-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.

111

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.

v

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

vi

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

vii

Six3 29

Conclusion 31

Appendix A: Tables 32

Appendix B: Figures 37

References 43

viii

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

ix

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

xii

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

xiii

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

1

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

2

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

3

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

4

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

5

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

6

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).

7

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

8

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

10

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

11

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

14

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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