Roles for Msx and Dlx homeoproteins in vertebrate development

Gene 247 (2000) 17–31 www.elsevier.com/locate/gene

Review

Roles for Msx and Dlx homeoproteins in vertebrate development A.J. Bendall, C. Abate-Shen * Center for Advanced Biotechnology and Medicine and Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA Received 15 November 1999; received in revised form 12 January 2000; accepted 7 February 2000 Received by A.J. van Wijnen

Abstract This review provides a comparative analysis of the expression patterns, functions, and biochemical properties of Msx and Dlx homeobox genes. These comprise multi-gene families that are closely related with respect to sequence features as well as expression patterns during vertebrate development. Thus, members of the Msx and Dlx families are expressed in overlapping, but distinct, patterns and display complementary or antagonistic functions, depending upon the context. A common theme shared among Msx and Dlx genes is that they are required during early, middle, and late phases of development where their differential expression mediates patterning, morphogenesis, and histogenesis of tissues in which they are expressed. With respect to their biochemical properties, Msx proteins function as transcriptional repressors, while Dlx proteins are transcriptional activators. Moreover, their ability to oppose each other’s transcriptional actions implies a mechanism underlying their complementary or antagonistic functions during development. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Embryogenesis; Gene regulation; Protein–protein interaction; Transcription factors

1. Introduction Homeobox genes illustrate the now prevailing themes that developmental control genes have been highly conserved throughout evolution and that complex organisms acquire multiple copies through duplication of individual ancestral genes. Thus, invertebrate species have relatively few homeobox genes, while vertebrate development is orchestrated by hundreds of homeobox genes that can be classified into subgroups based on the sequence relationships of their homeobox motifs. Msx and Dlx genes, the subject of this review, form two distinct, but closely related, sub-families of homeobox genes that play essential roles during vertebrate development (Davidson, 1995; Stock et al., 1996). Vertebrates have three Msx and at least six Dlx genes ( Table 1 and Fig. 1), in contrast to invertebrates that have single msh/Msx and dll/Dlx genes (Gehring, 1987; Cohen et al., Abbreviations: AER, apical ectodermal ridge; dpc, days postcoitum. * Corresponding author. Tel.: +1-732-235-5161; fax: +1-732-235-4850. E-mail address: [email protected] (C. Abate-Shen)

1989; Holland, 1991; Holland et al., 1996). The accumulation of additional family members in vertebrates has led to expression in new regions as well as the acquisition of novel functions. Consequently, Msx and Dlx genes are primarily expressed in regions that give rise to highly derived or vertebrate-specific structures such as the skull, teeth, limbs, axial and appendicular skeleton, and the tripartite brain. Here, we review the genetic, cellular, and biochemical evidence, which suggests that Msx and Dlx genes function during multiple phases of vertebrate development, as exemplified by their expression patterns and actions during early, middle, and late stages of craniofacial, limb, and nervous system development. Thus, initially, the differential expression patterns of Msx and Dlx genes confer spatial information on the mesenchyme of the branchial arches and limbs. Later, during orofacial and limb morphogenesis, overlapping expression of Msx and Dlx genes mediates signaling between the branchial arch and limb mesenchyme and the adjacent epithelia. In the central nervous system, expression of Msx and Dlx genes at early stages contributes to the spatial restriction of neural induction, their expression at inter-

0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 0 0 ) 0 0 08 1 - 0

18

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

Table 1 Vertebrate Msx and Dlx genesa Mouse/ratb

Human

Msx1= Hox7= Hox7.1 Msx2= Hox8= Hox8.1

Chicken/quail

Frog

Newt/axolotl

Zebrafish

References

MSX1= Msx1= HOX7 G-Hox7

Xmsx1= Xhox7.1

NvMsx-1 AmMsx1

(MsxA) (MsxD=msh-D) (MsxE)

MSX2= Msx2= HOX8 GHox8 Quox-8c

Xhox7.1∞

AmMsx2

(Hill et al., 1989; Robert et al., 1989; Ivens et al., 1990; Su et al., 1991; Suzuki et al., 1991; Akimenko et al., 1995; Crews et al., 1995; Simon et al., 1995; Ekker et al., 1997; Koshiba et al., 1998) (Takahashi and Le Douarin, 1990; Coelho et al., 1991a; Monaghan et al., 1991; Su et al., 1991; Hodgkinson et al., 1993; Jabs et al., 1993; Carlson et al., 1998; Koshiba et al., 1998) (Ekker et al., 1992; Akimenko et al., 1995; Shimeld et al., 1996; Wang et al., 1996) (Price et al., 1991; Simeone et al., 1994; McGuinness et al., 1996; Stock et al., 1996) (Porteus et al., 1991; Robinson et al., 1991; Dirksen et al., 1993; Papalopulu and Kintner, 1993; Selski et al., 1993; Akimenko et al., 1994; Simeone et al., 1994; Fang and Elinson, 1996; McGuinness et al., 1996; Stock et al., 1996; Begbie et al., 1999) (Beauchemin and Savard, 1992; Morasso et al., 1993; Papalopulu and Kintner, 1993; Akimenko et al., 1994; Dirksen et al., 1994; Robinson and Mahon, 1994; Shirasawa et al., 1994; Price et al., 1998) (Papalopulu and Kintner, 1993; Akimenko et al., 1994; Simeone et al., 1994; Zhao et al., 1994; Ferrari et al., 1995; Chen et al., 1996a; Fang and Elinson, 1996) (Asano et al., 1992; Simeone et al., 1994; Chen et al., 1996a; Stock et al., 1996) (Beauchemin and Savard, 1992; Weiss et al., 1994; Nakamura et al., 1996; Stock et al., 1996; Morasso et al., 1997; Quinn et al., 1997)

Msx3 Dlx1=Dlx

DLX1

Dlx2=Tes-1

DLX2

Dlx3 rDlx3

DLX3

Dlx5 rDlx

DLX5

Dlx6

DLX6

Dlx7

DLX7= DLX8= DLX4

MsxB MsxC=msh-C zfdlx1 Dlx2

Xdll4/X-DLL1 EcDlx2

Xdll2/Xdll-2

Dlx5

zfdlx2/zfdlx5

NvHbox4

zfdlx3

Xdll3 EcDlx4

zfdlx4

Xdll

zfdlx6 NvHbox5

zfdlx7/zfdlx8

a As this compilation makes clear, unifying the nomenclature of Msx and Dlx genes suffers from the usual problems of dealing with historical precedence and the belated recognition of orthologous relationships between genes from different organisms. Assignment of zebrafish msx genes to orthologous groups is particularly difficult, based on sequence information and expression patterns ( Ekker et al., 1997), and MsxA, MsxD, and MsxE have been placed in parentheses arbitrarily with Msx1 here. Synonyms for the same gene are separated by an ‘=’. The existance of two very closely related genes in Xenopus and zebrafish is likely to be the result of large-scale genome duplications in those organisms ( Wolf et al., 1969; Kobel and Du Pasquier, 1986). Pseudoallelic genes in these organisms are separated by a ‘/’. b Organisms: Am=axolotl, Ambystoma mexicanum; Ec=frog, Eleutherodactylus coqui; G=chicken, Gallus gallus; Nv=newt, Nopthalmus viridescens; Qu=quail, Coturnix coturnix japonica; r=rat, Rattus norvegicus; X=toad, Xenopus laevis; zf=zebrafish, Danio rerio. c The quail Msx2 gene was initially thought to be orthologous to the murine Msx1/Hox7 gene and so was called Quox7 in the cited reference but was renamed Quox8 in later publications.

Fig. 1. Msx and Dlx genes encode closely related homeodomains. Comparison of the murine Msx and Dlx homeodomains showing residues shared between all members (green), Msx-specific residues (blue) and Dlx-specific residues (yellow). The homeodomain consensus sequence is shown.

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

mediate stages to regional subdivision and morphogenesis, and their expression at later stages to cell-type specification. A notable feature of Msx and Dlx expression patterns is their relationship to cellular proliferation and differentiation in multiple cell lineages that contribute to embryonic and adult structures. Thus, in general, Msx expression is restricted to cells that are proliferating or dying, whereas Dlx expression is primarily found in regions undergoing differentiation, or capable of doing so. Accordingly, Msx and Dlx proteins appear to have opposing transcriptional properties since Msx proteins function as transcriptional repressors, whereas Dlx proteins are activators. Finally, we discuss evidence suggesting that execution of at least some of these biochemical functions requires the homeodomain as a protein interaction interface rather than as a mediator of DNA binding. We conclude with a model in which Msx proteins function as inhibitors of differentiation through transcriptional repression wherein homeodomain-mediated protein–protein interactions preclude productive DNA binding by activators, such as Dlx proteins.

