Reduced fertility of Drosophila melanogaster hybrid male rescue (Hmr) mutant females is partially complemented by Hmr orthologs from sibling species

Abstract

The gene Hybrid male rescue (Hmr) causes lethality in interspecific hybrids between Drosophila melanogaster and its sibling species. Hmr has functionally diverged for this interspecific phenotype because lethality is caused specifically by D. melanogaster Hmr but not by D. simulans or D. mauritiana Hmr. Hmr was identified by the D. melanogaster partial loss-of-function allele Hmr1, which suppresses hybrid lethality but has no apparent phenotype within pure-species D. melanogaster. Here we have investigated the possible function of Hmr in D. melanogaster females using stronger mutant alleles. Females homozygous for Hmr mutants have reduced viability posteclosion and significantly reduced fertility. We find that reduced fertility of Hmr mutants is caused by a reduction in the number of eggs laid as well as reduced zygotic viability. Cytological analysis reveals that ovarioles from Hmr mutant females express markers that distinguish various stages of wild-type oogenesis, but that developing egg chambers fail to migrate posteriorly. D. simulans and D. mauritiana Hmr+ partially complement the reduced fertility of a D. melanogaster Hmr mutation. This partial complementation contrasts with the complete functional divergence previously observed for the interspecific hybrid lethality phenotype. We also investigate here the molecular basis of hybrid rescue associated with a second D. melanogaster hybrid rescue allele, In(1)AB. We show that In(1)AB is mutant for Hmr function, likely due to a missense mutation in an evolutionarily conserved amino acid. Two independently discovered hybrid rescue mutations are therefore allelic.

Figure 1.—

Map of Hmr region and transgenic constructs and expression analysis of P{Hmr^2t8.0} transgenes in hybrids. (A) CG2124 and Rab9D are on the opposite strand relative to Hmr. For the five constructs shown the relevant Hmr genotype is written above the name of the transgenic construct. D. melanogaster Hmr contains an AvaI restriction site. The D. melanogaster Hmr² construct contains the two indicated amino acid substitutions; the second substitution mutates the AvaI site. This restriction site is absent in D. simulans and D. mauritiana Hmr. The D. melanogaster Hmr² construct is described in this study. The other D. melanogaster constructs are described in Barbash et al. (2003); the D. simulans and D. mauritiana constructs are described in Barbash et al. (2004a). (B) RT–PCR analysis of hybrids containing P{Hmr^2t8.0} transgenes. M, DNA marker showing bands of indicated sizes; lanes 1–3, AvaI-digested PCR products amplified from genomic DNA; lanes 4–7, AvaI-digested PCR products amplified from cDNA samples. Samples were run on a 2% agarose gel. Template DNAs and cDNAs were: (1) y; cn bw sp D. melanogaster DNA (wild-type control); (2) D. simulans w⁵⁰¹ DNA (wild-type control); (3) D. melanogaster In(1)AB, w DNA; (4) FM6,w/X_sim, w⁵⁰¹; P{Hmr^2t8.0}1-7/+ hybrid female cDNA; (5) FM6,w/X_sim, w⁵⁰¹; +/+ hybrid female cDNA; (6) FM6,w/X_sim, w⁵⁰¹; P{Hmr^2t8.0}2-1/+ hybrid female cDNA; and (7) FM6,w/X_sim, w⁵⁰¹; +/+ hybrid female cDNA. X_sim indicates the D. simulans X chromosome. Flies from lanes 4 and 5 are siblings from a single cross, as are those from lanes 6 and 7. The 323-bp region of Hmr amplified from D. melanogaster contains an AvaI site that when cleaved produces fragments of 240 and 79 bp, excluding single-strand overhangs (lane 1). This AvaI site is absent from the Hmr allele carried by In(1)AB (lane 3) due to one of the missense changes described in Barbash et al. (2003) (see Figure 1A); it is also absent from D. simulans Hmr (lane 2). cDNAs in lanes 5 and 7 show an approximately equal expression of the D. melanogaster allele from the FM6,w chromosome and the D. simulans allele. cDNAs in lanes 4 and 6 show an increase in the amount of the uncleaved 324-bp band relative to the 240-bp band, indicating expression derived from the P{Hmr^2t8.0} transgene.

Figure 2.—

RT–PCR analysis of Hmr mutants. RT–PCR from the indicated genotypes was performed with three sets of primer pairs that span the indicated adjacent exons in Hmr, and with Gapdh1 as a control. Expected sizes of products are indicated to the left of each gel. RT − and + refers to absence and presence of reverse transcriptase enzyme during cDNA preparation. Canton-S and Oregon-R are wild-type D. melanogaster strains. The first lane of each gel contains a DNA molecular weight ladder in 100-bp increments.

Figure 3.—

Egg structure defects in Df(1)Hmr⁻ females. Five eggs each from 9- to 10-day-old Df(1)Hmr⁻/Df(1)Hmr⁻; P{Hmr^+tp72}11-1/+ females (A) and Df(1)Hmr⁻/Df(1)Hmr⁻ females (B) collected from experiment 2 of Table 5. Eggs from Df(1)Hmr⁻/Df(1)Hmr⁻ females are slightly shorter than from Df(1)Hmr⁻/Df(1)Hmr⁻; P{Hmr^+tp72}11-1/+ females and have substantially shorter chorionic appendages that are fused at their distal ends. Note that one of the eggs from Df(1)Hmr⁻/Df(1)Hmr⁻; P{Hmr^+tp72}11-1/+ females in A also has a chorionic appendage defect.

Figure 4.—

Two classes of ovariole morphology in 15- to 17-day-old P{EPgy2}Hmr³/P{EPgy2}Hmr³ females. (A, C, and E) Ovarioles that resemble wild-type or P{EPgy2}Hmr³/+ controls. (B, D, and F) Ovarioles with severe morphological defects. Anterior is marked with an asterisk in all images. (A and B) Ovarioles stained with anti-Vasa (green), anti-Hts 1B1 (red), and TO-PRO3 (DNA, blue). (A) Ovariole showing the characteristic wild-type pattern where at the anterior of the ovariole developing cysts are present in which anti-Vasa stains the germ cells and anti-Hts 1B1 stains the fusome, which gets larger as the cyst grows [for example, see Zaccai and Lipshitz (1996)]. Anti-Hts 1B1 also stains the cytoskeleton. Egg chambers budding from the germarium show a normal pattern of polyploid nuclei. (B) Ovariole showing egg chambers squished into anterior end. Several cysts are not correctly enveloped by a layer of follicle cells. (C and D) Ovarioles stained with anti-Orb (green) and TO-PRO3 (blue). (C) Ovariole showing younger cysts with Orb evenly dispersed and older egg chambers where Orb has accumulated in the oocyte, as described by Lantz et al. (1994). (D) Despite obvious morphological defects in the ovariole, younger cysts in the anterior of the ovariole with nonpolyploid nuclei show Orb correctly present throughout and older egg chambers where polyploid nuclei are present show an accumulation of Orb in one or two cells. (E and F) Ovarioles stained with anti-Vasa (green), anti-Hts-RC (red), and TO-PRO3 (blue). (E) Ovarioles showing the accumulation of Hts-RC in postmitotically active cysts and in the ring canals of egg chambers as observed in Robinson et al. (1994). (F) While egg chambers are squished into the anterior of the ovariole and in some cases disrupted, Hts-RC can still be seen at ring canals of intact egg chambers as well as near the nuclei of dispersed egg chambers.