Identification of mushroom body miniature, a zinc-finger protein implicated in brain development of Drosophila

Abstract

The mushroom bodies are bilaterally arranged structures in the protocerebrum of Drosophila and most other insect species. Mutants with altered mushroom body structure have been instrumental not only in establishing their role in distinct behavioral functions but also in identifying the molecular pathways that control mushroom body development. The mushroom body miniature(1) (mbm(1)) mutation results in grossly reduced mushroom bodies and odor learning deficits in females. With a survey of genomic rescue constructs, we have pinpointed mbm(1) to a single transcription unit and identified a single nucleotide exchange in the 5' untranslated region of the corresponding transcript resulting in a reduced expression of the protein. The most obvious feature of the Mbm protein is a pair of C(2)HC zinc fingers, implicating a function of the protein in binding nucleic acids. Immunohistochemical analysis shows that expression of the Mbm protein is not restricted to the mushroom bodies. BrdUrd labeling experiments indicate a function of Mbm in neuronal precursor cell proliferation.

Fig. 2.

Analysis of the adult mbm¹ phenotype. Frontal sections of paraffin-embedded heads of WT females (A–D), mbm¹ females with a moderate (E–H)ora strong (I–L) phenotype, and Df(2L)A1/mbm¹ females carrying a single copy the rescue construct P[TW115] (M–P). Photographs were taken after staining with the anti-Leonardo antibody (33) at the level of the Kenyon cell (Kc) bodies/calyx (A, E, I, and M), the peduncle (B, F, J, and N), and the α/β (C, G, K, and O), and the γ (D, H, L, and P) lobe system. In the WT, the Kenyon cell bodies are located in the dorsal-posterior cortex. Kenyon cell dendrites and extrinsic input fibers form the calyx (ca). The Kenyon cell fibers form the peduncle (ped) extending anterior-ventrally where they divide to form the dorsally and medially projecting lobe system. Two major dorsally projecting lobes (α and α′) and three medially projecting lobes (β, β′, and γ) are visible; the additional subdivisions described by Strausfeld et al. (9) are not discernible at this resolution. Despite the severe reduction in the number of Leonardo-positive cells (E and I), the structural subdivision of the MBs is maintained in mbm¹ (F–H). Even in the most severe cases (J–L), a rudimentary lobe system can be detected. Note also the many unstained cell bodies surrounding the Leonardo-positive Kenyon cells in mbm¹ (I Inset). The MB defect of Df(2L)A1/mbm¹ females is rescued to nearly WT appearance by the genomic construct P[TW115] (M–P).

Fig. 1.

Premature degeneration of MB axons in mbm¹. Shown are the dorsal lobes of WT (A and C) and mbm¹ (B and D) MBs. (A) In late third-instar larvae, axon branches of the γ-neurons that form the dorsal lobe are uniformly labeled with the anti-fasciclin II antibody. Six hours after puparium formation (APF), degeneration of γ-neuron axons in WT animals becomes evident by the appearance of large unstained holes (arrows in C). The mbm¹ mutation is characterized by a general size reduction of the lobe system and the appearance of unstained holes already at third larval instar (arrows in B). (D) Six hours APF, only residual anti-fasciclin II staining can be detected in mbm¹.

Fig. 3.

Genomic organization and identification of the mbm gene. The thick horizontal line represents a physical map of the genomic scaffold sequence AE003590 (position 263820–283640) (36). Restriction sites for AatII (A), EcoRI (E), HindIII (H), and HpaI (P) are indicated. The lines above the genomic sequence indicate the extension of the deficiencies Df(2L)net-PMF in the distal direction and Df(2L)A1 in the proximal direction. The proximal breakpoint of Df(2L)net-PMF and the distal breakpoint of Df(2L)A1 are located in the hatched boxes. Df(2L)al is located outside the map in proximal direction. Below the genomic map, the rescue constructs used in this study are shown. Their ability to rescue the mbm¹/Df(2L)A1 phenotype is indicated with + or –. Predicted or known genes in this region are shown by arrows. The exon–intron structure of the mbm transcription unit CG11604 is diagrammed at the bottom. Filled boxes represent the ORF. In mbm¹, aCtoT transition is found in the 5′ untranslated region.

Fig. 4.

Staining of brains from third-instar larvae with the anti-Mbm anti-serum. (A) The projection view of one brain hemisphere shows the nuclear localization of the Mbm protein in many cells. The arrows point to the anlagen of the optic lobes. In addition, the antibody labels the neuropil of the larval MBs (arrowhead). In a group of three large cells (*), the Mbm protein localizes to the cytoplasm. (B) Costaining of brains with the anti-Mbm antiserum (green) and an antibody against phospho-histone H3 (red). A single confocal section (0.8 μm) at the level of the Kenyon cell body layer is shown. The Kenyon cell bodies are barely visible below the three outlined neuroblasts. The arrowhead points to a dividing cell at anaphase.

Fig. 5.

Altered cell proliferation in brains of mbm¹/Df(2L)A1 larvae. Dorsal views of brains of late third-instar larvae labeled with BrdUrd (brown) and the D-Mef2 antibody (blue). In the central brain of the WT larvae (A), the large neuroblasts are faced at one side by two to four BrdUrd-positive ganglion mother cells or neurons. The Kenyon cells (blue) are localized beneath the MB neuroblasts and, therefore, most of them are out of focus. Only scattered BrdUrd-positive cells and small D-Mef2 cell clusters are seen in mbm¹/Df(2L)A1 brains (B).