USING RNAi TECHNOLOGY TO

DOWN-REGULATE Six2 EXPRESSION IN VITRO

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

BIOMEDICAL SCIENCES (pHYSIOLOGy)

MAY 2008

By Thomas Eugene Hynd, Jr.

Thesis Committee:

Scott Lozanoff, Chairperson Richard Allsopp

Yusuke Marikawa

We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope

and quality as thesis for the degree of Master of Science in Biomedical Sciences

(Physiology).

THESIS COMMITTEE

C· on

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ii

ACKNOWLEDGMENTS

The author wishes to thank Dr. Ben Fogelgren, Dr. Keith Fong, Dr. Sheri Fong, Beth

Lozanoft' and Marl Kuroyama for their assistance and continued support throughout this

project. Dr. Yusuke Marlkawa generously supplied the Pl9 cells and performed the

shRNA transfections. Special appreciation is given to Dr. Scott Lozanofl', without whom

this project could not have been possible.

iii

ABSTRACT

The Brachyrrhine (Br) mutant mouse, which displays frontonasal dysplasia and renal

hypoplasia, has previously been described. Linkage analysis mapped the semi-dominant

Br mutation close to the homeobox transcription factor Six2, which is normally expressed

in the facial and metanephric mesenchyme during embryonic development. The purpose

of this study is to evaluate the role of Six2 in craniofacial and renal branching

morphogenesis by quanti tying its expression level in the facial prominences and using

immunohistochemistry to visualize branching of the ureteric bud in Br mice. In addition,

an in vitro system utilizing RNA interference (RNAi) technology was developed to

determine whether Six2 could be experimentally down-regulated. Medial nasal (MNP),

lateral nasal (LNP) and maxillary prominences (MAX) of EII.5 embryos were dissected

and Six2 expression was measured with qRT-PCR. Six2 expression was highest in the

MNP, with about 2-fold less in the MAX, and 3-fold less in the LNP of wild-type

embryos. Our data indicate a haploinsufficient pattern of Six2 expression in each of the

three sets of facial prominences. In addition, kidney organ explants were dissected from

E13.5 mouse embryos and immunostained to reveal differences in ureteric bud branching

patterns between the three genotypes (+1+, Brl+ and BrIBr). We confirmed a down­

regulation of Six2 in a cell culture system utilizing five RNAi constructs. These data

indicate a lack of Six2 expression may playa role in the development of a median facial

cleft and its reduced effect in the kidney may lead to renal hypoplasia Additionally, an

in vitro system was established that will allow experimental down-regulation of Six2 to

assess effects on morphogenesis.

iv

TABLE OF CONTENTS

List of Tables ........................................................................................ vi

L· fF· .. 1st 0 19ureS .................................•....•.•.•.••••..............••••...•....•...••••.•.. V11

Introduction .......................................................................................... 1

Materials and Methods ............................................................................ 1 0

Results ............................................................................................... 17

Discussion ........................................................................................... 31

References ........................................................................................... 36

v

LIST OF TABLES

I. Microsatellite recombination data ....................................................... 8

2. Data obtained from qRT-PCR to quantify Six2 expression in facial prominences

among genotypes .......................................................................... 22

3. Data obtained from qRT -PCR to quantify Six2 expression in facial prominences

among facial prominences ................................................................ 24

4. Data obtained from qRT -PCR to quantify Six2 expression in PI9 cells

resulting from a serial dilution .......................................................... 27

5. Data obtained from qRT-PCR to quantify Six2 expression in PI9 cells

following shRNA transfections .......................................................... 29

vi

LIST OF FIGURES

Figure

1. Wild-type ElLS mouse embryo during dissection of the facial prominences ...... 18

2. Photograph of 4% Metaphor agarose gel used for genotyping ....................... .19

3. Melting curve analyses to confirm specific amplification of Six2 and GAPDH

in facial prominences ......................................................................... 19

4. Differences in Six2 expression among facial prominences in El1.5 +1+, Brl+

and BrlBr embryos ........................................................................... 21

5. Differences in Six2 expression among genotypes in ElLS facial prominences ..... 23

6. Schematic of dissection protocol for removing E13.5 kidneys ........................ 25

7. Branching of the ureteric bud by stage 13.5 ............................................. 26

8. Plasmid serial dilution used to quantify the expression of Six2 in stock P19

cells ............................................................................................. 27

9. Confirmation of specific amplification of Six2 in P 19 cell line ........................ 28

10. Relative expression of Six2 in P19 cells following transfection with shRNA ....... 29

11. Melting curve analysis to confirm specific amplification of GAPDH in P19

cells ............................................................................................. 30

vii

INTRODUCTION

The development of the mammalian primary palate is crucial for the natura1 formation

of the mid-face. Malformations of this structure result in catastrophic results for the

fetus, including difficulty in breathing, suckling, mastication and speech formation.

Frontonasal dysplasia (FND) was explained as an umbrella term for two or more of the

following: (I) ocular hypertelorism, (2) broadening of the nasal root, (3) median facial

cleft affecting the nose and/or upper lip and palate, (4) unilateral or bilateral clefting of

the alae nasi, (5) lack of formation of the nasal tip, (6) anterior cranium bifidum occultum

and (7) a V-shaped or widow's peak frontal hairline (DeMyer, 1967; Sedano et al., 1970;

Sedano and Godin, 1988). FND has an occurrence of 0.43-0.73% in the human cleft

population (Apesos and Anigian, 1993).

Roizenblatt et al. (1979) has shown FND can be associated with renal hypoplasia

(RH), a developmental disturbance resulting in an abnormally small kidney. Renal

hypoplasia is described as an insufficient number of nephrons secondary to defective

branching of the ureteric bud during renal morphogenesis (Glassberg, 2002). Renal

failure results due to inadequate filtering, improper excretion of wastes, failure to retain

electrolytes and inability to properly concentrate urine.

Development of the face

The mid-face develops as a result two processes: (I) the fusion of three sets of facial

prominences: the medial nasal prominences (MNP), lateral nasal prominences (LNP) and

the maxillary prominences (MAX) to form the future primary palate, consisting of the

1

upper lip. alveolus and the anterior hard palate. and (2) the merging of the bilateral MNP

to form the philtrum. The secondary palate, derived from a different embryologic origin

and responsible for the development of the hard palate posterior to the incisive foramen

and soft palate, forms later.

The neuroectodermally derived trigeminal neural crest mesenchyme from the

midbrain and the first two rhombomeres migrates into the upper jaw primordia where the

mesenchyme proliferates to form the orofacial prominences (Johnston, 1964, 1966;

Lumsden et al., 1991; Schilling and Kimmel. 1994; Rossel and Capecchi, 1999). The

high rate of proliferation of the neural crest mesenchyme is maintained by an interaction

at the epithelial-mesenchymal interface (Minkoff and Kuntz, 1977, 1978). This

interaction, mediated by developmental factors. is significant for the sustained growth of

the facial primordia (Minkoff, 1991).

The morphogenesis of the human primary palate begins at approximately 41 days

post-fertilization (O'Rahilly. 1978) and in the mouse at 10 days and 18 hours

postfertilization (Reed, 1933; Trasler. 1968). The basis of the primary palate. the upper

lip, forms from the MNP (Diewert and Lozanoff, 1993). The bilateral MNP merge at the

facial midline to form the central tuberculum of the upper lip (Diewert and Shiota, 1990;

Diewert et al., 1993a; Diewert and Lozanoff. 1993; Rude et al.. 1994). The continued

growth and migration of the mesenchyme underlying the epithelium of the MNP

eIiminates the distance between the paired MNP, distending the epithelium and merging

the two structures (Patten, 1961). Completion of the sides of the lip and closure of the

palate requires the fusion of the MNP with the MAX at the nasal fin and the breakdown,

2

by apoptosis, of the epithelial seam to achieve mesenchymal confluence and provide

continuity of the upper lip (Shuler, 1995). This completion of the lip also separates the

nasal pit from the stomodeum.

