the function and evolution of msx genes: pointers and paradoxes

he m&-like genes are an ancient family, represented in animals ranging from coelenterates to mammals (Table 1). On the basis of a few specific differences in the otherwise very highly conserved homeodomain and neighbouring regions of the proteins, three subclasses can be distinguished by their similarity to the products of the vertebrate M.s& or Msx2, or the invertebrate msh genesl. The mouse genes, Msxl and Msx2 (previously Hox7 and Hox~), and their homologues in other species have been widely studied. (A third mouse gene, M&$ has been partially characterizedz; its homeobox places it in the Msxl subclass.) The proteins encoded by the Msxl and Msx2 genes from different species have some subclass-specific features and some features that are shared by all the genes (Fig. 1). These features can be recognized in mammalian and avian species (and, with less conservation, in Xenopus) and enable at least seven regions of the proteins to be distinguished that might reflect domains with discrete functions. The products of the zebrafish msxA, B, C and D genes (not shown in Fig. 1) do not show clear subclass-specific differentiation: only those parts of the protein that are conserved across subclasses show large stretches of similar sequence. However, genes more closely re- sembling the Mm1 and the Msx;Z subclasses might remain to be discovered in zebrafish. In the higher vertebrates, regions that are similar in all subclasses might represent ancestral functions, while those that are subclass-specific might have gained new functions, for example, by acquiring novel interactions with other proteins. Specific genetic and biochemical analyses of these putative functional domains will clearly be a focus of interest for molecular, developmental and evolutionary biologists alike.

&XI and M&z,2 are expressed during embryogenesis and organogenesis (summarized in Table 2; reviewed in

x genes of vertebrrties comprise a small family of cbromosomally unlinked bomeobox-containing genes related to tbe Drosopbika gene muscle-segment

(msb]. Despite tbeir aucieutpedigree, tbe are expressed in a range of vertebrate-specific kuiing ueural crest, cranial sensory pLaco&s,

bone and teetb. Tbey are active in numerous systems, wbicb bave been used as models to study pattern forma&u aud tissue interaction, and are, tberefore, attracting a growing interest among develh@neutal biologists. But beyond tbeirpresumed role as transcription factors, we do not know wbat tbeir fuuctious are in tbe cell or tbe embrya Here, I review recent ev&nce tbat is beginning to address tbis problem

aUy iucrease our undkrstandhg of bow tbe vertebrate embryo bas evolved

Ref. 3). Generally, both genes are expressed in overlapping or related patterns from the early stages of tissue differentiation, in a position-related, rather than cell- type-specific, manner (e.g. Fig. 2). During early development, both genes are transcribed in the mesoderm and ectoderm of the primitive streak. Later, expression becomes restricted to the lateral surface ectodem, lateral mesoderm and the neuroepithelium that will form the dorsal part of the neural tube, including the region that gives rise to migratory neural crest cells. From

TABLE 1. msb-like genes hrn nt species

nf&4ike genes Chromosomal location

Man

Mouse

Chick Quail xenopus Zebrafish As&kin (Ciona intestinali. Amphioxus Sea urchin (Sttvngylocentmtus puqmratus~ Lhsopbika Honeybee Hydra (Chlombydra viridissimu~

MSXl 4~16.1 52,53 MSX2 5q34q35 Msxl Proximal end of chromosome 5 P5 Msx2 Distal region of chromosome 13 1:63 Ma3 2 MS& Mm2 54-56 Mm2 6 Msxl, Mm2 7a mswl-& 44,57

ml? msb

nlsb msb tib

mb

2 50

: 58

59

%ee legend to Fig. 1.

TIG OCTOBER 1995 VOL. 11 No. 10

0 1995 Elsevier Science Ltd 0168 - 9525/%/$O!XSO 40s

these early domains develop more complex, focused patterns of expression in diverse organs, These temporal and spatial patterns of expression are highly conserved in mammals*~s and birds6 and, at least, broadly similar in amphibians’. It is not yet clear how similar are the patterns of expression in fish. Preliminary data on mouse embryos suggest that Msx3 has a simpler pattern of expression, confined mainly to the dorsal part of the neural tube in a domain that overlaps those of Mm2 and Msx2, but is more ventral and excludes the roof plate (J. Grindley, R. Hill and D. David- son, unpublished; P. Sharpe, pers. commun.).

