PAUL HUNT AND ROBB KRUMLAUF PATTERN FORMATION

Hex genes coming to a head Mice with a mutated Hox gene provide the first direct

evidence that Hox proteins are involved in the molecular mechanisms that generate pattern in the vertebrate head and thorax.

In vertebrate developmental biology there has been a renaissance in the field of pattern formation, exempli- fied by studies on the development of the head. Over 160 years ago it was first suggested that the hindbrain and other parts of the central newous system are orga- nized into segmental units, but the significance and even the existence of these metameric units had remained an open question. Recently, reevaluation of the peri- odic swellings observed in the hindbrain, termed rhom- bomeres, has shown that they are segmental units of or- ganization, which define lineage-restricted cellular com- partments (reviewed in [ 1,2] >. Molecular studies have also provided support for segmental organization of the diencephalon in the forebrain [3]. But the entire central nervous system is not structured in the same manner, as there is no evidence for segmentation of the spinal cord. Rather, in the trunk the somites seem to be the critical unit of segmentation as they regulate many aspects of pat- tern formation in this region [ 41. Therefore the head and the trunk employ different developmental strategies for generating regional diversity. Classic embryology experiments based on grafting re- gions of the chick hindbrain and somites to ectopic sites have indicated another important difference between the head and the trunk. In the hindbrain region most of the mesenchyrne of the facial structures and connective tis- sue is of neural crest origin, derived from cells that mi- grate from the dorsal edge of the neural epithelium into the bran&al arches 151. Figure 1 shows the location and relationship between rhombomeres and bran&al arches in an early mouse embryo. The cranial neural crest has an. intriguing property in that it carries a form of pre- patterning or molecular imprinting which enables it not only to specify a particular structural pattern, but also to instruct surrounding mesodermal and ectodermal tissue [6]. In ectopic locations, the crest and its new surround- ing tissue will form identical structures characteristic of the crest’s position of origin on the antero-posterior axis. Neural crest from the trunk does not display this prop- erty, but trunk somites are pre-patterned [7]. As sub-pop- ulations of cranial neural crest are derived from specific rhombomeres, it has been suggested that an important function of rhombomeric segmentation in the hindbrain

. . is to establish the pre-patterning cues of neural crest. In this way rhombomeres could have a fundamental role not just in organization of the central and peripheral nervous system, but in the specification of craniofacial structures in general. Recent molecular approaches have begun to identify genes that could be involved in vertebrate pattern for- mation. One group of such genes encodes a family of

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transcription factors, the Hox homeobox proteins, which may be important in head development. In vertebrates there are four different Hox gene clusters on separate chromosomes which are related to each other by dupli- cation and divergence from a common ancestral cluster (reviewed in [ 81). A great deal of speculation has focused attention on the Hox proteins as candidate molecules for the specification and interpretation of regional. dif- ferences. These arguments are based on the striking similarity in structure, chromosomal organization, and ordered domains of expression along the embryonic axis shared between the vertebrate Hox and Dmc@ih HOM-C homeotic clusters [9,10]. By analogy with their Drasopbika homologues, the Hox genes may play a part in the regulation of segment identity, and extensive stud- ies on patterns of gene expression during mouse devel- opment strongly support this idea [8]. The Hox-2 genes have patterns of expression in the hindbrain that map to distinct rhombomere boundaries [ll] and are also restricted to specific populations of cranial neural crest cells [12]. Similar ordered domains of Hax expression are observed in the somites, limbs, mesxlermal organs and peripheral nervous system. The current data suggest that in many embryonic contexts the Hox network is part of an evolutionarily conserved mechanism for specifying regional differences along the embryonic axis 111-141. This is achieved by overlapping domains of Hex expres- sion, representing a molecular combinatorial code that generates positional values and regional diversity.

