Pursuing the functions of vertebrate homeobox genes: Progress and prospects

Pursuingthe funcb'onsof vertebrate homeoboxgenes:progressandprospects Peter W. H. Holland

Departmentof Zoology, Universityof Oxford,5outhParks Road,Oxford OXl 3PS,UK.

uch progress has been made towards elucidating the celM lular and molecular mechanisms that generate body organization during vertebrate embryogenesis. However, the regulatory genes presumed to underly these processes have long remained obscure. It is therefore significant that recent research suggests that some of the genes responsible for controlling critical developmental events, such as body regionalization, neuronal diversification and neural crest cell fate, may be in hand, and open to molecular investigation. Strong candidates are several gene families evolutionarily related to known development-regulating genes from Drosophila. These include some genes containing the conserved homeobox, paired-box and zinc-finger motifs (for review see Ref. 1). In Drosophila, functional analysis of these genes has relied primarily on the well-established genetics of this animal, supplemented by molecular biology. For example, description of mutant phenotypes suggests that some Drosophila homeobox genes (e.g. ftz, eve) have roles in controlling both segmentation and neuronal differentiation, whereas others (the homeotic genes) play a very different role, to control positionspecific characteristics in the early embryo and in the CNS2. By contrast, most of the members of these gene families in vertebrates were isolated solely by virtue of structural similarity to Drosophila genes, in the absence

of genetic information. How then can the functions of these genes be investigated? At present, three general strategies appear feasible (Table I). The first is descriptive, relying on data concerning molecular evolution and gene expression to give insight into function. The second and third approaches aim to disrupt gene activity experimentally, either indirectly or via genome modification. Description of gene sequence, organization and expression is an essential prerequisite to experimental disruption. Furthermore, at least for some of the mammalian homeobox genes, recent descriptive data have allowed the formulation of surprisingly specific hypotheses regarding function. The mammalian homeobox genes characterized to date include two genes given the prefix 'En', containing homeobox sequences similar to Drosophila engrailed, and at least 25 'Hox' genes, in four clusters, with closer sequence similarity to homeotic genes within the Drosophila Antennapedia and Bithorax complexes 3'4. As reviewed elsewhere 5'6, these genes are expressed in precise rostrocaudal regions of the early embryo, the developing CNS, and, often, the PNS and mesoderm. These observations have led to the suggestion that the Hox and En classes of mammalian homeobox genes, like Drosophila homeotic genes, act in combination to control positionspecific differences in cell behaviour and differentiation. Examples

TABLE I. Strategies for investigating developmental roles of vertebrate genes Strategy

Examples

Description Gene expression Sequence and organization

In situ hybridization, immunocytochemistry Comparison with Drosophila

Somatic disruption of gene product Sense RNA injection Antisense RNA injection Antibody injection

Xenopus Xhox- IA RNA 8 XenopusXlHbox-1 antibodies 9

Mutation of the genome Test for allelism with known mutations Transgenes Homologous recombination 206

Mouse Pax-I allelic with undulated 12 Creation of dominant, gain-of-function mutation 13 Disruption or modification of endogenous gene 21'22 © 1989,ElsevierSciencePublishersLtd,(UK) 0166-2236189/$02.00

of such roles may include the control of neuronal differentiation in the CNS, and the specification of fate in some populations of neural crest cells. In the light of these suggestions, it is particularly exciting that recent DNA sequence analyses4'7 suggest that the Drosophila homeotic gene complexes and the four clusters of mammalian Hox genes have evolved from a common ancestral gene cluster. Although the pathways of gene duplication have been different in the two lineages, the order of genes within the clusters has been maintained to a striking degree. Thus, in several cases, particular mammalian genes can be identified as homologues of particular Drosophila genes. For example, mammalian Hox-2.5 and Hox-l.7 are likely to be homologues of Drosophila iab-7, whereas Hox-2.6, Hox-l.4 and Hox-5.1 may have shared an ancestral gene with Drosophila Deformed. These observations, together with the continually refined expression data, greatly strengthen the suggestion that vertebrate Hox genes have similar functional roles to Drosophila homeotic genes, in controlling the regional diversification of the embryo and its organ systems. In order to test these suggestions experimentally, the cellular activity of specific genes or gene products must be perturbed. A variety of strategies, using both somatic and inherited components, could be considered (Table I). Furthermore, several recent reports indicate that many of these strategies are feasible, and even informative. The concentration of a gene product can be modulated by injecting specific macromolecules into embryos, which may then be observed for developmental defects. For example, levels of mRNA from a particular gene could be enhanced by injection of mRNA synthesized in vitro, or reduced by injection of 'antisense' RNA designed to hybridize to, and interfere with, endogenous mRNA molecules. In a recent report by Harvey and Meltons, the former approach was used to disrupt the levels of mRNA from the Xenopus gene Xhox-lA; a homeobox gene homologous to the 'Deformed-like' group of mareTINS, VOI. 12, NO. 6, 1989

