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Keywords:

  • Pbx;
  • homeodomain proteins;
  • limb development;
  • TALE;
  • girdle;
  • scapula;
  • pelvis;
  • Exd;
  • Meis;
  • hth

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES

Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen-encoding genes like Shh in limb posterior mesoderm, and transcription factor-encoding genes like Alx1 in pre-scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5′ HoxA/D gene expression. Developmental Dynamics 240:1063–1086, 2011. © 2011 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES

Limb development has long provided an excellent model for understanding the genetic and molecular principles driving embryogenesis. Studies utilizing both the chick and the mouse as model systems have led to new insight into fundamental biological processes that play important roles in limb patterning and morphogenesis, including cell fate specification, gene regulation, signal transduction, proliferation, migration, and adhesion (Niswander, 2003). Over the past two centuries, research in these two systems, coupled with studies in atypical models, has managed to address seminal questions of limb anatomy, function, and evolution (Stopper and Wagner, 2005). More recent work has centered on the genetic and transcriptional control of limb bud development, as well as the gene regulatory networks controlling appendicular skeleton and soft tissue formation (Bismuth and Relaix, 2010; Butterfield et al., 2010; Schweitzer et al., 2010; Zeller, 2010).

LIMB AND GIRDLE ANATOMY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES

In vertebrates, each limb is connected to the body axis via a bony girdle along with associated musculature (Romer and Parsons, 1986), and it is during embryogenesis that the developmental integration of limb and girdles occurs. However, the integration of limbs to their girdles has received less attention than deserved in the past. Therefore, in this review we will concentrate on both, given their developmental and functional interdependence.

In vertebrates, the limb proper consists of three main developmental domains: the stylopod (upper arm or humerus; thigh or femur), the zeugopod (forearm or radius and ulna; leg or tibia and fibula), and the autopod (hand or carpals, metacarpals, and digits; foot or tarsals, metatarsals and digits) (Zeller et al., 2009). In the forelimb, the humerus proximally articulates with the scapula, a large triangular-shaped bone consisting of an expanded blade, head, and neck that in most tetrapods sits dorsolaterally on the thorax and is enveloped by musculature. The scapula, together with the clavicle, forms the pectoral girdle. In the hindlimb, the femur is proximally articulated via the hip or femoral-acetabular joint to the pelvic bone, which together with the sacral vertebrae form the complete pelvic girdle (Romer and Parsons, 1986). Limb and girdle development comprise all the processes by which the appendicular compartments form and their skeletal and soft tissue fates are realized.

LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES

Limb development begins when a portion of the lateral plate mesoderm (LPM) at stereotypic positions along the embryonic axis undergoes lateral outgrowth. The mechanisms that control when and where this initial budding occurs remain mostly unknown. During this stage, the early limb bud or prominence consists of a proliferating mass of apparently undifferentiated mesenchymal cells enveloped by a layer of ectodermal cells (see Zeller et al., 2009). This prominence next emerges to form a true bud due to additional outgrowth and the establishment of several signaling centers that act to define cell fates along the three main limb axes (Niswander et al., 1993, 1994; Parr and McMahon, 1995; Loomis et al., 1996; Cygan et al., 1997; Towers and Tickle, 2009; Zeller et al., 2009). These axes are: the proximal-distal (P-D) axis (e.g., from shoulder to finger tip), the anterior-posterior (A-P) axis (e.g., from thumb to digit five), and the dorsal-ventral (D-V) axis (e.g., the back of the hand to the palm). Essential to the formation of the three axes, and the resulting skeletal elements that are specified within the bud, are the coordinated and controlled expression of numerous genes, most notably from the Hox, Fgf, Shh, Wnt, and Bmp gene families (Parr and McMahon, 1995; Zuñiga et al., 1999; Sun et al., 2000, 2002; Khokha et al., 2003; Scherz et al., 2004; Zakany et al., 2004; Verheyden and Sun, 2008; Benazet et al., 2009; reviewed in Zeller et al., 2009).

At the distal tip of the limb bud, a specialized epithelium, the apical ectodermal ridge (AER) signaling center forms and controls growth and patterning along the P-D axis via the production of growth factors belonging to the FGF family of signaling molecules (Niswander et al., 1993; Lewandoski et al., 2000; Mariani et al., 2008). Within the flank and proximal limb, Hox genes are also expressed, and together with proximalizing factors, such as retinoic acid and distally expressed Fgfs, set up a P-D coordinate system that helps specify cellular identity along this axis (see Zeller et al., 2009). Concurrently, the A-P axis also develops due to the specification of a subset of cells along the posterior mesoderm of the limb, producing a second signaling center, the Zone of Polarizing Activity (ZPA) (Saunders and Gasseling, 1968). ZPA cells are the source of another signaling molecule, Sonic hedgehog (Shh), which plays a critical role in maintaining the A-P axis, preserving AER integrity (and thus P-D growth), and is essential for distal limb and digit patterning and morphogenesis (Echelard et al., 1993; Riddle et al., 1993). Along the D-V axis, patterning is directed by the ectoderm lining the dorsal and ventral surfaces of the limb bud, which produce signaling molecules of the Wnt and Bmp families, respectively (Niswander et al., 1993, 1994; Parr and McMahon, 1995; Loomis et al., 1996; Cygan et al., 1997). Gene expression within the three signaling networks is controlled by tightly coordinated and integrated feedback loops and this coordination leads to the formation of the bony and soft tissue structures of the vertebrate limb (see Niswander, 2003; Zeller et al., 2009).

Concomitant with the formation of the limb and its axes, the mesodermal portion of the somatopleure or medial portion of the LPM houses the cells that will give rise to each of the girdles (Huang et al., 2006; Malashichev et al., 2008). In the forelimb, cells from the dermomyotome of the somites migrate to the somatopleure, and together with nascent cells within this tissue form the scapula blade, head, and neck (Huang et al., 2000; Valasek et al., 2010). In the hindlimb, it has recently been shown that the entire pelvis forms from the somatopleure proper with no somitic contribution (Malashichev et al., 2008), while the sacral vertebrae form from the sclerotome (Christ et al., 2000).

TALE Homeodomain Proteins

During the past few years, we have reported that Pbx members of the TALE (Three Amino acid Loop Extension) superfamily of atypical homeodomain-containing transcription factors (Bürglin, 1994, 1997) are essential for multiple developmental processes, including limb and girdle development (Fig. 1). Vertebrate Pbx genes, together with their orthologous Drosophila exd and C. elegans ceh-20 genes, belong to the gene family encoding the PBC subclass of TALE proteins, which sets itself apart for having a conserved protein-protein interaction domain, the PBC domain (Burglin, 1998). This domain serves as an interaction surface with members of two other known subclasses of TALE proteins: MEIS/MEINOX (Burglin, 1997; Mukherjee and Burglin, 2007) and PREP (Fognani et al., 2002). Vertebrate genomes contain four Pbx genes: Pbx1, which was discovered first, due to its involvement in a chromosomal translocation in human pre-B cell acute lymphoid leukemia; Pbx2 and Pbx3, which were subsequently identified because of their sequence homology to Pbx1; and Pbx4, which was initially isolated via a forward genetic screen for mutations disrupting the segmental patterns of gene expression in the developing zebrafish brain (Nourse et al., 1990; Monica et al., 1991; Kamps and Wright, 1994; Pöpperl et al., 2000). Since their discovery, TALE transcription factors and in particular Pbx proteins have been implicated in crucial developmental processes, largely from the perspective of their function as co-factors to members of the Hox family of transcriptional regulators (reviewed in Moens and Selleri, 2006; Laurent et al., 2008). Recently, though, a number of studies have indicated that Pbx proteins may act more broadly to include also Hox-independent functions in the control of pattern formation and organogenesis (see below).

thumbnail image

Figure 1. Phases of Pbx gene expression in limb mesenchyme during early bud development. As shown in the schemata, Pbx1, Pbx2, and Pbx3 expression domains are highlighted at various stages of limb development. In the earliest phase (I) Pbx1 and Pbx2 genes exhibit overlapping expression domains in the LPM and early limb field (purple domains), as well as within the mesodermal component of the somatopleure that gives rise to the girdles. Pbx3 is absent from the LPM and early limb field. During phase II, the domains of Pbx1 and Pbx2 expression diverge and only partially overlap in the most proximal domain of Pbx2 expression during girdle and limb bud development. Interestingly, in phase II, Pbx3 expression is restricted to only the forelimb bud mesenchyme (until E11.0). Phase III marks the return of Pbx overlapping expression domains, in the anterior and posterior limb mesenchyme, just proximal to the autopod. FL, forelimb; HL, hindlimb. In panels, E9–E11.5, proximal is to the left, anterior is up.

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Here, we will review recent findings on the function of Pbx genes in vertebrate limb and girdle development, as genetic analyses in the mouse have started to reveal critical roles for this gene family in appendage formation. Indeed, Pbx genes are essential in determining limb bud positioning, early bud formation, limb bud axis establishment and coordinated regulation, and patterning and morphogenesis of limb skeletal elements. In addition, Pbx transcription factors also co-regulate girdle formation and the integration of each girdle with the limb proper. Before discussing in detail the functions of Pbx proteins in limb and girdle developmental processes, we will review their transcriptional and biochemical features as well as illustrate the molecular landscape in which they act.

PBX PROTEINS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES

The Perfect Partners for Many a Situation

A wealth of studies over the past two decades has unveiled physical and functional interactions between Pbx proteins and a number of other transcription factors and transcriptional co-regulators. This has led to the conclusion that Pbx proteins are central to multiple gene regulatory networks. The most-studied interaction partners of Pbx proteins are Hox proteins. Hox proteins belong to a family of evolutionarily highly conserved transcription factors that control regional identities and cell fates along the A-P body and limb axes (Alexander et al., 2009; Wellik, 2009; Mallo et al., 2010). Hox protein functions in development reveal an intriguing paradox. Indeed, while Hox display an exquisite degree of functional specificity in vivo, being able to selectively direct developmental programs in a gene-specific manner, their DNA binding capabilities, as analyzed in vitro, appear to be relaxed. In fact, most Hox proteins recognize different DNA sequences, characterized by a TAAT core consensus, with essentially the same binding affinity, and individual TAAT-containing sequences can be bound by different Hox proteins with similar efficiency. To explain this paradox, the intervention of co-factors was invoked. Co-factors, by physically interacting with Hox proteins, would raise their DNA-binding specificity/selectivity to in vivo standards (Chan et al., 1994; Van Dijk and Murre, 1994; reviewed in Mann, 1996). One of the first co-factors identified was the product of extradenticle (exd) in D. melanogaster (Rauskolb et al., 1993); the ortholog to the vertebrate Pbx. exd was shown to act genetically in parallel to Hox genes in patterning the segmental body plan of the fly (Peifer and Wieschaus, 1990), and its gene product was found to form stable heterodimers onto specific DNA sequences, whose complexity was considerably higher than that of the sites bound by the Hox or Exd proteins alone (Mann, 1996). Soon it was shown that Pbx proteins could also heterodimerize onto DNA with a large subset of vertebrate Hox proteins to enhance their target binding site selectivity (Chang et al., 1995; Knoepfler and Kamps, 1995; Phelan et al., 1995; Peltenburg and Murre, 1996).

