Pax6 and DIx2 Regulate the Extracellular Matrix Molecule Tenascin-c in vertebrate Forebrain Development

by

Yongyao Tan

A Thesis submitted to the Faculty of Graduate Studies

of

The University of Manitoba

in partial fi.llfilment of the degree requirements for

MASTER OF SCIENCE

Deparhnent of Biochemistry & Medical Genetics

University of Manitoba

Winnipeg

Copyright @ 2008 yongyao Tan TIIE IINTVERSITY OF MANITOBA

FACI]LTY OF GRÄDUATE STUDIES frt!**rt COPYRIGHT PERMISSION

Pax6 and Dlx2 Homeobox Genes Regulate the Extracellular Matrix Molecule Tenascin-C in Vertebrate Forebrain Development

BY

Yongyao Tan

A ThesislPracticum submitted to the Faculty of Graduate Studies of The University of

Manitoba in partial fulfillment of the requirement of the degree of

MASTER OF SCIENCE

Yongyao Tan @ 2008

Permission has been granted to the University of Manitoba Libraries to lend a copy of this thesis/practicum, to Library and Archives Canada (LAC) to lend a copy of this thesis/practicum, and to LACrs agent (tIMI/ProQuest) to microfilm, sell copies and to publish an abstract of this thesis/practicum.

This reproduction or copy of this thesis has been made available by authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws or with express written authorization from the copyright owner. Table of Contents Acknowledgements""' Abstract ...... iv ...... v List of Abbreviations...... vii List of Tab'es...... ix List of Figures""""""' ...... x List of Copyrighted Material for which permission was Obtained...... 1' Introduction ...... xii ...... t 1.1. Forebrain: structure and development...... , 1.2. Dlx genes in development...... 10 1.2.1. Dlx family...... t0 1.2.2. Expression pattern of Dk genes ...... I I 1.2.3. The functionof DIx genes...... _...... 12 1.2.4. Transcriptional targets of Dlx genes...... 1.3. 1g genes ...... 20 1.3.1. Theexpressionpatternofthepax6gene...... ZI 1.3.2. The functio n of p ax6 in forebrain development...... 1'4' ...... 2l Tenascin-c ...... 30 1.5. Hypothesis and Researbh Aims ...... 3g 2. Materials and Methods...... 41 2.1. Animal and tissue preparation ...... 41 2.2. Tissue embedding and sectionirrg...... 42 2.3. Histological staining, immunohistochemishy (IHC) and immunofluorescence (IF)...... 42 2'4' chromarin Immunoprecipitation (chrp) and chrp-rechrp assays ...... 44 2.5. Elecrrophoreric Mobiliry Shift Assays @MSA) ...... 47 2.6. Co-Immunoprecipitation ...... 4g 2.7. Luciferase reporter gene assays ...... 4g 2.8. Site-directed mutagenesis and DNA sequencing...... 3...... 50 Results ...... 52 pattems 3-1' Expression of DLX2, pAX6 and renascin-c Forebrain in the Deveroping ...... 52 3.2. DLX2andpAX6bindto theTenascin_Cgenepromoter ..... 59 3' 2' I' DLx2 and pAX6 bind to the Tenas cin-c promote r in vivo...... 59 3.2.2. DLX2 binds to the Tenascin_C promoter in vitro...... 65 3'3' DLx2 and PAX6 transfection promotes Tenascin-Cpromoter invitro...... expression ...... 69 3.4. DLX2interactswithpAX6 invitroand,invivo...... 4' Discussion' ...... j4 ...... 77 js 4.L Pax6 essential for the expression of Tenascin_C...... j7 4'2' Dlx2 and Pax6 tanscription factors bind to the same region of Tenascin-C promoter"" ...... 7g 4.3- PAx6 and DLX2 interact physically and function in forebrain development ...... g0 5. Conclusions and Future Directions ...... gz 6. Literature Cited. ...,...... g3 Acknowledgements

This thesis just represents not my typing at the keyboard; it is the pinnacle of all of my experiences at the Manitoba Institute of cell (MICB). I have had an unforgettably amazingexperience at MICB. MICB has provided me with lots of opportunities of which to t¿ke advantage. Throughout these years at MICB, I have learned that passion and dedication are indispensable to obtain even small achievements in research. This thesis could not have been accomplished without the help and support from the kind people who are close to or even får from me, and whom I wish to acknowledge.

First and foremost I would like to thank my advisor, Dr. David D. Eisenstat, Director of Neuro-Oncology, CancerCare Manitoba. Dr. Eisenstat is the most outstanding and the busiest professor I have seen, and he is also a diligent medical doctor. He has been extremely supporlive since the day I began to work in the lab. Dr. Eisenstat has supported me not only by guiding me on the research project but also by encouraging me ,.don,t emotionally. I remember so many times when he said to me worry, *" *itt figure it out". I sincerely thank him for supporting me through the rough road to finish this thesis. During the most difücurt times in these years, tre atways gave me the timely and valuable support I needed to move on. My graduate committee has been guiding me in the past three years. Thank you, Dr. Barbara Triggs-Raine and Dr. David Merz for being my committee members. Thanks for all the advice and the encouragement.

A lot of other people have helped and taught me at MICB. shunzhen always is a source of help. Thanks for teaching me techniques and always providing suggestions about experiments and daily life. Mario has been helping me out, not just with purification' Trung is a nice lab mate and a true friend. Qi was my roommate before he was my worlcrnate and I learned a lot from him. Vanessa is much better than my timer' Thanks for all the kind reminders. Mehdi and Miten are other lab mates who have been supportive. Thank Nina for having done some previous work on the project. I also thank other students and researchers who have left the lab: eingping, Jimmy, Fen, Molly, Andrew, Nikki, and Guoyan.

Thanks to the CancerCare Manitoba Foundation, Manitoba Institute of Child Health, and the CIHR training program for financial support.

Last, but by no means least, I wourd like to thank my wife Jiemin for her personal support and great patience. My parents, sister and brother-in-law have given me their never-ending love and support throughout, for which my mere of thanks likewise does not "*pr.rri-on suffice. while falling leaves return to their roots, I am always looking forward to being with you all.

Yongyao Tan, 2008, Winnipeg

lv Abstract

The forebrain is required for complex functions of the vertebrate brain, and d.efects in

human forebrain development can lead to severe neurodevelopmental disorders, such as

autism and epilepsy. The extracellular matrix protein Tenascin-C (Ten-egene promoter

contains candidate binding sites for containing homeodomains, paired domains

and paired class homeodomains. Our hypothesis is that DIx2 and pax6 genes may control

the expression of Ten-C at the striataVneocortical bowrdary, thereby affecting neuronal migration during forebrain development. Our objective is to understand the gene

regulatory network between the factors DLX1/DLX2 andpAx6, and the

candidate target gene Ten-C.

To determine TEN-G expression in both wild fype and mutant mouse embryos, we performed immunohistochemistry (ErC) and immunofluorescence (IF). To study the

protein-DNA interactions of DLX1/DLX2 and/orPAx6 and the Ten-Cpromoter, we

carried out chromatin immunoprecipitation (ChIP) and electrophoretic mobilify shift (EMSA) assays' To study the functional consequences of DLX I/2 andp¡X6-Ten-C promoter interactions, we performed luciferase gene reporter assays.

TEN-C protein expression is unaffecte dnfhe DlxI/Dtx2 double knockout mouse, whereas it is not detected in the forebrains of the PaxÍhomozygous null mouse. Both DLX2 and

PAX6 bind to regions of the Ten-C promoter in vitro utd in vivo and hansactivate expression of a Ten-C reporter gene construct in vitro. Of interest, using ChIp-reChlp assays' both DLX2 and PAX6 bind to a small region of the Ten-Cpromoter in vivo and

DLX2 and PAX6 form protein-protein complexes in vitro and, in situ.

It is unclear why DLX2 transactivat es Ten-C expression in vitro yet loss of DIxl/Dtx2 function does not affect Ten-C expression at the striataVneocortical boundary in vivo.It js possible that PAX6 and DLX2 compete for binding to the Ten-Cpromoter. Further

chatactenzation the of interactions between these transcription factors and their target genes will improve our understanding of forebrain development and help elucidate the mechani sms underlying human neuro deveiopmental disorders.

vt List of Abbreviations ChIP : chromatin immunoprecipitation

CNS : central nervous system

DAB : diaminobenzidine tetrahydrochloride

Dlx: Dlx gene

DLX: DLX protein

DLXllz: DLXI and DLX2

DNA : deoxyribonucleic acid

E#: embryonic day # (i.e. E13.5 : embryonic day 13.5)

ECM : extracellular matrix

EMSA: electrophoretic mobility shift assay

GABA : gamma-aminobutyric acid

IHC : immunohistochemistry

IF: immunofluorescence

LGE: lateral ganglionic eminence

Luciferase: luciferase reporter gene assay

MGE : medial ganglionic eminence

MZ: mantle zone

Pax: Pax gene

PAX: PAX protein

vlt PSB : pallio-subpallial boundary

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

SY Z : subventricular zone

Ten-C: Tenascin-C gene

TEN-C : TENASCIN-C protein

YZ: ventricular zone

WT: wild-type

vll¡ List of Tables

Table 1 : Primary antibodies for IHC and IF...... 44

Table2: Secondaryantibodies forIHC andIF...... 44

Table 3: Oligonucleotide primer sets for ChIp assays ...... 47

Table4: Primersforsite-directedmutagenesis...... 51

Table 5: Mutated candidate binding sites of the Ten-Cpromoter...... 51

Table 6: The putative binding sites of the Ten-Cpromoter...... 77

1X List of Figures

Figure 1:Anatomy of the embryonic mouse forebrain...... 2

Figure 2: Prosomeres are defined by ...... 4

Figure 3: Structure of Tenascin-C

Figure 4: Schematic diagram of the Ten-C gene promoter ...... 37

Figure 5: Expression pattern of DLX2, pAX6, and TEN-G in the developing

forebrain...... 54

Figure 6: The expression of Tenascin-C in Dlxl/2 mutants...... 55

Figure 7:The expression of Tenascin-C inpax6 mutants (IHC)...... 56

Figure 8: The expression of Tenascin-Cinpaxímutants (F)...... 57

pax6 Figure 9: The expression of DLX2 in mutants...... 5g

Figure 10: Sequence of four fragments selected from the Ten-c gene

promoter

Figure 1 1: chrP assays of DLX2 or pAX6 and the Ten-c promoter...... 62

63

Figure 13: EI\{SA of recombinantDLX2 and Ten-C promoter

sequences. 65

Figure 14: Luciferase reporter assays with the Tenascin-c full-length

promoter...... 69 Figure i5: Luciferase repofer assays with four selected fragments of the

Tenascin-C promoter. 77

Figure 16: Luciferase reporter assays with mutated ren-c3 promoter

constructs. -.. -... ..72

Figure 17:Co-IPofDLX2andpAX6...... 74

Figure 18: Models for PAX6 and DLX2 binding to fhe Ten_C

promoter...... g2

Figure 19: updated schematic diagram of the Ten-c gene promoter...... ß2 List of copyrighted Material for which permission was obtained

The following figures are reproduced with written permission from the institution(s) and/orjournals which hold the copyright to the articles which they are from:

Figure i (Wigle, 2008), Blackwell publishing

Figure 2 (Rubenstein, 1994), the American Association for the Advancement of Science

Figure 3, 4 (Jones, 2000), Elsevier Limited

xl¡ 1. Introduction

The forebrain is a vital part of the vertebrate brain. Transcription factors, such as DIx2

and Paxt,play roles in the development of vertebrate forebrain. It is interesting to

find that the expression of DLX2 and PAX6 is separated by a border that contains the

adhesion molecule Tenascin-C. General background information about the vertebrate

forebrain, the transcription factors, Dlx2 and pax6, andthe extracellular matix

protein Tenascin-C will be introduced in this chapter.

1.1. Forebrain: structure and development

In vertebrates, the forebrain is the largest and most complex part of the whole brain.

The forebrain contains an elaborate set of structures, which are required for some

complicated functions of the vertebrate brain (Marin and Rubenstein, 2003). The

forebrain is involved in such diverse tasks as integration of sensory and motor processing, thought and emotion (wilson and Houart, 2004; Marin and Rubenstein,

2003).In forebrain development, cell migration, which disperses neurons across multiple functionally distinct areas to their final destinations, plays an extremely important role. Defects in the development of the forebrain may lead to significant neurodevelopmental diseases, such as autism, epilepsy, and severe learning disabilities (Marin and Rubenstein, 2003).