2. Msx and Dlx expression confers spatial information in branchial arch and limb mesenchyme In mammals and birds, the cranial neural crest is a site of early Msx gene expression; indeed, Msx1 and Msx2 expression demarcates the area from which neural crest cells will emigrate ( Hill et al., 1989; Robert et al., 1989; MacKenzie et al., 1991a,b; Graham et al., 1993). Expression in the cranial neural crest continues during cell migration and colonization of the branchial arches where Msx2-expressing cells are restricted to more distal regions of all four branchial arches compared with those expressing Msx1 (MacKenzie et al., 1992; Catron et al., 1996). Dlx genes are expressed in the branchial arches in complex spatiotemporal patterns; broadly speaking, though, Dlx1 and Dlx2 are expressed throughout the first and second arches, whereas Dlx3, Dlx5, and Dlx6 expression is restricted more distally (Dolle´ et al., 1992; Bulfone et al., 1993a; Dirksen et al., 1993; Akimenko et al., 1994; Robinson and Mahon, 1994; Qiu et al., 1997). The nested proximodistal expression patterns of Msx and Dlx genes in the branchial arches can be used to account for the craniofacial phenotypes in mutant mice since proximal arch structures are more sensitive to Msx and Dlx gene deletion than distally derived structures (see below). Msx and Dlx genes are therefore expressed early enough in the cranial neural crest to specify differential fates as these cells populate the branchial arches and subsequently shape the skull and its associated sensory structures. Differential expression of Msx and Dlx genes

19

in the visceral arches suggests that they confer positional identity on arch ectomesenchyme, particularly in the dental field (Sharpe, 1995). However, the craniofacial phenotypes of Msx and Dlx mutant mice can be interpreted with respect to two different models: one in which Msx and Dlx genes confer spatial information in a cellautonomous fashion, and one in which they mediate instructive signaling between ectomesenchyme and arch epithelium. Adding to the complexity, a number of Dlx genes are expressed in craniofacial ectoderm as well as ectomesenchyme (Bulfone et al., 1993a; Robinson and Mahon, 1994; Simeone et al., 1994) where they may contribute to this instructive signaling. Targeted gene disruption of Msx1 in mice affects the shape of several of the membranous calvarial bones of the skull as well as chondrogenic craniofacial bones, both of which are derived from the first branchial arch (Satokata and Maas, 1994). Like Msx1−/− mutant mice, the skulls of Dlx2−/− and Dlx5−/− mutants also contain a number of misshapen bones that are derived from the first and second branchial arches, as well as ectopic bony elements that arise from an excess of cartilaginous mesenchyme migrating into the first arch (Satokata and Maas, 1994; Qiu et al., 1995; Acampora et al., 1999; Depew et al., 1999). It is not clear from the phenotypes whether these defects in craniofacial development result from incorrect positional specification in the visceral arches or whether there is a breakdown in the ectomesenchymal–epithelial signaling that is necessary for cartilaginous condensation (Hall, 1987). In addition, the phenotypes are complex, and there are also secondary effects. For example, Msx1−/− mutant mice have a cleft secondary palate despite the fact that Msx1 is not normally expressed in the affected bones (Satokata and Maas, 1994). Detailed analyses of the ontogenetic processes that lead to skull assembly are therefore needed in order to attribute the phenotypic effects of Msx and Dlx gene disruption to specific functions. The identification of target genes that are regulated by Msx and Dlx proteins in the cranial neural crest will be crucial in this regard. In zebrafish, Msx and Dlx expression appears to regulate three distinct steps of inner-ear development ( Ekker et al., 1992). First, dlx-3 expression in a cluster of ectodermal cells may specify those cells as precursors of the otic vesicle or render them responsive to signals that induce formation of the otic vesicle. Secondly, dlx3 and msh-D expression may specify positional information within the epithelium that orients the subsequent growth of the semicircular canals. Finally, msh-D and msh-C expression in sensory neurons and dlx-3 in epithelial cells may contribute to the specification of inner ear cell types ( Ekker et al., 1992). Inner-ear defects in Dlx5−/− mutant mice are consistent with a role for Dlx5 in the ectoderm, regulating semicircular canal

20

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

Fig. 2. Expression patterns of Msx and Dlx genes during development. Schematic diagram of (A) molar tooth and (B) distal limb development in the mouse showing spatial localization of Msx1 (blue), Msx2 (purple), Dlx1(yellow), Dlx2 (orange), Dlx3 (green), and Dlx5 (red ) gene expression. The apical ectodermal ridge of the 11.5 dpc limb bud is shaded brown to indicate that all six genes are expressed in this tissue. Stripes of color are shown separately for clarity but represent overlapping expression in other areas. A, ameloblasts; dp, dental papilla; EEE, external enamel epithelium; ek, enamel knot; h, humerus condensation; O, odontoblasts; r, radius; SI, stratum intermedium; SR, stellate reticulum; u, ulna.

growth and epithelial cell specification in mammals as well (Acampora et al., 1999; Depew et al., 1999). Non-uniform expression of Msx and Dlx genes in mouse and chick limb mesenchyme also demarcates regions with different cell fates. Msx1 expression is restricted to distal mesenchyme, which ultimately forms the chondrogenic condensations (Hill et al., 1989; Robert et al., 1989; Davidson et al., 1991; Suzuki et al., 1991). Msx2 expression overlaps with Dlx5 along the anterior limb margin in cells that will not contribute to chondrogenic condensations and in a localized region of the posterior mesenchyme, the posterior necrotic zone (Ferrari et al., 1995 and references therein; Zhang et al., 1997). Notably, Msx2 expression in the chick mandible also delineates a non-chondrogenic region that undergoes programmed cell death (Mina et al., 1995). The anterior expression domain of Msx2 and Dlx5 may also demarcate the anterior limit of the AER ( Ferrari et al., 1995). Targeted gene disruption in mice has not yet been informative with respect to limb functions of Msx or Dlx genes.

3. Msx and Dlx genes regulate epithelial–mesenchymal signaling during orofacial and limb development The most compelling evidence for Msx and Dlx gene functions in reciprocal epithelial–mesenchymal signaling comes from studies of tooth development wherein the expression of Msx (MacKenzie et al., 1991a,b, 1992) and Dlx (Bulfone et al., 1993a; Weiss et al., 1994, 1995; Thomas et al., 1995) genes is dynamic ( Fig. 2). Msx2 expression in the neural crest-derived mandibular and maxillary mesenchyme becomes progressively focused until it underlies the epithelium that subsequently thickens to form the dental lamina; Msx2 expression then switches to this epithelial layer (MacKenzie et al., 1992). The dental epithelium now contains instructive signals for tooth formation and Msx1, Msx2, Dlx1, and Dlx2 are each activated in dental mesenchyme in response to BMP and FGF signals from the overlying epithelium (Bei and Maas, 1998). The Bmp4-mediated induction of Msx1 expression and the subsequent Msx1-dependent activation and maintenance of Bmp4 expression in the

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

dental mesenchyme are key steps in conferring odontogenic potential on this tissue. Thereafter, the epithelium loses its ability to instruct naı¨ve mandibular mesenchyme and assumes a more passive role in odontogenesis (Jowett et al., 1993; Vainio et al., 1993; Chen et al., 1996b; Tucker et al., 1998 and reviewed in Thesleff and Nieminen, 1996; Peters and Balling, 1999). The failure of tooth development to progress past the early bud stage in Msx1−/− mutant mice (Satokata and Maas, 1994) and the earlier arrest at the laminar stage in the absence of both Msx1 and Msx2 (Bei and Maas, 1998) emphasizes the essential roles played by Msx genes in mediating these signaling events. While mice lacking either Dlx1 or Dlx2 show no defects of tooth development, Dlx1−/−; Dlx2−/− compound mutant mice specifically lack maxillary molars, indicating sensitivity to Dlx1 and Dlx2 protein levels in a limited subset of teeth (Qiu et al., 1997). Failure of molar development can be attributed to a signaling defect in which the Dlx1−/−; Dlx2−/− mutant mesenchyme cannot respond to the initial odontogenic signals from the maxillary molar epithelium and adopts a cartilaginous fate instead ( Thomas et al., 1997). In humans, a point mutation in the MSX1 homeobox results in inherited autosomal dominant agenesis of second premolars and third molars in affected individuals ( Vastardis et al., 1996). This mutation results in a loss of MSX1 protein function (Hu et al., 1998), which demonstrates the sensitivity of certain teeth to levels of Msx as well as Dlx proteins. Limb outgrowth depends upon reciprocal signaling between a specialized epithelium at the distal tip of the limb bud, the apical ectodermal ridge (AER), and the underlying limb mesenchyme (the progress zone) (reviewed in Cohn and Tickle, 1996; Johnson and Tabin, 1997). Msx1 and Msx2 are expressed in the AER, as are all six Dlx genes ( Fig. 2; Dolle´ et al., 1992; Bulfone et al., 1993a; Akimenko et al., 1994; Zhao et al., 1994; Ferrari et al., 1995; Morasso et al., 1995; Zhang et al., 1997), which suggests a role for these genes in formation of the AER or in regulation of its activities. Conversely, the AER is a source of instructive signals for Msx expression in the underlying mesenchyme, since Msx1 and Msx2 expression is lost from the progress zone of limb buds in which an AER fails to form (Coelho et al., 1991b) or following experimental removal of the AER (Robert et al., 1991). Loss of Msx expression in the progress zone is accompanied by reduced proliferation of mesenchymal cells and a cessation of limb outgrowth. Accordingly, the continued expression of Msx1 and Msx2 in the distal region of tetrapod limbs (Hill et al., 1989; Robert et al., 1989, 1991; Takahashi and Le Douarin, 1990; Coelho et al., 1991a; Davidson et al., 1991) and fish fins (Akimenko et al., 1995) is compatible with outgrowth of the appendages.

21

4. Msx and Dlx genes contribute to multiple stages of vertebrate central nervous system development Development of the vertebrate central nervous system can be thought of as being divided into three phases: (1) neural induction, (2) regional subdivision and morphogenesis, and (3) cell type specification and differentiation. A role for Msx and Dlx genes at each of these stages can be inferred from their normal expression patterns and experimental perturbation of that expression. 4.1. Neural induction: patterning the ectoderm The earliest stage of Msx1 expression in vertebrate embryogenesis has been demonstrated during dorsoventral patterning of the Xenopus gastrula, where it mediates the action of BMP4 (Maeda et al., 1997). In this context, Msx1 induces the ectoderm to adopt an epidermal, rather than neural, fate (Suzuki et al., 1997). More recently, Dlx3 has been shown to be expressed in an overlapping domain with Msx1 in the ventral Xenopus gastrula and to have a complementary function of inhibiting neurogenic genes ( Feledy et al., 1999a). Equivalent roles for Msx in dorsoventral patterning of mammalian gastrulae have not been described, but Dlx5 expression in the presumptive anterior neural ridge of the gastrulating mouse embryo defines the rostral boundary of the neural plate, suggesting an early role in patterning of the mammalian neuroepithelium ( Yang et al., 1998). This may represent a conserved function of Dlx5 genes since equivalent expression is detected in Xenopus (Papalopulu and Kintner, 1993) and chicken (Pera et al., 1999) embryos. This function appears to be compensated for in Dlx5−/− mutant mice, however, since they display no phenotype at this stage (Acampora et al., 1999; Depew et al., 1999). The early phase of Dlx5 expression appears to be discontinuous with its later expression in the forebrain of vertebrate embryos since the neural plate has a distinct embryological origin ( Yang et al., 1998 and references therein). Whether these early functions of Msx and Dlx genes are mediated by the same molecular mechanisms that operate later in development remains to be determined. 4.2. Dlx genes in the vertebrate forebrain: regional specification supports prosomeric models of fore- and midbrain morphogenesis Vertebrate Dlx genes were first isolated in attempts to find homeobox genes that pattern the rostral central nervous system (Porteus et al., 1991; Price et al., 1991; Robinson et al., 1991). Indeed, all Dlx genes, with the exception of Dlx3, are expressed in anterior neural structures (Price, 1993). Beginning around 9.5 dpc in the ventral forebrain of the mouse, the expression pat-

