-UNIVERstry'OFHAWAl'iLiB-RARY · phenotypic features offrontonasal dysplasia (FND) and multicOystic...
Transcript of -UNIVERstry'OFHAWAl'iLiB-RARY · phenotypic features offrontonasal dysplasia (FND) and multicOystic...
-UNIVERstry'OF HAWAl'i LiB-RARY
EXPRESSION OF SIX3, ZFP161 AND ALK GENES IN THE BRAIN AND
KIDNEYS OF 3H1 Br MUTANT MICE
A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY
OF HAWAI'IIN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF
MASTER OF SCIENCE
IN
BIOMEDICAL SCIENCES (CELL AND MOLECULAR BIOLOGY)
DECEMBER 2002
By
Edith Margaryan
Thesis Committee:
Scott Lozanoff
Andre Theriault
John Hardman
ACKNOWLEDGEMENTS
This study was curried out at the Department of Anatomy and
Reproductive Biology, University of Hawaii School of Medicine, between 2000
2002. I would like to thank all those who have contributed to this work, in
particularily:
My supervisor Dr S. Lozanoff, who gave me an opportunity to work on this
project, encouraged an independent work and generously supported my
research interests and activities, including participation and presentation of my
work in the scientific conference.
My committee member and also former supervisor, Dr A. Theriault, who very
cordially, enthusiastically and patiently helped me in the beginning of my carrier;
encouraged and trained to extensive skills, valuable in research.
He also provided me with technical help and advice in my current work.
My committee member Dr J. Hardman, for his advice and instructional support in
the pathology sessions.
Dr C. Jourdan - Le Saux and for her advice and help in the genetic experiments;
Oana Bollt for her instrumental and courageous help.
Dr G. Bryant-Greenwood and her group for their hospitality and instructive
support; Kristie Okasaki, Sandy Yamamoto and Lisa Webster, for their technical
help and advise.
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My team member Mari Kuroyama, for her invaluable assistance and support.
Allison Richards, for kind support and help.
Drs C. Boyd and K. Csiszar, for their considerate support and participation in my
carrier; Keith Fong and Tongyu Cao, for their help in my work.
The experiments were supported by HCF20012653 to Scott Lozanoff.
IV
ABSTRACT
A newly identified mouse mutation, called Br, displ~ys heritable
phenotypic features of frontonasal dysplasia (FND) and multicOystic renal
hypodysplasia (MRHD). The Br mutation is mapped to distal murine
chromosome 17, but the causative gene remains unknown. The objective of this
project was to analyze expression of three positional candidate genes, Six3,
Zfp161 and ALK, in their target tissues. Two of these genes, Six3 and the
putative mouse TGIF homologue, Zfp161, are candidates for the
neurodevelopmental disorder, holoprosencephaly (HPE). Previous reports
indicate a possible pathogenic relationship between HPE and FND, implicating
HPE candidate genes in development of FND. The third gene of interest, ALK, is
a neural developmental gene, also involved in renal morphogenesis. Embryos
were collected on gestational day (GD) 18, and brain and kidney tissues were
extracted. RT-PCR experiments were performed and expression was visualized
with ethidium bromide staining. All three genes were found to be expressed in all
examined tissues. Expression of the Six3 gene in the kidney was not previously
described in the literature. This implies a novel role of Six3 in the renal
development. Detailed mutational analysis of identified Six3 expression will be
useful for evaluating its role in Br mutation.
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TABLE OF CONTENTS
Acknowledgements iii
Abstract v
List of Tables ix
List of Figures x
List of Abbreviations xi
Introduction 1
Craniofacial Morphogenesis 2
Pathogenesis of HPE and FND 3
FND in Br Mice 4
Genetic Mechanisms of FND 5
HPE Candidate Genes and FND 5
Renal Morphogenesis 6
Pathogenesis of Renal Cystic Hypodysplasia 7
MRHD in BrMice 8
Genetic Mechanisms of MRHD 8
Purpose 10
Part I. In Silico Analysis of Br Candidate Genes 13
Methods 13
Candidate Gene Search 13
Results and Discussion 14
ALK 14
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Zfp161 14
Six3 15
Part II. Expression of BrCandidate Genes in Target Tissues 17
Methods 17
Animals and Tissue Collection 17
RNA Extraction 18
Reverse Transcription 19
Primer Design for PCR 20
PCR Amplification 20
Statistical Analysis 21
Results 23
Control Experiment 23
RNA Preparation and Gene Expression 23
Gene Identity 24
Experiment using 3H1 +1+, Brl+ and BrlBr Mice 25
RNA Preparation and Statistical Data 25
Expression of Genes , 26
ALK 26
Zfp161 26
Six3 27
Discussion 27
ALK 27
Zfp161 28
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Six3 29
Conclusion 31
Appendix A: Tables 32
Appendix B: Figures 37
References 43
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LIST OF TABLES
Table Page
1. Key Developmental Genes Associated with Renal Cystic Hypodysplasia 32
2. Candidate Genes within the Br Locus 34
3. i. Characterization of Experimental Genes in NCBI Database 35
ii. Experimental Primers 35
iii. GAPDH Control Gene and Primer 35
4. RNA Analysis and RT-PCR Results 36
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LIST OF FIGURES
Figure
1. Sequence Alignment of the Amplified Target with Mouse ALK cDNA 37
2. Sequence Alignment of the Amplified Target with Mouse Zfp161 cDNA ......38
3. Sequence Alignment of the Amplified Target with Mouse Six3 cDNA 39
4. Sequence Alignment of Mouse Six3 and Six2 cDNA .40
5. RT-PCR Expression of ALK, Zfp161 and Six3 Genes in the Kidneyof 3H1+1+ mouse. Control Experiment. .41
6. RT-PCR Expression of ALK, Zfp161 and Six3 Genes in the Brainand Kidney of 3H1 +1+, Brl+ and BrlBr Mice .42
x
LIST OF ABBREVIATIONS
aa amino acid
ALK Anaplastic lymphoma kinase gene
Bcl2 B-cell lymphoma 2 gene
bHLH basic helix loop helix
BLAST Basic Local Alignment Search Tool
BMP Bone morphogenic protein
BOR Branchio-oto-renal (syndrome)
bp base pair
Br Brachyrrhine
cDNA complementary DNA
Chr chromosome
cM centiMorgan
c-myc v-Myc avian myelocytomatosis viraloncogene homologue
c-ret proto-oncogene tyrosine-protein kinasereceptor ret precursor
DEPC diethyl pyrocarbonate
DI de ionized
DNA deoxyribonucleic acid
DNAase deoxyribonuclease
dNTP deoxynucleotyde triphosphate
DTT dithiothreitol
Xl
FGFB
FND
FNP and MXP
GAPDH
GD and E
GDNF
Grg5
HPE2; HPE4
MET
MM
MRHD
mRNA
NCBI
n-Myc
OD
Pax
PBS
PCR
PKD
RAR
Fibroblast growth factor 8
frontonasal dysplasia
frontonasal and maxillary processes
Gglyceraldehyde3-phosphatedehydrogenase
gestational and embryonic days
Glial cell line-derived neurotrophicfactor
Groucho-related gene protein 5
Holoprosencephaly type 2 and 4
mesenchymal-epithelial transformation
metanephric mesenchyme
multicystic renal hypodysplasia
messenger RNA
National Center for BiotechnologyInformation
avian v-myc myelocytomatosis viralrelated oncogene, neuroblastomaderived
optical density
Paired box
phosphate buffered saline
polymerase chain reaction
polycystic kidney disease
Retinoic acid receptor
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ret9, ret51
retk
RNA
RNAase
rpm
RT
RT-PCR
Shh
Ski
TGF-B1
TGIF
Tm
TSG taq
us
VUR
Wnt
WT1
Zf5
Zfp
Zic 2
c- ret gene isoforms
receptor tyrosine kinase encoded by cret
ribonucleic acid
ribonuclease
revolutions per minute
reverse transcription
reverse transcriptase polymerase chainreaction
Sonic hedgehog
Sloan Kettering Institute gene
Transforming growth factor-B1
Transforming growth interacting factor
melting temperature
Thermus sp thermis aquaticus(thermostable DNA polimerase)
ureteric bud
vesicoureteral reflux
Wingless gene
Wilms tumor gene 1
Zinc finger gene 5
Zinc finger protein
Zinc finger protein homologous toDrosophila odd-paired
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INTRODUCTION
Br is inherited as a semidominant lethal mutation with phenotypic features
of midline craniofacial defects and renal dysplasia (Ma and Lozanoff, 1993;
Lozanoff et aL, 2001; McBratney et aL, 2002). Newborn 3H1 BrlBr mice display
typical features of FND, characterized by widely set eyes (hypertelorism) and
deficiencies of midline structures, including agenesis of the nasal septum,
absence of the presphenoid bone and a midline facial cleft. Adult 3H1 Brl+
heterozygotic mice develop midfacial retrognathia and a short anterior cranial
base arising from deficient cellular proliferation (Ma and Lozanoff, 1999; 2002).
