Our research seeks to investigate the fundamental question of how cardiac cells sense and respond to their environment. Focusing on tissue interactions we seek to understand the mechanisms underlying the regulation of morphogenic and identity transformations that occur during development and disease. We use the assembly of the heart tube in zebrafish as our model with which to elucidate these mechanisms. Some of the specific research questions we are interested in include, but are not restricted to: how multiple tissues interact to regulate large movements? How intercellular adhesions are tuned during collective movements? How lumen formation is intrinsically and extrinsically encoded? and How the plasticity of cardiovascular identity is regulated? These challenging questions require we take an interdisciplinary approach, combining the genetic and imaging strengths of zebrafish with both biomechanics and systems-level methodologies.
Are these the type of difficult challenges that excite you? We are recruiting graduate students to join our laboratory. Contact Josh directly at josh@olemiss.edu.
-More information about the laboratory can also be found at joshuabloomekatz.wordpress.com.
-Additional positions are available in our interdisciplinary graduate program in the department of Biology at the University of Mississippi. For more information about our graduate program including rotations please see biology.olemiss.edu
Applications are invited from highly motivated and enthusiastic individuals for an MRC funded PhD position in the laboratory of Dr. Raman Das at the Faculty of Biology, Medicine and Health at the University of Manchester. This position will commence in September 2018.
The successful candidate will have or expect to obtain a first or upper-second class degree (or equivalent) in the biological sciences and will additionally have a strong interest in cell and developmental biology and in vivo imaging.
This exciting project builds on our recent discovery of a new form of cell sub-division (apical abscission) that regulates shedding of the apical tips of newborn neurons, leading to an acute loss of cell polarity and retention of the centrosome (Das and Storey, Science, 2014). How these neurons re-establish their polarity and subsequently extend an axon in the correct orientation is now a key question in the field. This project will focus on the role of the retained centrosome in re-establishment of polarity in the new-born neuron using a highly interdisciplinary approach integrating pioneering cell and developmental biology techniques. The successful candidate will utilise cutting-edge live-tissue imaging techniques to visualise centrosomal dynamics and microtubule architecture rearrangements during neuronal differentiation in the embryonic spinal cord. This approach will be complemented by super-resolution microscopy to visualise the fine sub-cellular architecture of differentiating neurons.
Overall this project lies at the critical interface between cell and developmental biology and is therefore likely to provide physiologically relevant insights into the molecular mechanisms leading to neuron polarisation and axon extension.
Further details and information on how to apply are available here
Further information about the University of Manchester MRC DTP programme is available here
Deadline for applications: 17th of November 2017
Applications from EU citizens are welcome
Informal enquiries are encouraged and should be directed to Dr. Raman Das at raman.das@manchester.ac.uk.
I was pumped about my project, examining the signaling and physiological role of a novel dopamine receptor complex. We had experienced research associates, state-of-the-art equipment, and bold hypotheses. I was ready to take the first step on the road of biomedical discoveries. Then I faced my first obstacle:
Which antibody should I use for my Western Blot?
It’s one of those things that nobody really teaches you. My lab had well established protocols for many antibody-based applications, ranging from Western Blot, to immunofluorescence, to FRET, but we never had an established “protocol” for finding the most suitable antibodies. Yet, antibodies can often be the main determining factor for successful and reproducible experiments.
In this article, I’d like to outline a 10-step “protocol” as a guide for every antibody search. (Hint: it’s more than just Googling.)
1. Identify target antigen and alternative gene names
Use GeneNames to find out the approved nomenclature for the protein in study, as different proteins may share a common name. Then, use GeneCards to identify aliases to expand the possible search queries, as antibody suppliers may name the same protein using different aliases.
2. Define antigen restrictions
Decide the intended specificity for isoforms, functional domains, processed forms, domains with different subcellular localization (extracellular vs. intracellular etc.) and post-translational modifications of the protein in study.
3. Obtain canonical protein sequence
Use Uniprot to find out the amino acid sequence for the protein (and its isoforms) in study.