Signal transduction pathways associated with facial development

The differentiation and growth of the mesenchymal cells composing the facial

primordia and the closure of the primary palate are under the control of spatial and

temporal signal transduction pathways. Growth factors directly stimulate cellular

differentiation, proliferation and migration. Bmp4, Bmp2 and Bmp7 appear to be

involved in early head development by inducing and determining the migration patterns

of the neural crest mesenchyme as well as their condensation and proliferation in the

facial primordia (Nie et al., 2006). Bmp2 and Bmp4 have been suggested to be required

for the migration of the cranial neural crest cells to the facial primordia (Kanzler et al.,

2000). Bmp4 and Bmp7 are expressed in epithelium of the facial primordia and are

associated with the expression of Bmp2 in the mesenchyme (Barlow and Francis-West.

1997, Bennett et al., 1995, Francis-West et al., 1998). Later, Bmp4 is also expressed in

the mesenchyme of the facial primordia (Barlow and Francis-West, 1997). Altered

expression of this signaling cascade leads to irregular development of the facial primordia

(Barlow and Francis-West, 1997). The Fgfgene family, including FgfJ, Fgf2, Fgf4,

Fgf5, Fgf8, Fgf9 and FgfJ2, are also expressed in the facial primordia, although their

expression patterns are not well understood (Francis-West et al., 1998; Colvin et al.,

1999). However, Richman and Crosby (1990) found bFGF is involved in the

3

development of the facial primordia by regulating the enlargement and stimulating an

increase in proliferation in the frontonasai mass mesenchyme.

The expression of TGF-p family of genes is pivotal for palatogenesis via a receptor

signaling complex, the Alk-S/Smad pathway. TGF-p3 has been suggested to be heavily

involved in palatal fusion, such that its expression is responsible for the disappearance of

the midline epithelial seam (Dudas et al., 2004). This disappearance can be achieved

through three different means: apoptosis, cell migration and epithelial-to-mesenchymal

transdifferentiation (Martinez-Alvarez et al., 2000).

These signal transduction pathways are under the control of specific transcription

factors. The Hox family of genes, a particular collection of homeobox genes. functions

heavily in patterning the body axis, however, Hox genes also mediate craniofacial

developmental patterns in both mice and humans (McGinnis et al., 1984; Krumlauf,

1993; Vieille-Grosjean et al., 1997). Hox7, Hox8 and Hox9 are expressed in several areas

where epithelial-mesenchymal interactions occur during embryogenesis (MacKenzie et

al., 1991a, 1991b). Their expression patterns form gradients, identified by DeRobertis et

al. (1991), suggesting they provide positional information through signals that influence

morphogenesis where epithelial-mesenchymal interactions occur.

Development of the kidney

The development of the kidney begins with the appearance of the primary nephric

duct on day 22 in humans and day 8 in mice (Gilbert, 2000). The ureteric bud (UB), an

outgrowth of the primary nephric duct, extends into the adjacent metanephric

4

mesenchyme (MM) where the invading bud epithelia begin branching morphogenesis.

Upon branching, MM cells condense around the tips of the branching DB before

undergoing a mesenchymal-to-epithelial transition, beginning nephrogenesis. Each

cluster of epithelial cells elongates into a "comma" shape followed by a characteristic S­

shaped tube.

Differentiation of the epithelial cells ensues to form the functional cells of the mature

nephron: capsule cells, podocytes and distal and proximal tubule cells. During

differentiation, a continuous lumen is formed between the UB, destined to become the

renal collecting ducts, and the newly formed nephron by the breakdown of the basal

lamina between the two structures, allowing material to pass from the nephron to the

collecting duct (Bard et aI., 2001).

Signal transduction pathways associated with lddney development

The embryonic kidney develops as the result of reciprocal induction from the

condensing MM and the DB. AB described by Saxen (1970) and Sariola (1982), if the

MM is induced by other tissues, such as embryonic salivary gland or neural tube tissue,

the MM responds by forming only kidney tubules and no other structures. GDNF,

synthesized in the condensing MM, causes the UB to branch from the primary nephric

duct. GDNF binds to and activates a receptor complex consisting of Ret receptor

tyrosine kinase and GDNF family receptor al (GFRaI) (Airaksinen and Saarma, 2002).

The GDNF receptor complex is synthesized in the primary nephric duct and becomes

concentrated in the DB (Schuchardt et aI., 1996). GDNF also induces secondary buds

5

once the UB has entered the MM (Sainio et aI., 1997). Gdnr'- mice demonstrated renal

agenesis and died shortly after birth, as did mice deficient for the GDNF receptor (Moore

et aI., 1996; Pichel et aI., 1996; Sanchez et aI., 1996; Gilbert, 2000).

The VB induces the MM to congregate at its tips and differentiate to generate the

functional cells of the nephron. Grobstein (1955) and Koseki et aI. (1992) showed if the

MM is not induced by the VB, the mesenchymal cells undergo apoptosis. The signals

from the VB resulting in induction of the MM include FGF2 and BMP7. Both factors

inhibit apoptosis and promote the condensation of MM cells around the tips of the

branching UB (perantoni et aI., 1995; Dudley et aI., 1995; Luo et aI., 1995).

The transcription factor Pax2 indirectly promotes the mesenchymaI-to-epitheliai

transition by upregulating the expression of Ret as part of the GDNF receptor complex on

the UB epithelium. It is this epithelium that induces the condensation and differentiation

of the MM. In PaxT- mice, Ret loses expression in the nephric duct by EI0.5 and

ureteric buds capable of inducing the MM do not form, resulting in rena1 agenesis (Torres

et aI., 1995). Hence, Pax2 is an important factor necessary for establishing and

maintaining the nephrogenic zone.

Six2, a transcription factor expressed in the nephrogenic zone MM, is required to

suppress the mesenchymal-to-epitheliaI transition initiated by the VB in order to maintain

an adequate number of progenitors. The failure to renew the mesenchymal cells

responsible for nephrogenesis results in severe renal hypoplasia (Self et aI .• 2006). Six2

appears to work in conjunction with Eya proteins, which loca1ize to the nucleus and may

act as a coactivator and up-regulator of Six2 transcription (Pignoni et aI., 1997; Ohto et

6

al.. 1999; lou et al .• 2004; Purcell et al .• 2005). Gong et al. (2007) identified a Six2

promoter binding site crucial for this activation and proposed a Hoxll-Eyal-Pax2

network that translates anterior-posterior positional information within the embryonic

kidney. Brodbeck et al. (2004) showed Six2 possesses a transcriptional activation

domain in the C-terminus and may activate various kidney morphogenetic factors. such

as GDNF. Thus. Six2 appears to play an important role in nephron development.

The Br mouse as a model for abnormal facial and kidney development

A mouse mutant on the 3Hl background strain with frontonasal dysplasia has been

identified (Lozanoff, 1993). This phenotype. associated with the semidominant

Brachyrrhine (Br) mutation, was induced during the testing of irradiation effects on

chromosome structure. The adult Br mouse displays a severe median facial cleft, as

described by DeMyer (1967). resulting from the failure of the MNP. LNP and MAX to

fuse. Linkage analysis showed that the likely candidate involved in the mutation is Six2.

located on distal chromosome 17 (Table 1; Fogelgren et al .• unpublished data).

Additionally, the Br mouse exhibits renal hypoplasia. This type of malformation is

coupled with a decrease in Six2 expression in the differentiating metanephric

mesenchyme.

The long-term goal of this research is to determine the role of Six2 in embryonic

development using the Br mouse model. To achieve this goal, we will first examine the

expression of Six2 in embryonic tissues. The first specific aim is to characterize the

expression of Six2 in the facial prominences in wild-type ( +/+). heterozygous (Br/+) and

7

Table 1. Microsatellite recombination data. 8r mutation mapping data using microsatellite markers along mouse chromosome 17. Shown are both Cast and Balb backcrosses where X is number of recombinants among N total backcrossed mice analyzed. The calculated di stance (in centimorgans) and LOD scores are shown on the right. Permiss ion to present data kindly provided by Fogelgren et a1. (unpubli shed data).

