Journal: Developmental biology

Article Title: Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function

doi: 10.1016/j.ydbio.2013.04.004

Figure Lengend Snippet: Panels A–H represent wandering third instar. In panels A–H and L–O, dome-Gal4, UAS-2xEGFP (wild-type, A, L and N) is used to express the specified genetic constructs in prohemocytes (green). In panels J–K, the protein trap line ZCL1973X labels endogenous Trol-GFP protein fusion expression. In all panels peroxidasin (Pxn) expression is shown in red. (A) Wild-type lymph gland. (B) Downregulating trol expression in prohemocytes induces their differentiation into Pxn+ hemocytes, increasing CZ size at the expense of the MZ. (C–H) Progenitor-specific downregulation of thisbe (thsRNAi, C) or pyramus (pyrRNAi ,E) or overexpression of heartlessDN (htlDN, G) reduces differentiation (red), impairing CZ formation. Downregulation of thisbe (thsRNAi; trolRNAi, D) or pyramus (pyrRNAi; trolRNAi ,F) or overexpression of heartlessDN (htlDN; trolRNAi, H) upon downregulation of trol is sufficient to block the increased differentiation induced by trol downregulation alone (B). (I) Quantification of Pxn+ hemocyte population distribution (% area) in the lymph gland. Data are represented as mean ± s.d., n=10. Asterisks denote statistical significance compared to wild-type (dome >), measured by Student’s t-test. (J) At wandering third instar stage (wL3), high levels of Trol are detected in the extracellular matrix amidst the population of Pxn hemocytes of the MZ, but Trol expression is not observed among Pxn+ hemocytes (red) of the CZ (inset). (K) During the second larval instar stage (L2), high levels of Trol are observed in the extracellular matrix throughout the lymph gland. Large pockets of Trol-negative tissue outline groups of early differentiating (Pxn+) hemocytes. (L–O) Wild-type lymph glands at second instar (L2, L) contain a small population of early differentiating hemocytes, which increases by early third instar stage (eL3, N) to begin forming the early CZ. Downregulation of trol in prohemocytes expands the population of differentiating hemocytes during second and early third instar stages (M and O, respectively) compared to wild-type. (P) Thisbe/Pyramus-Heartless signaling in Drosophila lymph gland hemocyte progenitors. Thisbe- and Pyramus-induced Heartless (Ht1) activation in lymph gland progenitors is modulated by Tro1/Perlecan heparan sulfate proteoglycan. Heartless activation by its ligands induces plasmatocyte differentiation in a Pointed/ETS-dependent manner through Ras activation, affecting the ratio of MZ progenitors and CZ differentiated hemocytes in the lymph gland. U-shaped/FOG is also required for Heartless- and Pointed-mediated effects on differentiation and functions either downstream of or in parallel to Heartless signaling. Thisbe-specific Heartless activation induces lamellocyte differentiation in a TORC1-dependent manner that is independent of Pointed or U-shaped gain-of-function and is also not observed upon pyramus overexpression. Scale bars=50 µm. Scale bar in A corresponds to panels A–H, except for the following additional magnifications: X0.75 for E, X0.8 for B and H, and X1.2 for D. Scale bar in L corresponds to L–M. Scale bar in N corresponds to N-O.

Article Snippet: The following Drosophila stocks were used: UAS-heartless DN , UAS-[d4EBP(LL )] and thisbe-Gal4 (Bloomington); UAS-heartless Act (A. Michelson); UAS-thisbe WT and UAS-pyramus WT (A. Stathopoulos); FRT82B heartless AB42 and FRT42B pyramus Ex36 (unpublished reagents; kindly provided by A. Stathopoulos); UAS-ras DN , UAS-ras85B Act , UAS-rolled (MAPK) Act , UAS-pointedP2 Act , and Trol-GFP protein trap line ZCL1973X (V. Hartenstein); UAS-u-shaped WT (N. Fossett); FRT82B ras85B ΔC40B and FRT82B pointed Δ88 (J. Bateman); UAS-vein, UAS-secreted-spitz, UAS-pvf1 , and UAS-pvf2 (U. Banerjee); UAS-secreted-gurken and UAS-secreted-keren (A. Simcox); UAS-jelly-belly (J. Weiss); UAS-branchless (M. Krasnow); hh-F4f-GFP (R. Schulz).

Techniques: Construct, Expressing, Over Expression, Blocking Assay, Activation Assay