Therefore, the Br craniofacial phenotype displays three non-overlapping
phenotypes including median facial cleft 3H1 BrlBr, midfacial retrognathia (3H1
Brl+) and wild type mice showing normal facial prognathism (3H1 +1+);
(McBratney et aL, 2002).
Associated with the 3H1 Brcraniofacial dysmorphology, renal
malformations also occur (Lozanoff et aL, 2001). The homozygous mutants
display severe multicystic renal hypodysplasia (MRHD). The kidneys of these
mutant mice are hypoplastic with defects in the collecting system. The
heterozygotes also show MRHD, but the phenotype is less severe than the 3H1
BrlBr condition while functional deficiencies are variable in penetrance compared
to the normal condition (unpublished data). Thus, the two target tissues affected
by Br include the craniofacial region and the kidney. An understanding of the
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craniofacial and renal morphogenesis is critical for analyzing and interpreting
expression of Br candidate genes in these target tissues.
Craniofacial Morphogenesis
Craniofacial pattern is established during early embryogenesis. During
neurulation, the neural plate, derived from the primitive ectoderm, folds into the
neural tube, establishing anteroposterior polarity of the embryo. With successful
closure of the neural tube, the anterior end, or rostrum, develops three out
pouchings (vesicles) which distinguish the territory for the forebrain
(prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon)
(Nanni et aI., 2000). Concomitant with neurulation, the mesoderm becomes
separated into notochord and paraxial mesoderm. Paraxial mesoderm gives rise
to the frontonasal prominence and bones of the skull base posterior to sella
turcica. Cranial neural crest cells, originating in the dorsal lip of the neural tube,
migrate to pharyngeal arches, proliferate, differentiate, and give rise to facial
structures (Couly and Douarin, 1990; Hall, 1987; Noden, 1988, 1991; Osumi
Yamashita, 1994; Tyler and Hall, 1977). The first pharyngeal arch forms the
maxillary and mandibular prominences, bones of the skull base in the skull vault
and anterior to sella turcica; cartilages and connective tissue. Maxillary and
mandibular prominences contribute to the upper and lower jaw, while medial and
lateral nasal processes, derived from the frontonasal prominence, contribute to
the medial part of the face and nose. Abnormal migration, proliferation,
differentiation or mesenchymal-epithelial interaction of the neural crest and/or
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paraxial mesenchyme may disrupt development of these structures and lead to
development of facial hypoplasia and clefts. Two craniofacial diseases that
result from abnormal growth of the midline structures are HPE and FND.
Pathogenesis of HPE and FND
The character of facial dysmorphology and clefting, occurring in HPE and
FND, is developmental stage dependent. During late neurulation, when dorso
ventral patterning occurs, the prosencephalon divides into diencephalon and
telencephalon, and the cerebral hemispheres arise as bilateral evaginations of
the lateral wall of the telencephalon. Optic vesicles arise laterally, as outgrowths,
before closure of the anterior neuropore. Epithelial tissues invade brain,
proliferate and separate two hemispheres, positioning eyes widely on the lateral
sites of the face. If cleavage fails to occur, the brain lobes fuse and eyes
become positioned closer to midline (hypotelorism) or, as extreme, converge
medially into one (cyclopia). In addition to this, a nose-like structure (proboscis)
forms above the eye, and maxillary and mandibular bones become hypoplastic,
resulting in the midline cleft. This phenotype is typical of HPE and it is
associated with many developmental genes. One of these, Sonic Hedgehog
(Shh), is considered to be the main candidate gene for HPE (Belloni et aI., 1996).
Shh is first expressed in the axial mesendoderm (Shimamura et aI., 1995), and
its mutations in the mouse lead to abnormal patterning of the neural plate
resulting in HPE and cyclopia (Chiang et aI., 1996; Cohen and Sulik, 1992;
Hammerschmidt et aI., 1997; Roessler et aI., 1996). Later Shh is expressed in
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the ectoderm of frontonasal and maxillary processes (FNP and MXP) (Helms et
aI., 1997; Wall and Hogan, 1995) and plays an important role in the
morphogenesis of facial structures. Experiments on chick embryos show that
deficiency of Shh causes HPE with narrowing of the frontonasal prominence,
hypotelorism and primary midline cleft. Conversely, excess Shh leads to
mediolateral widening of the frontonasal prominence, hypertelorism (Hu and
Helms, 1999) and formation of the secondary midline cleft (Gorlin et aI., 1990;
Vermeij-Keers et aI., 1984); features characteristic of FND.
After cleavage of the brain, frontonasal and maxillary prominences
proliferate and differentiate into midfacial structures. During embryonic week 8
12 in human (GO 11-14 in mice), the nasal septum forms and the eyes become
positioned closer to the midline. Abnormal growth and differentiation of midline
structures at this stage can result in hypertelorism and midline hypoplasia,
characterized by secondary cleft palate and deficiency of bone and cartilaginous
elements (Vermeij-Keers et aI., 1984; Kjaer et aI., 2002). These elements
include nasal septum and presphenoid bone in the skull base and frontal bones
in the cranial vault. Dysplasia of the frontal bones results in the frontal bossing
(anterior cranium occultum), which, in association with nasal malformations and
other midline defects, comprise FND.
FND in Br Mice
FND, also known as Median Cleft Face Syndrome, is a very rare disorder,
characterized by hypertelorism, frontal bossing, flat broad nose, and/or a vertical
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groove down the middle of the face. All these features, in addition to occasional
eye malformations, are present in 3H 1 BrlBr mutant mice (McBratney et aI.,
2002). The brain in these mice displays two discrete hemispheres and does not
demonstrate HPE (unpublished observations). Thus disruption of genes
regulating formation of midline craniofacial structures, but not the brain, appears
to occur in conjunction with the Br mutation.
Genetic Mechanisms of FND
Interestingly, HPE and FND have been viewed as separate clinical entities
since certain phenotypic features appear as opposite extremes. For example,
eye position is in contrasting positions (hypotelorism versus hypertelorism); HPE
typically involves brain structures while FND does not. However, certain
similarities also exist such as median facial clefting and nasal dysgenesis or
agenesis. Based on experimental data indicating Shh overexpression in chick
embryos causes FND, mutant genes may functionally upregulate expression of
Shh in FND, and Shh deficiency/downregulation causes HPE, it can be
hypothesized that FND is related to HPE, and they both represent opposite
extremes along a spectrum of the same developmental disorder. Therefore,
HPE causative genes may also playa role in the development of FND.
HPE Candidate Genes and FND
HPE is one of the most common of all congenital neurological diseases in
humans (Muenke and Beachy, 2000). As a result, many recent advances have
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been made regarding gene mutations in HPE due to the availability of human
pedigrees. FND on the contrary is a very rare disease, making pedigree
identification impossible and virtually no information exists concerning candidate
genes for this disease. Four primary genes have been reported to be associated
with HPE. These include Shh, Zic2, Six3, and TGIF (Muenke and Beachy, 2000;
Nanni et aI., 1999; Brown et aI., 1998; Wallis et aI., 1999; Gripp, 2000). Two of
these genes, Six3 and TGIF, are cosyntenic in the mouse and are in close
proximity of Bron the distal end of chromosome 17. Thus it is tempting to
speculate that these genes are candidates for Br. Alternately, Br may be a
different gene but affected by either of these two genes resulting in the
development of FND in the 3H1 mouse strain. The long-term aim of this
research is to eventually sequence Br and determine how these genes contribute
to the morphogensis of FND in 3H1 mice.
Renal Morphogenesis
Mouse kidneys develop from intermediate mesoderm through the
formation of a rudimentary pronephros, transitory mesonephros and permanent
metanephros (reviewed by Horster et aI., 1999; Schedl and Hastie, 2000).