Cast Marker X

D17Mit122 14 D17Mit155 7 D17Mit189 3 D17Mit56 1 D17Mit76 1 D17Mit21 1 0

Br MS3 0

MS34 4 MS25 5 MS6 5

D17Mit123 17

Balb Marker X

D17Mit155 5 D17Mit76 1

Br

N Distance (cM) 513 2.73 ± 0.72 513 1.36 ± 0.51 513 0.58 ± 0.4 720 0.14 720 0.14 720

720 720 0.56 720 0.69 720 0.69 513 3.31 ± 0.79

N Distance (cM) 220 2.27 ± 1.00 248 0.40 ± 0.40

LOD 127 138 146 213 213

206 204 204 122

LOD 56 72

D17Mit1 23 18 250 7.20 ± 1.63 47

homozygous mutant (8rI8r) embryos. Previous work in our laboratory showed that Six2

express ion is reduced in the craniwll of mutant mouse embryos and thi s work will build

upon the previous findings by examining express ion In individual facial

prominences. We expect that expression will be reduced most III the MNP of BrlBr

embryos since these mice show median fac ial clefts, with intermediate expression in Brl +

mice, and greatest expression in +1+ aninlals. The significance of thi s work is that it

will show whether Six2 expression is associated with specific facial prominences and

8

median facial clefts. At the same time, it will provide justification for establishing an in

vitro system for experimental testing of Six2 function in embryonic tacial tissues.

The second specific aim is to determine whether the UB branching pattern in the

mutant embryos is reduced using an in vitro system. Previous work in our laboratory has

already shown conclusively that Six2 expression is reduced in the kidney of mutant

embryos. Thus, we expect that branching will be reduced in Brl+ mice and severely

reduced in the BrlBr condition when compared to the wild-type (WT) kidney. The

significance of the current work will show whether this reduced expression is associated

with decreased UB branching and establish a whole organ kidney culture system as an

experimental basis for future work assessing the effects of Six2 down-regulation on UB

branching.

The third specific aim is to apply RNA interference (RNAi) technology to determine

whether Six2 expression can be reduced in an in vitro system. This objective will be

accomplished by determining whether Six2 is expressed in an established cell line using

qRT-PCR methodology. If present, RNAi, including Six2 shRNA transfection into the

cell line, will be applied with the expectation that Six2 expression will be reduced. If so,

then this work will establish a method to decrease Six2 transcription in an in vitro

system. The significance of this work is that it will lay the foundation for future

experiments to determine the effects of Six2 on embryonic tissues in vitro.

9

MATERIALS AND MEmODS

Animals

All procedures were carried out in accordance with IACUC specifications and were

approved by the Laboratory Animal Services, University of Hawai'i. Adult 3Hl mice

were housed under standard conditions with a 12-hr light cycle and supplied with tap

water and Purina Mouse Chow ad libitum. Embryos were obtained via reciprocal crosses

of Br adults. Females were examined for a vaginal plug; if present, the day was

designated EO.5. Embryos were obtained on day ElLS for analyses regarding facial

development, since this is the critical point during which the MNP merger occurs.

Kidneys were collected at E13.5, since this is the developmental period when the ureteric

bud interacts with the metanephric mesenchyme and has begun its initial subdivisions.

All were staged using Theiler criteria (TS) ensuring the developmental stage of each

embryo was similar to the conception day (E) designation (Theiler, 1989). Only animals

of the same E designation and TS were compared. At the appropriate embryonic stage,

the gestational female was anesthetized with an isoflurane inhalant, cervical dislocation

performed and embryos collected via Caesarian section.

Genotyping

Previous physical mapping analysis showed the Br mutation is located in an

approximately 171 kb region of murine chrornsome 17 that includes only one known

gene; namely Six2. Microsatellites were tested for recombination to establish a primer

suitable for genotyping (Fogelgren et al., unpublished data). To generate animals that

10

could be genotyped successfully, 3HI Brl+ mice were outbred with inbred lines of

Castaneous (3HI Brl+ x Cast) and Balb (3HI Brl+ x Balb) mice. DNA samples were

obtained from each embryo using extraneous tissue after the kidneys or facial

prominences were dissected free from surrounding structures. Genotyping was

conducted using primers to amplify D17Mit76 (D17Mit76-f: 5'-AGC AAA OCT TAG

TGT TIC OC-3'; and D17Mit76-r: 5'-GOG GAT GCA AGT TAC TCC TC-3'). All

primers were synthesized at the University of Hawai'i Biotech Core, Honolulu, HI, UH

core facility. Pairs of oligonucleotides were amplified using a Thermo Electron

thermocycler with a PCR profile consisting of an initial denaturation at 94°C for 4

minutes, then 35 cycles of 30 seconds at 94°C (denaturation), 30 seconds at 55-60°C

(annealing), and 30 seconds at 72°C (extension), with a final extension at 72°C for 4

minutes. The PCR products were separated by electrophoresis in 4% Metaphor agarose

gels and stained with ethidium bromide. The gels were photographed with a Kodak Gel

Logic 200 photographic module. Each gel included a 25 bp size ladder, blank water

control, control3HI, control Balb or Cast and the embryos for analysis.

Genotyping was based on the number of amplimers present. An embryo that

displayed only one 3Hl amplimer was scored as a homozygous mutant (BrIBr) since it

only possessed the 3HI resulting from the outcross. If two amplimers were present, it

was identified as a heterozygous mutant (Brl+) since it possessed both a 3Hl and

outcross allele for D17Mit76 (one 3Hl and one of either Balb or Cast). If one amplimer

consistent with the Balb or Cast allele was present, the sample was identified as a

homozygous WT animal (+I+).

11

qRT-PCR ofSix2 in facial prominences of WT and Br mice

Previous work in our laboratory demonstrated the kidney in Br mice demonstrates a

baploinsufficient expression pattern of Six2 utilizing the quantitative real-time

polymerase chain reaction (qRT-PCR). However, the Br mutant mouse strain had not

been tested for Six2 expression in the facial prominences. Thus, we undertook a qRT­

PCR analysis of the facial prominences to determine whether Six2 is expressed in a

baploinsufficient pattern in the facial prominences of homozygous mutant, heterozygous

and homozygous normal 3HI x Balb and 3HI x Cast mice.

Dissection of the facial prominences for mRNA extraction was carried out at EII.S, as

this is the stage at which the MNP merger occurs and contact is established between the

MNP and MAX, beginning the continuity of the upper lip (Wyszynski, 2002). The

paired MNP, LNP and MAX were dissected using microforceps and placed in RNA/aler

(Ambion) until genotypes could be confirmed. The dissection was accomplished by first

staging the embryo, placing it laterally, and then removing the MAX. The embryo was

then placed in the frontal position and the MNP and LNP were separated from the

remaining cranium. The nasal pits were then transected at the superior and inferior points

separating the LNP from the MNP. After each of the prominences were dissected away

from the surrounding structures, any extraneous tissue seen still adhering to the

prominence edges was removed. This ensured mRNA was extracted only from

prominence tissue and not surrounding structures. Each pair of facial prominences were

placed immediately and individually in 400 ilL of RNA/ater and stored at 4°C for one to

three weeks before processing. A total of 22 embryos from 4 litters derived from

12

reciprocal 3Hl x Balb Brl+ or 3Hl x Cast Brl+ crosses were collected. Each litter

contained +1+, Br/+ and BrlBr embryos. A separate set of litters (2) were obtained from

stock 3Hl, Balb or Cast matings to provide additional (12) control embyros.

mRNA from individual pairs of facial prominences was extracted using Qiagen's

RNeaY}' Mini Kit according to the included protocol for animal tissues. Total RNA was

reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad) and the

included protocol. qRT-PCR reactions (25 ilL final volume) were performed in triple

replicates with 1 ilL of cDNA, 1 ilL of each primer and 12.5 ilL of Bio-Rad's IQ SYBR

Green Supermix with the MyiQ iCycler thermocycler and single color real-time PCR

detection system (Bio-Rad). Primers to amplify Six2 (Six2-f: 5'-CTC ACC ACC ACG

CAA GTC AGe AAC-3'; and Six2-r: 5'-CAC CGA CTT GeC ACT GeC ATT GAG-

3') and the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH;

GAPDH-f: 5'-TGe ACC ACC ACC TGe TTA Ge-3'; and GAPDH-r: 5'-GGC ATG

GAC TGT GGT CAT GAG-3') were used. The thermocycle profile used was an initial

denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 15 sec (denaturation),

59°C for 30 sec (annealing), noc for 60 sec (extension) and 80°C for 6 sec

(quantification of fluorescence). Product-specific amplification of Six2 and GAPDHwas

confirmed by melting curve analysis. The threshold cycle, C(t), was established at the

linear portion of the log scale curve and the ratio of Six2 to GAPDHwas calculated using

the 2-M C(t) method (Livak and Schmittgen, 2001).

