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. 2010 Sep-Oct;3(9-10):628-38.
doi: 10.1242/dmm.005058. Epub 2010 Jun 2.

UDP-galactose 4' epimerase (GALE) is essential for development of Drosophila melanogaster

Rebecca D Sanders  1 , Jennifer M I Sefton, Kenneth H Moberg, Judith L Fridovich-Keil
Affiliations

UDP-galactose 4' epimerase (GALE) is essential for development of Drosophila melanogaster

Rebecca D Sanders et al. Dis Model Mech. 2010 Sep-Oct.

Abstract

UDP-galactose 4' epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose in the final step of the Leloir pathway; human GALE (hGALE) also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. GALE therefore plays key roles in the metabolism of dietary galactose, in the production of endogenous galactose, and in maintaining the ratios of key substrates for glycoprotein and glycolipid biosynthesis. Partial impairment of hGALE results in the potentially lethal disorder epimerase-deficiency galactosemia. We report here the generation and initial characterization of a first whole-animal model of GALE deficiency using the fruit fly Drosophila melanogaster. Our results confirm that GALE function is essential in developing animals; Drosophila lacking GALE die as embryos but are rescued by the expression of a human GALE transgene. Larvae in which GALE has been conditionally knocked down die within days of GALE loss. Conditional knockdown and transgene expression studies further demonstrate that GALE expression in the gut primordium and Malpighian tubules is both necessary and sufficient for survival. Finally, like patients with generalized epimerase deficiency galactosemia, Drosophila with partial GALE loss survive in the absence of galactose but succumb in development if exposed to dietary galactose. These data establish the utility of the fly model of GALE deficiency and set the stage for future studies to define the mechanism(s) and modifiers of outcome in epimerase deficiency galactosemia.

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Figures

Fig. 1.
Fig. 1.
The Leloir pathway of galactose metabolism. In both humans and D. melanogaster, GALE catalyzes the interconversion of UDP-gal and UDP-glc, as well as UDP-galNAc and UDP-glcNAc.
Fig. 2.
Fig. 2.
dGALE alleles used in this study. Introns are shown as thin lines and exons as thick bars, with coding regions in filled black and non-coding regions cross-hatched. Transposon insertion sites are denoted by triangles, and deleted regions are indicated.
Fig. 3.
Fig. 3.
Compromised viability of dGALE mutants. (A) Some zygotic dGALEf00624/dGALEΔy mutants (open circles) died as embryos, whereas others survived to mid-larval stages; however, all dGALEf00624/dGALEΔy germline clone mutants (filled squares) died as embryos. By contrast, the survival rates of germline clone dGALEf00624/+ heterozygotes (filled triangles) and their germline clone homozygous wild-type counterparts (filled circles) were indistinguishable. (B) GALE-deficient zygotic larvae (top three animals), which were hatched and maintained on grape juice agar plates with yeast paste for 48 hours, were smaller than their wild-type counterparts (bottom three animals).
Fig. 4.
Fig. 4.
dGALE is required throughout development. Each row represents a cohort of animals in which dGALE knockdown was initiated on a particular day in development by shifting the animals from the GAL80ts permissive temperature of 18°C, to the restrictive temperature of 28–29°C. Shaded boxes represent days at the permissive temperature (dGALE expressed) and open boxes represent days at the restrictive temperature (dGALE knockdown). Of note, knockdown was not complete and varied by age, as presented in Table 1 and described in the Results. The arrows at the left of the figure indicate cohorts of larvae and pupae that were harvested for enzymatic assays (Table 1). Slashed and crossed boxes denote the outcomes observed for a given cohort on a given day, as indicated in the key. Final outcomes are summarized to the right of each row.
Fig. 5.
Fig. 5.
Hypomorphic dGALE mutants are sensitive to dietary galactose. Crosses between homozygotes and heterozygotes for both the precise excision dGALEd allele (dark columns) and the hypomorphic dGALEh allele (light columns) yielded the indicated percentages of homozygous offspring on food containing glucose, glucose plus galactose, and glucose plus mannose. The number above each column represents the total number of progeny scored for that condition. The expected proportion of 50% homozygotes is represented by a dashed line. Crosses involving the dGALEh hypomorphic allele produced significantly fewer homozygotes on food containing galactose than crosses involving the control dGALEd allele.
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