Development of Planar Cell Polarity in the Mammalian Vestibular Sensory Organs Open Access

Etheredge, Jack Nixon (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/1544bp28w?locale=en
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Abstract

Abstract


Development of Planar Cell Polarity in the Mammalian Vestibular Sensory
Organs
By Jack Nixon Etheredge
Epithelial planar cell polarity (PCP) refers to the coordinated orientation of
neighboring cells, resulting in a polarity axis parallel to the plane of the
epithelium. The vertebrate PCP pathway consists of a set of membrane-
associated proteins, and primary cilia and their associated basal bodies. The
underlying mechanism in vertebrate PCP signaling, however, has yet to be
revealed. Each sensory hair cell of the inner ear contains a polarized hair bundle
comprising an asymmetrically positioned primary cilium, the kinocilium, and rows
of microvilli-derived stereocilia of graded height. Hair cells within each inner ear
organ are oriented coordinately. In particular, the hair cells in the saccule and
utricle show reverse polarity along a line of polarity reversal, providing a unique
opportunity to dissect PCP signaling. To explore the mammalian maculae as
PCP models, we established the timeline for cell proliferation, differentiation, and
hair bundle formation in the mouse maculae. We further analyzed the subcellular
localization of membrane associated PCP proteins, Vang-like 2 (Vangl2) and
Frizzled 3 (Fz3). We found asymmetric and polarized membrane distribution of
Vangl2 and Fz3. Moreover, asymmetric distribution of both Fz3 and Vangl2
precedes formation of stereocilia. Similar to published results of another PCP
protein Pk2, confocal microscopy of embryonic day (E)18 mouse embryos shows
that Vangl2, or Vangl2-GFP, and Fz3 appear to colocalize on one side of the
boundary formed by a sensory hair cell and the adjacent non-sensory supporting
cell in both maculae, independent of the orientation of the hair cells. Adenoviral
transfection of eGFP-Vangl2 fusion protein in cultured utricles and saccules
showed that Vangl2 can be enriched in both the hair cell and the supporting cell
to the same boundary. These observations suggest that PCP proteins are
distributed asymmetrically in both sensory hair cells and their neighboring
supporting cells, and that the polarity of PCP proteins alone is not sufficient to
determine the orientation of hair cells.

Table of Contents

Table of Contents

Chapter 1 General Introduction 1
Planar Cell Polarity 1
Intrinsic Cellular Polarity 2
Invertebrate PCP Signaling 2
Vertebrate PCP Signaling 4
Ciliary Genes and Relative Centriole Orientation in Vertebrate PCP Regulation 5
Development of the Utricle and Saccule 6
Outstanding Issues in Vertebrate PCP Signaling 8
Chapter 2 Development of the utricular and saccular maculae of mouse 14
Introduction 14
Results 15
The saccule and utricle grow in size and change shape during development 15
Wildtype (WT) sensory hair cell morphology and development in mouse utricle and saccule 15
WT intrinsic cell polarization and coordinated polarization occur simultaneously 16
Discussion 16
Chapter 3 Vangl2 is asymmetrically localized in the utricle and saccule 23
Introduction 23
Results 24
Vangl2 is asymmetrically localized in the utricle and saccule 24
Vangl2 is enriched along the medial side of hair cell membranes between hair cells and supporting cells 24
Vangl2 is enriched along supporting cell membranes parallel to the line of reversal 25
Vangl2 is enriched on the medial side of hair cell and supporting cell membranes in single-cell labeling by viral transfection 25
Discussion 26
Chapter 4 Fzd3 is asymmetrically localized in the saccule and utricle 34
Introduction 34
Results 34
Fzd3 is asymmetrically localized in both utricle and saccule. 34
Fzd3 colocalizes frequently with Vangl2-GFP on the apical membrane of supporting cells and hair cells. 35
Fzd3 is enriched at the medial edges of hair cells across both sides of the line of reversal in utricle and saccule 35
Discussion 36
Chapter 5 Overall Conclusions and Future Directions 41
Future directions 43
Chapter 6 Materials and Methods 46
Mouse Strains and animal care 46
Viral Transfection of Organ Culture 46
Whole mount analysis of inner ear tissues 46
Chapter 7 References 48

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