Welcome

Hey lab!

This is the new lab blog site.  We will give this site a trial run over the 2015 calendar year.  Though it could evolve into something else, my intention is that it serves as a way to interact with the lab, provide some history for new folks to see what we’ve been up to, and provide a context for those away from the lab to see what’s going on.  Blog entries will be open for all lab members and will be (eventually) password protected so that only lab affiliates can access.

What to discuss?  I want to see some brief entries on papers of significant impact, including journal articles discussed at lab meetings.  Hosts for the lab meetings will be expected to make an entry before lab meeting, listing the basic why, how, what of the article and the relationship to our work.  If you read significant articles that you think would be good for lab meeting, subgroup meetings, or general information, it would be great to post here.  In addition, I would like to see the site used during scientific conferences, and possibly after you attend other seminars on campus.  Have other ideas?  Post.

Lastly, I would like to see how we might use this or another site as a WIKI for lab protocols, antibody use, etc.

No worries if this ends up failing.  As always, the goal is better communication and I want to try any tool that improves this.

Keith

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3.11.2016 – Comparative, transcriptome analysis of self-organizing optic tissues

Andrabi, M., Kuraku, S., Takata, N., Sasai, Y., and Love, N.R. (2015). Comparative, transcriptome analysis of self-organizing optic tissues. Sci. Data 2, 150030.

Summary: This paper discusses an RNASeq study of optic organoids, focusing on describing in-depth methods. The goal was to examine transcriptional regulation downstream of Fgf and Wnt signaling during differentiation of neural retina (NR) and retinal-pigmented epithelium (RPE). These pathways were already known to play important roles in NR and RPE development, but transcriptional effects had not been elucidated. Several known targets of Wnt and Fgf and markers of NR and RPE were found to be modulated according to expectations. Furthermore, publication of the RNASeq dataset may lead to discovery of additional targets not previously identified. This could guide developmental studies and lead to further optimization of optic organoid method.

Basic methods: The optic organoid method was the basis of the inner ear organoid method and begins similarly with the dissociation of mouse ES cells and reaggregation in serum-free medium using U-bottom 96-well plates (3000 cells per well) on day 1. Matrigel was added at 4% (v/v) on day 2. (Interestingly, the original method published in 2011 called for 2% Matrigel, but this paper reports that an increased concentration results in a continuous epithelium of retinal precursors (Rx+) rather than a confined, budding optic cup-like structure.)

On day 10, this epithelium was dissected via forceps and either collected for RNASeq or cultured further in a maturation medium. Wnt or Fgf signaling was modulated between day 10 and day 15 using Wnt pathway agonist CHIR99201 (3 uM on day 10, then 1 uM from day 11 on) or Fgf2 (5 ng/mL) + 10% FBS. Media was exchanged on days 11, 12, and 14, and additional samples were collected for RNASeq on days 12 and 15.

Important RNASeq parameters: 3 replicates per sample, 100 bp paired-end reads, RINs > 7, Reference genome mm10

Main experimental questions:

  • Day 10 vs day 12 comparison: What genes change expression levels after stimulation of competent Rx+ tissue with Wnt or Fgf?
  • Day 12 vs day 15 comparison: What genes change expression during maturation?
  • Day 12 Wnt vs day 12 Fgf2 or day 15 Wnt vs day 15 Fgf2: How are transcriptome profiles of RPE- and NR-like tissues different?

Why it’s important: We are planning several RNASeq experiments, and this study is a useful model for our analyses. Since the inner ear organoid method was based on the optic organoid method, it makes sense to refer to this study as a model for RNASeq experiment design in an organoid system. The journal (Scientific Data) seeks to publish datasets and in-depth descriptions of the methods used to generate them in order to improve reproducibility. Therefore, this paper is a good resource as I’m learning to do the bioinformatics to analyze and present the results from our RNASeq experiments. I also think it’s important to note the rationale: They already knew Fgf and Wnt would be important but wanted to know more about downstream transcriptional effects.

 

Andrabi et al 2015 Sci Data

Andrabi et al 2015 Sci Data_supplement

Lab meeting 3.11.2016

01082016 Transplantation of neural stem cells into the modiolus of Mouse cochleae injured by cisplatin

Tetsuya T, Takayuki N, Fukuichiro I, et al. Acta Otolaryngol 2004; Suppl. 551: 65-68.