Although ectopic expression of Hox genes in transgenic mice results in transformations of vertebral phenotype 1141, presumably because of changes to the Hox code, until now there has been no direct evidence that the Hox proteins actually have a normal function in positional specification. This link has finally been made by Chisaka and Capecchi [ 151. They have generated a germline mu- tation in the mouse Hex-2.5 gene using homologous recombination to disrupt the endogenous gene in em- bryonic stem cells. The mutation was made by inserting a dominant selectable marker (neol) and its associated regulatory elements into the homeodomain of the Hex- 1.5 gene. This results in a truncated form of the pro- tein, missing critical DNA-binding elements of the home- odomain. Although this altered Hox-1.5 protein could it- self have dominant effects, no phenotypic abnormalities were observed in mice heterozygous for the mutation, suggesting that it was a non-functional or null allele. Anal- ysis of the mutant phenotypes of mice homozygous for this disrupted allele, however, provides clear evidence that Hox genes have an important developmental role in the organization of the head structures.

@ 1991 Current Biology

Fig. 1. Scanning electron micrographs of a 5-somite mouse embryo. (a) A dorsal view into the hindbrain and midbrain before closure of the neural tube, illustrating the periodic undulations that correspond to hindbrain rhombomeres. fb) A lateral view of the same embryo showing a developing first branchial arch. The arrows in both panels indicate the same axial level and correspond to the junction in the neural plate and surface ectoderm that delineates the first and second branchial arches. Anterior is to the left and posterior to the right. Magnification X 93 and x 58 (a) and fb), respectively. (Photographs courtesy of Liz Hirst.)

Mice homozygous for this mutant Hex-1.5 gene are not viable and die shortly after birth [ 151. Severe deformities in the head and throat of such new born mice result in both respiratory and circulatory problems, which are the most likely cause of death. Among the defects observed are: loss of the thymus and parathyroid glands, reduced thyroid gland, size reduction in the mandible and laryr- geal cartilages, loss of the lesser horn of the hyoid bone, compression of upper cervical vertebrae, poor muscular organization of the face, and the loss or deformation of veins, arteries, chambers and valves associated with the heart. The abnormalities are clearly not conIined to a sin- gle tissue, cell type or structure, but they are concentrated in lower regions of the head, face and upper trunk. Such a complex array of phenotypes arises from a progres- sion of abnormal patterning events and makes it very dif- ficult to identify and separate primary from secondary de- fects. It is therefore important to examine early stages of development to find the first appearance of phenotypic differences between the mutant and wild type embryos. The earliest reported stage at which such Hox- 1.5 homo- zygous mutants display abnormalities is 10.5 days of em- bryogenesis [15]. The third and fourth bran&al arches and the underlying branchial pouches have an abnor- mal morphology and are reduced in size. This observa- tion is intriguing, as the pattern of expression of Hox- 1.5, summarized in Fig. 2, shows that the gene is ex- pressed in surface ectoderm, ganglia, and cranial neural crest derivatives of the third and fourth branchial arches and not in first or second branchial arch structures. The boundary of expression in the hindbrain maps to rhom- bomere 415 and the expression in the branchial arches to B 213 (Fig. 2). The disparity in the anterior boundaries of Hox- 2.5 expression in rhombomeres versus bran&al arches arises as rhombomere 5 does not generate crest that can migrate into the arches. The early phenotype in specific branchial arches combined with the fact that many .of the malformations observed in newborn mice are in tissues derived from neural crest, mesoderm, pha- ryngeal endoderm and ectodenn of the third and fourth

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bran&al arches, argues that Hox-1.5gene plays a critical part in patterning head structures at this axial level. These findings also support the idea that Hex genes could be providing the pre-patterning information for cranial

neural crest.

