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First published online 29 April 2008
doi: 10.1242/jcs.021931

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Haemocyte-derived SPARC is required for collagen-IV-dependent stability of basal laminae in Drosophila embryos

Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario, M5S 3G5, Canada

View larger version (86K): [in this window] [in a new window]   Fig. 1. Expression of SPARC transcript and SPARC protein during embryonic development. Transcript and protein expression overlap during gastrulation. (A-N) Wild-type embryos; A, D-G are in situ hybridizations (blue) and B, C, H-N are immunostains. (A) The ventral view of an ES7 embryo shows that SPARC mRNA is expressed by invaginating mesoderm (m) along the ventral midline and the cephalic furrow (cf). (B) Lateral view of an ES8 embryo shows SPARC protein expression (brown) in mesoderm (m), anterior midgut (am) and posterior midgut (pm) primordia. (C) Lateral view of an ES9 embryo shows SPARC protein expression (brown) throughout mesoderm and anterior (am) and posterior (pm) midgut primordia. (D) Lateral view of an ES11 embryo shows SPARC expression (blue) in midgut rudiments as they begin to migrate along the underlying mesoderm. (E) Lateral view of an early ES12 embryo shows SPARC expression at the onset of germ band retraction in growing midgut rudiments and mesoderm. Several neurons are also highlighted at this stage by 22C10 immunostaining (brown, arrow). (F) Dorsal view of an ES12 embryo during germ band retraction. SPARC is observed in migrating midgut rudiment epithelia (blue) and haemocytes (arrows). (G) Dorsal view of an ES13 embryo with fused midgut and haemocytes (arrow) expressing SPARC. (H) Ventral lateral view of haemocytes in an ES13 wild-type embryo. Haemocytes populate the head and begin posterior migration along the ventral nerve cord (VNC) (arrows). (I) The dorsal view of an ES14 embryo shows SPARC-positive haemocytes present throughout the embryo. (J) SPARC immunostains basal laminae around the developing brain (left arrow) and midgut (right arrow) at ES15. (K) Lateral view of an ES17 embryo expressing SPARC in the fat body (arrow) and haemocytes. (L) The same embryo as in K shows SPARC immunostaining in the basal lamina around the midgut (upper arrow), VNC channels and around the VNC (lower arrow). (M,N) Magnified views of an ES14 wild-type embryo immunostained with SPARC antibody (lateral view). (M) Haemocytes (arrows) migrating along the VNC express SPARC. Low to no SPARC immunostaining is observed in haemocyte nuclei. (N) A different focal plane reveals that SPARC colocalizes with VNC channels (arrows) and ventral epidermal (arrowheads) basal laminae. All confocal images are single sections.

View larger version (63K): [in this window] [in a new window]   Fig. 2. Loss of SPARC function results in embryonic lethality and compromised basal lamina stability. (A-H) A-D Wild-type embryos A-D and SPARC-mutant embryos (E-H) immunostained with anti-laminin (red) and anti-neurotactin (green) antibodies to highlight the developing central nervous system (CNS). (A) Lateral view of a late ES11 wild-type embryo shows that laminin accumulates along the mesoderm-neuroepithelium interface (arrowhead). (B) Laminin sheets form around the CNS and internal organs by ES13. (C) Thicker laminin sheets surround the CNS, body-wall muscles and the digestive tract by ES17. (D) A different confocal view of the embryo shown in C. Note that the entire VNC can be seen in a single confocal section. (E) Laminin is loosely associated with the VNC because spaces are observed between the deformed CNS and the discontinuous laminin sheet (arrow) at ES15. (F) The ventral view of an ES16 embryo shows fragmented laminin immunostaining around the VNC. (G) Laminin immunostaining around the CNS and other tissues is discontinuous at ES17 in the SPARC-mutant embryo. (H) A different confocal plane of the embryo in shown in G shows a distorted and uncondensed VNC. (I) Ventral view of an ES17 wild-type embryo immunostained with anti-perlecan antibodies (red) shows continuous perlecan distribution around the condensed VNC. Commissures of the VNC are highlighted by anti-CNS axons antibodies (green). Perlecan immunostaining around the ventral cord is continuous. (J) The ventral view of an ES17 SPARC-mutant embryo also shows continuous perlecan immunostaining around the VNC. (K) Lateral view of an ES16 deficiency embryo that lacks both collagen IV genes immunostained with laminin. Laminin distribution is also discontinuous around the VNC (green). (L) Lateral view of a SPARC-mutant embryo showing a complete absence of ventral and head cuticle. (M) Ventral view of SPARC-mutant embryonic cuticle highlighting cuticle holes along the ventral surface. All images of immunostains are single confocal sections.

