Table Of ContentUniversity of Iowa
Iowa Research Online
Theses and Dissertations
Fall 2009
Control of synaptogenesis and dendritic
arborization by the γ-Protocadherin family of
adhesion molecules
Andrew Garrett
University of Iowa
Copyright 2009 Andrew Garrett
This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/362
Recommended Citation
Garrett, Andrew. "Control of synaptogenesis and dendritic arborization by the γ-Protocadherin family of adhesion molecules." PhD
(Doctor of Philosophy) thesis, University of Iowa, 2009.
https://doi.org/10.17077/etd.u3pmq9ss
Follow this and additional works at:https://ir.uiowa.edu/etd
Part of theNeuroscience and Neurobiology Commons
CONTROL OF SYNAPTOGENESIS AND DENDRITIC ARBORIZATION BY THE
γ-PROTOCADHERIN FAMILY OF ADHESION MOLECULES
by
Andrew Garrett
An Abstract
Of a thesis submitted in partial fulfillment of the requirements for the Doctor of
Philosophy degree in Neuroscience in the Graduate College of The University of
Iowa
December, 2009
Thesis Supervisor: Assistant Professor Joshua A. Weiner
1
ABSTRACT
During development, the mammalian nervous system wires into a precise
network of unrivaled complexity. The formation of this network is regulated by an
assortment of molecular cues, both secreted molecules and cell-surface proteins.
The γ-Protocadherins (γ-Pcdhs) are particularly good candidates for involvement
in these processes. This family of adhesion molecules consists of 22 members,
each with diverse extracellular adhesive domains and shared cytoplasmic
domains. Thus, cellular interactions with varied adhesive partners can trigger
common cytoplasmic responses. Here we investigated the functions of the γ-
Pcdhs in two processes involved in neural network formation: dendrite
arborization and synaptogenesis.
We first asked how γ-Pcdhs regulate synaptogenesis in the spinal cord.
We found that the γ-Pcdhs are differentially expressed by astrocytes as well as
neurons. In astrocytes, the proteins localize to perisynaptic processes where
they can mediate contacts between neurons and astrocytes. In an in vitro co-
culture system in which either only astrocytes or only neurons were null for the γ-
Pcdhs, we found that astrocytic γ-Pcdh is required for an early stage of
synaptogenesis in a contact-dependent manner, while neuronal γ-Pcdh is
sufficient for later stages. Conversely, if neurons lacked the adhesion molecules,
very few synaptic contacts formed at all. By deleting the γ-Pcdhs from astrocytes
in vivo, we demonstrated that these contacts are required for the normal
progression of synaptogenesis.
We also investigated the function of the γ-Pcdhs in the cerebral cortex. We
found that cortical-restricted loss of the adhesion molecules resulted in a severe
reduction in thickness of layer 1. By crossing the mutant mice to a line in which
scattered layer 5 neurons express YFP, we saw that this thinning resulted from a
reduced complexity in the apical tufts of dendrites from layer 5 neurons. Sholl
2
analysis demonstrated that the arbor reduction existed throughout the cell, a
phenotype that was recapitulated in vitro. Using the in vitro system, we found that
the arborization defect was caused by hyperphosphorylation of the PKC
substrate, MARCKS, indicating that the γ-Pcdhs may function by inhibiting PKC
activity. Thus, we provide new information about the mechanisms through which
the γ-Pcdhs influence neural network development.
Abstract Approved: _____________________________________
Thesis Supervisor
_____________________________________
Title and Department
_____________________________________
Date
CONTROL OF SYNAPTOGENESIS AND DENDRITIC ARBORIZATION BY THE
γ-PROTOCADHERIN FAMILY OF ADHESION MOLECULES
by
Andrew Garrett
A thesis submitted in partial fulfillment of the requirements for the Doctor of
Philosophy degree in Neuroscience in the Graduate College of The University of
Iowa
December, 2009
Thesis Supervisor: Assistant Professor Joshua A. Weiner
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
PH.D. THESIS
This is to certify that the Ph.D. thesis of
Andrew Garrett
has been approved by the Examining Committee for the thesis
requirement for the Doctor of Philosophy degree in Neuroscience at the
December 2009 graduation.
Thesis Committee:
Joshua Weiner, Thesis Supervisor
Kevin Campbell
Michael Dailey
Steven Green
Jim Lin
To Stephanie and Violet
ii
Reasonʼs last step is the recognition that there are an infinite number of things
which are beyond it. It is merely feeble if it does not go as far as to realize that.
− Blaise Pascal
iii
ACKNOWLEDGEMENTS
I would like to thank my mentor, Dr. Joshua Weiner, first and foremost.
Anything worthwhile that I know to do as a scientist I learned from him; any
shortcomings are from my own failure to pay attention. Working with Dr. Weiner
has been a pleasure that I would recommend to anyone. I would also like to
thank Leah Fuller and Claire Vernon for assistance in several of the experiments
described in this thesis, as well as their invaluable work maintaining the mouse
colony. To Dr. Dietmar Schreiner and the other graduate students from the
Weiner lab: Tuhina Prasad, Karry Jannie, Lindsey Helsper, and Mark Lobas,
thank you for all of the help along the way and for making the lab a great place to
work. Thanks also to Dr. Michael Dailey for always being willing to spare a few
minutes and expertise, and to the rest of my thesis committee, Drs. Jim Lin,
Kevin Campbell, and Steven Green for their helpful comments and for taking the
time to serve on my committee.
Finally, I would like to thank all of my friends and family for supporting my
efforts throughout graduate school. Particular thanks are due to my wife,
Stephanie, for her patience, strength, and understanding through the whole
process, and to my daughter, Violet, for being beautiful. Thank you for making it
easy to make my family my greatest success.
iv
ABSTRACT
During development, the mammalian nervous system wires into a precise
network of unrivaled complexity. The formation of this network is regulated by an
assortment of molecular cues, both secreted molecules and cell-surface proteins.
The γ-Protocadherins (γ-Pcdhs) are particularly good candidates for involvement
in these processes. This family of adhesion molecules consists of 22 members,
each with diverse extracellular adhesive domains and shared cytoplasmic
domains. Thus, cellular interactions with varied adhesive partners can trigger
common cytoplasmic responses. Here we investigated the functions of the γ-
Pcdhs in two processes involved in neural network formation: dendrite
arborization and synaptogenesis.
We first asked how γ-Pcdhs regulate synaptogenesis in the spinal cord.
We found that the γ-Pcdhs are differentially expressed by astrocytes as well as
neurons. In astrocytes, the proteins localize to perisynaptic processes where
they can mediate contacts between neurons and astrocytes. In an in vitro co-
culture system in which either only astrocytes or only neurons were null for the γ-
Pcdhs, we found that astrocytic γ-Pcdh is required for an early stage of
synaptogenesis in a contact-dependent manner, while neuronal γ-Pcdh is
sufficient for later stages. Conversely, if neurons lacked the adhesion molecules,
very few synaptic contacts formed at all. By deleting the γ-Pcdhs from astrocytes
in vivo, we demonstrated that these contacts are required for the normal
progression of synaptogenesis.
We also investigated the function of the γ-Pcdhs in the cerebral cortex. We
found that cortical-restricted loss of the adhesion molecules resulted in a severe
reduction in thickness of layer 1. By crossing the mutant mice to a line in which
scattered layer 5 neurons express YFP, we saw that this thinning resulted from a
v
Description:Page 1 the arborization defect was caused by hyperphosphorylation of the PKC substrate, MARCKS . APPENDIX B: PCR PRIMER SEQUENCES .
