Mouse Hox genetic functions

Robb Krumlauf

National institute for Medical Research, London, UK

Functional analysis of the genetic roles of the mammalian Hox homeobox family has not been facilitated by the analysis of disease-associated or classically derived mutant loci. However, an increasing number of mouse /-/ox mutations, generated by homologous recombination, have now demonstrated that the Hox genes are key components in regulating

the patterning of axial structures.

Current Opinion in Genetics and Development 1993, 3:621-625

Introduction

The four clusters of Hox homeobox genes in vertebrates have come to represent one of the best known exam- ples of conservation in structural and genetic organi- zation in animal species (reviewed in [l-d]). There is intense interest in these chromosomal complexes be- cause the genes encode transcription factors that pre- sumably have been selected for their ability to regulate common processes in diverse vertebrate and inverte- brate species. It is the elusive nature of this conserved functional role that has drawn the attention of many evo- lutionary and developmental biologists. The Ho.x genes were originally isolated as a result of their homology to Drosophifa homeotic genes in the HOMC (ANT-C BX-C) complex, and most of the current ideas on how they function are derived from the roles of their Drosophila counterparts [2,4,6,7]. The HOM-Cgenes are genetically functional early in Drosophila embryogenesis in the specification of segmental identity, where ‘loss of function’ mutations can result in the transformation of one segment into an adjacent identity [8,9]. The vertebrate Hox genes are also expressed in segmental units in the hindbrain (rhombomeres) [lo] and pat-axial mesoderm (somites) [ 11,121, but there is widespread expression in tissues that are not segmented. It is pos- sible that segmentation is the common functional link; however, clusters of the HodHOM-type have been ob- served in non-segmented organisms [ 131 and it is widely believed that segmentation arose independently in pro- tostomes and deuterostomes. Therefore, the Ho-dHOM complexes may have initially been important for some other process or processes, such as patterning in the nervous system, and then independently co-opted for use in segmentation.

A fundamental property of the Hox/HOM complexes (termed colinearity) is a key element in the most widely accepted suggestion that the common role for this gene family is the specification of regional identity along em-

bryonic axes [2,14,15]. In the Hox clusters there is a di- rect linear correlation (colinearity) between the order of the genes along the chromosome and their temporal ap- pearance and anteroposterior CAP) boundary of expres- sion along the embryonic axis [16,171. This is observed in the central nervous system (CNS), peripheral nervous system (PNS), par-axial mesoderm, cranial neural crest, limbs and genitalia [7,10,16-261. This qridespread colin- ear relationship suggests the HodHOM complexes are a molecular reflection of the embryonic axis that provides a coordinated system of axial cues which form part of a conserved mechanism for generating different positional identities in the embryo. The term Hex code has been used to describe such a complex combinatorial pattem- ing system [7,18] and much of the current experimental work is directed at investigating this model. The purpose of this review is to examine how the phenotypes aris- ing from manipulation of Hoxgenes in mice are provid- ing critical insight into the genetic function of this gene family.

The axial skeleton and similarity in HoxlHOM function

A major site of Hex expression is in the sclerotome derivatives of developing somites, which generate the axial skeleton. Ectopic expression of Hex-a7 in trans- genie mice leads to perturbations of craniofacial and skeletal structures, and also to the homeotic transforma- tion of the first cervical vertebrae into a posterior iden- tity [27,28]. These experiments lead Kessel and Gruss to propose a Hox code for specifying the identity of individual prevertebrae [7] that has been strongly sup- ported by the recent experimental evidence. All mouse mutants generated by ectopic expression of Hox genes [28,2900-310*] or targeted disruption [32-34,35**,36**] have been examined for skeletal abnormalities, because

Abbreviations AP-anteroposterior; C-cervical; CNS-central nervous system; l-lumbar; PNS--peripheral nervous system;

RA-retinoic acid; RAR-RA receptor; T-thoracic.

@ Current Biology Ltd ISSN 0959437X 621

622 Pattern formation and developmental mechanisms

phenot)rpic alterations to the skeleton are easily assayed. In all cases, clear changes to the skele@ components arise and a large subset of these result in homeotic transformations. Mutation of the Hex-CL? gene provided the first example of a homeotic transformation result- ing from loss of function, demonstrating that the Hox genes have a normal role in patterning of prevertebrae [35**]. Transformations occurred between the seventh thoracic (T), l7 and first lumbar CL), Ll, vertebrae, with the mice most notably displaying an extra pair of ribs derived from Ll. An important finding from this experi- ment is that the loss of function phenotype represents an anterior transformation of posterior structures, as found in loss of function mutations for the Drosophila HOM-C homeotic loci.