Interactions Between PBC and Hox Proteins

PBC TALE proteins interact with their Hox partners by making contact with a small, evolutionarily conserved, tryptophan-containing motif, located at a variable distance N-terminal to the homeodomain of Hox proteins, which was variably christened “hexapeptide,” “pentapeptide,” or “YPWM” (Mavilio et al., 1986; Krumlauf et al., 1987; Johnson et al., 1995; Chan et al., 1996). PBC proteins form a hydrophobic pocket, comprising TALE family's characteristic three extra amino acids, which accommodates the “YPWM” motif, and thus stabilizes the physical interaction between the two proteins (Chang et al., 1995; Passner et al., 1999; Piper et al., 1999). Additional analyses, though, suggested that Hox-PBC interactions may be more versatile than previously thought. For example, Exd was shown to be able to heterodimerize with the Ubx and Abd-A Hox protein, in the absence of a “YPWM” motif, which was also shown to be sufficient to stabilize the Hox-PBC complex (Chan et al., 1994; Johnson et al., 1995), indicating that other contact surfaces may participate in the interaction between PBC and Hox proteins (Galant et al., 2002).

As mentioned above, the interaction between Hox and PBC proteins leads to the formation of DNA-bound heterodimers that recognize sequences with a higher complexity with a higher complexity than single proteins. Site selection experiments, together with the identification of a number of naturally occurring regulatory elements within the promoters of target genes, have led to the definition of a consensus binding sequence for Hox-PBC complexes, 5′-TGATNNATNN-3′, where the TGAT motif represents the core half site occupied by PBC proteins, while the remaining nucleotides vary in base composition according to the Hox partner entering the complex (Chan et al., 1994; Johnson et al., 1995; Phelan et al., 1995; Chang et al., 1996; Phelan and Featherstone, 1997; Passner et al., 1999; Piper et al., 1999). As to the capability of Hox-PBC heterodimers to regulate transcription, the transcriptional effector domain of the dimeric complex appears to be mainly contributed by the Hox (or other non-Hox, see below) partner, as in the case of the Hoxb1-Pbx1 complex, which has been shown to activate transcription via an activation domain located within the Hoxb1 protein N-terminal portion (Di Rocco et al., 1997). Pbx proteins, on the other hand, do not appear to have intrinsic capabilities to activate or repress transcription (Van Dijk et al., 1993; Lu et al., 1994; LeBrun et al., 1997), despite the fact that they are able to recruit transcriptional co-repressors (Asahara et al., 1999; Saleh et al., 2000). The analysis of the transcriptional outcome (i.e., activation or repression) of Hox-PBC complexes binding at regulated target promoters has led to somewhat contradictory results. While earlier experiments suggested a simple scenario in which heterodimerization with PBC proteins converts Hox proteins from transcriptional repressors into activators (see Pinsonneault et al., 1997), more recent data suggest that the transcriptional effect of a given Hox-PBC heterodimer depends on several factors such as: the structure of the bound regulatory sequence, the presence of specific co-activators or co-repressors, and the activity of cell-specific signaling pathways (Kobayashi et al., 2003; Gebelein et al., 2004). Lastly, it was shown that for the fly proteins Sex Comb reduced (Scr) and Deformed (Dfd), Exd positions a normally unstructured portion of the Hox protein so that two basic amino acid side chains can insert into the minor groove of a Scr- or Dfd-specific DNA-binding site. Also, subtle differences in the way Scr or Dfd recognize their specific binding sites, in conjunction with non-DNA-binding domains, influence whether the target gene is transcriptionally activated or repressed (Joshi et al., 2010).

Furthermore, an additional level of complexity in Hox-PBC interactions was demonstrated in Drosophila. For example, it was demonstrated that in vivo specificity often depends on extended and unstructured regions that link Hox homeodomains to Exd. Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, it was shown that Hox proteins recognize specific Hox-Exd binding sites via residues located in the extended regions that insert into the minor groove, but only when presented with the correct DNA sequence (Joshi et al., 2007). This work suggests that these specific residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence. In summary, the concept has been put forth that PBC reveals a latent specificity that is then built into the Hox N-terminal arm/linker region.

Trimeric Interactions of PBC, MEIS/PREP, and Hox Proteins

An additional level of complexity was added when it was discovered that PBC proteins are almost always associated with members of two other sub-families of TALE proteins, MEIS and PREP (Bürglin, 1997; Fognani et al., 2002; Mukherjee and Bürglin, 2007). The MEIS sub-family of TALE proteins includes the products of the vertebrate Meis1-3, the fly hth, and the C. elegans unc-62 (ceh-25) genes, while the PREP sub-family includes the vertebrate Prep1 and Prep2. MEIS/PREP proteins interact in solution with PBC proteins via a conserved bipartite domain, the MEIS or HM domain, located N-terminal to the homeodomain (Bürglin, 1997; Rieckhof et al., 1997). This domain directly contacts the PBC domain (Moskow et al., 1995; Chang et al., 1997; Chen et al., 1997; Knoepfler et al., 1997; Berthelsen et al., 1998). As PBC proteins interact with Hox and MEIS/PREP proteins via different surfaces, it is recognized that heterotrimeric Hox-PBC-MEIS/PREP complexes can assemble on specific DNA sequences, adding a further level of binding selectivity to Hox proteins (Mann and Chan, 1996; Berthelsen et al., 1998; Mann and Affolter, 1998; Jacobs et al., 1999; Ryoo and Mann, 1999; Ferretti et al., 2000; Ebner et al., 2005). This interaction with MEIS/PREP proteins appears a means for controlling PBX protein function post-translationally. Specifically, both Exd and Pbx proteins have been shown to require MEIS/PREP for their nuclear import in specific cell contexts such as limb mesenchymal cells in vertebrates (see below) or limb imaginal disc cells in flies (Abu-Shaar et al., 1999; Berthelsen et al., 1999; Mercader et al., 1999; Jaw et al., 2000; Kilstrup-Nielsen et al., 2003).

Interactions of PBC Proteins With Non-Hox Homeodomain and Non-Homeodomain Transcription Factors

Pbx proteins have also been shown to interact with other homeodomain-containing and non-homeodomain DNA-binding proteins, demonstrating their involvement in the control of cellular processes other than regional cell fate specification. A number of non-Hox homeodomain-containing proteins indeed possess a “YPWM” motif, or variant forms of it, making them suitable candidates for Pbx interactions (reviewed in Moens and Selleri, 2006).One of the best studied non-Hox Pbx heterodimerization partners is Pdx1 (Peers et al., 1995), which plays a critical role in pancreas development by regulating promoters of pancreas-specific genes, such as those encoding somatostatin and insulin (Peers et al., 1995; Goudet et al., 1999). The somatostatin gene promoter is indeed bound by a Pdx1-Pbx1 heterodimer and becomes activated via a cooperative interaction between the Pbx1-Prep1 complex and Pdx1 bound at two adjacent regulatory elements (Peers et al., 1995; Goudet et al., 1999). Additionally, pancreatic acinar cell–specific expression of the elastase 1 gene (ELA1) is controlled by a trimeric Pdx1-Pbx1b-Meis2 complex (Swift et al., 1998; Liu et al., 2001). Recently, another non-Hox homeodomain protein, Emx2, was found to heterodimerize with Pbx1 and to control co-operatively with Pbx1 the expression of Alx1, a gene required for scapular blade development (see below) (Capellini et al., 2010).

Among non-homeodomain Pbx interaction partners, MyoD, of the bHLH family, is a notable example. Pbx1 has been shown to physically interact with several members of the bHLH transcription factor family, including myogenin, Myf-5, Mrf-4, and MyoD, via a tryptophan-containing motif, resembling the Hox “YPWM,” located in proximity of the bHLH domain (Knoepfler et al., 1999). This interaction between MyoD and Pbx-Meis complexes has been shown to be important for the transcriptional regulation of the Myogenin gene, where MyoD is recruited through an interaction with a Pbx-Meis complex constitutively bound at a regulatory element within the Myogenin promoter (Berkes et al., 2004). Pbx proteins have also been found to physically interact with Smad transcription factors, which represent intracellular mediators of the TGFβ family of signaling molecules. A Pbx1-Prep1 heterodimer was shown to form trimeric complexes with Smad proteins. Indeed, a Smad4-Pbx1-Prep1 complex regulates the promoter of the FSHβ gene (Bailey et al., 2004).

Also members of the nuclear receptor superfamily of transcription factors have been reported to functionally interact with Pbx proteins. The malic enzyme gene, whose expression is activated by the thyroid hormone (T3), contains in its promoter a regulatory region, which is bound by several Pbx/Meis complexes, one of which contributes to its transcriptional activation. A physical interaction between the thyroid hormone receptor TRa, a nuclear receptor Zn-finger transcription factor, and Pbx1 has also been shown to be responsible for transcriptional activation as well as formation of a DNA-bound complex containing TR/RXR and Pbx1/Meis (Wang et al., 2001). Likewise, an interaction between Pbx1 and another nuclear hormone receptor, the glucocorticoid hormone receptor (GR), was inferred to explain the glucocorticoid responsiveness, via a Hoxb1-Pbx1 complex, of the Hoxb1-ARE regulatory element (Pöpperl et al., 1995) in cultured cells (Subramaniam et al., 2003).

Lastly, a member of the forkhead transcription factor protein family, FoxC1, involved in the formation of neural crest–derived tissues and mesenchymal mesoderm, has also been shown to interact with Pbx1 (Berry et al., 2005). It has been reported that Pbx1 represses FoxC1 transcriptional activity and co-localizes with FoxC1 in heterochromatin-rich, transcriptionally silent regions of the cell nucleus (Berry et al., 2005).

Pbx Proteins Have Distinct Isoforms

Pbx proteins, more specifically Pbx1 and Pbx3, come in different flavors, as both can be found in distinct isoforms, a and b for Pbx1, and a through d for Pbx3, which arise via alternative splicing and differ in length and amino-acid composition at their N-terminal (Pbx3) and C-terminal (Pbx1 an Pbx3) ends (Monica et al., 1991; Milech et al., 2001). Pbx isoforms show tissue- and/or stage-specific expression patterns during development (Swift et al., 1998; Milech et al., 2001; Schnabel et al., 2001), which points at potentially differential activities. In practice, functional differences between Pbx isoforms have been only partially explored. However, the sparse findings available currently suggest that distinct isoforms have differential activities, adding further versatility to the Pbx universe. The Pbx3c and Pbx3d isoforms, for instance, which lack a portion of the N-terminal PBC domain, have been found to heterodimerize weakly with Meis1 and not with Prep1 (Ferretti et al., 1999). Also differences were reported between Pbx1a and Pbx1b as to their capability to modulate the transcriptional activity of Pdx1-Pbx1 heterodimers via the opposed recruitment of chromatin remodeling complexes (see below). Further work in this direction via selective inactivation of Pbx-specific isoforms in mouse models is needed to uncover novel aspects of the complexity of Pbx-mediated gene regulation.

