The Hox gene family in transgenic mice Robb Krumlauf MRC National Institute for Medical Research, London, UK Evidence is accumulating which suggests that the vertebrate Hox homeobox gene family plays an important role in pattern formation, particularly in the specification of regional diversity. In the last year important advances in the understanding of their regulation and function have been provided using transgenic mice. Current Opinion in Biotechnology 1991, 2:796-801
Introduction
Regulation of Hox transgenes
The vertebrate Hox homeobox gene family has been the increasing focus of attention with respect to both its potential roles for patterning axial diversity and the regulation of its spatially-restricted domains of expression. There is some confusion as to the term Hox, which many believe to be a generic means of referring to h o m e o b o x containing genes; however, Hox actually refers to a specific family o f vertebrate homeobox genes and the properties of the Hox family are distinct from other genes. In mammals and most vertebrates Hox genes are organized in clusters; there are four clusters that share many conserved structural features with homeotic genes in the Drosopbi/a ANT-C and BX-C complexes [1-3]. The overall structure of the mouse H o x network, its relationship to Droscpbi/a homeotic genes, and some of the expression properties are summarized in Fig. 1. This similarity between species initially led to the idea that H o x genes could be involved in axial specification based on the roles of their Drosophila homologues. Further support for this concept has come largely from in situ hybridization analyses (for review see [4"']) that showed that genes in the Hox clusters had graded overlapping domains of expression that correlated with their gene order along the chromosome, which is termed collinearity.
The primary method used to examine the spatial regulation o f Hox genes in transgenic mice involves using the bacterial /ac_Z gene as a reporter. In initial experiments very limited domains o f expression were observed [14,15o16-18]. In one case, a Hox-23/lacZ transgene was expressed in the kidney, but major sites of mesodermal and ectodermal expression were absent [14]. In another study [16], a 5' flanking region of the Hox-L3 gene directed expression of a /acZ gene on a heterologous promoter in a small region of the neural tube but only for a brief time during development. This result was extended to show that an enhancer region in the 5' flanking sequences originally from Hox-1.3 was responsible for this expression in the neural tube [17]. The relationship o f all of these patterns to expression of the endogenous genes was not clear, and it was impossible to determine whether these represented genuine subsets of the normal Hox gene pattern or ectopic expression. The tentative conclusion, generally drawn, was that the tmnsgenic constructs contained some but not all of the c~acting regulatory requirements, and that the missing regions might be distributed throughout the clusters. In view of these results and the collinear relationships between gene order and expression, one speculation was that an intact Hax cluster might be required to preserve the order of regulatory regions and generate the normal patterns of expression. In such cases, a region similar to the 13-globin locus control region would be required by the Hoxclusters ([19], see also review by Enver and Greaves pp 787-795).
Collinear spatially-ordered domains were observed in the limb [5,6"'], axial mesoderm [7], developing nervous system [1,8] and branchial arches [9"']; and recently, coUinearity was found to apply to the relative sensitivity of H o x g e n e s to retinoic acid in cell culture [10..,11 °.] and the timing of embryonic activation [12.]. The collinear relationship between gene order and embryonic expression appears to be a basic conserved feature of Hoxclusters and has led to the belief that the tightly regulated domains of expression represent part of a molecular combinatorial code for the differential specification o f axial structures [9°.,13..]. In this review particular attention will be devoted to recent transgenic mouse experiments that have made significant advances towards our understanding o f both the function and regulation of the H o x network in embryogenesis.
796
New data on two Hox genes, however, reveal that it is possible to reproduce the correct overall pattern of expression for a gene isolated from its normal context in a cluster and in a new chromosomal location [20..,21-.]. In an initial study by Puschel et aL [22] a Hox-1.1/lacZ transgene displayed the correct timing and anterior boundaries o f expression, but was unable to undergo the same spatial and lineage restricted expression in mesodermal derivatives as the endogenous gene. In a further study, Puschel et aL [2I..] also identified
(~) Current Biology Ltd ISSN 0958-1669
The Hox gene family in transgenicmice Krumlauf
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Fig. 1. Conservation between Hox and Hom-C homeobox complexes. Alignment of the mouse (Hox) and Drosophila (HOM-C) homeobox gene clusters. The vertical rows of boxes indicate genes which are highly related to each other, forming subfamilies. The brackets above the mouse genes identify the Drosophila homologue. Note that not all mouse Hox clusters have members in each subfamily. Shaded boxes are sequenced genes and the dashed boxes are genes present in the human clusters but not as yet identified in mouse. The large arrow beneath the clusters refers to the trends in collinear expression where successive genes from the left to right have increasing anterior boundaries, earlier stages of expression and sensitivity to retinoic acid (RA). the missing regulatory components, many of which are within the transcription unit itself, and demonstrated that a transgenic construct was capable of reproducing the endogenous Hox-l.1 pattern. These experimenters concluded that the promoter was able to regulate the early phase of Hox-l.1 expression with respect to the appropriate tissue distribution and anterior boundaries of expression; but that multiple elements are independently required to restrict the expression from this promoter in a lineage-specific manner. In an independent study by Whiting et aL [20-'], a Hox26,//acZ transgene was also found to be capable of duplicating the normal expression pattern on the basis that it had an identical time course, tissue distribution and spatial domains as the endogenous gene. In addition, the levels of the transgene expression were comparable to those of the endogenous H o x - 2 6 gene. Fig. 2 shows a comparison between the transgene and the endogenous Hox-2Ggene expression, to illustrate their similarity and the type and position of regulatory regions mapped in the transgenic experiments. Deletion analysis revealed that three regulatory regions were involved, and two of these functioned as spatially-specific enhancers [20..]. In contrast to the H o x l . 1 enhancers, the H o x - 2 6 enhancers were capable of imposing the same spatially restricted domains of expression on heterologous prorooters. Hence there is little specificity in the H o x - 2 6 promoter. Whereas the experiments using these two genes clearly demonstrate that it is possible for Hox genes to be appropriately expressed independently of their clusters, it is equally apparent that the two genes use quite differ-
ent strategies to establish and maintain their expression domains: In both cases multiple regulatory regions were involved in controlling expression, and several of these dements were located in the introns, exons, or 5' and 3' untranslated regions. This suggests that simply using flanking regions of a gene to assay for regulatory regions is not sul~cient, and it is important to maintain as much of the structural integrity of a Hox gene as possible in transgenic constructs. This is probably one reason why some of the early studies were unsuccessful. It is not surprising that multiple regulatory regions or enhancers are required for normal Hox expression and some of these may be shared between different members of a cluster. Therefore, it still remains possible that one of the reasons why Hox clusters have been conserved is to maintain the organization of dispersed regulatory regions, despite the fact that some genes can be regulated independently. The new data, however, clearly rule out an extreme model that requires a type of locus control region [19] for the proper expression of all genes In the cluster. Many of these issues can be resolved by using large regions conmining an entire Hox cluster on a yeast artificial chromosome [23"], and the development of tedmology for making transgenic animals with these chromosomes will be an important tool for those interested in Hox regulation.
