Ciliated epithelium timecourse

Differentiation of basal cells to tracheal ciliated cells

Time course ID: mouse_Tracheal_epithelial_cells
Sample provider:  Mitsuru Morimoto

Introduction

The vertebrate cilium is a sensory and mechanical organelle critical for a broad array of homeostatic mechanisms. Although such a biological importance of ciliogenesis is well investigated, molecular processes of ciliogenesis has been remained in mystery. Notch signaling plays a crucial role in alternative ciliated or secretory cell fate determination from bi-potential progenitor in developing lung epithelium. Using mouse tracheal epithelial primary culture cells (mTECs) that allow us to examine airway mucociliary epithelial cell differentiation from its progenitor cells in in vitro, we successfully established high-efficient ciliated cell induction culture system with pharmacological inhibition of Notch signaling. Taking advantage of this culture system, we harvested time-course samples from differentiating ciliated cells at 18 time points after the differentiation induction and harvested total RNA from these.

Samples

The detailed protocol for the mTECs culture has been published by Vladar and Brody, 2013. mTECs are isolated from the tracheas removed from female mice between 12~20 weeks of age. mTECs were cultured on 12-well plates, 1.00 x 105 /well, containing Transwell permeable membrane supports (Corning). Within 5 days, the cells become confluent and differentiation was induced by removing serum and growth factors from the medium. For hyper induction of ciliated cells, 10 uM of gamma secretase “DAPT” was added into the medium. At the time points after differentiation induction, total RNA was extracted by lysing mTECs with QIAzol Lysis Reagent and purified by the miRNeasy kit (QIAGEN).

Quality control

Known marker genes: FoxJ1 [2] and Mcidas [3].

Figure 1: CAGE expression of marker genes in TPM.

References

[1] Vladar EK and Brody SL, Analysis of ciliogenesis in primary culture mouse tracheal epithelial cells. Methods Enzymol. 2013;525:285-309.

[2] You Y, Huang T, Richer EJ, Schmidt JE, Zabner J, Borok Z, Brody SL. Role of f-box factor foxj1 in differentiation of ciliated airway epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2004 Apr;286(4):L650-7. Epub 2003 Jun 20.

[3] Stubbs JL, Vladar EK, Axelrod JD, Kintner C. Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation. Nat Cell Biol. 2012 Jan 8;14(2):140-7. doi: 10.1038/ncb2406.

Beginning of non-public section

...

Zenbu configurations and status

• Ciliated epithelium differentiation

Expression profiles

• Gene and CAGE cluster expression for the Ciliated epithelium differentiation series
  • https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/mouse_Tracheal_epithelial_cells/expression_tables/

MARA based network results

• https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/mouse_Tracheal_epithelial_cells/mara/

Related samples

• ..

References

• ..

Quality control

https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/mouse_Tracheal_epithelial_cells/

Marker gene expression

• ..

ISMARA analysis results

All samples: http://ismara.unibas.ch/timecourses/Ciliatedepdiff/ismara_report/

Replicate averaged: http://ismara.unibas.ch/timecourses/ciliated-avgd/averaged_report/index.html

For more information, see ISMARA.