Pbx Proteins and Chromatin Remodeling

Chromatin remodeling protein complexes have been involved in the process of transcriptional regulation since it was recognized that some transcriptional co-activators and co-repressors function by inducing local changes in chromatin structure to favor or prevent gene transcription (Naar et al., 2001). Like numerous other transcription factors, Pbx proteins were shown to recruit chromatin remodeling complexes to promoters, thereby facilitating the binding of other transcription factors, thus enhancing or repressing transcription. For instance, Pbx1 was reported to physically interact, in the context of the myogenin promoter, together with MyoD, and with Brg1, a member of the SWI/SNF ATP-dependent chromatin remodeling complex (see above) (de la Serna et al., 2005). A Pbx isoform-specific recruitment of chromatin modifiers was also reported. A Pbx1b-Pdx1 complex was shown to recruit the histone acetyl transferase (HAT) CBP to activate transcription of the somatostatin promoter, while a Pbx1a-Pdx1 heterodimer recruited two co-repressor complexes NcoR and SMRT (Asahara et al., 1999). It was reported that recruitment of co-repressors by Pbx proteins can be controlled via cell signaling. PKA signaling, for instance, was shown to mediate the switch of the Hoxb1-Pbx1 complex from a transcriptional repressor to an activator (Saleh et al., 2000). In this context, repression was mediated via the association of the N-terminus of Pbx1 with a co-repressor complex containing histone deacetylases (HDACs), such as HDAC1 and HDAC3, NCoR/SMRT, and mSIN3b (Saleh et al., 2000). In summary, Pbx proteins could be considered as “pioneer factors” that poise tissue-specific loci for activation by downstream master regulators.

Our Current View on PBC Proteins: From Ancillary Co-Factors to “Equal Partners”

The first evidence that Pbx proteins function as Hox co-factors in vertebrate development came with the identification of paired Hox/Pbx regulatory elements in the promoters of mammalian Hox genes themselves. Hox gene expression, particularly in the vertebrate hindbrain, is auto- and cross-regulated (reviewed in Alexander et al., 2009). For example, amongst the Hox paralog group 1 genes, Hoxa1 is required for the normal onset of Hoxb1 expression, while Hoxb1 is required for maintaining its own expression (Pöpperl et al., 1995; Studer et al., 1998). In these and other examples of Hox-dependent gene regulation in vertebrates, the Hox recognition element consists of a paired Hox/Pbx binding site (often with a nearby Meis/Prep site), and mutating either the Hox- or the Pbx-binding element prevents reporter expression in transgenic mice (Pöpperl et al., 1995; Maconochie et al., 1997; Ferretti et al., 2000; Manzanares et al., 2001; Samad et al., 2004). Additionally, Pbx mutants in fish and mouse partially recapitulate Hox loss-of-function phenotypes. Indeed, zebrafish pbx4 mutants were originally identified as resembling Hox mutants previously described in animal models (Pöpperl et al., 2000). They phenocopy mouse Hoxb1 mutants (Goddard et al., 1996) and hoxb1a morpholino-injected zebrafish embryos (Studer et al., 1996; McClintock et al., 2002; Arenkiel et al., 2004). Furthermore, targeted mutations in Pbx1, Pbx2 and Pbx3 genes in the mouse cause less severe patterning defects than those of zebrafish pbx4 or pbx2; pbx4 mutants, indicating functional redundancy between the mouse genes at early developmental stages (see below). Interestingly, some aspects of mouse Pbx1 homozygous mutants recapitulate Hox loss-of-function phenotypes. For example, Pbx1−/− embryos do not display dramatic patterning defects in the hindbrain (F. Rijli, personal communication), but they exhibit morphological transformations of neural crest–derived second pharyngeal arch cartilages to structures similar to first arch cartilages (Selleri et al., 2001), a phenotype partially resembling that observed in Hoxa2 mutants (Gendron-Maguire et al., 1993; Rijli et al., 1993). Phenotypic overlaps with Hox loss-of-function mutants are also observed in caudal pharyngeal pouch–derived organs of Pbx1−/− embryos, such as thymus, parathyroids, and ultimobranchial bodies. For example, the thymus in Pbx1−/− embryos is mostly ectopic; the lobes do not fuse and do not descend into the mediastinum (Manley et al., 2004). A similar thymic phenotype is observed in mice that are compound mutants for three paralogous group 3 Hox genes (i.e., Hoxa3+/−;Hoxb3−/−; Hoxd3−/− mutants) (Manley and Capecchi, 1998), consistent with a scenario in which Pbx1 acts together with multiple Hox proteins and in multiple cell types to regulate pharyngeal development. However, the above evidence for Pbx roles as in vivo Hox co-factors is circumstantial, as no clear-cut, unequivocal demonstration for such roles has been put forth in vertebrate development, with the only exception of the vertebrate hindbrain. Only in the hindbrain have Pbx functions as co-factors for 3′ Hox proteins been demonstrated with some stringency in fish and mouse models (Pöpperl et al., 1995, 2000; Ferretti et al., 2000; Samad et al., 2004). Interestingly, mice with mutations in the Hoxa1 hexapeptide, which mediates Pbx contact, display hindbrain and cranial nerve defects reminiscent of those reported for Hoxa1 loss-of-function (Remacle et al., 2004).

In light of the widespread presence of Pbx proteins in multiple developing tissues and organs (Schnabel et al., 2001; Selleri et al., 2001, 2004; Kim et al., 2002; Rhee et al., 2004; Capellini et al., 2006, 2008, 2010; Di Giacomo et al., 2006), as opposed to the more restricted expression patterns of Hox interaction partners, a different perspective has emerged, which posits that Hox should actually be considered as the co-factors, while Pbx proteins would play the leading role (Mann and Affolter, 1998; Laurent et al., 2008). This view, while perhaps appealing, does not stand up to the fact that at least some Pbx partners, such as select Hox proteins, have been reported to display patent PBC/MEIS-independent functions (Galant et al., 2002; Joshi et al., 2010). Not to mention that a subset of Hox proteins, such as those belonging to paralogy groups 11–13, appear unable to interact directly with PBC proteins (Chang et al., 1995; Knoepfler and Kamps, 1995; Phelan et al., 1995; Neuteboom and Murre, 1997). Thus, it would be too extreme to consider Hox proteins, and possibly other interaction partners, solely as co-factors to PBC and MEIS/PREP proteins. In sum, we believe that deciding who is the co-factor and who is the main player based on the extent of expression in embryonic development is a simplistic criterion that is ultimately misleading. This semantic issue set aside, it is becoming clear in light of mounting experimental evidence in animal models that Pbx homeoproteins and their multiple partners all exert both autonomous and co-operative functions in developmental programs and should thus be regarded as “equal partners” with equal dignity in their transcriptional relationship.

ROLES OF PBX IN ORGANOGENESIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES

Commonalities of TALEs

Using a mouse line with global loss of Pbx1 (Pbx1−/−), we established that virtually every organ of Pbx1−/− embryos is markedly hypoplastic and malformed leading to late-gestational lethality (Selleri et al., 2001). This observation led us to hypothesize that Pbx1 controls progenitor cell proliferation in mammalian embryonic development, possibly through direct control of the cell cycle. Indeed, S-phase Common Myeloid Progenitors (CMPs) are reduced in number (DiMartino et al., 2001) and proliferation rates are similarly diminished in chondrogenic, pancreatic, urogenital, adrenal, thymic, and splenic progenitors of Pbx1−/− embryos (Selleri et al., 2001; Kim et al., 2002; Schnabel et al., 2003; Manley et al., 2004; Brendolan et al., 2005). Interestingly, Pbx1 was originally identified in acute leukemia by the t(1:19) translocation, which results in Pbx1 dysregulation leading to aberrant proliferation of hematopoietic progenitors and leukemic transformation (Kamps et al., 1990; Nourse et al., 1990). Similarly, the inappropriate expression of Hox genes encoding binding partners of Pbx proteins, such as Hoxa9 (Shen et al., 1999; LaRonde-LeBlanc and Wolberger, 2003), was also implicated in leukemogenesis. Furthermore, the presence of Pbx/Meis as co-factors has been shown essential for Hox proteins to prevent differentiation or cause aberrant proliferation and transformation of cultured cells (Sauvageau et al., 1997; Krosl et al., 1998; Wang et al., 2010). Overall, these studies demonstrate a role for Pbx1 in promoting cellular proliferation, both in normal organ development and uncontrolled growth in cancer (Moens and Selleri, 2006). Interestingly, mice with global loss of Pbx2 exhibited no overt developmental defects (Selleri et al., 2004) and mice with global loss of Pbx3 succumbed to perinatal death due mainly to respiratory defects (Rhee et al., 2004), underscoring at least some unique and distinctive roles of Pbx homeoproteins in mammalian development.

PBX GENES IN LIMB DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES

Expression of Pbx and Other TALE-Encoding Genes During Axis and Limb Development

Research conducted in zebrafish suggested that the functional differences among pbx genes are likely to be due to differences in their expression rather than in their biochemical activities (Pöpperl et al., 2000). Indeed, ectopic expression of any of the zebrafish pbx genes was able to completely rescue the embryonic phenotype of pbx4 mutants. As it turns out, Drosophila exd can accomplish the same rescue, underscoring the high degree of functional conservation across this protein family. Consistent with these findings, the in vitro DNA-binding properties of different Pbx proteins appear very similar: intriguingly Pbx1, Pbx2, and Pbx3 exhibit identical DNA cooperative binding with a subset of Hox proteins in vitro (Chang et al., 1995). These findings predicted that where the expression of Pbx family members tightly overlaps, their functions may be partially redundant. Different Pbx proteins are expressed in temporally and spatially distinct patterns during organogenesis and in adult mice, and in these contexts mouse single mutant phenotypes have been extremely informative. However, overlapping expression patterns of more than one Pbx protein in multiple developing tissues and organs has been observed, including the appendage (Schnabel et al., 2001; Selleri et al., 2001, 2004; Wagner et al., 2001; Kim et al., 2002; Waskiewicz et al., 2002; Rhee et al., 2004). Therefore, analysis of Pbx compound mutants was required to understand Pbx function in these contexts. The limb is a prime example (see below).

Like many transcription factor–encoding genes, Pbx and their TALE-encoding family members have dynamic expression patterns during development reflecting their multi-faceted roles in embryogenesis. Within the developing limb, the dynamic nature of Pbx expression provides the backdrop in which to envisage the multiple genetic and molecular interactions described above. Before discussing their expression patterns prior to and during limb and girdle development, it is important to highlight some general commonalities in Pbx expression in the vertebrate embryo. To begin, mRNA transcripts for Pbx and other TALE-encoding genes, such as Meis and Prep, are not restricted to a single germ layer (i.e., mesoderm, endoderm, or ectoderm), nor to a single histological type (e.g., mesenchyme versus epithelium), but present in all tissue types. Additionally, their expression is not confined to one developmental stage but found dynamically from early embryogenesis (e.g., gastrula) through organogenesis and into adulthood (G. Di Giacomo and L. Selleri, unpublished observations). Pbx and Meis family members often considerably overlap in their expression territories, likely reflecting their known function as Pbx heterodimers as described above. Accordingly, their expression domains can also, either separately or collectively, overlap with those of other homeodomain transcription factors, such as members of the Hox family, reflecting their ability, as discussed above, to form either heterodimeric (e.g., Pbx-Hox or Meis-Hox) or heterotrimeric (Pbx-Meis-Hox) complexes.

With regard to Pbx genes proper, Pbx1–3 exhibit wide expression territories including the limb and girdle, while Pbx4 is expressed solely in the testes (Wagner et al., 2001). However, despite Pbx1–3's broad and often overlapping expression patterns in limb domains, the loss of any one Pbx gene does not substantially alter the expression of the others. This is specifically the case for the limb and girdle, as in these structures, single Pbx gene loss is not compensated for by spatial up-regulations in other Pbx genes (i.e., at least as assessed by in situ hybridization) (Capellini et al., 2006, 2010). The expression of Meis was also examined in single Pbx1 and Pbx2, as well as compound Pbx1−/−; Pbx2+/−, mutant limbs and their expression remained unaltered spatially in the proximal forelimb and hindlimb in all mutant genotypes listed above (Selleri et al., 2001; Capellini et al., 2006).