F u n c t i o n a l analysis in t r a n s g e n i c a n i m a l s
Descriptive studies were essential in putting forward the idea of a combinatorial Hox code for the specification
797
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Mammalian gene studies
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F Main enhancer
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lqeural crest Spinal corcl Gut Somites Stomach Kidney
Neural enhancer "Only hindbrain" and spinal cord
Fig. 2. Reconstruction of the endogenous pattern of expression by a Hox-2.6/lacZ transgene. Diagram of the Hox-2.6 gene and the location of regulatory regions. Regions A and C are spatially-specific.
of regional diversity. Experiments in transgenic animals, however, have played an important role in verifying these ideas and actually examining the function of Hox genes in development. The over-expression of a Hox-l.4 gene in the colon of transgenic mice leads to an over-proliferation of intestinal epithelial cells resuhing in a megacolon phenotype [24]. Ectopic expression of the mouse Hox1.1 gene using a chicken actin promoter results in severe craniofacial abnormalities and the transformation of anterior vertebrae in the axial skeleton of transgenic mice [25,26-.]. Recently, it has been shown that the application of retinoic acid to mouse embryos results in similar craniofacial and vertebral transformations, and that it may do so by altering the patterns of Hoxexpression in these embryonic regions [13°.]. All these experiments show that the dominant, ectopic expression of a gene from the Hox network can change the identity and pattern of specific regional structures, presumably by changes in the Hox code. The most convincing evidence that Hoxgenes have a normal function in development has been elegantly provided by using embryonic stem cells (see review by Bradley pp 823--829) to generate tmnsgenic mice. In two cases, endogenous Hoxgenes ( - 1.5 and - 1.6) have been mutated by homologous recombination in embryonic stem cells; mice carrying the mutated aUeles in the germIine were generated by blastocyst injection and embryo transfer [27.*,28°*]. For both genes, heterozygous mice bearing the mutation displayed no visible phenotypes, but homozygous animals were non-viable and died shortly after birth. This suggests that the phenotypes had mutations resulting in a loss of function. Mutants of the Hox1.5 gene had a range of phenotypes largely apparent in tissues from the region of the head and thorax [27.°]. In the homozygous mutants the patterning of the head and thorax was altered so that the anterior cervical vertebrae were compressed, many cranial neural crest derivatives were malformed or missing, and the pharyngeal arches were abnormal in size and shape. It is difficult to separate primary from secondary and tertiary effects, and
the abnormalities were not extensively examined in early embryos. It is clear, however, that the perturbations arise in tissues where the endogenous Hox-l.5 gene is normaUy expressed, strongly suggesting that this gene is required for patterning in the branchial region of the head and thorax [27°°]. Homozygous mouse mutants for the Hox-l.6gene had a phenotype that was also restricted to the branchial region of the head, but in this case it was focused in cells derived from rhombomeres 4-7 [28-*]. Abnormalities are not confined to these regions and occur in many tissues and cell types, defects are observed in neuroectoderm, surface ectoderm, neural crest and paraxial mesoderms. The phenotypes themselves are extremely interesting but in terms of Hox function, it is the similarities and differences between the two phenotypes (wild type and mutant) which are most informative. Hox-l.5 and Hox-l.6 are expressed in posterior regions of the embryo, but no defects in these regions were observed [27",28-.], thus indicating that not all sites of Hoxexpression appear to require this expression for normal patterning. This is important because the four vertebrate Hox clusters are related by duplication and divergence from a common ancestor, which raises the possibility of functional overlap between highly related genes. But the fact that phenotypes do arise demonstrates that there cannot be a complete overlap or compensation between Hox genes. The neural crest is abnormal in both mutants, but in the case of Hox-l.5 it is the mesenchymal derivatives that are altered and the nerves and cranial sensory ganglia appear normal [27"]. In contrast, in the Hox-I.6mutants it is the neurogenic and not the mesenchymal derivatives that are missing [28--]. This suggests that different Hox genes from the same cluster can be used in the differential specification of the neural crest Finally, in neither mutant was there a simple transformation of structures similar to those observed in somites or cervical vertebrae upon ectopic expression of the Hox-l.1 [26-]. This may reflect basic differences in the way that the head and tnmk are patterned, and suggests that Hox genes may
The Hox gene family in transgenicmice Krumlauf 799 play different roles in different embryonic contexts (for reviews see [29",30",31]). Why are the mutant phenotypes concentrated in the branchial region and does this make sense based on the patterns of Hox expression? It has been postulated that Hox genes pLaya major role in the patterning formation of the embryonic head [9-*,29",51]. The Hox-2genes display segmentally-restricted patterns of expression which map to rhombomere boundaries [8], and these patterns extend to the surface ectoderm, cranial ganglia and mesenchymal neural crest (derived from the rhombomeres) in the branchial arches [9"*]. The relationships between Hoxgenes and the organization of the branchial head are illustrated in Fig. 3. It has been suggested that one important funcdon of Hox genes and rhombomeric segmentation in the hindbrain is to provide a means of linking positional information in the central and peripheral nervous system with the craniofacial structures and branchial arches [9",29",32"']. These recent studies on Hox mutants provide direct support for this theory.
