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  • Bruno Vellutini 13:06 on 2014/09/23 Permalink
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    4D with U0126 Membranipora 

    15 °C, 15 µM

    BCV_Mm_2014_09_23 (old Mmem1_23_09_2014)

  • Bruno Vellutini 13:44 on 2014/09/22 Permalink
    Tags: Terebratulina retusa   

    Terebratulina spawning 

    In the site site as Novocrania we collected shells and rocks with Terebratulina (likely retusa). I cut their pedicle to remove from the substrate and with tweezers it was quite easy to open the valves and check if gonads were ripe. I selected the three first females, split the valves apart, and removed the gonadal tissue from the mesenteries holding it in the inner part of the valves. I passed the oocytes through a 150 µm mesh and washed 3 times with a 70 µm mesh by reverse filtering.

    Oocytes dissected at 1200. They seem to be at stage 5 and 6 as described by (10.1007/BF01319413) since they are large and most have a complex microvillous border:


    I kept the males alive until it is time to prepare the sperm. There are still a few females in case I need to try the fertilization again.

    1400 – Oocytes look the same.
    1600 – Germinal vesicle still there.
    1800 – Looking the same. Dissected and prepare sperm of 3 males in 40 mL.
    2000 – Fertilized with 15 mL.


    1030 – After 3 washes I realized that there were swirling blastula near or at the bottom of the dish.
    1800 – Swimming blatulas haven’t changed much. No sign of blastopore.


    1000 – There were radial and asymmetric gastrulas. I picked them into a clean new bowl.


    1000 – There were different stages, from asymmetric gastrula until trilobed larva with a long mantle lobe. I will fix tomorrow.


  • Bruno Vellutini 15:38 on 2014/09/20 Permalink
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    Membranipora Endo-IWR-1 

    Trying the Endo-IWR-1 to inhibith the Wnt Pathway. Setting 2 plates with 10, 20, and 40 µM.

    For 24h

    DMSO 10 µM
    20 µM 20 µM

    For 48h

    DMSO 10 µM
     40 µM 40 µM

    I miss calculated the well and put 20 µM on the last well of the first plate. So I set 40 µM for the third and fourth wells of the second plate.


    I checked the embryos and it is hard to see if there is any phenotype. I’m letting it go another day to fix.


    Fixed at 48h.

  • Bruno Vellutini 15:34 on 2014/09/20 Permalink
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    Membranipora Azakenpaullone for in situ 

    Prepared large spawning of Membranipora for Azakenpaullone treatment at 1, 2.5 and 5 µM in 4 mL volume of sea water.

    DMSO 1 µM
    2.5 µM 5 µM

    To be fixed at 24 and 48h.

  • Bruno Vellutini 13:00 on 2014/09/20 Permalink
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    4D with U0126 Membranipora 

    BCV_Mm_2014_09_20 (old Mmem1_20_09_2014)

    10 µM, 15 °C

  • Bruno Vellutini 15:34 on 2014/09/19 Permalink
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    Membranipora U0126 inhibitor time windows 

    I set the U0126 inhibitor 10 µM with Membranipora at 15 °C. I am washing every 2 hours after 1h30 hours after activation as shown below (2x, for 24h and 48h fixation).

    • Activated: 1100
    • Inhibited: 1230
    • 1st wash: 1430
    • 2nd wash: 1630
    • 3rd wash: 1830
    DMSO 3.5 hpa
    5.5 hpa 7.5 hpa


    Fixed 24h.


    Fix 48h.

  • Bruno Vellutini 17:07 on 2014/09/18 Permalink
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    Membranipora 4D U0126 10 µM 

    Started a recording BCV_Mm_2014_09_18 (old Mmem1_18_09_2014) with Membranipora in 10 µM U0126 MEK inhibitor at 15°C. Embryo is looking quite good and stable.


    Embryo is still lokking good and healthy (?). Letting it on… Stopped and saved the embryo. It moved out of the field of view around 1700.

  • Bruno Vellutini 16:34 on 2014/09/17 Permalink
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    Membranipora Wnt inhibitors 

    Second round of testing inhibitors and activators of the Wnt pathway in Membranipora. Firt one was last year and they all showed a disrupted phenotype. I am repeating some treatments. Started with 2h post activation. 15 °C.

    Azakanpaullone (x2)

    DMSO 1 µM Azakanpaullone
    2.5 µM Azakanpaullone 5 µM Azakanpaullone


    DMSO 10 µM IWR
    20 µM IWR 40 µM IWR


    DMSO 1 µM PNU
    10 µM PNU 25 µM PNU


    Fixed one plate of Azakanpaullone at 24h. Leaving the others to be fixed at 48h.

    Azak: seems to give a concentration dependent phenotype, but it is quite hard to identify what is wrong.

    IWR & PNU: Although higher concentrations seem to have disturbed the development somehow, it is not clear what is wrong. Maybe there is nothing wrong. I’m leaving them for 48h, maybe a phenotype becomes clearer.


    Azak: 1 µM has a mild phenotype with a reduced early larva. 2.5 µM shows ciliated swimming balls or some partly differentiated reduced larvae. I am not sure if it is a ventralized phenotype, but it seems that the apical organ is missing. 5 µM balls of cells, not swimming, maybe already dead.

    IWR & PNU: Not sure if there is any phenotype…

    Fixed all for 1 hour at room temperature.

  • Bruno Vellutini 16:22 on 2014/09/17 Permalink
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    Membranipora temporary MEK inhibitor 

    Trying to understand better the effect of U0126 on Membranipora development I am starting a treatment with long pulses of the inhibitor first. Inhibitor will be washed every 8h.

    DMSO control 10 µM U0126
    10 µM U0126 washed after 9.5h 10 µM U0126 washed after 19h


    Interestingly, constant concentration shows reduced phenotype as usual, but the sample that was washed after 9h shows a higher number of relatively control like embryos. This might indicate that the effect of the MAPK inhibitor is either reversible, or, most probably, or unrelated to the observed phenotype which might be only a toxic general effect of the drug.

    Fixed one plate at 24h. The other for 48h.


    Same as previous sample. 9.5 hpa showed a higher proportion of normal larva then the 19hpa or the constant treatment. Fixwed with 4 % FA at RT for 1h.

  • Bruno Vellutini 16:15 on 2014/09/17 Permalink
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    Membranipora 4D U0126 10 µM 

    Started a recording BCV_Mm_2014_09_17 (old Mmem1_17_09_2014) with Membranipora in 10 µM U0126 MEK inhibitor at 15°C. Embryo is looking quite good and stable.


    Well, all the embryos were dead. Cells exploded and all. I think this was due to the high temperature of the room which made the slide temperature not be 15 °C as the ring should be, but more, killing the embryos.

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