THE ROLE OF UXS1 IN CHONDROCYTE ORGANIZATION,
MORPHOGENESIS, AND SIGNALING PATHWAYS DURING
SKELETOGENESIS
by
GABRIEL A. SMITH
THE POSTLETHWAIT LAB
INSTITUTE OF NEUROSCIENCE
Presented to the Department of Biology and the Honors College
of the University of Oregon in partial fulfillment of the
requirements for the degree of Bachelor of Science
February 2016
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PREFACE
This manuscript is formatted in accordance to the standards for submission
guidelines of the journal Development1. There are a few formatting conventions that
should be highlighted before perusal of this manuscript. Herein, the genes of model
organisms like zebrafish and chicken are in lower-case italics (uxs1). Human genes are
all capitals in italics (UXS1). Functional proteins or enzymes are capitalized with no
italics (Uxs1).
ABSTRACT
The role proteoglycans play in molecular-genetic mechanisms of skeletogenesis
is not completely understood. UDP-glucuronic acid decarboxylase 1 (Uxs1) converts
UDP-glucuronic acid to UDP-xylose which is used by xylosyltransferase 1 to initiate
assembly of a common tetrasaccharide linker critical to the biosynthesis of chondroitin
sulfate, dermatan sulfate, and heparan sulfate proteoglycans, all of which are found in
abundance within the extracellular matrix of cartilage. In this paper, we present two
alleles, hi3357 and mow, that have mutations in uxs1. Zebrafish embryos with uxs1
mutations presented an absence of Alcian staining from cells in the pharyngeal
cartilages. Fluorescent confocal microscopy revealed improper chondrocyte
organization and morphogenesis in cartilage elements of the neurocranium and
pharyngeal skeleton. Additionally, we observed reductions in Alizarin red staining at
endochondral and intramembranous ossification centers indicating improper bone
development. Whole-mount in situ hybridization experiments revealed uxs1 expression
to be ubiquitous in developing embryos until 2 dpf and thereafter localized to the
1
These guidelines can be found online at
http://www.biologists.com/web/submissions/dev_information.html
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pharyngeal skeleton, suggesting a critical role for uxs1 expression during
skeletogenesis. These results suggest that chondrocyte organization and
morphogenesis, endochondral ossification, and intramembranous ossification are all
dependent upon uxs1 function. In mutants homozygous for uxs1hi3357 and uxs1mow, wheat
germ agglutinin staining revealed a reduction in proteoglycans in the extracellular
matrix. Moreover, antibodies against heparan sulfate revealed deletion of heparan
sulfate proteoglycans in the pharyngeal cartilage elements. The deletion of heparan
sulfate proteoglycans in uxs1 mutants suggests that Wnt, Fibroblast growth factor, or
Hedgehog signaling cascades may be disrupted as previously shown to occur in the fruit
fly Drosophila. These findings reveal an absence of UDP-xylose dependent
proteoglycans from the extracellular matrix due to mutations in uxs1. Experiments also
showed a deletion of type II collagen from the extracellular matrix of chondrocytes,
suggesting a role for Uxs1 or proteoglycans in the secretory or localizing mechanisms.
Additionally, expression of col10a1 was absent from endochondral ossification centers
and reduced at intramembranous sites, suggesting that uxs1 is necessary for proper
reciprocal signaling pathways between perichondrial cells and chondrocytes critical for
bone formation. Thus, we conclude that UDP-xylose dependent proteoglycans are
absent in uxs1 mutants which is altering the composition of the extracellular matrix and
disrupting reciprocal signaling between cells. This study provides new insight into the
role proteoglycans play in skeletogenesis and the evolutionarily conserved role of uxs1
across life on earth.
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ACKNOWLEDGEMENTS
Special thanks to: Nathan Tublitz, and Joseph Fracchia for the advising on this
manuscript; Yilin Yan, Xinjun He, Ruth BreMiller, Zac Wood, Amber Starks, and
Amanda Rapp for assistance in this project; Amy Singer and John Postlethwait for
being incredible mentors who have taught me everything I know. This work was
supported by NIH grant: Resources for Teleost Gene Duplicates and Human Disease.
NIH Grant Number: 5R01RR020833-03, UO Grant Fund Number: 212180
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TABLE OF CONTENTS
ii: Preface
ii: Abstract
iv: Acknowledgements
v: Table of Contents
1: Introduction
2: The mechanism of skeletogenesis2 in vertebrates
3: The role of extracellular matrix macromolecules in chondrogenesis
4: Defects in proteoglycan-mediated pathways can lead to serious problems
during embryogenesis and later in life
5: Skeletogenesis and dysgenesis3 are dependent upon molecular-genetic
networks that establish reciprocal signaling pathways
7: The mechanism of proteoglycan biosynthesis
10: Understanding molecular-genetic networks involved in skeletogenesis
12: Approaches to investigate the molecular-genetic defect in mow
13: Results
13: Chondrogenesis and osteogenesis require mow function
17: Identification of the molecular genetic defect of mow
20: Characterization of the molecular genetic defect of mow
23: Conserved syntenies for uxs1 in human and zebrafish genomes
25: The uxs1 expression4 pattern during zebrafish development
27: Craniofacial defects of uxs1mow and uxs1hi3357
31: Both uxs1hi3357 and uxs1mow disrupt type II collagen (Col2a1) localization to
the extracellular matrix
32: Uxs1hi3357 and uxs1mow disrupt col10a1 expression
34: Proteoglycans are reduced in uxs1hi3357 and uxs1mow
2
Skeletogenesis is a term for the process by which cartilage and bone develop in vertebrates to make the
skeleton figure.
3
Skeletal Dysgenesis is the breakdown of skeletal elements later in adult life.
4
Expression is a term used for the transcription of a gene from the DNA template into a messenger RNA
transcript.
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35: Heparan sulfate proteoglycans are absent in homozygous uxs1hi3357 and
uxs1mow
38: Uxs1 disruption does not affect expression of reciprocal signaling molecules
known to be involved in skeletogenesis
39: Uxs1 disruption does not appear to alter expression of runx2 or sox9
40: Expression of uxs1 is not regulated by sox9
41: Discussion
41: Evaluation of the molecular-genetic defect of uxs1hi3357 and uxs1mow
43: Evaluating the neurocranial and pharyngeal skeleton defects in uxs1hi3357 and
uxs1mow
44: The role of uxs1 in proteoglycan biosynthesis and localization
46: The role of uxs1 in skeletogenesis
48: Heparan sulfate proteoglycan deletion in uxs1hi3357 and uxs1mow suggests
evidence for disruption of FGF, Wnt, and Shh signaling cascades
52: The evolutionarily conserved role of proteoglycans and uxs1
53: Experimental Procedures
53: Alcian blue and Alizarin red staining of zebrafish
54: Mapping, cloning, and identification of mow
56: RNA extraction assays
56: RT-PCR protocol for uxs1 message
57: BODIPY®FL C5-ceramide confocal microscopy
58: Tg(fli1:EGFP)y1 and Alizarin red dual-channel confocal microscopy
58: Whole-mount in situ hybridization
59: Whole-mount and dissections of wheat germ agglutinin staining
59: Whole-mount antibody staining
60: References