Further support that the Hex genes can function in an analogous manner to HOM-C has recently been pro- vided by the ectopic expression of the Hox-d4 gene [29”]. Regulatory regions from the Hex-al gene linked to the Hex-d4 gene promote its expression in mesoderm in regions anterior to its normal boundary of expres- sion. This causes the posterior transformation of the occipital bones of the skull. Combined with the previ- ous observation that the ectopic expression (‘gain of function’) experiments in transgenic mice with Hex-a7 also results in posterior vertebral transformations [28], it appears that gain of function mutations in both the murine Hex and Drosophila HOM genes generate recip- rocal phenotype transformations as compared with their loss of function alleles.

A Hex code model

This strong experimental correlation between the genetic functions of HodHOM genes is compelling evidence that specification of axial identity is a common feature of this ancient complex. However there are important differences between Hox and HOM-C In Drosophila, there is not as extensive an overlap in the expression of homeotic genes that regulate the identity of adjacent seg- ments. For example Anp expression in one parasegment is repressed by Ubx in the adjacent parasegment. Conse- quently, ectopic expression of Ubx in the Anp domain represses Anp function resulting in the more posterior Ubx mediated identity.

Conversely, loss of Ubx allows Anp to be expressed in the posterior segment generating an anterior transforma- tion [37]. However, the Hox genes have domains of ex- pression that usually extend from the posterior regions of the embryo to sharp anterior boundaries. This gen- erates extensive degrees of overlap in posterior regions, raising the question as to which gene takes functional precedence in the presence of other members of the network.

To account for the overlap enigma, Hox genes are be- lieved to exert their biological role in a restricted portion of their expression domains, normally confined to their most anterior domains of expression. This phenomenon

is termed ‘posterior prevalence’, asserting that the next gene or genes in the complex(s), which has an ante- rior boundary posterior to the gene in question, will supersede its function and take the dominant role in patterning. Simply stated this means that a gene would function primarily in the domain where it is unique or does not overlap with other members of the same cluster. This issue is of course complicated by the fact that there are four clusters, and a unique domain for a gene from one cluster will overlap with unique domains for genes from complexes. The Hox code is therefore interpreted to mean that a given regional identity is determined by the diverse combinations of these unique anterior domains of gene expression. At present there is no clear indica- tion whether the pattern is set because all cells need to express the full combination of genes, or whether spe- cific subsets of cells are marked by the separate genes and they cooperate to form the final structure. The data marginally favour the latter hypothesis, but more data is required to distinguish between the alternatives or their intermediates.

Complications in the HoxlHOM functional model

While the experimental mutant phenotypes appear to support the general model, it was surprising that pheno- types in the Hex-c8 mutant mice did not correspond to the anterior limit of expression for the gene, which is nor- mally T5 [35-l. Recent ectopic expression experiments have also challenged the simple interpretation of loss and gain of function phenotypes for the Hoxgenes. Over-ex- pression of Hex-cc8 [31-l results in similar transfomra- tions of the thoracic and lumbar vertebrae as observed in loss of function alleles [35-l. This is clearly inconsistent with previous predictions of the Hox model and d@ers from the Drosophila model. To account for these results it is postulated that Hoxgenes may function in an antipo- dal manner where over- or under-expression results in similar phenotypes. However, this is also perplexing as it predicts that levels of expression are critical and all of the null alleles only display recessive phenotypes. Perhaps in the heterozygous state the wild-type allele can compen- sate or modulate its level of expression. This problem is further complicated by the finding that over-expression of the Hex-&gene also causes the same additional-rib phenotype [30*-l. This could theorectically have been explained by assuming that Hox-c6 achieves the same phenotype as Hex-c8 because it alters endogenous ex- pression of HOX-c8, however, this has been excluded by monitoring the boundaries of Hex-c8 expression in the ectopic expression experiments. Part of the confu- sion in attempting to understand these conflicting results is that the appearance of lumbar ribs is a frequently ob- served phenotype in normal mice [38,39] and in those treated with retinoic acid (RA) [7] or other epigenetic factors [40]. One explanation could be that a general process or tissue involved in intluencing the patterning of vertebral identities in this region is particularly sensitive

Mouse Hex genetic functions Krumlauf 623

to multiple changes in Hex expression or other environ- mental stress factors. As a consequence, multiple events that perturb this common process might display the same phenotypic output. It may be that there exist underlying changes in vertebral identity that are distinctly different in these experimentally induced Hox phenotypes and that they are being confused by the appearance of ex- tra lumbar ribs. However, caution must be exercised in basing a Hox model for genetic function centred on such thoracic/lumbar transformations.

