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First published online 22 February 2006
doi: 10.1242/dev.02299

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Steroid hormone-dependent transformation of polyhomeotic mutant neurons in the Drosophila brain

Jian Wang^1,*, Ching-Hsien J. Lee^2,*, Suewei Lin³ and Tzumin Lee^3,

¹ Department of Entomology, University of Maryland, College Park, MD 20742, USA.
² Division of Hematology Oncology, Department of Medicine, University of Massachusetts Memorial Medical Center, Worcester, MA 01655, USA.
³ Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

View larger version (55K): [in a new window]   Fig. 1. Identification of l(X)MB342 based on presence of ectopic unrecognizable MARCM clones. (A) Composite confocal images of an adult brain showing labeling of the paired MBs by GAL4-OK107-driven expression of mCD8-GFP (arrows). GAL4-OK107 labels a few non-MB structures in the central brain (arrowheads). (B) Composite confocal images of a wild-type mosaic adult brain showing one MB Nb clone in the left hemisphere and multiple single-cell/two-cell clones of MB neurons in the right hemisphere. (C) Summary of the genetic crosses for the MARCM-based genetic screen. The star represents a mutagenized chromosome. (D) Composite confocal images of a mosaic adult brain containing clones of l(X)MB342 homozygous mutant neurons. Multiple ectopic clones with rudimentary projections are present. Clones were induced in newly hatched larvae unless otherwise indicated. Scale bar: 20 µm. Genotypes: (A) UAS-mCD8-GFP/+;GAL4-OK107/+; (B) FRT19A/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;GAL4-OK107/+; and (D) FRT19A,l(X)MB342/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;GAL4-OK107/+.

View larger version (32K): [in a new window]   Fig. 2. Loss of Ph accounts for l(X)MB342 mutant phenotypes. (A) l(X)MB342 carries a lethal mutation within the internal 2D3 to 2E1, defined by the distal breaking points of Df(1)64c18 and Df(1)Pgd-kz. All rescuing regions of duplication are shown in green, while the l(X)MB342-complemented (purple) and l(X)MB342-non-complemented (red) deleted segments are shown. In addition, three (in red) out of five lethal complementation groups around this region failed to complement with l(X)MB342. (B) Composite confocal images of a mosaic adult brain containing MARCM clones of ph602 homozygous mutant neurons. Phenotypes are similar to l(X)MB342 mutant clones shown in Fig. 1D. (C) Composite confocal images of a mosaic adult brain containing P{ph-d+}-rescued l(X)MB342 mutant neuronal clones. Genotypes: (B) FRT19A,ph602/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;GAL4-OK107/+; and (C) FRT19A,l(X)MB342/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;P{ph-d+}/+;GAL4-OK107/+. Scale bar: 20 µm.

View larger version (90K): [in a new window]   Fig. 3. MARCM analysis of ph, using various GAL4 drivers, before and after metamorphosis. Composite confocal images of mosaic brains examined at the wandering larval (WL) (A-H) or adult (I-P) stages. MARCM clones of wild-type or ph mutant neurons were labeled using various GAL4 drivers. (A-D,I-L) In wild type, different GAL4s permit labeling of distinct types of neurons before metamorphosis (A-D), and usually the labeling patterns are preserved at the adult stage (I-L). (E-H) Before metamorphosis, various GAL4 drivers label different clones of ph mutant neurons; distinct ph mutant clones, like their wild-type controls (A-D), acquire different characteristic projection patterns. (M-P) By contrast, at the adult stage, GAL4-OK107 (N) and GAL4-NP225 (O), like the pan-neuronal driver elav-GAL4 (M), labels many clones, while ato-GAL4 is completely suppressed (P). Moreover, without characteristic projection patterns, ph mutant clones are no longer identifiable in adult brains. Genotypes: (A,I) FRT19A/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;elav-GAL4/+; (E,M) FRT19A,ph602/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;elav-GAL4/+; (B,J) FRT19A/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;GAL4-OK107/+; (F,N) FRT19A,ph602/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;GAL4-OK107/+; (C,K) FRT19A/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/GAL4-NP225; (G,O) FRT19A,ph602/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/GAL4-NP225; (D,L) FRT19A/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/ato-GAL4; (H,P) FRT19A,ph602/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/ato-GAL4. Scale bar: 20 µm.

