Updates from August, 2013 Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 15:12 on 2013/08/31 Permalink
    Tags: ,   

    Extraction of Membranipora neuropeptides 

    Collected thousands of cyphonautes larvae from early (3d) to older than a week into a plastic syringe with 5 µm mesh immersed in FSW. Once all the larvae were concentrated I immersed the syringe in mqH2O and pipetted the larvae to a clean extraction tube with 300 µL of extraction buffer.

    I used the screwdriver with a pistil to intensively smash the larvae and completed the volume of the tube with extraction buffer. Tube was vortexed for 2 minutes and spinned down for 20 min at 4 °C. Supernatant was transferred to a clean tube and kept at -20 °C.

    I noticed that the larvae were not completely into pieces (although they were crushed), but I did what I could do with the screwdriver.

     
  • Bruno Vellutini 18:34 on 2013/08/30 Permalink
    Tags: , , ,   

    4D Membranipora Azakenpaullone 5 µM 

    Folder: BCV_Mm_2013_08_30

    Another recording with Az. Kind of lateral view and optics are good.

    31/08/13

    Looking good. It became something.

    02/08/13

    Stopped the recording. Deleted from lineage station stack X3201 onwards, embryo was getting bad.

     
  • Bruno Vellutini 19:27 on 2013/08/29 Permalink
    Tags: , , ,   

    4D Membranipora Azakenpaullone 5 µM at 15 °C 

    Folder: BCV_Mm_2013_08_29

    Set a recording with azakenpaullone 5 µM. Embryo at 2 cell, looking ok.

    30/08/13

    Checked the history and during the second division 1 blastomere fails to divide. Subsequent divisions look kind of normal, but I presume this is not due to the inhibitor.

    Stopped the recording in the afternoon.

     

     
  • Bruno Vellutini 18:04 on 2013/08/28 Permalink
    Tags: , , ,   

    Novocrania en, wnt1, wnt5 

    Set PCR with 50 °C annealing temperature and 40 cycles with new primers.

    29/08/13

    2013-08-29 11.55.15

    Not good enough, I cut the top band of wnt1 anyway…

     
  • Bruno Vellutini 17:37 on 2013/08/28 Permalink
    Tags: ,   

    4D Membranipora 15 °C normal 

    Folder: BCV_Mm_2013_08_28

    Very good optics showing animal pole.

    29/08/13

    Still looking good. I just tapped down once the light to see one of the edges better.

    The larva swam away and I stopped the recording. It was rescued and kept at 10 °C.

    31/08/13

    Fixed!

     
  • Bruno Vellutini 19:50 on 2013/08/27 Permalink
    Tags: ,   

    4D Membranipora normal at 15 °C 

    Folder: BCV_Mm_2013_08_27

    Set new recording with tilted animal pole view. It seems that the whole scope is vibrating, maybe due to the camera? I could not stop the recording to check (because the embryo looks good), but will do it after this.

    28/08/13

    Checked the whole overnight recording and the embryo is not well stabilized. It is moving around… I’ll stop it soon and put a new one.

     
  • Bruno Vellutini 18:35 on 2013/08/26 Permalink
    Tags: , , ,   

    Submitted abstract to SICB2014 

    Beyond boundaries: expression of “segment polarity” genes during larval lobe development in brachiopods

    VELLUTINI B.C. & HEJNOL A.
    Sars International Centre for Marine Molecular Biology, Univ. of Bergen, Norway
    bruno.vellutini@sars.uib.no

    Brachiopods are sessile bivalved spiralians closely related to annelids, molluscs, and nemerteans. Despite having an unsegmented adult body, the larval body of many brachiopods is divided in lobes disposed along the anterior-posterior axis. This morphology and presence of partitioned coeloms in some larvae have been treated as evidence that brachiopods evolved from a segmented ancestor. We approached this hypothesis by characterizing the development of brachiopod larval lobes and the expression of genes commonly expressed in segments of arthropods and annelids (i.e. “segment polarity” genes) in the trilobed larva of Terebratalia transversa and the bilobed larva of Novocrania anomala. We have cloned Engrailed, Wnt genes, and components of the Hedgehog pathway and analysed their expression by in situ hybridization. The three lobes of T. transversa larva were delimited by an anterior ectodermal groove and a posterior less prominent constriction. We detected adjacent stripes of wnt1 and engrailed transcripts in the ectoderm delimiting the anterior boundary. At the posterior boundary, wnt1 and engrailed were co-expressed on a ventral and a dorsal band. Genes of the Hedgehog pathway were not expressed on the larval lobes. Adjacent stripes of wnt1 and engrailed are also found at parasegment and segment boundaries of arthropods and annelids, respectively, while co-expression is observed in the chordate mid-hindbrain boundary and hemichordate collar-trunk boundary. Thus, our results suggest that engrailed and wnt1, but not hedgehog, might be involved in the development of lobe boundaries of T. transversa larvae in a similar manner as observed in other morphological boundaries.

     
  • Bruno Vellutini 20:28 on 2013/08/21 Permalink
    Tags: , , , ,   

    4D Membranipora with 10 µM U0126 

    Folder: BCV_Mm_2013_08_21

    Set an animal view of the embryo at 15 °C with 10 µM U0126. There was some weird blebbing in the beginning, so I thought it was over.

    22/08/13

    Looking ok, disruption in the 8 to 16 cell stage. Let it go.

    23/08/13

    Sabrina said it was fine.

    24/08/13

    Looking ok, still some cellular movements so letting it go.

    25/08/13

    ?

    26/08/13

    Someone turn the cooler and recording off around 15h. I rescued and fixed the embryo at 19h.

    Deleted from workstation stack X3001 onwards.

     
  • Bruno Vellutini 20:43 on 2013/08/20 Permalink
    Tags: ,   

    4D Membranipora normal 

    Folder: BCV_Mm_2013_08_20

    Another recording at 15 °C, normal development, lateral view.

    Not good, ends before gastrulation since embryos begins to be squeezed by coverslip.

     
  • Bruno Vellutini 17:29 on 2013/08/20 Permalink
    Tags: , ,   

    10 µM U0126 for in situ, second try 

    Since the first try failed, I’m repeating it. Large batch of embryos put into 10 µM in 3 mL of FSW 3h post activation.

    22/08/13

    Fixing embryos after 33h for in situ.

     
c
Compose new post
j
Next post/Next comment
k
Previous post/Previous comment
r
Reply
e
Edit
o
Show/Hide comments
t
Go to top
l
Go to login
h
Show/Hide help
shift + esc
Cancel