1.1.1. Anatomical structure of the forebrain

The forebrain consists of two major divisions: the diencephalon and the telencephalon. The diencephalon is corurected superiorly and rostrally (toward the snout) by the cerebral hemispheres; the midbrain is immediately caudal (toward the tail). The telencephalon comprises the largest part of the brain, containing two major regions: the pallium and the subpallium (Figure l) (Marin and Rubenstein,2003; corbin et al., 2001). The pallium primarily expresses dorsal molecular markers; whereas the subpallium primarily expresses venhal molecular markers (Toresson et a1',2000). Between pallium and subpallium there is a pallial-subpallial boundary

(PSB) containing both progenitor and postrnitotic cells in the mouse embryo. These cells can be subdivided into several regions that express particular sets oftoanscription factors (Toresson et al., 2000).

D (b)

5ub-Pollîum

Figure 1: Anatomy of the embryonic mouse forebrain. (a) A schematic diagram of the developing mouse forebrain. In this diagram, the forebrain is separated into the pallium and the subpallium. There is a boundary (anowhead), the pallio-subpallial boundary, between these regions. The pallium contains the neocortex (NCÐ and the subpallium contains the medial ganglionic eminence (MGE) and the lateral ganglionic eminence (LGE). Proliferating progenitor ceils arise from the ventricular zone (Z), with a secondary proliferative population from the subventricular zone (SVZ). þ) Pax6 is expressed in the pallium, whereas DlxI, Dlx2, and Gsh2 are expressed in the subpallium. D: dorsal; V: ventral;LV: lateral ventricular zone;MZ: mantle zone (adapted from Wigle and Eisenstat,2008. Reprinted with permission from Blackwell Publishing). 1.I.2. The prosomeric model of forebrain development

How does the nervous system develop into mature tissues, for example, the forebrain?

There are many models to describe the mechanism of development of the vertebrate

forebrain, among which the prosomeric model is best described and accepted (Puelles

and Rubenstein, 2003; Rubenstein et al., 7994). In the prosomeric model, the

anterior/rostral longitudinal axis of part of the brain from the midbrain and hindbrain

is compartmentalized. The prosomeric model proposes that there are six segments,

named prosomeres, forming the forebrain (Figure 2), similar to rhombomeres of the

developing hindbrain. The entire neuraxis consists of three zones, the ventricular zone

(YZ),the subventricular zone (s\az) and the mantle zone (MZ) (Figure l). These

three zones are located along the longitude of the neuraxis. The majority of neural

progenitors arise from theYZ which is adjacent to the ventricular surface (Puelles and

Rubenstein, 2003; Rubenstein eta1.,1994). Prosomeres pl-p3 are located in the

primary prosencephalon or diencephalon, which includes the pretectum, thalamus and pre-thalamus, and prosomeres p4-p6 are in the secondary prosencephalon, which

includes the telencephalon and hypothalamus (Puelles and Rubenstein, 2003;

Rubenstein et al., 1994).

The prosomeric model hypothesizes that morphological characteristics result from the topographic pattern of expression of diverse genes in the forebrain. It gives emphasis to topological gene expression, which is evolutionarily conserved in the neural tube

(Puelles and Rubenstein, 2003; Rubenstein etal., 1994). This useful model provides a conceptual framework to comprehend the complexities of vertebrate forebrain

development.

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?trr.â,â.W {:*.t;;þ $ift.¡¡æ örz-êm €s#âhhffi }.ti.r.Z.ti;i fitÍ,#ffi

Figure 2: Prosomeres are defined by gene expression. The expression of six genes-Dlx-2,Gbx-2, Nk-2, OÞ.-z and (Sonic hh) in the neural tube (E12.5). The provisional transverse and longitudinal boundaries are indicated as thin black lines. M, mesencephalon-midbrain; os, optic stalk; p, prosomere; r, rhomobomere (adapted from Rubenstein et al, 1994. Reprinted with permission from AAAS).

1.1.3. Comparftnents and boundaries in forebrain

The importance of cell lineage restriction boundaries was first recognized in research on invertebrate development, which showed cellular comparfments were segregated by boundaries in the development of both the abdomen and the wing anlagen (the initial clustering of embryonic cells from which a part or an organ develops) of insect embryos (Kiecker and Lumsden, 2005). Comparftnent boundaries were proposed to have two functions: first, inducing cells to differentiate into different parts of the

embryo and, second, preventing false information to flanking cell populations. Thus,

boundaries are essential for growth and patterning in embryos (Kiecker and Lumsden,

2005). In vertebrates, compartrnent boundaries \ryere first revealed in the development

of chick hindbrain (Fraser et al., 1990). The expression of f/o.r genes in the

developing hindbrain in the chick is much like that inD. melanogaster, showing a

nested pattern of expression and separated by boundaries (Lumsden, 2004; Gavalas et

a1.,7997; Fortin et al., 1995; Heyman et al., 1993).

The skucture of the forebrain is much more complicated than that of the hindbrain. In the telencephalon, it is revealed that a dorsoventral lineage restriction boundary exists between the cortex and the lateral ganglionic eminence (LGE), named the pallio-subpallial boundary (PSB), in mouse embryos (Fishell et al., 1993). The psB only prevents cells entering the ventricular zone,while postmitotic neurons can migrate across this boundary in the mantle zone. Although Hox genes are only expressed in the hindbrain, other genes encoding hanscription factors are expressed in restricted pattems in the forebrain-midbrain area. These genes includes members of the distalless (Dlx), empty spiracles (E**), forkhead (Fox), orthodenticle (otx),paired

(Pax) and sine oculis (.Sr-r) gene families (simeone et a1., 1992; Rubenstein et al.,

Lee4).

However, forebrain markers are expressed in a highly dynamic pattern in the forebrain, both temporally and spatially (Bell et a1.,2001). Based on research regarding the expression of Emxl, Emx2, Ùfl, and Pax6, itwas proposed that

expression domains share some overlapping areas and do not have sharp boundaries

between each other in the telencephalon. It further demonstrated that the pSB is one

of the true cell lineage restriction boundaries in the forebrain (Puelles and Rubenstein,

2003).

So far, there are several descriptions of the function of the PSB during forebrain

development. It was repofed that either Tlx or pax6 mutants have abnormal

formation of the development of this boundary, and the was significantly

worsened when one allele of Paxó was removed from the Tlx homozygous mutant.

This data supported the hypothesis that I'lx and Pax6 regalate the establishment of the

PSB in a cooperative relationship (Stenman et a1,2003).

Studies on the function of the PSB showed that cell movements were restricted at the

PSB, and cell migration was inhibited directly by the boundary (Neyt et al., 1997).lt was also shown that the boundary separated dorsal and ventral telencephalon by either a contact-dependent or a short-range diffrrsible mechanism (Neyt et al., 1997).

Research on the molecular mechanisms of boundary formation also revealed that cadherin superfamily members, cell adhesion molecules, were expressed in a restricted pattern in subdivisions of the brain, suggesting that cadherins play arole in development of the vertebrate brain (Redies et al., 2000). In fact, differential expression pattern of two cadherins in the embryonic telencephalon is responsible for establishing the PSB (Inoue etal.,200l) 1.1.4. Cell migration in the development of the forebrain

cell migration plays an important role in patteming during development of the

vertebrate forebrain. Neurons in vertebrates are generated in the proliferative zone

lining the surface of the cerebral ventricle (Rubenstein and Beachy, 1998). After their

genesis, corlical neurons migrate to different destinations (Marin and Rubenstein,

2003).

Two general types of migration have been identified in the forebrain on the basis of its orientation, radial migration and tangential migration. Radial glia are the usual

substrate for radial migration, which extends from the ventricular zone out to the surfäce of the cortex. Radial glia is used as a scaffold by neocortical cells bom in the cortical ventricular zone (Kriegstein and Noctor, 2004). Radial glia was first reported by Dr. Rakic who pointed out that there was migration of neuroblasts from their origin to their destination (Rakic, 1972). Radial glial cells are generated from the ventricular zone during the early development of the neural tube (Gotz e|a1.,2002).

Radial glial precursor cells may not only generate newbom neurons, but also provide guidance for daughter neurons to migrate to their destinations (Kriegstein and Noctor,

2004; Noctor et al., 2002; Noctor et al., 2001).

Radial migration can be described as four sequential steps: initiating movement; aftaching to the radial glial fiber; migration along the fiber that involves nucleokinesis; and detaching from the radial glial fiber and acquiring the correct destination position (Marin and Rubenstein, 2003). Atthough the radial glial scaffold acts as the primary guidance system for cell migration in the central nervous system

(CNS), there exists another pattem in which cells movement in the forebrain doesn't

use this glial fiber system (Marin and Rubenstein, 2003).

Tangential migration, also called non-radial migration, is another form of cell

migration in forebrain development, in which cell movement is perpendicular to radial

glia (wozriak, 1999). Tangential migrating cells may not use radial glia as their

substrate. Some neurons use each other as the scaffold to promote migration, for

example, olfactory bulb interneuron pïecursors. Some neurons precede tangential migration by following the growing to approach their destination. other neurons that migrate tangentially may disperse in a rather individual manner instead

of following speciflrc cellular substrates, such as the cells migrating from the

subpallium to the pallium (Marin and Rubenstein, 2003). It is also shown that tangentially migrating cells travel long distances to their residence sites, which raises the question as to the identity of the molecular cues that guide these cells to their

ultimate destinations (Zhu et al., 1999) .

A large majority of cells originate from the embryonic subpallium and migrate tangentially toward and hippocampus in development (Cobos et al.,

2001; Anderson et a1.,2001). The subpallium generates gaÍrma-aminobutyric acid

(GABA)-ergic interneurons and cortical oligodendrocytes. Interneurons destined for the striatum originate from the MGE (cobos et al., 2006; Marin and Rubenstein,

2003). The tangential migration of intemeurons from the subpallium and pallium is influenced by three different types of factors including factors that stimulate the

movement of interneurons, structural elements that constitute the substrate for their

migration, and cues that direct intemeurons toward their target through the

appropriate pathways (Marin and Rubenstein, 2003).

The¡e are very restricted routes for intemeurons to migrate towards the cortex. When

they initiate their migration, interneurons whose destination is cortex have to avoid

entering the striatum. It is observed that neuropilins are receptors for semaphorins,

which are inhibitory cues, and migrating intemeurons expressing neuropilins are

guided to the cortex; those lacking them enter the striatum. These observations

provide evidence that these,receptors also control intemeuron migration in the CNS

(Marin etaL.,2007; Le et al., 2007).

Adhesion molecules are proposed to play a role in cell migration in the developing

forebrain. Adhesion molecules have been observed in the migratory route of olfactory interneuron precursors, which migrate via the rostral migratory stream (RMS). The extracellular matrix (ECM) may also play a role in olfactory interneuron precursors migration (Murase and Horwitz,200zb). Taking the ECM protein Tenascin-c as an example, it is a for ovB3 and ovp6 integrins (yokosaki etal., 1996). olfactory precursors migrate in the adult RMS through a pathway formed by astrocytes that express high levels of Tenascin-c (Jankovski and Sotelo, 1996), Moreover, cv-, p3-, and p6-integrin subunits are also expressed in the postnatal RMS. Additionally, ol and Bl integrins receptors and o5 and yl laminins ligands are also expressed in the route of olfactory interneuron precursors in embryos (Murase and Horwitz ,2002),

which suggests that alBl integrins can be used by migrating cells.

1.2. DIx genes in development

DIx genes are the vertebrate homologs of the homeobox gene Distal-less

gene (Dl[), which is the ear]iest known gene specifically expressed in developing

insect limbs and maintains expression throughout limb development. Tlhe Dlx gene

encodes a homeodomain that binds to DNA with ATTA/TAAT

containing sequences, similar to other homeodomain proteins. The DIx genes are

found in all vertebrates. The Dlx genes of vertebrates are involved in a variety of developmental pro""r.", ranging from ,r"urogenesis to hematopoiesis (Merlo et al.,

2000; Panganiban and Rubenstein,2002; Zrtou et a1.,2004).