Conclusions The recent transgenic experiments leave little doubt that the Hox network is a critical factor in the molecular mechanism of the specification of regional diversity. The mapping of regulatory regions has provided an insight into how the spatially-restricted domains of expression are generated in embryogenesis. Perhaps more importan@, they provide a means of specifically altering the patterns of Hox expression in transgenic animals to directly test predictions of the Hox code. In the absence of convenient genetic screens these regulatory regions will also permit biochemical approaches to identify and isolate the upstream factors in the regulatory hierarchy that is responsible for regulating Hox gene regulation. A great deal of information has already been obtained from just two mutant Hox genes, and as other members of the family are examined it appears possible that many of the interac~'e processes used to pattern vertebrate structures, particularly the head, will at last begin to fit into a clear molecular framework.
Acknowledgements Branchial arches
Nervous system
Surface Arch Cranial ectoderm mesenchyme ganglia
Neural tube
I ~x)uld like to thank members of my laboratory for extended discussions, in particular Paul Hunt, Jenny Whiting, Stefan Nonchev, and Mai Har Sham. I also thank Peter Rigby and Rudy Allemmm for discussion about our collaborati,.e work on Hox.2.6 and David Wflldnson, Peter Thorogood and Andrew Lumsden for ideas on head patterning.
-"
B1
References and recommended reading Papers of spedal interest, published within the annual period of re~e~,
havebeen higl~ghtedas: • •,
of interest of outstanding interest
1.
GR.,dO,.MA, PAPALOPULUN, KRUMI.AUF R: The Murine and Drosophila Homeobox Clusters have Common Features of Organisatinn and Expression. Cell 1989, 57:367-378.
2.
DtmOU1.ED, DOIZE P: The Structural and Functional Organization of the Murine Hox Gene Family Resembles that of Drosophila Homeotic Genes. EMBO 1989, 8:1497-1505.
3.
2.0
112.8
2.7
1--12.6
Fig. 3. Collinear and segmental expression Hox-2 codes for the branching region. Summary of the segmentally restricted pattern of Hox expression in the branchial region of the head. The bulges in the neural tube represent rhombomeres (r), and B1-B3 represent the branchial arches. Note Hox expression is restricted to specific axial levels in the rhombomeres, cranial ganglia, arch mesenchyme, and surface ectoderm [9"].
BONCLN'ELLI E, AC&~PORA D, P&N'NESEM, D'EsPOSITO M, SOYLVL~ R, GUAD~¢OG, STOmZ~a'UOLOA, CAHXROM, FAmLLAA, S~tEO.',Z A: Organization of Human Class I Homeobox Genes. Genome 1989, 31:745-756. 4. KE.~ELM, GRUSS P: Murinc D~'elopmental Control Genes. Science 1990, 249:374-379. ~ ' s is art excellent recent review which provides the reader with a reasonable idea of the important areas of research in the Hox field. It hightlights the s-altre of identifying genes by evolutionary homology and also details proportion of other genes implicated in the regulation of vertebrate development. 5.
DOI.I.E P, IZHSUA-BELMON'rEJC, FALKLXSaX~ H, RENUCO A, DtmOULE D: Coordinate Expression of the Murine Hox.5 Complex Homeobox-Containing Genes During l lmh Pattern Formation. Nature 1989, 342:767-772.
6. •,
IZPISUA-BELMONTE J-C, TICKLEC, DOLLEP, WOLPERTL, DUBOUtE D: Expression of Homeobox Hox.4 Genes and the Specification of Position in Chick Wing D~'elopment. Nature 1991, 350:585-589.
800
Mammalian gene studies A key paper for those interested in the limb and Hox genes. Demonstrates beautifully the collinear spatial regulation of Hoxin the limb and correlates their expression to retinoic acid and patterning. Suggests how a Hox code for the limb could be used for differential patterning. 7.
8.
DRESSLER GR, GRUSSP: Anterior Boundaries of Hox Gene Expression in Mesoderm-derived Structures Correlates with the Linear Gene Order Along the Chromosome. D~'erentiation 1989, 41:193-201. WILK~3OND, BHA'['r S, COOK M, BONCLN'ELUE, KRUMIAUF PC Segmental Expression of Hox 2 Homeobox-Containing Genes in the Developing Mouse Hindbrain. Nature 1989, 341:405-409.
9. •.
HUNT P, WmKLXSOND, KRU,Va~UFPC Patterning the Vertebrate Head: Murine Hox 2 Genes Mark Distinct Subpopulations of Premigratory and Migrating Neural Crest. Development 1991, 112:43-51. A crucial study that links Hoxgenes to patterning in the neural crest and branchial head in addition to the previously described rhombomeres in the hindbrain. Details the idea ofa Hox code for the head, and suggests that Hoxgenes could be part of the molecular pre-patteming signal for cranial neural cresL 10. •.
SIMEONEA, ACAMPORAD, ARCION1L, A~'DP,EWS FW, BONCB,'ELU E, MAV11JO F: Sequential Activation of Hoxl Homeobox Genes by Retinoic Acid in Human Embryonal Carcinoma Cells. Nature 1990, 346:763-766. Provides an important extension of the basic properties of Hox regulation, by showing that retinoic add induction in cultured ~ is collinear ~ith gene order. This suggests a means whereby retinoic acid can set up overlapping domains of Hox expression in vertebrate embryos. 11. •.