The striking sequence conservation of the Msx genes in and around the homeobox, and their expression early in the differentiation of diverse organs suggests that they might have a fundamental role in development, which has

EVIEWS

1 2 3 Ml mouse Msxl

~~s-T._ *.; - I;GVKVBD---SAFAKPAGGGVGQAPGAAAATATAMGTDEEGAKPKVPASLLPFSVEALMAD HI human MSXI ii f>* *p;y +++;** *_+ - - - I _z: ***G**““*“A”“**S**“***A***A*“*“*”*ffSpX************

Cl chick Msxl ** I~~~~~"~"~~~~~~~,~~PPA***S**G** _________LPV*A***GE*XSD**ffSP"P****+***"** i."

Xl Xenopus Msxl .I < F) -1 D _j* _q*:t* ?~~lr~*~_~YQP?~~~~~~------- R*LNR------MQQTGVKPSL *__*D__*""f*_GIf"""**"**f+

M2 mouse Msx2 -VLAGPGPG-----------PGGAEGSAEER---RVKVSSLPFSVEALMSD H2 human MSX2 _*V******__-______-_L***A*A****-__*****”***~*******

C2 chick Msx2 __________-__-__---__ -_-A”AE*HH--_K*“***+**“**““X””

Q2 quail Msx2 _______--_____--- -----__A*AE”HH_-_K”+“X+**+****“********

X2 Xenopus M&2 ___m * ~~--~~-_~--~~--~-~~~PS*DH_-~Kf*I+***”***~~~~~~~~

4 5 Ml HRKPGAKESVLVASEGAQAAGGSVQHLGTRPGS-- LGAPDAPsSPRPLG--HFSV~~~~~~~~~P~~~R~P~Q

******++*A*AP***V******A*p***-_**************-- * * * *‘** *?t* *‘rkV*,*>** * *<,* *;**:**&$*:e** * * r _ _ / “;., *“- :. j : I > , * * >;;+: i $ ̂. II R*SR---mj)Gp-f------ **GPP*AP*ANLG-ALTTE T L ** ** ***GHFP"X*A"G_**"'"*_*~~*'~'~_*ix"*

Xl -**"*RDRDLSSPTG----SPLAGTSHSP*V** --IA*GET*N**IS**-NRYP*,*ASMQ;*-~~~~,~~~,"F~~~.~~~-~*~~S*I*

Ml

:: Xl

M2 H2

z X2

Ml

E Xl

TLRKHKTNRKPRTPFTTSQLLALERKFRQKQYLSIAERAEiFSSSLNLTETQVKIWFQNRRAKAKR ***********************************************************S****~ ***************************************************************** *******************************t************************************ *****************************************************************

7 LQEAELEKLKMAAKPMLPPAAFGLsFPLGG ----PAAAGASLYSASGPFQRAALPVZiPVGLYTAHVGYSMYI+T--* ******************~*,**~~~~~***ipAAVA**"******~**~*~*****~~~,.~*,~,***~~*~*~~**~*~*___ ^^ ********************iik*h**e%. __,_**V""**~~*i3**Siij***Gi*;**'i****~*~*~'*~~___'~~__ / * *******************;*,$+++*+*$_____

. -“,Y PVPT*“**GT*N**r*Q***~S*~~*~**_***L*,***i*___

.-. 1 ” .

FIGURE 1. Amino acid sequences of Msxl and Msx2 proteins in the higher vertebrates. Comparisons are based on a clustal multiple- sequence alignment of predicted amino acid sequences published in the HGMP genome database (see also Table 1 for Refs). Wenopus Msxl is Xhox-7.1 in Ref. 7; Xeno@s Msx2 is Xhox-7.1’ in Ref. 7.) ??Shows amino acid residues identical to the corresponding mouse gene. Strikingly, there is significant sequence identity not only in the homeodomain and flanking sequences, but also in other parts of the proteins. Three kinds of region can be recognized on the basis of sequence similarity, assumed to be due mainly to conservation. (1) Regions showing conservation between all Msx proteins (grey regions 3 and 6 - which include the homeodomain). (2) Regions showing conservation within a subclass, but numerous consistent differences between subclasses (regions 1, 5 and 7; Msxl, light blue; Msx2, dark blue). (3) Regions showing little or no conservation (regions 2 and 4). The precise alignments and boundaries between these regions are open to debate, but the broad features are clear. Interestingly, the Msxl-specific and Msx2-specific regions (light blue and dark blue) are in equivalent parts of the sequence; this pattern is broken only by two small domains (the conserved PPKE-P sequence in region 4 of Msx2 proteins and a poorly conserved part of the sequence of Msxl proteins in region 7).