7.2.1. Dlx gene family

There are six known DIx genes found in mice and humans: DIxI, Dlx2, Dlx3, Dlx4

(also known as DIxT or DlxS), Dlx5, and Dlx6 (ozcetik et al., 1992;pangaHiban and

Rubenstein, 2002;Yanget al., 1998c; Quinn et al., l99g; Nakamura et a1.,1996;

Robinson and Mahon, 1994; v/eiss et al., 7994; ozcelik et al., 1992; Robinson et al., i991). In mouse and human, the Dlx genes are found to form three convergent

(tail-to-tail arrangement) transcribed pairs or bigenic clusters. These pairs include

Dlxl and Dlx2, Dlx3 and Dlx4, and Dlxï and DIx6. Each 5' to 3', 3' to 5' bigenic gene pair is linked to a Hox cluster: Dlxl and Dlx2 are linked to Hoxd, Dlx3 and, Dlx4

l0 are linked to Hoxb, and Dlxï and Dlx6 are linked to Hoxa (McGuinness et al., 1996;

Nakagawa et al., 1996; stock et al., 1996). Each of the DIx geneshas a similar

-intron organization with 3 and 2 introns. The homeobox domain is

encoded by exons 2 and3 which are located within the homeobox coding site (Ellies

et al., 1997; Liu et a1.,7997).It has been shown that several Dlx genes can have

multiple transcripts due to altemative transcriptional initiation (i.e. Dtxl) or

alternative splicing (i.e. Dlx4, Dlx5) (Yang et al., 1998; Liu et al.,1997;Nakamura et

a1.,1996). Dlxï, specifrcally, has been reported to also encode variants without either

the homeodomain or a nuclear localization signal (Yang et al., 1998; Liu et a1., 1997).

Normally, the DLX proteins arelocalized to the nucleus; however, DLX5 also can be

observed in the cytoplasm of some cells, which suggests a potential developmental

firnction for DIx5 (Eisenstat et al., 1999).

I.2.2. Expression pattem ofDlx genes

During midgestational stages in the mouse, all six Dlx genes are primarily expressed in the nervous system and the surface ectoderm. Four of the genes, DlxI, Dlx2, Dlx;

andDlx6, are expressed in the cNS (Merlo etal,z007; saino-saito eta1.,2003;

Panganiban and Rubenstein,2002; zerucha and Ekker, 2000; zhao et al., 2000;

Eisenstat et al., 1999' Price, 1993; Robinson et al., 1991). Their expression is shown to be highly restricted to the forebrain in diencephalon and telencephalon. There is overlapping expression of four DIx genes in the subpallium in the forebrain: Dlxl and

DIx2 are localized to the ventricular zone (Z); DlxI, Dlx2, and Dh5 to the

ll subventricular zone (SVZ) and Dlxí and Dlx6 to the mantle zone (MZ). DlxI and,

Dlx2 expression form a distinct boundary at the PSB (Stuhmer et ar.,2002a; stuhmer

et a1.,2002b; Quint et al., 2000; Robinson and Mahon, lgg4).It is reported that their

expression follows a temporal sequence: DIx2, Dlxl and Dlx5, then Dlx6 @anganiban

and Rubenstein,2002; Zerucha and Ekker, 2000; Eisenstat et al., 1999; Liu et al.,

1997). DlxI, Dlx2 and Dlx5 are expressed together in most of the svZ, while Dtx7

and Dlx6 are expressed in many of the postmitotic differentiating neurons @isenstat et

a1.,7999; Liu et a1.,1997). Dlx3 is expressedin the anterior neural ridge andolfactory

placode. Dlx2, Dlx3,Dlx5 and Dlx6 are also expressed in the otic placode and laterin

the otic vesicle (sumiyama and Ruddle ,2Xl3;Panganiban and Rubens tein,2002;

Roberson et a1.,2007; Quint et al., 2000).

At later developmental stages, Dlx gene expression is foundin differentiating skeletal

tissues. Expression patterns of physically lìnked D/x genes overlap extensively. The

DIx genes are expressed in both ectodermal and mesenchymal comparfnents of

developing teeth (Depew et a1.,2002; Robledo et a1.,2002). In particular , Dlxs and.

Dlx6 arc essential for craniofacial, axial, and appendicular skeletal development

@obledo eta1.,2002) . DIx4 þreviously DlxT) is expressed in other mesodermally derivedtissues (hematopoietic cells), where its function is implicated in proliferation and surr.ival (Davideau et al., 1999; Nakamura et al., 1996; Sumiyama et al., Z00Z).

1.2.3. The function ofDi.r genes

The function of Dlx gene family members in vertebrate development has been

t2 revealed in large part through the study of loss of function in various Dlr mutants in

mice. some gain-of-function information is also available. The mice with

homozygous mutations in individual D/x genes die just after birth; however, there are

no distinct in some tissues that express these genes, such as the CNS, as

other Dlx genes are also expressed in the same regions (Saino-saito et al., 2003;

Anderson et a1., 7999; Anderson et al., 1997). The functional redundancy exists

particularly amongst those D/x genes forming bigenic pairs: DlxI-Dlx2 and DIx5-Dlx6

(de Melo et a1.,2005; saino-saito et a1.,2003; Anderson et al., 1999; Anderson et al.,

1997b; Qiu et al., 1997). As a result, double mutants are utilized in order to generate

more severe phenotypes. This strategy has been facilitated by the distinct bigenic

pairing of Dk genes.

Dlr genes in the forebrain

Four members of the Dlx family, DlxI, Dtx2, DIxS, and DIx6, are expressed in the developing CNS. There is considerable overlap between the expression ofDh genes and that of nearly all GABAergic inhibitory interneurons, which suggests a role of

genes Dh in generating this subclass of neuron (stuhmer et al.,200za; stuhmer et al.,

2002b; A¡derson et al., r997a; Anderson et al., l99Tb). GABA is converted from glutamic acid by the enq¡me glutamic acid decarboxylase (GAD). Neocortical neurons arise from the corlical proliferative zone. There is also a subpopulation of neocortical intemeurons which originates within the subcorrical telencephalon. It is shown that GABA-expressing cells migrate from the subcortical telencephalon into

l3 the neocortex. Dlxl/2 double mutant mice show no detectable cell migration from the

subcorlical telencephalon to the neocortex and still express GABA in the subcortical

telencephalon (Anderson et al., rggTb). Ectopic expression of DIx2 andDIx5

induces the expression of glutamic acid decarboxylases (GADs) in embryonic brain

slices (Stuhmer et al., 2002a). These frndings suggest an important function of Dlx

genes in the forebrain developmen! especially for GABAergic interneurons.

The expression of DIx genes in the developing forebrain occurs in overlapping sets of

cells, which suggests that there may be functional redundancy for D/x genes

(Eisenstat et al., 1999; Bulfone et aI,1993). As mentioned before, mutafion of a

single Dlx gene results in subtle defects in developing forebrain. For exampl e, Dlx2

single mutation leads to a loss of dopaminergic neurons in the olfactory bulb.

However, DlxI/2 double mutants show severe blocks in (Anderson et

al.,1997).In these double muknt mice, the first wave of neurogenesis from the

primary proliferative population (PPP) withintheYZof the embryo is not affected,

while the generation of later-born neurons is terminated. This results in abnormalities in the SVZ, in which exists the secondary proliferative population (Spp). While the

PPP in theYZ appears normal, the spp is not able to mature. The mutant spp expresses high levels of Notchl and its ligand Deltal, which are characteristic of the

PPP. As a result, an increase in Notch signaling may result in an increased expression of Hesí, a bHLH transcription factor, inhibiting neurogenesi s intheDlxl/2 double mutants (Panganiban and Rubenstein,2002). The fact that the SPP cells fail to mature leads to the loss of DIxS and DIx6 expression and a block in radial migration of these

14 cells to the mantle zone (Anderson et a1.,7997b).

The failure ofthe SPP to differentiate also results in the decreased differentiation of

several types of GABAergic, dopaminergic, and cholinergic interneurons (Anderson

et a1.,200I; Marin et al., 200i; Pleasure et al., 2000). Since Dlx genesplay important

roles in generating GABAergic, cholinergic, and dopaminergic neurons, it has been

hypothesized that regionally restricted defects in DIx function may result in non-lethal

phenotypes in the forebrain. Therefore, this may lead to disorders such as seizures and

defects in cortical circuit function or effects on learning, motor conhol, and cognitive

behaviour (Panganiban and Rubenstein,2002). Previous research in the mouse has

shown that the majoqty of telencephalic inhibitory neurons are generated from

subcortical telencephalon progenitors. As a result, there is a severe reduction in the

numbers of GABAergic interneurons in the cerebral cortex found inthe DIxl/Dlx2

double mutant (Anderson et a1.,2001; Marin and Rubenstein, 2001; pleasure et al.,

2000; Anderson et al.,I997b).All of these deficits result from failure of tangential

migration of interneurons from the subcortex (subpallium) to the cerebral cortex

þallium).

There are at least two streams of tangential migration from the subcortical telenchephalon (Anderson et al., I999a).One stream is a rosftomediodorsal stream from the lateral ganglionic eminence (LGE) to cerebral cortex. The other is a laterodorsal stream from the medial ganglionic eminence (MGE) from which interneurons migrate to the striatum and cerebral cofex (Marin and Rubenstein, 2003;

l5 Anderson eT al., 1999; Miyashita-Lin et al., 1999). Dlxl /2 double mutants contain defects in both streams, resulting in loss of striatal, olfactory bulb, and corfical intemeurons (Marin and Rubenstein, 2003; Marin et al., 2000; Bulfone et a1.,1999;

Anderson et aI.,1997b). The regulation of mechanism of long-distance migrations from the subcortical telencephalon to the striatum, olfactory bulb and cortex is another important goal of Dlx gene reseatch. As mentioned earlier, neuropilin/semaphorin signalling is involved in migrating subcortical telencephalic interneurons to other regions of the brain (Marin and Rubenstein, 2003). Le has demonstratedthat Dlxl and Dlx2 directly regulate the expression of Neuropilin-2, providing apartial molecular mechanism for failure of tangential interneuron migration to the neocortex in DIx mutantmice (Le et a1.,2007).

Dlxl/Dlx2 null mice result in compromised migration of GABAergic interneurons to the neocortex (Anderson et al., 7997a). However, there is little known about the molecular basis for the multiple defects due to the loss of Dkl/Dlx2 function.

Neuropilin-2 (l\rRP-2) expression is affected n DlxI/2 mutants (Le et a1.,2007).

NRP-2 is a for Class III semaphorins, which are inhibitors of neuronal migration. It is important to point out that Dlxl/Dlx2 null mice show increased and aberrant expression of NRP-2 in the forebrain and this is the first demonstration that

DLXI or DLX2 can function as transcriptional repressors in vivo. Down-regulation of

Nrp-2 expression by DLXIl}may facilitate tangential interneuron migration from the basal forebrain (Le et a1,2007), which provides further knowledge about the role of

Dlx genes in forebrain development.

ló DIx genes can also affect cell fate in forebrain development. A study investigating the

combinatorial expression and function of DIxI &2, Olig2, and, Mashl transcription

factors in the ventral telencephalon suggests a new role for Dlxgenes (petyniak et al.,

2007).It was shown that DIx homeobox transcription factors, required for

GABAergic interneuron production, repress the formation of oligodendrocyte

precursor cells (oPC) (Petryniak et a1.,2007). This is due to Dh genes affecting the

cell fate of a common progenitor between acquiring neuronal or oligodendroglial cell

fates. DlxI/2 down-regulate the formation of orig2-dependent opc, while Mashl

up-regulates OPC formation by repressing the generation of D/-r-positive progenitors

@efiyniak et a1.,2007). when progenitors were toansplanted from DIxI/2 mutant ventral telencephalon into newborn wild-type mice, there were no neurons produced but myelinating oligodendrocytes differentiated and survived into adulthood. These results identiff another role for Dlx genes distinct from regulation of tangential migration, as modulators of neuron versus oligodendrocyte development in the development of embryonic forebrain (Petryniak et a1.,2007). This ¡esult is supported when studyinga DIx2t^ù"Z knock-in mouse model, which showed a lineage relationship between Dlx2-expressing cells and glia in the dorsal telencephalon. It is demonstrate d'that Dlx2-expressing cells derived from subpallium differentiated into astrocytes and oligodendrocytes in cerebral cortex (Marshall and Goldma n,2002).