22

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

terns of Dlx1 and Dlx2 are virtually indistinguishable in the basal telencephalon and ventral diencephalon (Porteus et al., 1991; Price et al., 1991, 1992; Robinson et al., 1991; Salinas and Nusse, 1992; Bulfone et al., 1993a,b; Price, 1993), where they broadly overlap with expression of Dlx5 and Dlx6 (Simeone et al., 1994; Liu et al., 1997). The rostrocaudal and dorsoventral expression boundaries of Dlx and other homeobox genes coincide with subsequent morphological boundaries in the fore- and midbrain, suggesting that these genes demarcate functional compartments in the rostral central nervous system (Salinas and Nusse, 1992; Bulfone et al., 1993b; Tole and Patterson, 1995 and references therein) in an analogous way to that in which Hox gene expression underlies segmentation in the hindbrain (Holland and Hogan, 1988; Graham et al., 1989; Kessel and Gruss, 1990). 4.3. Msx genes pattern the neural tube and hindbrain The expression domain of Msx1 at 9.5 dpc in the mouse extends along the entire length of the anteroposterior axis in dorsal neural structures including the cephalic fold, dorsal neural tube, cranial neural crest, and neural pore (Robert et al., 1989). Dorsolateral expression of Msx1 continues in the brain during neurulation where its expression domain becomes more lateral, and in the neural tube where it remains restricted to the dorsal mid-line. Msx3 expression in rhombomeres 3 and 5 is delayed compared with its expression in the rest of the hindbrain (Shimeld et al., 1996) and is complementary to Msx2 expression, rhombomeres 3 and 5 being the earliest sites of Msx2 expression in the hindbrain (Graham et al., 1993). Given that rhombomeres 3 and 5 are specifically depleted of neural crest cells through programmed cell death (Lumsden et al., 1991), Msx2 and Msx3 may have opposing functions in the neurectoderm. 4.4. Specification of neural identity While the anteroposterior and dorsoventral expression boundaries of Dlx1, Dlx2, Dlx5, and Dlx6 are the same, direct comparison of their expression domains reveals differences along the axis of cell differentiation in the developing brain. Thus, Dlx1 and Dlx2 are expressed in the ventricular and subventricular zones, where neuroblasts are proliferating; Dlx5 is expressed in the subventricular zone and mantle, where postmitotic cells are beginning to differentiate; and Dlx6 expression is largely restricted to the mantle, which is the site of terminally differentiated neurons and glia (Porteus et al., 1994; Liu et al., 1997). Recent evidence further suggests that the order of gene activation in these Dlx-positive lineages is Dlx2, Dlx1, then Dlx5 and that individual cells can co-express combinations of Dlx

proteins ( Eisenstat et al., 1999). Functional studies in the mouse demonstrate a specific requirement for Dlx2 in the differentiation of periglomerular neurons in the olfactory bulb (Qiu et al., 1995). Antisense inhibition of Dlx expression in primary cultures of embryonic telencephalon further suggests that Dlx2, but not Dlx1, is required for the normal progression of immature neurons to an intermediate differentiated phenotype (Ding et al., 1997). In mice lacking both Dlx1 and Dlx2, a subset of neurons in the basal telencephalon fail to complete differentiation in, and migration from, the subventricular zone of the basal ganglia (Anderson et al., 1997a,b). Dlx1 and Dlx2 are required for the expression of Dlx5 and Dlx6 in these cells, although they may repress the expression of other homeobox genes, such as Lhx2 (Anderson et al., 1997b) and Lhx5 (Sheng et al., 1997). Msx1, Msx2, and Msx3 are coexpressed in the dorsal neural tube until 10.5 dpc, after which, Msx1 and Msx2 become restricted to the roof plate, and Msx3 becomes restricted to the dorsal ventricular zone, where it has a more ventral expression boundary compared with Msx1 and Msx2 ( Wang et al., 1996). This resolution of Msx gene expression coincides with the time when dorsal spinal cord neurons are being born ( Wang et al., 1996). Thus, like Dlx genes in the forebrain, Msx gene expression is partitioned in spatially discrete domains of the spinal cord that correspond to regions of differential neuronal maturation. Together, these observations suggest the existence of a functional hierarchy of Msx and Dlx genes in neuronal development in which different Msx and Dlx genes have either earlier or later roles in neuron specification and in which the same Msx or Dlx gene can carry out different functions at different times during neurogenesis.

5. Msx and Dlx genes regulate cellular proliferation, differentiation, and death Morphogenesis and histogenesis of embryonic structures represent a balance between the proliferation of precursor cells and their differentiation into mature cell types. This balance must be finely regulated such that progenitor populations are just large enough to supply a sufficient number of cells for appropriately sized tissues. The elimination of cells by programmed cell death also contributes to appropriately sized and shaped structures. Msx1 and Msx2 are expressed in regions of mesenchyme undergoing cellular proliferation as well as those undergoing cell death, suggesting roles in both processes ( Table 2). 5.1. Osteogenesis in the head and limbs While targeted deletion of Msx and Dlx genes has revealed essential roles in regionalization and morpho-

23

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31 Table 2 Expression and function of Msx and Dlx genes in the mouse Gene

Expression patterns

Msx1 Ectoplacental cone, amnion, allantois, uterus, primitive streak, cranial neural crest, branchial arches I–V, cranial sensory placodes, frontonasal process, cranial sutures, eye, tooth, heart, progress zone of limb, migrating myogenic precursors, dorsal ectoderm, apical ectodermal ridge (AER), dorsal neural tube, roof plate Msx2 Primitive streak, cranial neural crest, branchial arches I–V, cranial sensory placodes, frontonasal process, cranial sutures, eye, tooth, heart, progress zone of limb, dorsal ectoderm, AER, dorsal neural tube, roof plate Msx3 Dorsal neural tube, dorsal ventricular zone Dlx1 Branchial arches, tooth bud mesenchyme dental laminae, AER, basal telencephalon, anterior diencephalon (ventricular and subventricular zone), nerve trunks (peripheral nervous system), myenteric parasympathetic ganglia, neural retina Dlx2 Cranial neural crest, branchial arches, dental mesenchyme, genital tubercle, dental epithelium (preameloblasts), frontonasal prominence, otic pit, AER, basal telencephalon, anterior diencephalon (ventricular and subventricular zone), olfactory bulb, cerebral cortex, retina (ganglional layer), dorsal otic vesicle Dlx3

Dlx5

Dlx6

Dlx7

Function/knockout phenotype

References

Perinatal death with cleft secondary palate, tooth development arrested at bud stage, bone defects in skull, middle ear, jaw, nasal region

(Hill et al., 1989; Robert et al., 1989; MacKenzie et al., 1991a,b; Monaghan et al., 1991; Suzuki et al., 1991; Lyons et al., 1992; Chan-Thomas et al., 1993; Pavlova et al., 1994; Satokata and Maas, 1994; Noveen et al., 1995; Friedmann and Daniel, 1996; Phippard et al., 1996; Stelnicki et al., 1997; Bendall et al., 1999; Houzelstein et al., 1999) ( Takahashi and Le Douarin, 1990; Coelho et al., 1991a; Monaghan et al., 1991; Yokouchi et al., 1991; MacKenzie et al., 1992; ChanThomas et al., 1993; Noveen et al., 1995; Friedmann and Daniel, 1996; Phippard et al., 1996; Stelnicki et al., 1997; Bei and Maas, 1998) (Shimeld et al., 1996; Wang et al., 1996) (Price et al., 1991; Dolle´ et al., 1992; Bulfone et al., 1993b; Price, 1993; Liu et al., 1997; Qiu et al., 1997)

Knockout phenotype not reported, tooth development arrested at laminar stage in Msx1; Msx2 compound mutants

Knockout phenotype not reported Death within 1 month of birth with abnormalities in proximal first and second branchial arch derivatives of the head

Perinatal death with abnormal differentiation in forebrain (periglomerular cells in olfactory bulb respecified ). Abnormalities in proximal first and second branchial arch derivatives. Disrupted differentiation of late born neurons in striatum, defect in migration of interneurons from basal forebrain to neocortex, and maxillary molars absent in Dlx1; Dlx2 compound mutants Ectoplacental cone, chorionic plate, labyrin- Early embryonic death from placental failure thine layer, placenta, branchial arches, inner ear, dental papilla (differentiating ameloblasts and ondontoblasts), otic placode and vesicle, epithelial and sensorial ectoderm, AER, whisker follicles, differentiated keratinocytes Branchial arches, nasal mesenchyme, condro- Perinatal death with variety of abnormalities genic condensations, otic vesicle, frontonasal affecting first four branchial arch derivatives prominence ectoderm, olfactory epithelia, ear including proximal mandible, calvaria, olfacossicles, AER, basal telencephalon, anterior tory and otic placodes, teeth diencephalon (strong in subventricular zone, weak in mantle), ganglionic eminence, hypothalamus, perichondrium/periosteum of developing skeletal elements, membrane bone osteoblasts, teeth primordia Generally same as Dlx5 but lower levels of Knockout phenotype not reported mRNA, anterior diencephalon (weak in subventricular zone, strong in mantle) Placenta, retina, trigeminal ganglion, fore- Knockout phenotype not reported brain, inner enamel epithelium of tooth germs, whisker follicles

genesis in the head, other genetic, biochemical, and cellular evidence implies that Msx and Dlx also function later during histogenesis of the bony parts of the skull. In particular, Msx and Dlx proteins appear to have antagonistic activities with respect to proliferation and differentiation of osteogenic precursors. Murine Msx2

(Porteus et al., 1991; Robinson et al., 1991; Bulfone et al., 1993a,b; Price, 1993; Robinson and Mahon, 1994; Qiu et al., 1995; Thomas et al., 1995; Anderson et al., 1997a,b; Liu et al., 1997; Qiu et al., 1997; Thomas et al., 1997)

(Robinson and Mahon, 1994; Morasso et al., 1999)

(Simeone et al., 1994; Zhao et al., 1994; Ferrari et al., 1995; Liu et al., 1997; Acampora et al., 1999; Depew et al., 1999)

(Simeone et al., 1994; Liu et al., 1997)

( Weiss et al., 1994, 1995; Quinn et al., 1997)

expression in osteoblasts in the developing skull bones and teeth precedes expression of Osteocalcin and prevents terminal differentiation of osteoblasts (Newberry et al., 1997a; Bidder et al., 1998). These observations highlight the role of Msx2 in maintaining a balance between proliferation and differentiation of osteoblasts.