PAPAI.OPULUN, LOVELL-BADGER, KRUMLAUFPc The Expression of Murine Hox-2 Genes is Independent on the Differentiation Pathway and Displays Collinear Sensitivity to Retinoc Acid. Nucleic Acids Res 19:5497-5506. This study extends the collinear s e n s i ~ t y of Hox genes to mouse cells and Xenopus embt3x3s. This demonstrates that the coUinear retinoic acid response is consetx'ed in vertebrate evolution and likely to be an important feature in viL~ 12.
IZPISUA-BELMO.WI'EJ, FALKENS'I'ELNH, DOLLE P, RENUCC! A, • DUBOULED: Murine Genes Related to the Drosophila AbdB Homeotic Gene are Sequentially Expressed During Development of the Posterior Part of the Body. EMBO J 1991, 10:2279-2289. An interesting study that deals vdth the evolution of 5' subfamilies in Hox clusters and also shows a temporal coUinearity for the tIox-4genes in the posterior embryo. 13. •.
KESSELM, GRUSS P: Homeotic Transformations of Murine Prevertebrate and Concomitant Alteration of Hox Codes Induced by Retinoic Acid. Cell 1991, 67:89-104. This is another key paper dealing with two issues. It proposes a Hox code for the somites and paraxial me.soderm which complements those proposed for the limb and head. It also demonstrates that retinoic acid can cause transformations to vertebrae presumably by changing the Hox code. 14.
E~.ss C, VOGELSP,, DE GRAAFFW, BO,~q~-ERO'fC, MEIJLtNXFNJF, DESCHAMPSJ: Hox-2.3 Upstream Sequences Mediate lacZ Expression in Intermediate Mesoderm Derivatives of Transgenie Mice. Development 1990, 109;776-786.
15. •
BIEBEmCHC, UTSETM, AWGULE~TI3CHA, RUDDLEF: Evidence for Positive and Negative Regulation of the Hox-3.1 Gene. Proc Natl Acad Sci USA 1990, 87:8462-8466. This paper describes some interesting features of the Hox-3.1 promoter. 16.
ZAK&NYJ, TUGGLE CK, PATEL MD, NGUYEN-HUUMC: Spatial Regulation of Homeobox Gene Fusions in the Embryonic Central Nervous System of Transgenic Mice. Neuron 1988, 1.679-691.
17.
TUGGLECK, ZAK~" J, CraWl'l"1 L, PESCH1.EC, NGUYEN-HUUMC: Region-Specific Enhancers Near Two Mammalian H o m e o -
Box Genes Define Adjacent Rostrocaudal Domains in the Central Nervous S),tem. Genes Dev 1990, 4:180-189. 18.
SOtUGHARTK, BIEBERICH C, Ell) R, RUDDLE F: A Regulatory Region from the Mouse Hox.2.2 Promoter Directs Gene Expression Into Developing Limbs. Development 1991, 112:807--812.
19.
GROSVELD F, VAN ASSENDELFT G, GS.EAVES D, Komm B: Position-lndependent, High Level Expression of the Human Beta.Globin Gene in Transgenic Mice. Cell 1987, 51:975--985.
20. *.
WHITLNGJ, MARSHALLH, COOK M, KRUMLAUFR, R/GBYP, STOTT D, ALLEMANNPc Multiple-Specific Enhancers are Required to Reconstruct the Pattern of Hox.2.6Gene Expression. Genes Dev 1991, 5: 2048-2059. This is an important study which demonstrates that it is largely possible to reconstruct Hox expression patterns in transgenic mice. It extends previous studies in identifying regulatory regions involved in this process which act as enhancers that set boundaries of expression even on heterologous promoters. Provides an interesting contrast to the Hox1.1 experiments showing different t)pes of regulatory controls. 21. **
PUSCHELA, BALLINGR, GRUSS P: Separate Elements Cause Lineage Restriction and Specify Boundaries of Hox. 1.1 Expression. Development 1991, 112:279--288. The first demonstration that a/ac.Z transgene can direct normal patterns of expression, and defines the progressive restriction of promoter expression by multiple dements. Contrasts with the finding on Hox-26 and shows that different mechanisms are used by different Hoxgenes to establish patterns. 22.
PUSCHELA, BALLLNGR, GRUSS P: Position-Specific Activity of the Hox 1.1 Promoter in Transgenic Mice. Development 1990, 108:435-442.
23.
R~K
.
LEHRACHH: A Yeast Artificial Chromosome Containing the
M, LARL'4Z, COOK M, PAPAJ.OPULUN, KRUMLAUFN,
Mouse Homeobox Cluster Hox-2. Proc Naa Acad Sci USA 1990, 87:4751-4755. A useful paper that s h o ' ~ that Hox complexes can be isolated in a single ~xmstartificial chromosome clone. 24.
WOmEMU'm D, BEHR~GER R, MOSTOILER M, BRD,'S'I'ER R, PMMITER Pc Transgenic Mice Overexpressing the Mouse Homeobox-Containing Gene Hox.l.4 Exhibit Abnormal Gut Development. Nature 1989, 337:464-467.
25.
BAILINGR, MUTTERG, GRUSSP, KESSELM: CraI~ofaclal Abnormalities Induced by Ectopic Expression of the Homeobox Gene Hox-l.1 in Transgenic Mice. Cell 1989, 58:337-347.
26. ..
KESSELM, BAILLNG R, GRUSS P: Variations of Cervical Vertebrae After Expression of a Hox 1.1 Transgene in Mice. Cell 1990, 61:301-308. A key paper with the first demonstration in tramgenic mice that specific alterations to vertebrae can be explained by changes in Hox expression. These data prosided important support of the basic idea of a Hox code. 27. ..
