Understanding the Mechanism of Neural Cell Fate Recovery in Inpp5eM2 Mice Mutant Open Access

Abstract

A crucial question in the study of vertebrate development is defining the cellular and molecular mechanisms of how embryonic cell fates are reproducibly patterned. For example, vertebrate hedgehog signaling is critical regulator of embryonic cell fate and patterning. Sonic hedgehog (Shh), one of three vertebrate Hh ligands, controls an essential signaling pathway that regulates ventral neural tube patterning. Previous research indicates that a phosphoinositide 5-phosphatase, Inpp5e, regulates Shh signaling. By using forward genetic screening, we isolated a novel allele of Inpp5e called Inpp5e^M2. Homozygous Inpp5e^M2 mice showed expanded vental neural tube cell fates during embryonic development (embryonic day 9.5 and 10.5). Surprisinly, using immunofluorescence, I found the abnormal patterning of Inpp5e^M2 at E10.5 partly recovered over time by E12.5. Previous work from the lab studied the conditional allele of Arl13b, which associates with Inpp5e, and showed Arl13b mutants can correct neural patterning over time. Furthermore, this "recovery" depends on Gli3, a negative effector of Shh signaling pathway. With accumulating evidence of Inpp5e being an effector of Arl13b, I hypothesize Inpp5e^M2 recovery is also dependent on Gli3 activity. To genetically test this, I aimed to generate double mutant of Inpp5e^M2 ; Gli3^flox. Using the immuno-fluorescent antibody staining at E12.5, we tried to observe whether the double mutant show mispatterning neural tube. Taken together, if the double mutant result shows mispatterning of neural tube, then the recovery of patterning in Inpp5e^M2 is due to Gli3.

Table of Contents

Introduction 1

Materials and Methods 6

Mouse Information 6

PCR (Genotyping) 6

Tamoxifen Injection 7

Mouse Embryo Dissection 7

Mouse Embro Embedding and Sectioning 7

Phenotypic Analysis - Immuno-fluorescent Staining 8

Quantitative Analysis 9

Results 9

Discussion 12

References 1 4

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics Health Sciences, Human Development Biology, General
Keyword	Neural Tube Cilia Recovery Sonic Hedgehog Inpp5e
Committee Chair / Thesis Advisor	Caspary, Tamara, Emory University
Committee Members	Kenney, Anna M, Emory University Corces, Victor, Emory University

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