Development of the metanephros in the mouse begins at GD 10.5 with outgrowth
of the ureteric bud (UB) from the mesonephric duct. After invasion into the
metanephrogenic blastema, UB branches and induces condensation of
surrounding loose undifferentiated mesenchymal cells. Signals from the
metanephric mesenchyme (MM) induce further dichotomic branching of UB and
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formation of primary collecting tubules. Upon initial condensation, MM generates
peritubular aggregates, undergoes mesenchymal-epithelial transformation (MET)
and forms comma and S-shaped bodies. The proximal portion of S-shape
bodies develops into glomeruli and proximal tubules; distal portions give rise to
the loop of Henle and distal convoluted tubules. On the opposite side, primary
collecting tubules, derived from UB, branch extensively and differentiate into
collecting ducts. Upon interaction with distal part of S body, they fuse with distal
tubules and form a functional nephron.
Pathogenesis of Renal Cystic Hypodysplasia
Correct fusion of the distal and proximal portions of the nephron is critical
for proper renal development (Gloor and Torres, 1999; Pope et aI., 1999). In the
case of malfusion, excrectory obstruction and consequent vesicoureteral reflux
(VUR) cause tubules to dilate and form cysts. Genetic mechanisms underlying
disjunction remain largely unknown. Directly, or through subsequent increase of
intrarenal pressure and toxic effects of refluxed urine, this type of malformation
interferes with epithelial maturation of the tubules and differentiation of
glomerular structures. A reduction of the number of developing glomeruli and
functional nephrons (oligonephronia), disorganization of renal parenchyma
(dysplasia) and retardation of the renal growth (hypoplasia) results. Congenital
obstructive nephropathy develops into a multicystic hypodysplastic phenotype
that properly describes the kidneys of Brmice (Lozanoff et aI., 2001).
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Multicystic renal hypodysplasia (MRHD) in Br mice
Multicystic renal dysplasia is the most common form of cystic disease in
infants (Glassberg et aL, 1987). Type liB cystic disease, described by Potter
(1982), is characterized by small kidneys, multiple cysts, primitive ducts and
sparsely present mature glomeruli. MRHD in Br is inherited as an autosomal
semidominant trait (McBratney et aL, 2002). Homozygotes die at birth from
severe hypodysplasia and acute renal failure. Heterozygotes survive and
develop a cystic phenotype and chronic renal disease. Data suggest that the
nephrogenic defect occurs in Brat midgestation (GD12-14), when MM forms
peritubular aggregates and undergoes MET. Histological sections show initial
MM condensations around UB tips. After E12, fewer branches of the UB develop
and fewer aggregates and S shaped bodies form. Neonatal kidneys have small
size, multicystic tubular dysmorphology, and reduced numbers of glomeruli and
nephrons. Renal parenchyma is dysplastic, filled with immature collecting ducts
and interspersed with loose undifferentiated mesenchyme. Preliminary
morphological analysis suggests that aberrant signal(s), mediating MM
differentiation into epithelium, initiates dysplastic changes in Br mice and leads to
development of obstructive nephropathy and multicystic hypodysplasia.
Genetic Mechanisms of MRHD
Pax2, BCL2, WNT4 and BMP? and n-Myc genes are involved in epithelial
differentiation of MM aggregates, as well as formation of comma and S shape
bodies (reviewed in Piscione et aL, 2000; Horster et aL, 1999). GDNF, c-ret and
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RARa /f3 genes are also expressed in the developing kidneys, and they are
essential for branching morphogenesis of US. Pattern of expression of these
groups of genes is tightly regulated and, when disturbed, leads to development of
multicystic renal hypodysplasia (Table 1). Thus MRHD mechanisms may include
aberrant MET, US branching or connection of S-bodies to US.
Abnormal fusion of the S-bodies with US derivatives results in VUR and
aberrant cystogenesis, and it is controlled by the genes of the S-shape bodies.
Pax2 plays a major role in maturation of S-shaped bodies and their fusion with
collecting ducts. It is widely expressed in the condensed MM and peritubular
aggregates. At the stage of comma and S-bodies, during MET, Pax2 is
downregulated (Horster et aI., 1999, Lipschutz, 1998). This downregulation
promotes formation of glomeruli, maturation of the tubules and their fusion with
US. Persistent expression of Pax2 is implicated in development of renal
dysplasia and VUR (Dressler and Woolf, 1999). Aberrant expression of Pax2
similar to this pattern is detected in 3H1 Br/Brkidneys (Lozanoff et aI., 2001),
suggesting the role of Pax2 in development of Br MRHD. bHLH, homeobox, and
SMAD transcription factors can regulate expression of Pax2 gene (Kuschert et
aI., 2001). One of them, a ubiquitous repressor, bHLH protein Groucho, is
expressed in the distal part of the comma shaped body. Upon expression, it
converts transcriptional activator Pax2 into a repressor and restricts Pax2 to the
tubular portion and Wt1 to the glomerular portion of the body. Thus groucho
appears to regulate Pax2 function in the proximal nephron and, presumably,
9
tubular fusion with Us. Interestingly, signals regulating Groucho expression are
currently unknown (Schedl and Hastie, 2000).
In addition to its role in nephrogenesis, Pax2 is implicated in epithelial
proliferation. Upregulation of Pax2, c-myc and bc/2 genes is characteristic of the
dysplastic kidney and it is specifically detected in the dysplastic epithelium of the
cystic tubules (Piscione et aI., 2000; Dressler et aI., 1999; Yang et aI., 2000;
Woolf and Winyard, 2000). The effect of Pax2 on cell proliferation is mediated by
c-myc transcription factor. Normally, c-myc decreases with differentiation, and
becomes absent in the mature epithelium by birth. Upregulation of c-myc is
detected in the cystic tubules of PKD (Trudel et aI., 1997). It represents the state
of increased cellular proliferation and apoptosis and is counterbalanced by TGF-
{3 signaling. Upregulation of TGF-{3 inhibits Pax2, bc/2 and c-myc in dysplastic
tubules, and, by repressing proliferation and apoptosis, MET and US branching,
it also leads to development of renal cystic hypodysplasia (Yang et aI., 2000).
Thus upegulation of either c-myc or TGF-{3 pathways can underlie development
of MRHD. It will be interesting to determine if one of these pathways is involved
in the pathogenesis of MRHD in Br mutant mice.
PURPOSE
The long-term goal of this research is to determine and sequence the Br
gene. The general approach for mapping genes in the mouse has been
reviewed by Silver (1995). The first step is to undertake a genome wide scan
thus localizing the mutation to a particular chromosome. High-resolution
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microsatellite linkage analysis is then performed with a large DNA sample to
localize the mutation to a specific region of the chromosome. Once a critical
region is identified, a small number of DNA samples, that define the critical
region, are used to further resolve the locus to a region approximately 1-2 cM
(megabases) in length. An in silico analysis is performed to identify candidate
genes within the region and target tissues are analyzed for expression.
Depending on the outcome of target tissue expression, gene sequence analysis
is pursued once the candidate region is sufficiently reduced in size in an attempt
to specifically define the nature of the mutation.
The 3H1 Br mutation arose in the 3H1 germ cell line as a result of over
exposure to gamma radiation and it was initially mapped to the distal portion of
chromosome 17 (Searle, 1966; Beechey et aI., 1997). The majority of radiation
induced mutations in mouse germ cells results in gene deletions by causing
double-stranded breaks in DNA that the germ cell is unable to rejoin (reviewed by
Abrahamson and Wolff, 1976; Sankaranarayanan, 1991,1999). However, the
telomere may heal some breaks and confer some degree of protection
particularly in the distal portions of a chromosome (Slijepcevic and Bryant, 1998).
Evidence suggests that the mammalian protein DNA polymerase micro (pol
micro) forms discrete clusters following radiation exposure which could stabilize
the free ends (Mahajan et aI., 2002). Even so, over-exposure to gamma
raditaion is likely to damage a chromosome, if not completely deleting a portion,
since the vast majority of radiation-induced germ line mutations result in loss-of
function and demonstrate haploinsufficiency in the heterozygote and lethality in
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the homozygous state (Sankaranarayanan, 1991,1999). Although base
substitutions, frameshifts and point deletions do occur, these types of mutations
are much less frequent as a result of radiation exposure while insertions seldom
occur (Grosovsky et aI., 1988). The radiation-induced nature of Bras well as its
inheritance pattern and its position near the telomere of chromosome 17 is
consistent with a repaired radiation induced double stranded break following a
deletion.