Specific Pbx and Meis Expression Domains in the Limb and Girdle

Our previous studies established that Pbx genes are expressed throughout girdle and limb development in five general phases (Phases I–V). During Phase I, Pbx1 and Pbx2 show overlapping expression domains as early as E7.5 in the developing somites, in the Lateral Plate Mesoderm (LPM) along the embryonic axis, and through E9.0 in the forelimb bud prominences (Fig. 1). In this phase, they also display overlapping expression in similar domains of the hindlimb, which are typically delayed in their development by approximately half a day (Fig. 1). In contrast, Pbx3 is not expressed in the LPM and is restricted solely to the early forelimb field (Di Giacomo et al., 2006). Less is known regarding Meis expression during this phase, although preliminary data indicate that Meis1 and Meis2 are also expressed in early somites and along the LPM, while Prep has a more restricted domain in the dermomyotome (unpublished results).

Phase II begins when the limb prominences become recognizable limb buds and ends at the first onset of a morphologically identifiable autopod. During this period, Pbx gene expression patterns dynamically change. As limb bud induction and initial outgrowth commence (E8.5–E9.5), Pbx1 transcripts are restricted to LPM and its derivative medial somatopleuric and lateral proximal forelimb mesenchyme, and by E9.25 are lost from the distal mesenchyme (Fig. 1). Henceforth, during this phase from E9.5–11, Pbx1 transcripts remain in flank and proximal limb bud mesenchyme only (Fig.1). Conversely, during this same phase, Pbx2 transcripts become localized distally once the forelimb bud is formed (E9.25). From E9.5 to E11, Pbx2 expression remains distal, and as the bud matures it is confined only along the peripheral rim of the distal mesenchyme (Fig. 1; unpublished data). However, Pbx2 expression still partially overlaps with Pbx1 (and Pbx3 in forelimb only) in the anterior proximal limb field, a domain important for girdle development. During this phase, besides their mesenchymal expression, Pbx1 and Pbx2 also display overlapping expression in the developing AER (our unpublished results). Unlike Pbx1 and Pbx2, Pbx3 expression is present in the flank of early (E9.5) embryos (Fig. 1) but shortly thereafter becomes restricted to only the forelimb bud mesenchyme (until E11.0–11.5). Interestingly, Pbx3 is not present in the AER (our unpublished observations). Regarding Meis, Meis1–3 expression is typically restricted during these stages to the proximal fore- and hindlimb mesenchyme (Capellini et al., 2006; unpublished data), although Prep overlaps in its distal domain with Pbx2 (our unpublished results). Overall, these early expression patterns for Phase I and Phase II suggest a scenario wherein Pbx1 and Pbx2 provide first an overlapping and then a complementary “code” along the early limb and girdle fields and in the entire limb bud (Fig. 1). In contrast, Pbx3 displays unique expression patterns that make this family member an excellent candidate for a highly specific role in early forelimb development (Di Giacomo et al., 2006). Experimental work on compound Pbx1/Pbx3 mutants suggests this may be the case (see below).

Despite their dynamic, and unique expression domains from E9.5 to E11, later in development, Pbx1–3 transcripts again substantially overlap in their spatial extent. During Phase III in the forelimb, expression of all three Pbx (1–3) overlaps in the anterior and posterior mesenchyme proximal to the autopod (E11–11.75, Fig. 1). In the hindlimb, transcripts for Pbx1 and Pbx2 are present at high levels within these domains, unlike Pbx3, which begins being expressed in the hindlimb only after E11. Phase IV occurs during handplate and footplate formation, when all three Pbx are co-expressed in the interdigital mesenchyme, suggesting that the three genes play important roles in later digit specification and morphogenesis (Di Giacomo et al., 2006). Lastly, during Phase V, once the skeletal elements of the limb are formed, Pbx1, Pbx2, and Pbx3 are similarly expressed in proliferative chondrocytes of long bones, while their expression wanes in prehypertrophic and hypertrophic chondrocytes in endochondral ossification (Selleri et al., 2001).

Limb Phenotypes of Pbx Mutant Mice

The multi-phase expression patterns described above suggest that Pbx family members have potentially multiple roles during limb positioning, induction, and outgrowth, as well as limb and girdle patterning, along with functions in digital patterning and endochondral ossification. The use of global mutant mice and the generation of compound mutant embryos, in which multiple Pbx alleles are concomitantly lost, have shed light on Pbx critical functions in these developmental processes.

With regard to the limbs and girdles of single Pbx mutant embryos, only Pbx1 homozygotes exhibit phenotypes (Selleri et al., 2001), while Pbx2 and Pbx3 homozygotes exhibit phenotypically normal appendages (Rhee et al., 2004; Selleri et al., 2004). This finding, along with those obtained from the study of compound Pbx mutants, demonstrate the prime role of Pbx1 in multiple developmental processes (Capellini et al., 2006, 2008, 2010). Pbx1 mutants exhibit forelimbs with a superior-to-inferior reduced scapular blade that is fused at the scapular head to an adjoining humerus, which bears a detached and rudimentary deltoid tuberosity (Fig. 2; Selleri et al., 2001). Their hindlimbs display a pelvis that often lacks an ilium or exhibits one that is detached from the pubis and ischium, and which is fused at the hip or femoral-acetabular joint to a truncated femur. Despite these proximal alterations, the distal fore- and hindlimbs of Pbx1 homozygous embryos appear phenotypically normal (Fig. 2; Selleri et al., 2001). Pbx1 roles in limb development are also evident in zebrafish, in which loss of Pbx1 ortholog pbx4 leads to the reduction and absence of the pectoral fin in the Lazarus mutant (Pöpperl et al., 2000). The roles of other pbx paralogs in zebrafish remain unknown.

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Figure 2. Girdle and limb phenotypes of single and compound Pbx mutant embryos at E13.5. FL, Cartilage preparations of forelimbs. Pbx1 homozygous mutants exhibit scapula with superior-to-inferior reduced blade (red arrowhead), and fusions of the scapula to the humeral head with an expanded coracoid process (black arrowhead). Pbx1−/−;Pbx2+/− mutant forelimbs additionally show exceptionally hypoplastic scapular blades (red arrowhead in red inset) with heads that appear duplicated (double black arrowheads in red inset). Distally, they display contorted humeral shafts with deltoid tuberosities that are expanded and projected at an angle to the shaft (black arrowhead). Both the radius and ulna are truncated (red arrows) and there is a significant reduction to loss of digits 1 (open red arrowhead) and 5 (open black arrowhead). Pbx1−/−;Pbx3+/− mutant forelimbs display scapular blade hypoplasia (red arrowhead in inset), fusions, and expansion in the proximal humerus (black arrowhead) and humeral head duplications (double black arrowheads in red inset). However, distal to the humerus, these mutant forelimbs appear grossly normal. HL, Cartilage preparations of hindlimbs. Pbx1 homozygous mutants exhibit reduced pelvic girdles, with an absent ilium (empty black arrowhead) and reduced pubis and ischium (red arrowheads), which are fused to the proximal femur. Distal to the femur, these mutants are grossly normal. Pbx1−/−;Pbx2+/− mutant hindlimbs bear pelves with only one remaining element, likely an ischium (red arrowhead) that is fused to the shortened and malformed femur. They display only one zeugopodial element, likely a malformed tibia, lack the fibula (red arrow), and show one single autopodial ray (open black arrowhead in inset). Pbx1−/−;Pbx3+/− mutants phenocopy single homozygous Pbx1 mutants in all proximal girdle and femur abnormalities (red and open black arrowheads), and like Pbx1 mutants are grossly normal distal to the femur. fe, femur; fi, fibula; FL, forelimb; HL, hindlimb; hu, humerus; pg, pelvic girdle; ra, radius; sc, scapula; ti, tibia; ul, ulna; WT, wildtype. In all panels, proximal is up and anterior is to the right.

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To date, analysis of available loss-of-function mouse models for the genes encoding the Pbx TALE dimerization partners Meis and Prep, has not been informative. Indeed, single Meis1 and Prep mutants, along with compound Meis1/Prep mutants, do not reveal limb skeletal phenotypes. All mutants listed above bear normal limbs, while Meis2 mutants have not been obtained yet (Fernandez-Diaz et al., personal communication). Interestingly, work conducted in chick reported that Meis genes are involved in limb development, since over-expression of Meis in distal limb buds leads to proximalization of the limb (Capdevila et al., 1999; Mercader et al., 1999, 2000, 2005). While these data do indicate that Meis homeoproteins are important for proximal limb patterning, their requirements in mammalian limb development are still mostly unknown, as they cannot be inferred solely from over-expression experiments. In sum, to date there is no functional study in the mouse demonstrating that Meis genes are sufficient and/or required for limb bud outgrowth and patterning in mammals.

While the analyses of single Pbx mutants reveal a role for Pbx1 in patterning proximal limb and girdle structures, by generating compound Pbx mutants, we have addressed Pbx roles in the entire appendage and girdle. In this regard, we have begun to investigate issues of functional cooperation and redundancy within domains in which multiple Pbx genes are co-expressed. Below we address Pbx involvement in limb positioning, induction, and outgrowth, as well as limb and girdle patterning, along with their functions during endochondral ossification.

Pbx and Limb Bud Positioning

One of the first events involved in limb formation is the specification of the axial levels where limbs form. It was demonstrated by the work of Harrison (1921) and Slack (1976), for example, using surgical manipulations of chick and salamander embryos, that limb bud position was determined well before bud induction and outgrowth. Yet, despite the seminal work of the early days, the determination of limb bud position along the embryonic rostral-caudal axis still remains enigmatic. A sparse number of mouse mutant models exhibit shifts in the axial position of their limb buds allowing the study of critical factors required for positional determination. Such mouse mutants, as for example single and compound Hox mice, exhibit hindlimbs that are caudally shifted along the embryo (McIntyre et al., 2007). These studies are supported by work in the chick, demonstrating that the expression of Hox9 paralogs along the axial LPM is important in maintaining the boundary conditions involved in limb positioning (Cohn et al., 1997). In these studies, however, little has surfaced regarding the molecular functions of these genes in axial specification. Interestingly, among Polycomb genes, compound loss of Eed and Bmi leads to modest rostral shifts in hindlimb axial position (Kim et al., 2006). As Eed and Bmi typically act as repressors of Hox in anterior axial domains, rostralization of the expression domains of multiple axial Hox genes is observed when these Polycomb genes are experimentally ablated (van der Lugt et al., 1994; Faust et al., 1995, 1998; Lessard et al., 1999; Kim et al., 2006). Thus, Polycomb proteins may provide essential genetic control of limb positioning along the axis, via the modulation of Hox territories.