13

Kidney organ explants

The uterus was removed and placed in PBS. Under an Olympus SZ-CTV dissecting

microscope, each EI3.5 embryo was dissected from the uterus and placed in PBS in its

own 35 rom tissue culture dish. Using microforceps, the viscera of the abdomen was

carefully removed as not to damage the posterior abdominal wall. Once the nephric duct

was identified, it was resected laterally to expose the kidney, gonad and adrenal gland.

With the forceps, the kidney was gently loosened from the underlying and surrounding

tissue and placed on a Nuclepore filter in an individual well of a 24-well culture plate

filled with 200 ).lL of PBS until all embryos in the litter had been dissected. Kidneys

were then fixed in 100% methanol at -20°C.

To visualize renal branching patterns, tissues were permeabilized with 0.1% Triton-X

solution and non-specific binding was blocked with 10% normal donkey serum (NDS).

Incubations with the polyc1onal primary antibody specific for ca1bindin-D28k (rabbit

polyc1onal anti-ca1bindin, I: 100 dilution in PBS; Cemines, Santa Cruz Biotechnology)

and secondary (Alexa 546, donkey anti-rabbit, 1 :2000 dilution in PBS; Molecular Probes)

antibodies were performed overnight at 4°C. Visualization was carried out under

fluorescent microscopy with an Olympus BX-41 microscope (TRITC filter) and an

Olympus DP-II photographic module.

cDNA synthesis / qRT-PCR / quantification ojSix2 in stock P 19 cells

P19 embryonic carcinoma cells were obtained from ATCC (Manassas, VA) and were

cultured in MEM Alpha Medium with 2.5% fetal bovine serum plus 7.5% calf serum.

14

mRNA was extracted and cDNA synthesis carried out as previously described. qRT­

PCR was perfonned to amplifY Six2. qRT-PCR reaction volumes (run in triple

replicates) were previously described. A serial dilution of a Six2 plasmid was used as a

standard by which the P19 cDNA was compared to establish a Six2 concentration. The

thennocyc1e profile used was an initial denaturation at 94°C for 2 min. followed by 35

cycles of 94°C for 15 sec (denaturation), 59°C for 30 sec (annealing), noc for 60 sec

(extension) and 80°C for 6 sec (quantification of fluorescence). Following every

extension step, fluorescence was measured using a MyIQ Single Color Real-Time PCR

Detection System. Following qRT-PCR, product-specific amplification of Six2 was

confinned by electrophoresis in a 1 % USB agarose gel.

Transfection ofSix2 shRNA into P 19 cells

Transfection of Six2 shRNA into Pl9 cells was accomplished using Lipofectamine

2000 Transfection Reagent (Invitrogen). One day before the transfection, lOS cells were

plated in 500 III of culture medium sans antibiotics and allowed to proliferate to 90-95%

confluence at the time of transfection. On the day of transfection, 0.8 Ilg of each of five

different plasmid shRNA constructs was diluted in 50 ilL of Opti-MEM and gently

mixed. 2 ilL of Lipofectamine was diluted in 50 ilL of Opti-MEM and mixed gently.

Both solutions were incubated at room temperature for 5 min. The DNA and

Lipofectamine solutions were combined, mixed by pipetting and incubated for at least 20

min. The resulting 100 ilL mixture was added to cell culture plate.

15

Quantification ofSix2 in P 19 cells following transfoction with shRNA

First strand cDNA synthesis, from an mRNA template (mRNA concentration, 200

ng!!!L), was performed as previously described. qRT-PCR was performed in triple

replicates to amplify Six2 and GAPDH. Primers to amplify Six2 and GAPDH were

previously described. The thermocycle profile and fluorescence quantification used was

the same as when Six2 expression was measured in the stock Pl9 cell line. In order to

analyze the knockdown efficiency of each of the shRNA constructs, the ratio of Six2 to

GAPDH was calculated using the 2-M C(t) method (Livak and Schmittgen, 2001). Specific

amplification of GAPDH was confirmed with melting curve analysis.

16

RESULTS

Quantification ofSix2 in the facial prominences

The anatomy of the facial prominences, from which mRNA was extmcted, is shown in

Figure 1. Of the microsateIlite markers analyzed in Table 1, D17Mit76 and D17Mit56

both had only 1 recombination out of 720 mice (LOD score of 213), thereby placing the

Br mutation just distal to DI7Mit76 (Table 1). Thus, DI7Mit76 was considered

informative and used to genotype mice in subsequent analyses since it is likely to be

nearest to the site of the Br mutation (Fogelgren et al., unpublished data). Genotypes

were assigned based on the scoring of gels following electrophoresis. An example of a

4% Metaphor agarose gel is seen in Figure 2. For 3Hl Br/+ x Balb Br/+ or 3H1 Br/+ x

Cast Br/+ crosses, a genotypic ratio of 1 :2: 1 (+/+ : Br/+ : Br/Br) was expected. If a gel

showed what appeared to be an unusual ratio, PCR amplification of D 17Mit76 was re-run

and a second gel photographed to confirm genotypes.

qRT-PCR of facial prominence mRNA began with melting curve analysis to confirm

the specific amplification of Six2 (Figure 3a) and GAPDH (Figure 3b). Melting curve

analysis also ruled out the possibility of primer-dimers. Each curve demonstrated a

single peak, confirming a single sequence had been amplified by each primer set and that

the primers were satisfactory for future qRT-PCR runs.

17

Figure 1. Wild-type El1.5 mouse embryo during dissection of the facial prominences. The MAX were di ssected first fol lowed by the LNP and MNP. (A,B) Anterior view showing the MNP, LNP and MAX. (C,D) Profile view showing the right MAX. (E) MNP and LN P are visible fo llowing dissection. (F) MAX following di ssection.

18

25 bp H20 3Hl Balb Br/+ Br/+ +/ + BrlBr Or/+ Or/+ Orl+ DrlBr , . ~ .

iii

-Figure 2. Photograph of 4% Metaphor agarose gel used for genotyping. Genotypes were scored based on the number of amplimers seen. One band at 3H I was scored BrIBr, one band at BaJ b (or Cast) was scored +/+, a band at 3H I and Balb (or Cast) was scored Br/+ ). Photograph taken with a Kodak Gel Logic 200 photographic module.

A a

• ••• ~ .•

;-, ;.co ~- -

JIi. • •

B

r If

'J .

- -

, !~ t!~\ In

'I

- A~

• • ,_ .... -11

R i

" ---

Figure 3. Melting curve analyses to confirm specific amplification of Six2 and GAPDH in facial prominences. A. Melt curve fo r Six2. Melting point - 88.S°C. B. Melt curve for GAPDH. Melting point - 84°C.

19

qRT-PCR was utilized to compare Six2 expression among facial prominences (Figure

4, Table 2) and genotypes (Figure 5, Table 3) using the 2,MC(I) method (Livak and

Schmittgen, 2001) and GAPDHfor standardization. Three sets of each prominence were

run in triple replicates and C(t) values averaged. Among the wild-type prominences, the

MNP showed the highest expression of Six2 and, thus, was set as the standard by which

the other prominences were compared throughout the study. The expression of Six2 in

wild-type MAX was less than 45% of that in the MNP while LNP displayed 29% the

expression level ofMNP. The heterozygous MNP displayed the highest expression level

of Six2, while the MAX and LNP expression was 53% and 27% of the MNP,

respectively. Among the homozygous mutant population measured for Six2 expression.

the expression pattern paralleled that of the wild-type and heterozygous conditions: the

MNP showed the highest expression of Six2 with the MAX expression 71% and LNP

expression 58% of the wild-type.

