Two Z stacks of coelomogenesis in vestibula larvae of the sea urchin Holopneustes purpurescens recorded by high resolution confocal laser scanning microscopy from Morris (2016)

Morris, Valerie B.

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Open Access

Field	Value	Language
dc.contributor.author	Morris, Valerie B.
dc.date.accessioned	2016-01-15
dc.date.available	2016-01-15
dc.date.issued	2016-01-14
dc.identifier.citation	Morris VB (2016) Analysis of coelom development in the sea urchin Holopneustes purpurescens yielding a deuterostome body plan. Biology Open (2016). 5, 348-358. doi:10.1242/bio.015925.	en
dc.identifier.uri	http://hdl.handle.net/2123/14231
dc.description	Morris (2016) describes early coelomic development in a sea urchin that develops directly. Early coelomic tissue is separated into hydrocoele and coelomic mesoderm and between them is the enteric channel, which connects with the cavity of the archenteron. The hydrocoele is formed on the aboral side of the archenteron and the coelomic mesoderm is formed on the lateral and oral sides of the archenteron. The hydrocoele develops five primary podia that form as a group of three, the C, D and E podia and a group of two, the A and B podia. The podia develop orally next to the epithelium of the vestibule. The coelomic mesoderm spreads around the hydrocoele and between the podia. The enteric channel and archenteron are assumed to be where the parts of the gut will form including, orally, the oesophagus and mouth. Core structures of this early coelom development were schematized to derive a deuterostome body plan. Adult H. purpurescens were collected from coastal waters of New South Wales, Australia. Ova released from excised ovaries were fertilized with a diluted suspension of sperm from excised testes. Embryos and larvae from a fertilization were cultured in filtered sea water (FSW) at 20 degrees celsius. Larvae were fixed at hourly intervals from 27 h to 40 h after fertilization for viewing by confocal laser scanning microscopy. For the fixation, larvae were immersed in 4% (w/v) paraformaldehyde in FSW for 2 h, washed in FSW, dehydrated in a series of methanols to 100% methanol and stored at -20 degrees celsius For viewing in the microscope, larvae were cleared in 2:1 (v/v) methyl benzoate/methyl alcohol and mounted in the clearant in a cover-slip enclosed chamber set within a microscope slide. Larvae were autofluorescent from the paraformaldehyde fixation. They were viewed in the Leica TCS SP5 MP multi-photon laser scanning confocal system with a tunable Mai Tai Deep See laser attached to a Leica DMI6000B-CS inverted microscope. Each specimen was imaged using multi-photon microscopy at lambda ex = 870 nm with pulses in the 100-200 femtosecond range and detected in a reflected non-descanned detector at lambda em 545-605 nm. A Z stack was collected, with default X flipped, averaged over two frames in a 1024 x 1024 pixel array, 12 bits/pixel, at a slice thickness of 0.5 micron using a Leica HCX PL APO 63x/1.30 GLYC CORR CS 21 degrees C objective lens. The default X was flipped to restore the reflected image created in the inverted microscope to the non-reflected true image. Each Z stack was saved as a .lif file by the SP5 Leica Microsystems software. The .lif files of the two Z stacks used for figures 4 and 7 in Morris (2016) were each exported as a folder of .tif files with the .lif metadata deleted. The filenames of the folders are Fig 4 and Fig 7, matching the figure names in Morris (2016). The .tif images in the folders can be viewed individually or the set of .tif images in each folder can be analysed by image analysis software such as ImageJ available from the National Institutes of Health site (http://rsbweb.nih.gov/ij/).	en
dc.description.abstract	The Z stacks are those used for figure 4 and figure 7 in Morris (2016). Figure 4 shows the development of the hydrocoele and the primary podia in a 34 hour larva. Figure 7 shows the development of the hydrocoele, the enteric channel, the primary podia and the coelomic mesoderm in a 40 hour larva.	en
dc.publisher	The University of Sydney	en
dc.rights	Other	en
dc.subject	serial imaging	en
dc.subject	morphogenesis	en
dc.subject	echinoderm	en
dc.subject	evolution	en
dc.title	Two Z stacks of coelomogenesis in vestibula larvae of the sea urchin Holopneustes purpurescens recorded by high resolution confocal laser scanning microscopy from Morris (2016)	en
dc.type	Dataset	en
usyd.faculty	Faculty of Science, School of Biological Sciences	en