Summary:

Sound stimulation is adapted by auditory hair cells, converted into electronic stimulation in hair cells and transmitted to Spiral gangolion neurons (SGNs). SGNs are auditory primary neurons in the Rosenthal’s canal in the modiolus of the cochlea.  In humans, degeneration of the auditory nerve in the inner ear is an irreversible which eventually reduces residual hearing in any hearing impaired patient. Cochleae implant (CI) is implantable device designed to stimulate SGNs, it is dependent on the proper transduction of signals and subsequently on the function of the auditory nerve (AN).  The survival of SGNs is therefore a critical issue for maintenance of hearing function, and for obtaining the clinical benefits of CI. For the lesions that primarily affect the AN such as acoustic neuroma, surgery, trauma and auditory neuropahy, CI doesn’t help. Regeneration of SGNs has thus been an important issue for restoration of hearing as their regenerative activity is very limited . New experimental approaches
including cell transplantation are, thus, being pursued to restore hearing .The aim of this study was to examine the potential of cell transplantation for restoration of SGNs.

They use fetal neural stem cells (NSCs) that have the potential for differentiation into neurons as donor cells, and adult mice affected by cisplatin, in which severe degeneration of SGNs is induced as the recipient animals.

Basic methods:

A cisplatin solution (2.5 mg/ml) was injected from the left posterior semicircular canal to deafen the animal. 14 days later the left cochlea of recipient animals was exposed, 2 μl NSC was injected into the cochlea through the round window toward the direction of the cochlear modiolus . 14 days after transplantation, the left cochlea was re-exposed and local perfusion was done, The animals were then sacrificed. The temporal bones were collected . Cryostat sections 10 μm thick were made, and the mid-modiolus sections were used for histological analysis.

The cell fates of grafted NSCs were determined by immunohistochemistry for Tuj 1 (marker for neurons) or GFAP (marker of glial cells). Counterstaining with DAPI. The numbers of transplant-derived cells in the modiolus in one section and the ratios of positivity for each marker in transplant-derived cells were examined. Robust survival of the injected cells was found in cochleae. NSC-derived cells expressing GFP located in the modiolus of cochleae or scala tympani, settled in the apical portion of the modiolus .The mean and standard deviation of numbers of NSC derived cells in the modiolus was 190.5 and 60.8. TuJ1-positive grafted cells were positioned on the periphery of grafted cells, GFAP-positive grafted cells were mainly located in the center of grafted cells.

Why it’s important:

From this paper I get some information about the fate of the fetal NSCs in the modiolus  of the mice and it shows  that TuJ1-positive grafted cells were positioned on the periphery of grafted cells while GFAP-positive grafted cells were mainly located in the center of grafted cells. For my further studies, as I would like to inject the NPCs into the guinea pig’s internal auditory canal bottom, the result of the paper give me inspiration for further study.

lab meeting.ppt

Transplantation of neural stem cells into the modiolus of Mouse cochleae injured by cisplatin

12.11.2015 – Mesenchyme-free culture of explanted embryonic mouse otic vesicle

Miura T, Shiota K, and Morriss-Kay G. 2004. A mesenchyme-free culture system to elucidate the mechanism of otic vesicle morphogenesis. J. Anat. 205 (4): 297-312.  PMID 15447689

Summary:

Culture of isolated otic vesicles (OV) can be used for many purposes, such as studying the effect of surrounding tissue on OV morphogenesis.  However, prior methods retained some mesenchymal cells, which precludes analysis of OV-intrinsic morphogenetic cues.  This paper describes a method for clean isolation of OV and culture in Matrigel, based on one designed for mesenchyme-free culture of lung epithelium (Nogawa and Ito, 1995).  They find that major morphological events still occur—to an extent—in E11.5 mouse OV explants: the cochlear duct elongates and turns, the CVG neuroblasts emigrate, and the semicircular canals emerge and invaginate.

They use this culture system to study several possible factors involved in cochlear and semicircular canal morphogenesis.  When they isolate the cochlear region at E11.5, it forms a semicircular canal-like structure, suggesting that cell fate has not yet been determined.  When they explant CVG and place it alongside the cochlea, it can affect turning and result in formation of an additional cavity from the cochlear epithelium.  By labeling proliferating cells, they find that turning may be driven by uneven proliferation of cells along the inner and outer curvatures of the cochlear duct.  By blocking actin polymerization, they find that contraction of actin fibers is involved in cochlear duct turning but not semicircular canal formation.  Finally, by using an enzyme to degrade extracellular matrix components (hyaluronic acid, chondroitin, and chondroitin sulfates), they show disruption of overall structure.

 

Basic methods:

Mesenchyme-free mouse OV culture in Matrigel: E10.5 or 11.5 OV dissected and treated with dispase to remove residual mesenchyme.  OV placed in 50 uL drop of Matrigel or Type I collagen in tissue culture dish and positioned with tungsten needles before gel solidification.  CVG and growth factor-soaked beads also placed in drop and positioned alongside OV explants.  Cultures maintained up to 72 hours.  Medium: DMEM (high glucose), 1% antibiotic, and 10% FBS (or 0.1% BSA for serum-free condition).