When Six2 expression was quantified among genotypes, consistent results confirmed

highest expression in each of the wild-type prominences with lower expression in the

Brl+ and lowest in the BriBr. Among the MNP, expression of Six2 decreased by 60% in

the Brl+ and 93% in the BriBr. LNP expression of Six2 decreased by 62% in the Brl+

and 87% in the BriBr. The MAX Brl+ prominences decreased expression 50% while the

BrlBr lost 89% expression of Six2.

20

A

B

c

1.'

8 1.2

';; .. e ~ 0.8

N ,tj o.e VI

" MNP > 0.' ',"

~ '" 0.2

0

Prominence

1.2 ,----------------------,

8 ';; .. e 0.8 +----1 ~ " ~ o.e +-----! VI

" ~ 0.' +------1 ~ '" 0.2 +-----{

MNP

o +-_--.L __ J.ilJ~~ Prominence

1.,----------------------, 8 1.2

';; .. ~ 08 +----1 .... • /:! 0 e +-----{ VI

.~ 04 +-----{ .. ~ 0.2 +-----{

MNP

O +-----~------~ Prominence

Figure 4. Differences in Six2 expression among facia l prominences in E lLS +1+, Brl + and BrlBr embryos. (A) +/+ (B) Br/+ (C) Bri Br. mRNA was extracted from fac ial prominences and Six2 expression measured by qRT­peR and standard ized GAPDH expression.

21

Table 2. Data obtained from qRT-PCR to quantify Six2 expression in facial prominences among genotypes.

Relative Genotype Prominence C(t) Six.2 C(t) GAPDH expression a

of Si,2 19.9 17 2

MNP 19.9 16.9 1.02 .27 19.2 169 ..................... ··ii:;r············i6:S····· .. ............. .

+/+ LNP 21 .1 16.7 .20 0.03

20.9 16.6 ············ ·········----2i·0--····.·······1':; .. 0······-,.", MAX 20.8 17.0 0."3 0.03

20.4 16.6 21.0 17.0

MNP 20.9 16.9 1.0 0.03

20.8 16.8 ········ ·· ····23."5 ····'1"8""······················ Br/+ LNP 23.5 17.7 0.27 D."

23.5 17.3 ................... 22" .. 1"· ··· ······ --':;7:3" ········ ················· ···· ··

MAX 22.0 17 2 ' .53 0.03

21.9 17.0 24.1 17.4

MNP 23.8 17.2 1.01 0.15

23.0 16.7 ..................... ····24j··· ··········171""····· ................................. . Br/Br LNP 24.6 17.3 0.58 0.10

24.5 16.9 .... ·············i5:S-····· ···iiiT ··

MAX 25.3 18.4 0.7 1 0.06

25.0 17 8

22

A

B

c

1.4.,----------------------,

.~ 1.2 +------+----------------1

" " ~ Co ~ 0.8 +-----1 '" ;)l 0.0 +-----1

.~ +-----1 _ 0.4 ~

.!! +-----1 0.2

.,. o +-----~--------~

Genotype

1.2,-----------------------,

,~ " " ~ 0.8 +----1 ~ ~ 0.6 +-----i II)

" ~ 0.4

" ex: 0.2 +-----i

O +-----L-------~ Genotype

1.2 ,--------------------,

.~ " .. ~ 0.8 +----1 ~ ~ 0.0+----1 II)

~ 0.4 +----1

i 0.2 +-----i

O +---~------~~~ Genotype

Figure S. Differences in Six2 expression among genotypes in EIl.S facial prominences. (A) MNP (B) LNP (C) MAX. mRNA was extracted from fac ial prominences and Six2 expression measured by qRT­peR and standard ized GA PDH expression.

23

Table 3. Data obtained from qRT-PCR to quantify Six2 expression in facia l prominences a mong facia l prominences.

Relative PromInence Genotype C(I) S .. 2 C{I) GAPDH expresston a

of Six2 19.1 .17.2

+/+ 19.' .16.9 1 02 027

; ......................... ~~~ ............. ;.'.~:~ ........................................... . ~, 21 .0 -' 7.0

MNP 8rl+ 20.9 .16.9 0.40 0,01

LNP

MAX

I····~~····· · ~:;· ···· ~~n ·····~:~; · · ···· ····~ .. ~·; ···· 2"

+/+ 211

.'68

.16.7 100 009

~ ......................... ~.~ ............. :~~ ~~ ........... ............................... . 23.5 -17.8

Brit 23.5 -17.7 0.38 006

r" ...................... ~·t· ........ .. -~ ~ i ~i--' -----_ .. -_ ... --- .. -_ .... -_ .... _ .... _ .. . Br/Br 2.1;8 .17.3 0.13 0.02

2" S .16.9 210 .17.0

+/+ 20& .17.0 100 0.06

l··· ··· ··············-····ii·; · ··--········~!~·~····· ·· ................................... . Br/+ 22' ·17.2 0.50 0 03

i 2U1 -17.0 } ........................................................................................... . j 25.6 -18.6

EkIBr 25~

25.'

.184 _17.8

0.11

Kidney whole mount immunohistochemical staining

Kidney dissections were carried out at E 13 .5 for the wild type, heterozygote and

homozygous mutants as shown in Figure 6. These kidneys were immediately fixed,

followed by staining for VB branches. Visualization of the UB branches was by indirect

immunofl uorescent microscopy using antibodies directed against the calcium-binding

protein Calbindin-D28k. While absent in the renal tubules derived from the MM,

metanephric UB branches contain Calbindin-D28k (McIntosh et aI., 1986). Thus,

CaJbindin-D28k is suitable for labeling only structures derived from the UB while MM

derived structures are not immunolabeled. Wild-type kidneys were expected to show the

24

A

c

CUT I

..­'''''

cur:z Hi'Id lionb

B

D

CUT 3 CUl •

~-CUTe

Figure 6. Schematic of dissection protocol for removing E13.5 kidneys. Cuts were made to iso late the abdomen from the rest of the embryo (A), the hind limbs removed (8) and somites removed and latera l resection of the nephric duct (C) allowed for visualization of the embryonic kidney. Kidneys were removed (0) with micro forceps and placed in PBS (based on an illustration provided by Dr. Carl Bates, Dept. Nephrology, The Ohio State University).

greatest number of branches from the VB, while fewer branches were expected in the

BrI+ and fewest in the BriBr.

Figure 7 shows the branching pattern of +1+ , BrI+ and BrlBr kidneys. The greatest

number of UB tips originate form the UB in the wild-type kidney (24). The number of

tips decreases in the Brl+ (16) and further decreases in the Brl Br (5).

25

Figure 7. Branching of the ureteric bud by stage E13.S. Following staining for Calbindin-D28k, newest buds can be seen in the renal cortex (arrowbeads) while older buds reside in the medulla. The ureter can also be seen (arrow). (A) +/ + , (B) BrI+ and (C) BriBr. Bar = IOOl!m.

Quanrijication ojSix2 in stock P 19 cells

In order to determine if embryonic carcinoma P 19 cells expressed Six2 at a level that

could be knocked-down subsequently, a plasmid serial dilution was carried out and qRT-

PCR performed (Figure 8). Following qRT-PCR, the equation y = -3.383(x) + 37.915

was used to calculate Six2 starting quantity in the cell line.

qRT-PCR to confim1 and establish Six2 starting quantity in Pl9 cells was perfom1ed

in triple replicates and the results displayed in Table 4. The P 19 cell culture system

showed a Six2 concentration of 4.2E+O I copiesil!LcDNA' Melting curve analysis for Six2

demonstrated duplex targets. This was confim1ed by gel electrophoresis (Figure 9).

26

35

30

• 1! 25 u ."

20 :g ~ 15

10

: • ' I.

~ ~

: ~

""'- ..... 5

o 2 6 8 10

l og Starting QUllntity,copv numb6

Figure 8. Plasmid serial dilution used to quantify the expression of Six2 in stock P19 cells. Scatterplot shows the negative correlation between starting quantity and threshold cycle. Equation for line used to calculate starting Six2 copy number in PI 9 cells: y = -3.383(x) + 37.915.

Table 4. Data obtained from qRT-PCR to quantify Six2 expression in P19 cells resulting from a serial dilution.