DIr genes in the

So far, research on the role of DIx genes in development of the eye is mainly focused

17 on the role of Dlx genes in the retina, a forebrain derivative. Tlne Dlx2 gene is first

expressed in the retina of mice embryos at El 1.5 (de Melo et al., 2008) . Dtx j is

expressed in the developing retina by approximately 812.5 (Eisenstat et al., 1999). It

was shown that expression is limited to the retina and not in the developing optic

nerve, pigment epithelium, , iris, or cornea. The proportion of Dlx2-positive cells

is greater than Dlxl-positive cells adjacent to the ophthalmic ventricle. The majority

of D/:r-positive cells were post-mitotic when labeling DlxI and DIx2 withmarkers for

M- and S-phase; however, a limited number of Dlxl-positive cells are in S-phase

when they were labeled by bromodeoxyuridine @rdU). A small portion of

Dlx2-positive cells was identified to be in M-phase by staining with propidium iodide

(Eisenstat et al., 1999).

T]¿e DlxI/DIx2 null mutant has a reduced ganglion cell layer (GCL), and late-bom differentiated retinal ganglion cells (RGCs) are affected (de Melo et al., 2008). It was also found that the expression of the neurotophin receptor TrkB is significantly

reduced in the retina of Dlx I /Dlx2 null mutant by E 1 6. 5. DLx2 binds to the TrkB promoter in vivo during embryogenesis and this binding causes functional consequenc es in vitro,which supports TrkB asa DLX transcriptional target (de Melo et a1., 2008). Thus, Dk2 expression regulates the expression of rrkB thatmay regulate the suwival of late-born RGC during development (de Melo et al., 2008).

1.2.4. Transcriptional targets of Dlx genes

Several genes have been proposed and identified as candidate targets of Dlx genes,

l8 including the Dk genes themselves. It has been reported that DLX2 can induce Dlx5

expression, and that DIxl, Dlx2 and DIx5 can activate transcription from a Dlx5/6

enhancer in both the mouse and the zebrafrsh (stuhmer et a1.,2002b; yu et a1.,2007;

Zerucha et a1.,2000). Optimized chromatin immunoprecipitation (ChIP) was utilized

to identifli that DLX1 and DLX2 directly bind to the DIxS/Dtxó intergenic enhancer

(MI56) and activate transcription in the mouse (Zhou er. a1.,2004). Ectopic expression

of Dlx2 and Dlxí activates the transcription of glutamic acid decarboxylases (GADs)

in mouse embryonic cerebral cortex (stuhmer et al.,200zb). Dlx2 also regulates

neuropilin-2 (Le et aL,2007) as well as GAD isoforms (Le et al., in preparation; Wigle

and Eisenstat,2008)

IthasbeendemonstratedthatDlx2alsobindstoHBs-l inÍheWntl enhancer(Ileret

a1.,1995).It was hypothesized that expression of DLX3 and DLx5 may regulate

wntl as well (Panganiban and Rubenstein, 2002). DLX3 has been reported to transcriptionally regulate expression of the alpha subunit gene in cells of trophoblast origin in the human placenta (Roberson et al., 2001) and that of profilaggrin in differenting keratinocytes (Morasso et al., 1996). DLX4 has been shown to regulate the expressi on of Gatal and c- in hematopoietic cells (Shimamoto et al., 1997).

Additionally,DLX4 overexjlression inhibits apoptosis, which is mediated by DLX4 activation of the intercellular adhesion molecule 1 (ICAM-l) (Shimamoto et al.,

2000). DLX5 regulates several targets during bone development, which includes osteocalcin (Ryoo et a1., 1997), collagen lAl (Dodig et ar., 1996), and bone sialoprotein (Benson et al., 2000).

19 1.3. Pax6 genes

The Pax gene family is another family of kanscriptional regulators characterized by

the presence of the paired box domain, usually with a homeodomain. play

key roles in tissue formation and organ development during the development and

growth of embryos (Buckingham and Relaix, 2007;Langetal.,2007;Tsonis and

Fuentes, 2006;Kozlrnik,2005). The paired box domain, which is highly conserved

through evolution, has a length of 384 base pairs (bp) and encodes a DNA-binding

domain of 128 amino acids. The consensus DNA binding site for the Paired domain is

(c/T) T (T/c) (c/A) (c/T) (c/c) (cic). There are nine pAX proreins in rhe pAX

protein family. PAX proteins are expressed in diverse tissues and organs during

organogenesis, for example, the skeleton (PAXI and 9), cNS (pAx2 ,3, 5, 6,7, and

8), kidney (PAX2 aJrd 8), B-cells (PAX5), thyroid (pAxg), (pAx4 and 6)

and skeletal muscle (PAx3 andT).It is not clear how pAX proteins affect

organogenesis (Lang et al., 2007)

Pax6 is a member of the Pax gene family and encodes a protein that contains two domains, a paired domain and a homeodomain. Pax6 was first identified as a master control gene for (Lang et a1.,2007). Moreover, pax6 also plays an important role in neurogenesis in the development of the vertebrate foreb¡an. pax6 functions in development of the forebrain include boundary formation, dorsal-ventral and anterior-posteriorpatterning, specification ofneuronal subtypes, neuronal migration and axonal guidance (Stoykova and Gruss, 1994; walther and Gruss, l99l;

20 Matsumoto and Osumi, 2008).

1.3.1. The expression pattem of the Pax6 gene

Pax6 is expressed in mouse embryonic development as early as embryonic day g

(88'0) in neuroepithelium of the prosencephalon that includes the prospective optic

vesicle, the telencephalon, and the diencephalon. Pax6 is expressed in several sites,

including the forebrain and hindbrain, the developing spinal cord, and a broad region

ofhead surface ectoderm covering the forebrain. Later in developm enr" pax6

transcripts are present in both the telencephalon and the diencephalon and in the

myelencephalon (a part of the hindbrain). Expression of Pax6 is observed in the

midbrain from day El i.5. Pax6 is also expressed in neuroepithelial regions of the eye

and nose, named the optic vesicle and the olfactory bulb, respectively (Callaerts et al.,

1997; Anderson et a1.,2002; Englund et a1.,2005). In the telencephalon (from E10.5 to El8-5), the expressionof Pax6 is restricted in the ventricular zone of the lateral and dorsal neural epithelium (Figure'1). There is little expression of pax6 in the basal telencephalon (callaerts eta7., 1997; Englund etal.,2oo5; Kawaguchi eta1.,2004).

1.3.2. The function of Pax6 in forebrain development

Pax6 geneis a master contool gene for eye development, which is affected in human patients with and in the mouse mutant Small eye (Sey) (Gehring and lkeo, lggg). rn Pax6 heterozygotes, the eyes are smaller than normal , and in pax6 homozygotes the eyes are absent. The ocular defects observed from mutations of the

21 Drosophila Paxíhomologue ey is similar to those found in the human and the mouse.

These findings were also supported by RNA interference experiments in planarians

and nemerteans (Gehring,2002; Gehring and Ikeo, 1999).

In addition to its role in eye , Pax6 is also involved in development of

the brain and olfactory organs. Pax6 homtozygous mutant mice lack eyes and nasal

structures and die at birth with serious brain abnormalities, including defects in

forebrain patterning and growth (Gehring, 2002).In humans, alarge proportion of

aniridia patients, who are Pax6heterozygotes, also display defects in cerebral

formation and olfactory function (Sisodiya et a1.,2001).

Pax6 in forebrain patterning

Pax6 also plays an important role in patterning of the telencephalon. As described

previously, the telencephalon has two major subdivisions, the pallium and subpallium.

Gsh2 isa homeobox transcription factor. There is overlapping early expression of

Pax6 and Gsh2 in the region of the dLGE. Pax6 and Gsh2 have complementary roles

in patterning the primordia flanking the PSB as demonstrated by analysis of Gsh2

mutant mice and Pax6 mutantmice. In Gsh2 mutant mice, the dLGE becomes a Vp

(Venfral pallium)-like structure, whereas in Pax6 mutant mice the VP is transformed

into a dlGElike structure (Yun et a1.,2001).More research demonstrated that the

Gsh2 gene plays an essential role in sustaining the molecular characteristics of other

striatal progenitors. For example, in Gsh2 homozygous mutants, the ventral telencephalic regulatory genes Mash| and Dtxl/2 are absent from the striatum, while

22 the dorsal regulators, Pax6, neurogenin-l andneurogenin-2 are expressed in striatum

(Toresson et al., 2000). Conversely, Pax6 is essential for maintaining the molecular

identity of cortical progenitors . ln Pax6 homorygote mutants, are absent

from the cortex, but ventral Gsh2, Mashl and Dlx genes are observed in cortex. This

converse expression of cortical and striatal progenitors leads to the abnormal

development of the cortex and striatum as reveale d in Pax6 (smatl eye) and. Gsh2

mutants, respectively. Moreover, double homozygous mutants for pax6 and Gsh2

exhibit signif,rcant improvements in both cortical and striatal development when

compared to their respective single mutants. Taken together, these results demonstrate

thatPax6 and Gsh2 play important roles in the development of cortex and striatum by

regulating the expression ofeach other, and by regulating regional expression of

developmental regulators Mashl, the neurogenins and Dlx genes in telencephalic progenitors (Toresson et al., 2000).

Pax6 is also involved in patterning the medial dorsal telencephalon. Emx2,another homeobox gene, promotes the development of the caudal-medial cortex, while pax6 promotes the rostral-lateral cortex. Both Emx2 and Pax6 genes are necessary and sufficient for the specification of neuroblasts in the dorsal telencephalon as cortical versus ganglionic neuroblasts (Muzio and Mallam aci,2003). Emx2is expressed in the

YZ of the dorsal telencephalon in a gradient level, expressed from high in rostral to low levels in lateral cortex, which is opposite to that of Pax6, from low in caudal to high levels in medial cortex. lnthe Emx2-l- mutant or paxfl-mutant, the cerebral cortex forms and is morphologically and molecularly distinguishable from adjacent structures such as basal ganglia. In double homozygous paxf/-/Emx2-t- m:uÍants,

defects are shown in embryonic forebrain. The entire LGE is converted into MGE,

while the entire cortex acquires morphological and molecular features reminiscent of

those of the striatum (Muzio and Mallamaci, 2003). Thus, it is necessary and

sufficient for at least one functional allele of either Emx2 or Pax6 to induce the

morphogenesis of the cerebral cortex (dorsal) and to repress that of a-djacent structures

such as striatum (ventral). Moreover, in Paxfl-lEmx2-/- mutants, cortical markers

(1d3, Otx2, Msxl) spread into a large part of the telencephalon. This patterning defect

in the mutants for both Emx2 anð,Pax6 indicates that Pax6 isinvolved in patteming of

the medial dorsal telencephalon (Muzio and Mallamaci,2003; Muzio etal.,200za;

Muzio etal.,2002b).

Pax6 in boundary formation

It has been established that the PSB boundary is composed by cells expressing both

DLx2 and PAX6, using double immunostaining techniques (carney etal.,2006).

There are certainly Paxí-expressing cells that coexpre ss Dlx2 existing in the

subpallium. The PSB is shown by the overlap of DLX2- and pAX6-expressing cells in the striatum and neocortex, respectively (Carney et a1.,2006). Dlx2 expressing cells enter the cortex tnfhe Pax6 homozygous mutant forebrain. This dorsal expansion of subpallial gene expression was also shown with other factors (Gsh2, Mashl, DhI,

vaxl , ) in Pax6 mutants (Manuel and Price, 2005; stoykova et al., 1997).In particular, the expression pattern of the homeobox gene Gsh2 inthe wild type mouse

24 forebrain is similar to that of Dlx2, in subpallium, and is complementary to that of

Pax6,in pallium (Yun et al.,ZXo];Toresson et al., 2000) . Gsh2expression is

restricted to the ventricular zone of the ventral telencephalon (the subpallium) with

highest levels of expression in the dLGE. As mentioned before, Gsh2 homozygous

mutants have a phenotype reciprocal to that of Pax6 homozygous mutants (Yun et ai.,

2001; Toresson et al.,2000)

At the PSB, PaxÍmutation causes a downregulation or a dorsal "retraction" of the

expression of other pallial markers (EmxI, Tbrl, Tbr2)and leads to the dorsal

expansion of subpallial gene expression (VaxI,,Srx3) into the pallium. Ngn2 and,Tbr2

are other pallial markers, while Dbxl is a marker for venhal pallium. ln pax6 -/-

embryos, the expressi on of Ngn2 and Tbr2is markedly reduced, and the expression of

DbxI is completely lost (Yun et a1.,2001; Toresson et al., 2000).

