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  • Bruno Vellutini 16:35 on 2013/11/05 Permalink
    Tags: , , segmentation,   

    Novocrania in situ wnts 

    Another in situ now using the newly synthesized probes.

    06/11/13

    Added Probes.

    08/11/13

    First washing day. Diluted 0.2 µL of Anti-DIG-AP in 1x blocking buffer and let overnight in cold room.

    09/11/13

    Washing off antibody. And started developing. Exchanged the substrate 4 times and no signal. Let overnight in cold room.

    10/11/13

    Wnt1 signal is visible! Ring around blastopore in gastrula and posterior ventral in later stages. No bands! Continued developing until 14h, then 4°C until 18h when I exchanged the substrate and let overnight again. Wnt5 had signal in the chaetae sacs.

    11/11/13

    Checked and signal looks a bit stronger so I developed 40 min more and stopped the reaction. Crystals were forming in the wnt5 well. Ethanol washes and will put them in glycerol with DAPI.

    12/11/13

    Mount and take pictures.

    Wnt1

    Wnt5

     
  • Bruno Vellutini 17:03 on 2013/10/27 Permalink
    Tags: , , segmentation,   

    Novocrania in situ 3rd try 

    Third try with engrailed, wnt1, and wnt5. This time using half-concentrated 5 min ProteinaseK treatment. They should not get damaged!

    • Fixed for 1h25min.
    • Washed normally and incubated in pre-hybe.

    28/10/13

    Added probes at 11h.

    30/10/13

    Washing day embryos looking good.

    31/10/13

    Washed out antibody with long 30 min washes.

    01/11/13

    Started developing. Engrailed began to appear after 2hs, but nothing in the wnts. I exchanged the AP substrate every 2 hours. At 18h I transferred to new wells because crystals were forming. At 19h30 I exchanged the AP and put them in the cold room.

    02/11/13

    Stopped engrailed and continued developing the wnts during the day. No signal came up except for background. Paused in AP without magnesium.

    04/11/13

    Restarted the wnts to see if something comes up…

    05/11/13

    Stopped wnts… Ethanol washes and added glycerol+DAPI overnight in cold room.

    06/11/13

    Mount slides. Engrailed has some shitty background… Remember next time to stop before overnight.

     
  • Bruno Vellutini 18:51 on 2013/10/11 Permalink
    Tags: , segmentation,   

    Terebratalia engrailed immuno staining 

    I will try using the engrailed/invected 4D9 antibody in Terebratalia.

    • Get radial, asym., bilateral, and trilobed larvae (used mixed stages from Kristineberg samples).
    • Permeabilize with 0.2% PTx (5x 5 min, 1x 30 min).
    • 2x 15 min PBT.
    • 1x 30 min 5% NGS.
    • 17h45: incubate with primary on a tube with 100 µL and dilution of 1:20.

    13/10/13

    • Wash out primary antibody along the day (3x 5 min PBT, 4x 30 min PBT, 1x 30 min PTx+NGS).
    • Incubate with secondary anti-mouse-pod 1:250 overnight.

    14/10/13

    • Washed 3x 5 min in PBT.
    • 4x 30 min PBT.
    • Develop with TSA and Cy5 fluorochrome (49 µL diluent + 1 µL fluorochrome).
    • Stop the reaction with 1x detergent solution at 60 °C and 2x at RT.
    • Washed 3x 5 min in 0.2% PTx.
    • Stained with phallacidin for 2h.
    • Washed 3x 5 min in PBS.
    • Grading series with TDE and mounted in 97% TDE with DAPI.

    15/10/13

    Confocal time. Engrailed antibody is indeed not working. I could try a higher concentration, maybe..

    Ttra DAPI-Phall TDE 1.lif - Series018 Ttra DAPI-Phall TDE 1.lif - Series021 Ttra DAPI-Phall TDE 1.lif - Series024 Ttra DAPI-Phall TDE 1.lif - Series027

     
  • Bruno Vellutini 18:36 on 2013/09/24 Permalink
    Tags: , , , segmentation,   

    Novocrania in situ en, wnt1, wnt5 

    Started an in situ with Novocrania! I picked 3 specimens of each stage making 12 embryos in total. The stages are: radial, bilateral gastrula, early larva and larva.

    Nano en Nano wnt1 Nano wnt5

    All 12 embryos per well survived the first day.

    25/09/13

    Since engrailed probe had low concentration I diluted what I had (13 ng/µL) into 200 µL of hybe (resulting in 0.5 ng/µL) and added anyway. I’m synthesizing more so that I can add tomorrow.