been conserved at least throughout the evolution of the vertebrates and possibly in all higher metazoans. If this

that distinguish the development of individual tissues

is so, it will be important to characterize the conserved and, in the wider view, might provide insights into the

molecular fknctions and to determine if conservation evolution of the vertebrate body. An understanding at

extends to higher levels, in the molecular pathways in this fundamental level can be expected to influence

which the genes act and the cell properties they con- profoundly our appreciation of the basis for human

trol. Superimposed on this conservation is a diversity congenital malformations. Moreover, because the very

that suggests that the different subclasses have assumed early steps in tissue differentiation might be closely

specific functions. Determining what these functions related to processes of oncogenesis and tissue repair,

are will help us to understand the molecular processes understanding their regulation might have implications for the treatment of a range of disease conditions.

TIC; OCTOBER 1995 VOL. 11 No. 10

406

Mouse M&Xl

Msxl and Mm2

Chick or quail Msxl and Msx2 Xenopus Msxl and Mm2

Zebrafish m, B, G D W.&Y, D

Drosophila msh

Uterus, cervix, vagina, uterine wall, ectoplacental cone, amnion, allantois, umbilical vein and Rathke’s pouch.

Primitive streak, somatopleural lateral mesoderm, dorsal ectoderm, neural plate, dorsal mesenchyme, dorsal region of the neural tube and of the brain (hindbrain, midbrain and forebrain), choroid plexus, cranial neural crest cells, bran&al arches, facial processes, tooth germs, eye, ear, nose, vibnssae, heart, pericardium, limb bud, genital tubicle, tail and tail ridge.

Similar to mouse where the tissues are applicable. Dorsal mesodermal mantle, lateral plate mesoderm, neural crest, dorsal

neural tube, dorsal region of brain and heart. Fin buds.

13,bo

Li3

6, 56,64 7

44 Inner ear. Segmental muscles and ventral nervous system.

Clearly, one issue for the immediate future is to determine in what molecular pathways the Msx genes function. In addition, we can ask if any primitive function is evident, for example, from the involvement of different Msx genes in a common pathway. Recent evidence is beginning to clarify the processes that act upstream of the Mm genes. The patterns of expression of Msxl and Msx2, and their correlation with inductive epithelial-mesenchymal interactions, suggest that they could function in the early response of cells to diffusible factors that control the growth and differentiation of diverse cell typess. This view is supported by grafting and tissue combination experiments, which show that Msx expression can be activated by tissue interactions that are otherwise known to induce differentiation, for example, in the limb bud@, facial processeslOJ1, tooth buds12 and epithelium of the perinatal Mullerian ductl3.

One group of diffusible growth factors implicated in tissue interactions is the bone morphogenetic proteins (BMPs), in particular the closely related BMP2 and BMP4. The genes encoding these proteins are coexpressed with the Mm genes at numerous sites, including the primitive streak, lateral mesoderm, limb bud, myocardium, hindbrain and tooth germl*J5. The addition of recombinant BMP2 or BMP4 protein to cultures of tooth mesenchyme can induce expression both of Mm1 and of Mm2, thereby mimicking the action of dental epithelium on the mesenchymel6. Mm2 expression is also induced in neural crest tissue of the hindbrain in rapid response to exogenous BMP4 (Ref. 17). Though there is, as yet, no molecular evidence to show how direct is the activation of the Mm genes in response to the binding of BMPs to their cell-surface receptors, these results do suggest that MsxZ and Ms.& function in a common pathway involving BMPs in different parts of the embryo. The reported domains of expression of B&W2 and BMp4 do not, however, account for all sites of Msx activity: they are apparently not expressed in the dorsal part of the neural tube, for example. BMps are members of the TGF-p superfamily, which includes the TGF_P, act&in and dorsalin gene families. Several members of these families, for example, TGF-_P and dolsalin-2, are also coexpressed

57 5

with the Msx genes *v5J8J9. Further studies of the effects of these proteins on Mm expression, coupled with more detailed analyses of the &-regulatory regions involved, will show whether, or not, the inductton of MSX genes by TGF-P-like proteins could reflect the diversification of a primitive control network.