[Six2 ] Avg [Six2] Cell fine C(t) Six2 (copies/~L.DNAl copies/~LeDNAl

32.7 3.4E+01 P19 32.4 4.2E+01 4.2E+01

32.1 5.1E+01

27

- - Qoo'I .... .,g-_ ... _-A :

/) ,

• . J, ~ ~ '0. .~r:/- - ' "

J' , ..• .

• " . • • •

B

150 bp

plasmid P19

Figure 9. Confirmation of specific amplification of Six2 in P19 cell line. (A) Melting curve analysis for Six2 showed two peaks indicating non-specific amplification. (B) Gel electrophoresis for PCR confimled the second peak. Weight of Six2 amplicon, 150 bp.

Quanlificalion ojSix2 in in P J 9 cells jollowing lransjeclion wilh shRNA

RNAi technology was applied to reduce Six2 expression in the P 19 cell line. Six

cultures of P 19 cells were transfected (five with different shRNA constructs and one

mock-transfected to provide a control) to determine if Six2 could be knocked down in a

cell culture system. C(t) values obtained by qRT-PCR for Six2 and GAPDHwere entered

into the T MC(t) method for normalizing gene expression (Table 4) (Livak and Schmittgen,

200 1). The knock-down efficiency of each of the Six2 shRNAs are shown in Figure 10

and Table 5 for the five constructs: 37%, 12%, 15%, 53% and 32% respectively (shRNA

28

1.2.-- ------------------,

~ 1~~_+_,-----._-----~ .~

!! c.. 0.8

" .. :;; 0." V)

~ 0 4

"i Q; '" 0 2

Figu re 10. Relative expression of Six2 in P19 cells following transfcction with shRNA. Five shRNA constructs (1-5) were all able to knock down Six2 expression to some degree, relati ve to the control, mock-transfected cells, as measured by qRT-PCR and standardized GAPDH expression. Knockdown percentages: (1) 34%, (2) 9%, (3) 12%, (4) 51% and (5) 30%.

Table 5. Data obtained from qRT-PCR to quantify Six2 expression in P19 cells following shRNA transfections.

Relative Identifier C(I) Six2 C(I) GAPDH Six2 a

ex£!:ession 23.4 13.6

mock 23.4 13.3 1.01 0.12

23.2 13.1 23.1 13.5 22.7 13.4 0.63 0.11

22.5 13.4 no 13.1

2 22.7 12.9 0.66 0.06 22.5 12.6 23.6 14.1

3 23.5 13.7 0.65 0.16 22.2 13.2 23.4 14.6

4 23.3 14.4 0.47 0.04 23.1 14 23.0 13.6

5 22.9 13.5 0.66 0.03 22.9 13.4

1-5). Specific amplifica tion ofGAPDH was confi mled by melting curve analysis (Figure

II ).

29

__ 0-.,0 _ _ .. ___

• rf'

• • I ' •• ,Ix

- .P \. \\. • .. ~ r • • • . •

Figure II. Melting curve analysis to confirm specific amplification of GAPDH in Pl9 cells. Melti ng point - 84°C.

30

DISCUSSION

Identifying phenotypes proved to be extremely difficult at EIl.5, therefore, grouping

of similar embryos was delayed until confinnation of genotypes could be achieved. Our

lab has previously linked the Br mutation to the homeobox gene Six2, located on distal

murine chromosome 17 (McBratney et aI., 2003). Microsatellites were analyzed for

recombination to identify a suitable primer for differentiating genotypes. Dl7Mit76 was

determined best suited for genotyping due to its low incidence of recombination (I

recombination from 720 total backcross mice analyzed, distance between D 17Mit76 and

Six2 = 0.14 cM) and its PCR products yielding a significant size difference between 3HI,

BaIb and Cast strains that could be differentiated by agarose gel electrophoresis. Even

though no recombination was observed from MS3 (720 total mice analyzed) and the peR

product size was significantly different between 3HI and Cast strains, the size difference

between 3HI and BaIb was too small to differentiate following gel electrophoresis. Thus,

after electrophoresis, a single 3Hl band was scored as a BrlBr, a single BaIb or cast band

was scored +1+, and bands at both 3HI and BaIb/Cast was scored Brl+.

qRT-PCR was performed on mRNA extracted from the MNP, LNP and MAX to

amplify and quantify the expression of the transcription factor Six2. Transcription factors

bind to promoter regions of DNA using ultra-specific binding domains and precisely

regl\late the transcription of relevant genes by initiating RNA polymerase. For example,

homeobox genes encode transcription factors, such as Six2, whose domain

(homeodomain) is responsible for binding to the promoter region and, in turn, initiating

transcription of the target gene. Thus, if a genetic mutation arises in the DNA template

31

and compromises the expression of Six2, the target gene of the transcription factor may

also be misexpressed.

The growth of the facial prominences is activated and regulated by specific

transcription factors (Diewert and Lozanoff, 2002). Oliver et al. (199S) found expression

of the transcription factor Six2 in the developing mid-face to be localized in the neural

crest-derived mesenchymal tissues of the cranium and these cells are responsible for the

proximodistal growth of the frontonasa1 prominence (Marcucio et al., 200S). In this

study, we have reported Six2 expression is highest in the MNP of wild-type embryos and,

in the developing MNP of Br/+ embryos, Six2 expression decreases by more than SO%

while the homozygous mutant shows a 96% decrease. Fogelgren et al. (unpublished

data) observed mesenchymal hypoplasia in Br mice, suggesting Six2 may promote

cellular proliferation in the neural crest mesenchyme. This proliferation of the neural

crest mesenchyme pushes the MNP and MAX together (Senders et al., 2003). Thus, we

suggest the reduction of Six2 expression in Br mice results in mesenchymal hypoplasia,

retarding the growth of the mid-face, leaving the merging of the paired MNP incomplete

and leading to FND with the possibility of cleft palate and/or lip if the fusion of the MNP

and MAX is incomplete as well.

Kidneys at EI3.S were dissected away from surrounding tissues and prepared for

staining for Calbindin-D28k to visualize branching of the VB. Six2 up-regulates

morphogenetic factors such as GDNF. which are secreted from the MM to initiate VB

branching (Brodbeck et al., 2004). Thus, based on the branching morphogenesis of the

E13.S Br kidney, we can propose the influence of Six2 expression on renal

32

morphogenesis. During embryonic days EIl.5 until EI4.5, the UB experiences

approximately 7.5 bifid branching events (Cebrilln et aI., 2004). The UB in the wild-type

kidney demonstrated 24 UB tips, or approximately 5 branching events. The heterozygote

kidney demonstrated 16 UB tips, or 4 branching events, and the Br kidney 5 tips, or

approximately 2.25 branching events. While this haploinsufficient branching pattern

parallels Six2 expression demonstrated in embryonic kidney (F ogelgren et aI.,

unpublished data), our observation of one explant per genotype will need further analysis

to conform our hypothesis. However, the reduced number of UB tips in the Br

embryonic kidney implies a fewer number of glomeruli and thus smaller (hypoplastic)

kidneys could be expected in the adult. This has been reported in BrlBr mice where

Lozanofi' et aI. (2001) found the nephrogenic zone and associated glomerular number was

greatly diminished in the homozygous mutant. Thus, an adult with a mutation in Six2

would be expected to display diminished glomerular development and numbers resulting

in features of chronic renal failure including hypertension, increased filtration rate but

diminished clearance and possibly polyuria. Six2 mutations in human adults might be

expected to predispose a patient to renal disease since a decrease in glomerular number is

associated with kidney disease (Hughson et aI., 2003). Further studies should be directed

to determine whether experimental animals display chronic renal failure.

shRNA transfection begins by introducing a vector containing the shRNA sequence

into a cell culture system. Lipofectamine 2000 (Invitrogen) was used as a transfection

reagent to alter the plasma membrane such that the shRNA could enter the cytoplasm.

Once inside the cell, shRNA is cleaved by dieer RNase into 20-25 nucleotide long small

33

interfering RNA (siRNA). Each fragment of siRNA can bind with RNA induced

silencing complex (RISC) proteins, where the siRNA fragment becomes the guide strand

to which complementary, endogenous mRNA can base pair. The endogenous mRNA is

then degraded via argonaute endonuclease, preventing the translation of the mRNA and,

thus, silencing the target gene, Six2. Therefore. a siRNA approach could be expected to

down-regulate Six2 in vitro.