24

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

The importance of MSX2 in craniofacial development is further evidenced by a naturally occurring autosomaldominant mutation that is responsible for Boston-type craniosynostosis (premature fusion of calvarial sutures in the pre-natal skull ) (Jabs et al., 1993). This mutant allele results in an amino acid substitution in the N-terminal arm of the homeodomain (Pro148His) that appears to represent a gain-of-function mutation since the mutated MSX2 protein has an increased affinity for DNA (Ma et al., 1996) and since elevated levels of either wild-type or mutant Msx2 in transgenic mice also leads to craniosynostosis (Liu et al., 1995, 1999). Although in-vitro and in-vivo evidence suggest that Dlx5 may repress the Osteocalcin gene (Ryoo et al., 1997; Acampora et al., 1999), other studies have demonstrated transcriptional activation by Dlx5 on Osteocalcin and other osteogenic promoters (Newberry et al., 1998; Miyama et al., 1999). The misshapen and poorly mineralized crowns of the molars of Dlx5 −/− mutant mice (Depew et al., 1999) are consistent with a requirement for Dlx5 in osteoblast differentiation. In humans, a four base pair deletion in the DLX3 gene is responsible for tricho-dento-osseous ( TDO) syndrome in which all affected individuals have enlarged dental pulp chambers and tooth enamel hypoplasia, and a majority have thickened cranial bones (Price et al., 1998). This deletion is predicted to render a truncated protein that is unable to activate transcription (Feledy et al., 1999b). The phenotype of affected individuals is consistent with a model in which a progenitor population in the teeth and skull has overgrown at the expense of differentiated osteoblasts, indicating that Dlx3 transcriptional activity is required for normal maturation of osteoblasts. Dlx5 (and Dlx6) expression is activated in all chondrogenic condensations in the embryo and continues during chondrogenesis, initially throughout the developing skeletal elements and subsequently localized to the perichondrium and periosteum of the axial and appendicular skeleton (Simeone et al., 1994; Zhao et al., 1994; Ferrari et al., 1995; Chen et al., 1996a; Acampora et al., 1999). The overall patterning of the axial skeleton is normal in Dlx5−/− mutant mice, but there are histological bone lesions in the periosteum (Acampora et al., 1999). While the possibility that Dlx5 has both positive and negative activities on the Osteocalcin promoter cannot be ruled out, overall, Dlx5 expression is clearly associated with chondrogenesis and the differentiation of osteoblasts. 5.2. Limb mesenchyme As cells leave the progress zone and differentiate during limb development, Msx1 expression is downregulated. In urodele amphibians, which are capable of limb regeneration as adults, reactivation of Msx1 and Msx2 accompanies the dedifferentiation of mesenchyme that

characterizes the formation of a blastema (Crews et al., 1995; Carlson et al., 1998; Koshiba et al., 1998). Thus, Msx1 appears to maintain the proliferative capacity of the distal mesenchyme by preventing the differentiation of those cells in which it is expressed. Indeed, forced expression of Msx1 efficiently blocks differentiation of multipotent mesodermal cells in the presence of various differentiation stimuli, but does not have a strong mitogenic effect (Song et al., 1992 and G. Hu, A.J.B., C.A.-S., in preparation). These observations suggest that Msx expression maintains cells in a mitotically active state by preventing their terminal differentiation. 5.3. Myogenesis It was unclear for some time whether the ability of Msx1 to block differentiation in myoblast cell lines (Song et al., 1992; Woloshin et al., 1995) was of biological significance since Msx gene expression was not found to be associated with myogenic precursors. Recently, however, transgenic and knock-in analysis using lacZ reporter genes (MacKenzie et al., 1997; Houzelstein et al., 1999) and in-situ analysis with digoxygeninlabeled probes (Bendall et al., 1999) have confirmed that Msx1 is expressed in migrating limb muscle precursor cells. Furthermore, Msx1 suppresses terminal differentiation in these myoblasts by repressing MyoD expression (Bendall et al., 1999). These observations provide a mechanism by which Msx prevents terminal differentiation of precursor cells. 5.4. Proliferative capacity in the adult Msx expression in human and chicken skin also correlates with the proliferative capacity of this tissue. Thus, while Msx1 and Msx2 are expressed in fetal epidermis, hair follicles, and fibroblasts, their expression is restricted to the epidermis in adults, and downregulation of Msx expression in the dermis parallels the loss of its regenerative capacity (Noveen et al., 1995; Stelnicki et al., 1997). Conversely, Dlx3 is expressed in embryonic ectoderm and continues in differentiated adult skin (Morasso et al., 1993, 1995; Dirksen et al., 1994), and forced expression of Dlx3 in basal skin cells induces their premature differentiation (Morasso et al., 1996). An essential role for Dlx3 in skin development cannot be evaluated at this time since Dlx3−/− mutant mice die around 10 dpc due to placental defects (Morasso et al., 1999). In addition to the skin, the mammary gland and uterus epithelium of the non-pregnant adult mouse are sites of post-natal expression of Msx1 and Msx2 (Pavlova et al., 1994; Friedmann and Daniel, 1996; Phippard et al., 1996). Msx expression in these tissues is consistent with the plasticity of uterine and breast epithelial cells and the requirement that both cell types

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

remain developmentally responsive to changes in hormone levels. Expression of Msx1 and Msx2 decreases in the mammary gland during late pregnancy, corresponding to the period of reduced proliferation (Phippard et al., 1996). An additional element of regulation involves the responsiveness of Msx2 to estrogen, which raises the interesting proposition that continued expression of Msx genes links hormonal-directed tissue remodeling with pre-existing pathways of epithelial– mesenchymal signaling (Phippard et al., 1996). 5.6. Msx genes and apoptosis For proliferating cells, death represents an alternative pathway to differentiation. Thus, the final shape of developing structures may also depend upon the selective elimination of cells from some regions, and is usually achieved by promoting apoptosis. Msx2 is expressed in regions of the hindbrain (Graham et al., 1993), cranial neural crest derivatives (Mina et al., 1995; Winograd et al., 1997), teeth (Jernvall et al., 1998), and in necrotic zones of the limb (Chen and Zhao, 1998; Ferrari et al., 1998; Ganan et al., 1998) that undergo programmed cell death in response to developmental cues. Upregulation of Msx2 precedes BMP4-mediated apoptosis in explants of neural crest (Graham et al., 1994) and in cellular model systems (Marazzi et al., 1997), and forced expression of Msx2 induces apoptosis in vivo ( Takahashi et al., 1998). Although Msx1 is coexpressed with Msx2 in some of these same regions, notably the interdigital necrotic zones in the distal limb (Fig. 2), it is not clear whether Msx1 also mediates apoptosis. Indeed, the extent to which Msx1 and Msx2 display similar biochemical and biological activities will require additional studies in which they are directly compared.

6. Msx and Dlx proteins and models of transcriptional control The various biological and cellular activities of Msx and Dlx genes described in the preceding sections are presumably mediated by the homeoproteins that they encode, through their actions as transcriptional regulators. Indeed, Msx and Dlx proteins bind to specific DNA sequences via their respective homeodomains (e.g. Catron et al., 1993). Critical determinants of DNAbinding specificity include residues in the N-terminal arm and helices II and III of the homeodomain ( Ebu Isaac et al., 1995; Zhang et al., 1997; Bendall et al., 1998, 1999). For those Msx and Dlx proteins for whom an optimal DNA-binding site has been identified — Msx1, Msx2, Dlx5, and Dlx3 — the T–A–A–T sequence constitutes the core of the optimal recognition sequence while additional contributions to specificity are con-

25

ferred by one to three flanking nucleotides on either side of the core motif (Catron et al., 1993; Ma et al., 1996; Yang et al., 1998; Feledy et al., 1999b). Transcription studies using reporter plasmids containing these optimal binding sites reveal that Msx proteins are potent transcriptional repressors (Catron et al., 1995, 1996; Semenza et al., 1995; Zhang et al., 1996, 1997; Newberry et al., 1997b) while Dlx proteins, so far as they have been tested, are transcriptional activators ( Zhang et al., 1997; Newberry et al., 1998; Feledy et al., 1999b). Msx and Dlx proteins share several regions of conserved amino acids outside their homeodomains that further distinguish these two subclasses of homeoproteins. Particularly well conserved is a region flanking either side of the Msx or Dlx homeodomains that defines an ‘extended homeodomain’; indeed, the core repression domain of Msx2 consists largely of the extended homeodomain (Catron et al., 1996; Newberry et al., 1997b). The significance of other conserved motifs, at least with respect to their transcriptional activities, is less clear. For example, a region of Msx1 that contains a 12 amino acid motif that is highly conserved among Msx proteins ( Ekker et al., 1997) and that has been identified as a conserved transcription repression domain among several different families of homeoproteins (Smith and Jaynes, 1996) does not contribute significantly to repression by Msx1 (Catron et al., 1995, 1996). Similarly, a conserved block of amino acids near the N-terminus of vertebrate Dlx proteins, termed the ‘Distal-less domain’, is dispensable for activation of transcription by Dlx3, although the activation domain maps to another conserved region (Feledy et al., 1999b). The functional significance of these conserved regions therefore remains conjectural based on these initial biochemical analyses. It remains possible, however, that these conserved domains impart other important functions that have not yet been examined, or that they contribute in the context of the full-length proteins in a way that is not obvious in assays using truncated polypeptides. Alternatively, the extended homeodomain may be sufficient to carry out most of the biological functions of these proteins. In this regard, it is interesting to note that Msx and Dlx orthologous proteins are poorly conserved outside their extended homeodomains (sometimes less than 30% identity among vertebrate genes) compared with other homeoprotein families in which identity over the entire protein may exceed 90% [between fish and mouse Pax proteins, for example ( Westerfield et al., 1993)]. The preferred DNA-binding sites for Msx1, Msx2, Dlx5, and Dlx3 are essentially the same (Catron et al., 1993; Ma et al., 1996; Yang et al., 1998; Feledy et al., 1999b), an observation that underscores the relatedness of Msx and Dlx homeodomains and suggests that these proteins may compete for regulatory elements in vivo. However, competition for DNA-binding sites does not

