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  • Bruno Vellutini 11:45 on 2013/05/24 Permalink
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    Selected remaining juveniles ~2 month and incubated with 20 µM EdU from Click-iT Kit for 3h (stock concentration is 10mM, 2µL/1000µL). Relaxed with MgCl2 for 30min in cold room and fixed with 4% PFA for 1h in cold room + 1h at RT. Permeabilized with PBS+Triton-X and stored at 4 °C in PBS.

     
  • Bruno Vellutini 11:26 on 2013/05/21 Permalink
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    Probe synthesis for Meara RNA binding proteins 

    Began the probe PCR for RNA binding proteins in Meara. Finished by May 24th.

    Gene ID ENZYME ng/µL
    Ms gus a BV226 T7 3409
    Ms boule BV229 SP6 501
    Ms staufen BV233 SP6 1141
    Ms ago a BV237 SP6 563
    Ms ago b BV238 SP6 1236
    Ms bru a BV242 SP6 1226
    Ms mago nashi BV244 SP6 336
    Ms orb2 BV247 T7 473

    Ms RNA binding

     
  • Bruno Vellutini 12:12 on 2013/05/15 Permalink
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    Submitted EMBO course application (MAMED 2013) 

    Abstract

    Also at Google Docs.

    Expression of engrailed and wnt1 demarcates the anterior lobe boundary in a brachiopod larva
    Bruno C. Vellutini and Andreas Hejnol
    Sars International Centre for Marine Molecular Biology, Bergen, Norway

    Segmentation of the body along the anterior-posterior (AP) axis is a characteristic feature of arthropods, annelids, and chordates. It is a long standing question whether segmentation had a common evolutionary origin or if it has evolved multiple times in bilaterians. Regulatory mechanisms of segmentation can be surprisingly conserved across taxa (e.g., segment polarity genes), but can also vary even within related groups (e.g., different annelids). Brachiopods are sessile spiralians with a bivalved shell closely related to annelids, molluscs, and nemerteans. Despite having an unsegmented adult body, the larval body of many brachiopods is divided in two or three lobes disposed along the AP axis. They are denominated apical, mantle, and pedicle lobes and define an anterior and a posterior boundary in the trilobed larvae. The objective of this study is to verify if segment polarity genes have a role in the development of the larval lobes of brachiopods. Therefore we have cloned and performed in situ hybridizations of engrailed, wnt genes, and components of the Hedgehog pathway in different developmental stages of the brachiopod Terebratalia transversa. We detected transverse stripes of wnt1 and engrailed transcripts adjacent to each other at the anterior boundary of the larva. wnt1 is localized along the posterior-most region of the apical lobe and engrailed along the anterior-most region of the mantle lobe. This expression is similar to the patterns found at parasegment and segment boundaries of arthropods and annelids, respectively. Thus, our preliminary results suggest that engrailed and wnt1 are involved in forming and maintaining the anterior boundary of T. transversa larvae. Succeeding functional experiments will help to clarify the degree of conservation of such segmentation mechanism during brachiopod development.

    Motivation

    Also at Google Docs.

    Dear admissions committee,

    I am a PhD student in Andreas Hejnol’s group at the Sars Centre for Marine Molecular Biology and associated to the Molecular and Computational Biology Research School of University of Bergen, Norway. My general research goal is to understand how changes in organism development are related to the evolution of form in metazoans. I am interested in evo-devo of less studied marine invertebrates especially concerning the evolution of larval body patterns.

    It was the work of the 19th century Russian biologist Élie Metchnikoff that triggered my current interest for marine invertebrates and developmental processes. His career was based on comparative embryological studies of invertebrates and on a compelling evolutionary thinking which influenced me during my undergraduate studies. This early contact to exciting evolutionary questions involving the evolution of multicellularity and ontogenetic processes directed my attention towards evolutionary developmental biology. Since then, the interface between evolution and development became the main axis of my academic pursuit.