4. Determine potential cross-reactivity with other species or proteins
Analyze the sequence using NCBI’s BLAST tool to define whether there are distinct regions of the chosen protein containing linear epitopes that are unique to the individual antigen, as well as regions of the target antigen where antibodies are likely to cross-react with other proteins with which they share sequence identity.
5. Define the ideal epitope(s)
Depending on the study interest, the ideal epitopes could be unique regions in the target antigen conferring specificity or those conferring cross reactivity.
6. Decide applications
Antibodies are often more suitable for specific applications depending on whether they bind to linear (Western Blot and paraffin-embedded immunohistochemistry) or native (immunoprecipitation, frozen-section immunohistochemitry, flow cytometry, and ELISA) epitopes.
In addition, an antibody that recognizes a formalin-resistant epitope for immunohistochemistry may also work in another technique using formalin fixation, such as ChIP.
7. Decide primary antibody isotype and host
When probing with multiple primary antibodies in a single experiment, it may be advantageous to choose antibodies of distinct hosts or isotypes, allowing for detection of multiple targets using isotype- or host-specific secondary reagents conjugated with different fluorescent labels.
Examples include co-localization or protein-interaction studies.
8. Decide clonality of antibody
The clonality of the antibody should be determined based on the intended applications.
Polyclonal antibodies are produced by immunizing host animals with the antigen, and each batch contains a mixture of antibodies targeting various epitopes on the same antigen. As a result, polyclonals can enhance detection signals by enabling more antibodies to bind the same antigen to form large precipitating lattices. However, this lack of specificity limits the use of polyclonals to mainly Western Blot, which allows off target bindings to be distinguished as bands at various molecular weights.
On the other hand, since monoclonal antibodies are identical clones produced from a single hybridoma (uniquely identified by clone ID), they target a single epitope and a single isotype, thereby conferring higher specificity than polyclonals. Monoclonals are ideal for applications using native tissues, such as IP and flow cytometry.
9. Identify antibodies from the literature
Once the desired antigen and antibody characteristics have been defined, conduct extensive literature searches to identify antibodies that have been published in similar experimental contexts of interest (ie. same application, tissue, or cell line).
There are many resources that can help save time on the literature search, including BenchSci.
Review publications and carefully scrutinize antibody usage data. Watch out for antibodies that show discrepancies across the literature, such as an antibody detecting proteins of different molecular weights or showing different protein expression patterns in the same tissue type. If validation data were not presented in the paper, contact the authors to request this information.
10. Prioritize and validate antibody candidates
Generate a list of commercially available antibodies following the literature search. Prioritize the antibodies based on product data sheet to match the antigen and antibody characteristics defined in Step 1-8.
Keep in mind to always perform validation experiments (using knock-out, IP-MS, or CRISPR) prior to applying the antibody in your study.
What are the steps you usually took when searching antibodies? Let me know in the comment section below if I missed anything and I’ll update the “protocol” accordingly.
And to help you specifically with Step 9, why not register a free account on BenchSci to save time looking back and forth between vendor sites and papers?
Postdoctoral Fellowship in the Center for Stem Cell and Organoid Medicine (CuSTOM)
A multidisciplinary team in the laboratory of Dr. Aaron Zorn is seeking to recruit a highly-motivated postdoctoral fellow to spearhead research investigating the molecular and cellular basis of trachea-esophageal birth defects using animal models and human organoids. The goal is to understand how the trachea and esophagus arise from the embryonic foregut during fetal development and to model how genetic mutations in patients can disrupt this process leading to life threatening congenital birth defects.
The Zorn lab (www.cincinnatichildrens.org/zorn-lab) is part of the new Center for Stem Cell and Organoid Medicine (www.cincinnatichildrens.org/custom) and the Division of Developmental Biology at Cincinnati Children’s Hospital, one of the top pediatric research institutions in the world. Qualified applicants will have a PhD with peer-review research publications, a demonstrated expertise in cell biology, morphogenesis or biomechanics, and a keen interest to establish an independent research program in development and stem cell biology.