CHL~
Introduction
Regulation of Hox transgenes
The vertebrate Hox homeobox gene family has been the increasing focus of attention with respect to both its potential roles for patterning axial diversity and the regulation of its spatially-restricted domains of expression. There is some confusion as to the term Hox, which many believe to be a generic means of referring to h o m e o b o x containing genes; however, Hox actually refers to a specific family o f vertebrate homeobox genes and the properties of the Hox family are distinct from other genes. In mammals and most vertebrates Hox genes are organized in clusters; there are four clusters that share many conserved structural features with homeotic genes in the Drosopbi/a ANT-C and BX-C complexes [1-3]. The overall structure of the mouse H o x network, its relationship to Droscpbi/a homeotic genes, and some of the expression properties are summarized in Fig. 1. This similarity between species initially led to the idea that H o x genes could be involved in axial specification based on the roles of their Drosophila homologues. Further support for this concept has come largely from in situ hybridization analyses (for review see [4"']) that showed that genes in the Hox clusters had graded overlapping domains of expression that correlated with their gene order along the chromosome, which is termed collinearity.
The primary method used to examine the spatial regulation o f Hox genes in transgenic mice involves using the bacterial /ac_Z gene as a reporter. In initial experiments very limited domains o f expression were observed [14,15o16-18]. In one case, a Hox-23/lacZ transgene was expressed in the kidney, but major sites of mesodermal and ectodermal expression were absent [14]. In another study [16], a 5' flanking region of the Hox-L3 gene directed expression of a /acZ gene on a heterologous promoter in a small region of the neural tube but only for a brief time during development. This result was extended to show that an enhancer region in the 5' flanking sequences originally from Hox-1.3 was responsible for this expression in the neural tube [17]. The relationship o f all of these patterns to expression of the endogenous genes was not clear, and it was impossible to determine whether these represented genuine subsets of the normal Hox gene pattern or ectopic expression. The tentative conclusion, generally drawn, was that the tmnsgenic constructs contained some but not all of the c~acting regulatory requirements, and that the missing regions might be distributed throughout the clusters. In view of these results and the collinear relationships between gene order and expression, one speculation was that an intact Hax cluster might be required to preserve the order of regulatory regions and generate the normal patterns of expression. In such cases, a region similar to the 13-globin locus control region would be required by the Hoxclusters ([19], see also review by Enver and Greaves pp 787-795).
Collinear spatially-ordered domains were observed in the limb [5,6"'], axial mesoderm [7], developing nervous system [1,8] and branchial arches [9"']; and recently, coUinearity was found to apply to the relative sensitivity of H o x g e n e s to retinoic acid in cell culture [10..,11 °.] and the timing of embryonic activation [12.]. The collinear relationship between gene order and embryonic expression appears to be a basic conserved feature of Hoxclusters and has led to the belief that the tightly regulated domains of expression represent part of a molecular combinatorial code for the differential specification o f axial structures [9°.,13..]. In this review particular attention will be devoted to recent transgenic mouse experiments that have made significant advances towards our understanding o f both the function and regulation of the H o x network in embryogenesis.
796
New data on two Hox genes, however, reveal that it is possible to reproduce the correct overall pattern of expression for a gene isolated from its normal context in a cluster and in a new chromosomal location [20..,21-.]. In an initial study by Puschel et aL [22] a Hox-1.1/lacZ transgene displayed the correct timing and anterior boundaries o f expression, but was unable to undergo the same spatial and lineage restricted expression in mesodermal derivatives as the endogenous gene. In a further study, Puschel et aL [2I..] also identified
(~) Current Biology Ltd ISSN 0958-1669
The Hox gene family in transgenicmice Krumlauf
Abd-B Abd-A Ubx
Antp
Scr
Dfd
2.1
2.6
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pb
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[6]
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Anterior
early high RA response
Fig. 1. Conservation between Hox and Hom-C homeobox complexes. Alignment of the mouse (Hox) and Drosophila (HOM-C) homeobox gene clusters. The vertical rows of boxes indicate genes which are highly related to each other, forming subfamilies. The brackets above the mouse genes identify the Drosophila homologue. Note that not all mouse Hox clusters have members in each subfamily. Shaded boxes are sequenced genes and the dashed boxes are genes present in the human clusters but not as yet identified in mouse. The large arrow beneath the clusters refers to the trends in collinear expression where successive genes from the left to right have increasing anterior boundaries, earlier stages of expression and sensitivity to retinoic acid (RA). the missing regulatory components, many of which are within the transcription unit itself, and demonstrated that a transgenic construct was capable of reproducing the endogenous Hox-l.1 pattern. These experimenters concluded that the promoter was able to regulate the early phase of Hox-l.1 expression with respect to the appropriate tissue distribution and anterior boundaries of expression; but that multiple elements are independently required to restrict the expression from this promoter in a lineage-specific manner. In an independent study by Whiting et aL [20-'], a Hox26,//acZ transgene was also found to be capable of duplicating the normal expression pattern on the basis that it had an identical time course, tissue distribution and spatial domains as the endogenous gene. In addition, the levels of the transgene expression were comparable to those of the endogenous H o x - 2 6 gene. Fig. 2 shows a comparison between the transgene and the endogenous Hox-2Ggene expression, to illustrate their similarity and the type and position of regulatory regions mapped in the transgenic experiments. Deletion analysis revealed that three regulatory regions were involved, and two of these functioned as spatially-specific enhancers [20..]. In contrast to the H o x l . 1 enhancers, the H o x - 2 6 enhancers were capable of imposing the same spatially restricted domains of expression on heterologous prorooters. Hence there is little specificity in the H o x - 2 6 promoter. Whereas the experiments using these two genes clearly demonstrate that it is possible for Hox genes to be appropriately expressed independently of their clusters, it is equally apparent that the two genes use quite differ-
ent strategies to establish and maintain their expression domains: In both cases multiple regulatory regions were involved in controlling expression, and several of these dements were located in the introns, exons, or 5' and 3' untranslated regions. This suggests that simply using flanking regions of a gene to assay for regulatory regions is not sul~cient, and it is important to maintain as much of the structural integrity of a Hox gene as possible in transgenic constructs. This is probably one reason why some of the early studies were unsuccessful. It is not surprising that multiple regulatory regions or enhancers are required for normal Hox expression and some of these may be shared between different members of a cluster. Therefore, it still remains possible that one of the reasons why Hox clusters have been conserved is to maintain the organization of dispersed regulatory regions, despite the fact that some genes can be regulated independently. The new data, however, clearly rule out an extreme model that requires a type of locus control region [19] for the proper expression of all genes In the cluster. Many of these issues can be resolved by using large regions conmining an entire Hox cluster on a yeast artificial chromosome [23"], and the development of tedmology for making transgenic animals with these chromosomes will be an important tool for those interested in Hox regulation.