View larger version (94K): [in a new window]   Fig. 4. ph mutant clones, induced before versus after the prepupal ecdysone peak, exhibit distinct phenotypes. Composite confocal images of mosaic adult brains containing clones of wild-type or ph mutant neurons that were induced at the mid-3rd instar stage (A-D) or two days after pupal formation (2D APF) (E-H). (A,B,E,F) Wild-type brains exhibit different patterns of clones with GAL4-OK107 (A,E) versus GAL4-NP225 (B,F). (C,D) Both GAL4s labeled analogous ph mutant clones following induction of mitotic recombination at the mid-3rd instar stage. (G,H) By contrast, GAL4-OK107 (G), but not GAL4-NP225 (H), permits labeling of mid-pupa-born ph mutant MB neurons. Genotype: (A,E) FRT19A/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;GAL4-OK107/+; (C,G) FRT19A,ph602/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;GAL4-OK107/+; (B,F) FRT19A/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/GAL4-NP225; (D,H) FRT19A,ph602/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/GAL4-NP225. Scale bar: 20 µm.

View larger version (53K): [in a new window]   Fig. 5. Phenotypic analysis of ph mutant clones through puparium formation. Composite confocal images of mosaic fly brains fixed during puparium formation (0D APF), 1D APF or 2D APF. In wild-type mosaic brains, GAL4-NP225 constantly labeled clones of projection neurons through pupal stages (A-C). By contrast, GAL4-NP225 labeled many ectopic clones of ph mutant neurons after 2D APF (D-F). Genotypes: (A-C) FRT19A/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/GAL4-NP225; (D-F) FRT19A,ph602/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/GAL4-NP225. Scale bar: 20 µm.

View larger version (34K): [in a new window]   Fig. 6. Ecdysone-dependent transformation of ph mutant neurons in cultured mosaic larval brains. Composite confocal images of variably cultured larval brains that contain clones of ph mutant neurons with GAL4-OK107 as a driver. Few GFP-positive cells exist after 4 days of culture in the absence of 20-hydroxyecdysone (A). By contrast, there are many GFP-positive cells when ecdysone was added at the beginning (B) or on the fifth day of the culture (C). Genotype: FRT19A/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;GAL4-OK107/+. Scale bar: 20 µm.

View larger version (29K): [in a new window]   Fig. 7. MARCM analysis of Pc and E(z) in the mosaic fly brains. (A-G) Composite confocal images of various MB clones at the wandering larval (A,B) or adult (C-G) stage. A gigantic loose structure is present in the larval Pc mutant Nb clone (arrow in A) versus the dense well-defined calyx in the larval E(z) mutant Nb clone (arrow in B). Numerous ectopic neurites are present in both Pc (C) and E(z) (D) mutant adult Nb clones, and the dendritic tree is over-elaborated in the adult Pc single-cell clone (arrowhead in F; compare with arrowheads in E and G). A calyx-like structure in the adult E(z) mutant Nb clone is shown in the inset in D. (H) Various glia-like E(z) mutant brain cells became ectopically labeled by GAL4-OK107. Genotypes: (A,C,F) hs-FLP,UAS-mCD8GFP/+; PcXT109,FRT2A/tubP-GAL80,FRT2A;GAL4-OK107/+; (B,D,G,H) hs-FLP,UAS-mCD8GFP/+;E(z)731,FRT2A/tubP-GAL80,FRT2A;GAL4-OK107/+; and (E) hs-FLP,UAS-mCD8GFP/+;FRT2A/tubP-GAL80,FRT2A;GAL4-OK107/+. Scale bar: 20 µm.

View larger version (77K): [in a new window]   Fig. 8. Derepression of abd-B in clones of ph mutant neurons. Composite confocal images of whole wandering larval CNSs (A,H) and MARCM clones of wild-type (B-D,I-K) or ph mutant (E-G,L-N) neurons in the central brains of wandering larvae. Green shows MARCM clones that are positive for mCD8-GFP (B,E,I,L), red shows anti-Abd-B (A,C,F) or anti-Ubx (H,J,M). (D,G,K,N) Merged images. (A-G) Endogenous Abd-B is restricted to the terminal one-third of the ventral ganglion (A), and ectopic expression of abd-B in MARCM clones of ph mutant neurons (compare F and G with C and D). (H-N) By contrast, endogenous Ubx is enriched in the middle segment of the ventral ganglion (H) and no ectopic expression of Ubx is detected (I-N). Genotypes: (A,H) wild type; (B-D,I-K) FRT19A/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;elav-GAL4/+; (E-G,L-N) FRT19A,ph602/FRT19A,hs-FLP,tubP-GAL80;UAS-mCD8-GFP/+;elav-GAL4/+. Scale bar: 20 µm.