malian Hox genes. These authors injected synthetic Xhox-lA mRNA into fertilized eggs immediately prior to the first cleavage, such that the introduced excess RNA was likely to remain restricted to one side of the resultant embryos. Strikingly, subsequent histological examination revealed unilateral disorganization of muscle blocks in a large proportion of embryos developing after injection of Xhox1A. Specific functional conclusions are difficult to draw at present, particularly since gene activity in ectopic sites may have caused some effects unrelated to the normal function of the gene. Nonetheless, the results are encouraging, and suggest that Xhox-iA plays some role in controlling spatial organization within the somitic mesoderm. Perhaps easier to interpret are data reported by Cho et al. 9, which provide experimental evidence for homeobox gene involvement in the development of the PNS. These authors injected specific antibodies into fertilized Xenopus eggs, to interfere with the cellular activity of the XlHbox-1 gene product. Tiffs gene, another member of the Hox family of vertebrate homeobox genes, is normally expressed in a specific rostrocaudal region of the spinal cord, and in some derivatives of the neural crest (dorsal root ganglia and dorsal fin). The exciting result reported by Cho et al. was that in several embryos injected with antibodies to X1Hbox1, but in none injected with control antibodies, specific neural crest derivatives did not develop. Thus, response to tactile stimuli in the anterior trunk region was often abolished, and, at low frequency, both dorsal fin and dorsal root ganglia were absent in the cervical region. The results imply that the X1Hbox-1 protein is essential for the development of some tissues in which it is expressed, for example, some dorsal root ganglia. Furthermore, the data support the hypothesis that this gene controls cell fate within a specific rostrocaudal region, rather than controlling either segmental or cell-type specific events throughout the embryo. Interestingly, Cho et al. do not report any abnormalities of the CNS in affected embryos, although XIHbox-1 is normally expressed with striking spatial restrictions in TINS, VoL 12, No. 6, 1989

this tissue. It will thus be a challenge for future work to ascertain whether subtle changes are produced, and how these may relate to the normal pattern of expression of the gene. Another intriguing observation is that in experimental embryos lacking cervical dorsal root ganglia, the migration of melanocytes, which also develop from neural crest cells, appears to be normal. Although requiring further investigation, this suggests that the fates of different subpopulations of neural crest cells are specified via different molecular mechanisms. This hypothesis is also consistent with patterns of homeobox gene expression in the mammalian head 1°. As demonstrated by the reports of Cho et al.9 and of Harvey and Melton8, somatic disruption of gene activity has excellent prospects for further investigation into the function of vertebrate homeobox genes. A general pitfall of these strategies, however, is that the phenotypes produced in a particular experiment may range greatly in severity, presumably due to variations in persistence and distribution of the injected macromolecules. An alternative approach, less prone to this problem, is to study genetic mutations in which expression or activity of the target gene is altered. Many potentially interesting mutant phenotypes have been described and maintained in inbred strains of laboratory mice H, although for nearly all of these the molecular basis is unknown. Hence, in the quest for homeobox gene mutants, several laboratories have tested for allelism between mouse homeobox genes and previously characterized genetic mutations. Although this strategy has not yet led to the identification of any mutant homeobox gene, success has been reported for one of the mouse genes containing a 'paired box' motif. Thus Bailing et al. 12 have recently demonstrated that Pax-1 is allelic with a mouse mutant known as undulated. Discovery of this allelism may well prove useful for functional analysis of Pax-l; however, for most candidate development-regulating genes a more directed strategy is deafly required, so that specific mutations can be created. Furthermore, directed approaches can

