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Science 332 (6034): 1210-1213

Copyright © 2011 by the American Association for the Advancement of Science

Interaction Between Notch and Hif-α in Development and Survival of Drosophila Blood Cells

Tina Mukherjee1, William Sang Kim1, Lolitika Mandal1,*, and Utpal Banerjee1,2,{dagger}

1 Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA.
2 Molecular Biology Institute, Department of Biological Chemistry, and Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, USA.


Figure 1
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Fig. 1. Sima functions with Notch during crystal-cell development. Crystal cells marked with ProPO (red). Scale bars indicate 20 μm. (A) Wild-type lymph gland, crystal cells show elevated levels of Sima (green; yellow because of ProPO co-localization). (Inset) Magnified view of crystal cells expressing Sima (green). (B) Overexpression or (C) single-copy loss of sima causes crystal-cell expansion or reduction, respectively. (D) Overexpression of activated Notch (Nact) that functions as a gain of function Notch or (E) dominant-negative Notch (NDN) that functions as a loss of function Notch causes crystal-cell expansion and reduction, respectively. (F) Wild-type Notch reporter activity [12xSu(H)-lacZ, green] (G) increases with sima gain of function and (H) decreases with single-copy loss of sima. β-Gal, β-galactosidase. (I) Single-copy loss of sima suppresses Nact-driven crystal-cell expansion. Compare with (D).

 

Figure 2
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Fig. 2. Sima stabilizes Notch in mature crystal cells, which is necessary for their maintenance and survival. Crystal cells marked with ProPO (red). Scale bars, 20 μm. (A) Wild-type (WT) lymph gland. (B) Control lymph glands expressing membrane GFP in crystal cells. Magnified in (B'). Overexpression of Hph (C) early does not affect crystal cells [compare with (A)]; (D) late expression in crystal cells causes their reduction and (D') rupturing [compared with (B')]. Quantification of (E) lymph gland (n = 8) and (F) circulating crystal-cell (n = 8) data. Error bars indicate standard deviation. Expressing NRNAi (G) early and (H) late causes reduction in crystal cells [compared with (A) and (B)]. Loss of Notch late from crystal cells causes their rupturing [(H') similar to (D') compared with (B')]. Quantification of (I) lymph gland (n = 12) data and (J) circulating crystal-cell data expressing NRNAi (n = 8) and OfutRNAi (n = 8). (K) Wild-type lymph glands with elevated Notch protein [Notch intracellular domain (Nicd), red] in crystal cells (arrowheads, magnified in inset). (L) sima overexpression causes further Notch (Nicd, red) accumulation. Antibody against the extracellular domain of Notch (Necd, red) antibody detects (M and M') Notch in vesicles (arrowheads) in crystal cells from control lymph glands expressing GFP and (N and N') overexpressing sima further increases Notch (Necd, red) accumulation in vesicles (arrows, compare with M and M'). (O to Q) Live endocytic trafficking assay: Notch antibody-recognizing epitope on Necd (red) in wild-type lymph glands marked with nuclear (Cut, green) and crystal-cell (ProPO, blue) markers. (O and O') Notch protein is detected on all membranes at 0 min. (P) and (P') At 300 min, endocytosed Notch is degraded from surrounding cells [compare Necd levels in the dashed areas in (O') and (P')] except in crystal cells (arrowheads) (Q) that retain Notch (red) in Hrs-positive (green, arrowheads) early endosomes. Necd (red) and Hrs (green) co-localize (yellow).

 

Figure 3
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Fig. 3. Sima promotes ligand-independent stabilization of full-length Notch protein for crystal-cell maintenance. (A to C) SerRNAi in signaling cells affects crystal cell [(A) and (B)] early [n = 5, 50, and 60 hours after egg laying (AEL)] but not [(A) and (C)] late (n = 5, 76 and 88 hours AEL). Crystal cells marked with ProPO (red). Scale bars, 20 μm. (D to F) fng overexpression [(D and (F)] early in signaling cells (n = 7) causes crystal-cell reduction but not [(D), (E), and (G)] late (n = 4). (H) Wild-type lymph gland. (I) Overexpressing Nfl increases crystal cells [compare with (H)]. (J) Expressing Nfl in mib1 background shows no reduction in crystal cells [compare with (I) and (H)]. (K) Expressing dominant-negative presenilin (PsnD447A) in crystal cells causes reduction.

 

Figure 4
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Fig. 4. Sima function in mature crystal cells is independent of Tgo. ProPO (red) marks crystal cells (red). Scale bars, 20 μm. (A) Wild-type lymph gland. (B) Single-copy loss of tgoEY03802 or (C) expressing tgoRNAi causes an increase in crystal cells. (D) Expressing HphRNAi or (E) exposing second instar wild-type larvae to 5% hypoxic stress increases Sima (green) stabilization and crystal-cell expansion. (F) Crystal cells from third instar WT lymph glands show elevated NOS1 (green, yellow because of overlap with ProPO; see inset). (G) NOS1RNAi in crystal cells causes bursting [compare with (A)]. (H and I) Feeding larvae with (H) L-NAME (NO inhibitor) shows reduction in Notch reporter activity (red), whereas (I) D-NAME (inactive isomer) has no effect.

 


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