Importantly, Pbx loss from the embryonic axis (i.e., in Pbx1−/−;Pbx2+/− mutants), from Phase I onwards, leads to reductions in the expression of the Eed and Bmi Polycomb genes and to simultaneous rostralizations in Hox expression territories within the somites and adjacent LPM (Capellini et al., 2008; Fig. 3). These changes are indeed coincident with rostral shifts in the position of the hindlimb bud and limb skeleton in Pbx mutants (Capellini et al., 2008; Fig. 3). For example, unlike single Pbx1−/− and other compound Pbx mutant embryos (including compound triple Pbx1+/−;Pbx2+/−; Pbx3+/− heterozygotes), Pbx1−/−; Pbx2+/− embryos show a rostral shift of the hindlimb buds along the somites and, subsequently, limb skeletons that are shifted rostrally by several lumbar-sacral vertebrae (Fig. 3; and data not shown). These findings suggest that Pbx1 and Pbx2 cooperate in domains wherein the two genes exhibit overlapping expression to position the limb bud. The lack of axial shifts in examined compound Pbx1/Pbx3 mutant embryos is in agreement with our findings that Pbx1 and Pbx2 but not Pbx3 are expressed in the posterior hindlimb embryonic field (Fig. 1). Thus, hindlimb positioning along the embryonic axis is controlled specifically by genetic interactions of Pbx1 and Pbx2, which may accomplish this task via their control of Polycomb gene expression. While this possibility is intriguing, it remains unclear whether Pbx also cooperate with Hox as co-factors in this process (see above). If this were the case, then the observed limb shifts in Pbx mutants could also be the result of a more marked and broad loss of Hox DNA-binding specificity within the LPM, coincidental with alterations in Polycomb gene expression.

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Figure 3. Pbx homeoproteins control limb bud and skeletal positioning during limb outgrowth. A: Hierarchical role of Pbx on Polycomb (Bmi and Eed) gene expression along the rostral-caudal LPM and paraxial mesoderm (PM) in the axis. In Pbx1−/−;Pbx2+/− mutants, Bmi and Eed expression is downregulated or perturbed, such that their activity is reduced across the rostral-caudal axis. These changes may be responsible for the shifts in Hox expression along the entire axis, which in turn may account for the vertebral patterning defects also present across the entire axis. Concomitantly, loss of Pbx may dysregulate Hox activity independently of Polycomb (dashed arrow). Importantly, these shifts in Hox expression may be responsible for the rostralization in the position of the hindlimb to more anterior somite levels in Pbx1−/−;Pbx2+/− mutants compared to WT embryos (red line drawing/red somite numbers indicate shifts in the mutant condition, versus black lines/black somite numbers in the WT condition). B: Cartilage preparations showing shifts in limb position leading to the more anterior or rostral location of the pelvic girdle and hindlimb skeleton in Pbx1−/−;Pbx2+/− mutants (right) compared to WT controls (left) (red dashed lines demarcate distance of girdle to the last rib-bearing thoracic vertebrae). Proximal is to the left; rostral is up. Adapted from Capellini et al. (2008).

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Pbx and Girdle Development

Prior to and during initial limb bud induction, the LPM along the flank is partitioned into a medial portion, the mesodermal somatopleure, and a lateral portion, which gives rise to the limb proper. The mesodermal somatopleure, at the level of the respective limb fields, gives rise to specific structures of both the pectoral and pelvic girdles (Huang et al., 2000; Malashichev et al., 2008). In the forelimb, cells from specific axial levels of the dermomyotome migrate into this somatopleure, and via cues within this tissue, as well as from the paraxial mesoderm and ectoderm, become specified to form superior to inferior aspects of the scapular blade (Huang et al., 2000). Also, cells of the LPM are specified during the early limb bud stages to form the scapular head and neck, bony structures that directly articulate with the proximal humerus of the limb proper (Huang et al., 2006). In contrast, at the level of the hindlimb, only cells originating from the mesodermal somatopleure have been shown to give rise to the three main elements of each pelvic bone, i.e., a rostral oriented ilium, a ventral facing pubis, and a dorsal positioned ischium (Malashichev et al., 2008). These three elements fuse during development at the location of the hip joint, whereby each pelvic bone articulates with the proximal femur of the limb proper (Pomikal and Streicher, 2010). A number of genetic pathways are known to govern patterning of the bony elements of both pectoral and pelvic girdle during development, though knowledge of the genetic control of girdle development is still rudimentary in comparison to that of limb development (Kuijper et al., 2005; Huang et al., 2006). In the following, we will discuss Pbx contributions to the genetic control of girdle development and how Pbx loss during Phases I–III has marked phenotypic consequences on the development of each girdle. Importantly, like in axial development and limb positioning, Pbx1, Pbx2, and Pbx3 appear to have distinct and differential functions in pectoral versus pelvic development, likely a consequence of differences in their respective expression patterns.

In the pectoral girdle, we have found that Pbx1−/−, Pbx1−/−; Pbx2+/−, and Pbx1−/−; Pbx3+/− embryos exhibit alterations of the scapular blade and head. The latter two genotypes show markedly more severe phenotypes compared to the former (Capellini et al., 2010). While the scapular defects are more severe in Pbx1−/−; Pbx2+/− compared to Pbx1−/−; Pbx3+/− mutants, both bear reduced blades, fusions of the scapular head to the humerus, and apparent duplications of the head/neck complex. We reported that blade patterning genes within the mesodermal somatopleure, but not within the dermomyotome, are severely down-regulated in compound Pbx mutants. Specifically, we identified marked down-regulation of genes that pattern the superior (Alx4, Alx1) (Kuijper et al., 2005), central (Tbx15) (Kuijper et al., 2005; Lausch et al., 2008), and inferior (Gli3) (Johnson, 1967; Kuijper et al., 2005) scapular blade. Also, within the mesodermal somatopleure (and LPM of the proximal limb bud) scapular head and neck markers, such as Hoxc6 (Oliver et al., 1990), were severely reduced and mis-expressed in Pbx mutants, along with genetic pathways controlling mesenchymal condensation and chondrogenesis (Wright et al., 1995; Bi et al., 2001) (i.e., the Emx2[RIGHTWARDS ARROW] Sox9 pathway; and Col2a expression) (Capellini et al., 2010). Importantly, while a major theme of research has focused on Pbx roles as Hox co-factors, we revealed that their functions in scapular development reside, at least in part, in their ability to form heterodimeric complexes with homeodomain proteins other than Hox. Within the context of the complex morphogenesis of the scapula, we identified critical genetic interactions of Pbx1 with Emx2, a gene responsible for scapular blade patterning, and established that both Pbx1 and Emx2 proteins form a heterodimeric complex capable of directly regulating in vivo and in vitro a conserved Alx1 non-coding element (Capellini et al., 2010). Thus, in the forelimb pectoral field, Pbx homeoproteins cooperates with Emx2 affecting the transcriptional activation of key pathways in scapular blade and head formation.

As for the pelvic girdle, we recently found that Pbx1−/−; Pbx2+/− mutants exhibit only one rudimentary ischial fragment that remains fused to the femoral head (Capellini et al., pages xxx–xxx, this issue). Compared to our findings in the pectoral girdle, we did not observe a strong genetic interaction of Pbx1 with Pbx3 or Emx2, but found that Pbx genes lie upstream of Emx2, controlling its expression in the mesodermal somatopleure. We also established that Pbx homeoproteins hierarchically regulate multiple genes and pathways involved in the formation of each element of the pelvic girdle. Notably, we demonstrated that Pbx genes act upstream of Pitx1, Tbx15, and Emx2, genes that control ilium formation (Lanctot et al., 1999; Pellegrini et al., 2001; Marcil et al., 2003; Lausch et al., 2008); Alx1, Prrx1, and Twist1, genes that affect pubis development (ten Berge et al., 1998; Kuijper et al., 2005; Krawchuk et al., 2010); and Pax1, a marker of ischium formation (Timmons et al., 1994; LeClair et al., 1999). These results suggest that Pbx homeoproteins play critical roles in the early somatopleure, likely underlying the substantial down-regulation of genes involved in pelvic girdle development. Through the use of novel bioinformatic analyses (McLean et al., 2010) we additionally revealed that Pbx genes control girdle and hindlimb morphogenesis by potentially regulating multiple genes (including those listed above) with expression domains within the pelvic field/hindlimb. By this approach, we also found potential Pbx1/Emx2-binding sites in non-coding, conserved regions adjacent to genes whose disruption (by loss of function mutations in mouse, or spontaneous mutation in humans) results in pelvic/hindlimb phenotypes, such Prrx1 and Tbx15 (Capellini et al., pages xxx–xxx, this issue).

Pbx roles in developmental programs as cell fate specification have long been considered primarily as those of Hox cofactors (reviewed in Moens and Selleri, 2006). However, it is of note that no known single or compound Hox mutant mouse model displays scapular blade defects. Only Hox5 mutants exhibit rostral blade shifts, with normal blade morphology (Capellini et al., 2010). Similarly, misexpression or loss of Hox genes result at best in mild alterations of the pelvic girdle, including defects in mice misexpressing Hoxd12 in lateral plate derivatives; modest malformations of pelvic bones and sacrum in Hoxc10 mutants; and lack of uterosacral ligaments in Hoxa11 mutant mice (Capellini et al., pages xxx–xxx, this issue). Therefore, it is unlikely that Pbx homeoproteins affect their roles in girdle formation solely as Hox cofactors, suggesting instead cooperation with other proteins, as Emx2, in scapula development. Thus, we cannot yet invoke a mechanism whereby loss of Pbx proteins, as Hox co-factors, leads to the girdle phenotypes of Pbx compound mutants via altered Hox function.

Roles of Pbx in Limb P-D, A-P, and D-V Axis Formation, Outgrowth, and Skeletal Patterning

When compared to single Pbx mutants, compound mutants exhibit significant alterations in most skeletal elements of each limb type (Capellini et al., 2006, 2010). At E13.5, Pbx1−/−;Pbx2+/− limbs display marked exacerbations of the proximal limb phenotype of single Pbx1−/− mutants as well as novel distal limb malformations (Fig. 2). For example, Pbx1−/−;Pbx2+/− forelimbs have severely dysmorphic humeri that are fused to the girdle at the scapulohumeral joint (Capellini et al., 2010). Distally, these mutants exhibit a truncated and dysmorphic radius and ulna and bear substantial reductions in digits one and five (Capellini et al., 2006). On the other hand, Pbx3 functions at this time are limited to the formation of the humerus and scapula, as Pbx1−/−; Pbx3+/− mutant forelimbs are affected only proximally (Fig. 2; (Capellini et al., 2010). Compound Pbx1/Pbx3 double homozygous mutants die during early limb formation, thus limiting a deeper assessment of Pbx3 roles in this process.

The severity of appendicular phenotypes due to compound Pbx loss is more apparent in the hindlimb, wherein only Pbx1 and Pbx2 are expressed during limb outgrowth (Phase I and II; Fig. 1). Therefore, in the hindlimb dosage reductions have more substantial effects, since loss of Pbx1 and Pbx2 enables a Pbx-null state. Accordingly, Pbx1−/−;Pbx3+/− hindlimbs do not present phenotypic exacerbations versus single Pbx1 null mutants, and exhibit normal limb skeletons distal to the femur (Capellini et al., pages xxx–xxx, this issue). On the other hand, Pbx1−/−;Pbx2+/− hindlimbs at E13.5 exhibit a markedly truncated femur fused to a residual pelvic rudiment (Fig. 2; Capellini et al., 2006). Distally, there is only one zeugopodial element, a moderately malformed but truncated tibia, and a single autopodial ray consisting of one digit, likely digit number one (Fig. 2). This severe digit reduction is similar to that observed in Sonic hedgehog (Shh) null mutants (Chiang et al., 2001). The absence of distal limb elements and the drastic reduction in digit number, along with the severe proximal limb malformations, reveals that both Pbx1 and Pbx2 are required for patterning skeletal elements within all limb compartments. Despite the documented early gestational lethality of Pbx1−/−;Pbx2−/− homozygous mutants at E10.5, these embryos have forelimb buds that are highly dysmorphic and hindlimb buds that are barely detectable (Capellini et al., 2006).

