. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. FERTILIZATION AND DEVELOPMENTAL BIOLOGY 289 t. Figure 1. Protease treatment removes the ECM from 36 h vegetal isolates (A and B). H 'hen these ECM-less isolates are cultured an additional 36 h and assayed for myogenic and endodermal markers, they express these markers to the same extent as controls (C and D). The expression l myogenie markers is inhibited however, when the ECM-less isolates are cultured in the presence ol fiAPN (E and E). (A and B) I'egetal isolates were prepared from 36 h Strongylocentrotus purpuratus embry
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. FERTILIZATION AND DEVELOPMENTAL BIOLOGY 289 t. Figure 1. Protease treatment removes the ECM from 36 h vegetal isolates (A and B). H 'hen these ECM-less isolates are cultured an additional 36 h and assayed for myogenic and endodermal markers, they express these markers to the same extent as controls (C and D). The expression l myogenie markers is inhibited however, when the ECM-less isolates are cultured in the presence ol fiAPN (E and E). (A and B) I'egetal isolates were prepared from 36 h Strongylocentrotus purpuratus embryos (cultured at 15°C) by sedimentation in a clinical centrifuge at low speed and resuspension in calcium-free seawaler (CFS\\1 three times and in 1 M glycine. It) mM EDTA. once. This procedure loosens the eclodermal cells from the underlying basal lamina. After repealed sedimentation and washing in CFSH'and transfer to filtered seawater (FSW'). the vegetal isolates are separated from most / the free eclodermal cells, and the endoderm and mesenchyme cells are retained in a "hal/oon- likc'' bag consisting of the basal lamina (arrows in A) and the extracellular matrix that once filled I lie blaslocoel. To remove the ECM. vegetal isolates were treated with either collagenasesF, H. L. or N or pancreatin (allfrom Sigma, Si. Louis. MO) at 1 mg/ml inFSW or a combination ofpancreat in and collagenase (1 mg/ml each) at 3 7°C. Throughout incubation in the proteases, isolates were examined on a Zeiss Axiovert microscope equipped with DIC optics to assess the degree of ECM removal. After 30-40 min at 37°C the ECM was no longer visible in pancreatin-trea/ed isolates (B). Control isolates (A) were incubated in FSWfor the same time and temperature as theprotease-trealed isolates. (C and D) Once the ECM was no longer visible, the isolates were sedimenled as above and washed two limes with /-"Sir Protease-trealed and control isolates were cultured in FSW with 5 ng/ml of I he antibio
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