IHC: Paraffin-embedded tissues sectioned at 10 um.  Labels: PCNA (primary antibody for proliferating cell nuclear antigen), Pax2 (cochlear region), Netrin-1 (semicircular canal region), NeuN (differentiated neurons), TUNEL (apoptosis).

 

Why it’s important:

In my inner ear organoid cultures, I have otic vesicle-like structures that develop into hair cell-containing epithelia.  I also have a lot of “other” tissue (including epidermis, adipose, and mesenchyme).  The impact of mesenchyme on development of my OVs is relevant to the design of my experiments.  I am able to isolate my OVs but don’t know what effect this might have on their development; for this reason, I have debated whether to include mesenchyme (and how much) and/or embed in Matrigel.  This study suggests that Matrigel can provide ECM components that are crucial for major events in normal epithelial morphogenesis.  We could use a similar culture design to identify factors we can apply to promote organoid-derived OV development without influence of all the “other.”

 

Lab meeting 12.11.2015 – PPT

Miura_et_al-2004-Journal_of_Anatomy – Paper PDF

Role of membrane cholesterol in exocytosis at the neuromuscular junction: interplay of Ca2+ and ROS

Petrov et al, Role of membrane cholesterol in spontaneous exocytosis at frog neuromuscular synapses: reactive oxygen species-calcium interplay, J Physiol, 2014, 592.22:4995-5009

Summary

Cholesterol is a ubiquitous component of the cell membrane, adjusting membrane fluidity and stiffness and directly interacting with membrane proteins to affect their localization and function.  Petrov et al. sought to explain how cholesterol depletion leads to an increase in exocytosis at the frog neuromuscular junction.  Methyl-beta-cyclodextrin (MBCD) was used to mobilize cholesterol.  Exposure to MBCD caused a rapid increase in reactive oxygen species (ROS) production, which was measured by a variety of fluorescence reporter molecules.  This increase in ROS could be inhibited by pre-treatment with antioxidants and oxidase inhibitors.  At the same time, MBCD increased intracellular calcium.  Several blockers were used to show that this primarily arose from TRPV channel activity, though the blockers used would also inhibit voltage-gated calcium channels.  Block of Ca2+ increase did not affect ROS production, so ROS was not produced via the increased intracellular Ca2+.  It is less clear to me whether the increased calcium can be attributed to ROS vs a direct action on TRPV.  And it is less clear whether TRPV is the sole source of increased calcium flux since the blockers used are also inhibitors of voltage-gated calcium channels.

Why It’s Important

Though there are some open questions about the specificity of blockers used in the study, the primary results are concrete, namely that cholesterol depletion has impact on excitability by activating several processes that are of special interest to our ototoxicity study.  We hypothesize that HPBCD-induced ototoxicity involves activation of stress pathways and modulation of membrane ion channels, culminating in the induction of cell death pathways.  It is unlikely that one specific mechanism is the link between HPBCD injection and OHC death.  This paper highlights the possibility that oxidative stress and calcium influx may play a role.

Link to PDF of paperPetrov_JPhys_2014-MBCD-ROS-antioxidants-synapse

PPT fileLabMeeting_111315

Tfap2a Promotes Specification and Maturation of Neurons in the Inner Ear through Modulation of Bmp, Fgf and Notch Signaling

Summary– Neurons of the statoacoustic ganglion (SAG) have a complex patter of development. This paper focuses on how the transcription factor Tfap2a coordinates multiple signaling pathways to promote neurogenesis of these SAG neurons in the Zebrafish inner ear. Previous research has shown that Fgf initiates formation of SAG progenitors, but high levels of Fgf can halt this process. Notch signaling is also observed to decreases SAG development. Overexpression of Tfap2a caused an increase in neuroblast formation and an increase in the rate of differentiation. A rise in mature neuron formation was also seen, however, neuron death resulted soon after. Knockout of Tfap2a saw a decrease neuroblast formation and a reduced rate of differentiation with fewer than normal mature neurons. Blocking and increasing expression of Fgf and Notch by other means showed similar results to the addition and knockout of Tfap2a. This shows that induction of Tfap2a inhibits Fgf and Notch signaling. It was found that this was due to activating expression of BMP7a, an antagonist to Fgf and Notch. This demonstrates that a balance of BMP7a, Fgf, and Notch signaling is required for sustained accumulation of mature SAG neurons.