The goal of this project is to extend current work aimed at characterizing
the Br gene. Specifically, the purpose of this study is to identify candidate genes
in the region of interest in chromosome 17 and then determine whether they are
expressed in the tissues that are affected in the Br phenotype. Thus, the
hypotheses to be tested are:
1. Candidate genes for Brthat are associated with HPE and/or MRHD exist the
region of interest on distal murine chromosome 17.
2. cDNA expression of these Br candidate genes is absent in the target tissues
of 3H1 Brembryos.
12
PART I
IN SILICOANALYSIS OF BrCANDIDATE GENES
Previous studies have shown that Br localizes to distal chromosome 17
between approximately 40 - 50 eM (Beechey et aL, 1997; McBratney et aL,
2002). To test the initial hypothesis an in silico analysis of the candidate gene
region was undertaken.
Methods
Three databases were utilized to identify known genes in the region of
interest and determine whether they are involved in craniofacial and/or renal
morphogenesis. These databases included the Jackson Laboratory database
(www.informatics.jax.org), the Celera Gene Discovery database
(www.celera.com) and the Sanger Ensembl database (www.ensembLorg).
Candidate Gene Search
Recombinant mapping experiments narrowed the region of Br mutation
and identified microsatellite markers D17Mit128 and D17Mit190, demarcating the
mutant region on Chr 17. This region was compared to the human genome, and
showed homology with human Chr 2 and, partially, with Chr 18. Detailed
analysis of genes, positioned on mouse Chr 17 around the mutant locus, and
consideration of their molecular function, pattern of expression and role in
embryonic development, was performed. Comparison with their human analogs,
13
based on chromosome position, description and function, specified selection of
candidate genes for the Br mutation.
Results and Discussion
In silica gene analysis revealed three genes of interest for the Br mutation
(Table 2). Two of them, Six3 and Zfp161, potential mouse homologue for TGIF,
are selected as candidates for HPE. The third gene, ALK, is involved in
development of nervous system and kidneys. Human analogs are mapped on
Chr 2 (Six3 and ALK) and Chr 18 (Zfp161, TGIF) and have similar functions.
ALK
Anaplastic lymphoma kinase (ALK) is positioned on mouse Chr17 at 50.0
cM and it is homologous to human gene ALK on Chr 2 p23-p23. ALK is highly
expressed in the mouse neonatal brain at GO 8.5 and presumably plays an
important role(s) in development of nervous system (Iwahara et aI., 1997). In the
kidneys, at GO 14, ALK is expressed in the metanephric mesenchyme (MM) and
functions as a tyrosine kinase receptor for the pleyotrophin gene, that is involved
in regulation of branching morphogenesis (Stoica et aI., 2001; Sakurai et aI.,
2001).
Zfp161
Zinc finger protein 161 (Zfp161) is positioned on mouse Chr 17 at 41.0 cM
and is homologous to human gene Zfp161 on distal region of Chr 18 p11.21. It is
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98% homologous to a murine zinc finger protein, ZF5, which is a putative
transcriptional repressor of c-myc and exhibits growth-suppressive activity in
mouse cell line (Numoto et aI., 1993). The human HPE candidate TGIF is
positioned on 18p11.3 (Gripp et aI., 2000), whereas mouse homologue of TGIF is
unknown. Based on positional homology between distal region of mouse
chromosome 17 and human chromosome 18 as well as functional homology with
TGIF as a transcription repressor, mouse Zfp161 gene is suggested to be
analogous to TGIF, hence a candidate for HPE4 (Sobek-Klocke et aI., 1997).
Six3
Six3, a member of mouse sine oculus-related homeobox gene homologue
(Drosophila), is HPE2 candidate gene, essential for development of anterior
neural plate and eye (Wallis, 1999). It has been mapped to distal Chr 17 at 45.5
cM (www.informatics.jax.org), and its human homologue corresponds to Chr 2
p21-p16. In mouse, expression of Six3 is detected after GO 7.5 in
neuroectoderm and derivatives, including the anterior forebrain and eyes (Olivier
et aI., 1995a; Kawakami et aI., 1996b). Other members of Six family include:
Six1 and Six2, expressed in head and body mesenchyme, limb muscles, and
tendons (Olivier et aI., 1995b); Six4, expressed in neural tissues and encoding
transcription factor regulating Na,K-ATPase alpha1 subunit gene (Kawakami et
aI., 1996a); and Six5, expressed in a wide variety of murine tissues (Kawakami et
aI., 1996b). During forebrain and eye development, Six3 acts as a Groucho
dependent repressor. Its interaction with mouse Groucho homologue, Grg5, is
15
essential for recognition and binding to specific DNA sequence, subsequent
autoregulation and activation of other genes. Grg5 is expressed ubiquitously
from midgestation, and can interact with Six3 protein in any of Six3 expressing
tissues (Zhu et aL, 2002). In zebrafish embryos, overexpression of Six3 protein,
associated with Groucho repressor, results in upregulation of Pax2 in the optic
stalk, enlargement of the rostral forebrain, and general disorganization of the
brain (Kobayashi et aL, 1998). Interestingly, in the absence of Groucho co
repressor, Six3 acts as an activator and downregulates Pax2, causing eye and
forebrain hypoplasia (Kobayashi et aL, 2001). Other isoforms of Six gene, Six2
and Six4 interact weakly or not at all with Groucho proteins in mammals (Zhu et
aL,2002). Instead, they interact with Eya proteins, and a defect in the Pax6
Eya1-Six2 signaling pathway, conserved in the eye, ear and kidney (Xu et aL,
1999), leads to development of branchio-oto-renal syndrome (BaR). In BaR
mutants, deficiency of Eya1 protein prevents outgrowth of UB and expression of
GDNF, and leads to development of renal dysplasia. The role of Six3 and
Groucho dependent repression in renal development remains unknown.
16
PART II
EXPRESSION OF Br CANDIDATE GENES IN THE TARGET TISSUES
Genes, identified by in silico analysis, served as candidates for Br and
were used to assess expression within target tissues. If the Br phenotype
resulted from a chromosomal deletion of region that contains one or more of
these genes, then it was expected that the corresponding mRNA would not be
expressed in the target tissue.
Methods
RT-PCR was applied to determine whether Six3, Zfp161 and ALKwere
expressed in kidney and brain tissue of 3H1 +1+, Brl+, and BrlBr mice. PCR
amplification of GAPDH gene with eDNA from all samples was performed as a
positive control.
Animal and Tissue Collection
Pregnant 3H1 Brl+ and +1+ (stock control) mice were mated at 16:00. The
following day, females were checked for a vaginal plug. If present, the animal
was designated E 0.5. At E 18, the pregnant female was euthanized with an
overdose of isoflurane and embryos were removed by Cesaerian section.
Phenotyping was performed based on craniofacial morphology following
McBratney et al. (2002). Embryos were placed in PBS, and brain and kidney
17
tissues were extirpated with sterilized scissors. Tissues were snap frozen in
liquid nitrogen and stored at -70°C.
RNA Extraction
RNA extraction was achieved by first homogenizing the tissue. Frozen
tissue was weighed and transferred into RNA free DEPC treated tubes. After
addition of 1.0 ml RNA-STAT to the sample with weight less than 0.1 g, tissue
was disrupted by polytron homogenization. Samples were incubated for 5 min
and 0.2 ml chloroform was added to 1.0 ml RNA-STAT. After shaking for 15 s,
samples were incubated at room temperature for 2-3 min and centrifuged at 4°C
at 12000 rpm (max) for 15 min. RNA was then precipitated. Aqueous RNA was
separated into a new centrifuge tube, 0.5 ml of isopropanol was added to 1.0 ml
of RNA-STAT. After incubation for 10 min at room temperature, samples were
centrifuged at 12000 rpm for 10 min. The supernatant was removed and the
pellet was washed by vortexing with 75% ethanol (1.0 ml). After centrifugation
for 15 min, the pellet was dried and aliquoted in 25 III of DEPC treated water.