26

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

Fig. 3. Models of Msx repression. Three potential modes of Msx-mediated repression of activated transcription are shown: direct repression via Msx interaction with the pre-initiation complex (PIC ), or DNA-bound activator (Act), and indirect repression via Msx interaction with activators, thereby preventing DNA binding.

appear to represent the primary mode of regulation, at least for Msx proteins, since transcriptional repression by Msx1 or Msx2 does not require cognate DNAbinding sites (Catron et al., 1995; Semenza et al., 1995). Moreover, a mutation in the N-terminal arm of Msx2 that disrupts DNA binding ( Thr147Ala) has no effect on Msx2-mediated repression (Newberry et al., 1997b). Instead, Msx proteins appear to repress transcription through protein–protein interactions that are mediated by the homeodomain. Msx proteins can bind to core components of the basal transcription machinery that mediate activation, namely TBP in TFIID and RAP74 in TFIIF (Zhang et al., 1996; Newberry et al., 1997b). In so doing, Msx-mediated repression may be direct, either by disrupting the normal assembly of the preinitiation complex or by blocking the accessibility of the basal machinery to interactions with transcriptional activators (Fig. 3). Importantly, though, Msx proteins may also repress transcription indirectly through other types of protein– protein interactions (Fig. 3). For example, Msx proteins can interact with other homeoproteins that are themselves transcriptional activators, forming transcriptionally inactive complexes that cannot bind to DNA. Indeed, heterodimer formation between Msx1 and members of the Dlx, LIM, and Pax homeoprotein families is not compatible with DNA binding of either Msx or its protein partner (Zhang et al., 1997; Bendall et al., 1998, 1999; Bendall and Abate-Shen, unpublished ). This is perhaps not surprising since, in each case, the protein interaction is mediated by the Msx1 homeodomain and the corresponding DNA-binding domains of its partner

( Zhang et al., 1997; Bendall et al., 1998, 1999). Thus, Msx1 and Msx2 can each form a protein complex with Dlx2 and Dlx5, and heterodimer formation has a neutralizing effect on the transcriptional activities of Msx and Dlx proteins on synthetic promoters ( Zhang et al., 1997). Based on these and other observations, we have proposed that such protein–protein interactions result in functional antagonism. The recent finding that Msx1 is present in a multi-protein complex containing a sequence-specific activator (Sp1), a co-activator (CBP/p300), and a basal transcription factor ( TBP) underscores the flexibility of Msx1-mediated repression (Shetty et al., 1999). Antagonism between Msx2 and Dlx5 on the Osteocalcin promoter (Newberry et al., 1998) is also consistent with this model. Msx2-mediated repression of Osteocalcin in osteoblasts appears to depend on its ability to interfere with DNA binding of an activator of Osteocalcin (Newberry et al., 1998). Subsequent expression of Dlx5 in these cells leads to derepression of Osteocalcin by a mechanism in which Dlx5 is proposed to bind to Msx2, freeing the activator complex to bind the Osteocalcin promoter (Newberry et al., 1998). Recent in-vivo studies further suggest that such protein– protein interactions have functional consequences during development. Indeed, protein complex formation between Msx1 and Pax3 may prevent the premature activation of myogenic genes ( like MyoD) in migratory limb muscle precursor cells during their migration from the somites to the growing limb buds (Bendall et al., 1999). In light of these studies, the identification of Msx- and Dlx-type binding sites in promoters and

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

enhancers may not be informative with respect to the identification of in-vivo target genes, although we cannot yet rule out that, at least in certain situations, Msx or Dlx proteins regulate transcription through direct DNA binding.

7. Conclusion Msx and Dlx genes are comparatively well studied, yet much remains to be discovered concerning their mode of action. Future progress in this area will depend upon the identification of target genes for these regulatory proteins in the different cell types and embryological regions where they are expressed. Given the unusual mechanism by which Msx proteins regulate transcription, a productive way to search for target genes would be via the identification of other protein partners for Msx (and Dlx) proteins. Deciphering the functional redundancies between Msx and Dlx genes will require the analysis of additional compound mutant mice as well as those having tissue-specific gene disruptions. Another question that arises from the consideration of redundancy is how much of the biological function of individual Msx and Dlx proteins is dictated by intrinsically different biochemical properties and how much is context-dependent, being influenced by the molecular environment of the tissues in which they are expressed. Since Msx and Dlx proteins are used in a variety of developmental processes, understanding their functions promises to be of general interest to those who want to understand the elaboration of homeoprotein function during vertebrate development.

Acknowledgements We thank members of the Abate-Shen and Shen laboratories for critical reading of the manuscript and the anonymous reviewers for their thoughtful comments. This work is supported by an NIH grant (HD29446) to C.A.-S. and a post-doctoral fellowship from the American Heart Association to A.B.

References Acampora, D., Merlo, G.R., Paleari, L., Zerega, B., Postiglione, M.P., Mantero, S., Bober, E., Barbieri, O., Simeone, A., Levi, G., 1999. Craniofacial, vestibular and bone defects in mice lacking the Distalless-related gene Dlx5. Development 126, 3795–3809. Akimenko, M.A., Ekker, M., Wegner, J., Lin, W., Westerfield, M., 1994. Combinatorial expression of three zebrafish genes related to Distal-Less: part of a homeobox gene code for the head. J. Neurosci. 14, 3475–3486. Akimenko, M.A., Johnson, S.L., Westerfield, M., Ekker, M., 1995. Differential induction of four msx homeobox genes during fin

27

development and regeneration in zebrafish. Development 121, 347–357. Anderson, S.A., Eisenstat, D.D., Shi, L., Rubenstein, J.L., 1997a. Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes. Science 278, 474–476. Anderson, S.A., Qiu, M., Bulfone, A., Eisenstat, D.D., Meneses, J., Pedersen, R., Rubenstein, J.L.R., 1997b. Mutations of the homeobox genes Dlx-1 and Dlx-2 disrupt the striatal subventricular zone and differentiation of late born striatal neurons. Neuron 19, 27–37. Asano, M., Emori, Y., Saigo, K., Shiokawa, K., 1992. Isolation and characterization of a Xenopus cDNA which encodes a homeodomain highly homologous to Drosophila Distal-less. J. Biol. Chem. 267, 5044–5047. Beauchemin, M., Savard, P., 1992. Two distal-less related homeoboxcontaining genes expressed in regeneration blastemas of the newt. Dev. Biol. 154, 55–65. Begbie, J., Brunet, J.F., Rubenstein, J.L., Graham, A., 1999. Induction of the epibranchial placodes. Development 126, 895–902. Bei, M., Maas, R., 1998. FGFs and BMP4 induce both Msx1-independent and Msx1-dependent signaling pathways in early tooth development. Development 125, 4325–4333. Bendall, A.J., Rincon-Limas, D., Botas, J., Abate-Shen, C., 1998. Protein complex formation between Msx1 and Lhx2 homeoproteins is incompatible with DNA binding activity. Differentiation 63, 151–157. Bendall, A.J., Ding, J., Hu, G., Shen, M.M., Abate-Shen, C., 1999. Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors. Development 126, 4965–4976. Bidder, M., Latifi, T., Towler, D.A., 1998. Reciprocal temporospatial patterns of Msx2 and Osteocalcin gene expression during murine odontogenesis. J. Bone Miner. Res. 13, 609–619. Bulfone, A., Kim, H.J., Puelles, L., Porteus, M.H., Grippo, J.F., Rubenstein, J.L., 1993a. The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos. Mech. Dev. 40, 129–140. Bulfone, A., Puelles, L., Porteus, M.H., Frohman, M.A., Martin, G.R., Rubenstein, J.L., 1993b. Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J. Neurosci. 13, 3155–3172. Carlson, M.R., Bryant, S.V., Gardiner, D.M., 1998. Expression of Msx-2 during development, regeneration, and wound healing in axolotl limbs. J. Exp. Zool. 282, 715–723. Catron, K.M., Iler, N., Abate, C., 1993. Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins. Mol. Cell. Biol. 13, 2354–2365. Catron, K.M., Zhang, H., Marshall, S.C., Inostroza, J.A., Wilson, J.M., Abate, C., 1995. Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites. Mol. Cell. Biol. 15, 861–871. Catron, K.M., Wang, H., Hu, G., Shen, M.M., Abate-Shen, C., 1996. Comparison of MSX-1 and MSX-2 suggests a molecular basis for functional redundancy. Mech. Dev. 55, 185–199. Chan-Thomas, P.S., Thompson, R.P., Robert, B., Yacoub, M.H., Barton, P.J.R., 1993. Expression of homeobox genes Msx1 (Hox7) and Msx-2 (Hox-8) during cardiac development in the chick. Dev. Dyn. 197, 203–216. Chen, X., Li, X., Wang, W., Lufkin, T., 1996a. Dlx5 and Dlx6: an evolutionary conserved pair of murine homeobox genes expressed in the embryonic skeleton. Ann. NY Acad. Sci. 785, 38–47. Chen, Y., Bei, M., Woo, I., Satokata, I., Maas, R., 1996b. Msx1 controls inductive signaling in mammalian tooth morphogenesis. Development 122, 3035–3044. Chen, Y., Zhao, X., 1998. Shaping limbs by apoptosis. J. Exp. Zool. 282, 691–702. Coelho, C.N., Sumoy, L., Rodgers, B.J., Davidson, D.R., Hill, R.E., Upholt, W.B., Kosher, R.A., 1991a. Expression of the chicken