Dlx2-positive subpallial cells undergoing tangential migration are not mitotically active (Anderson et al., 2001). However, in pax6 mutant embryo, pallial cells that express the subpallial marker DLx2 are mitotically active (yun et al., 2001). It indicates that at least some of the pallial progenitors ectopically express subpallial markers. These studies suggest rhat Pax6 mutations lead to a compromise of the pSB, which further promotes subpallial cells to undergo tangential migration.

Similarly, NgnI and Ngn2 are involved in the specification of dorso-ventral patteming in the telencephalon, and play roles in the establishment of pallial properties and the inhibition of subpallial properties, such as the expression of the venhalizing factor Mashl (Fode et al., 2000) .NSn2 has been established as a direct target regulated by

PAX6 in the dorsal telencephalon (Scardigli et al., 2003). Thus, pax6 could activate

the expression of Ngnl and Ngn2, which results in repression of ventral

characteristics, such as expression of Mash|.

Pax6 regulates adhesion molecules

In brain development, Pax6 has been shown to be involved in the establishment of

regional cell adhesion molecule expression and guidance. R-cadherin (also

known as Cdh4) is a cell adhesion molecule implicated in axon guidance. ln pax6

mutant mice, the expression of the R-cadherin is lost in a region of the ventral

thalamus, where forebrain pioneer axons have pathfinding abnormalities. R-cadherin

substrate is able to promote pioneer axon outgrowth in vitro. Addittonally, pioneer

axon outgrowth is rescued in vivo when R-cadherin was replaced into culture d pax6 mutant embryos by electroporafion. Therefore, Pax6 isimplicated as an early brain patteming gene that regulates the expression of regional adhesive cells to guide pioneer axons (Andrews and Mastick, Z0O3).

Expression of R-cadherin (Rcad) in the pallium is essential to maintain the pSB by conferring specific adhesive properties to cortical progenitors (Stoykova et al., 1997).

The expression of Rcad is much ¡educed in the cortex and almost completely disappears at the PSB in Pax6 mutant embryos (stoykova et ar., 1997). The formation of the PSB is ma¡ked by a barrier of radial glial fascicles, separating cells in the pallium and in the subpallium (stoykova etal.,1gg7).rn pax6 mutant embryos, the mechanical PSB formed by the radial glial cells disappears and the adhesive

properties ofpallial cells are changed. In short term aggregation assays, cortical cells

of Pax6 mutant embryos separate from those obtained from wild-type embryos but are

unable to separate from wild-type or mutant skiatal cells, as the adhesive properties of

striatal cells do not change (Stoykova et al., 7997). The expression pattern of the

adhesion molecule R-cadherin (Rcad) overlaps with the expression pattern of Pax6 jn

theYZ of the dorsal telencephalon, forming a sharp border at the PSB (Stoykova et

a1.,1997).

Pax6 affects tangential migration

The mammalian neocortex contains two major neuronal subtypes, intemeurons and

projection neurons. Intemeurons arise from the ganglionic eminences (GE) and

projection neurons from the cortical ventricular zone (YZ).These separate origins

require two distinct pathways of migration, radial migration and tangential migration, previously discussed. Recent research showed That pax6 mutant mice (,SøyJ-) have increased numbers of cortical inhibitory interneurons, suggestin gthat Pax6 coald, induce development of inhibitors of interneurons or alternatively play arepressive role in guiding tangential migration and/o¡ speci$ring interneurons (Gopal and

Golden, 2008). Unexpectedly, there was a ¡eduction of the migration dist¿nce of Sey/- neurons, suggesting a disorganized migration, with frequent changes in direction. In contrast, the number of non-radial migrating GE cells was not different in Sey4- mice.

Thus, loss of Pax6 results in an increased number of intemeurons, which is not due to

27 an increased rate or number of tangential migrating cells, but to the ectopic

specification of interneurons in the cortical VZ. This research also indicate s fhat pax6

may play a role in axonal organization (Gopal and Golden, 200g).

Pax6 also affects GABAergic interneuron migration. In Pax6 mutant (sey/se) mice,

progenitors in the dorsal telencephalon (dTel) are progressively ventralized, and

regulatory genes nonnally expressed in GE progenitors are expressed in the

ventricular zone (YZ). Accompanying this ventralization of the dTelyZ,markers for

GABAergic interneurons (GABA) are expressed. Abnormally expressed GABA INs

originate from a cortical Emxl lineage. These INs are normally generated in the dTel

YZ, rcvealing that drel progenitors and progeny arc ventralized, jn pax6 mutants.

Taken together, Pax6 eslablishes the boundaries of an appropriate proliferative zone

for GABA INs and regulates their quantities and diskibution by suppressing the ventral fates of drel progenitors and progeny (wen et al., 200g; Nomura et a1.,2006;

Kroll and O'Leary, 2005).

Pax6 determines neuron cell fate

Pax6 is reported to be a key regulator in the determination of neuronal fate as well as the proliferation of neural stem cells, but the molecular mechanisms are still largely unknown. A recent research paper reportedthat Pax6 regulates the proliferation of neural progenitor cells of the cortical SVZ by directly modulating. (SRy Gex determining region Y)-box 2) expression during late developmental stages in mice. In

El8.5 Pax6/- mice, the number of sox2+neural progenitor cells in the SVZ was

28 dramatically decreased. A ChIP assay confirmed that Pax6 canbind to the Sox2

promoter, and luciferase reporter gene assays showed that Pax6 acttvafed Sox2

expression. Moreover, decreased neurosphere (non-adherent spherical clusters of cells

cultured in vitro) formation and proliferation was shown in neural progenitors in the

Pax6/- embryos (Wen et al., 2008).

Many neurons and glia in the cerebral cortex are produced by apical progenitors,

which divide at the ventricular surface of the dorsal telencephalon. Other neurons

derive from basal progenitor cells, originating from apical progenitors, which divide

away from the venhicular surface. Expressed in apical progenitors , pax6 is

downregulated in basal progenitors, which upregulate the transcription factor Ibr2.lt

is shown that most progenitor cells dividin g away from the ventricular surface in

Paxït- embryos fail to express the hanscription factor Tbr2 andthat pax¡is required

for Tbr2 expression in the cortex in developin g paxf/*<_ chimeras. Thus,

Pax6 functions in the early stage of development of cortical cells, represses the expression of several hanscription factors that are normally expressed in the LGE, and ensu¡es the terminal differentiation and maintenance of progenitor cells in order to induce normal development of cortical basal progenitor cells (wen et al., 200g;

Quinn eta1.,2007).

Tlhe Paxígene is one of the regulatory genes that are expressed in the earliest stage of diencephalon development (warren and price, rggT).rn paxímutants, abnormal morphology is observed in the diencephalon at all studied embryonic ages. These

29 abnormal phenotypes include loss of regional differences in cell density in the

diencephalon, a deficient diencephalon/mesencephalon boundary, and an enlarged

third ventricle. In the wild-type diencephalon at E14.5, pax6, DIxz and l[/nt3 are

expressed in distinctregions along the rostrocaudal and dorsoventral axes. Inpax6

mutants, the expressi on of Pax6 expanded in the ¡oshal domain, forming an

abnormally obscure caudal border; the expressi on of Dlx2 at the caudal boundary

became diffuse and the expression of Wnt3 normally at high levels in dorsal midline

disappeared. Thus, Pax6 is essential for the development of diencephalic precursors

(Warren and Price, 1997).

In summary, the Pax6 gene has a key role not only in the earliest steps of eye

developmenl but also in the developing central nervous system. Our knowledge of

the role of Pax6 in development is primarily obtained from the analysis of mutant mice. In vertebrate forebrain development, Pax6 plays critical roles in regionalizing the major domains of the telencephalon and diencephalon in the developing vertebrate forebrain, as well as in establishing boundaries, axon organization, and determining neuronal cell fate.

1.4. Tenascin-C

Tenascin is a family of exfracellular matrix (ECM) proteins, which includes

Tenascin-c, -x, -R, and -w. Tenascin-c (Ten-c) is a large ECM glycoprotein, consisting of individual polypeptides. The molecular weights of these peptides range from 180 to 300 kDa. The pattern of expression of Ten-c is tuansient and highly restricted during embryonic development. Ten-c is expressed again in some

pathological tissues such as those involved in wound healing, involution and

tumorigenesis (Jones and Jones, 2000b; Mackie, 1997; pas et al.,2006).

1.4.1. The structure of Tenascin-C protein

Ten-C has a multimeric, six-armed structure, called a hexabrachion (Figure 3). Each

arm of the hexabrachion contains an amino-terminal assembly (TA) domain that helps

individual Ten-C polypeptides interact at their amino termini through a number of

cysteine residues and heptad repeats. Following the TA domain is a group of

epidermal growth factor-like (EGFL) repeats. The EGFL repeat contains six cysteine

residues that are likely to form disulfide bonds. The function of the EGFL regions in

the Ten-C molecule is considered to be counteradhesive for fibroblasts, neurons and glia. Moreover, the EGFL may be invorved in neuronal migration and axon pathfinding during forebrain development @ischer et al., 1997; Gotz et a1.,1997). Ã series of fibronectin type IrI (FN-rfÐ domains are adjacent to the EGFL repeats.

FN-III consists of a highly elastic region of the Ten-C molecule and facilitates rapid stretching and refolding (oberhauser et al., l99g; Jones et al., 1997). A globular fibrinogen- domain is in the distal region of the Ten-c peptide, which harbors a calcium-binding loop. This fibrinogen domain has been found to interact with other ECM and cell surface proteins, including collagen fibrils, integrins, heparin and phosphacan (Milev et al., 1997; Jones and Jones, 2000a) .

The structure of Ten-C is determined by alternative splicing of mRNA, in which the

31 Figure 3: structure of renascin-c: A: The model of the Ten-c hexabrachion. B: Schematic representation of an individual Ten-C polypeptide. The tenascin assembly (TA) domain helps six polypeptides to associate via coiled-coil interactions that occur at the heptad repeat region. Conserved and alternatively-spliced FN.III repeats are represented by dark and light boxes, respectively. The C-terminal knob is made up of a fibrinogen globe circle (Adapted from Jones and Jones, 2000a. Reprinted with permission from Elsevier Limited).

number of FN-III repeats is diverse in different species and cell types (Ghert et al.,

2001;JonesandJones,2000b).Forinstance,itisrevealedthatthereare atleast2T

different FN-III variants of Ten-C that are expressed in the developing brain of the

mouse (Joester and Faissner, 1999).

Tenascin-C isoforms are normally present in almost all normal adult tissues but are highly expressed in fetal, regenerating, andneoplastic tissues. Scientists reported a human antibody fragment, TNll, derived from a phage library with high affinity for the spliced repeat C. They demonstated that this repeat is undetectable in normal adult tissues,barely detectable or undetectable in breas! lung and gastric carcinomas, meningioma, and low grade astrocytoma, but extremely abundant in high grade

astrocytoma (grade III and glioblastoma), especially around vascular structures and

proliferating cells. The antibody provides a potential method to develop a therapeutic

agent for patients with high grade astrocytoma, a common brain tumor in adults

(Camemolla et.al., I 999).

1.4.2. Expression pattem of Tenascin-C

Tenascin-C is first expressed at the stage of gastrulation and somite formation, and it

is expressed again in the rostral half of the somite at later stages (Tucker and McKay,

1991). In the developing cNS, Ten-C is also produced by glia, and is expressed at a

high level on radial and Bergmarur glial fibers during neuronal migration in the

developing forebrain (Yuasa, lgg6).In the peripheral nervous system, Ten-c is

expressed by Schwann cells during myelination, and in non-neural tissues it is expressed during branching morphogenesis of mammary gland and the lung. Ten-c is also expressed in the developing skeleton, cardiovascular system and in connective tissues (Ekblom and Aufclerheide, 1989). Of note, Ten-C expression is low in normal adult tissues, however, it is induced to be expressed at much higher levels during tissue involution, neovascularization and wound healing (Jones et al., 1995).

