Bällchen participates in proliferation control and prevents the differentiation of Drosophila melanogaster neuronal stem cells

Abstract

Stem cells continuously generate differentiating daughter cells and are essential for tissue homeostasis and development. Their capacity to self-renew as undifferentiated and actively dividing cells is controlled by either external signals from a cellular environment, the stem cell niche, or asymmetric distribution of cell fate determinants during cell division. Here we report that the protein kinase Bällchen (BALL) is required to prevent differentiation as well as to maintain normal proliferation of neuronal stem cells of Drosophila melanogaster, called neuroblasts. Our results show that the brains of ball mutant larvae are severely reduced in size, which is caused by a reduced proliferation rate of the neuroblasts. Moreover, ball mutant neuroblasts gradually lose the expression of the neuroblast determinants Miranda and aPKC, suggesting their premature differentiation. Our results indicate that BALL represents a novel cell intrinsic factor with a dual function regulating the proliferative capacity and the differentiation status of neuronal stem cells during development.

Keywords: Bällchen; Drosophila; Neuroblasts; Stem cells.

Fig. 1.. Expression of ball in embryonic neuroblasts.

(A–E) ball mRNA expression detected by in situ hybridization of whole mount embryos. ball mRNA is contributed maternally to cleavage division stage embryos (A) and ubiquitously expressed up to stage 10 of embryogenesis (B). ball mRNA is enriched in the nervous system starting at stage 11 (C). In late embryos, ball mRNA staining fades in the nervous system (D) and becomes visible in the gonads (arrowhead in D). Orientation of embryos (A–D) is anterior to the left, dorsal side up. (E) ball mRNA is expressed in neuroblasts, which are arranged in a stereotyped pattern along the ventral nerve cord of stage 11 embryos. Enlarged ventral region of the embryo is shown; anterior region is up. (F–K) ball mRNA and Prospero (PROS) protein localization in the ventral nerve cord of a stage 11 embryo. ball mRNA expression is high in the large neuroblast cells (dashed circles) and low in the PROS expressing GMCs. The overlay shows the DNA channel to better visualize the cells (H). Enlarged ventral region of the embryo is shown; anterior region is up. Scale bar in A–E, 100 µm. Scale bar in F–K, 10 µm.

Fig. 2.. Miranda expressing pNBs are lost from ball² mutant larval brains.

(A–C) Miranda (MIRA) expression detected by antibody staining of larval brains. The brackets indicate the position of thoracic pNBs in the ventral ganglion. (A) Wild type larval brains contain MIRA expressing pNBs at 96 h ALH. (B) ball² mutant brains carry MIRA expressing pNBs at 36 h ALH. (C) At 96 h ALH, ball² mutant brains lack MIRA expressing pNBs. (D) MIRA expressing pNBs of ball² mutant brains were initially functional, as evidenced by the asymmetric distribution of MIRA (arrowhead) and generation of Prospero (PROS) expressing GMCs. Scale bars in A–C, 100 µm. Scale bar in D, 10 µm.

Fig. 3.. ball² mutant thoracic pNBs proliferate at a reduced rate.

(A–A″) Control wild type cell lineage labeled by tub-GAL4 dependent β-Galactosidase (β-Gal) expression through the MARCM system. (A) 3D reconstruction from confocal image sections. Also shown are tilted views of the reconstruction (A′) and a single focal plane (A″) of which the position is indicated by a red dashed line in (A). An individual cell lineage is marked by a white dashed outline. (B) Counterstaining for DNA and the neuronal marker Elav (ELAV). Asterisks indicate the position of the pNBs, arrowheads point at GMCs, which lack ELAV expression. (C–C″) ball² mutant cell lineages stained, analyzed and displayed the same way as control wild type lineages (A–A″). (D) Counterstaining as in (B). (E) Quantification of neuronal cell numbers in wild type control (green) and ball² mutant (red) cell lineages at 96 h and 72 h ALH. The Number of ELAV expressing cells per cell lineage is displayed on the y-axis (neurons). Mean values and the total number of cell lineages analyzed (n) are given above the bars. (F) Quantification of mitotic pNBs in wild type control (green) and ball² mutant (red) cell lineages at 96 h and 72 h ALH, respectively. The fraction of pNBs that were in mitosis, based on H3S10ph positive staining, is indicated on the y-axis. Mean values and the total number of marked pNBs (n) are given above the bars. (G) Quantification of GMCs lacking ELAV expression in wild type control (green) and ball² mutant (red) cell lineages at 96 h and 72 h ALH, respectively. The number of ELAV negative cells below pNB size per marked cell lineage is indicated on the y-axis. Mean values and the total number of cell lineages (n) are given above the bars. Significance levels from Student's t-tests: p <0.005 (**), p <0.0005 (***). Scale bar in A–D, 10 µm.

Fig. 4.. Untimely differentiation of ball² mutant thoracic pNBs.

Wild type and ball² mutant cell lineages were labeled by tub-GAL4 dependent β-Galactosidase (β-Gal) expression through the MARCM system. β-Gal staining is left out for clarity in (A,B). (A) Miranda (MIRA, green) localizes to the basal cortex of mitotic pNBs that express H3S10ph (red). In interphase pNBs, MIRA is cytoplasmic. DNA counterstain is shown in blue. The percentage of marked wild type pNBs that express MIRA at 72 h (97%) and 96 h (97%) ALH is indicated. The percentage of ball² mutant pNBs that express MIRA drops from 72 h (56%) to 96 h (2%) ALH. The total number of pNBs analyzed per time point (n) is indicated. (B) Atypical protein kinase C (aPKC, green) localizes to the apical cortex of mitotic pNBs which express H3S10ph (red). In interphase pNBs, aPKC is ubiquitous. DNA counterstain is shown in blue. The percentage of marked wild type pNBs that express aPKC at 72 h (100%) and 96 h (99%) ALH is indicated. The percentage of ball² mutant pNBs that express aPKC drops from 72 h (67%) to 96 h (27%) ALH. The total number of pNBs analyzed per time point (n) is indicated. (C) pNBs from β-Galactosidase (β-Gal, green) marked ball mutant lineages at 96 h ALH stained for Miranda (top panel, MIRA, red), atypical protein kinase C (lower panels, aPKC, red) and DNA (blue). The channels for MIRA/aPKC and DNA are also shown separately. The positions of pNBs are indicated by dashed outlines. In addition to MIRA/aPKC loss from the pNBs (note staining of neighboring non mutant pNBs), ball mutant pNBs were found to be reduced in size (small pNBs) and symmetric pNB divisions were observed. Scale bar, 5 µm.