Basic Methods– Zebrafish embryos were heat shocked to allow for mis-expression of different genes, mainly Tfap2a . For the loss of function experiments, tfap2a was knocked out through injecting embryos at the 1-cell stage. Mutants were identified by characteristic phenotypes showing expected Mendelian frequencies. Treating embryos with LY411575 blocked Notch signaling and BMP was blocked by treatment with Dorsomorphin. Both in situ hybridization and antibody staining were used for visualization and statistical analysis was based on studet’s t-tests and one-way ANOVA.

Significance– We are currently trying to push our hESCs into an otic placode prior to differentiation into neurons. We are doing this through modulating BMP. This research shows us how different factors such as BMP, FGF, and Notch affect neurogenesis after we induce the placode. It is something to keep in mind while trying to induce the placode and when taking the next step of differentiation. Based upon how the overexpression of neurogenin produces neurons, the effects of BMP, Fgf, and Notch will be important in forming neurons.

Tfap2a Promotes Specification and Maturation of Neurons in the Inner Ear through Modulation of Bmp, Fgf and Notch Signaling

Lab Presentation

Static stretch affects neural stem cell differentiation in an extracellular matrix-dependent manner

Summary: This paper explores the role of static stretch in neural stem cell differentiation. It is well known that the fate of neural stem and progenitor cells (NSPCs) is strongly influenced by substrate stiffness, but the consequences of other mechanical stimuli such as tensile strain (stretch) have not previously been reported. After delivering a 10% static equiaxial stretch to NSPCs, the authors found that such a stretch specifically impacts NSPC differentiation into oligodendrocytes, but not neurons or astrocytes. They reported over a 2.6-fold decrease in O4 positive oligodendrocytes in mouse cortical NSPCs and a 3.2-fold decrease  in O4 positive oligodendrocytes in rat hippocampal NSPCs. Additionally, they found that the stretch-induced reduction in oligodendroctyte differentiation occurred only on laminin coated membranes, leading to further investigation indicating that the change was mediated by alpha6 laminin-binding integrin.

Basic Methods: Using a custom device called the J-Flex, a 10% static equiaxial stretch was applied to laminin (or fibronectin) coated silicone elastomer membranes at the onset of differentiation. Controls were ran on glass, unstretched membrane, and prestretched membrane (meaning the membrane was stretch before applying cells). Markers used to identify oligodendrocyte, neuron, and astrocyte differentiation included O4, MAP2, GFAP, and DCX+.

Why it’s important: The effects of static stretch and substrate stiffness on NSPC differentiation are important to the design of the nanofiber scaffolds in our tissue regeneration experiments. In the development of future scaffolds, consideration should be made for stiffness and possible strain that could occur in vivo.

Static stretch affects neural stem cell differentiation in an extracellular matrix-dependent manner PDF

Powerpoint Presentation

Differential palmitoylation regulates intracellular patterning of SNAP25

SUMMARY:

This paper explores the capacity of palmitoylation to localize the protein SNAP25 between the cellular membrane and intracellular domains, in specifically recycling endosomes (RE) and the trans Golgi network (TGN). SNAP25 is a peripheral membrane protein responsible for regulating exocytosis and belongs to the SNARE family of proteins. With four cysteines, which comprise the active sites for palmitoylation, these researchers manipulated the availability of these sites to palmitoylation and subsequently measured fluorescent expression of intracellular localization. They tested fluorescence after inhibiting protein synthesis in intracellular regions, mutating cysteine residues, and attached motifs outside of Cys-rich domain. They were able to conclude that palmitoylation can dictate patterning of SNAP25 protein movement across intracellular domains. Additionally, they noted that the cysteine-rich domain is “autonomous and sufficient” for inracellular localization.

BASIC METHODS:

Used PC12 cells to modify SNAP25 proteins. Utilized eGFP-tagged SNAP25b proteins after determining equal regional expression between eGFP-SNAP25b proteins and WT SNAP25 proteins. To inhibit protein synthesis, they used Cycloheximide before radiolabelling [30]palmitate and measuring palmitoylation activities of mature proteins. They mutated individual cysteines (85, 88, 90, and 92) with leucine to measure localization with decreased palmitoylation binding sites. To measure effects of motif association to SNAP25, they used a CAAX motif attached after the cysteine-rich domain (1-92). Flueorescent overlap was determined with statistical calculations like quantitative colocalization analysis and triple labelling.

WHY IT’S IMPORTANT:

We are interested in the dynamic action of protein palmitoylation and its specific effects of localizing proteins on the membrane. In our case, we would like to apply this analysis to ion channels in the inner ear hair cells and test how localization may affect the onset of hearing. The next step is applying a similar course of action to the auditory system and more specifically cochlear tissues. This paper also shows good controls as it continues to revisit ideas established early in the experiment, like Golgi localization and increased intracellular localization with cysteine mutation.

Palmitoylation of SNAP25 Paper

Palmitoylation SNAP25 Presentation