    26/09/13

    Changed engrailed hybe with nice new probe. However, embryos are really transparent. Larval morphology also does not look normal. I think something is wrong.

    27/09/13

    Engrailed well has not any normal looking embryos anymore. They are all falling apart. In the other 2 wells there are still 4-5 embryos that look ok, but fragile.

    I continued with the washes anyway and incubated with the antibody overnight at 4 °C.

     
  • Bruno Vellutini 14:52 on 2013/09/22 Permalink
    Tags: , , segmentation,   

    Probe synthesis of Novocrania segmentation 

    After successfully cloning Novocrania genes using Andi’s primers it is time for probe synthesis.

    Set Probe PCR for en, wnt1 and wnt5.

    23/09/13

    Gel and extraction went fine, except that I forgot the dna ladder…

    2013-09-23 23.37.45

    Extracted the probe PCR.

    24/03/13

    Began probe synthesis transcription reaction at 10h and stopped at 16h. Everything normal.

    25/03/13

    After centrifuging there was a very small pellet for engrailed. Wnt1 and wnt5 had normal pellets. Here are the nanodrop:

    Plasmid Gene Enzyme ng/µL
    BV267 engrailed SP6 13.08
    BV271 wnt1 SP6 156.92
    BV274 wnt5 SP6 54.99

    Since engrailed was so low I started a new transcription reaction at 12h.

    26/09/13

    Good engrailed concentration! 1334 ng/µL.

     
  • Bruno Vellutini 17:33 on 2013/09/13 Permalink
    Tags: , , , , , , , segmentation, ,   

    Terebratalia in situ for FGFR, Frizzleds and others 

    Starting an in situ for additional Terebratalia genes related to the segmentation project. Also a double with wnt1+wnt5.

    FGFR Pax2/5/8 Frizzled2 Frizzled4
    Frizzled10 LFNG ALDH2 Wnt1+Wnt5

    14/09/13

    Put probe.

    16/09/13

    Washed in situ and added anti-dig-ap 1:5000 to the first seven genes and anti-dig-pod 1:250 for the double fluorescent.

    17/09/13

    • Washed antibody.
    • Set Cy3 TSA to double fluorescent for 1h45.
    • Started developing regular NBT/BCIP at 16h.
    • Stopped Fz2 around 20h, let the rest overnight.

    18/09/13

    • Stopped FGFR and Fz4. Let the rest continue, almost stopped Pax2/5/8, since it is almost there.

    22/09/13

    • Ethanol washes.
    • Glycerol added.
     
  • Bruno Vellutini 18:35 on 2013/08/26 Permalink
    Tags: , , segmentation,   

    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 10:30 on 2013/06/25 Permalink
    Tags: , , , segmentation, ,   

    mamed2013_bcv

     
  • Bruno Vellutini 13:35 on 2012/11/12 Permalink
    Tags: segmentation,   

    Started organizing the expression patterns of Terebratalia for the segmentation project.

     
  • Bruno Vellutini 18:43 on 2012/04/13 Permalink
    Tags: , , , , , , segmentation, , ,   

    Katrine/Andi Terebratalia segmentation genes, miniprep specs 

    Since the previous sequencing for these minipreps did not work I am trying to sequence them again. These are the reads from the minipreps, they look ok.

    Gene ID ng/µL
    Tt gli 1 BV105 569.21
    Tt gli 2 BV106 755.79
    Tt gli 3 BV107 858.49
    Tt net 1 BV108 1080.2
    Tt net 2 BV109 416.45
    Tt net 3 BV110 452.18
    Tt net 3 BV111 474.99
    Tt nk6 1 BV112 1898.64
    Tt nk6 2 BV113 1217.41
    Tt nk6 3 BV114 1002.05
    Tt ptc 1 BV115 577.31
    Tt ptc 2 BV116 588.24
    Tt ptc 2 BV117 585.43
    Tt ptc 2 BV118 578.18
    Tt ptc 2 BV119 492.78
    Tt ptc 3 BV120 929.99
    Tt smo 1 BV121 631.31
    Tt smo 2 BV122 332.49
    Tt smo 3 BV123 1290.73
    Tt wnt7 1 BV124 968.39
    Tt wnt7 2 BV105 557.44
    Tt wnt7 3 BV106 2036.43
    Tt wnt7 4 BV107 554.82
    Tt wnt7 5 BV108 446.68

     
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