RNA concentration was determined by diluting 1.0 III with 499 III of 01
water. RNA counting was performed on spectrophotometer within the range of
absorbance wave (00) of 240-300. RNA was analyzed for purity (00 260/280,
1.6-2) and quantity (00 260 x 500 x 40 Ill/ml). DNAase treatment of RNA was
performed to purify the sample by removing traces of DNA. Samples with less
than 10 Ilg of total RNA were treated with 0.1 volume of 10x DNAase buffer and
1 III of DNAasel (2units) (DNA-free tm, Ambion), mixed and incubated at 37°C for
18
20-30 min. Samples with more than 10 ~g of total RNA were diluted to 100 ~g
Iml and treated with 0.1 volume of 10x DNAase buffer and 2-3 ~I of DNAase (4-6
units), followed by incubation at 37°C up to 1 hour. Following DNAase treatment,
0.1 volume or 5 ~I (whichever was greater) of DNAase inactivating reagent was
added to the samples, incubated at room temperature for 2 min and centrifuged
at 10.000 rpm for 1 min in order to pellet the DNAase inactivating reagent. The
RNA concentration was determined again after DNAase treatment (as above).
Reverse Transcription
Reverse transcription was performed by using 0.05 ~g of total RNA mixed
with 1 ~I of random hexamers (50 ng/~I), 1 ~I of dNTP, and DEPC treated water
up to 10 ~1. (Superscript 1m First strand synthesis system for RT-PCR (Invitrogen,
Life Technologies). Samples were incubated at 65°C for 5 min and placed on ice
for at least 1 min. Each reaction volume (9 ~I) included: 2 ~I of 10 x RT buffer, 4
~I of 25 mM MgCI2, 2 ~I of 0.1 M OTT, 1 ~I of RNase OUT recombinant
ribonuclease inhibitor. After incubation at 25°C for 2 min, 1 ~I (50U) of
Superscript II was added to each tube, mixed and incubated at 25°C for 10 min,
then transferred to 42°C for 50 min, finally terminated at 70°C for 15 min and
subsequently chilled on ice. Negative controls without Superscript II were
prepared for each set of reactions to control for genomic DNA contamination.
After transcription, reactions were collected by brief centrifugation, 1 ~I of
19
RNAase H was added to each tube and samples were incubated at 3rC for 20
min. cDNAs were stored at -20°C and used for ten PCR reactions.
Primer design for PCR
Primers were chosen for mouse ALK, Zfp161 and Six3 genes, using their
nucleotide sequences from NCBI database (National Center for Biotechnology
Information, Genbank, ncbi.nlm.nih.gov). Their accession number, length of the
gene, base pair position of cDNA coding sequence and chosen primers are listed
in Table 3i. PCR products were expected to amplify cDNA fragments of 162, 272
and 130 bp (Table 3).
Selection of primer sequences was based upon GC content (about 50%),
length (about 20 nucleotides) and melting temperature (Tm, 55-57°C; Table 3ii).
This was followed by analysis with IDT DNA database (www.idtdna.com) for
cross reaction and hairpin formation. Chosen primers were synthesized by Dr.
Gabor Mocz (University of Hawaii Biotechnology core).
Primers for GAPDH were supplied with mouse GAPDH primer set kit
(Maxim biotech). Mouse GAPDH gene accession number and expected product
size (532 bp) are shown in table 3iii.
PCR Amplification
Master mix for ALK, Zfp161 and Six3 samples was prepared with 2.5 III
10x buffer, 2 III dNTP, 1.25 III forward primer, 1.25 III reverse primer, 1.51l1
MgS04, 0.25 III TSG Taq (Lambda biotech), 14.25 III DEPC treated water (per
20
sample). 23 f..ll of each sample was aliquoted in PCR tubes and 2 f..ll of cDNA
was added. Master mix for control GAPDH samples was prepared with 40 f..ll of
optimized, premixed with GAPDH primers, PCR buffer (Maxim biotech), 0.25 f..ll
TSG Taq (Lambda biotech) and 7.75 f..ll DEPC treated water (per sample). 48 f..ll
of each sample was aliquated in PCR tubes and 2 f..ll of cDNA was added.
Samples with experimental and control primers were amplified together on
GeneAmp® PCR system 2400 (Perkin-Elmer). The amplification profile
consisted of initial denaturation at 94°C (2 min); 35 cycles of denaturation at 94
°C (30 s), annealing at 56°C (30 s) and extension at 72°C (30 s); followed by
termination at 72°C (7 min). PCR products were run in 5% polyacrylamide gels,
visualized with ethidium bromide staining, bands of expected size were detected
and photographed. To confirm the identity of the amplified genes, PCR products
were sequenced (Gabor Mocz, University of Hawaii Biotechnology Core) and
results were aligned in NCBI database, using Basic Local Alignment Search
Tool, BLAST.
Statistical Analysis
As indicated above, the assumption is that the radiation induced nature of
Br as well as its inbred semidominant inheritance pattern and its position near the
telomere on chromosome 17 is consistent with a repaired radiation induced
double-stranded break following a deletion. If the gene is deleted then the
expectation is that it is expressed in the 3H 1 +/+ tissue and absent in the 3H 1
Br/Brtissue. Thus, the expected incidence ratio of 3H1 +/+:Br/Br (amplimer
21
presence for +/+:amplimer absence for BrlBr) is 1:0 in the case of a deletion. If
the gene is not deleted then the ratio will be 1:1 (amplimer presence in +/+:
amplimer presence in +/+). Since the 3H1 Brl+ phenotype has at least one
normal allele associated with it, then it too should have an amplimer. To
determine the sample size needed to detect a true difference between two ratios,
a G test of independence can be used. Thus, the sample size necessary to
achieve an 80% probability that one is sure of detecting that the expected ratio
(1 :0) at the a = 0.05 level of significance occurs instead of the alternate ratio, i.e.,
1:1 is calculated following Sokal and Rohlf, 1981 :766-77
P1 = expected ratio of 1:0 or 100 % or 1.0
P2 = alternate ratio of 1:1 or 50 % or 0.5
ta[~]= 1.96 at infinity
h~ [=]=t 2(1-0.8) = t 0.4 =0.842 at infinity
A = [ta[~]v'P1(1-P1) + t2~[=]v'P2(1-P2]2
A=[1.96v'1 (1-1) + 0.842v'0.5(1-0.5)] 2=[0.842v'0.5(0.5)] 2=0.8422x 0.52
n = {A[1 + v'[1 + 4(P1 - P2)/A)]]2} + {4(P1-P2)2}
n={0.8422x OK{ 1+v'1+ 4(~/0.8422X 0.~]2} + {4x(},5'2}=
={0.8422x [1 +v'1 + 8/0.8422] 2} + {4}=
={0.8422x [1 +v'(0.8422+8)/0.8422] 2} + {4}=
={0.8422x [1 +v'9/0.8422] 2} + {4}=
={0.8422 x [1 +3/0.842] 2} + {4}=
={O.~ x [(0.842+3)/0.842f} + {4}=
=3.8422+ 4=3.6922
n = 3.69 or approximately 4 3H1 +1+ or 3H1Brl+ and 4 3H1 BrlBr.
Therefore four animals from each group are required.
Results
In order to examine the effect of mutation on expression of ALK, Zfp161
and Six3 genes in Br mice, a control experiment was done using tissue from a
3H1 +1+ mouse.
Control Experiment
RNA Preparation and Gene Expression
Total RNA was extracted from the kidney of 3H1 +1+ mouse embryo (litter
#1). Qualitative and quantitative analysis was performed after DNAase treatment
(Table 4, litter #1). In spite of low level of RNA purity (00 260/280 was about
1.1), this RT-PCR experiment amplified three products of expected sizes,
corresponding to ALK, Zfp161 and Six3 (Fig 5). Product sizes were estimated by
comparing them to a 100 bp ladder (Fig 5, first lane). An ALK gene product of
expected size 162 bp, and Zfp161 gene product of expected size 272 bp were
expressed highly (Fig 5, second and third lanes). A Six3 gene product of
expected size 130 bpwas expressed moderately (Fig 5, fourth lane). Negative
control RT-PCR, without reverse transcriptase, yielded no products and thus
confirmed absence of genomic contamination (data not shown). PCR products
23
were sequenced and gene identity was confirmed by alignment of their
sequences in NCBI database (BLAST).