1.4.3. The function of Tenascin-C

As mentioned before, the expression of renascin-c is quite high in neural development at critical stages, which suggests it functions during development.

JJ Research has shown that Ten-C has opposing fi.rnctions as adhesive or anti-adhesive,

as suppofer or inhibitor of cell migration, or as a promoter or inhibitor of neurite

outgrowth @artsch, 1996). Diverse functions of Ten-C could be due to different

functional domains in the protein, to interacting with different cellula¡ receptors, or to

being regulated by different signal transduction pathways (Bartsch, 1996).

For example, Ten-C is normally co-localized with fibronectin, a prominent adhesive

molecule of the ECM. In Ghert's (Ghert et a1.,2001) research, it was shown that the

Ten-C splice variant of small size (220 kDa) binds to fibronectin, while the Ten-C

splice variant of large size (320kDa) does not. Moreover, it was ¡evealed that the

small Ten-C splice variant reduced adhesion for cells when bound to fib¡onectin, but

promoted adhesion when bound to plastic in fibronectin-coated wells. It was also

confirmed that both the small splice variant of Ten-C and fibronectin assisted cell adhesion, while the large splice variant of Ten-c did not induce specific cell attachment. These results led to the conclusion that the function of Ten-c was dependent on its tissue-specific spricing pattem. The smaller Ten-c isoform contributed to adhesion, while the larger isoform played a role in cell metastasis

(Ghert et a1.,2001).

Tenascin-c regulates both cell proliferation and migration in oligodendrocyte precursors during development (Garcion et al., 2001) . rn Ten-c knockout mice, there were increased rates of migration of oligodendrocyte precursors along the optic nerve, while reduced rates of oligodendrocyte precursor proliferation in different regions of

34 (Garcion the CNS et al., 2001). At later developmental stages, there was reduced level

of programmed cell death in areas of myelination. In addition to the alpha(v)beta3

integrin mediated cell proliferation described previously, both CNS ECM and integrin

grorvth factor interacfion play roles in the regulation ofneural precursor behaviour

(Garcion et al., 2001).

Tenascin-c plays an important role in cell adhesion during peripheral nerve

morphogenesis as well, such as allowing axonal growth and inducing the compaction

of nerve fascicles (wehrle-Haller and chiquet, 1993). Generally, Ten-c inhibits

migration in most cells. The inhibition of migration mediated by Ten-C was induced

by modulation of interactions between the individual cell and ECM (Frost et al.,

1996).

Ten-C is also involved in the formation of the neural crest in vertebrate development.

During earþ stages of vertebrate development, shortly after the fusion of neural folds, neural crest cells delaminate from the neural tube and migrate ventrally along specific pathways to form the neurons and glia of the peripheral nervous system. Neural crest cells express Ten-C when they leave the neural tube during the initial stages of their migration, which indicates a possible role for Ten-C in conhibuting to the invasive behayiour ofneural crest cells (Tucker, 2001).

1.4.4. Regulation of Tenascin-C expression

Tenascin-C is induced or repressed by multiple soluble factors that are expressed,

35 activated or released from embryonic tissues, as well as in fully developed tissues that

are remodeling, injured, reactive or neoplastic. A number of growth factors secreted

by cells induce synthesis of Ten-C. It has been shown that fetal bovine serum (¡BS)

and transforming growth factor-pl (TGF-0l) variably regulate Ten-c. In some

instances, induction of Ten-c by these sorubre factors appears to depend upon the

grofih state of the target cell (Rettig etal.,1994). As mentioned previously, the

Ten-C gene promoter is activated by multiple transcription factors, which includes

those of the homeobox protein family (Jones and Jones, 2000a). As shown in Figure

4,the Ten-C promoter contains a homeodomain binding site (HBS), which is a_djacent

to the transcriptional start site (+1) and located at nucleotid e -57. prxl and prx2

encode paired-related homeodomain-containing transcription factors that interact with

the Ten-c promoter via binding to its HBS (copertino et al., 1997; Jones and Jones,

2000a ). Upstream from the HSB, there is an octamer motif that is a binding site for the homeobox protein Bm2 (copertino et ar.,1997; Jones and Jones, 2000a). Four other functional promoter elements are: a binding site for nuclear factor 1 (NF-l); a tenascin control element (TCE, related to NF-kB); a binding site for Krox24ÆGR-l at

-250, and anotu2 response element (ors, a binding site for the homeobox protein

OTX2) at -550 (Copertino et al., 1997; Jones and Jones, 2000a).

For example, Prxl and Prx2 are homeobox transcription factors that are expressed during vasculogenesis (Jones et al., 2001). Their pattems of expression appear to overlap with the expression of Ten-C, especially during epithelial-mesenchymal transitions, and in the developing skeleton and cardiovascular system. prxl and prx2 Iça\24 'rCE OtT t¡F.1 HBS

llsr.l NF-kB f Bm2l iâ-x1rr¿¡

Figure 4: schematic diagram of the Ten-c gene promoter. The mouse Ten-c

promoter has several transcription factor binding sites, such as orS (an oft2

response element, a binding site for oú2), aKtox} /Egr-l binding site, TCE

(tenascin control element, a binding site for NF-48), an octamer motif (ocr),

an NF-l binding site, and a homeodomain binding site (HBS). Adapted from

Jones and Jones, 2llÌa.Reprinted with permission from Elsevier Limited.

interact with A/T-rich gene sequences in vivo.It is also shown that express ion of prxl

significantly enhances Ten-C gene promoter activity by 20 fold, and the N-terminal porlion and the homeodomain of PrxI are essential to induce the bulk of Ten-C promoter activity (Jones et al., 2001). Furthermore, overexpression of either prxl or

Prx2 acttvates the Ten-C gene promoter in Prxl/2-null smooth muscle cells, therefore supporting the statementthat Ten-Cis regulated by the homeobox transcription factors PrxI andPrx2 (Jones et a1.,2001).

1.4.5. Phenotypes of Ten-C knockoutmice

Although Ten-c controls many cellular functions in tissue development and remodelling, it is surprising that Ten-c knockout mice do not have any gross

5t anatomical abnormalities. It seems that Ten-C is not essential for development, as

these mice are born alive, and in the original study showed no abnormalities.

However, in some more recent studies, it has been revealed that Ten-Cknockout mice

have several defects, such as abnormal behaviour and abnormalities in

neurochemistry as well (Mackie and Tucker, 1999).In these knockout mice, skin

wounds heal normally; however, ttrese mutant mice have defects in healing after

suture injury of corneas. The expression of fibronectin is abnormally low in Ten-C

knockout mice, and there is defective haemopoietic activity in bone marïow obtained

from these mice (Mackie and Tucker,1999).

Ten-c knockout mice appear to have-neurological defects that include poor

sensorirnotor coordination, hyperactivity in an open field test, and poor performance

in passive avoidance leaming tests (Fukamauchi et al., 199g). Fukamauchi

demonstrated that hyperlocomotion is observed in mutant mice that lack the Ten-C

gene, and these mice have difficulties in habituating to unfamiliar environments.

Moreover, these mice exhibit several deficits, such as poor appetite, abnormal circadian rh¡hms and low pregnancy rates. It was also suggested that some behavioural abnormalities exhibited by Ten-C mutant mice may partially result from the reduced level of expression of neuropeptide Y in the limbic system (Fukamauchi et al., 1998), possibly due to interneuron migration defects.

1.5. Hypothesis and Research Aims

It is hypothesized that Pax6 and DIx2 genes may contuol the expression of the adhesion molecule Tenascin-C (TEN-C) at the pallio-subpallial boundary, and that

regulation of TEN-G may affectdevelopment of the vertebrate fo¡ebrain.

Specific Aim 1: I)emonstration of TEN-C expression in normal development as

well as in Dkl/Dk2 or Paxlmutant embryos.

The expression patterns of PAX6, DLx2, and rEN-c will be analyzed.by

immunohistochemistry and immunofluorescence. All studies will be done on mouse

forebrain tissue sections, using specific antibodies to TEN-G, DLX2 and pAX6.

Specific Aim 2: Characterization of molecular interactions between pAX6 or

DLX2 and the Tenascin-C gene promoter.

Chromatin immunoprecipitation (ChIP) studies will be performed to determine

whether PAX6 and DLX2 bind to the promoter regions of Tenascin-c. To confirm this binding, electrophoretic mobility shift assays (EMSA) using recombinant pAX6 and DLX2 proteins and embryo forebrain nucleoprotein extracts will be carried out.

ChIP-reChIP experiments will be performed to test whether DLXzand pAX6 bind to the Tenascin-Cpromoter simultaneously. Co-immunoprecipitation experiments will be carried out to test whether DLX2 and PAX6 proteins inte¡act with each other in vitro and tn sítu.

specific Aim 3: Examination of the functional consequences pAX6 of DLX2 and , interactions with the Tenascin-C promoter.

39 The functional consequences of binding of PAX6 andDLX2 proteins to the

Tenascin-Cpromoter will be studied by luciferase reporter gene assays using the human embryonic kidney HEK293 cell line. Site-directed mutagenesis will be done to identifu which putative transcription factor binding sites are affected by Dtx2 or

Pax6 co-transfection.

40 2. Matenals and Methods

2.1. Animal and tissue preparation

All studied tissues were obtained from the mouse (Mus musculzs). Wild type tissues

were obtained from the CD-l (ICR) BR Swiss strain of albino mice (Charles River

Laboratories, worcester, MA, usA). Dlxl/Dlx2 knockout mice were a gift of Dr.

John Rubenstein, University of California at San Francisco, USA. Embryonic tissues

derived from wild type mice andpax6 knockout mice were obtained from Dr.

Anastassia stoykova, Max-planck Institute for Biophysical chemistry, Göttingen,

Germany.

Timed-pregnant mice were used to obtain embryos at the appropriate developmental

stages, usually E13.5 and E14.5. Embryonic age was determined by the appearance of

the vaginal plug after breeding, which is taken as E0.5, and confirmed by

morphological criteria' Adult animals were euthanizedby cervical dislocation before

embryos were dissected, while embryonic animals were euthanized,by decapitation

before dissection. Tissues were kept in phosphate-buffered saline (pBS) on ice, and

the forebrain \ryas dissected under a stereomicroscope. Genotyping was done to verifu

DlxI/2 genotype in embryos using specific primer pairs (eiu, rggT).For genotyping,

mice tails were added to 80 pl of tail lysis buffer (100 mM Nacl, 50 mM Tris-HCl, 50 mM EDTA,0.5yo sDS, pH 8.0) containing 0.1 pglpl proteinase K and incubated overnight at65"c. on the second day, they were heated in boiling water for l0 minutes and then centrifirged at maximum speed (13,000 rpm) for 2 minutes.

4l Supematant was collected and I pl of this solution was used for each PCR reaction.

All animal protocols were conducted in accordance with guidelines set by the

Canadian Council on Animal Care and were approved by the University of Manitoba

Animal Care Committee.

2.2. Tissue embedding and sectioning

After dissection, whole embryos were washed once with lX PBS. Tissues were fixed

in %oparaformaldehyde on a rotating shaker at 4oCovernight. The tissues were then

transferred to a I0o/o sucrose solution in PBS and incubated on a rotating shaker at 4

oC until they sedimented at the bottom of the tube. This step was repeated using 15%

sucrose and then 20olo sucrose.-Tissue was then incubated for at least 2 hours in a 1:1

mixture of 2f%sucrose and o.C.T. Compound (Sakura USA) in 4 oC, then quickly

frozen in O.C.T. Compound in plastic embedding molds, by placing them on a

dry-icel2-methylbutane bath. Embedded tissues were stored at -80oC. Frozen tissues

were section ed at 12 pm on a Microm HM 5I0 cryostat or a ThermoShandon

Cryotome@ cryostat. Sections were transferred onto Superfrost Plus slides (Fisher

Scientific) and stored at -80oC.