Roles of Pbx in P-D Outgrowth

The process by which the limb prominences undergo lateral, distal outgrowth occurs through the coordinated regulation of the three main P-D, A-P, and D-V developmental axes (see Zeller et al., 2009). Disruptions in any one of these axes typically alters the proper development of the others and often leads to cessation of limb bud outgrowth and/or mis-patterning of soft tissue structures and skeletal elements. One of the earliest events in the establishment of the P-D axis is the induction of a fibroblast growth factor, Fgf10, in the early mesenchyme subjacent to the limb ectoderm of both limb prominences (E8.5–9.5) (Cohn et al., 1997; Ohuchi et al., 1997; Min et al., 1998; Sekine et al., 1999). While being induced by Tbx5 and Tbx4 in the forelimb and hindlimb fields, respectively (Agarwal et al., 2003; Minguillon et al., 2005; Koshiba-Takeuchi et al., 2006; Saito et al., 2006), Fgf10 expression in these domains may also be under the control of collinear Hox gene expression in the early limb bud (Zakany et al., 2007). Once induced, however, Fgf10 expression promotes the expression of Fgf8 within a maturing strip of specialized ectodermal epithelium at the distal tip of the limb bud, the pre-AER and AER (Barrow et al., 2003). Fgf8 expression in the AER then further promotes cellular outgrowth and patterning in the limb via a feedback mechanism on Fgf10 expression in the subjacent mesenchyme (Fig. 4) (Xu et al., 1998; Lizarraga et al., 1999). This Fgf10-Fgf8 loop likely aids the early specification of the skeletal elements of the three limb compartments, and promotes and/or maintains the expression of multiple genes critical for limb development, such as Hox, Wnt, and Shh (Niswander et al., 1994; reviewed in Niswander, 2003; Zeller et al., 2009). For example, Hox expression territories along the P-D axis are also under the influence of Fgf expression in the distal limb (Sun et al., 2002) and are essential for P-D limb outgrowth, as evinced by extreme distal limb truncations in mutants lacking entire HoxA/HoxD cluster genes (Kmita et al., 2005). In these mutants, there is also a coordinated down-regulation and cessation of the Fgf10-Fgf8 feedback loop demonstrating the complex circuitry between these factors.

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Figure 4. Model of Pbx gene functions during early limb development. E8.5–E9.0: During early limb field induction, the proximal-to-distal (Pr-D) axis becomes established as Pbx genes control Fgf10-Fgf8 signalling in limb mesoderm (black arrows) and/or possibly in pre-AER ectoderm (light blue arrow; light blue line represents pre-AER). E9.0–E9.5: During Pr-D axis establishment, AER formation and limb outgrowth, Pbx continue to regulate Fgf8-Fgf10 signaling, at least in part due to their upstream control of Hox expression in the early limb bud (black arrows) and possibly via their control of Fgf8 in the AER (blue arrow; blue line represents AER). This upstream regulation leads to Hox expression throughout the limb bud and establishment of the pre-Shh ZPA domain (pink) in the posterior limb. E10–E10.75: Pbx control of Hox expression leads to the direct regulation of Shh expression onset and maintenance (black arrows) in the ZPA (terracotta-colored zone). Shh expression in turn promotes Fgf expression in the AER (blue) and maintains the ZPA-AER signalling network (black arrows). This genetic network leads to the proper control of Shh-dependent distal limb (digital) development and proximal-distal patterning and growth. A, anterior; D, distal; P, posterior; Pr, proximal; ZPA, zone of polarizing activity.

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As the proper spatio-temporal regulation of Hox genes and Fgfs is critical to the establishment of the P-D axis in the early limb prominence, we have identified essential roles for Pbx as upstream regulators of Hox gene expression in the early limb bud (Fig. 4). Indeed, in Pbx1−/−;Pbx2+/− mutants, transcripts of both 5′ HoxA and 5′ HoxD genes are substantially reduced and dysregulated in the forelimb bud, while they are absent in the early hindlimb bud specifically prior to and during initial P-D limb outgrowth (during Phase I and II; Fig. 1) (Capellini et al., 2006). Pbx1−/−;Pbx2+/− embryos exhibit truncated and mispatterned proximal-to-distal hindlimbs, defects that partially phenocopy those observed when entire HoxA and HoxD clusters are simultaneously lost from the limb (Kmita et al., 2005). Furthermore, in addition to the observed reductions in Hox expression in the hindlimb, we also identified reductions in Fgf10 and Fgf8 expression in the distal limb mesenchyme and AER, keeping in mind that the residual expression of Fgf10 and Fgf8 in their respective domains likely results from the remaining copy of Pbx2 in this mouse model (Capellini et al., 2006; data not shown). Accordingly, a hierarchical role for Pbx on P-D axis formation and the Fgf10-Fgf8 loop is evinced by the failure of hindlimb formation in compound Pbx1−/−;Pbx2−/− mutants (Capellini et al., 2006). Interestingly, in this mutant genotype Fgf8 expression is completely lost in the hindlimb bud and extremely reduced in the forelimb bud (unpublished data; Fig. 5). It is of note that loss of Fgf8 expression is not observed in other tissues of these mutant embryos, and is maintained, for example, at the midbrain to hindbrain junction (Fig. 5; unpublished data).

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Figure 5. Pbx hierarchically regulate early limb P-D and D-V axis establishment. Top left: In situ hybridization at E11 reveals that Fgf8 expression remains mostly unchanged (white arrowhead) at the mid-to-hindbrain junction in Pbx1−/−;Pbx2+/− embryos (30 som.) compared to somite-matched E10 WT (30 som.) and littermate E11 WT (44 som.) embryos (black arrowheads). Littermate WT embryos are included to demonstrate that Pbx1−/−;Pbx2−/− mutants are delayed in somitogenesis. Middle left: Fgf8 expression is barely detectable in Pbx1−/−;Pbx2−/− forelimb buds (red arrowhead) compared to somite-matched WT and littermate WT forelimb buds (black arrowheads). Bottom left: Fgf8 expression is absent in Pbx1−/−;Pbx2−/− hindlimb buds (blue arrowhead) compared to somite-matched WT and littermate WT hindlimb buds (black arrowheads). Top right: In situ hybridization at E10.25 reveals that En1 expression is absent from the anterior and posterior ventral ectoderm in Pbx1−/−;Pbx2+/− forelimbs (blue arrowheads) compared to controls. Middle right: In Pbx1−/−;Pbx2+/− hindlimbs at E10.25, En1 expression is only lost from the posterior ventral ectoderm (blue arrowhead) compared to controls. Bottom right: In hindlimbs at E10.75, Lmx1b expression is significantly up-regulated in the ventral domain in Pbx1−/−;Pbx2+/− mutants (red arrowhead), although such up-regulation may reflect a delay in limb bud outgrowth (E10 limb bud on right). Left, panels show distal limb bud tip with anterior to the top and dorsal to the left; right, panels show ventral limb bud with anterior to the top. A, anterior; FL, forelimb; HL, hindlimb; P, posterior.

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Roles of Pbx in Limb A-P Axis Formation

Concurrent with the initiation of the Fgf10–Fgf8 feedback loop, early asymmetry in the A-P axis occurs due to inhibitory interactions in the anterior and posterior limb field mesoderm, specifically between Gli3 and Hand2, respectively (Fig. 4). This mutual antagonism ultimately leads to the restriction of Hand2 expression to the posterior aspect of the early limb prominence (te Welscher et al., 2002). Simultaneously, collinear HoxA and HoxD gene expression patterns overlap at first in a wide A-P distributed pattern along this field. However, as the limb bud undergoes P-D outgrowth, transcripts of HoxA and HoxD genes become increasingly restricted to the distal and posterior limb bud (Zakany et al., 2004). This expression pattern, along with the presence of Hand2 (Charite et al., 2000), leads to the activation of Shh along the posterior limb bud mesenchyme in the ZPA (Fig. 4) (Zakany et al., 2004), which is essential to distal limb and digital patterning (Riddle et al., 1993). Shh signaling in the ZPA mesenchyme promotes a cascade of gene expression leading to the induction of Fgf4 in the AER (Chiang et al., 1996, 2001). Fgf4 further promotes ZPA function, maintains Shh and Hox gene expression along the A-P and P-D axes, and thus facilitates further outgrowth and patterning of the distal limb (Niswander et al., 1993, 1994; Fallon et al., 1994) In this regard, Hox genes serve to coordinate early development of the AER and ZPA via their effects of Fgf10 and Shh, both of which then serve to maintain positive regulatory interactions in the limb bud along both A-P and P-D axes (Fig. 4).

By showing that Pbx genes are required for regulating Hox gene expression in the limb, we have revealed a critical control of A-P axis formation by Pbx. Indeed, we have observed that the loss of Pbx1 and Pbx2 in the hindlimb field (i.e., in Pbx1−/−;Pbx2−/− mutants) leads to the complete failure of Shh induction in the ZPA, unlike in other tissues such as the cloaca and notochord wherein Shh is expressed at normal levels in these mutants (Capellini et al., 2006, 2008) (Fig. 4). Interestingly, in Pbx1−/−;Pbx2+/− hindlimbs, Shh absence is not coupled with marked reduction of Fgf8 expression in the posterior AER at early days, or of Hand2 expression in the mesenchyme, but is correlated with the near complete loss of all HoxA and HoxD transcripts in the pre-ZPA. Furthermore, the down-regulation of Hox and absence of Shh expression leads expectedly to the complete breakdown of the Shh-Fgf4 loop and the cessation of limb bud development. We have revealed that Fgf4 is not expressed in Pbx1−/−;Pbx2+/− hindlimb AER and that all effectors of this feedback loop are substantially disrupted (for review see Zeller et al., 2009). Not surprisingly, the distal hindlimb zeugopodial and autopodial phenotype of compound Pbx1−/−;Pbx2+/− mutants phenocopies that observed in Shh null mutants (Chiang et al., 2001), while the striking proximal-to-distal phenotype across all limb compartments results at least in part from significant disruption of HoxA/HoxD expression (Kmita et al., 2005). In the forelimb, Hox genes are down-regulated and mis-expressed, but not as severely as in the hindlimb, and Shh is only mildly down-regulated in the Pbx1−/−;Pbx2+/− mutant genotype, likely a consequence of Pbx3 expression in the forelimb. Notably, the importance of Hox control by Pbx in the pre-ZPA was further substantiated by our finding that both Hox and Pbx proteins are directly bound in vivo to a Shh long-range limb enhancer (Capellini et al., 2006). This enhancer, when experimentally deleted in mice, recapitulates a global Shh null limb phenotype (Lettice et al., 2002, 2003; Sagai et al., 2005). Thus, Pbx homeoproteins are essential upstream regulators of both P-D and A-P axes formation in the limb, in part via their coordinated control of Hox expression in the bud mesenchyme.

