Ellenberg Group - Echinoderms

<BODY bgcolor=#2A2558 text=#F6F845 link=#21C629 vlink=#E78C31> <font face="Arial, Helvetica, sans-serif"> <basefont size=3> <H1>Echinoderms</H1> <p align=justify> The oocytes of echinoderms such as starfish and sea urchin are a classic system to study cell division (Boveri, 1900). This oocytes are extremely useful because they</p> <UL> <LI><p align=justify>are autonomous and develop normally in sea water, an easily reproduced environment.</p> <LI><p align=justify>are optically clear, so that changes in organization deep in the cytoplasm can be viewed by transmitted light and fluorescence microscopy.</p> <LI><p align=justify>can be obtained as a homogeneous population in large quantities for biochemical studies.</p> <LI><p align=justify>are easy to inject quantitatively and support the expression of exogenous mRNAs and plasmids. Their maturation and fertilization is reliable and sets into motion several rounds of highly synchronized, rapid divisions.</p> </UL> <p align=justify>We keep the animals in custom built saltwater tanks in the lab in natural seawater from the Biologische Anstalt Helgoland, Germany. The tanks house sea urchin (<I>Lytechinus variegatus</I>) and starfish (<I>Asterina miniata</I>).</p> <BR> <table border=0 cellpadding=0 cellspacing=10 align="center" > <TR> <TD><IMG SRC="images/asterina_miniata.jpg" WIDTH=320 HEIGHT=245 ALIGN=LEFT ALT="Asterina miniata"> <TD><IMG SRC="images/lytechinus_variegatus.jpg" WIDTH=320 HEIGHT=245 ALIGN=LEFT ALT="Lytechinus Variegatus"> </TR> <TR> <TD WIDTH=320 ALIGN="CENTER"><font face="Arial, Helvetica, sans-serif"><B><I>Asterina miniata</I></B></FONT> <TD WIDTH=320 ALIGN="CENTER"><font face="Arial, Helvetica, sans-serif"><B><I>Lytechinus Variegatus</I></B></FONT> </TR> </table> <BR> <p align=justify> <FONT SIZE=-1> Boveri, T. 1900. Über die Natur der Centrosomen. <I>Zellen-Studien</I>. Vol. 4. T. Boveri, editor. Gustav Fischer, Jena. 29-61.<BR> </FONT> </P> <BR> <BR> <H2>Starfish oocyte preparation for microinjection<BR> <FONT SIZE=-2><A HREF="main_protocols.html" TARGET="main">(Get a PDF-version in the <I>Protocols section</I>.)</A></FONT></H2> <p align=justify>Species: <I>Asterina miniata</I> (batstars, California, Bodega Marine Station; Marinus Inc., Long Beach)</p> <UL> <LI><p align=justify>punch a 4 mm diameter hole in the tough skin of the animal on one side of the arm close to the madrepore (every arm can be used twice, but it is good to go around the animal clockwise from the madrepore and use each arm once only until all arms are used once).</p> <LI><p align=justify>If the animal is female yellow/orange ovaries will be close to the surface. In a male the gonads are white.</p> <LI><p align=justify>From a female take a piece of the ovary out (~ 10 mm in length) with a curved tweezer and put it into calcium free artificial sea water (cfASW) in a small beaker. In the cfASW shred the ovary with two tweezers into small pieces or use scissors to mince it finely. Allow the oocytes and follicle cells to flow out of the ovary for 2-3 min. This is speeded up by swirling the beaker.</p> <LI><p align=justify>When most oocytes are in suspension, swirl the beaker and decant the oocyte suspension into a fresh beaker leaving the ovary tissue behind. Wait for another 5-15 min for the follicle cells to detach. Check perdiodically on a microscope.</p> <LI><p align=justify>When most follicle cells are sheared from the egg replace the cfASW by regular ASW. Too long incubation in cfASW kills the eggs!</p> <LI><p align=justify>Oocytes should now be stored between 18-22°C and can be used for 12 h for microinjection.</p> </UL> <BR> <IMG SRC="images/starfish_oocyte_prep.jpg" WIDTH=400 HEIGHT=469 ALIGN=LEFT ALT="Starfish oocyte preparation" HSPACE=15> <BR><BR><BR><BR><BR><BR><BR> <p align=justify><B>1.</B> A female starfish on the prep area. Péter Lénárt (right) is holding the hole puncher, Jan Ellenberg (left) watching.</p> <p align=justify><B>2.</B> A closeup on the hole in the starfish arm.</p> <p align=justify><B>3.</B> Taking out a small piece of ovary with a tweezer.</p> <p align=justify><B>4.</B> Closeup of the ovary in a drop of seawater.</p> <p align=justify><B>5.</B> The starfish before punching the hole</p> <p align=justify><B>6.</B> The starfish oocyte, free of follicle cells.</p> <BR CLEAR=ALL> <BR> <HR> <address>Jan Ellenberg <BR> <A HREF="http://www.embl-heidelberg.de" TARGET=_top><IMG SRC="icons/embl_home.gif" WIDTH="38" HEIGHT="38" BORDER=0 ALT="EMBL Homepage" ALIGN=RIGHT></A> <A HREF="http://www-db.embl-heidelberg.de:4321/emblGroups/g_42.html" TARGET=_top><IMG SRC="icons/embl_group.gif" WIDTH="38" HEIGHT="38" BORDER=0 ALT="EMBL Grouppage" ALIGN=RIGHT></A> <A HREF="http://www.embl-heidelberg.de/ExternalInfo/ellenberg/homepage/" TARGET=_top><IMG SRC="icons/home1.gif" WIDTH="38" HEIGHT="38" BORDER=0 ALT="Home" ALIGN=RIGHT></A> Gene Expression and Cell Biology/Biophysics Programmes, <A HREF="http://www.embl-heidelberg.de/ExternalInfo/GeneralInfo/Heidelmaps.html" TARGET=_top>Location</A><BR> European Molecular Biology Laboratory, Meyerhofstrasse 1,D-69117 Heidelberg, Germany<BR> Email: <A HREF="MAILTO:Jan.Ellenberg@embl-heidelberg.de">Jan.Ellenberg@embl-heidelberg.de</A>, Fax: +49-6221-387-518<BR> Web design by <A HREF="MAILTO:Thorsten.Klee@web.de">Thorsten Klee</A>. Last modified 22.12.2001.</address> </BODY>