28

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

homeobox-containing gene GHox-8 during embryonic chick limb development. Mech. Dev. 34, 143–154. Coelho, C.N.D., Krabbenhoft, K.M., Upholt, W.B., Fallon, J.F., Kosher, R.A., 1991b. Altered expression of the chicken homeoboxcontaining genes GHox-7 and GHox-8 in the limb buds of limbless mutant chick embryos. Development 113, 1487–1493. Cohen, S.M., Bronner, G., Kuttner, F., Jurgens, G., Jackle, H., 1989. Distal-less encodes a homoeodomain protein required for limb development in Drosophila. Nature 338, 432–434. Cohn, M.J., Tickle, C., 1996. Limbs: a model for the pattern formation within the vertebrate body plan. Trends Genet. 12, 253–257. Crews, L., Gates, P.B., Brown, R., Joliot, A., Foley, C., Brockes, J.P., Gann, A.A., 1995. Expression and activity of the newt Msx-1 gene in relation to limb regeneration. Proc. R. Soc. Lond. B Biol. Sci. 259, 161–171. Davidson, D.R., Crawley, A., Hill, R.E., Tickle, C., 1991. Positiondependent expression of two related homeobox genes in developing vertebrate limbs. Nature 352, 429–431. Davidson, D., 1995. The function and evolution of Msx genes: pointers and paradoxes. Trends Genet. 11, 405–411. Depew, M.J., Liu, J.K., Long, J.E., Presley, R., Meneses, J.J., Pedersen, R.A., Rubenstein, J.L.R., 1999. Dlx5 regulates regional development of the branchial arches and sensory capsules. Development 126, 3831–3846. Ding, M., Robel, L., James, A.J., Eisenstat, D.D., Leckman, J.F., Rubenstein, J.L., Vaccarino, F.M., 1997. Dlx-2 homeobox gene controls neuronal differentiation in primary cultures of developing basal ganglia. J. Mol. Neurosci. 8, 93–113. Dirksen, M.L., Mathers, P., Jamrich, M., 1993. Expression of a Xenopus Distal-less homeobox gene involved in forebrain and craniofacial development. Mech. Dev. 41, 121–128. Dirksen, M.L., Morasso, M.I., Sargent, T.D., Jamrich, M., 1994. Differential expression of a Distal-less homeobox gene Xdll-2 in ectodermal cell lineages. Mech. Dev. 46, 63–70. Dolle´, P., Price, M., Duboule, D., 1992. Expression of the murine Dlx1 homeobox gene during facial, ocular and limb development. Differentiation 49, 93–99. Ebu Isaac, V., Sciavolino, P., Abate, C., 1995. Multiple amino acids determine the DNA binding specificity of the Msx-1 homeodomain. Biochemistry 34, 7127–7134. Eisenstat, D.D., Liu, J.K., Mione, M., Zhong, W., Yu, G., Anderson, S.A., Ghattas, I., Puelles, L., Rubenstein, J.L., 1999. DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation. J. Comp. Neurol. 414, 217–237. Ekker, M., Akimenko, M.A., Bremiller, R., Westerfield, M., 1992. Regional expression of three homeobox transcripts in the inner ear of zebrafish embryos. Neuron 9, 27–35. Ekker, M., Akimenko, M.A., Allende, M.L., Smith, R., Drouin, G., Langille, R.M., Weinberg, E.S., Westerfield, M., 1997. Relationships among msx gene structure and function in zebrafish and other vertebrates. Mol. Biol. Evol. 14, 1008–1022. Fang, H., Elinson, R.P., 1996. Patterns of distal-less gene expression and inductive interactions in the head of the direct developing frog Eleutherodactylus coqui. Dev. Biol. 179, 160–172. Feledy, J.A., Beanan, M.J., Sandoval, J.J., Goodrich, J.S., Lim, J.H., Matsuo-Takasaki, M., Sato, S.M., Sargent, T.D., 1999a. Inhibitory patterning of the anterior neural plate in Xenopus by homeodomain factors Dlx3 and Msx1. Dev. Biol. 212, 455–464. Feledy, J.A., Morasso, M.I., Jang, S.I., Sargent, T.D., 1999b. Transcriptional activation by the homeodomain protein distal-less 3. Nucleic Acids Res. 27, 764–770. Ferrari, D., Sumoy, L., Gannon, J., Sun, H., Brown, A.M., Upholt, W.B., Kosher, R.A., 1995. The expression pattern of the Distalless homeobox-containing gene Dlx-5 in the developing chick limb bud suggests its involvement in apical ectodermal ridge activity, pattern formation and cartilage differentiation. Mech. Dev. 52, 257–264.

Ferrari, D., Lichtler, A.C., Pan, Z.Z., Dealy, C., Upholt, W.B., Kosher, R.A., 1998. Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP4 expression, inhibiting cell proliferation and promoting apoptosis. Dev. Biol. 197, 12–24. Friedmann, Y., Daniel, C.W., 1996. Regulated expression of homeobox genes Msx-1 and Msx-2 in mouse mammary gland development suggests a role in hormone action and epithelial–stromal interactions. Dev. Biol. 177, 347–355. Ganan, Y., Macias, D., Basco, R.D., Merino, R., Hurle, J.M., 1998. Morphological diversity of the avian foot is related with the pattern of msx gene expression in the developing autopod. Dev. Biol. 196, 33–41. Gehring, W.J., 1987. The homeobox: structural and evolutionary aspects. In: Firtel, R.A., Davidson, E.H. (Eds.), Molecular Approaches to Developmental Biology. Alan R. Liss, New York, pp. 115–129. Graham, A., Papalopulu, N., Krumlauf, R., 1989. The murine and Drosophila homeobox gene complexes have common features of organization and expression. Cell 57, 367–378. Graham, A., Heyman, I., Lumsden, A., 1993. Even-numbered rhombomeres control the apoptotic elimination of neural crest cells from odd-numbered rhombomeres in the chick hindbrain. Development 119, 233–245. Graham, A., Francis-West, P., Brickell, P., Lumsden, A., 1994. The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest. Nature 372, 684–686. Hall, B.K., 1987. Tissue interactions in the development of the vertebrate head. In: Maderson, P.F.A. ( Ed.), Developmental and Evolutionary Aspects of the Neural Crest. Wiley, New York, pp. 215–259. Hill, R.E., Jones, P.F., Rees, A.R., Sime, C.M., Justice, M.J., Copeland, N.G., Jenkins, N.A., Graham, E., Davidson, D.R., 1989. A new family of mouse homeo box-containing genes: molecular structure, chromosomal location and developmental expression of Hox-7.1. Genes Dev. 3, 26–37. Hodgkinson, J.E., Davidson, C.L., Beresford, J., Sharpe, P.T., 1993. Expression of a human homeobox-containing gene is regulated by 1,25(OH )2D3 in bone cells. Biochim. Biophys. Acta 1174, 11–16. Holland, P.W., Hogan, B.L., 1988. Expression of homeo box genes during mouse development: a review. Genes Dev. 2, 773–782. Holland, P.W.H., 1991. Cloning and evolutionary analysis of msh-like homeobox genes from mouse, zebrafish and ascidian. Gene 98, 253–257. Holland, N.D., Panganiban, G., Henyey, E.L., Holland, L.Z., 1996. Sequence and developmental expression of AmphiDll, an amphioxus Distal-less gene transcribed in the ectoderm, epidermis and nervous system: insights into evolution of craniate forebrain and neural crest. Development 122, 2911–2920. Houzelstein, D., Auda-Boucher, G., Che´raud, Y., Rouaud, T., Blanc, I., Tajbakhsh, S., Buckingham, M.E., Fontaine-Pe´rusRobert, B., 1999. The homeobox gene Msx1 is expressed in a subset of somites, and in muscle progenitor cells migrating into the forelimb. Development 126, 2689–2701. Hu, G., Vastardis, H., Bendall, A.J., Wang, Z., Logan, M., Zhang, H., Nelson, C., Stein, S., Greenfield, N., Seidman, C.E., Seidman, J.G., Abate-Shen, C., 1998. Haploinsufficiency of MSX1: A mechanism for selective tooth agenesis. Mol. Cell. Biol. 18, 6044–6051. Ivens, A., Flavin, N., Williamson, R., Dixon, M., Bates, G., Buckingham, M., Robert, B., 1990. The human homeobox gene HOX7 maps to chromosome 4p16.1 and may be implicated in Wolf–Hirschhorn syndrome. Hum. Genet. 84, 473–476. Jabs, E.W., Mu¨ller, U., Li, X., Ma, L., Luo, W., Haworth, I.S., Klisak, I., Sparkes, R., Warman, M.L., Mulliken, J.B., Snead, M.L., Maxson, R., 1993. A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis. Cell 75, 443–450.

