Jeannotte, & Dasen, 2012). This clearly demonstrates that the DV restrictions to expression in later stages are functionally meaningful in the spinal cord; however, little is known about whether and how these dynamic DV patterns of expression translate to regulation of neuronal differentiation programs in the hindbrain. Dissecting these cellular programs during the establishment and differentiation of neuronal populations will be critical for understanding how the hindbrain generates the components and circuits associated with its role as a complex coordination center of the CNS. Finally, another important direction for the future is to understand how hindbrain segmentation and Hox genes play roles in later stages of elaboration of the CNS to generate adult structures and circuits. Insight is arising from conditional mouse mutants that reveals roles in somatosensory circuits, respiratory circuits, and generation of neural progenitor pools (Briscoe & Wilkinson, 2004; Chatonnet et al., 2003; Davenne et al., 1999; del Toro et al., 2001; Di Bonito, Glover, & Studer, 2013; Di Bonito, Narita,
HoxB cluster DV expression A D
V
Time B
HoxA
HoxC
C r4
r4
8.5 dpc
r4
13.5 dpc
11.5 dpc
r4
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Figure 2 Dorsoventral changes in Hox expression in the neural tube. (A) Cross-sectional diagrams of the developing neural tube showing how HoxB genes become enriched in dorsal regions. (B) Comparison of the different dorsoventral distributions of genes from separate Hox clusters. (C) Sketch showing that the r4 expression of Hoxb1 becomes restricted to small longitudinal stripes of neurons in r4.
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et al., 2013; Oury et al., 2006; Pasqualetti, Diaz, Renaud, Rijli, & Glover, 2007; Pattyn et al., 2003). With the development of new tools and technologies, there is hope that efforts will be successful in understanding the full implications of how hindbrain segmentation eventually leads to the full elaboration of this region of the CNS and assess how variations in the process impact development, disease, and evolution.
ACKNOWLEDGMENTS I would like to thank Mark Miller for the illustrations and the many friends, colleagues, and lab members who over the years have participated in the research and exchange of ideas that have made this such a wonderful area to study.
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