The Fribourg Graduate School of Life Sciences (FGLS) is an interdisciplinary, international graduate school, which offers a coordinated doctoral program in life sciences at the University of Fribourg, Switzerland. It addresses doctoral students in the fields of biology, biochemistry, molecular medicine, chemistry, physics, bioinformatics, and mathematics who have a life science focus. State-of-the-art theoretical and experimental research will lead to a PhD in science.
Currently, we are recruiting students in the fields of:
Molecular and Behavioral Neurogenetics | Sprecher Lab
We offer an integrated research and training program which leads to a PhD after three to four years. The entire program is run in English. It includes supervision and mentoring, as well as courses on novel technologies and soft skills. We expect applicants to have an excellent university degree and to be motivated and interested in interdisciplinary research subjects. Excellent communication skills in English are of benefit.
Candidates are requested to send by email a SINGLE PDF application including a CV, a brief statement of their research interests and the preferred lab, a copy of their Master diploma (with grades), and names of three referees to
We are seeking a highly motivated and ambitious candidate to join the Semb group as soon as possible upon agreement. The animal technician will focus on the set-up and practical delivery of in-vivo studies.
The position is time-limited to 1 year with a possible extension.
Job description
The animal technician will focus on the set-up and practical delivery of in-vivo studies.
Write and update applications for ethical permissions, breeding licenses and project plans.
Performing in-vivo procedures in-line with ethical permissions e.g. blood samples and testing (blood glucose measurements), injections.
Ensuring that all studies are in full compliance with protocols, policy and practice and scientists expectations.
Animal breeding of transgenic mice, genotyping and interpretation of results.
Working in conjunction with the scientists, lab staff and animal caretakers to ensure efficient scheduling of study related work.
Ordering of animals, lab consumables.
Data reporting.
Other tasks related to the animal facility and laboratory.
The key skills and educational/experience-related criteria for this position are as follows:
An educational background as animal technician or similar.FELASA B Certificate is a must.
Extensive professional hands-on experience gained from in-vivo studies working with rodents.
It is an advantage if you can carry out minor surgical procedures in rodents.
A team-orientated approach, together with excellent interpersonal and communication skills.
Ability to work independent within the Semb group.
High level of attention to detail, conducive to the delivery of timely and accurate data.
Committed to ongoing professional development, including a willingness to acquire and utilize new skills
A good command of English.
Familiar with MS word and Excel.
We offer
stimulating, challenging and multifaceted research environment.
possibility for continued education and training.
attractive employment conditions.
centrally located work place.
Employment conditions
The salary for the position will be according to agreement between the Ministry of Finance and OAO-S and organizational agreement for Lager- og handelsarbejdere I hovedstaden 3F (www.3fkbh.dk). A supplement could be negotiated, dependent on the candidate’s experiences and qualifications.
The application must include:
1. Motivation letter
2. Curriculum vitae incl. education, experience, previous employments, language skills and other relevant skills
3. Copy of diplomas/degree certificate(s)
Questions
For further information about the position please contact professor Henrik Semb (semb@sund.ku.dk).
The application must be submitted in English, by clicking on “Apply online” below. Only online applications will be accepted.
The closing date for applications is 23.59 pm, October 28, 2017.
Interviews will be held concurrently.
The University of Copenhagen wishes to reflect the diversity of society and welcomes applications from all qualified candidates regardless of personal background.
Founded in 1479, the University of Copenhagen is the oldest university in Denmark. It is among the largest universities in Scandinavia and is one of the highest ranking in Europe. The University´s eight faculties include Health Sciences, Humanities, Law, Life Sciences, Pharmaceutical Sciences, Science, Social Sciences and Theology. www.ku.dk
I am looking for talented and driven candidates for a 4yr-PhD programme to join my laboratory at the European Cancer Stem Cell Research Institute at Cardiff University. The studentship is funded by the GW4 BioMed Doctoral Training Partnership of the MRC, starting October 2018.