Gene Identity
Alignment of ALK PCR product revealed highest degree of homology with
mouse ALK gene. Comparison of their nucleotide sequences showed 98%
identity (Fig 1). Alignment of Zfp161 PCR product revealed 91 % homology with
mouse Zfp161gene (Fig 2). A similar degree of homology was found with Zic5
gene, and similarity between Zfp161 and Zic5 nucleotide sequence reached 100
% in the aligned region. Alignment of the Six3 PCR product revealed 100%
homology with Six3 gene (Fig 3). To eliminate the possibility of cross reactivity of
Six3 primers with other members of Six family, the homologue of the Six3
product sequence was also searched in Celera and Sanger databases.
Alignment yielded the highest degree of homology with the Six3 gene. To test
the misidentification of Six3 PCR product with Six2, implicated in renal
malformations of BOR syndrome, two pair BLAST search was performed with
Six3 and Six2 genes. Nucleotide comparison of their full cDNA sequences
revealed 78% identity within two portions of Six3 gene, corresponding to 1-115
bp and 181-567 bp segments (Fig 4). Nucleotide comparison of the Six3 PCR
product revealed homology with an expected fragment of Six3 cDNA, located
between 129-259 bp (Fig 4, shaded area; table 3). But only part of that fragment,
within 129-159 bp (Fig 4, boxed area) was recovered by the BLAST search (Fig
24
3). It rendered 100% homology exclusively to Six3 gene and determined the
specificity of the Six3 PCR product.
Experiment using 3H1 +1+. Brl+ and BrlBr mutant mice
RNA Preparation and Statistical Data
Based on results of the control experiment, further experiments were
designed to test the expression of ALK, Zfp161 and Six3 in the mutant mice.
Brl+ and BrlBrsiblings of control 3H1 +1+ embryo (litter #1); 3H1 +1+, two Brl+
and four BrlBr embryos (litter #2); and additional control normal 3H 1+1+ stock
mice (litter #3) were used (Table 4). Since sample size, necessary to achieve
80% reliability at a significance level of a =0.05 required a total of four
homozygous specimens, litters from two 3H 1 Brl+ females and 3H 1 stock control
mice were considered sufficient for statistical analysis. Each experiment,
including cDNA synthesis and PCR amplification was repeated at least twice to
confirm the consistency of results.
Analysis of purified brain and kidney RNA from most embryos revealed a
high yield and quality with 00 260/280 mean of 2.0 and standard deviation of 0.6
(table 4). RNA quantity varied significantly with 0.7 ~g/~l mean and standard
deviation of 2.0. However, the amount appeared to be adequate to perform
analysis on most specimens.
As additional qualitative control, GAPDH primers were amplified for all
samples, and their PCR product was loaded on the gel simultaneously with ALK,J
25
Zfp161 or Six3 samples. Negative RT-PCR control without reverse transcriptase
confirmed absence of genomic contamination.
Expression of Genes
ALK, Zfp161 and Six3 gene products were found to be expressed in the
brain and kidney tissues of both mutant and control normal mice, including 3H1
stock (litter #2 and 3, table 4; Fig 6).
ALK
ALK was expressed in the brain of all BrlBr, Brl+ and +1+ mice (Fig 6 A).
Control GAPDH was also expressed in all samples. Both ALK and GAPDH were
absent in the negative control (-rt). In the kidney, ALK was expressed in all BrlBr
mice, one Brl+ mouse (Fig 6 B), and +1+ stock control mice (data not shown).
Other experiments had detected ALK in the kidney of 3H1stock, +1+, and Brl+
mice in litter #1 (Fig 5 and other data not shown). GAPDH was also expressed in
the normal and mutant animals.
Zfp161
Zfp161, along with GAPDH, was expressed in the brain of all 3H1 BrlBr,
Brl+ and +1+ mice (Fig 6 C). Both were absent in the negative control (data not
shown). A similar type of expression was detected in the kidneys of all mice (Fig
6, D). RT-PCR for Zfp161 was repeated more than twice, included analysis of all
three litters and yielded a consistent expression of Zfp161 in all samples.
26
Six3
Six3 along with GAPDH was expressed in the brain of all 3H 1 Br/Br, Br/+
and +/+ mice (Fig 6 E), but both were absent in the negative control (-rt). In the
kidney expression of Six3 was detected in all samples and accompanied by
control GAPDH expression (Fig 6 F). Six3 was also expressed in all brain and
kidney tissues of litter #1 (data not shown). All RT-PCR experiments were
repeated more that twice, and results were consistent.
DISCUSSION
The aim of this project was to determine whether a deletion of a candidate
gene occurred in animals with 3H1Br/Brphenotype. Although the results were
not consistent with a deletion, the functional analysis of expressed candidate
genes shall further determine their potential role in Br mutation.
ALK
RT-PCR experiments revealed that ALK gene was expressed in the brain
of all 3H1 +/+, Br/+ and Br/Brembryos. Based on results from all three litters,
ALKwas expressed in the kidney of 3H 1stock, +/+, two Br/+ mice and in all four
Br/Br mutants. Thus ALK is present in control and mutant mice and is not
deleted by a mutation. However, an expressed ALK gene may be functionally
abnormal, which, considering its role in neural development and regulation of
renal branching morphogenesis, may contribute to Br malformations.
27
Zfp161
Zfp161 was expressed in the brain and kidney of all 3H1 +/+, Br/+ and
Br/Brembryos. Results were consistent and indicated that Zfp161 is present in
control and mutant mice and it is not deleted by a mutation. Expression of
Zfp161 may mediate TGF-~ signaling and/or c-myc repression, and activity of
Zfp161 can be regulated by phosphorylation and ubiquitin mediated proteolysis,
described for other members of TGF-f3 pathway (Massague and Chen, 2000).
Both TGF-f3 and e-mye are important for proliferation and differentiation of
developing tissues. In the head, TGF-f3 pathway regulates craniofacial
development and mutations of one gene in this pathway, Ski, results particularly
in FND (Colmenares et aI., 2001). Thus abnormal function of Zfp161 may be
essential for development of FND in Br mice. In the kidney both e-mye and TGF
f3 may mediate development of MHRD, and altered function of Zfp161 can reflect
activation of either pathway. Deficiency of Zfp161 could be associated with
activation of e-mye, while upregulation of Zfp161 and downregulation of e-mye
could be related to activation of TGF-f3 signaling. Since TGF-f3 inhibits e-mye,
and does so by an unknown pathway (Yagi et aI., 2001), Zfp161 can potentially
mediate this action. It can bind to identified TGF-f3 responsive elements in the e
mye promoter and repress e-mye expression. To test whether the activation of
TGF-f3, but not e-mye pathway is the primary mechanism of Br MRHD, and if it is
mediated by Zfp161, further analysis of Zfp161, TGF-f3 and e-mye expression in
Br kidneys should be done.
28
Six3
Six3 gene was found expressed in the brain and kidney of all 3H1 +1+,
Brl+ and BrlBr embryos. Results were consistent, demonstrating that Six3 is
present in control and mutant mice and gene is not deleted by gamma irradiation.
In spite of expression, Six3 gene function may be altered, resulting in Br
malformations. Function of Six3 can be tested for its DNA binding activity by gel
shift essay, for protein expression and interaction with other proteins by Western
blot and co-immunoprecipitation. Chromosomal location of Six3 and its function
make it an important candidate for the Br mutation. Six3 is positioned in the
distal end of the mouse Chr 17 within the Br locus (Celera and Sanger
databases), while in human patients, Six3 mutations cause HPE with
malformations of forebrain and eye.
The role of Six3 in HPE can be explained by the structure of the gene and
the character of its mutations. Mouse Six3 coding region predicts a protein of
332 amino acids (aa), containing a SIX domain of 115 aa and a homeodomain of
60 aa. This gene consists of three exons, but only the terminal two encode a
protein. One exon encodes the SIX domain and the homeodomain; another
exon encodes the carboxy terminus. Mutational analysis of HPE patients
identified many mutations in the homeodomain region (Wallis et aI., 1999).
Mutations were translocation breakpoints, deletions and missence mutations.
Deletions in the homeodomain resulted in the loss of amino acids, disrupted
helical structures of the Six3 protein, affected its interaction with DNA and
expectedly interfered with transcriptional activation. Missence mutations also
29
changed protein structure and function. One study identified a frameshift
mutation in the SIX domain, which lead to a nonsense mutation downstream in a
homeodomain region (Pasquier et aI., 2000). In general, the character and
position of a mutation of the Six3 gene specifies the phenotype of HPE patients.