Although this evidence suggests a common sig- nalhng pathway upstream of Mm1 and Mm2 in different parts of the embryo, recent evidence also points to regional differences in the control of the Mm genes. The discovery that a small part of the Mm2 regulatory region is responsible for specific expression in the apical ectodermal ridge of the limb bud20 implies at least some regional specificity in the pathways upstream of Msx2. Preliminary data on the regulatory sequences of Mm1 also suggest region-specific control (R. Hill, A. Mackenzie, M. Collinson, pers. commun.). The expression of the Mm genes in limbless and talpid (Ref. 2) mutant chicks, and in tissue combination experiments in vivo and in vitro, also reveals differences in the signal pathways that control the expression of a single Msx gene at different sites W21J2 and of Ms.rcl and Msx2 at the same site 12y22-24. The molecular nature of these functional differences is unresolved; it is not yet clear if they reflect the operation of unrelated signalling pathways, or variations on a theme. Recent evidence suggests that growth factors other than BMPs might also control Mm expression. In particular, exogenous FGE2 and FGF4, as welI as BMP4, can induce low levels of Mm1 expression in dissociated limb mesenchyme cells in vitm25. These observations suggest possibilities for complex regulation of the Ma genes by the independ- ent, or co-operative, action of TGF-p-like factors and members of the FGF family.

So far, there has been little progress in characteriz- ing the molecular processes downstream of MSXI and Msx2. One approach has been to identify direct targets of transcriptional regulation by Msx proteins. Homeo- domain-DNA-affinity cleaving analysis using the MSX~ homeodomain26 and oligonucleotide-binding analysis using the Msxl protein 27 have identified the consensus homeodomain-binding sequence IC/GlTAATI%. The binding of Msx2 to this sequence has not been described. This sequence has been found in the promoters of the gene encoding the signalling molecule


407

EVIEWS

wntl (Ref. 26) and the osteocalcin gene, which is expressed specifically in mature osteoblasts during bone development28, as well as in the promoter of Msx.2 itself29 (suggesting autoregulation or cross-regulation with M.s.x~). However, as with other similar homeodomain-binding sequences identified in vitro, this simple sequence might not account for the specificity of DNA binding in vivo 30 where protein interactions might also be involved. In the absence of homeodomain-DNA interaction, Msxl functions as a negative regulator of gene expression from artificially constructed promoters, apparently through direct interactions with proteins of the core transcription complex31. This function is mediated by parts of the Msxl protein both N-terminal and C-terminal to the homeodomain within regions (1+2), (4+5), and 7 of Fig. 1. The presence, within these regions, of differences between the Msxl and the Msx2 proteins might reflect differences in repressor activity; indeed the repressor function might be Msxl-specific raising the intriguing possibility that

Msxl and Msx2 could act antagonistically to control transcription.

Numerous other appropriately expressed genes could, in principle, act in the same pathways as M&xl and A&2. These include members of the Wnt family of signalling molecules32, retinoid-binding proteins and retinoid receptor@, and extracellular molecules, for example, the heparan sulphate proteoglycan, syndecan34. Indeed, the Msxl and Msx2 proteins themselves might interact directly, or by competition in DNA- or protein- binding. Expression data alone do not implicate these other gene products in the Msx pathway; but there is clearly an exciting time ahead as information from gene knockouts and ectopic expression experiments becomes available to test if any of these genes function upstream or downstream of the M. genes. An important step forward will be the characterization of the wr.sh genes and their regulatory regions in the invertebrates, particularly in moph& and perhaps in Caenohabditis elegans. This will facilitate the exploration of putative

FIGURE 2. Msxl and Msx2 expression during the early stages of organogenesis. The figure shows transverse sections through part

of the facial region of a 12.5 d mouse embryo, in general view for orientation (a), and in more detailed views of the nasal region (b, c)

chosen to illustrate the general features of expression of iM.1 (red) and Msx2 (yellow). Abbreviations: n, nasal epithelium; s, mesenchyme forming the nasal septum; v, vomeronasal organ.

Here, as in many other parts of the embryo, the genes are expressed in regions of epithelial or mesenchymal interaction. Notice that in neither case is expression uniformly confined to mesenchymal or epithelial tissue, though such restrictions are apparent locally (shown in this example by Msxl expression).

