William McGinnis

Homeobox genes and axial patterning

William McGinnis* and Robb Krumiauft *Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut 06511 tNationai institute for Medical Research Mill Hill London NW7 1AA England Animal embryos need to specify different cell types, and the genetic functions that provide such controls are easy to comprehend since they result in the formation of overtly differentiated tissues such as muscle, bone, and nerves. Embryos (and other fields of developing ceils) also need a more abstract system of genetic controls to respond to signals that establish asymmetry during early embryogen- esis (reviewed by St. Johnston and Niissiein-Volhard, 1992 [this issue of Ce//)), and transform those signals into different positional fates. On the anterior-posterior (A-P) axis of most embryos, such a system provides ceils with specific positional identities that eventually result in the development of structures appropriate to the position within the field, i.e., head structures from the anterior part of the field and so on. A fascinating surprise of recent years is that a Drosophila genetic system that specifies A-P positional identities is conserved in many other animals, where it apparently serves a similar function. The core of this system consists of a set of structurally similar genes that were orginaiiy discovered in Drosophila through the homeotic transformations (duplicated A-P positional vai- ues) that resulted from their mutation (reviewed by Lewis, 1976; Sanchez-Herreroet al., 1965; Kaufman et al., 1990). The proteins encoded by these homeotic selector genes are related at the structural level by their conservation of similar versions of the homeodomain motif. Over the past seven years, the term “homeodomain” has evolved to define a class of protein domains that have recognizable similarity to a 60 amino acid motif (encoded by 160 bp homeobox sequences) originally recognized in three Drosophila homeotic and segmentation proteins (reviewed by Scott et al., 1969). Those homeodomains that have been tested contain sequence-specific DNA binding activities and are part of larger proteins that function as transcriptional regulators (reviewed by Levine and Hoey, 1968). Recent nuclear magnetic resonance and crystaiio- graphic studies on two homeodomains have shown their structures to be related to the helix-turn-helix motif of pro- karyotic DNA-binding proteins (Otting et al., 1999; Kis- singeret al., 1990). Using amino acid sequence similarities within the homeodomain and in flanking protein sequence, it is possible to divide homeodomain proteins and the genes that encode them arbitrarily into various classes. One such is the Antp class, which includes homeodomains that share 69% or more identity with the Antennapedia

Human Hox-4.2 and Drosophila Deformed encode similar regulatory specificities in Drosophila embryos and larvae

Within the serial array of vertebrate homeobox genes in the Hox complexes, it is possible to define a subgroup that is structurally homologous to the Drosophila homeotic gene Deformed (Dfd). We wished to test whether a vertebrate Dfd-like protein could substitute for any of the regulatory functions of the Dfd protein in Drosophila embryos, including its ability to transcriptionally activate the Dfd transcription unit. A fusion gene consisting of a heat shock promoter attached to the human Hox-4.2 gene was introduced into the Drosophila genome, and its regulatory and developmental effects were assayed after heat shock. In developing embryonic and larval cells, we find that human Hox-4.2 specifically activates ectopic expression of the endogeneous Dfd transcription unit and phenocopies a dominant mutant allele of Dfd. Thus, human Hox-4.2 can specifically substitute for a normal regulatory function of its Drosophila homolog, Dfd.

Autoregulation of a Drosophila homeotic selector gene.

The Deformed (Dfd) gene is a homeotic selector that functions in specifying the identity of the mandibular and maxillary segments. We have constructed transformed fly strains carrying a Dfd cDNA under the heat-inducible control of the hsp70 promoter. With these strains we can induce the ectopic expression of Dfd protein in other segments at various stages of embryonic development. We find that both early and persistent synthesis of the protein is required for the transformation of other body segments toward head segmental identity. The persistent expression of the Dfd protein requires an endogenous copy of the Dfd gene, and we show that the expression of the endogenous copy can be induced by hsDfd expression. This implies that the Dfd protein autoactivates expression from the Dfd locus during normal development. The autoactivation circuit supplies a simple mechanism that can account, in part, for the stability of the determined state controlled by Dfd.

Homeo box genes of the Antennapedia and Bithorax Complexes of Drosophila

The Antennapedia, Ultrabithorax, and fushi tarazu genes of Drosophila melanogaster each contain a very similar protein coding sequence, the homeo box. Previously cloned homeo box sequences were used to isolate additional well conserved members of the homeo box gene family. The most strongly conserved members of the homeo box gene family map within either the Antennapedia or Bithorax gene complexes. The tissue distribution of transcripts encoded by the two rightmost homeo box genes of the Bithorax complex are compared with the iab-2 and iab-7 phenotypes.

