Molecular investigations into complex behavior: lessons from sexual orientation studies.

A review of recent molecular genetic investigations into sexual orientation is presented and used as a model to identify unique challenges encountered in research on complex behavior. Five main themes are presented that are generally applicable to most behavioral genetics investigations: (1) A precise definition of the phenotype that, as much as possible, unambiguously differentiates the study group from the control group must be achieved; (2) environmental and social contributions to a trait may exhibit significant sex differences; (3) subsets within the study sample may reveal important information about genetic and environmental contributions to the trait that otherwise would be missed; (4) the high degree of specificity required to define a study sample severely restricts the application of results to general populations; and (5) studies attempting to replicate data must strictly parallel the original methodology if valid comparisons are to be made.

A crossover interaction between sex, sexual orientation, and handedness.

The distribution of handedness scores was analysed in 1414 heterosexual, bisexual, and homosexual men and women. Hand preference was assessed by a questionnaire that distinguishes consistently right-handed from partially or completely left-handed individuals, and sexual orientation was measured by the Kinsey scales. As expected from population-based studies, heterosexual men were, on average, more left-handed than heterosexual women. By contrast, gay men were more right-handed than lesbians or heterosexual men, and lesbians were more left-handed than gay men or heterosexual women. This crossover interaction suggests that a common variable influences sex, sexual orientation, and hand preference.

Linkage between sexual orientation and chromosome Xq28 in males but not in females.

We have extended our analysis of the role of the long arm of the X chromosome (Xq28) in sexual orientation by DNA linkage analyses of two newly ascertained series of families that contained either two gay brothers or two lesbian sisters as well as heterosexual siblings. Linkage between the Xq28 markers and sexual orientation was detected for the gay male families but not for the lesbian families or for families that failed to meet defined inclusion criteria for the study of sex-linked sexual orientation. Our results corroborate the previously reported linkage between Xq28 and male homosexuality in selected kinships and suggest that this region contains a locus that influences individual variations in sexual orientation in men but not in women.

Development and familiality of sexual orientation in females.

The development and familial clustering of sexual orientation were studied in 358 heterosexual, bisexual, and homosexual women. Sexual orientation, as measured by the Kinsey scales, was diverse yet showed statistical congruity and stability over a 1- to 1.5-year time span. Developmental patterns, as measured by retrospective reports on the ages of first sexual or romantic attraction and of self-acknowledgement of sexual orientation were very similar in the heterosexual and lesbian subjects except for the difference in object choice. The bisexual subjects displayed intermediate patterns that were more similar to the heterosexuals' on most facets yet closer to the lesbian subjects' on other dimensions. Familial clustering of nonheterosexual orientation was significant. Using two criteria, elevated rates of nonheterosexuality were found in four classes of relatives: sisters, daughters, nieces, and female cousins through a paternal uncle. The current data are not sufficient to distinguish between genetic and shared environmental sources of this familial aggregation. We discuss the possibility of using developmental criteria to differentiate between inherited and cultural sources of variation in female sexual orientation.

A linkage between DNA markers on the X chromosome and male sexual orientation.

The role of genetics in male sexual orientation was investigated by pedigree and linkage analyses on 114 families of homosexual men. Increased rates of same-sex orientation were found in the maternal uncles and male cousins of these subjects, but not in their fathers or paternal relatives, suggesting the possibility of sex-linked transmission in a portion of the population. DNA linkage analysis of a selected group of 40 families in which there were two gay brothers and no indication of nonmaternal transmission revealed a correlation between homosexual orientation and the inheritance of polymorphic markers on the X chromosome in approximately 64 percent of the sib-pairs tested. The linkage to markers on Xq28, the subtelomeric region of the long arm of the sex chromosome, had a multipoint lod score of 4.0 (P = 10(-5), indicating a statistical confidence level of more than 99 percent that at least one subtype of male sexual orientation is genetically influenced.

Ectopic expression of the Drosophila homeotic gene proboscipedia under Antennapedia P1 control causes dominant thoracic defects.

A deletion mutation in the Antennapedia Complex of Drosophila melanogaster, Df(3R)SCBXL2, induces both dominant and recessive loss-of-function phenotypes. The deletion is associated with diminished function of proboscipedia (pb), a homeotic gene required for mouthparts formation. Df(3R)SCBXL2 also has associated dominant thoracic defects related to diminished expression of the homeotic Antennapedia (Antp) gene copy on the homologous chromosome. This is shown to be a consequence of ectopic pb expression in the thorax. Newly juxtaposed Antp sequences provide the pb gene on the deletion bearing chromosome with a second promoter, Antp P1, in addition to its own. Ectopic pb protein expression occurs under Antp P1 control, by alternate splicing, and results in diminished accumulation of Antp protein in the imaginal disc cells where Antp P1 is normally expressed. The analysis of this mutant chromosome thus demonstrates that pb protein is capable of participating in the negative regulation of a more posteriorly expressed homeotic gene, as well as serving a homeotic "selector" function in the head.

brahma: a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SWI2.