In this regard, recent results on the analysis of null or DNA-binding mutations in the HOX-64 gene indepen- dently provide direct support for the similarity in modes of function between the Hox and HOM-C genes in an- other region of the axial skeleton [36]. Two mutant al- leles of the Hex-64 gene display recessive anterior trans- formations of the second cervical (C) vertebrae, C2. The transformation is not complete because a dens charac- teristic of C2 is still present on the transformed vertebra, suggesting that the another Hox gene may be involved in regulating this aspect of the vertebral identity. The C2 to Cl transformation in Hex-&4 mutants is interesting in that it also suggests a further link between Hox genes and RA. Recent experiments on the analysis of mutant phenotypes in the retinoic acid receptor (RAR)-y gene indicate that a similar partial transformation of C2 to Cl with variable expressivity is observed in recessive RARy mutants [41**]. RARE may therefore mediate its effects on C2 by regulating Hex-64.

Another interesting aspect of the Hex-64 mutant study is that it demonstrates that Hox phenotypes have vari- able expressivity or penetrance that is dependent on the genetic background of the mutation [36**]. This argues that changes in timing of expression or modi- fier loci, which could include the Hoxgenes themselves, can influence the ability/requirement for a particular Hox gene in pattering regional identity. Redundancy or func- tional compensation within the Hox network has always been a concern in light of the evolutionaty similarity be- tween each of the four complexes. However, there may also be alternative processes which are able to ensure that the appropriate regional identity is established in cooperation with, or independent of, the Hox system. An important area of future research will involve using molecular genetic approaches to map and identify these modifier loci, which should enhance our understanding of components in the Hox regulatory cascade.

The hindbrain and branchial arches

It has also been suggested that a Hex code has a role in the specification of regional identity in the hindbrain and branchial arches [ 18,201. Mutational analysis of the Hex-al [33,34] and Hex-a.? [32] genes in mice demon- strates the genes have a role in the patterning of many branchial arch components, and in particular the neural crest derivatives. Despite the argument that rhombomeric

segmentation in the hindbrain is important, as it could serve to provide positional information to the develop- ing head through the migratory population of neural cells, no phenotypes in the rhombomeres themselves have been previously identitied. However, recent eval- uation of Hex-al mutants has revealed that there are changes in rhombomeres (t-4 and t-5) in hindbrain which precede neural crest migration [42**]. These changes correlate well with the later defects in neural crest and provide support for the importance of rhombomeres in neural-crest/branchial-arch patterning. However, no homeotic transformations were observed in the hind- brain or branchiaf arches. A possible explanation for this is that in the branchial region components from ectoderm, mesoderm and endoderm need to cooper- ate and interact to produce the final structures, and altering expression of a single component in multiple tis- sues would be more likely to result in a dysmorphology rather than a transformation [6]. Recently, it has been shown that it is possible to induce regional transforma- tions in the hindbrain that are associated with Hox ex- pression. Treatment of mouse embryos with RA induces shifts in Hoxexpression [43,44**,45**] that can ultimately result in transformation of rhombomere r2/3 into an r4/5 identity [44*-l as evidenced by changes in the fa- cial motor nerve [43,&i**]. This phenotype is dependent on the concentration and time of exposure to RA, which achieves this transformation by altering the expression of many genes, and it is presumably the induction of multiple changes in Hex expression that is required to generate the transformation, rather than loss of a single gene.

Conclusions

During the past two years understanding of the normal roles of Hox genes in vertebrate embryogenesis has been significantly advanced by genetic analysis in the mouse. There is little doubt that the HodHOM complexes play a vital role in the axial patterning of multiple tissues. The combinatorial Hex-code models provide a useful working hypothesis for evaluating how regional iden- tity is regulated. However, we have only a rudimentary understanding of how such a model actually functions at the molecular level.

Future research goals are now focus& on how the Hex family fits into a larger regulatory cascade. Thus far, little is known about factors that act upstream in the cascade to establish Hox expression or of the downstream HOX targets. RA seems to be one signalling molecule and the recent finding that Krox-20 directly regulates Hod2 is an important first step in the upstream pathway [46**]. However, further work will need to concentrate on target genes if we are to ultimately understand the true genetic functions of Hex genes. The future certainly promises to be a exciting time for those interested in the molecular mechanisms that establish the basic vertebrate body plan.

624 Pattern formation and develoDmental mechanisms

Acknowledgements 20.

I wish to thank David Wilkinson, Peter Rigby, h;ichael Kessel, Tom Lufkin, Pierre Chambon and Andrew Lumsden for valuable discus sions and Leanne Wiedemann for reading of the manuscript. 21.

References and recommended reading

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