The Expression of Signaling Molecules Lefty-1, Lefty-2, and Pitx-2 in HFH-4 Knockout Mouse Embryos.

Vertebrate body structure is outwardly symmetric about the midline. Internally, however, left-right asymmetry of visceral organs is a characteristic of normal vertebrates. For example, the heart normally tilts to the left, the liver is located on the right, and the stomach and spleen are on the left. In humans, as in all vertebrates, internal left-right axis formation is determined during embryogenesis and has been found to occur in a conserved, nonrandom fashion. Defects in normal left-right organ positioning, termed heterotaxy, occur in approximately 1:10,000 live births. As a result of observations made by Afzelius, it was found that mirror-image reversal of visceral organs, or situs inversus, was often characteristic of individuals with Immotile Cilia Syndrome (ICS). In this syndrome, it appears that the formation of the left-right axis occurs randomly, producing individuals with either situs inversus or situs solitus (normal organ positioning). The relationship between left-right axis malformations and absent or dysfunctional cilia has been observed in animal models as well, but the specific genes responsible for regulating the phenomenon have yet to be identified.

Winged helix transcription factors play important roles in cellular differentiation and cell-specific gene expression. One member of this family, hepatocyte nuclear factor/forkhead homologue 4 (HFH-4), is expressed in ciliated cells in the respiratory, reproductive, and nervous systems. Targeted mutation of the mouse hfh-4 gene results in a complete lack of cilia, as well as a random determination of left-right asymmetry.

Other investigators have identified a number of genes that are expressed asymmetrically in the developing embryo, which are thought to be crucial to normal left-right axis determination. Lefty-1 and Lefty-2 are members of the TGF-ß family and are expressed in the left midline and left lateral plate mesoderm respectively. Pitx-2 is a bicoid homeobox gene, which is expressed in the left lateral plate mesoderm and later, in the left side of the developing heart and gut. My research focused on the expression of these genes in HFH-4 knockout (hfh-4 -/-) mice.

Whole mount in situ hybridization was used to examine the patterns of expression of Lefty-1, Lefty-2, and Pitx-2 in wild type (hfh-4 +/+), heterozygous (hfh-4 +/-), and knockout (hfh-4 -/-) embryos (e7.5 to e8.5). Embryos were obtained by mating hfh-4 heterozygous male mice with heterozygous female mice. The genotype of each embryo was determined by PCR analysis of DNA, which had been isolated from embryonic membranes. Whole mount in situ hybridization was performed with anti-sense digoxigenin labeled RNA probes.

The results of whole mount in situ hybridization revealed an absence of Lefty-2 expression in hfh-4 -/- embryos. In wild type and heterozygous embryos, expression of Lefty-2 was detected in the left lateral plate mesoderm during early somite stages. Preliminary whole mount in situ hybridization also suggested an absence of Lefty-1 expression in hfh-4 -/- embryos. The number of embryos examined thus far, however, is limited.

The results suggest that HFH-4 plays a critical role in the determination of left-right asymmetry during the early stages of embryonic development. Future studies will examine additional embryos for Lefty-1 expression, as well as for expression of homeobox gene pitx-2. Eventually, scientists hope to determine the function of each gene in left-right axis development so as to prevent or greatly decrease the incidence of left-right axis malformations.