The successful candidate will have a 1st or 2:1 class degree in Biomedical or Biological Sciences, or a related discipline, and an interest in basic biomedical research and in vivo approaches. Applications from EU citizens are welcome.
The project aims at understanding the molecular mechanisms underlying the chronic radiation injury, which is an important limitation to the efficacy of cancer radiotherapy. The project combines transcriptional profiling, functional assays and microscopy and histology, in collaboration with Dr Pablo Orozco ter Wengel.
For further questions and informal enquiries contact Dr Joaquín de Navascués at deNavascuesJ@cardiff.ac.uk.
For further details of the partnership, including eligibility and expected grades of the candidates, visit the DTP website at http://www.gw4biomed.ac.uk/
I am looking for talented and driven candidates for a 4yr-PhD programme to join my laboratory at the European Cancer Stem Cell Research Institute at Cardiff University. The studentship is funded by the South-West Doctoral Training Partnership of the BBSRC, starting September 2018.
The successful candidate will have a 1st or 2:1 class degree in Biological Sciences or a related discipline, an appetite for technological development as well as an interest in quantitative approaches to stem cell biology. Applications from EU citizens are welcome.
The project aims at understanding how adult stem cells respond to the local needs for cell replacement through lineage tracing, genetic manipulation, confocal microscopy and the development of a new, Gal4-compatible, drug-inducible method for the temporal control of transgene expression in Drosophila. This will be done in collaboration with Prof Helen White-Cooper’s lab in Cardiff and that of Dr Edward Morrissey in Oxford (who will bring in his expertise in mathematical modelling).
For further questions and informal enquiries contact Dr Joaquín de Navascués at deNavascuesJ@cardiff.ac.uk.
For further details of the partnership, including eligibility and expected grades of the candidates, visit the DTP website at http://www.bristol.ac.uk/swbio/
The Journal of Developmental Biology are inviting applications for the 2018 JDB Travel Award. The award is for postdoctoral researchers and PhD students to attend a conference of their choice in 2018).
The award will consist of 800 CHF (Swiss Francs), and nominations must be in by 15/10/17
CTCF binds to chromatin and is thought of as an architectural protein in the genome. If the genome were a text, CTCF would act like the punctuation marks, so that words are grouped together becoming meaningful sentences.
When I started my PhD, the Manzanares lab had been fruitfully collaborating with that of Jose Luis Gómez-Skarmeta at the CABD in Seville for several years in different projects. In one of them, they were studying IrxA gene regulation in the developing embryo, and previous results they had generated suggested that CTCF had a role in the regulation of this cluster. This is a fascinating gene cluster, duplicated during vertebrate evolution, which comprises only three homeobox-containing genes spread over more than one megabase of DNA. It has three genes, the two first (Irx1 and Irx2) showing nearly identical expression patterns, while the third gene, Irx4, shows distinct prominent expression in the heart.
The ideal experiment we first thought of was to delete a particular CTCF binding site. Another option was to prevent CTCF protein from binding there. This could be achieved by using a conditional mouse mutant allele, where Ctcf is flanked by two loxP sites (1), which allows the specific deletion of this gene, in our case using a Cre-driver specific for the developing heart. Other groups had already used this Ctcf floxed line and conditionally deleted the gene in several contexts, such as the developing limb or T-cells, finding that CTFC had roles in processes such as apoptosis or proliferation.
Although we initially aimed to study the function of a particular CTCF binding site, I started characterizing the phenotype derived from the lack of CTCF in the developing heart. Quickly, this experiment acquired life of its own, when we saw that deleting CTCF during heart development led to embryonic death. This highlighted the importance of CTCF in the process, and thus we decided to focus on the overall function of CTCF in the developing heart.
In order to obtain a global view of the effects on gene regulation by Ctcf-deletion we carried out RNA sequencing of control hearts and of other two scenarios: one or two copies of Ctcf deleted. We found that only one copy of Ctcf is enough for correct gene expression and normal heart development (mice reach adulthood with no problems) so we focused on the comparison of gene expression between wild-type and Ctcf homozygous mutant embryonic hearts.