The gene region amplified in Br mice encoded a part of the SIX domain.
This 130 bp product sequence, expressed in the brain and kidney tissues of all
mice, may be part of the gene that contains a mutation downstream, and thus
encodes a nonfunctional protein. It is possible that a new type of Six3 mutation
has occurred in Br mice, which disrupted transcriptional activity or interaction with
other proteins and lead to development of the Br specific phenotype. Further
analysis of the complete Six3 gene sequence in Br mice is needed to evaluate
the role Six3 in the Br mutation.
In the kidney, expression of Six3 has not been recorded previously.
Based on recent discovery of Six3 interaction with bHLH family proteins
(Tessmar et aI., 2002; Zhu et aI., 2002), it is tempting to predict the novel function
of Six3 in the kidney development. Particularly, interaction of Six3 with Groucho,
expressed during nephrogenesis, can be essential for regulation of Pax2
expression, maturation of S-shaped bodies and their fusion with collecting ducts.
Abnormal function of Six3 can result in development of VUR, obstructive
nephropathy and MRHD. In addition to this, the Six3 promoter was found to be
regulated by MSX proteins (Lengler and Graw, 2001), which are involved in
craniofacial development. Expression of Six3 in the kidney, found in this work,
30
adds a new member in the molecular net of renal development and a new link
with craniofacial development.
CONCLUSION
Expression of Zfp161, Six3 and ALK genes in Br brain and kidney tissues
has several implications. It rejects the involvement of these genes in the
potential chromosomal deletion in Br mice. No direct relation between HPE
candidate genes Six3 and Zfp161 (mouse TGIF candidate) and FND in Br mice
is found. This does not exclude the possible role of these genes in Br mutation
as result of alternative mutation such as frameshift, missence or point deletion, or
as result of indirect interaction with Br locus. A novel expression of Six3 gene in
the kidneys suggests a new function of Six3 in the renal development and a
potential role in the Br mutation.
31
Wtv
APPENDIX A
Table 1. Key developmental genes associated with renal cystic hypodysplasia
Genes
Type ofmutation Pax2 Bcl2 TGF-p 1BMP7 Wnt4 c-Myc n-Myc RAR alP GDNF c-Ret
Retk Ret9 Ret51Transgene Cystic kidneys, PKD, upregulated Multicystic Multicystic Normal
not differentiated proliferation of dysplasia dysplasiaepithelium cystic epithelium,
apoptosis (bcl2,p53 independent);cysts fromglomerules,collecting ducts,
R later- from proximale nephrons.n Haplo- Hypoplasia-less Hypoplasia: Hypoplasiaa insufficiency Ub branches, normalI less cortical structure, few
nephrogenesis, nephrons, noless MET cysts
Mutation Smallp (deletion, disorganizedh missence kidneys,e etc.) vesiculoureteraln reflux.0
t Knockout -1- Hypoplastic Development of Small, dysgenic, Multicystic Agenesis Agenesis! Multicystic Normaly polycystic glomeruli, undifferentiated hypo- severe dysplasiap kidney with epithelium is MM, dysplasia dysplasiae increased affected, early interdispersed
apoptosis; induction normal, with UBperinatally but genes (pax2, branches. Initialcystic wnt4, wnt1, ret) nephrogenesis isdilations of expressed normal. Noproximal abberantly. growth by E15.tubules, Later- no MM in MM is induced,postnatally - periphery, no but does notof collecting aggregates, undergoducts. apoptosis, peritubular
medullar region aggregation andfilled with MET- no comma,collecting ducts, S bodies. MMinterdispersed condenseswith loose around UB, Pax2stromal cells. present, Pax8
absent.
ww
Table 1. (Continued) Key developmental genes associated with renal cystic hypodysplasia
Genes
Pax2 Bcl2 TGF-~ Wnt4 c-Myc n-Myc RAR aI~ GDNF c-RetIBMP?
Expression Only in induced MM: MM Upregulated in BMP? is expressed On condensed MM, then Highly Upregulated in early Stromal MM UB, MD
pattern condensates and aggregates, condensing MM and from E9 to in glomeruli. Secreted by expressed in induced MM. cells
comma and S-shape bodies. UB (prevents their adulthood. induced MM uninduced Downregulated after
Downregulated in S-shaped bodies apoptosis) and at S- At E14.5 in comma, Induction- MM MM, epithelial polarization
on the site of podocyte stage. Bcl2 S shaped bodies. condensation- developing
development. Downregulation is regulated apoptosis aggregation (Wnt4 up), tubules; up
required for complete maturation of is important at this mature nephron (Wnt4 with induction;
S-shaped bodies and their fusion stage. down.) later only in the down by birth;
with collecting ducts. Downregulated in cortical layer. notmost mature cells, downregulatedterminal epithelia in PKD.and olomerular cells.
Downstream Unknown c-Myc, bcl2, pax2 Wnt4 encodes
factors glycoprotein, which mayinteract with BMP?WNT4 receptor isunknown.
Gene Homeobox TGF-~
regulation ISix3?\by transcrip SMAD TGF-~
tion factors (TGF-~, downregulates Pax2BMP?)bHlH Grouchois(Groucho) stimulated by
unknown signal.Expressed in the
R distal part of thee coma shaped bod,g and turnsu transcriptionalI activator Pax2 into aa repressor andt restricts Wt1 to thei proximal part and
0 Pax2 to the tubulen formino oart.
Indirect? GDNF Pax2t- GDNFt- Wt1, c-ret, pax2, GDNF- IWnt4t- MET- c-RetMET-GDNF.!.- Wnt4 c-ret GDNF.!.- c-Pax2.!. ret.!.- Wnt4.!.
FGF8 TGF-~.!. - BMP? and SHHFGF8.!.-cystickidnevs
SHH TGF-~.!.-
SHHtUpstream factors Wt1 Wt1 regulates Pax2, TGF ITGF-~t - Wt1, Pax2, c-Ret
TGF-~, p53, GDNF -~ bcl2.!., but noapoptosis
t -upregulaled; L-downregulaled
Table 2. Candidate Genes within the Br Locus
Genes ALK TGIF ZfD161 Six3HPE candidates + ? +Essential deveJoomental oenes in the head + +Essential developmental genes in I Ureteric Bud (UBl +the kidney Metanephric ? ?
