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Epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases

Hiroshi Shimizu1, Xiaoming Zhang2,*, Jinsong Zhang2, Alexey Leontovich2,{dagger}, Kaiyin Fei2,{ddagger}, Li Yan2,{ddagger} and Michael P. Sarras, Jr.2,§

1 National Institute of Genetics, Mishima, Japan
2 University of Kansas Medical Center, Kansas City, KS 66160-7400, USA
* Present address: Children’s Mercy Hospital Medical Center, Kansas City, MO 64108, USA
{dagger} Present address: Department of Experimental Pathology, Mayo Clinic, Rochester, MN 55904, USA
{ddagger} Present address: Harvard University, Boston, MA 02115, USA



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  		Fig. 1. Initial morphological events within 1 hour of decapitation as monitored by whole-mount immunofluorescence using antibody to hydra laminin ß1 chain (LM) (A-C) and hydra type I collagen (Col) (D-F), light microscopy (G-I) and transmission electron microscopy (TEM) (J). As shown in A,D,G, the ECM (arrow) is continuous along the head pole. Immediately after decapitation, the epithelial bilayer is separated into two halves and as indicated by the arrows in B,D,H, the ECM is contained within each half. The cut edge of the ECM can be visualized in whole mounts by immunofluorescent staining of both LM (localized to basal lamina) (B) and Col (localized to the interstitial matrix) (E). One hour after decapitation, the two separated halves of the bilayer have fused (I) creating a closed head pole that lacks the morphological features of an adult polyp (no hypostome or tentacles). The arrow in I indicates that the ECMs of each epithelial bilayer half are still not fused at this time as shown by TEM analysis in J (region indicated by the box in I). As also shown in J, the cut edge of the ECM is thickened (white arrow in J) when compared with the normal thickness of the ECM more distal to the cut edge (white arrowhead in J). The thickened cut edge of the ECM 1 hour after decapitation is seen in whole-mount immunofluorescence as a bright circular signal (white arrows in C and F) at the apical pole of the body column as monitored by staining for LM (C) or Col (F). The epithelium at the apical pole that has fused, but lacks an ECM, is flattened (arrowhead in I) when compared with the epithelium that is associated with an ECM (epithelium in the left half of the box shown in I). Scale bars: in F, 250 µm for A-F; in I, 100 µm for G-I.

 


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  		Fig. 2. Morphological and biosynthetic events occurring within 3 to 96 hours after decapitation as monitored by whole-mount in situ hybridization for hydra collagen I (Col) (A) and hydra laminin ß1 chain (LM) (B), whole-mount immunofluorescence for LM (C-F) and Col (G-J) and Northern blot analysis (K). As shown in A,B, upregulation 3 hours after decapitation of hydra collagen is associated with the ectoderm (A, arrow) while upregulation of hydra laminin is associated with the endoderm (B, arrow). While the epithelial bilayer has already fused at the apical pole (asterisk in C-J), a hiatus in the ECM still exists 3 hours after decapitation (C,G). The original cut edge of the ECM can still be detected up to 24 hours after decapitation, as monitored with antibodies to LM (C-E) and Col (G-I). Reformation of a continuous ECM at the regenerating head pole is first observed with antibodies for LM between 7 and 12 hours after decapitation (D, arrowhead) and this signal continues for 24-48 hours of regeneration (E,F, respectively; arrowhead). By contrast, an ECM-associated signal for hydra Col is only weakly detected by 15-24 hours (not evident at the magnification shown in I), while an easily observed signal is seen between 24 and 48 hours at this same magnification (J, arrowhead). Upregulation of mRNA for LM (K) and Col (data not shown) precedes the appearance of immunofluorescent signals for proteins associated with the reforming ECM. Elongation factor {alpha}1 (Ef{alpha}1) is used as a loading control for northern blot analysis of the mRNA lanes shown in K; rimes above each lane are in hours. The relative fluorescent and northern blot signals for LM and Col over 72 hours after decapitation are shown in L. Scale bars: in B, 200 µm for A,B; in J, 250 µm for C-J.

 


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  		Fig. 3. Whole-mount in situ hybridization for hydra laminin ß1 chain (LM), hydra type I collagen (Col), and hydra matrix metalloproteinase (MMP) mRNA monitored at 24, 48 and 72 hours after decapitation. The progression of the in situ signal (arrows) for LM, Col and MMP mRNA over this time frame changes from a general signal along the apical pole at 24 hours (A,D,G) to one associated with erupting tentacles at 48 hours (B,E,H) and 72 hours (C,F,I). Scale bar: 250 µm.

 


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  		Fig. 4. Reformation of an ECM is also associated with wound healing along the body column. An incision along the body column (at 90° to the longitudinal axis) results in creation of a gap in the ECM (A,C) that is still present 3 hours after wounding, as monitored by whole-mount immunofluorescent staining for laminin (LM) and collagen (Col). The cut edge of the ECM at the wound site is indicated by the arrows in A,C. As observed during head regeneration, the epithelium has already fused by this time (arrowheads in A,C). Upregulation of mRNA occurs 3 hours after wounding (LM shown in E; a similar signal for Col and MMP also occurs, data not shown). Twenty-four hours after wounding, reformation of the ECM has occurred, as monitored with antibody to LM. At this time, a signal for Col is not apparent at this magnification. As with head regeneration, a signal for Col is more easily observed between 24 and 48 hours (data not shown). Upregulation of ECM mRNA continues for 24 hours after wound healing, as monitored for LM (F). Upregulation of Col and MMP mRNA also continues through this time (data not shown). Scale bars: in D, 100 µm for A-D; in F, 200 µm for E,F.

 


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  		Fig. 5. Reformation of an ECM is also observed at the incision site during grafting experiments (A-C) and with morphogenesis during head regeneration (D-F), as monitored by whole-mount immunofluorescence. (A-C) Staining with mAb for hydra laminin (m52). (D-F) Staining for hydra fibrillar collagen (m39). As observed 24 hours after grafting of body segments, the region between the graft halves is the site of ECM biogenesis (region indicated by the arrowheads in A-C). The cut edges of the ECM from each graft appear as two transverse signals at the graft site (only one edge of the ECM is indicated by the arrows in A-C). The region of fusion of the epithelium at the graft site where ECM biogenesis is occurring correlates with deformation in the bilayer. This deformation may be observed as a narrowing or bulging in the body wall at the graft site (indicated by arrowheads in A-C). After decapitation, head regeneration always occurs apical to the original cut edge of the ECM (arrows in D-F), where matrix biogenesis is occurring. Scale bar: 250 µm.

 


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  		Fig. 6. Localized electroporation (LEP) of antisense thio-oligonucleotides to hydra laminin ß1 chain (LM) or hydra matrix metalloproteinase (MMP) into the apical pole of decapitated polyps results in a reversible inhibition of head regeneration. As monitored by whole-mount immunofluorescence, antisense oligos to LM blocked the appearance of laminin protein at the apical pole at 24 hours after LEP and decapitation (A). Sense oligos did not block the appearance of LM protein at the regenerating pole (B, arrows indicate reforming ECM). Antisense oligos to LM also blocks the appearance of hydra type I collagen (Col-I), as monitored as early as 36 hours and as late as 48 hours after decapitation with antibody to Col-I (C). Sense oligos to LM had no effect on the appearance of Col-I (D, arrows indicate reforming ECM). The area between the two arrows in B and D represents 100 µm.

 

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