F u n c t i o n a l analysis in t r a n s g e n i c a n i m a l s
Descriptive studies were essential in putting forward the idea of a combinatorial Hox code for the specification
797
798
Mammalian gene studies
HOXo2.2
HOX-2.1
HOX-2.6 * !
HOX-2.7 | !
o,y,
F Main enhancer
Lung
lqeural crest Spinal corcl Gut Somites Stomach Kidney
Neural enhancer "Only hindbrain" and spinal cord
Fig. 2. Reconstruction of the endogenous pattern of expression by a Hox-2.6/lacZ transgene. Diagram of the Hox-2.6 gene and the location of regulatory regions. Regions A and C are spatially-specific.
of regional diversity. Experiments in transgenic animals, however, have played an important role in verifying these ideas and actually examining the function of Hox genes in development. The over-expression of a Hox-l.4 gene in the colon of transgenic mice leads to an over-proliferation of intestinal epithelial cells resuhing in a megacolon phenotype [24]. Ectopic expression of the mouse Hox1.1 gene using a chicken actin promoter results in severe craniofacial abnormalities and the transformation of anterior vertebrae in the axial skeleton of transgenic mice [25,26-.]. Recently, it has been shown that the application of retinoic acid to mouse embryos results in similar craniofacial and vertebral transformations, and that it may do so by altering the patterns of Hoxexpression in these embryonic regions [13°.]. All these experiments show that the dominant, ectopic expression of a gene from the Hox network can change the identity and pattern of specific regional structures, presumably by changes in the Hox code. The most convincing evidence that Hoxgenes have a normal function in development has been elegantly provided by using embryonic stem cells (see review by Bradley pp 823--829) to generate tmnsgenic mice. In two cases, endogenous Hoxgenes ( - 1.5 and - 1.6) have been mutated by homologous recombination in embryonic stem cells; mice carrying the mutated aUeles in the germIine were generated by blastocyst injection and embryo transfer [27.*,28°*]. For both genes, heterozygous mice bearing the mutation displayed no visible phenotypes, but homozygous animals were non-viable and died shortly after birth. This suggests that the phenotypes had mutations resulting in a loss of function. Mutants of the Hox1.5 gene had a range of phenotypes largely apparent in tissues from the region of the head and thorax [27.°]. In the homozygous mutants the patterning of the head and thorax was altered so that the anterior cervical vertebrae were compressed, many cranial neural crest derivatives were malformed or missing, and the pharyngeal arches were abnormal in size and shape. It is difficult to separate primary from secondary and tertiary effects, and
the abnormalities were not extensively examined in early embryos. It is clear, however, that the perturbations arise in tissues where the endogenous Hox-l.5 gene is normaUy expressed, strongly suggesting that this gene is required for patterning in the branchial region of the head and thorax [27°°]. Homozygous mouse mutants for the Hox-l.6gene had a phenotype that was also restricted to the branchial region of the head, but in this case it was focused in cells derived from rhombomeres 4-7 [28-*]. Abnormalities are not confined to these regions and occur in many tissues and cell types, defects are observed in neuroectoderm, surface ectoderm, neural crest and paraxial mesoderms. The phenotypes themselves are extremely interesting but in terms of Hox function, it is the similarities and differences between the two phenotypes (wild type and mutant) which are most informative. Hox-l.5 and Hox-l.6 are expressed in posterior regions of the embryo, but no defects in these regions were observed [27",28-.], thus indicating that not all sites of Hoxexpression appear to require this expression for normal patterning. This is important because the four vertebrate Hox clusters are related by duplication and divergence from a common ancestor, which raises the possibility of functional overlap between highly related genes. But the fact that phenotypes do arise demonstrates that there cannot be a complete overlap or compensation between Hox genes. The neural crest is abnormal in both mutants, but in the case of Hox-l.5 it is the mesenchymal derivatives that are altered and the nerves and cranial sensory ganglia appear normal [27"]. In contrast, in the Hox-I.6mutants it is the neurogenic and not the mesenchymal derivatives that are missing [28--]. This suggests that different Hox genes from the same cluster can be used in the differential specification of the neural crest Finally, in neither mutant was there a simple transformation of structures similar to those observed in somites or cervical vertebrae upon ectopic expression of the Hox-l.1 [26-]. This may reflect basic differences in the way that the head and tnmk are patterned, and suggests that Hox genes may
The Hox gene family in transgenicmice Krumlauf 799 play different roles in different embryonic contexts (for reviews see [29",30",31]). Why are the mutant phenotypes concentrated in the branchial region and does this make sense based on the patterns of Hox expression? It has been postulated that Hox genes pLaya major role in the patterning formation of the embryonic head [9-*,29",51]. The Hox-2genes display segmentally-restricted patterns of expression which map to rhombomere boundaries [8], and these patterns extend to the surface ectoderm, cranial ganglia and mesenchymal neural crest (derived from the rhombomeres) in the branchial arches [9"*]. The relationships between Hoxgenes and the organization of the branchial head are illustrated in Fig. 3. It has been suggested that one important funcdon of Hox genes and rhombomeric segmentation in the hindbrain is to provide a means of linking positional information in the central and peripheral nervous system with the craniofacial structures and branchial arches [9",29",32"']. These recent studies on Hox mutants provide direct support for this theory.