Mesenchvme (MMlPositional candidates + + +Chromosomal location Mouse 17 (50.0 eM) UN 17p11.2(41.0 17 (45.5 eM)
eM)Human 2023-023 18011.3 18011.2 2021- 16
Gene expression Head Head mesenchyme 8.5; RNA in situ(by gestational day derived from neuralGO unless crestspecified) l' arch 8.5' RNA in situ 9.5' RNA in situ
Ectoderm 9.5' RNA in situMesenchvme 8.5; RNA in situNeuroectoderm 8.5; RNA in situ 7.5-8.5; RNA in
situForebrain 10-11.5;RNAin
situOienceohalon 9.5; RNA in situTelencephalon 11.5-14.5; RNA
in situBrain 9.0-13.5; RNA in 12,15-16; RNA in 7.5-14.5; RNA in
situ situ situKidney Metanephric 14.0; RNA in situ
mesenchvmeReference 1 Morris, 1997; 1 Bertolino, 1996 NA 1 Martinez-
2 Iwahara, 1997 Barbera, 2000;2 Martinez-Morales, 2001;3 Suda, 19994.0Iiver, 1995
34
Table 3
i. Characterization of experimental genes in NCBI database
CodingGene definition Accession Gene sequence Forward primer Reverse primer PCR prooduct
(CDS)Mus musculus similar to ALK tyrosine XM_123195 5439 bp 119-2722 bp 566-583 bp 728-710 bp 162 bpkinase receptor precursor(Anaplastic Ivmphoma kinase. ALK) mRNAMus musculus zinc finger protein NM _009547 1755 bp 61-1410 bp 276-296 bp 528-548 bp 272 bp1611Zfp161) mRNAMus musculus sine oculis-related homeobox XM_140104 774 bp 774 bp 129-147 bp 259-243 bp 130 bp3 homolog (Drosophila)(Six3) mRNA
ii. Experimental primers
Primers Sequence Number of bases GC content % TmoCALK (fwd) 5'-GGC GAG GAG ACG ATT CTT-3' 18 55.6 54.8ALK (rev) 5'-CCA CTT CCG ATG CCT TCT T-3' 19 52.6 55.8Zfp 161 (fwd) 5'-CTC GTC CGT CAT AGA GAT AGA-3' 21 47.6 53.5Zfo 161 (rev) 5'-CAT CAT CGT CCT GAG CAT CA-3' 20 50.0 55.5Six3 (fwd) 5'-GTG CGA GGC CAT CAA CAA-3' 18 55.6 56.7Six3 (rev) 5'-GCT TGC CGT GAG ACT CCT TA-3' 20 55.0 57.9
iii. GAPDH control gene and primers
Gene definition Accession PCR Primers Sequence Number of Tm °cprooduct bases
Mouse GAPDH M32599 532 bp GAPDH (fwd) 5'-GGGTGGAGCCAAACGGGTC-3' 19 70GAPDH (rvs) 5"-ACGCTGAAGTTGTCGTTGAGG-3' 21 67
35
Table 4. RNA analysis and RT-PCR results
Litter # Mice Tissue RNA quality RNA quantity Expression Repeated(3H1) (OD260/280) (l!g/(J!l) (ALK, Zfp161, Six3)
1 +1+ kidney 1.1 8.8 ves 2 timesBr/+ kidnev 1.9 2.0 ves 2 timesBr/Br brain 1.7 1.3 yes 2 times
2 +1+ kidney 1.6 0.5 ves 2 timesbrain 1.8 1.45 ves 2 times
Br/+ kidnev 1.4 0.45 ves 2 timesbrain 2 1.6 yes 2 times
Br/+ kidney 2.2 0.5 ves 2 timesBr/Br kidnev 4 0.06 ves 2 times
brain 2.25 1.9 ves 2 timesBr/Br kidnev 1.6 0.18 yes 2 times
brain 1.8 1.0 ves 2 timesBr/Br kidnev 1.75 0.05 ves 2 times
brain 2.2 1.5 yes 2 timesBr/Br kidnev 2 0.05 ves 2 times
brain 2 0.3 ves 2 times3 +1+ stock kidnev 1.7 0.56 yes 2 times
brain 1.85 2.3 ves 2 timesMean 2.0 0.7Standart dev 0.6 2.0
36
APPENDIX B
Alk
cDNA 565 ggcgag gagacgattc ttgaaggctg tattggtccc ccagaggagg tagcggc tgt1111111111111111111
product ccagaggagg cagcggc tgt
cDNA 622 gggga tac tc cag t tcaacc tcagcgagc t 9 t tcagc tgg t gga tt ct cc1111111111 1111111111 1111111111 1111111111 1111111 II
product gggga tac tc cag t tcaacc tcagcgagc t 9 t tcagc tgg ngga tt ct cc
cDNA 662 acggcgaagg gaggctgagg at ccgcc tg a tgcc t gaga ag1111111111 111111111 1111111111 111111111 II
product 9 acggcgaagg gaggctgagg a tccgcc tg a tgcc t gaga ag
cDNA 722 aaggca tcgg aag tggI111111111 111111
product aaggca tcgg aag tgg
Fig 1. Sequence alignment of the amplified target with mouse ALK cDNA(BLAST result). Nucleotide identity is 98%.
37
Zfp161
cDNA 281 ccgtcataga gatagatttc ctccgctctg acatatttga agaggtgctg aactacatgt
cDNA 341 acaccgccaa gatttccgtg aaaaaggagg acgtcaact t gat gat gt cgIII 1III111111
product gaa c tac t acagc
cDNA 391 aggcncgt ct tag 9 tgcca ta ggcc aaaaac ga taaactgt gttctcagaa1111 II1I1 II III I II 1111111111 1I111
product tccgggcaga t t c t -cgg ta t ccggt t t t t 9 ct at t
cDNA 441 gcgcgatgtg tctagtccgg atgaaagtaa cggccagtcg aagagtaagt attgcctcaa
cDNA 501 actaaaccgc cccatcggag acgctgctga tgctcaggac gatgatg
Fig 2. Sequence alignment of the amplified target with mouse Zfp161 cDNA(BLAST result). Nucleotide identity is 91 %.
38
Six3
eDNA 129 gt gcgaggccat caacaaacac gagt cgat cc tgcgcgcgcg gccttccacaII 1111I11111 11111I1111 1111111111 I
product ca cgc t ccggta 9 t tg t t tg tg c t cagc tagg a
cDNA 181 ccggcaactt ccgcgacctg taccacatcc tggagaacca caagttcact aaggagtctc
cDNA 251 acggcaagc
Fig 3. Sequence alignment of the amplified target with mouse Six3cDNA (BLAST result). Nucleotide identity is 100%.
39
Six3/Six2
Six3 eDNA 1 atgtteeagt tg eeeaeeet eaae tt eteg eeggageagg tggeeagegt etgegagaeg11111111 I 1111111 II I 111111 I 1I1I 1111 111111 I
Six2 eDNA 306 tg eeeaeet t egge tteaeg eaggageaag tggegt gegt gtgegaggtg
Six3 eDNA 61 etggaggaga egggegaeat egageggetg ggeegettee te tggteget geeegtggeeIII II III 1111111111 1111111111 1111111111 111111111111111
Six3 eDNA 366 etgeageagg egggegaeat egageggetg ggeegettee te tggteget geeeg
Six3 eDNA 121 eeegggg~Six3 eDNA 181
111111111 11111111 I11111 II 11111 1111 111111 1111 I 11111 ISix2 eDNA 464 geetteeaee ggggeaaett eegegagete taeaaaatee tggagageea eeagttet eg
Six3 eDNA 241 t geaageeatg tggetegagg egeaetaeea ggaggeegagI I I III 1I11I1 III II 111111 III 11111111 11111 III
Six2 eDNA 524 eegeaeaaee aegeeaaget geageagttg tggeteaagg egeaetaeat egaggeggag
Six3 eDNA 301 aagetgegeg geegeeeget eggeeeggtg gaeaagtaee gegtgegeaa gaagtteeet1111111111 1111 1111I III I III I 11111111 1111111 11111111
Six2 eDNA 584 aagetgegeg geeggeeget gggegeegtg ggeaagtaee gegtgeggeg eaagtteeeg
Six3 eDNA 361 etgeegegea eeatetggga tggegageag aagaeeeatt getteaagga geggae teggI1111 III 1111111111 1111I1 II I I I I I 1111111111 I II I I
Six2 eDNA 644 etgeeeeget eeatetggga eggegaggag aeeaget aet getteaagga gaagageege
Six3 eDNA 421 ageetgetge gggagtggta eetgeaggat eeetaeeeea aeeeeageaa gaaaegegaaII I 111111 I I1111I11 I II I II111111 II I I III 11111
Six2 eDNA 704 agegtgetge gegagtggta egeteaeaae eeetaeeegt egeeaegaga gaagegegag
Six3 eDNA 481 etggegeagg eeaeeggeet eaeeeeeaea eaagtaggea aetggtttaa gaaeeggega11111 III I111I11111 11I1 1111 11111 III 1111111 II II11111 II
Six2 eDNA 764 etggeegagg eeaeeggeet eaeeaeeaeg eaagteagea aetggtteaa gaaeeggegg
Six3 eDNA 541 eagegegaee gegeagegge ggeeaag 56711I111111 I II II I 1111I11
Six2 eDNA 824 eagegegaea gggeggeega ggeeaag 850
Fig 4. Sequence alignment of mouse Six3 and Six2 cDNA (paired Blast results).Nucleotide identity is 78%. Fragment homologous to Six3 amplified product isrepresented by shaded area. Part of this fragment, that determines specificity ofSix3 product, is shown in the box.
40
ALK, Zfp161 and Six3
Kidney
300 bp
200 bp
100 bp
ALK Zfp161 Six3
Fig 5. RT-PCR expression of ALK, Zfp161 and Six3 genes in the
kidney of 3H 1 +/+ mouse. Control experiment.
41
Brain
ALK
Kidney
GAPDH
ALK
GAPDH
ALK
Brain
Zfp161
Kidney
GAPDH
Zfp161
GAPDH
Zfp161
Brain
Six3
Kidney
GAPDH
Six3
GAPDH
Six3
Fig 6. RT-PCR expression of ALK, Zfp161 and Six3 genes in the brain and
kidney of +1+, Brl+ and BrlBr mice. GAPDH expressed as a positive control.
No reverse transcriptase added in +/+ brain samples as a negative control (-rt).
42
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