Figure 1.
Generating a DBHD null allele.
(A) The DBHD genomic locus and the targeting strategy. P1–P4 represent the PCR primers. (B) PCR analysis of the genomic DNA. In DBHD−/− larvae (−/−), a 4.8 kb fragment and a 5 kb fragment could be amplified by the corresponding primer pairs. (C) rtPCR analysis of the DBHD transcripts in various tissues. LB: larval brain; disc: mixtures of larval imaginal discs. α-tubulin (at 84B) was used as the positive control. (D) A DBHD antibody recognizes a band at about 55 kDa (arrow) of the whole larval extracts, which is absent in the DBHD−/− larvae. w1118 was used as the wild-type control (WT). (E) Statistical analysis of the developmental profiles of two strains. The +/TM3 flies contains a healthy 3rd chromosome and a GFP-marked third chromosome balancer (TM3, Kr::GFP). The −/TM3 flies contains the same balancer and the DBHD− allele. Animals survived to different stages were counted. Numbers in the parenthesis are the theoretical values according to the Mendel rules. *: all the survived adults are heterozygotes. (F) Comparison of DBHD−/− (−/−) and the sibling heterozygotes (−/+) at different days after egg laying. Embryos collected within three hours and cultured in the same food vials were picked for images at each time point. All heterozygotes have eclosed by 14 days after egg laying and thus were not pictured.
Figure 2.
Mitosis and endoreplication are suppressed in DBHD−/− larvae.
PH3 marks the mitotic cells. EdU marks the cells undergoing DNA synthesis. DAPI marks the nuclei. (A and B): Eye imaginal discs. (C, D, K, L): Brains. (E, F, I, J): Fat bodies. (G and H): Salivary glands. The sibling heterozygotes (−/+) were taken as the wild-type controls. Note all the DBHD−/− samples (−/−) are reduced in size, the polyploidy are also reduced in cells from fat body (F) and salivary gland (H).
Figure 3.
Autophagy is elevated in the DBHD−/− larvae.
(A–C) LysoTracker staining (red) of unfixed larval fat bodies. The GFP-positive tissues (green) are from heterozygotes (Kr::GFP). (A) The LysoTracker signal is much stronger in the DBHD−/− (−/−) fat bodies than in the heterozygotes (−/+). The 4-day-old larvae were picked form the same food vial, processed in the same staining tube and imagined in one optical field. (B) LysoTracker signal became strong in the heterozygotes starved for 3 hours by supplying with distilled water only. The same staining of fat body from starved DBHD−/− is also shown. (C) 3-Methyladenine (3-MA) suppresses autophagy in DBHD−/− fat body. (D) Foraging assay by feeding larvae with colored food (the baker’s yeast powder mixed with black ink). Green: GFP (A–C); Blue: DAPI (C).
Figure 4.
Rescue of the DBHD−/− growth defects by nutrients.
(A) DBHD−/− larvae were sensitive to yeast supply. NF: normal food; Star: starvation, normal food without yeast. All samples were picked at Day 8 after hatching from eggs. (B) The developmental profiles of flies cultured on two kinds of nutritious foods. yeast paste: the baker’s yeast powder was mixed with water and supplied on agar plate. leucine: normal food supplemented with 100 mM leucine. Fifty newly hatched larvae for each genotype were picked. Numbers of the heterozygotes (left) and DBHD−/− were separated by slashes. (C) Examples of four dead DBHD−/− pharates cultured on yeast pastes. (D) Rescue effects of leucine. Animals aged for 7 days after hatching were imaged. NF: normal food; leu: normal food with 100 mM leucine; rapa: normal food with 1 µM rapamycin; leu+rapa: normal food with 100 mM leucine and 1 µM rapamycin. (D) Free amino acids analysis of the larvae. The amino acids levels are displayed as milligram per gram of body weight (mg/g). The amounts of larvae for each experiment are listed in the parenthesis.
Table 1.
The developmental profiles of flies cultured on various yeast foods.
Table 2.
Rescue of the DBHD−/− larvae by supplemented nutrients.
Figure 5.
The human FLCN could partially rescue the DBHD−/− larvae.
(A) Dorsal view of pupae. The heterozygote (−/+) is revealed by Sb (marked with short and thick bristles on the notum, arrow), see materials and methods for the cross scheme. (B) The genotypes of the pupae were confirmed by PCR analysis of genomic DNA. The fly CG10414 gene was used as a positive control.
Figure 6.
Expression patterns of DBHD-res.
The expression of DBHD-res was detected by the EGFP staining (green). Genotype in all panels: DBHD-res; DBHD−/−. (A) Larval fat body. (B) Larval salivary gland. (C) eye-antennal disc. (D) Magnified view of eye disc. (E) Larval brain. (F) Ring glands. (G) Epithelium of larval midgut. (H) Epithelium of adult midgut. (I) Magnified view of adult midgut. (J) Testis. (K) Germarium. (L) Egg follicles. Arm (red) marks the cell borders; Prospero (Pros, red, in nuclei) marks the intestinal EE cells; Dpn marks the neuroblasts in the brains; DAPI (blue) marks the nuclei in G–I, L. Note the DBHD-res was also expressed in some EC cells in the gut (E–G, marked by the DAPI-labeled polyploid cells).
Figure 7.
Clonal analyses of DBHD−/− cells in the larval imaginal discs.
(A–C) GFP signals. (C’) PH3. Random clones were generated in the wing (a) and eye (b) imaginal discs. eyeless-flipase induced large clones in the eye disc (C, C’). The DBHD−/− cells are absent of GFP (green) and circled with solid lines. The wild-type twin spot cells are marked with double GFP signals and enclosed with dashed lines. Note the DBHD−/− and the twin spots are similar in clone size. The amount of PH3-positive cells is not clearly declined or increased in the DBHD− clones.