potentially allow gene activity to be altered in prespecified ways, to create a range of mutations of known molecular basis. Strategies have been conceived that should allow the creation of either dominant 'gain-of-function' or recessive 'loss-of-function' mutations in any vertebrate gene, again primarily using mice as the experimental organism. If successful, these approaches will herald a new era in developmental biology, and, although it is still early days, several recent reports suggest the prospects are good. The now standard strategy to create gain-of-function mutations is via microinjection of recombinant DNA sequences into fertilized mouse eggs, to create transgenic strains of mice. These animals have the injected DNA sequence (for example, a modified gene) stably incorporated into their nuclear genome, although with no predictability regarding the site of DNA integration. It is important to note that under these conditions the animal will carry the modified gene in addition to its two endogenous copies. For example, Wolgemuth et al. 13 report the production of transgenic mice carrying an extra copy of the Hox-l.4 gene. Inter° estingiy, these mice develop an unusual postnatal phenotype: a grossly enlarged hindgut. However, although this suggests that Hox-l.4 expression can affect gut development, it is still difficult to draw precise functional inferences. An alternative application of transgenic technology involves the use of 'hybrid' transgenes, which allow gene expression to be deliberately targeted to inappropriate cells. For example, constructs could be introduced conraining the coding sequence from a homeobox gene under the control of regulatory DNA sequences from cell- or tissue-specific genes. An alternative experiment, which may prove particularly informative, will be to target gene expression to inappropriate spatial regions within tissues where normal expression is region-specific; for example, the expression of Hox genes in the CNS and PNS. However, this can only be achieved when DNA sequences capable of directing region-specific gene expression are characterized. A recent paper by Zakany et 207

al. 14 is significant in this respect.

These authors have produced transgenic strains of mice carrying DNA sequences from upstream of the H o x - l . 3 gene fused to the coding sequence for the readily detected histochemical marker, [5galactosidase. By utilizing varying lengths of upstream DNA sequence, Zakany et al. identified a cis-acting regulatory region, between 300 and 800 base pairs 5' of the H o x - l . 3 transcription start site, which directs gene expression to a particular rostrocaudal region of the embryonic spinal cord. The transgene is only expressed in a subset of the cells normally expressing the endogenous Hox-l.3; a result with interesting implications for understanding the regulation of homeobox gene expression. However, the most significant result from this work must be the identification of the regulatory element itself, which may prove an invaluable tool for misregulating other homeobox genes in transgenic mice. For example, hybrid genes could be constructed containing this regulatory sequence fused to the coding region of Hox-1.1 or Hox-3.1, genes normally expressed in more caudal regions of the CNS than is H o x - l . 3 (Refs 5, 15, 16). Analysis of transgenic mice carrying such constructs may allow a test of the hypothesis that different vertebrate Hox genes control cell fate within different rostrocaudal regions of the CNS. However, a potential problem with this approach is highlighted by the results of an analogous experiment performed on Drosophila. By expressing an introduced Antennapedia gene under the control of a heat shock promotor, Gibson and Gehring 17 have clearly demonstrated that different Drosophila tissues vary greatly in their sensitivity to ectopic homeotic gene expression. This feature, if also true for mammalian embryos, may make interpretation of gain-offunction mutants difficult. By contrast, the complementary approach of targeting loss-offunction mutations to particular endogenous Hox genes may allow more direct functional analysis. It is, however, only in the past four years that this strategy has become a less than impossible objective. The practical problem which had always precluded this experi208

ment is that when cloned DNA is introduced into the mammalian genome (for example, in the production of transgenic mice), the site of integration is essentially random. However, in 1985, Smithies et al. TM reported that, following transfection of DNA into tissue culture cells, recombination can occur between an introduced DNA molecule and identical sequences in an endogenous gene. Thus, by introducing a modified gene comprising wild-type sequences flanking a mutation, it is possible for a copy of the endogenous gene to be replaced by a mutant copy19. However, in these experiments such 'homologous recombination' occurred in only about one in a thousand cases of DNA integration, a frequency far too small to be directly applicable to the production of transgenic mice. Hence, the task now created was to transfer the technology for large-scale manipulation, and screening for rare events, from the tissue culture dish into the organism. A possible solution pursued by several laboratories relies on the use of pluripotent cell lines, known as embryonal stem (ES) cells. These mouse embryo-derived cells, when injected into host blastocysts, can contribute descendant cells to embryonic tissues and, in some cases, to the germ cell lineage 2°. Hence the general strategy involves microinjection or transfection of modified gene sequences into cultured ES cells, followed by in vitro selection for cells in which homologous recombination has occurred, and introduction of these into blastocysts. Mice developing to term will be chimaeric, with both wild type and heterozygous mutant cells; thus inbreeding will be required to produce animals homozygous for the desired mutation. However, there are several technical hurdles to overcome in this scenario, including the design of in vitro selection methods, first to identify cells in which DNA integration has occurred, and second, to recognize among these any cases of homologous recombination. Several elegant strategies have been described, and in two recent studies by Joyner et al. 21 and Zimmer and Gruss z2, have been used in attempts to disrupt homeobox genes.