When P19 embryonic carcinoma cells are isolated into culture, they grow indefinitely

in a monolayer as an undifferentiated cell line (Martin and Evans, 1974, 1975; Martin,

1975). As shown in Figure 7, P19 cells indeed express the transcription factor Six2.

These attributes make P19 cells a suitable candidate for shRNA mediated knock-down of

Six2. However, the presence of a non-specific product may have improperly augmented

the fluorescence in the qRT-PCR, and, in tum, the calculated Six2 concentration. We

hypothesized that a preliminary reduction of gene expression in the P 19 cell culture

system by shRNA by at least 50% should be effective enough to use on whole organ

cultures. Of the five shRNA constructs we tested, one construct did, in fact, reduce Six2

expression by about 50%.

Future experiments will involve the transfection of shRNA into facial prominence cell

culture systems. We have already begun work on establishing cell culture systems for

MNP, LNP and MAX to perform knock-down experiments. Using the most effective

shRNA construct (and combinations of constructs), we will attempt to transfect the facial

prominences and quantifY the reduction in Six2 expression in vitro. When we can

confirm knock-down in all of these cell culture systems, we will attempt whole organ

34

knock-downs of the kidneys and face. We have already established a successful cultw"e

system for E 12.5 kidneys.

Self et al. (2006) has shown the knockout effects Six2 in mice in vivo. These

knockout mice demonstrated the renal hypoplasia phenotype associated with the Br

mutation, however, no observation of FND was reported. Furthermore, Six2+/· mice did

not display any difference in phenotype from wild type mice and Six2 expression in the

craniofacial tissues of knockout mice was not reported. In fact, little is known of the

mechanisms by which Six2 is active in the craniofacial tissues. lfwe can incorporate our

RNAi technology into an in vivo situation, we could quantify and compare Six2

expression in the developing mid-face and kidney of Six2 knock-down and BrIBr and

Brl+ embryos to better understand the relationship between Six2 expression, frontonasal

dysplasia and renal hypoplasia

35

REFERENCES

Airaksinen M, Saarma M. 2002. The GDNF family: signaling, biological functions and therapeutic value. Nat Rev Neurosci 3:383-394.

Apesos J, Anigian GM. 1993. Median cleft of the lip: its significance and surgical repair. Cleft Palate Craniofac J 30:94-96.

Bard JBL, Gordon A, Sharp L, Sellers W. 2001. The early stages of nephron formation in the developing mouse kidney. J Anat 199:385-392.

Barlow AJ, Francis-West PH. 1997. Ectopic application of recombinant bmp-2 and bmp-4 can change patterning of developing chick facial primordia. Development 124:391-398.

Bennett JH, Hunt P, Thorogoood P. 1995. Bone morphogenetic protein-2 and -4 expression during murine orofacial development. Arch Oral Bioi 40:847-854.

Brodbeck S, Besenbeck B, Englert C. 2004. The transcription factor Six2 activates expression of the Gdnfgene as well as its own promoter. Mech Dev 121:1211-1222.

Cebrilln C, Borodo K, Charles N, Herzlinger DA. 2004. Morphometric index of the developing murine kidney. Dev Dyn Nov;231:601-608.

Colvin JS, Feldman B, Nadeau JH, Goldfarb M, Omitz DM. 1999. Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene. Dev Dyn 216:72-88.

Davies JA, Ladomery M, Hohenstein P, Michael L, Shafe A, Spraggon L, Hastie N. 2004. Development of an siRNA-based method for repressing specific genes in renal organ culture and it use to show that the Wtl tumour suppressor is required for nephron differentiation. Hum Mol Genet 13:235-246.

DeMyer W. 1967. The median cleft face syndrome. Differential diagnosis of cranium bifidum occultum, hypertelorism, and median cleft nose, lip, and palate. Neurology 17:961-971.

DeRobertis EM, Morita EA, Cho KWY. 1991. Gradient fields and homeobox genes. Development 112:669-678.

Diewert VM, Shiota K. 1990. Morphological observations in normal primary palate and cleft lip embryos in the Kyoto collection. Teratology 41:663-677.

36

Diewert VM, Lozanoff S. 1993. Growth and morphogenesis of the human embryonic midface during primary palate formation analyzed in frontal sections. J Craniofac Genet Dev Bioi 13:193-201.

Diewert VM, Lozanoff S, Choy V. 1993a Computer reconstruction of human embryonic craniofacial morphology showing changes in relations between the face and brain during primary palate formation. J Caraniofac Genet Dev Bioi 13:193-201.

Diewert VM, Lozanoff S. 2002. Animal models of facial clefting: experimental, congenital and transgenic. Understanding Craniofacial Anomalies: The Etiopathogenesis of Craniosynostosis and Facial Clefting. Mooney M and Siegel MI (eds.). New York: Wiley Liss.

Dudas M, Nagy A, Laping NJ, Moustakas A, Kaartinen V. 2004. Tgf-beta3-induced palatal fusion is mediated by AJk-5/Smad pathway. Dev Bioi 266:96-108.

Dudley AT, Lyons KM, Robertson EJ. 1995. A requirement for bone morphogenesis protein-7 during development of the mammalian kidney and eye. Genes Dev 9:2795-2807.

Fogelgren B, Kuroyama M, McBratney-Owen B, Spence AA, Melahn LE, Anawati MK, Cabatbat C, Alarcon V, Marikawa Y, Lozanoff S. Misexpression of Six2 is associated with heritable frontonasal dysplasia and renal hypoplasia in 3Hl Br mice. Developmental Dynamics, manuscript in revision.

Francis-West P, Ladher R, Barlow A, Graveson A. 1998. Signalling interactions during facial development. Mech Dev 75:3-28.

Gilbert SF. 2000. Developmental Biology. Sunderland, MA: Sinauer Associates, Inc.

Glassberg, KI. 2002. Normal and abnormal development of the kidney: a clinician's interpretation of current knowledge. J UroI167:2339-2350.

Gong KQ, Yallowitz AR, Sun H, Dressler GR, Wellik DM. A Hox-Eya-Pax complex regulates early kidney developmental gene expression. 2007. Mol Cell Bioi 27:7661-7668.

Grobstein C. 1955. Induction interaction in the development of the mouse metanephros. J Exp Zool130:319-340.

Hughson M, Farris AB 3rd, Douglas-Denton R, Hoy WE, Bertram JF. 2003. Glomerular number and size in autopsy kidneys: the relationship to birth weight. Kidney Int 63:2113-2122.

37

Johnston MC. 1964. Facial malformations in chick embryos resulting from removal of neural crest. J Dent Res 43:822.

Johnston MC. 1966. A radiographic study of the migration and fate of the craniofacial neural crest cells in the chick embryo. Anat Rec 156:143-155.

Kanzler B, Foreman RK, Labosky PA, Mallo M. 2000. Bmp signaling is essential for development of skeletogenic and neurogenic cranial neural crest. Development 127:1095-1104.

Koseki C, Herzlinger D, Al-Auqati Q. Apoptosis in metanephric development. J Cell Bioi 119:1327-1333.

Krurn1auf R. 1993. Hox genes and pattern formation in the branchial region of the vertebrate head. Trends Genet 9:106-112.

Levashova ZB, Plisoz SY, Perantoni AO. 2003. Conditionally Immortalized cell line of inducible metanephric mesenchyme. Kidney Int 63:2075-2087.

Livak KJ, Schrnittgen, TD. 2001. Analysis of relative gene expression data using real time quantitative PCR and the 2,MCt method. Methods 25:402---408.

Lozanoff S. 1993. Midfacial retrusion in adult Brachyrrhine mice. Acta Anat (Basel) 147:125-132.

Lozanoff S, Johnston J, Ma W, Jourdan-Le Saux C. 2001. Immunohistochemical localization ofPaxl and associated proteins in the developing kidney of mice with renal hypoplasia. J Histochem Cytochem 49: 1 081-1 097.

Lumsden A, Sprawson N, Graham A. 1991. Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development 113: 1281-1291.

