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

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