2.3. Histological staining, immunohistochemistry (IIIC) and

immuno fl uo rescence (IF)

To make reference slides, tissues were stained with cresyl violet dye for two minutes, and then washed by transfer through graded alcohols (50%to 100%) and

42 xylene. Then tissues were mounted with Permount (Fisher Scientifrc) and cover slips.

oC, For IHC experiments, frozen sections were taken from -80 transferred on dry ice,

and air dried for 10 minutes at room temperature. Tissues were pre-incubated in

blocking solution (PBS, 5% normal horse serum,0.zyo Triton x-l00, 0.1% BSA

fraction V, and 0.02 % sodium azide).Incubation was performed at room temperature

for two hours. Sections were then incubated with a primary antibody (diluted in

blocking solution) at 4oc ovemight, washed three times (3x) in pBS/0.05% Triton

x-l00 (PBS-T) @H 7.4) and incubated for 2 hours at room temperature with biotinylated secondary antibodies. Sections were again washed 3X in pBS-T and then treated with0.3%. HzOz in PBS-T for 30 minutes. Slides were washed 3X in PBS_T and then developed using the Vectastain ABC system (Vector Laboratories). Slides were again washed 3X in PBS-T and stained with diaminobenzidine tetrahydrochloride (DAB) or vIP (very intense purple) substrate reagent (vector

Laboratories). Negative controls were performed by omitting the primary antibody.

In IF experiments, tissue was incubated in blocking solution and incubated with the primary antibody as described above. Slides were washed 3X in pBS-T and secondary antibody incubations were for 2 hours in the dark at room temperature. Secondary antibodies were directly conjugated to a fluorophore. All sections were mounted using

Vectashield fluorescence mounting medium (Vector Laboratories). Negative controls were performed by omitting the primary antibody. Primary antibodies and secondary antibodies are listed in Tables 1 and2.

43 Table 1: Primary antibodies used for immunohistochemistry and immunoflourescence

Pri An Dilution Source Purified Rabbit anti-DLX2 l:1000 in-house by Dr. Eisenstat and Dr.J. Rubenstein Rabbit anti-PAX6 1:1000 Covance

Rat anti-TEN-C l:1000 R&D Systems

Table 2: Secondary antibodies used for IHC and IF

Secondary Antibodv Dilution Source Biotinylated goat anti-rabbit 1:200 Vector Labs Biotinylated rabbit anti-rat I:200 Vector Labs FITC-conjugated donkey anti-rabbit l:200 Jackson Immunoresearch FlTC-conjugated donkey anti-rat l:200 Jackson Immunoresearch

2.4. Chromatin Immunoprecipitation (ChIp), and Chlp-reChlp

The chromatin immunoprecipitation (ChIP) assay is a method used to determine whether proteins, such as transcription factors, bind to a particular region of DNA, for example, a gene promoter. This method can be used to show that the binding between protein and DNA takes place in vivo. Embryonic tissue was obtained from timed pregnant mice. Forebrain was used as a positive control. Snce Dlx2 is not expressed in the hindbrain, hindbrain was used as a negative control for DLX2 chrp experiments. Pax6 is expressed in the hindbrain but not in the hearl so heart was used as a negative control in ChIP experiments for pAX6.

44 Tissue was dissected in PBS on ice under the stereomicroscope. The cells were

dispersed by pipetting up and down th¡ou gh a9'glass pipette tip and loading tip, and

then incubated in l%o paraformaldehyde (PFA in PBS) with protease inhibitors (pI)

for 45 minutes at room temperature. Samples were then washed twice in pBS and

collected at2000 rpm for 5 minutes by microcentrifuge at 4oc. The cells were

resuspended in 400 pl of sDS Lysis-Buffer (1% sDS, 10 mM Tris-HCl pH g.l, l0

mM EDTA) with PI and incubated on ice for 10 minutes. The lysates were then

sonicated (10-15 times, 15 pulses at4\%opulse strength, <4 ouþut control settings)

using a B¡andon cell disruptor. Samples were kept on ice during sonication and kept

sitting on ice for I minute between sets of pulses. After sonication, the lysate was

checked by running on a I .0 %o agarose gel to verifu that the size of the major DNA band was approximately 400 bp. Lysate was collected by cenfifugation at 13000 rpm for i0 minutes at 4oC' The OD26e value of the lysate was measured. Four A26e units of the sample were diluted with i ml of dilution buffer (0.01% SDS, 1.1% Triton X-100,

1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM Nacl) and pI in a fresh 1.5 ml tube.

Sixty pl prewashed of Protein A-sepharose beads þrepared to be a S}%oslurry) were added to the lysate and incubated for 60 minutes at 4oc on a rotating shaker. The solution was centrifuged at 2000 rpm for 2 minute at room temperature. The supernatant was transferred into a fresh 1.5 ml tube. BSA and tRNA were added to a final concentration of 500 ug/ml. Thirty pl of porycronal antibody was added and incubated with the supernatant ovemight at 4oc on the rotating shaker. Negative conûols',vithout antibody were incubated separately.

On the following day, 60 pl of prepared Protein A*G-sepharose beads (50% slurry)

with PI, BSA and tRNA were added to the tube, and tubes were kept on the shaker for

3 hours' The tubes were then centrifuged at 4000 rpm for 5 minutes. The supernatant

was aspirated, and the beads were washed with the following buffers in sequence:

Low Salt solution (0.1% sDS, l% Triron x-100, z nMrEDTA, 20 mM Tris-HCl pH

8.1, 150 mM Nacl) for 5 minutes; High salt solutio n (0.\o/osDs, 1% Triton x-100,2

mM EDTA, 20 mM Tris-HCI pH 8.1, 500 mM Nacl) for 30 minutes; Licl Buffer

(0.25 M Licl l% deoxycholare, I mM EDTA, i0 mM Tris-HCl pH g.i, 1% Np-40)

for 30 minutes, TE buffer (pH 8.0) for 5 minutes twice. Following the washes, the bound complexes were eluted by adding I 00 pl of freshly prepared elution buffer (1 % sDS, oc. 0.lM NaHCo3, preheated to 65 "c) and incubated for 10 minutes at 65 The lysate was centrifuged at 13,000 rpm at 2 minutes at room temperature. The supernatant was collected into a fresh tube.

To reverse the cross-linking, 25 pl of 5 M Nacl and 1pl RNase A were added to the eluate and incubated oc. for 4 hours at 65 z0 ¡tl of 0.25 M EDTA, 20 pl of I M

Tris-IICI (pH 6.5), and2 ¡il of proteinase K were added to the eluates and incubated at

oC 55 for 2 hours. DNA was then extracted using the Qiaquick PCR purification kit according to the protocol. DNA samples were analyzed with specific pcR primers

(Table 3) designed from putative binding site regions inthe Ten-cpromoter

(GeneBank:Ul9460). PCR products were sequenced for verification and were further

46 used for EMSA and luciferase reporter gene assays.

Table 3: Oligonucleotide primer sets for ChIp assays

Name (position) Forward Primer (5'-3') Reverse Primer (5'-3')

cagaatcagatggcaaatgg Ten-Cl (-3708 to -3356) gggaactglccatttagc tggtga Ten-C2 (-2773 to -2495) gtatggagggçaãgggt ccaaccaaccaaaacçtcc

Ten-C3 (-2014 to -1718) ggafacl.gggatgtggag glaagctgagagaglctg

Ten-C4 (-1465 ro -1106) ctgaatcagcccaaatgg!1 ctgctttgctggactgtg

ChIP-reChIP was a modified ChIP assay based on the ¡outine ChIp protocol. The

second primary antibody was used before reversing the crosslinking. Eluent generated

from the first ChIP experiment was diluted 10 times with dilution buffer (0.01% SDS,

1.1% Tritonx-100, 1.2 mM EDTA, 16.7 mMTris-HCl pH 7.0, 167 mM Nacl) and incubated for 10 minutes at 65"c. The second primary antibody was added and processed as above. Two types of ChIP-reChIP experiments were carried out. On one hand, anti-DLX2 antibody was used first and then the anti-pAX6 antibody. For the reverse experiment anti-PAX6 antibody was used first and then anti-DLX2 antibody was used for the re-ChIP procedure.

2.5. Electrophoretic Mobility Shift Assays (EMSA)

Electrophoretic mobility shift assays (EMSA) were carried out as reported previously

(Ztou et a1.,2004)' There were four fragments of the Ten-Cpromoter cloned from the full length promoter using oligonucleotide primer sets. Each fragment was digested by

47 Ecok[' As the¡e is one EcokI resfnction digestion site in each of the promoter

fragments labeled Ten-C1 and Ten-C3, both Ten-Cl and Ten-C3 were separated into

two parts' Hence, there were six fragments after digestion in total, named Ten-C lA

2, lB, 34, 38, 4. Each fragment was radiolabeled with a-32p-dATp (perkin Elmer) by

using the Klenow (large fragment) of DNA pol l (#1g012-ozl,Invitrogen).

Radiolabeled promoters were then incubated with recombinant protein (DLX2 or

PAX6, purified in laboratory) for 30 minutes at room temperature and then run on an

"EMSA acrylamide gel" (Protean-Il apparatus, Biorad) as described (promega

technical bulletin #110 "Gel shift Assay system"). The gel was then dried under

vacuum using a BioRad gel drier for I hour and exposed overnight to autoradiography

-80 oC. film at The same procedures were carried out with the tissue extracts obtained

from El3.5 mouse embryos.

2.6. Co-Immunoprecipitation assays

co-immunoprecipitation (co-Ip) is a common technique for research on protein-protein interactions. When Co-IP was performed, an antibody specific to the protein to be studied was added to the cell lysate. The protein-A sepharose was added into the lysate to precipitate any antibody-protein complexes. other proteins or molecules that were bound to the first protein would be precipitated as well. The co-precipitated protein can then be identified by western blotting using specific antibodies.

Nuclear extracts were obtained from El3.5 embryos of wild type mice. Nuclear exfract (500 pg) was treated with I ml of RIPA buffer (1% Triton, 158 mM NaCl, 10

mM Tris-HCI pH 7.6,0.1yo5DS, 1% Na-Deoxycholate, lmM EDTA). 50 ¡rl of 1:1

slurry of Protein A-Sepharose was added and incubated for 30 minutes with rotation

at 4oC. The beads were pelletted by a quick spin. The supernatant was removed to a

1.5 ml microfuge tube. 2 pg of polyclonal antibody was added and incubated, at 4oC

ovemight. on the following day, 50 ¡rl of Protein A-sepharose was added and

incubated at 4oC for 3 hours. Beads were subsequently washed th¡ee times with I ml

of RIPA buffer. Supernatants were used for the western blotting procedure.

Samples rvere run on an appropriate (generally l0 or I2%;o) acrylamide gel

(Mini-Protean, Biorad) at l66Y. Proteins were transferred to a nitrocellulose

membrane (Biorad) at l00V for 1.5-2 hours. The memb¡ane was then blocked for 1

hour with ívomllk in PBS and then incubated with the specific primary antibody

ovemight. The membrane was then washed with TBS-T for three times, incubated

with a horseradish-peroxidase-conjugated secondary antibody for t hour, washed with

TBS-T three times again, incubated for 1 minute',vith ECL Plus (Amersham) and then

exposed to the auto¡adiography film (Kodak).

2.7. Luciferase reporter gene assays

Samples were analyzed for luciferase and p-galactosidase (F-gal) activities as described in technical bulletins 281 ("Luciferase Assay system") and,097

("p-Galactosidase Enzyme Assay system with Reporter Lysis Buffet''), both from

Promega. Constructs for reporter gene assays were made. Tlne Tenqscin-C fulllength promoter was cloned using PCR with the forward primer of Ten-Cl and the reverse

primer of Ten-C4. Ten-C fulI length promoter and four selected promoter regions

were inserted in the pGL3 basic vector. T¡ansfection experiments were performed in

the IIEK293 human embryonic kidney cell line. IßK293 cells were cultured in

DMEM (#12800-017 Gibco with 3.7 g NaHCo3lL and l0o/o fetal bovine serum). 24

hours prior to transfection, cells were seeded uniformly into l2-well plates at adensity

of I x 107 per 36 mm2 dish. Plasmid pRSV-ß-gal (Promega, Madison, WI) DNA was

used to determine transfection efficiency. On the foltowing day, cells were transfected

with 0.4 pg of plasmid DNA (0.2 pe pcDNA3 and}.z pg pGL3 consrrucrs) and 0.1

pg plasmid pRSV-B-gal. Transfections were done using Lipofectamine 2000 reagent

(Invitrogen). Cells were-harvested24 hours after tansfection, using reporter lysis

buffer (Promega). Luciferase activity was measured using the Luciferase Reporter

Assay System (Promega) and read out by a standard luminometer. Luciferase activity

was measured following the methods outlined in technical bulletins 281 ("Luciferase

Assay System") and p-galactosidase (Þ-gal) activity in as described in technical bulletin 097 ("p-Galactosidase Enzyme Assay System with Reporter Lysis Buffer), both from Promega.