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31 ˚ berg, T., Kettunun, P., Kera¨nen, S., Thesleff, I., 1998. Jernvall, J., A The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125, 161–169. Johnson, R.L., Tabin, C.J., 1997. Molecular models for vertebrate limb development. Cell 90, 979–990. Jowett, A.K., Vainio, S., Ferguson, M.W., Sharpe, P.T., Thesleff, I., 1993. Epithelial-mesenchymal interactions are required for msx 1 and msx 2 gene expression in the developing murine molar tooth. Development 117, 461–470. Kessel, M., Gruss, P., 1990. Murine developmental control genes. Science 249, 374–379. Kobel, H.R., Du Pasquier, L., 1986. Genetics of polyploid Xenopus. Trends Genet. 2, 310–315. Koshiba, K., Kuroiwa, A., Yamamoto, H., Tamura, K., Ide, H., 1998. Expression of Msx genes in regenerating and developing limbs of axolotl. J. Exp. Zool. 282, 703–714. Liu, Y.H., Kundu, R., Wu, L., Luo, W., Ignelzi Jr., M.A., Snead, M.L., Maxson Jr., R.E., 1995. Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull. Proc. Natl. Acad. Sci. USA 92, 6137–6141. Liu, J.K., Ghattas, I., Liu, S., Chen, S., Rubenstein, J.L., 1997. Dlx genes encode DNA-binding proteins that are expressed in an overlapping and sequential pattern during basal ganglia differentiation. Dev. Dyn. 210, 498–512. Liu, Y.H., Tang, Z., Kundu, R.K., Wu, L., Luo, W., Zhu, D., Sangiorgi, F., Snead, M.L., Maxson Jr., R.E., 1999. MSX2 gene dosage influences the number of proliferative osteogenic cells in growth centers of the developing murine skull: a possible mechanism for MSX2-mediated craniosynostosis in humans. Dev. Biol. 205, 260–274. Lumsden, A., Sprawson, N., Graham, A., 1991. Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development 113, 1281–1291. Lyons, G.E., Houzelstein, D., Sassoon, D., Robert, B., Buckingham, M.E., 1992. Multiple sites of Hox-7 expression during mouse embryogenesis: comparison with retinoic acid receptor mRNA localization. Mol. Reprod. Dev. 32, 303–314. Ma, L., Golden, S., Wu, L., Maxson, R., 1996. The molecular basis of Boston-type craniosynostosis: the Pro148His mutation in the N-terminal arm of the MSX2 homeodomain stabilizes DNA binding without altering nucleotide sequence preferences. Hum. Mol. Genet. 5, 1915–1920. MacKenzie, A., Ferguson, M.W.J., Sharpe, P.T., 1991a. Hox-7 expression during murine craniofacial development. Development 113, 601–611. MacKenzie, A., Leeming, G.L., Jowett, A.K., Ferguson, M.W.J., Sharpe, P.T., 1991b. The homeobox gene Hox 7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth development in vivo and in vitro. Development 111, 269–285. MacKenzie, A., Ferguson, M.W.J., Sharpe, P.T., 1992. Expression patterns of the homeobox gene, Hox-8, in the mouse embryo suggest a role in specifying tooth initiation and shape. Development 115, 403–420. MacKenzie, A., Purdie, L., Davidson, D., Collinson, M., Hill, R.E., 1997. Two enhancer domains control early aspects of the complex expression pattern of Msx1. Mech. Dev. 62, 29–40. McGuinness, T., Porteus, M.H., Smiga, S., Bulfone, A., Kingsley, C., Qiu, M., Liu, J.K., Long, J.E., Xu, D., Rubenstein, J.L., 1996. Sequence, organization, and transcription of the Dlx-1 and Dlx-2 locus. Genomics 35, 473–485. Maeda, R., Kobayashi, A., Sekine, R., Lin, J.J., Kung, H.-F., Mae´no, M., 1997. Xmsx-1 modifies mesodermal tissue pattern along dorsoventral axis in Xenopus laevis embryo. Development 124, 2553–2560. Marazzi, G., Wang, Y., Sassoon, D., 1997. Msx2 is a transcriptional

29

regulator in the BMP4-mediated programmed cell death pathway. Dev. Biol. 186, 127–138. Mina, M., Gluhak, J., Upholt, W.B., Kollar, E.J., Rogers, B., 1995. Experimental analysis of Msx-1 and Msx-2 gene expression during chick mandibular morphogenesis. Dev. Dyn. 202, 195–214. Miyama, K., Yamada, G., Yamamoto, T.S., Takagi, C., Miyado, K., Sakai, M., Ueno, N., Shibuya, H., 1999. A BMP-inducible gene, Dlx5, regulates osteoblast differentiation and mesoderm induction. Dev. Biol. 208, 123–133. Monaghan, A.P., Davidson, D.R., Sime, C., Graham, E., Baldock, R., Bhattacharya, S.S., Hill, R.E., 1991. The Msh-like homeobox genes define domains in the developing vertebrate eye. Development 112, 1053–1061. Morasso, M.I., Jamrich, M., Sargent, T.D., 1993. The homeodomain gene Xenopus Distal-less-like-2 (Xdll-2) is regulated by a conserved mechanism in amphibian and mammalian epidermis. Dev. Biol. 162, 267–276. Morasso, M.I., Mahon, K.A., Sargent, T.D., 1995. A Xenopus Distalless gene in transgenic mice: conserved regulation in distal limb epidermis and other sites of epithelial–mesenchymal interaction. Proc. Natl. Acad. Sci. USA 92, 3968–3972. Morasso, M.I., Markova, N.G., Sargent, T.D., 1996. Regulation of epidermal differentiation by a Distal-less homeodomain gene. J. Cell Biol. 135, 1879–1887. Morasso, M.I., Yonescu, R., Griffin, C.A., Sargent, T.D., 1997. Localization of human DLX8 to chromosome 17q21.3–q22 by fluorescence in-situ hybridization. Mamm. Genome 8, 302–303. Morasso, M.I., Grinberg, A., Robinson, G., Sargent, T.D., Mahon, K.A., 1999. Placental failure in mice lacking the homeobox gene Dlx3. Proc. Natl. Acad. Sci. USA 96, 162–167. Nakamura, S., Stock, D.W., Wydner, K.L., Bollekens, J.A., Takeshita, K., Nagai, B.M., Chiba, S., Kitamura, T., Freeland, T.M., Zhao, Z., Minowada, J., Lawrence, J.B., Weiss, K.M., Ruddle, F.H., 1996. Genomic analysis of a new mammalian distal-less gene: Dlx7. Genomics 38, 314–324. Newberry, E.P., Boudreaux, J.M., Towler, D.A., 1997a. Stimulusselective inhibition of rat osteocalcin promoter induction and protein–DNA interactions by the homeodomain repressor Msx2. J. Biol. Chem. 272, 29607–29613. Newberry, E.P., Latifi, T., Battaile, J.T., Towler, D.A., 1997b. Structure–function analysis of Msx2-mediated transcriptional suppression. Biochemistry 36, 10451–10462. Newberry, E.P., Latifi, T., Towler, D.A., 1998. Reciprocal regulation of osteocalcin transcription by the homeodomain proteins Msx2 and Dlx5. Biochemistry 37, 16360–16368. Noveen, A., Jiang, T.X., Ting-Berreth, S.A., Chuong, C.M., 1995. Homeobox genes Msx-1 and Msx-2 are associated with induction and growth of skin appendages. J. Invest. Dermatol. 104, 711–719. Papalopulu, N., Kintner, C., 1993. Xenopus Distal-less related homeobox genes are expressed in the developing forebrain and are induced by planar signals. Development 117, 961–975. Pavlova, A., Boutin, E., Cunha, G., Sassoon, D., 1994. Msx1 (Hox-7.1) in the adult mouse uterus: cellular interactions underlying regulation of expression. Development 120, 335–346. Pera, E., Stein, S., Kessel, M., 1999. Ectodermal patterning in the avian embryo: epidermis versus neural plate. Development 126, 63–73. Peters, H., Balling, R., 1999. Teeth. Where and how to make them. Trends Genet. 15, 59–65. Phippard, D.J., Weber-Hall, S.J., Sharpe, P.T., Naylor, M.S., Jayatalake, H., Maas, R., Woo, I., Roberts-Clark, D., Francis-West, P.H., Liu, Y.H., Maxson, R., Hill, R.E., Dale, T.C., 1996. Regulation of Msx-1, Msx-2, Bmp-2 and Bmp-4 during foetal and postnatal mammary gland development. Development 122, 2729–2737. Porteus, M.H., Bulfone, A., Ciaranello, R.D., Rubenstein, J.L.R., 1991. Isolation and characterization of a novel cDNA clone encod-

30

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31

ing a homeodomain that is developmentally regulated in the ventral forebrain. Neuron 7, 221–229. Porteus, M.H., Bulfone, A., Liu, J.K., Puelles, L., Lo, L.C., Rubenstein, J.L., 1994. DLX-2, MASH-1, and MAP-2 expression and bromodeoxyuridine incorporation define molecularly distinct cell populations in the embryonic mouse forebrain. J. Neurosci. 14, 6370–6383. Price, M., Lemaistre, M., Pischetola, M., Di Lauro, R., Duboule, D., 1991. A mouse gene related to Distal-less shows a restricted expression in the developing forebrain. Nature 351, 748–751. Price, M., Lazzaro, D., Pohl, T., Mattei, M.G., Ruther, U., Olivo, J.C., Duboule, D., Di Lauro, R., 1992. Regional expression of the homeobox gene Nkx-2.2 in the developing mammalian forebrain. Neuron 8, 241–255. Price, M., 1993. Members of the Dlx- and Nkx2-gene families are regionally expressed in the developing forebrain. J. Neurobiol. 24, 1385–1399. Price, J.A., Bowden, D.W., Wright, J.T., Pettenati, M.J., Hart, T.C., 1998. Identification of a mutation in DLX3 associated with trichodento- osseous ( TDO) syndrome. Hum. Mol. Genet. 7, 563–569. Qiu, M., Bulfone, A., Martinez, S., Meneses, J.J., Shimamura, K., Pedersen, R.A., Rubenstein, J.L., 1995. Null mutation of Dlx-2 results in abnormal morphogenesis of proximal first and second branchial arch derivatives and abnormal differentiation in the forebrain. Genes Dev. 9, 2523–2538. Qiu, M., Bulfone, A., Ghattas, I., Meneses, J.J., Christensen, L., Sharpe, P.T., Presley, R., Pedersen, R.A., Rubenstein, J.L., 1997. Role of the Dlx homeobox genes in proximodistal patterning of the branchial arches: mutations of Dlx-1, Dlx-2 and Dlx-1 and -2 alter morphogenesis of proximal skeletal and soft tissue structures derived from the first and second arches. Dev. Biol. 185, 165–184. Quinn, L.M., Johnson, B.V., Nicholl, J., Sutherland, G.R., Kalionis, B., 1997. Isolation and identification of homeobox genes from the human placenta including a novel member of the Distal-less family, DLX4. Gene 187, 55–61. Robert, B., Sassoon, D., Jacq, B., Gehring, W., Buckingham, M., 1989. Hox-7, a mouse homeobox gene with a novel pattern of expression during embryogenesis. EMBO J. 8, 91–100. Robert, B., Lyons, G., Simandl, B.K., Kuroiwa, A., Buckingham, M., 1991. The apical ectodermal ridge regulates Hox-7 and Hox-8 gene expression in developing chick limb buds. Genes Dev. 5, 2363–2374. Robinson, G.W., Wray, S., Mahon, K.A., 1991. Spatially restricted expression of a member of a new family of murine Distal-less homeobox genes in the developing forebrain. New Biol. 3, 1183–1194. Robinson, G.W., Mahon, K.A., 1994. Differential and overlapping expression domains of Dlx-2 and Dlx-3 suggest distinct roles for Distal-less homeobox genes in craniofacial development. Mech. Dev. 48, 199–215. Ryoo, H.M., Hoffmann, H.M., Beumer, T., Frenkel, B., Towler, D.A., Stein, G.S., Stein, J.L., van Wijnen, A.J., Lian, J.B., 1997. Stagespecific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression. Mol. Endocrinol. 11, 1681–1694. Salinas, P.C., Nusse, R., 1992. Regional expression of the Wnt-3 gene in the developing mouse forebrain in relationship to diencephalic neuromeres. Mech. Dev. 39, 151–160. Satokata, I., Maas, R., 1994. Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat. Genet. 6, 348–355. Selski, D.J., Thomas, N.E., Coleman, P.D., Rogers, K.E., 1993. The human brain homeogene, DLX-2: cDNA sequence and alignment with the murine homologue. Gene 132, 301–303. Semenza, G.L., Wang, G.L., Kundu, R., 1995. DNA binding and transcriptional properties of wild-type and mutant forms of the