Both genes are expressed in position-related patterns, overlapping in some regions and not in others.


conserved pathways, as well as functions that might have diverged in the protostome and deuterostome lineages. It will be interesting to discover, for example, if decapentaplaegic (dpp), the insect gene most similar to BMP2 and BMP4, regulates msh in Drosophila.

e genetic

The most direct evidence concerning the function of the MSX genes at the tissue level comes from mutations. Mice homozygous for a targetted insertion in Msx.2 fail to form teeth and have craniofacial abnormalities, which include absence of the alveolar bones in the jaw and lesser abnormalities in parietal, frontal and nasal membrane bones, and in the malleus in the ear35. The mutant mice also have cleft palate, which, though possibly a secondary effect, could provide an important model for some human cleft palate syndrome&. A dominant mutation in the human MSX2 is also associated with craniofacial abnormalities, in particular craniosynostosis (premature fusion of the skull bones)37. Though the Boston-type craniosynostosis associated with this mutation is rare, analysis of the function of MSX2 in the development of the disease could help in understanding the more common forms. Mutations in the genes encoding the fibroblast growth factor receptors FGFRl and FGFR2 also result in craniosynostosis, while specific mutations in FGFR2 result in Apert syndrome, which includes both craniosynostosis and bony syndactyly3s. It will be interesting to determine if the FGFR genes act in the same pathways as Mm2. In view of the expression of Mn2 in the limb bud, it is also interesting that minor abnormalities of the distal limb bone pattern have been observed in some individuals carrying the MS” mutation.

It is striking that mutations in Msxl and in MSX2 affect the development of bones in the head, suggesting a direct or indirect role for both genes in bone development. Additional evidence supports this view. MSX2 has been identified in cDNA from adult human bone cells and its expression can be stimulated in bone- derived cells in vitro by the vitamin D derivative 1,25(OH),D3, which promotes bone differentiation39. Msx2 has also been implicated in the development of the spinous processes of the vertebrae in bird@. The Mm genes do not, however, have a universal role in bone formation; they are not expressed, for example, in the sclerotomes. One of the most puzzling questions, then, is why do some bones require Mm activity for their formation while others do not? In experiments in which mesenchyme from the avian jaw (which expresses Mm2 in viva) was cultured with, or without, epithelium, cartilage formed in either case, but membrane bone formation and the maintenance of M&x2 expression required the presence of epithelium? This result might implicate Mm2 in the formation of membrane bones rather than cartilage; however, there is, as yet, no strict inclusive or exclusive relationship of this type with regard to the bones affected by the known mutations.

If the Msx genes are not required for bone differentiation per se, might they function in determining the characteristic shapes of specific bones? Indeed, mutations in Msxl and MAX2 affect the very early

processes that precede the differentiation of particular bones. Teeth and alveolar bone are derived from delltal follicular mesenchyme, which normally expresses Mm1 and is depleted in Msxl-mutant mice35. The association Of craniosynostosis with a point mutation in MS+ suggests that this gene might also be involved in the control of bone differentiation. Mice carrying an Msx;? transgene with the same mutation, fused with a promoter that drives widespread expression, also display craniosynostosis, establishing the link between the human phenotype and the gene defect?*. Interestingly, wide-spread expression of additional copies of the wild-type gene also results in craniosynostosis, arguing against the mutant phenotype being caused by a loss of protein function 41. In neonatal mice, M.9~2 is expressed at the margins of the skull bones in differentiating cells that will eventually lay down bone matrix. Under the influence of tissue interactions, the bones grow at the margins but do not normally fuse until skull growth is completej7. One interpretation is, therefort , .! +b mutation results in precocious different;- v : M=2-expressing cells.

The control of bone formation also involves tht BMPs and it has been suggested that the local distribution of different BMP proteins controls the pattern and form of individual bone&. The evidence that BMP2 and BMP4 induce Msx expression suggests that Msxl and Ma2 could be part of such a system regulating the balance between the undifferentiated and differentiated states. However, the control of bone formation by the BMPs is complex and, as yet, poorly understood. It will be important to develop cellular assays to explore this problem. Do Msxl and Ma2 act antagonistically or in successive stages of the process? The BMPs stimulate bone healing and the formation of ectopic bone42 - it will be interesting to discover if these phenomena are also affected in Msx-mutant mice. The speculation that Mm1 activity functions to suppress differentiation has received attention43 because it might account for Msxl expression in the distal ‘progress zone’ mesenchyme in the limb bud, where the skeletal pattern is established while cell-type-specific differentiation, including chondro- genesis, is held in check. Interestingly, MSX gene expression also correlates with plasticity of differentiation during regeneration of the appendicular skeleton following amputation, in the blastema of regenerating fins in zebra&he and in the regenerating tips of digits in mice45.