    Before starting my master’s thesis I had the opportunity to take two courses at Friday Harbor Laboratories. The practical contact with the wide diversity of embryonic and larval forms of marine invertebrates as well as the enlightening discussions about evolution and development had a major influence on my project. For my master’s thesis I investigated the developmental origins of the morphology of sea biscuits and sand dollars. I established a detailed morphological description from fertilization to late juvenile stages of a tropical sea biscuit species with emphasis on the juvenile patterning. During the project I gained experience with documentation techniques for live samples with light microscopy using photography, video, and timelapse imaging. I also experimented with 3D reconstruction which sparkled my interest for image processing and programming.

    Having a strict morphological background I felt the necessity of expanding my approaches for evo-devo research with molecular techniques and gene expression studies as well as more advanced imaging techniques. In my PhD position at the Hejnol Lab I am working with the evolution of larval body patterns using a comparative approach with non-model marine invertebrates such as bryozoans, brachiopods, and nemerteans. My project includes a survey for segmentation mechanisms in brachiopod larvae and identifying the origin and fate of pluripotent larval tissues in different spiralian species. I am mainly using gene expression patterns of candidate genes and confocal imaging.

    The course “Marine animal models in evolution & development” fits my immediate research interests in several aspects. Because of my inclination for comparative studies developing a solid set of skills for establishing molecular techniques and genomic resources of new organisms is paramount. Getting insights about in situ hybridization and antibody staining as well as the analysis of gene expression patterns and gene regulatory networks will be extremely useful for my current PhD projects. I also have a strong interest for experimenting with more advanced imaging techniques, specially SPIM, since they make a crucial aspect of development accessible to us, the dynamics of ontogenetic processes.

    Other topics offered that I want to incorporate to my research repertoire, but have no previous experience include functional approaches and its many techniques such as microinjection of embryos, gene knockdown, transgenesis, and other reverse genetics methods. Having a primer during the course will certainly be of good value for developing future projects.

    Finally, I am excited to explore the fjord diversity and particularly attracted by the scheduled discussions which will provide grounds and stimuli for new research ideas and positively impact my work. This EMBO Practical Course is definitely aligned with my career goals and is a perfect opportunity to learn and be up-to-date with several state-of-art methods in evo-devo.

    Best regards,

    Bruno

    CV

    Sent this one.

    Referees contacts

    Andreas Hejnol
    Group leader
    Sars International Centre for Marine Molecular Biology
    andreas.hejnol@sars.uib.no
    Tlf (47) 55 58 43 28
    Fax (47) 55 58 43 05

    Alvaro Esteves Migotto
    Professor
    Marine Biology Center of University of São Paulo
    aemigott@usp.br
    Tlf (55 12) 3862 8416
    Fax (55 12) 3862 8454
    I’m searching for developmental data and patterns of marine invertebrates which may add
    new insights on how and why metazoan evolved.

    Registration

    You have successfully submitted an abstract!
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    User-ID: 38
    Password: n9edk9z7

     
  • Bruno Vellutini 20:47 on 2013/05/07 Permalink
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    Selected 12 juveniles ~1 month and incubated with… 

    Selected 12 juveniles ~1 month and incubated with 20 µM EdU from Click-iT Kit for 3h (stock concentration is 10mM, 2µL/1000µL). Relaxed with MgCl2 for 30min in cold room and fixed with 4% PFA for 1h in cold room + 1h at RT. Permeabilized with PBS+Triton-X and stored at 4 °C in PBS.

     
  • Bruno Vellutini 12:54 on 2013/05/06 Permalink
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    Fixing 32d Lineus viridis juveniles 

    After failing to properly relax juveniles yesterday, I did the following.

    1. 45 min in MgCl2 in cold room
    2. 2 min in %2 cystein diluted in MgCl2
    3. 2 washes with MgCl2
    4. 15 min in MgCl2 in cold room
    5. Fix with ice cold 4% PFA in MgCl2

    Standard fixation time ~3h in cold room + ~30 min at RT.