The severe alterations of 5′ HoxA/HoxD gene expression, especially in posterior hindlimb bud domains of Pbx1/2 mutant embryos, occurred in limbs that exhibited relatively normal early AP patterning. Therefore, at least in the limb, Pbx1/Pbx2 act upstream of Hox genes and not solely as Hox-cofactors, in a manner that appears to occur independently from known molecular regulators required for the establishment of early AP asymmetry. Furthermore, this Pbx-mediated Hox gene control occurs early, perhaps in the limb field, because both 3′ and 5′ HoxA/HoxD genes were already altered at the onset of limb bud development. The mechanisms by which Pbx hierarchically control and maintain Hox genes' spatial distribution in early limb mesenchyme remain elusive. Pbx cooperative roles with other transcription factors upstream of Hox genes (Berkes et al., 2004) (reviewed by Moens and Selleri, 2006) may have bearings. Indeed, we cannot exclude the possibility that in the limb Pbx might directly regulate Hox gene colinearity, given their co-expression with Hox genes in the limb bud.

Roles of Pbx in Limb D-V Axis Formation

Unlike for the P-D and A-P axes and their coordinated regulation, fewer molecular regulators of D-V axis formation have been identified. It is also unclear whether Hox genes have roles in D-V axis establishment. Two known genetic regulators of the D-V axis are Engrailed1 (En1), expressed in the ventral ectoderm and essential for ventral fates, and Wnt7a, expressed in the dorsal ectoderm and critical for dorsal fates. Genetic studies using single and compound loss-of-function models for En1 and Wnt7a demonstrated roles for these genes in D-V patterning and AER formation (Loomis et al., 1998). Specifically, En1 has been shown to inhibit Wnt7a signaling ventrally, restricting it to the dorsal ectoderm, whereas Wnt7a spatially limits the expression of the transcription factor Lmx1b to the dorsal mesoderm (Loomis et al., 1996). In turn, Lmx1b has been shown to be critical for dorsal mesenchymal and ectodermal patterning (Riddle et al., 1995; Vogel et al., 1995; Chen et al., 1998). Interestingly, loss of proper D-V axis establishment, particularly via the disruption of Wnt7a in the dorsal ectoderm, does lead to the reduction of Shh expression in the ZPA and failure of A-P axis formation. This finding indicates that some degree of developmental coordination between these axes exist. Likewise, as En1 and Wn7a also genetically interact to direct proper AER development in the distal limb ectoderm (Loomis et al., 1998; Kimmel et al., 2000), they are important factors in P-D axis establishment and limb bud outgrowth (Fig. 4).

Recent results (unpublished) in our laboratory suggest that Pbx genes may also have a role in the formation of the D-V axis. Indeed, we examined the expression of the key D-V regulators discussed above in Pbx1−/−; Pbx2+/− embryos from E10 to E11 (note: we could not examine double Pbx1−/−;Pbx2−/− embryos as they succumb in utero prior to these gestational days). In E10.25 Pbx1−/−; Pbx2+/− forelimbs, we found that En1 expression was reduced in the most anterior and posterior domains of the ventral ectoderm, while in hindlimbs it was specifically down-regulated in the posterior aspect of the ventral ectoderm (Fig. 5). However, despite En1 reduction, we found that the expression of genes such as Fgf8 remained mostly unperturbed in the posterior AER of the early bud (Capellini et al., 2006). The expression of Lmx1b was also examined. Lmx1b is normally expressed throughout the early mesenchyme prior to E9.5–10.5, but later becomes restricted to the dorsal mesenchyme. At E10.75, we found that Lmx1b expression was restricted to the dorsal mesenchyme of Pbx1−/−;Pbx2+/− forelimbs as in wildtype littermates, indicating that the D-V axis was relatively well established in this mutant limb type. Furthermore, no overt skeletal or soft-tissue phenotypes, which could reflect mis-patterning along the D-V axis, were identified in the mutant forelimb. In Pbx1−/−;Pbx2+/− hindlimbs, Lmx1b expression was up-regulated compared to somite-matched littermate controls (Fig. 5). Specifically, this up-regulation was evident not only throughout the dorsal limb mesenchyme but also in the posterior ventral mesenchyme, suggesting a possible localized perturbation of Wnt7a signaling in the overlying ectoderm (Fig. 5). However, we observed only a minor down-regulation of Wnt7a expression in this domain (data not shown). Interestingly, we found that Lmx1b was also expressed in ventral posterior and anterior domains in earlier staged wildtype hindlimb buds. This observation suggested that the ectopic Lmx1b expression in posterior Pbx1−/−;Pbx2+/− hindlimbs potentially results from a developmental delay (Fig. 5). Currently, it remains unclear if control of P-D and A-P axes formation by Pbx factors leads to a concurrent disruption in early D-V polarity, or if the latter D-V disruption is cell autonomous and results from Pbx loss in the ectoderm.

Overall, the loss of multiple Pbx genes from the limb substantially affects the establishment of all three developmental axes and disrupts major signaling centers. It is also highly probable, given the alteration of limb bud positioning and loss of key signaling centers in the early limb bud of Pbx compound mutants, that Pbx homeoproteins exert their roles in early development during limb initiation, prior to the formation of the A-P, P-D, and likely D-V axes (Fig. 4). Experiments by tissue-specific and temporally controllable Pbx deletion in the mouse are ongoing to address all of these open questions.

Pbx Limb Phenotypes and Models of Limb Specification

We have established that the dosage of Pbx1 is paramount, compared to that of Pbx2 and Pbx3, in the developmental control of multiple organs, including the limb and girdle (Capellini et al., 2006, 2008, 2010). We have also observed that Pbx2 is the only Pbx family member that overlaps markedly with Pbx1 in the early limb field (and ectoderm) and is the only Pbx expressed in the distal limb mesoderm after initial bud formation (Fig. 1). Of all compound mutants analyzed, including Pbx1+/−;Pbx2+/−, Pbx1+/−;Pbx2−/−, Pbx1−/−;Pbx3+/−, Pbx1+/−;Pbx3−/−, Pbx2−/−;Pbx3+/−, Pbx2+/−;Pbx3−/−, and Pbx1+/−;Pbx2+/−; Pbx3+/−, only compound Pbx1−/−; Pbx2+/− and Pbx1−/−;Pbx2−/− mutants have drastic P-D limb defects in which each compartment of the forelimb and hindlimb is markedly affected. Multiple known markers of early limb P-D and A-P axis formation are markedly perturbed in these mutants. These experimental findings are in conflict with our observations that single Pbx2−/− and compound Pbx1+/−;Pbx2−/− mutants have phenotypically normal limbs (Selleri et al., 2004; Capellini et al., 2006), and lack changes in limb gene expression. Taken together, these data constitute strong genetic evidence that Pbx1 and Pbx2 control early limb bud formation, in domains wherein they overlap and that Pbx1 plays a prime role in this interaction. We argue that they most likely govern specification and patterning of the limb skeletal elements early in development within the limb field proper.

As several models have been put forth over the last five decades describing how cells become specified to form the individual P-D segments of the limb skeleton (for a detailed review, see Benazet and Zeller, 2009), each of these models (e.g., progress zone model, early specification model, two signal gradient model, and differentiation front model) has specific predictions as to when and where cells of each of the limb compartments are specified, as well as to the roles that specific limb centers (i.e., proximal limb, PZ, AER) have during development (Summerbell et al., 1973; Dudley et al., 2002; Tabin and Wolpert, 2007; Benazet and Zeller, 2009). In this regard, it will be relatively straightforward to test against these temporal requirements using conditionally targeted Pbx alleles, together with specific Cre deleter lines that permit excision of Pbx at specific time points (prior to, or during, early limb field induction versus during limb bud formation) and in select limb spatial compartments (mesoderm versus AER). It is clear from predictions based on preliminary experimental evidence that two key features of Pbx expression (i.e., localization in mesoderm versus AER and differential temporal specificity) must be addressed in depth by genetic dissection. These critical experiments are currently underway. They will pinpoint Pbx functions in the limb and reveal when and where the dominant control of limb axes formation by Pbx homeoproteins occurs during development.

Pbx Roles in Limb Endochondral Ossification

Soon after generating the single Pbx1 loss-of-function mouse model, we reported that the affected embryonic domains of limbs (and ribs) in these mutants displayed markedly diminished chondrocyte proliferation, associated with a notable increase in the number of hypertrophic chondrocytes, accompanied by premature bone ossification (Selleri et al., 2001). Intriguingly, the pattern of expression of genes known to regulate chondrocyte differentiation was not perturbed in Pbx1-deficient cartilage at early days of embryonic skeletogenesis. However, precocious expression of Col1a1, a marker of bone formation, was found in single Pbx1 homozygous mutant embryos. These initial studies established an iterative role for Pbx1 in multiple developmental programs in skeletal formation and revealed a novel function in coordinating the extent and/or timing of proliferation with terminal differentiation during limb endochondral ossification. These later functions of Pbx impact on the rate of endochondral ossification and bone formation (Selleri et al., 2001).

Additionally, it was recently shown that Pbx1 represses osteoblastogenesis by blocking Hoxa10-mediated recruitment of chromatin-remodeling factors in mesenchymal cells. Conversely, targeted depletion of Pbx1 by short hairpin RNA (shRNA) in cultured cells increased expression of osteoblast-related genes. Chromatin-associated Pbx1 and Hoxa10 were present at osteoblast-related gene promoters preceding gene expression, but only Hoxa10 was associated with these promoters during transcription. These interesting results further revealed that Pbx1 is associated with histone deacetylases normally linked with chromatin inactivation. Loss of Pbx1 from osteoblast promoters in differentiated osteoblasts was associated with increased histone acetylation and CBP/p300 recruitment, as well as decreased H3K9 methylation. Based on these studies, conducted in cultured cells, it was proposed that Pbx1 plays a central role in attenuating the ability of Hoxa10 to activate osteoblast-related genes in order to establish temporal regulation of gene expression during osteogenesis (Gordon et al., 2010). It will now be important to corroborate these exciting findings in a system in vivo, possibly in animal models deficient for Pbx proteins.

PBX FUNCTION IN EVOLUTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES

Comparisons of gene sequence data for coding regions of Pbx orthologs indicate that they are evolutionarily conserved among Metazoan taxa (Moens and Selleri, 2006). For example, vertebrates such as mice and humans, and invertebrates, such as flies, share at least 90% identity in coding regions of Pbx1 orthologs. This degree of conservation is even higher at the protein sequence level. This high conservation among Pbx1 genes across different species is also present, though to a lesser extent, among the other orthologs (e.g., Pbx2 across different vertebrates) and even between family members (e.g., between Pbx1 and Pbx2 in vertebrates). These findings suggest that Pbx proteins have remained conserved in terms of general functionality over the course of Metazoan evolution (Moens and Selleri, 2006). However, it is possible that cis- and trans-regulation of Pbx has undergone evolutionary changes leading to differences in the spatio-temporal expression of these genes and their functional domains. Unfortunately, the regulatory elements for Pbx remain unknown and are the subject of current investigations. Regardless of this limitation, it is important to address if there exist functional differences in Pbx genes across different Metazoan taxa, and how such differences could relate to morphological divergence and specialization. At the same time, while comparative data on Pbx gene expression and function in different mammals remain unknown, knowledge of Pbx function during limb and girdle development can be used to address issues of modularity and the manners in which variation is generated by developmental systems. This knowledge can serve as a framework for interpreting evolutionary changes in limb and girdle morphology and function.

exd/Pbx and the Evolution of Invertebrate and Vertebrate Limbs

Comparisons of the expression of Pbx to its invertebrate ortholog, exd, indicate some similarities and differences that may stem from common heritage as well as from functional constraints during evolution (Fig. 6). For example, exd is expressed in the wing and leg imaginal disc, ectodermal tissues critical for appendage patterning in flies (Rieckhof et al., 1997; Mercader et al., 1999). While exd is expressed in other fly embryonic tissues, it does not appear to be expressed in the epithelial-like cap surrounding the imaginal disc. These findings suggest that exd may not be required for regulating tissue-to-tissue interactions in the fly appendage. Conversely, Pbx1 (and Pbx2) are expressed in both the vertebrate limb mesenchyme, a tissue of mesodermal origin, and in the surface ectoderm and AER of the limb and embryo proper (Schnabel et al., 2001; Selleri et al., 2001; unpublished results). Thus, Pbx1 may have essential roles for mesenchymal-epithelial interactions in vertebrate limb development. Other Pbx genes have been discovered only in vertebrates, and their additional domains of expression include both the proximal (Pbx3) as well as the distal (Pbx2) limb mesenchyme throughout limb development (Fig. 1). These vertebrate innovations suggest that heterotopic shifts in the expression of TALE-encoding genes may have occurred during vertebrate evolution as Pbx2 and Pbx3 also play important roles in limb patterning and morphogenesis (Fig. 6). In sum, we propose that de novo use of additional vertebrate Pbx homologs may have led to the evolution of the autopod and its acquisition of specialized functions.