The brahma (brm) gene is required for the activation of multiple homeotic genes in Drosophila. Loss-of-function brm mutations suppress mutations in Polycomb, a repressor of homeotic genes, and cause developmental defects similar to those arising from insufficient expression of the homeotic genes of the Antennapedia and Bithorax complexes. The brm gene encodes a 1638 residue protein that is similar to SNF2/SWI2, a protein involved in transcriptional activation in yeast, suggesting possible models for the role of brm in the transcriptional activation of homeotic genes. In addition, both brm and SNF2 contain a 77 amino acid motif that is found in other Drosophila, yeast, and human regulatory proteins and may be characteristic of a new family of regulatory proteins.

A functional and structural analysis of the Sex combs reduced locus of Drosophila melanogaster.

We have undertaken a developmental genetic analysis of the homeotic gene Sex combs reduced (Scr) of Drosophila melanogaster by examining embryonic and adult phenotypes of mutations affecting Scr gene function. Molecular mapping of Scr breakpoint lesions has defined a segment of greater than 70 kb of DNA necessary for proper Scr gene function. This region is split by the fushi tarazu (ftz) gene, with lesions affecting embryonic Scr function molecularly mapping to the region proximal (5') to ftz and those exhibiting polyphasic semilethality predominantly mapping distal (3') to ftz. Gain-of-function mutations are associated with genomic rearrangements and map throughout the Scr locus. Our analysis has revealed that the Scr locus encompasses genetic elements that are responsible for functions in both the embryonic and larval to adult periods of development. From these studies, we conclude that Scr is a complex genetic locus with an extensive regulatory region that directs functions required for normal head and thoracic development in both the embryo and the adult and that the regulation of Scr during these two periods is distinct.

The homeotic gene Sex combs reduced of Drosophila melanogaster is differentially regulated in the embryonic and imaginal stages of development.

The Sex combs reduced (Scr) locus is unique among the genes contained within the Antennapedia complex (ANT-C) of Drosophila melanogaster in that it directs functions that are required for both cephalic and thoracic development in the embryo and the adult. Antibodies raised against protein encoded by Scr were used to follow the distribution of this gene product in embryos and imaginal discs of third instar larvae. Analysis of Scr protein accumulation in embryos hemizygous for breakpoint lesions mapping throughout the locus has allowed us to determine that sequences required for establishment of the Scr embryonic pattern are contained within a region of DNA that overlaps with the identified upstream regulatory region of the segmentation gene fushi tarazu (ftz). Gain-of-function mutations in Scr result in the presence of ectopic sex comb teeth on the first tarsal segment of mesothoracic and metathoracic legs of adult males. Heterozygous combinations of gain-of-function alleles with a wild-type Scr gene exhibit no evidence of ectopic protein localization in the second and third thoracic segments of embryos. However, mesothoracic and metathoracic leg imaginal discs can be shown to accumulate ectopically expressed Scr protein, implying a differential regulation of the Scr gene during these two periods of development. Additionally, we have found that the spatial pattern of Scr gene expression in imaginal tissues involved in the development of the adult thorax is governed in part by synapsis of homologous chromosomes in this region of the ANT-C. However, those imaginal discs that arise anteriorly to the prothorax do not appear to be sensitive to this form of gene regulation. Finally, we have demonstrated that the extent of Scr expression is influenced by mutations at the Polycomb (Pc) locus but not by mutant alleles of the zeste (z) gene. Taken together, our data suggests that Scr gene expression is differentially regulated both temporally and spatially in a manner that is sensitive to the structure of the locus.

Developmental and evolutionary implications of labial, Deformed and engrailed expression in the Drosophila head.

Prior developmental genetic analyses have shown that labial (lab) and Deformed (Dfd) are homeotic genes that function in the development of the embryonic (larval) and adult head. Using antibody probes to reveal the spatial distribution of the lab and Dfd proteins in embryonic and imaginal tissues, we have assessed the respective roles of these genes through an analysis of the correspondence of their expression patterns with their mutant phenotypes. With regard to imaginal development, lab and Dfd occupy adjacent non-overlapping expression domains in the peripodial cell layer of the eye-antennal disc, in patterns that are consistent with their adult mutant phenotypes and published fate maps. During embryogenesis, lab and Dfd exhibit limited overlapping expression in areas that are of no obvious significance to the development of larval head structures, but also in areas that may have consequences for imaginal development. The head of Drosophila and other cyclorrhaphous Dipterans is characterized by an extreme morphological difference between the larval and adult stages. Given this unique ontogenetic and phylogenetic history and the observation that homeotic transformations produced by the lab, Dfd, and proboscipedia (pb) loci are manifested only in the adult, we suggest that distinct regulatory paradigms evolved for homeotic gene function in the development of the larval versus adult head. Finally, a detailed examination of the engrailed (en) expression pattern in the embryonic head strengthens the view of insect morphologists that the clypeolabrum evolved from the fusion of paired labral appendages.