Joyner et al. transfected into ES cells a recombinant DNA sequence containing a neomycin resistance gene (neo r) inserted into, and therefore disrupting, the coding sequence of a cloned E n - 2 gene. Since the inserted neo r gene is under the control of a promoter active in ES cells, and since neo ~ confers resistance to G418 in eukaryotic cells, treatment with this drug will select for cells in which the construct has integrated into the genome. To identify the rare cases of homologous recombination among the background of cells with random integration sites, Joyner et al. used the polymerase chain reaction (PCR) method, a technique which vastly amplifies DNA segments between two pre-specifled unique sequences. Thus by "choosing one sequence from the introduced neo r gene, and a second adjacent to the desired recombination site in En-2, PCR amplification should only be possible if the pool of analysed cells includes one with a correctly targeted mutant gene. Repeated subdivision of the cell cultures, followed by further PCR analysis, allowed Joyner et al. to obtain lines of ES cells carrying the mutated E n - 2 gene, which were used to produce chimaeric mice via injection into blastocysts. Zimmer and Gruss 22 employed a similar strategy to disrupt the Hox1.1 gene in ES cells, and then produce chimaeric mice, although in this case drug resistance was not involved. The only modification made to the introduced construct was the insertion of a short oligonucleotide into the coding sequence of Hox-l.1, creating a premature stop codon. After microinjection into ES cells, PCR was again used to identify cases of homologous recombination, in this case among pools of all injected cells. That Zimmer and Gruss succeeded in isolating ES cells carrying the targeted mutation, without first selecting for all cases of DNA integration, reflects the high frequency of homologous recombination observed in this experiment (1 in 150 injected cells). This achievement is particularly significant, since if this frequency can be routinely attained it will be possible to target and identify even the most minor sequence modifications, in perhaps any gene. The future for vertebrate TINS, Vol. 12, No. 6, 1989

developmental genetics, therefore, looks better than ever. Recent technical advances have made possible targeted mutation of specific genes, thus potentially allowing thorough genetic analysis of any vertebrate gene. This may sound like the ultimate wish of many developmental biologists, but what would it really achieve? Will homologous recombination enable precise functional analysis, and reveal specific roles for homeobox genes in, for example, neurulation, neuronal differentiation and neural crest cell fate? It is already clear that if these aims are to be fulfilled, several complicating factors must be taken into account. For example, if a mutation created via homologous recombination exerts a donfinant, deleterious effect on gametogenesis or embryogenesis, offspring might not be obtainable from chimaeric mice. It may be significant that Joyner et al. and Zimmer and Gruss have not yet reported obtaining heterozygous mutant offspring from their chimaeric mice, although the reasons for this are unclear. Another complication for strategies aimed at abolishing the activity of a gene is that descriptions of gene expression and evolution suggest that homeobox genes have multiple roles, at different stages of development. In addition, since development itself involves hierarchies of tissue interactions, disruption of one event is likely to have many indirect consequences. It is, therefore, critical that these, and many other, predictions are taken into account when specific gene disruption experiments are designed. Only by combining analyses of gene evolution and expression, somatic disruption of gene activity, experimental embryology, transgenic technology, and homologous recombination, will an understanding of the roles of vertebrate homeobox genes eventually emerge.

