Interaction between Notch and Hif-alpha in development and survival of Drosophila blood cells

Abstract

A blood cell type termed crystal cell in Drosophila functions in clotting and wound healing and requires Notch for specification and maintenance. We report that crystal cells express elevated levels of Sima protein orthologous to mammalian hypoxia-inducible factor-α (Hif-α) even under conditions of normal oxygen availability. In these platelet-like crystal cells, Sima activates full-length Notch receptor signaling via a noncanonical, ligand-independent mechanism that promotes hemocyte survival during both normal hematopoietic development and hypoxic stress. This interaction initiates in early endosomes, is independent of Hif-β (Τangο in Drosophila), and does not activate hypoxia response targets. Studies in vertebrate myeloid cells have shown a similar up-regulation of Hif-α protein in well-oxygenated environments. This study provides a mechanistic paradigm for Hif-α/Notch interaction that may be conserved in mammals.

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 (N^act) that functions as a gain of function Notch or (E) dominant-negative Notch (N^DN) 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 N^act-driven crystal-cell expansion. Compare with (D).

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 N^RNAi (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 N^RNAi (n = 8) and Ofut^RNAi (n = 8). (K) Wild-type lymph glands with elevated Notch protein [Notch intracellular domain (N^icd), red] in crystal cells (arrowheads, magnified in inset). (L) sima overexpression causes further Notch (N^icd, red) accumulation. Antibody against the extracellular domain of Notch (N^ecd, 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 (N^ecd, red) accumulation in vesicles (arrows, compare with M and M′). (O to Q) Live endocytic trafficking assay: Notch antibody-recognizing epitope on N^ecd (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 N^ecd 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. N^ecd (red) and Hrs (green) co-localize (yellow).

Fig. 3

Sima promotes ligand-independent stabilization of full-length Notch protein for crystal-cell maintenance. (A to C) Ser^RNAi 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 N^fl increases crystal cells [compare with (H)]. (J) Expressing N^fl in mib1 background shows no reduction in crystal cells [compare with (I) and (H)]. (K) Expressing dominant-negative presenilin (Psn^D447A) in crystal cells causes reduction.

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 tgo^EY03802 or (C) expressing tgo^RNAi causes an increase in crystal cells. (D) Expressing Hph^RNAi 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) NOS1^RNAi 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.