One puzzle arising from the study by Chisaka and Capec- chi [ 15 1 is that many of the regions that normally express the gene [ 161 have no detectable abnormalities. Posterior regions of the embryos such as the spinal cord, kidney, lung, stomach, spleen and spinal ganglia all seem normal. One explanation for this may be made by analogy with the Drosopbilu homeotic genes (reviewed in [ 171). The Drasopbika Ultrabitborax (Ubx) gene is expressed in a broad domain and is at its highest level at the anterior borders of segments. Mutations in the U&xgene only af- fect structures that are derived from the anterior domains of V&x expression, and ectopic expression of the gene in posterior regions of the embryo has no effect. Other Droscphla homeotic genes therefore seem to dominate in the posterior regions and protect them from the action of genes that would normally dominate in the anterior. In the mouse many other Hox genes are expressed in posterior tissues, which are normal in homozygous Hox- 1.5 mutants, and they may dominate over m&-expressed Hox1.5 or compensate for its loss.

Not all sites of Hm-1.5 expression are affected; it is. in- teresting that rhombomeric segmentation and some cra- nial neural crest derivatives appear normal in the mutant mice. Cranial sensory ganglia appear completely normal. Hex-I.5 is highly related to two other genes, Hex-2.7 and Hex-4. I (Fig. 2). The three genes share several re- gions of homology over their entire protein sequence and have similar spatially restricted domains of expression in the hindbrain and branchial arch neural crest. Therefore, there is the potential for functional redundancy between these genes, which could account for normal pattem- ing of the rhombomeres and some neural crest struc- tures in the mutant mice. However, little is known about

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interactions between these genes or whether they can even be coexpressed in the same cells.

Midbrain Hindbrain Spinal cord

/\ /B3

Bl/ kf “L.

1.6 1.11 1.5 1.4 Hox- 1

2.9 2.7 .-fl--$+&-,,‘;” Hox-2

A Hox-3 4.1 4.2

>--la.- Hex-4

Fig. 2. Region of expression of Hox-1.5 in the hindbrain, spinal cord and branchial arches of a 9.5day mouse embryo (24 somites). The branchial arches fBl-B4) are shown fully formed, ie. at a stage slightly later than that in Fig.1. The structural rela- tionship to genes in other Hox complexes kindicated below, the coloured boxes indicate closely related genes. Rhombomeres are labelled rl-r8.

Perhaps the most surprising findings to come from these studies [ 151 are that abnormal phenotypes are also ob- served in regions that are not thought to correlate with normal domains of expression for this gene. An example of this is the hyoid bone of mutant animals. The greater horn is derived from the third btanchial arch, which does express Hex-1.5 but is normal, whereas the lesser horn derived from the second branchial arch which does not express Hox- 1.5 is missing. A trivial explanation is that previous in situ hybridization studies on Hex- 1.5 expres- sion failed to detect expression because of low levels of sensitivity, or that expression is transient and was sim- ply missed. However, it is also possible that the insertion of the selectable marker and its control regions has al tered the expression of other linked Hex genes, such as Hoxl.12 and Hex-1.6 One clear linding was that there is no simple homeotic transformation in the homozygous mutant mice. In con- trast, n&expression of the Hp-2.1 gene in somites re- sults in an anterior transformation of cervical vertebrae [ 141. This may reflect intrinsic differences in the way that the head and trunk use Hox codes to establish pattern. In the head, the iinal pattern is likely to be a result of interactions between neural plate, neural crest, surface ectoderm, mesoderm and pharyngeal endoderm, which are not derived from a single axial level. It is too much to expect that the alteration of one component of this network would generate a simple transformation. Many of the questions arising from these data can be ad- dressed by a more detailed examination of the mutant

phenotypes from this gene and analysis of the patterns of expression of other members of the Hex network in the mutant background. Studies of mutations ln other Hex genes are underway ln many laboratories and they will help to address the problems of functional overlap and redundancy in the HCAX network. Although gene target- ting experiments firmly link Hax genes with elaboration of pattern in head and ttunk development, they also serve to highlight the complex nature of cell movement and tis- sue interactions involved in specification of cranial struc- tures. It will be both an exciting and daunting task to begln to sort out the primary and secondary pathways uncovered by the Hox mutants.

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Paul Hunt and Robb Krumlauf, Laboratory of Eukaryotic Molecular Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 lAA, UK.

@ 1991 Current Biology