homeodomain protein Msx2. Biochem. Biophys. Res. Commun. 209, 257–262. Sharpe, P.T., 1995. Homeobox genes and orofacial development. Connect. Tissue Res. 32, 17–25. Sheng, H.Z., Bertuzzi, S., Chiang, C., Shawlot, W., Taira, M., Dawid, I., Westphal, H., 1997. Expression of murine Lhx5 suggests a role in specifying the forebrain. Dev. Dyn. 208, 266–277. Shetty, S., Takahashi, T., Matsui, H., Ayengar, R., Raghow, R., 1999. Transcriptional autorepression of Msx1 gene is mediated by interactions of Msx1 protein with a multi-protein transcriptional complex containing TATA-binding protein, Sp1 and cAMPresponse-element-binding protein- binding protein (CBP/p300). Biochem. J. 339, 751–758. Shimeld, S.M., McKay, I.J., Sharpe, P.T., 1996. The murine homeobox gene Msx-3 shows highly restricted expression in the developing neural tube. Mech. Dev. 55, 201–210. Shirasawa, T., Sakamoto, K., Tkahashi, H., 1994. Molecular cloning and evolutional analysis of a mammalian homologue of the Distalless 3 (Dlx-3) homeobox gene. FEBS Lett. 351, 380–384. Simeone, A., Acampora, D., Pannese, M., D’Esposito, M., Stornaiuolo, A., Gulisano, M., Mallamaci, A., Kastury, K., Druck, T., Huebner, K., Boncinelli, E., 1994. Cloning and characterization of two members of the vertebrate Dlx gene family. Proc. Natl. Acad. Sci. USA 91, 2250–2254. Simon, H.G., Nelson, C., Goff, D., Laufer, E., Morgan, B.A., Tabin, C., 1995. Differential expression of myogenic regulatory genes and Msx1 during dedifferentiation and redifferentiation of regenerating amphibian limbs. Dev. Dyn. 202, 1–12. Smith, S.T., Jaynes, J.B., 1996. A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2-, and msh-class homeoproteins, mediates active transcriptional repression in vivo. Development 122, 3141–3150. Song, K., Wang, Y., Sassoon, D., 1992. Expression of Hox-7.1 in myoblasts inhibits terminal differentiation and induces cell transformation. Nature 360, 477–481. Stelnicki, E.J., Ko¨mu¨ves, L.G., Holmes, D., Clavin, W., Harrison, M.R., Adzick, N.S., Largman, C., 1997. The human homeobox genes MSX-1, MSX-2, and MOX-1 are differentially expressed in the dermis and epidermis in fetal and adult skin. Differentiation 62, 33–41. Stock, D.W., Ellies, D.L., Zhao, Z., Ekker, M., Ruddle, F.H., Weiss, K.M., 1996. The evolution of the vertebrate Dlx gene family. Proc. Natl. Acad. Sci. USA 93, 10858–10863. Su, M.W., Suzuki, H.R., Solursh, M., Ramirez, F., 1991. Progressively restricted expression of a new homeobox-containing gene during Xenopus laevis embryogenesis. Development 111, 1179–1187. Suzuki, H.R., Padanilam, B.J., Vitale, E., Ramirez, F., Solursh, M., 1991. Repeating developmental expression of G-Hox-7, a novel homeobox-containing gene in the chicken. Dev. Biol. 148, 375–388. Suzuki, A., Ueno, N., Hemmati-Brivanlou, A., 1997. Xenopus msx1 mediates epidermal induction and neural inhibition by BMP4. Development 124, 3037–3044. Takahashi, Y., Le Douarin, N., 1990. cDNA cloning of a quail homeobox gene and its expression in neural crest-derived mesenchyme and lateral plate mesoderm. Proc. Natl. Acad. Sci. USA 87, 7482–7486. Takahashi, K., Nuckolls, G.H., Tanaka, O., Semba, I., Takahashi, I., Dashner, R., Shum, L., Slavkin, H.C., 1998. Adenovirus-mediated ectopic expression of Msx2 in even-numbered rhombomeres induces apoptotic elimination of cranial neural crest cells in ovo. Development 125, 1627–1635. Thesleff, I., Nieminen, P., 1996. Tooth morphogenesis and cell differentiation. Curr. Opin. Cell Biol. 8, 844–850. Thomas, B.L., Porteus, M.H., Rubenstein, J.L., Sharpe, P.T., 1995. The spatial localization of Dlx-2 during tooth development. Connect. Tissue Res. 32, 27–34. Thomas, B.L., Tucker, A.S., Qui, M., Ferguson, C.A., Hardcastle, Z.,

A.J. Bendall, C. Abate-Shen / Gene 247 (2000) 17–31 Rubenstein, J.L., Sharpe, P.T., 1997. Role of Dlx-1 and Dlx-2 genes in patterning of the murine dentition. Development 124, 4811–4818. Tole, S., Patterson, P.H., 1995. Regionalization of the developing forebrain: a comparison of FORSE-1, Dlx-2, and BF-1. J. Neurosci. 15, 970–980. Tucker, A.S., AlKhamis, A., Sharpe, P.T., 1998. Interactions between Bmp-4 and Msx-1 act to restrict gene expression to odontogenic mesenchyme. Dev. Dyn. 212, 533–539. Vainio, S., Karavanova, I., Jowett, A., Thesleff, I., 1993. Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75, 45–58. Vastardis, H., Karimbux, N., Guthua, S.W., Seidman, J.G., Seidman, C.E., 1996. A human MSX1 homeodomain missense mutation causes selective tooth agenesis. Nat. Genet. 13, 417–421. Wang, W., Chen, X., Xu, H., Lufkin, T., 1996. Msx3: a novel murine homolgue of the Drosophila msh homeobox gene restricted to the dorsal embryonic central nervous system. Mech. Dev. 58, 203–215. Weiss, K.M., Bollekens, J., Ruddle, F.H., Takashita, K., 1994. Distalless and other homeobox genes in the development of the dentition. J. Exp. Zool. 270, 273–284. Weiss, K.M., Ruddle, F.H., Bollekens, J., 1995. Dlx and other homeobox genes in the morphological development of the dentition. Connect. Tissue Res. 32, 35–40. Westerfield, M., Akimenko, M.A., Ekker, M., Pu¨schel, A., 1993. Eyes ears and homeobox genes in zebrafish embryos. In: Bernfield, M. ( Ed.), Molecular Basis of Morphogenesis. Wiley-Liss, New York, pp. 69–77.

31

Winograd, J., Reilly, M.P., Roe, R., Lutz, J., Laughner, E., Xu, X., Hu, L., Asakura, T., van der Kolk, C., Strandberg, J.D., Semenza, G.L., 1997. Perinatal lethality and multiple craniofacial malformations in MSX2 transgenic mice. Hum. Mol. Genet. 6, 369–379. Wolf, U., Ritter, H., Atkin, N.B., Ohno, S., 1969. Polyploidization in the fish family Cyprinidae, order Cypriniformes. I. DNA-content and chromosome sets in various species of Cyprinidae. Humangenetik 7, 240–244. Woloshin, P., Song, K., Degnin, C., Killary, A.M., Goldhamer, D.J., Sassoon, D., Thayer, M.J., 1995. MSX1 inhibits MyoD expression in fibroblast×10T cell hybrids. Cell 82, 611–620. 1/2 Yang, L., Zhang, H., Hu, G., Wang, H., Abate-Shen, C., Shen, M.M., 1998. An early phase of embryonic Dlx5 expression defines the rostral boundary of the neural plate. J. Neurosci. 18, 8322–8330. Yokouchi, Y., Ohsugi, K., Sasaki, H., Kuroiwa, A., 1991. Chicken homeobox gene Msx-1: structure, expression in limb buds and effect of retinoic acid. Development 113, 431–444. Zhang, H., Catron, K.M., Abate-Shen, C., 1996. A role for the Msx-1 homeodomain in transcriptional regulation: residues in the N-terminal arm mediate TATA binding protein interaction and transcriptional repression. Proc. Natl. Acad. Sci. USA 93, 1764–1769. Zhang, H., Hu, G., Wang, H., Sciavolino, P., Iler, N., Shen, M.M., Abate-Shen, C., 1997. Heterodimerization of Msx and Dlx homeoproteins results in functional antagonism. Mol. Cell. Biol. 17, 2920–2932. Zhao, G.Q., Zhao, S., Zhou, X., Eberspaecher, H., Solursh, M., de Crombrugghe, B., 1994. rDlx, a novel distal-less-like homeoprotein is expressed in developing cartilages and discrete neuronal tissues. Dev. Biol. 164, 37–51.