View larger version (69K): [in this window] [in a new window]   Fig. 3. Collagen IV is absent from basal laminae in SPARC-mutant embryos. (A) ES15 wild-type embryo immunostained with DN-cadherin (blue), SPARC (red) and collagen IV (A', green). Channel glia are highlighted by arrows. A''. The merged image shows that SPARC and collagen IV colocalize in the basal lamina surrounding the VNC (arrows), haemocytes (arrowheads) and channel glia (lines). Images in A and C are single confocal sections. (B) Projection of a magnified view of haemocytes from wild-type embryos coimmunostained with SPARC and collagen IV. (C) In an ES17 SPARC-mutant embryo, collagen IV is only observed in haemocytes (arrowhead). DN-cadherin highlights the deformed commissures. The intensity of collagen IV immunostaining in haemocytes (arrowheads) is similar to that in wild-type embryonic haemocytes. The inset shows a higher magnification of collagen IV-positive haemocytes. Collagen IV appears as puncta within haemocytes. (D) SPARC immunostains the fat body (arrow) in SrpHemo-GAL4 embryos expressing the UAS-SPARC RNAi transgene. (D') GFP is expressed under the control of the SrpHemo-GAL4 driver only in haemocytes. (D'') The merged image shows the absence of SPARC protein from haemocytes. (E) Collagen IV immunostaining (red) is only detected in haemocytes (arrowheads) in embryos of the same genotype as D. (E') The same embryo as D shows GFP-positive haemocytes (green). (E'') Collagen IV immunostaining colocalizes with haemocytes and basal lamina immunostaining is not observed. The inset shows a higher magnification of collagen IV puncta (red) in haemocytes (green). (F) The SPARC-positive fat body (arrows) is disorganized and fragmented in an ES17 embryo of the same genotype as D. Expression of SPARC is also observed in the channel glia (arrowheads). (F') Haemocytes are GFP positive (green). (F'') The merged image shows that SPARC is not expressed by haemocytes. (G) The SPARC-positive fat body (arrows) is disorganized and fragmented in the ES16 SPARC RNAi embryo. (G') GFP is expressed exclusively in the haemocytes under the control of a collagen-GAL4 driver. (G'') The merged image shows that SPARC is not expressed by haemocytes. (H) A ES16 SrpHemo-GAL4 embryo expressing UAS-ricin. SPARC (red) immunostaining in fat body (arrow) and the remaining haemocytes (arrowhead). (H') Faint GFP expression is observed in a decreased population of haemocytes (green). (H'') The merged image shows SPARC immunostaining in a few haemocytes (arrowhead). Images D-H are confocal projections. Scale bar, 10 µm. (I) S2R+ cells immunostained with SPARC (red, I) and collagen IV (green, I') show colocalization of SPARC and collagen IV in intracellular vesicles (yellow, I''). All images in I are single confocal sections. The following genotypes were used: C: w;; H2AvD-GFP, Df (3R)nm136/H2AvD-GFP, Df (3R)nm136 D-F: w; SrpHemo-GAL4, UAS-eGFP/+; UAS-SPARC RNAi/+ G: w; Collagen-GAL4, UAS-GFP/+; UAS-SPARC RNAi/+. H: w; SrpHemo-GAL4, UAS-eGFP/UAS-ricin.

View larger version (119K): [in this window] [in a new window]   Fig. 4. Collagen IV associates with basal laminae in SPARC mutants that express transgenic SPARC. Embryos transgenically expressing SPARC were immunostained with anti-collagen IV (ColIV, red) and anti-SPARC (dSPARC, green) antibodies. (A) ES16 embryo expressing the SPARC transgene in a SPARC mutant background in haemocytes (UAS-SPARC; gcm-GAL4) shows SPARC colocalization with collagen IV in basal laminae (arrow, yellow). No morphological defects are observed. Endogenous SPARC expression and colocalization with collagen IV is observed in the fat body (arrowhead). (B) ES16 rescued embryo (UAS-SPARC/+ or y; gcm-GAL4/+; H2Av-GFP, Df(3R)nm136/H2Av-GFP, Df(3R)nm136) immunostained for SPARC (green). (C) The same embryo as in B immunostained for collagen IV (red). GFP expression is not shown. (D) The merged image (yellow) shows their colocalization in haemocytes (arrowhead) and basal laminae (arrows) around the brain, VNC, midgut chambers and along the VNC channels. (E) Lateral view of an ES16 SPARC-mutant embryo that expresses transgenic SPARC in haemocytes (UAS-SPARC/+ or y; SrpHemo-GAL4 UAS-GFP/+; H2Av-GFP, Df(3R)nm136/H2Av-GFP, Df(3R)nm136) immunostained for collagen IV (red) and SPARC (green). Intense coimmunostaining is observed in haemocytes (arrowheads) and basal laminae (arrows). (F) ES17 embryo with the same genotype as in E. Collagen IV and SPARC colocalize in basal laminae. (G) UAS-SPARC/+ or y; gcm-GAL4/+; H2Av-GFP, Df(3R)nm136/H2Av-GFP, Df(3R)nm136 embryos immunostained for laminin (red). Laminin distribution is continuous around the VNC. (H) Lateral view of an ES17 SPARC-mutant embryo expressing SPARC (blue) in all neuroblast- and glia-derived cells and sensory organ precursor cells (UAS-SPARC/+ or y; sca-GAL4/+; H2Av-GFP, Df(3R)nm136/H2Av-GFP, Df(3R)nm136). Motorneurons are disorganized and the VNC twists out of focus. All images are single confocal sections.

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