The Drosophila melanogaster Cajal body

Abstract

Cajal bodies (CBs) are nuclear organelles that are usually identified by the marker protein p80-coilin. Because no orthologue of coilin is known in Drosophila melanogaster, we identified D. melanogaster CBs using probes for other components that are relatively diagnostic for CBs in vertebrate cells. U85 small CB-specific RNA, U2 small nuclear RNA, the survival of motor neurons protein, and fibrillarin occur together in a nuclear body that is closely associated with the nucleolus. Based on its similarity to CBs in other organisms, we refer to this structure as the D. melanogaster CB. Surprisingly, the D. melanogaster U7 small nuclear RNP resides in a separate nuclear body, which we call the histone locus body (HLB). The HLB is invariably colocalized with the histone gene locus. Thus, canonical CB components are distributed into at least two nuclear bodies in D. melanogaster. The identification of these nuclear bodies now permits a broad range of questions to be asked about CB structure and function in a genetically tractable organism.

Figure 1.

dU2 snRNA and dU85 scaRNA occur in the same nuclear body. (A) In situ hybridization of ovarian follicle nuclei with an antisense probe against dU2 snRNA (red). dU2 occurs diffusely throughout the nucleus with a single bright focus that is associated with the unlabeled nucleolus. (B) dU85 scaRNA is detected with an antisense probe in a single discrete focus (green). (C) The overlay shows precise colocalization of dU2 snRNA (red) and dU85 scaRNA (green). DNA labeled with DAPI (blue).

Figure 2.

dSMN-YFP expression in ovarian nurse cells and the oocyte. (A) As in other organisms, dSMN is primarily a cytoplasmic protein. Each of the 15 nurse cell nuclei contains one or occasionally two or three discrete foci of dSMN-YFP. Because this image is a projection of multiple sections, the relatively small GV is obscured by material lying above and below it. The inset shows a single section through the GV from another egg chamber. The GV from young egg chambers, up to about stage 9, invariably displays a single brightly labeled nuclear body. (B–D) dU85 scaRNA colocalizes with dSMN-YFP in nurse cell nuclei. (B) A nurse cell nucleus from a fly expressing dSMN-YFP (green), showing a single positive nuclear body. (C) In situ hybridization with an antisense probe against dU85 scaRNA (red). (D) dSMN-YFP and dU85 scaRNA precisely colocalize in the nurse cell nucleus.

Figure 3.

Fibrillarin occurs primarily in the nucleolus but also in the CB in ovarian follicle cells and nurse cells. (A) In follicle cells, fibrillarin (red) is detected by mAb 72B9 in the fibrillar part of the nucleolus. DNA in the rest of the nucleus appears blue after staining with DAPI. (B) In situ hybridization with an antisense probe against dU85 (green) reveals a single CB in each nucleus. (C) Merge of the fibrillarin (red) and dU85 (green) images shows that the majority of CBs are at the periphery of the fibrillar part of the nucleolus. Some CBs appear to be inside the nucleolus, although it is difficult to be sure if they are simply above or below the fibrillar region. (D) Two follicle cell nuclei at higher magnification, stained for fibrillarin. The nucleus on the right displays a large patch of stain in the fibrillar region of the nucleolus and a smaller focus near the periphery of the nucleus. (E) In situ hybridization with dU85 reveals a CB in each nucleus. (F) The merge shows that the extra focus of fibrillarin in the right nucleus colocalizes with dU85 in the CB. (G) In this nurse cell nucleus, mAb 72B9 (red) stains the fibrillar region of the highly lobulated nucleolus, as well as a single, more intense focus at the periphery of one lobe. (H) In situ hybridization for dU85 (green) identifies a single CB. (I) The merged image clearly shows that the CB overlaps the most intense focus of the fibrillarin stain. DNA appears blue after DAPI staining.

Figure 4.

dLsm10 and dLsm11 in larval brain and salivary gland cells. (A) Cells in the brain of a third instar larva expressing YFP-dLsm11. In each nucleus, YFP-dLsm11 (green) is restricted to a single nuclear body (arrows). It is also expressed throughout the cytoplasm, with a single very bright focus in many cells (arrowhead). (B) Brain cell nuclei stained with a polyclonal serum against dLsm10 (green), mAb 72B9 against fibrillarin (red), and DAPI (blue). The focus of dLsm10 often lies near the fibrillar zone of the nucleolus. (C) A single confocal section through a nucleus in an intact, unfixed salivary gland from a third instar larva. The highest concentration of YFP-dLsm11 (green) occurs in the polygonal body near the nuclear envelope. The next highest concentration is in the large nucleolus (N), with detectable levels throughout the rest of the nucleus. (D) Salivary gland nucleus stained with a polyclonal serum against dLsm10 (green), mAb 72B9 against the nucleolar protein fibrillarin (red), and DAPI (blue). As in the smaller brain cells shown in B, the focus of dLsm10 often lies near the fibrillar part of the nucleolus (left nucleus), but not invariably so (right nucleus).