     
  • Bruno Vellutini 18:02 on 2013/05/05 Permalink
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    Fixation of 26d Lineus 

    I had an egg mass with many worms. I tried relaxing for 20 min and they were not contracted, but also not fully extended. I then did a 2min 2% cystein wash followed by a FSW wash to remove the cystein. Worms started to move again, so I re-added MgCl2 and waited 5 min. After adding the fixative they contracted and became small balls :/

     
  • Bruno Vellutini 20:37 on 2013/05/04 Permalink
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    RNA extraction for 2nd series of Priapulus stages and L. longissimus 

    Starting an extraction for another series of Priapulus stages. Using Phase Gel Lock tubes, LPA carrier and overnight precipitation.

    Embryos were at the bottom so it was a little easier to pick, but in the end I had to spin them many times to dry out the collecting tube to 100 µL. Another problem was using 1 mL for separating the phases in the gel lock tubes. They only support 750 µL. For this reason the gel was at the top in most tubes. So I collected the aqueous phase with Chema’s help and mixed an equal volume of chloroform to take the phenol out. Only after this we added the carrier (1 µL) and let it precipitate overnight.

    05/05/13

    Some tubes had a transparent bottom phase. After spinning this unknown droplet trapped the pellet, nothing changed after the ethanol wash. When I added 22 µL of DEPC water the pellet and droplet seem to have dissolved. In the tubes where there was a clear and nice pellet a solid case remained where the pellet was. These are the results of the NanoDrop:

    Sample ID ng/ul 260/280 260/230
    Pc2 oocytes 106,74 1,78 0,41
    Pc2 1d 186,70 1,95 0,38
    Pc2 2d 236,72 2,18 0,50
    Pc2 3d 246,45 2,04 0,50
    Pc2 4d 834,65 1,93 0,43
    Pc2 5d 215,32 2,13 0,43
    Pc2 6d 112,33 1,68 0,39
    Pc2 7d 303,38 1,85 1,37
    Pc2 9d 283,21 2,04 0,55
    Pc2 12d 390,20 1,92 0,52
    Pc2 15d 485,06 1,94 0,96
    Pc2 2nd larvae 131,70 1,74 0,40
    Llon mixed 288,17 1,85 0,59

    Candidates for re-extraction are: oocytes, 7d, 2nd larvae

     
  • Bruno Vellutini 13:11 on 2013/05/03 Permalink
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    Re-extraction of Priapulus stages 

    Re-extracted a couple of samples from Priapulus stages using gel-lock, carrier, and overnight precipitation and here are the curves.

    For the 4d sample the curves are the same, just lower.

    5d sample looks better with a lower 270 and 230 peaks although the 260/280 ratio is the same. 260/230 has improved.

    Concentration was lower in both.

    So, I’ll do the extraction for the second series using the gel-lock, carrier and o/n precipitation. If these curves look much better than the first series than it might be worth to re-extract them all. But maybe not..

     
  • Bruno Vellutini 13:55 on 2013/05/02 Permalink
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    Cystein treatment for 12/04/13 (20d) egg mass. Most embryos were still inside the larval epidermis…

    03/05/13

    Fixed, but some did not look so good.

     
  • Bruno Vellutini 13:29 on 2013/05/01 Permalink
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    Lineus viridis in situ background 

    First in situs in Lineus viridis showed an epithelial background of what it looks like mucus cells:

    Epidermis_insitu

    To avoid this I’m fixing with a cystein treatment pre-fixation in juvenile stages (details later on). However, to be sure that the background cells are the mucus cells and that cystein will work I had a look at some basic descriptions. Below it is a DIC image of the epidermis with some gland cells.

    Epidermis

    Comparing this image with the in situ pictures it looks like the background cells are below the epidermis (dermis) and that these droplets in the epidermis are serous cells of protein content. A diagram from (10.1093/icb/25.1.37) seems to confirm this for heteronemerteans:

    Norenburg1985_integument

    Bibliography

     
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