Conclusions The recent transgenic experiments leave little doubt that the Hox network is a critical factor in the molecular mechanism of the specification of regional diversity. The mapping of regulatory regions has provided an insight into how the spatially-restricted domains of expression are generated in embryogenesis. Perhaps more importan@, they provide a means of specifically altering the patterns of Hox expression in transgenic animals to directly test predictions of the Hox code. In the absence of convenient genetic screens these regulatory regions will also permit biochemical approaches to identify and isolate the upstream factors in the regulatory hierarchy that is responsible for regulating Hox gene regulation. A great deal of information has already been obtained from just two mutant Hox genes, and as other members of the family are examined it appears possible that many of the interac~'e processes used to pattern vertebrate structures, particularly the head, will at last begin to fit into a clear molecular framework.
Acknowledgements Branchial arches
Nervous system
Surface Arch Cranial ectoderm mesenchyme ganglia
Neural tube
I ~x)uld like to thank members of my laboratory for extended discussions, in particular Paul Hunt, Jenny Whiting, Stefan Nonchev, and Mai Har Sham. I also thank Peter Rigby and Rudy Allemmm for discussion about our collaborati,.e work on Hox.2.6 and David Wflldnson, Peter Thorogood and Andrew Lumsden for ideas on head patterning.
-"
B1
References and recommended reading Papers of spedal interest, published within the annual period of re~e~,
havebeen higl~ghtedas: • •,
of interest of outstanding interest
1.
GR.,dO,.MA, PAPALOPULUN, KRUMI.AUF R: The Murine and Drosophila Homeobox Clusters have Common Features of Organisatinn and Expression. Cell 1989, 57:367-378.
2.
DtmOU1.ED, DOIZE P: The Structural and Functional Organization of the Murine Hox Gene Family Resembles that of Drosophila Homeotic Genes. EMBO 1989, 8:1497-1505.
3.
2.0
112.8
2.7
1--12.6
Fig. 3. Collinear and segmental expression Hox-2 codes for the branching region. Summary of the segmentally restricted pattern of Hox expression in the branchial region of the head. The bulges in the neural tube represent rhombomeres (r), and B1-B3 represent the branchial arches. Note Hox expression is restricted to specific axial levels in the rhombomeres, cranial ganglia, arch mesenchyme, and surface ectoderm [9"].
BONCLN'ELLI E, AC&~PORA D, P&N'NESEM, D'EsPOSITO M, SOYLVL~ R, GUAD~¢OG, STOmZ~a'UOLOA, CAHXROM, FAmLLAA, S~tEO.',Z A: Organization of Human Class I Homeobox Genes. Genome 1989, 31:745-756. 4. KE.~ELM, GRUSS P: Murinc D~'elopmental Control Genes. Science 1990, 249:374-379. ~ ' s is art excellent recent review which provides the reader with a reasonable idea of the important areas of research in the Hox field. It hightlights the s-altre of identifying genes by evolutionary homology and also details proportion of other genes implicated in the regulation of vertebrate development. 5.
DOI.I.E P, IZHSUA-BELMON'rEJC, FALKLXSaX~ H, RENUCO A, DtmOULE D: Coordinate Expression of the Murine Hox.5 Complex Homeobox-Containing Genes During l lmh Pattern Formation. Nature 1989, 342:767-772.
6. •,
IZPISUA-BELMONTE J-C, TICKLEC, DOLLEP, WOLPERTL, DUBOUtE D: Expression of Homeobox Hox.4 Genes and the Specification of Position in Chick Wing D~'elopment. Nature 1991, 350:585-589.
800
Mammalian gene studies A key paper for those interested in the limb and Hox genes. Demonstrates beautifully the collinear spatial regulation of Hoxin the limb and correlates their expression to retinoic acid and patterning. Suggests how a Hox code for the limb could be used for differential patterning. 7.
8.
DRESSLER GR, GRUSSP: Anterior Boundaries of Hox Gene Expression in Mesoderm-derived Structures Correlates with the Linear Gene Order Along the Chromosome. D~'erentiation 1989, 41:193-201. WILK~3OND, BHA'['r S, COOK M, BONCLN'ELUE, KRUMIAUF PC Segmental Expression of Hox 2 Homeobox-Containing Genes in the Developing Mouse Hindbrain. Nature 1989, 341:405-409.
9. •.
HUNT P, WmKLXSOND, KRU,Va~UFPC Patterning the Vertebrate Head: Murine Hox 2 Genes Mark Distinct Subpopulations of Premigratory and Migrating Neural Crest. Development 1991, 112:43-51. A crucial study that links Hoxgenes to patterning in the neural crest and branchial head in addition to the previously described rhombomeres in the hindbrain. Details the idea ofa Hox code for the head, and suggests that Hoxgenes could be part of the molecular pre-patteming signal for cranial neural cresL 10. •.
SIMEONEA, ACAMPORAD, ARCION1L, A~'DP,EWS FW, BONCB,'ELU E, MAV11JO F: Sequential Activation of Hoxl Homeobox Genes by Retinoic Acid in Human Embryonal Carcinoma Cells. Nature 1990, 346:763-766. Provides an important extension of the basic properties of Hox regulation, by showing that retinoic add induction in cultured ~ is collinear ~ith gene order. This suggests a means whereby retinoic acid can set up overlapping domains of Hox expression in vertebrate embryos. 11. •.