One situation where inhibition of cell differentiation has been correlated more directly with MaI expression, is during myogenesis in vitm46. Constitutive expression of transfected MaI in myogenic cells inhibits muscle differentiation (including MyoD expression) and promotes a transformed phenotype (though the effects of different quantities of Msxl protein remain to be explored). These effects were not seen in cells constitutively expressing Msx2. MSXJ is not widely expressed in myogenic cells, though some myoblasts (e.g. in the limb bud) probably do express the gene. The effect of MaI expression on myogenesis in situ warrants investigation.

As I have outlined above, the expression of Msa9 and M& coincides with, and is stimulated by, inductive tissue interactions at numerous sites in the embryo. However, with the exceptions of tooth, bone and


409

EVIEWS

muscle, the sole instance where the evidence gives more than a broad suggestion of any functional involvement is in programmed cell death (apoptosi:isj. MSX~ and hf.2 expression precedes apoptosis at several locations, including the limb bud and the hindbrain. A tight correlation has been established in the chick hindbrain through an elegant series of experimental manipulations, which permit, or prevent, the apoptosis that occurs specifically in the neural crest region of rhombomeres 3 and 5 during normal development17T47. Both apoptosis and Mm2 expression depend on interaction of rhombomeres 3 and 5 with adjacent rhombomeres, but both can be induced by exogenous BMP4 in isolated tissue from rhombomere 3. This accords with the finding that, during normal development, BMP4 is transcribed specifically in rhombomeres 3 and 5. A causal relationship between Mm2 expression and apoptosis remains to be shown. If apoptosis is indeed a result of Ma2 expression, then the gene could either be part of a specific apoptosis pathway, or be acting less directly, According to one current mode1‘t8, apoptosis is a general default for cells that have initiated growth or differentiation in the absence of critical factors. Msx2 could perhaps function in an abortive pathway initiated by BMP4 whose failure at this location leads to apoptosis. Alternatively, Msx2 might promote apoptosis indirectly by preventing differentiation.

In the light of the suggested functions of the Msx genes in tissue interactions throughout the embryo, and in muscle differentiation and apoptosis, it is paradoxical that neither of the Ms.x mutations reported, so far, dis- plays phenotypic effects other than on the development of bones and teeth. If Mm1 and Msx2 do have wider roles, why is this not revealed in the phenotypes of existing mutants? Clearly, this could be a result of redundancy, either ‘functional’ (the function of the mutant gene is substituted by another process or by a related gene), or ‘non-functional’ (the gene products have no function in some regions where the gene is transcribed; see Ref. 49). Alternatively, the phenotypes reported thus far might not represent the null condition - more subtle abnormalities might remain to be discovered or the mutant proteins might retain some functional capabilities. The recessive MaI mutation truncates the gene 3’ to a site in the homeobox, which might leave residual activity involving the N-terminal part of the protein? The MSX2 mutation37 changes a single highly conserved residue in the homeodomain (Pro148 to His), which could affect DNA binding or protein interaction, but is unlikely to abolish both.

Further advances will depend on progress in pre- dictable directions - the creation of definitive null mutants, functional analyses of regulatory elements combined with reporter genes in transgenic mice, biochemical and genetic identification of downstream genes, and the development of molecular and cellular assays of gene function both in epithelial and in mesenchymal tissues. Bone and teeth are vertebrate-specific tissues, but m&-like genes function in invertebrate and even diploblastic organisms. During the evolution of primitive vertebrates, it is most likely that the formation of these new tissue types would build on existing mechanisms that control cell-type differentiation, including those local interactions that control the distribution

of differentiated cells. (Candidate mechanisms might be those controlling neural, or perhaps muscle, development.) It will be interesting, therefore, to examine the functions of M.1 and Msx2 in different cell types in higher vertebrates and to compare these with the sup- posed primitive functions of the progenitor gene.

in evolutionary side to the story The invertebrates and primitive chordates examined

to date have single m&like genes, whereas the vertebrates have severaP0. This has led to the suggestion that m&like genes duplicated early in vertebrate evolution, probably between the periods when lines leading to the present-day Amphioxus and hagfish arose (Ref. 50; but see Ref. 1 for a different view). Indeed, Mm1 and Mm2 are expressed in structures that are considered to be primary in vertebrate evolution, in particular, neural crest and cranial sensory placodes. The implication that the Mm genes could have played a part in this evolution emphasizes the need to identify the functions of the archetypal w&-like gene.