A homeodomain substitution changes the regulatory specificity of the Deformed protein in drosophila embryos

Homeodomain proteins are believed to direct developmental pathways during Drosophila embryogenesis by the specific regulation of other genes. An unresolved issue is whether it is the homeodomain or the other regions of such proteins that confer target specificity. To test the role of the homeodomain in determining target specificity, we replaced the homeobox of Deformed with the homeobox of Ultrabithorax. The resulting chimeric protein cannot activate transcription from the Deformed gene, as does the normal Deformed protein. Instead, the chimeric protein activates ectopic transcription of Antennapedia, a gene normally regulated by Ultrabithorax. Our results indicate that in the context of the developing embryo, even closely related homeodomain sequences have different target specificities.

Homeo box gene complex on mouse chromosome 11: Molecular cloning, expression in embryogenesis, and homology to a human homeo box locus

The homeo box is a 180 bp protein-coding domain found within homeotic genes of Drosophila and conserved in a variety of invertebrate and vertebrate species. It has been suggested that the mammalian homeo box sequences may play a role in controlling pattern formation during embryogenesis. We report findings that support this hypothesis. We have cloned three overlapping recombinant phage clones that cover a region of mouse chromosome 11 that contains a cluster of four homeo boxes (the Hox-2 locus). This locus encodes multiple transcripts that are expressed during embryogenesis. Forty kilobases of the Hox-2 region is devoid of repetitive elements and shows extensive homology with the human Hox-2 locus. These results provide direct evidence for genetic expression during embryonic development, a conserved organization in comparison to the cognate human locus, and a complexity of organization and transcript expression similar to that found in Drosophila.

Region-specific expression of two mouse homeo box genes.

Mammalian homeo box genes have been identified on the basis of sequence homology to Drosophila homeotic and segmentation genes. These studies examine the distribution of transcripts from two mouse homeo box genes, Hox-2.1 and Hox-3.1, throughout the latter third of prenatal development. Transcripts from these genes are regionally localized along the rostro-caudal axis of the developing central nervous system, yielding expression patterns very similar to patterns of Drosophila homeotic gene expression.

Altering the regulatory targets of the Deformed protein in Drosophila embryos by substituting the Abdominal-B homeodomain

The homeotic selector genes of Drosophila melanogaster encode transcriptional regulatory proteins that control the determination of different segmental fates. Binding of selector proteins to regulatory DNA sequences is mediated by an evolutionary conserved protein domain, the homeodomain. Although homeodomains encoded by the selector genes are very similar in their amino acid sequence and in vitro DNA-binding properties, here we provide additional evidence that the homeodomain is responsible for most of the regulatory specificity of the entire protein. A heat-shock promoter/selector gene was constructed that encodes a Deformed/Abdominal-B chimera in which the Abdominal-B homeodomain is substituted for that of Deformed. Expression of this chimeric protein throughout the embryo causes morphological transformation of anterior segments toward more posterior identities. A number of other homeotic selector genes, all normally repressed by Abdominal-B, are ectopically activated by the chimeric protein. These results support the hypothesis that the target specificity of similar homeodomain proteins is largely determined by the amino acid sequence of the homeodomain.

Functional analysis of the mouse homeobox gene HoxB9 in Drosophila development

Mammalian genomes contain clusters of homeobox genes (Hox-C, HOX-C) which are structurally similar to the homeotic genes of the Drosophila HOM complex. One method for assessing the functional similarity of particular Drosophila HOM and mammalian Hox genes is to test the ability of Hox genes to induce homeotic phenotypes when expressed in developing Drosophila. Here we describe such functional tests using mouse HoxB9 (formerly Hox-2.5), whose closest structural relative in Drosophila is Abdominal-B. When expressed from a heat shock promoter, HoxB9 induces transformations of head towards more posterior identities in Drosophila larvae and adults. These transformations share some similarities with the phenotypic effects produced by ectopically expressed Abdominal-B, but are also similar to the transformations induced by Antennapedia and mouse HoxB6 (Hox-2.2), suggesting that HoxB9 specifies a positional identity that is intermediate between Antennapedia and Abdominal-B.

Ectopic expression from the Deformed gene triggers a dominant defect in Drosophila adult head development

Drosophila adults carrying a dominant allele of the Deformed locus (DfdD) have a mutant phenotype in which lower eye and orbital structures of the adult head are eliminated. The molecular defect responsible for this dominant mutation is a large, transposon-flanked, fragment of DNA inserted downstream of the Deformed (Dfd) transcription unit. The downstream transposon-flanked insert causes the inappropriate activation of Dfd gene expression in a subset of the cells of the eye imaginal disc, resulting in their abnormal development, and in the loss of lower eye structures from the adult head. A chromosome selected for its reversion of the dominant phenotype is missing a portion of the transposon-flanked insert and reverts to a normal expression pattern of Dfd in the eye-antennal disc. Additional evidence that the dominant head phenotype is caused by ectopic expression of Dfd in the eye disc derives from experiments showing that a DfdD-like phenotype can be induced by ectopic expression of a Dfd gene under heat shock promoter control.