PAPAI.OPULUN, LOVELL-BADGER, KRUMLAUFPc The Expression of Murine Hox-2 Genes is Independent on the Differentiation Pathway and Displays Collinear Sensitivity to Retinoc Acid. Nucleic Acids Res 19:5497-5506. This study extends the collinear s e n s i ~ t y of Hox genes to mouse cells and Xenopus embt3x3s. This demonstrates that the coUinear retinoic acid response is consetx'ed in vertebrate evolution and likely to be an important feature in viL~ 12.
IZPISUA-BELMO.WI'EJ, FALKENS'I'ELNH, DOLLE P, RENUCC! A, • DUBOULED: Murine Genes Related to the Drosophila AbdB Homeotic Gene are Sequentially Expressed During Development of the Posterior Part of the Body. EMBO J 1991, 10:2279-2289. An interesting study that deals vdth the evolution of 5' subfamilies in Hox clusters and also shows a temporal coUinearity for the tIox-4genes in the posterior embryo. 13. •.
KESSELM, GRUSS P: Homeotic Transformations of Murine Prevertebrate and Concomitant Alteration of Hox Codes Induced by Retinoic Acid. Cell 1991, 67:89-104. This is another key paper dealing with two issues. It proposes a Hox code for the somites and paraxial me.soderm which complements those proposed for the limb and head. It also demonstrates that retinoic acid can cause transformations to vertebrae presumably by changing the Hox code. 14.
E~.ss C, VOGELSP,, DE GRAAFFW, BO,~q~-ERO'fC, MEIJLtNXFNJF, DESCHAMPSJ: Hox-2.3 Upstream Sequences Mediate lacZ Expression in Intermediate Mesoderm Derivatives of Transgenie Mice. Development 1990, 109;776-786.
15. •
BIEBEmCHC, UTSETM, AWGULE~TI3CHA, RUDDLEF: Evidence for Positive and Negative Regulation of the Hox-3.1 Gene. Proc Natl Acad Sci USA 1990, 87:8462-8466. This paper describes some interesting features of the Hox-3.1 promoter. 16.
ZAK&NYJ, TUGGLE CK, PATEL MD, NGUYEN-HUUMC: Spatial Regulation of Homeobox Gene Fusions in the Embryonic Central Nervous System of Transgenic Mice. Neuron 1988, 1.679-691.
17.
TUGGLECK, ZAK~" J, CraWl'l"1 L, PESCH1.EC, NGUYEN-HUUMC: Region-Specific Enhancers Near Two Mammalian H o m e o -
Box Genes Define Adjacent Rostrocaudal Domains in the Central Nervous S),tem. Genes Dev 1990, 4:180-189. 18.
SOtUGHARTK, BIEBERICH C, Ell) R, RUDDLE F: A Regulatory Region from the Mouse Hox.2.2 Promoter Directs Gene Expression Into Developing Limbs. Development 1991, 112:807--812.
19.
GROSVELD F, VAN ASSENDELFT G, GS.EAVES D, Komm B: Position-lndependent, High Level Expression of the Human Beta.Globin Gene in Transgenic Mice. Cell 1987, 51:975--985.
20. *.
WHITLNGJ, MARSHALLH, COOK M, KRUMLAUFR, R/GBYP, STOTT D, ALLEMANNPc Multiple-Specific Enhancers are Required to Reconstruct the Pattern of Hox.2.6Gene Expression. Genes Dev 1991, 5: 2048-2059. This is an important study which demonstrates that it is largely possible to reconstruct Hox expression patterns in transgenic mice. It extends previous studies in identifying regulatory regions involved in this process which act as enhancers that set boundaries of expression even on heterologous promoters. Provides an interesting contrast to the Hox1.1 experiments showing different t)pes of regulatory controls. 21. **
PUSCHELA, BALLINGR, GRUSS P: Separate Elements Cause Lineage Restriction and Specify Boundaries of Hox. 1.1 Expression. Development 1991, 112:279--288. The first demonstration that a/ac.Z transgene can direct normal patterns of expression, and defines the progressive restriction of promoter expression by multiple dements. Contrasts with the finding on Hox-26 and shows that different mechanisms are used by different Hoxgenes to establish patterns. 22.
PUSCHELA, BALLLNGR, GRUSS P: Position-Specific Activity of the Hox 1.1 Promoter in Transgenic Mice. Development 1990, 108:435-442.
23.
R~K
.
LEHRACHH: A Yeast Artificial Chromosome Containing the
M, LARL'4Z, COOK M, PAPAJ.OPULUN, KRUMLAUFN,
Mouse Homeobox Cluster Hox-2. Proc Naa Acad Sci USA 1990, 87:4751-4755. A useful paper that s h o ' ~ that Hox complexes can be isolated in a single ~xmstartificial chromosome clone. 24.
WOmEMU'm D, BEHR~GER R, MOSTOILER M, BRD,'S'I'ER R, PMMITER Pc Transgenic Mice Overexpressing the Mouse Homeobox-Containing Gene Hox.l.4 Exhibit Abnormal Gut Development. Nature 1989, 337:464-467.
25.
BAILINGR, MUTTERG, GRUSSP, KESSELM: CraI~ofaclal Abnormalities Induced by Ectopic Expression of the Homeobox Gene Hox-l.1 in Transgenic Mice. Cell 1989, 58:337-347.
26. ..
KESSELM, BAILLNG R, GRUSS P: Variations of Cervical Vertebrae After Expression of a Hox 1.1 Transgene in Mice. Cell 1990, 61:301-308. A key paper with the first demonstration in tramgenic mice that specific alterations to vertebrae can be explained by changes in Hox expression. These data prosided important support of the basic idea of a Hox code. 27. ..
CHL~