Can we recognize a primitive domain of Mm activity in the embryos of present-day vertebrates? Holland has proposed that expression characteristics shared by related homeobox genes within a species probably reflect the functions of an ancestral genesl. On this view, the most primitive Msx function is to be found in the dorsal neural tube in a domain, which, perhaps significantly, excludes the neural crests. Moreover, as Mm.3 expression appears to be restricted almost entirely to this domain, it might be most closely related to. the archetypal m&like geneso. Though this suggestion is clearly speculative, it does open up questions about how the functions of the Mm genes in higher vertebrates arose. How did Mm1 and Mm2 come to be so widely expressed? Why did the functions of the present- day Msx genes diverge from this origin? One possible explanation is that, following duplication of an Msx3- like gene, changes in the regulatory sequences of one of the genes led to an expansion of the expression domain into the lateral part of the neural plate to encompass the neural crest region. The diverged Msx gene would have duplicated and, in the novel embry- onic environments to which the neural crest cells were exposed, those genes now represented by the Mm2 and Msx2 subclasses diverged, acquiring new functions, for example in the development of bone and teeth. What led to the expression of Msxl and Mm2 in the primitive streak, heart, lateral mesoderm and limbs? It is not clear whether responsiveness of Mm to BMP, rather than to other TGF-p-related molecules, is an ancient or more recent affiliation. But it is possible that the expression of BMP4 in these regions15 led to an expansion of MaI and Msx2 expression that was independ- ent of their encroachment on the neural crests.

Investigation of these problems is in its infancy. The pioneering studies of Holland, and others, on the msb- like genes in primitive chordates and primitive vertebrates promise exciting insights. The hagfish, in particular, will be important targets for this research because they might, or might not, show duplication of the msh- like genesjo. The Msxl and Msx2 subclass characteristics are not well-defined in the multiple zebrafish genes. If this holds true for other bony fish, analyses of


15

16 17 18 19 20 21

, 22

23 24

25

26

27

28

;; 31

cis-regulatory sequences and mutational studies in this species will be a promising area for understanding the functional evolution of the Msx genes. The next gener- ation of problems concerns the extent to which entire molecular pathways that underlie supracellular organ- ization are subject to change or are static modules, ‘developmental units’, in evolution. YVhat kinds of link between pathways are most likely to be made or broken during evolution in periods of environmental or genetic upheaval? The molecular and developmental studies of the Msx genes that are on the immediate agenda will have implications for these apparently distant issues.

AC nt§ I thank my colleagues at the MRC Human Genetics Unit,

particularly Bob Hill, Martin Collinson and Ralph Holme, for stimulating discussions and critical comments on the text, Drs Yi-Hsin Liu and R.E. Maxson kindly made their results on the ectopic expression of mutant and wild-type M.2 available before publication.

ferences 1 Bell, J.R. etal. (1993) Genomics 16, 123-131 2 Holland, P.W. (1991) Gene 98,253-257 3 Davidson, D.R. and Hill, R.E. (1992) Semin. Dev. Biol. 3,

405-412 4 Hill, R.E. eE al. (1989) Genes Deu. 3, 26-37 5 Robert, B. et al. (1989) EMBO J. 8,91-100 6 Takahashi, Y. and Le-Douarin, N. (1990) Proc. Nut1 Acad.

Sci. USA 87,7482-7486 7 Su, M.W. et al. (1991) Development 111, 1179-1187 8 Davidson, D.R. et al. (1991) Nature 352,429-431 9 Robert, B. ec al. (1991) Genes Dev. 5, 2363-2374

20 Takahashi, Y., Bontoux, M. and Le-Douarin, N.M. (1991) EM..0 J. PO, 2387-2393

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the function and evolution of msx genes: pointers and paradoxes

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