Luo G, Hofinann AL, Bronckers JJ, Sohocki A, Bradley A, Karsenty G. 1995. BMP-7 is an inducer of nephrogenesis and is also required for eye development and skeletal patterning. Genes Dev 9:2808-2820.

Ma W, Lozanoff S. 1999. Spatial and temporal distribution of cellular prolifemtion in the cranial base of normal and midfacially retrusive mice. Clin Anat 12:315-325.

MacKenzie A, Ferguson MWJ, Sharpe PT. 1991a. Hox-7 expression during murine craniofacial development. Development 113:601-611.

38

MacKenzie A, Leeming GL, Jowett AK, Ferguson MWJ, Sharpe PT. 1991b. The homeobox gene 7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth development in vivo and in vitro. Development 111:269-285.

Marcucio RS, Cordero DR, Hu D, Helms JA. 2005. Molecular interactions coordinating the development of the forebrain and face. Dev Bioi 284:48-61.

Martin GR. 1975. Teratocarcinomas as a model system for the study of embryogenesis and neoplasia. Ce1l5:229-243.

Martin GR, Evans MJ. 1974. The morphology and growth of a pluripotent teratocarcinoma cell line and its derivatives in tissue culture. Cell 2: 163-172.

Martin GR, Evans MJ. 1975. Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc Nat! Acad Sci USA. 72: 1441-1445.

Martinez-Alvarez C, Tudela C, Perez-MiguelsanzJ, O'Kane S, Puerta J, Ferguson MW. 2000. Medial edge epithelial cell fate during palatal fusion. Dev BioI 220:343-357.

McBratney BM, Margaryan E, Wenbin M, Urban Z, Lozanoff S. 2003. Frontonasal dysplasia in 3Hl BrlBr mice. Anat Rec 271A:291-302.

McGinnis W, Levine MS, Hafen E, Kuroiwa A, Gehring WJ. 1984. A conserved DNA sequence in homoeotic genes of the Drosophila Antennapedia and bithorax complexes. Nature 308:428-433.

Mcintosh JE, Bourdeau JE, Taylor AN. 1986. Immunohistochemica11oca1ization of ca1bindin-D28k during the development of the rabbit nephron. Anat Rec 215:383-389.

MinkoffR. 1991. Cell proliferation during formation of the embryonic facial primordia. J Craniofac Genet Dev Bioi 11:251-261.

MinkoffR, Kuntz AJ. 1977. Cell proliferation during morphogenetic change; analysis of frontonasal morphogenesis in the chick embryo employing DNA labeling indices. J Embryol Exp MorphoI40:101-113.

Minkoff R, Kuntz AJ. 1978. Cell proliferation and cell density of mesenchyme in the maxillary process and adjacent regions during facial development in the chick embryo. J Embryol Exp MorphoI46:65-74.

39

Moore MW, Klein RD, Fariftas I, Sauer H, Armanini M, Phillips H, Reichardt LF, Ryan AM, Carver-Moore K, Rosenthal A. 1996. Renal and neuronal abnormalities in mice lacking GDNF. Nature 382:76-79.

Nie X, Luukko K, Kettunen P. 2006. BMP signaling in craniofacial development Int J Dev BioI 50:511-521.

Ohto H, Kamada S, Tago K, Tominaga SI, Ozaki H, Sato S, Kawakami K. 1999. Cooperation of Six and Eya in activation of their target genes through nuclear translocation ofEya. Mol Cell BioI 19:6815--6824.

Oliver G, Wehr R, Jenkins NA, Copeland NG, Cheyette BN, Hartenstein V, Zipursky SL, Gruss P. 1995. Homeobox genes and connective tissue patterning. Development 121 :693-705.

O'Rahilly R. 1978. The timing and sequence of events in the development of the human digestive system and associated structures during the embryonic period proper. Anat EmbryoI153:123-136.

PattenBM. 1961. The Normal Development of the Facial Region. Congenital Anomalies of the Face and Associated Structures. Pruzansky S. (ed.) Springfield, IL: Thomas.

Perantoni AO, Dove DF, Karavanova I. 1995. Basic fibroblast growth factor can mediate the early inductive events in renal development Proc Natl Acad Sci USA 92:4696-4700.

Pichel JG, Shen L, Sheng HZ, Granholm AC, Drago J, Grinberg A, Lee EJ, Huang SP, Saarma M, Hoffer BJ, Sariola H, Westphal H. 1996. Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 382:73-76.

Pignoni, F, Hu B, Zavitz KH, Xiao J, Garrity PA, Zipursky SL. 1997. The eye­specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. CeIl91:881-891.

Purcell P, Oliver G, Mardon G, Donner AL, Maas RL. 2005. Pax6-dependence of Six3, Eyal and Dachl expression during lens and nasal placode induction. Gene Expr Patterns 6:110-118.

Reed SC. 1933. An embryological study of harelip in mice. AnatRec 56:101-110.

Richman JM, Crosby Z. 1990. Differential growth of facial primordia in chick embryos: responses of facial mesenchyme to basic fibroblast growth factor (bFGF) and serum in micromass culture. Development 109:341-348.

40

Roizenblatt J, Wajntal A, Diament AJ. 1979. Median cleft face syndrome or frontonasal dysplasia: a case report with associated kidney malformation. J Pediatr Ophthalmol Strabismus 16:16-20.

Rossel M, Capecchi MR. 1999. Mice mutant for both Hoxal and Hoxbl show extensive remodeling of the hindbrain and defects in craniofacial development. Development 126:5027-5040.

Rude FP, Anderson L, Conley D, Gasser RF. 1994. 1bree dimensional reconstructions of the primary palate. Anat Rec 238:108-113.

Sainio K, Suvanto P, Davies J, Wartiovaara J, Wartiovaara K, Saarma M, ArumlIe U, Meng X, Lindahl M, Pachnis V, Sariola H. 1997. Glial cell derived neurotrophic factor is required for bud initiation from ureteric epithelium. Development 124:4077-4087.

Sanchez MP, Silos-Santiago I, Fris6n J, He B, Lira SA, Barbacid M. 1996. Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382:70-73.

Sariola H, Ekblom P, Saxen L. 1982. Restricted development options of the metanephric mesenchyme. In Embryonic Development, Part B: Cellular Aspects. Burger M, Weber R (eds.). Alan R Liss, New York, pp.425-431.

Saxen L. 1970. Failure to demonstrate tubule induction in heterologous mesenchyme. Dev Bioi 23:511-523.

Schilling TF, Kimmel CB. 1994. Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo. Development 120: 483-494.

Sedano HO, Cohen MM Jr, Jirasek J, Godin RJ. 1970. Frontonasal dysplasia. J Pediatr 76:906-913.

Sedano HO, Godin RJ. 1988. Frontonasal malformation as a field defect and in syndromic associations. Oral Surg Oral Med Oral PathoI65:704-710.

SelfM, Lagutin OV, Bowling B, Hendrix J, Cai Y, Dressler GR, Oliver G. 2006. Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney. EMBO J 25:5214-5228.

Senders CW, Peterson EC, Hendrickx AG, Cukierski MA. 2003. Development of the upper lip. Arch Facial Plast Surg 5:16-25,.

41

Shuler CF. 1995. Programmeel cell death and cell transformation in craniofacial development. Crit Rev Oral Bioi Meel 6:202-217.

Theiler K. 1989. The house mouse: atlas of embryonic development. New York: Springer-Verlag.

Torres M, Gomez-Pardo E, Dressler OR, Gruss P. 1995. Pax-2 controls multiple steps of urogenital development. Development 121:4057-4065.

Trasler DO. 1968. Pathogenesis of cleft lip and its relation to embryonic face shape in AlJ and C57BL mice. Teratology 1:33-50.

Vieille-Grosjean I, Hunt P, Oulisano M, Boncinelli E, Thorogood P. 1997. Branchial HOX gene expression and human craniofacial development. Dev Bioi 183:49-60.

Wyszynski DF (eel.). 2002. Cleft Lip and Palate. New York: Oxford University Press.

Zou D, Silvius D, Fritzsch B, Xu PX. 2004. Eyal and Sixl are essential for early steps of sensory neurogenesis in mammalian cranial placodes. Development 131:5561-5572.

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