Selected references 1 Dressier, G. and Gruss, P. (1988) Trends Genet. 4, 214-219 2 Doe, C. Q, and Scott, M, P. (1988) Trends Neurosci. 11, 101-106 3 Martin, G. R. etal. (1987)Nature325, 21-22 4 Boncinelli, E. et al. (1988) Hum. Reprod. 3,880-886 5 Holland, P. W. H. and Hogan, B. L. M. (1988) Genes Devel. 2, 773-782

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6 Stern, C. D. and Keynes, R. J. (1988) Trends Neurosci. 11, 190-192 7 Graham, A., Papalopulu, N. and Krumlauf, R. Cell (in press) 8 Harvey, R. P. and Melton, D. A. (1988) Cell 53,687-697 9 Cho, K. W. Y. etal. (1988) EMBOJ. 7, 2139-2149 10 Holland, P. W. H. (1988) Development 103 (Suppl.), 17-24 11 Green, M. C. (1981) Genetic Variants

and Strains of the Laboratory/House Gustav Fischer Verlag 12 Bailing, R., Deutsch, U. and Gruss, P. (1988) Cell 55, 531-535 13 Wolgemuth, D. et al. (1989) Nature 337, 464-467 14 Zakany, J. et aL (1988) Neuron 1, 679-691

15 Gaunt, S. J., Sharpe, P. T. and Duboule, D. (1988) Development 104 (Suppl.), 169-179 16 Mahon, K. A., Westphal, H. and Gruss, P. (1988) Development 104 (Suppl.), 187-195 17 Gibson, G. and Gehring, W. J. (1988) Development 102, 657-675 18 Smithies, O. etal. (1985) Nature 317, 230-234 19 Thomas, K. R. and Capecchi, M. R. (1987) Cell 51,503-512 20 Bradley, A. et al. (1984) Nature 307, 255-256 21 Joyner, A. L., Skarnes, W. C. and Rossant, J. (1989) Nature 338, 153-156 22 Zimmer, A. and Gruss, P. (1989) Nature 338, 150-153

Downonchromosome21 ? ost cases of Down's synM .drome are caused by the presence of an extra copy of chromosome 21 (trisomy 21). It is still unclear why an extra copy of a normal chromosome 21 should lead to the Down's phenotype. Presumably, over-expression of genes encoded on this chromosome underlies the condition and leads to alterations of expression of genes encoded on other chromosomes. The alteration in gene expression leads to a distortion in the balance of biochemical pathways important for the proper development and functions of organs affected in the syndrome 1. However, it is not clear what proportion of chromosome 21 genes is important in determining the characteristic phenotype of the syndrome. Do individual genes underlie specific features (e.g. the dermatoglyphics, the facies, the mental retardation, the susceptibility to congenital heart abnormalities or to leukaemia)? Or does imbalance in the expression of many or all of the genes on chromosome 21 act in a non-specific fashion to produce the phenotype? The relative importance of different genes on chromosome 21 in determining the Down's phenotype can be investigated using one of two main approaches. The first is the careful molecular and clinical investigation of that small number of individuals who have unbalanced translocations of part of chromosome 21 to determine the minimal extent of chromosome triplication necessary to produce the Down's phenotype. The second is the pro-

duction and examination of experimental animals that have extra copies of genes expressed on human chromosome 21. Down's syndrome cases with partial trisomy 21 A very small proportion (1-5%) of individuals with Down's syndrome have an unbalanced translocation of part of the long arm of chromosome 21. Careful molecular analyses of such cases ~-5 have determined that the distal third of the long arm of the chromosome is all that is required for the development of the Down's phenotype. This has led to this part of the chromosome region being defined as the Down's 'obligate' region2-5 (see Fig. 1). However, there are two problems with this type of careful correlation between genotype and phenotype. First, the definition of the Down's phenotype is unsatisfactory. Clearly, the characteristic facies and the mental retardation are essential aspects of the phenotype. However, other variable features, while no less characteristic of the phenotype, are more difficult to study using individual cases. For example, the increased prevalence of leukaemia in trisomy 21 Down's cases cannot be assessed in individual transiocation 21 cases. This prevents assignment of this characteristic to the Down's obligate region. Furthermore, it is not clear whether persons who are trisomic only for the 'obligate' Down's region develop Alzheimer's disease in the same way as the full trisomy cases. One

© 1989, ElsevierSciencePublishersLtd,(UK) 0166- 2236/89/$02.00

John Hardy Nick Irving Anna Kessling Departmentof Biochemistryand MolecularGenetics, StMary's Hospital MedicalSchool, London W2 1PG,UK.

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