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Figure 6. Comparative expression of TALE-encoding genes and their functions in invertebrate and vertebrate appendage development. Schemata depict comparative Exd/Pbx-mediated invertebrate (left panels) and vertebrate (right panels) proximal-distal appendage development. Blue text denotes ectodermal gene expression, while black text denotes mesenchymal (mesoderm) gene expression. During larval imaginal disc development (top), Exd (and hth, not shown) restrict Dll signaling distally in the invertebrate ectoderm (imaginal disc) setting up distinct proximal and distal zones. This expression pattern is different in vertebrates, in which Pbx genes in the ectoderm are, at this time, upstream regulators of Dlx genes, which are Dll homologs. Furthermore, the entire vertebrate limb is Pbx positive, due to Pbx2 in the distal limb at this stage (i.e., E10). During later-appendage development (middle panels), hedgehog (Hh) signaling in the invertebrate ectoderm acts to repress Exd (hth) to proximal domains and activates other genes distally (not shown), further establishing and compartmentalizing proximal and distal appendicular domains. Also, the fly leg P-D axis develops independently of Hox. In contrast, in vertebrates Pbx genes in the limb mesoderm hierarchically regulate Hox expression, which leads to Shh activation and promotes later distal limb patterning (i.e., at E10–E11). Lastly, once adult limb morphology is established, only the proximal domains of the invertebrate limb are specified by Exd (hth), as based on expression and experiments conducted during imaginal disc stages (see text for details), whereas the entire vertebrate limb requires Pbx function. Proximal (left); anterior (top). Illustrations adapted from Pueyo and Couso (2005).

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Interestingly, the appearance of novel Pbx expression domains in vertebrate appendages, especially throughout limb development, is coupled with the novel expression of other genes encoding proteins that are critical for the proper function of Pbx factors. For example, as in the fly in which hth (the ortholog of vertebrate Meis) is expressed proximally together with exd in the larval imaginal disc (Rieckhof et al., 1995; Capdevila et al., 1999; Mercader et al., 1999), in the mouse, Meis1 and Meis2 are also expressed proximally in E10.5 to E12 limb mesenchyme with Pbx1 (Mercader et al., 1999; Capellini et al., 2006). In addition, both Meis genes are expressed in the ectoderm and AER with Pbx1 and Pbx2 (unpublished results). Furthermore, in the mouse, Pbx2 and Prep1 proteins additionally co-localize in the distal limb mesenchyme at E10.5 (our unpublished data), while in flies during larval imaginal disc development and maturation, the distal appendage is devoid of nuclear Exd and hth protein (data not shown). These comparative data indicate that Pbx/Exd and Meis/hth may have shared a common evolutionary heritage in the ectoderm and only later have appeared in the limb mesenchyme of vertebrates. Future work will have to clarify these relationships throughout evolution.

From an evolutionary point of view, the observed co-localization of both Pbx/Exd and Meis/hth indicates that their functions (and their underlying expression domains) may have co-evolved during Metazoan evolution due to constraints placed by their functional cooperativity. For example, one of their conserved functions may involve Meis/hth ability to control the nuclear importation of Pbx/Exd (discussed above). In a similar fashion, since Pbx/Exd and Meis/hth co-localization results in their ability to form dimeric and trimeric complexes (with or without Hox) that are critical for the regulation of multiple target genes in developmental processes, such a constraint may have limited the functions of these proteins in domains where they overlap.

The findings on TALE gene functions indicate that the mechanisms of P-D and A-P limb patterning may be only partially conserved between invertebrates and vertebrates (Fig. 6). There are indeed some interesting similarities and differences. First, unlike in vertebrates in which biphasic Hox gene expression underlies the development of distal limb domains (Capecchi, 1997; Freitas and Cohn, 2006; Freitas et al., 2007), fly leg P-D axis establishment develops independently of Hox (although the identity of nascent imaginal disc cells may require early Hox activity). Second, mutations in fly exd result in homeotic transformations without altering the expression patterns of Hox genes themselves, thereby suggesting that exd acts in parallel to Hox genes (Peifer and Wieschaus, 1990; Rauskolb et al., 1993; Rieckhof et al., 1997). However, in vertebrates loss of Pbx leads to alterations in Hox expression (Capellini et al., 2006), indicating an additional upstream role. Third, it was once thought that in the fly, exd and hth are restricted to proximal limbs throughout entire wing/leg development (Rieckhof et al., 1997; Capdevila et al., 1999; Mercader et al., 1999) and that exd/hth are required for specification of proximal, but not distal, limb identities (Gonzales-Crespo et al., 1998). However, McKay and colleagues (2009), found that the cells that give rise to the entire distal leg coexpress hth and exd in late embryos, and it is only later, during larval imaginal disc development, that hth is turned off from the distal leg. In this manner, both Exd and hth may be required for overall outgrowth prior to imaginal disc maturation. This may be similar to the early colocalization of Pbx and Meis in the limb bud at induction and initial outgrowth, the period during which Pbx1 and Pbx2 may cooperate to govern development of the entire limb via control of A-P and P-D axes. Fourth, in the fly, exd and hth are restricted to proximal limbs during late larval imaginal disc development (Rieckhof et al., 1997; Capdevila et al., 1999; Mercader et al., 1999) and accordingly Exd has been shown to be required for specification of proximal, but not distal, limb identities at this stage (Gonzales-Crespo et al., 1998). This process occurs partially through their distal repression, and thus restriction of Distaless (Dll) gene expression (Abu-Shaar and Mann, 1998). In vertebrates, such as mice, Pbx appear upstream of Dlx, homologs of Dll, as loss of Pbx1 and Pbx2 causes drastic alterations in the expression of Dlx genes in E10.5 and E11.5 embryos (our unpublished results). Fifth, despite their colocalization early, the roles of hth and Exd serve to antagonistically restrict hedgehog signaling to distal domains leading to P-D and D-V axis establishment (Abu-Shaar and Mann, 1998; Gonzales-Crespo et al., 1998) (Fig. 6). In vertebrates, the situation is different, as Pbx1 and Pbx2 control Hox gene expression and Shh activation (Fig. 6). This vertebrate innovation has, in turn, crucial bearings on A-P formation and digital patterning, as well as on the origins of morphological complexity of distal appendages in tetrapods.

CONCLUDING REMARKS AND FUTURE DIRECTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES

We have discussed the hierarchical, overlapping, and iterative functions of Pbx family members in appendicular skeleton development. Pbx1 and Pbx2 collaborate to control critical effectors of girdle and limb development along all three axes. We have found that such regulation affects many levels of girdle and limb patterning and morphogenesis, from directly controlling morphogens secreted by signaling centers, such as Shh from the limb ZPA, to downstream transcription factor-encoding genes, such as Alx1 in pre-scapular domains. At least in the limb bud, Pbx proteins appear to act not only as Hox cofactors, but also in the upstream control of 5′ HoxA/HoxD gene expression. Our work has additionally established that Pbx1/Pbx2 genetically regulate the development of all post-cranial axial and appendicular skeletal structures and that Pbx1 is the linchpin of these genetic interactions. On the other hand, the unique expression patterns of Pbx3 make this protein an excellent candidate for a highly specific role in early forelimb development. It is of note that Pbx primal control of multiple genetic pathways is not limited to the limb, but involves most developing organs. In many tissues, including the limb, Pbx proteins appear to have critical roles in the control of cell number, by regulating the expression of proliferation and/or apoptosis genes in populations whose fates have already been specified.

Concomitantly, Pbx factors may control directional cell movements in tissues such as the early limb field, where both segmental identity and shape depend on the growth and migration patterns of mesenchymal progenitors. Cutting-edge technology such as live imaging (Nowotschin et al., 2010), lineage tracing in vertebrate models, and computer simulations (Boehm et al., 2010), have recently facilitated an unprecedented analysis of rapidly changing cellular dynamics and intercellular relationships during limb morphogenesis. For example, such technologies have uncovered the importance of oriented cellular behaviours and directional movements of mesenchymal cells migrating into the limb bud (Boehm et al., 2010; Wyngaarden et al., 2010). Application of these new technologies to the characterization of limb defects in Pbx loss-of-function models will shed light into the cellular behaviors that are governed by Pbx homeoproteins in early limb bud development.

In other tissues like muscle and pancreas, Pbx proteins have been shown to act as “pioneer” transcription factors that poise tissue-specific loci for activation by their downstream master regulators. Given the co-localization of multiple partner proteins with Pbx1–3, it is likely that the differential regulation of target genes by Pbx factors requires the formation of context-specific complexes, a process that is still poorly understood. As new imaging, molecular, and biochemical techniques are brought to bear, along with the use of Pbx, Meis, and Prep tissue-specific loss-of-function mouse models, the Pbx-dependent molecular circuitry in different limb compartments at specific developmental times, as well as their control of specific cellular behaviors in limb development, will hopefully gain clarity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES

We are especially grateful to Robert Aho for the preparation of the artwork and to Richard Mann for critical reading of the manuscript and insightful comments. We are thankful to Giuseppina Di Giacomo for performing some of the experiments discussed in this review; Karen Handschuh and Xiao P. Peng for contributing ideas and thoughtful critiques; Liz Lacy, Ann Foley, Filippo Rijli, and James Sharpe for helpful discussions; Michael Cleary for anti-Pbx antibodies; and researchers of the Mouse Genetics Community for in situ probes that facilitated our research. T.C. was the recipient of the CUNY Carole and Morton Olshan Fellowship. All research that made this review possible was supported by grants from: the NIH (2RO1HD043997, 1RO1HD061403, and R21DE018031-02S1 to L.S); March of Dimes and Birth Defects Foundation (6- FY03-071 to L.S.); the AIRC, Italian Association for Cancer Research and the ASM Foundation to V.Z. L.S. is an Irma T. Hirschl Scholar and recipient of research awards from The Alice Bohmfalk Trust and The Frueauff Foundation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. LIMB AND GIRDLE ANATOMY
  5. LIMB AND GIRDLE EMBRYOLOGY: ESTABLISHMENT OF SIGNALING CENTERS
  6. PBX PROTEINS
  7. ROLES OF PBX IN ORGANOGENESIS
  8. PBX GENES IN LIMB DEVELOPMENT
  9. PBX FUNCTION IN EVOLUTION
  10. CONCLUDING REMARKS AND FUTURE DIRECTIONS
  11. Acknowledgements
  12. REFERENCES