When we carried out functional annotation of the list of approximately two thousand genes with significant changes in expression between wild-types and mutants, the Gene Ontology (GO) term “heart development” stood out in all different GO tools we used. What also caught our attention was that the “heart development” and related GO terms were only present in the down-regulated gene set. To corroborate this finding we analyzed the expression pattern of some of the genes belonging to the “heart development” GO term (such as the master cardiac regulator Nkx2-5) by in situ hybridization, and we could observe clear downregulation of all genes examined. This led us to think that CTCF controlled heart development by directly regulating those genes critical for embryonic patterning.
Our next question was pretty straightforward: what is the relationship between CTCF binding to the genome and our set of differentially expressed genes in the Ctcf knockout? Taking advantage of the previously described genome-wide distribution of enhancers and CTCF binding generated by ChIP-seq in the mouse heart by Bing Ren’s lab, we tried to tackle this question. The analysis showed that both up and down-regulated genes were closer to a CTCF binding site than expected, but that only down-regulated genes were closer to a heart enhancer. This observation suggested that CTCF is acting to bring together gene promoters and developmental enhancers to achieve proper expression in this developing system.
Following this last idea, we wanted to study in more detail if the alteration in transcription of selected genes in the absence of CTCF was due to changes in the 3D chromatin structure. When we were developing this work, chromatin conformation capture techniques (3C) and its derivatives were the way to go to study 3D chromatin structure, so we decided to use this emerging technology to address the issues we had in hand. At this point, two questions came up. First, how do we choose interesting candidates for further studies among 2000 genes. And second, which 3C-derived technique should we use? For the first, we chose Irx4, as it is one of the members of the IrxA gene cluster, on which the Manzanares and Gómez-Skarmeta labs had been already working, and it has an important role in ventricle identity (2,3). Most importantly, we had seen that it was strongly downregulated in our RNA-seq data, had predicted heart enhancers and ChIP-seq CTCF binding sites in its vicinity, and in situ hybridization confirmed its down-regulation in mutant hearts. Regarding the 3C-derived technique to use, we chose 4C-seq because we knew very little about Irx4 regulatory landscape and in this way we could address all the interactions occurring from specific regions in the locus, as this is a “one-versus-all” approach.
Standard 4C-seq protocols are supposed to use 1×107 cells, and this was a challenge given that we planned to carry out the analysis using E10.5 embryonic hearts, that are composed of roughly 30-40 thousand cells. For example, in a previous work showing p300 ChIP-seq in E11.5 embryonic hearts, 270 hearts were used (4). 270!! Obtaining wildtype hearts for setting up the technique was relatively straightforward, but Ctcf heart-specific mutants would only be one fourth of the litters. We managed to obtain reproducible 4Cs using pools of 45-60 for each genotype and replicate, but each pool took 2-3 months and long hours on the dissecting scope to collect. Nevertheless, the help of Claudio Badía-Careaga made this enormous effort, and the project in general, much more bearable.
Our 4C-seq showed that the Irx4 promoter interacted with surrounding CTCF binding sites. One of them was located between Irx1/Irx2 and Irx4, and therefore a prime candidate to be mediating the differences in expression of these two IrxA genes in the developing heart. To see if this site was important for regulating Irx4 in vivo, we deleted it with the new CRISPR-Cas9 system that Isabel Rollan in our lab had been working to set up. We could observe a subtle decrease of Irx4 expression levels in the heart when we compared the deletion mutant to controls. But, we were so focused in heart expression that we almost miss the fact that we had Irx4 ectopic expression. And it was in an Irx1 expression territory!! Thus, this CTCF binding site is crucial for proper Irx4 expression, both by aiding in its regulation by heart specific enhancers, and also by prevents its ectopic expression in territories where Irx1/Irx2 are normally expressed, possibly by not allowing other tissue-specific enhancers to activate the gene.