Figure 5.

YFP-dLsm11 foci during the blastoderm stage of embryogenesis. (A) Surface view of blastoderm during the interphase after the 14th cell cycle. Most nuclei contain a single nuclear body, whereas a few have two. (B) Lateral view of the blastoderm from an embryo of the same stage. Note that the single nuclear body is located near the apical end of the nucleus. One nucleus has two nuclear bodies. (C) Pole cells at the posterior end of an embryo, at the time when they first begin to migrate dorsally. Unlike the blastoderm nuclei in the same embryo, many of the pole cells contain two nuclear bodies. Embryos were fixed in formaldehyde and stained with a polyclonal rabbit anti-GFP for YFP-dLsm11 (green) and DAPI for DNA (blue). These images are projections of several individual confocal sections taken through the entire thickness of the nuclei.

Figure 6.

The distribution of dU7 snRNA and dU85 scaRNA in nurse cell nuclei. (A) YFP-dLsm11 (green) is concentrated in multiple bodies in the large polyploid/polytene nuclei of nurse cells. (B) In situ hybridization with an antisense probe against dU7 snRNA (red) reveals a similar pattern of multiple nuclear bodies. (C) Overlay of YFP-dLsm11 (green) and dU7 snRNA (red) images shows precise colocalization. (D) In situ hybridization with an antisense probe against dU7 (green) labels multiple nuclear bodies. Simultaneous in situ hybridization with an antisense probe against dU85 (red) shows a single focus of dU85 scaRNA, which lies near one of the larger dU7 nuclear bodies. (E) In this nucleus, there are two dU85 foci (red), both of which are next to dU7 nuclear bodies (green).

Figure 7.

YFP-dLsm11 is localized at the histone gene loci in brain cell nuclei. (A) YFP-dLsm11 stained with a rabbit polyclonal antibody against GFP (green). A single nuclear body is evident in each nucleus. (B) In situ hybridization of the same cells with a probe against D. melanogaster histone H4 (red). (C) Merge of YFP-dLsm11 (green) and histone genes (red), also stained with DAPI to show DNA (blue). Precise colocalization of histone genes and YFP-dLsm11 foci in the nucleus is evident. There is a single histone gene cluster on chromosome 2L. Although there are two clusters in a diploid nucleus, there is only a single focus of in situ hybridization, which is presumably due to somatic pairing of homologous chromosomes.

Figure 8.

Associations between CBs and HLBs. (A) Double in situ hybridization with antisense probes against dU2 (red) and dU7 (green) snRNA. dU2 has a diffuse nuclear distribution with a single prominent focus associated with the nucleolus (unstained area). dU7 is limited to a single discrete focus in each nucleus. dU2 and dU7 foci are often adjacent to each other but can be widely separated. (B) Double in situ hybridization with antisense probes against dU85 scaRNA (red) and dU7 snRNA (green). Each probe is limited to a single discrete focus. The dU85 and dU7 foci, like those of dU2 and dU7, are often close together but may be distantly separated.

Figure 9.

A diagram summarizing the distribution of CB components and other features of a “typical” D. melanogaster nucleus. The HLB, here identified by dLsm10, dLsm11, and dU7 snRNA, is present in all nuclei and is invariably associated with the histone gene locus. The CB is identified by dU85 scaRNA, dU2 snRNA, dSMN, and fibrillarin. It is frequently, although not invariably, associated with the HLB. Both the CB and the HLB lie near the heterochromatin—the HLB because the histone gene cluster is at the base of chromosome 2L near the euchromatin/heterochromatin boundary, and the CB because it is associated with the nucleolus, which itself lies in the middle of the heterochromatin of the X chromosome (and the short arm of the Y). The diagram includes “Rabl orientation” of the chromosomes (centromeres clustered at one point on the nuclear periphery) and somatic pairing of homologues, features common to many D. melanogaster nuclei.