Top panel, Irx4 expression in the heart. Bottom panel, Irx4 ectopic expression in the oral-esophageal region of foregut, an Irx1 expression territory.
We were very pleased with these results, but we struggled for a while in publishing our data. We presented the work at several meetings and in one of them, somebody asked what happened with the upregulated genes, what are those? And why are there no heart development genes among them? Sure enough, we had looked at them but up to then merely described the functional enrichments. Therefore, we went back and re-checked the GO terms associated with up-regulated genes in Ctcf mutant hearts. Two categories stood out by the number of genes up-regulated: translation and mitochondria. The great majority of ribosomal protein-coding genes from both small and large subunits were among this subset, and more than 300 genes labelled with mitochondrial GO terms were up-regulated. When cardiomyocytes mature, they require two things in abundance: protein to build sarcomeres and energy to contract. Therefore, loss of Ctcf appeared to push cardiomyocytes towards maturation.
Looking at the mitochondrial genes differentially expressed in our RNA-seq, we found both functional and structural genes. We wanted to see if this upregulation led to more mitochondria and if these were functional. This was a new territory for us, and we were extremely lucky that in our same institute we had colleagues that are world- experts in mitochondrial biology. Ana Victoria Lechuga-Vieco, Rocio Nieto-Arellano and Jose Antonio Enriquez guided us through the analysis of the mitochondrial phenotype in our mutants. The RNAseq analysis showed an increase in several OXPHOS (oxidative phosphorylation) system components. We also saw this increase at protein level. However, the assembly of some OXPHOS super-complexes was impaired. One of my favorites results were the pictures Ana Lechuga-Vieco took with TEM (transmission electron microscopy). Not only they were absolutely beautiful and looked like a biology text book, but they showed that loss of CTCF lead to immature and sick-looking mitochondria, and that the sarcomeres assembled earlier than expected in the Ctcf mutant hearts. The 6 month-long wait for the TEM to be available had been worth it! Therefore it appears that despite increased transcription of maturation genes, proper assembly of cellular components does not follow leading to non-viable cardiac cells.
Transmission electron microscopy (TEM) showing swollen and larger mitochondria in the Ctcf KO hearts (Ctcf fl/fl ; Nkx2.5-Cre) in comparison to the control.
We concluded that CTCF was controlling two transcriptional programs in opposite directions, and this dynamic was necessary for proper formation of the heart. It is curious how the attempt to close a story opened a new one. And also, how a fresh look at your data can change the course of the story.
Numbers show part of the CTCF team in Miguel Manzanares lab. 1, Claudio Badía-Careaga. 2, Isabel Rollán. 3, Miguel Manzanares. 4, Melisa Gómez Velázquez. 5, Alba Alvarez
This post was written by Melisa Gómez-Velázquez and Miguel Manzanares
Helen Heath, Claudia Ribeiro de Almeida, Frank Sleutels, Gemma Dingjan, Suzanne van de Nobelen, Iris Jonkers, Kam-Wing Ling, Joost Gribnau, Rainer Renkawitz, Frank Grosveld, Rudi W Hendriks, and Niels Galjart. CTCF regulates cell cycle progression of αβ T cells in the thymus. EMBO J. 2008 Nov 5; 27(21): 2839–2850. doi: 10.1038/emboj.2008.214. Epub 2008 Oct 16.
Matthew J. Blow, David J. McCulley, Zirong Li, Tao Zhang, Jennifer A. Akiyama, Amy Holt, Ingrid Plajzer-Frick, Malak Shoukry, Crystal Wright, Feng Chen, Veena Afzal, James Bristow, Bing Ren, Brian L. Black, Edward M. Rubin, Axel Visel, and Len A. Pennacchio. ChIP-seq Identification of Weakly Conserved Heart Enhancers. Nat Genet. 2010 Sep; 42(9): 806–810. Published online 2010 Aug 22. doi: 10.1038/ng.650.
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