Statistics were done by SPSS 10.0 for Windows and Microsoft Excel 2003 software.

Charts were generated using Excel.

2.8. Site-directed mutagenesis and DNA sequencing

Ten-C prontoter TAAT/ATTA mutant constructs were generated by site-directed mutagenesis (QuikChange, Stratagene). Three pairs of primers were designed as

shown in Table 4.The mutated binding sites are listed in Table 5.

Table 4: Primers for site-directed mutagenesis

Name Forward Primer (5'-3') Reverse Primer (5'-3')

Ten-C3-M1 ggatactgggatgfggag Egatactgggatglggaglgaataa afaaaggaatgg

Ten-C3-M2 c gcttlc c a gtaglaggltaagc ag ctgcttaacctactactggaaagcg

Ten-C3-M3 ggþagctgagagagtctgctaaa cagactctctca gctlac agccgtglqattgatagg

Table 5: Mutated candidate binding sites of the Ten-C promoter.

Name Wild fype Mutant

Ten-C3-Ml ATTAAT AGGAAT

Ten-C3-M2 TAAT TAGT Ten-C3-M3 ATTAAT ATCAAT

All constructs were cloned into the basic pGL3 vector and sequenced for verification.

Luciferase reporter gene assays were performed by co-transfecting plasmids into the

HEK293 cells.

5l 3. Results

3.1. Expression Patterns of DLX2, PAX6 and TEN-C in the Developing

Forebrain

D/; homeobox genes are expressed in interneurons in the embryonic forebrain

(Anderson eta1.,1997b). During early stages of the development of embryonic telencephalon, the subpallium expresses Dlx2 primarily intheYZ and SVZ (Eisenstat et al., 1999). The pallio-subpallial boundary GSB) is formed between pallium and subpallium, separating neocortex and striatum (Yun et al., 2001). Protein expression was examined by IHC and IF in mouse embryos at E13.5 and 814.5. The expression of DLX2 and PAX6 in the developing forebrain was restricted to specific regions.

DLxz is expressed mainly in embryonic shiatum, both in LGE and MGE, while

PAX6 is expressed in neocortex, and is also expressed at low levèls intheYZ of the

LGE. The expression domains of DLX2 and PAX6 in the forebrain of the embryo are divided by the PSB, in which rEN-c is highly expressed (Figure 5). The overlapping expression of TEN-C, DLx2 and PAX6 in the PSB suggests that these genes may play imporlant roles in controlling the formation of the PSB and the development of the anatomic subdivisions that flank it.

The expression of DLX2, PAX6 and TEN-C in the developing forebrain of mice was examined in our research. We observed that the expression of TEN-C in the forebrain was unaffec ted in Dtx I /2*/*, Dl* I /2*/-' and, Dlx I /2-l- mice, as shown by IF (Figure 6).

Similar results were observed in both E13.5 and E14.5 mouse embryos. These results indicate that Dlx2 does not play a key role in regulating the expression of TEN-C at

the PSB. On the other hand, Ten-C is expressed in the forebrain in Pax6 wlld,type and

heterozygous mice, while this expression is lost n Pax6 mutant mice. Similar results

are shown in both IHC and IF experiments (Figures 7, 8). This data suggests that

TEN-C synthesis at the PSB is compromised during development of the forebrain in

Pax6 homozygous mutant mice. We come to a conclusion that Pax|is a key regulator

of expression of Ten-C at the PSB. of interest, at the pSB, the p ax 6 null mutation

leads to the dorsal expansion of the subpallial domain of expression of DLX2 into the

pallium/neocortex (Figure 9). Thus, subpallial genes (ventral) are expressed in the

cortex (dorsal) inlhe Pax6 homozygous mutant. These results suggest that1he pax6

function might be related to establishment of the formation of the pallio-subpallial boundary, and cell migration from the striatum to the neocortex.

53 E13.5 814.s

DLX2

PAX6

TEN-C

Figure 5: Expression patterns of DLX2, PAX6, and rEN-c in the developing forebrain. Immunochemistry with specific antibodies shows thatDLXZ is expressed in the striatum (LGE and MGE) (4, D), PAX6 is expressed in neocortex, and is expressed at lower levels in the ventricular zone of the LGE (8, E), and Tenascin-C is expressed at the pallio-subpallial boundary (C, F). Similar expression pattems áre shown at both 813.5 (,{, B, and c) and El4.5 (D, E, and F). Black arrows point to the PSB. Scale bar,222 pm.

54 DlxI/2*/* DlxI/2*/- DlxI/2t-

E13.5

814.s

Figure 6: The expression of renascin-c in DlxL/2mutants. At E13.5 (A, B, c) and 814.5 (D, E, F), the expression of rEN-c is shown at the pSB in wild type (A, D) and DIxI/2 heterozygotes (8, E). However, in the Dlxl/2mutants, the expression of TEN-C is unaffected at the PSB. White arrovüs point at the PSB. Scale bar, 250 ¡tm.

55 Paxfh Pox6 *t-

E13.s

814.5

Figure 7: IHC. The expression of renascin-c in Pax6 mufants (IHC). Ar 813.5 (A, B, c) and 814.5 (D, E, F), the expression of Ten-c is shown at the psB in wild type (A, D) and Pæ6heterozygotes (8, E). However, inthe pax6L(c, F), TEN-C expression is not pSB. detected in the forebrain. Black anows point to the Scale bar, 222 ¡tm.

56 Paxfl* Pax6*/' Pax6/-

E13.s

814.5

Figure IF. The expression pax6 8: of Tenascin-c in mutants (IF). At E13.5 (A, B, c) and 814.5 (D, E, F), the expression of ren-c is shown at the psB in wild type (A, D) and Pø6heterozygotes (8, E).However, inthe pø6/- mutant (c, F), the expression of Ten-C is not detected in the forebrain. White arrows point to the pSB. Scale bar, 250 pm.

57 Paxf/* Pax6/-

E13.s

A

/

irlt.îiioÌ.l .l;t:h ' .:i.:i;*;: ' :-4ì::l!?"!:: 1 ïl¡', Et4.s :È ..::jl:r. :i;,' ' i ...ii:.. I : ... , .t1 :..\ ..

Figure 9: The expression of DLX2 in Pax6 mutants. DLX} expression is resfricted in embryonic striatum in the forebrain of wild type mice, both at El3.5 (A, B, c) and E14.5 (D, E, F). The same expression pattern is shown in paxft* (A, D) and pæ6*t- tissue sections (8, E), while Dlx2-expressing cells enter cortex tnthe PaxCL mutant (c, F).Black affows point to theyz limit of DLX2 expression. scale bar,222 pm.

58 3.2. DLX2 and PAX6 bind to the Tenøscin-C gene promoter

Tenascin-C is strongly expressed in the developing CNS, and is especially enriched in

radial glia during neuronal migration in the cortex (Jones and Jones, 2000b). Ten-C is

regulated by growth factors, integrins and other transcription factors. However, little is

known about which transcription factors regulate Ten-C expression.By arnlyzing the

sequence of the promoter of Ten-C, we discovered that this promoter contains several

putative binding sites for both DLX2 and PAX6 transcription factors @igure 10).

3.2.1. DLX2and PAX6 bind to the Tenasctn-C pro,moter i.n vivo

It was hypothesized that Ten-Cis a candidate transcriptional target of members of the D/x

and Pax gene families. To test this hypothesis, a modified ChIP assay was performed to

veriff the binding to the promoter of Ten-cbyDLX2 and pAX6 in vivo.In chrp

experiments with DLXZ antibody, the ganglionic eminences of mouse embryos at E13.5

were coilected and treated with paraformaldehyde, crosslinking protein-DNA complexes.

Sonicated fragments were immunoprecipitated with anti-DLX2antibody. The polymerase

chain reaction (PCR) was used to ampliff the candidate-binding regions of the Ten-C

promoter' Four regions of the Ten-C promoter were selected. ChIP assays demonstrated

thatDLX2 bound to each selected region of the Ten-cpromoter. DLX2 was not

expressed in the hindbrain, so extracts from the embryonic hindbrain were used as

negative controls in this ChIP assay. The ChIP assays without primary antibody or with

DLXZ antibody and chromatin derived from embryonic hindbrain tissues were both negative (Figure i 1, left panel).

A similar ChIP procedure was ca:ried out to test whether PAX6 bound to the Ten-C

59 promoter. Embryonic cortex was used as positive control tissue, while embryonic heart was selected as a negative tissue control since PAX6 is not expressed in the heart but is expressed in hindbrain tissue. The ChIP assay also revealed that PAX6 bound to all of the four selected regions of Ten-c promoter in vivo (Figure 1 1, right panel).

To veriÛr whether both DLX2 and PAX6 interact at the same region of the Ten-C promoter, we performed ChIP-reChIP experiments. In one "direction", the primary ChIp was carried out with PAX6 antibody, and the secondary ChIP was performed with DLX2 antibody. In the other "direction", ChIP was performed with DLX2 antibody first and

PAX6 antibody second. Purified chrp DNA was analyzed by pcR using primers

'We designed for the respective Ten-C promoter regions observed. concluded that DLX2 and PAX6 bo'nd to one rQgron of ren-c, termed ren-c3 (Figure 12).

60 Ten-Cl:

CAGAATCAGATGGC AA ATGGTGGTGAGAGGTGGÀAATAAAGGGATTAAG TTAATTTCAGCACAAGTAAAGGCTTATGTCTTGAGGAGCAC GTGATCTAC ACAGACCTAC CTGCACAGAATGGAAGCTCTGAúAAGGAATTCAGTGGC CAT TAGATGAC CATGAG GAACTC CTGCATAGC A.TA.T{TAGAAGTGTTAGTTGCCC A AGGG AAT AA ACAACCTGTGGCAAGGGAGAJA AA.TCCTCACTATTTATTCA TCTTATAGGAATT AATATC CATGGTATATAAAAATATTTTTTTAAAAATCC fu\A AGAGC fuqú\TTCATTCAGCATATGGGC AA AAGAGCTAJAATGGACAGTT CCC

Ten-C2:

GTATGGAGGGCAAGGGTAGGCAAGGCATTAGAAGACTGATTATCACAGT ATTATGTGCACTATGG A AACAGCACAACAAATGCAATCACTCAGTATTAT TACTCTATGT AúqAGTAT AA AGAGTC CAGGGGGGACAGAACAGTGTCTAGT CTGAGCAGTC CTCCTCACTGT AAGGCTGAAGTATAGCTAGAATTATGATGT TTCTCAGCACTTTGG GAGAJ{ÁACTTTTTTT GTTAGTGAJA"{GGGTCTCTTGTT TGTTCTTGGAGGTTTTGGTTGGTTGG

Ten-C3:

GGATACTGGGATGTGGAGTGAATAJqAT fuA ATT A*{TGGAJAJAJAJAJqr{GATAG ATCTCAGGTGTCATGTGACACATTTCTGTGACCACTGATGGTTATAGTCTT ATGTGTCAGGATCACCATCCTCAGGGCCTCCCATCTGACTTCGCTTTCCAG TAATAGGTTAAGCAGTTTGTGGTAGCTGTGAATTCATGATTGACTATAGAA CACTAAGAATTGTTGGGGACCAGGGATGTCAAGTCTGGAACATTC TTAGA AGC CCTATTAATCACACGGCTTTTAGCAGACTCTCTCAGCTTAC

Ten-C4:

CTGAATCAGCCC AA ATGGTTTAJM AGTTC AJAATGAGTCGTTTTACGCAAT GACTATTTATGGCTGTTC CTGATGTCTGAGCGGTGTGTGTGTGTGTGTGTGT GGGGGGGGGGGGGATAGGGAACACTTAAGCAGGCAGTCTTCGAGTCCTG G AATCGGTGCCACCCTAGGGATAGCCTAATTTTGTCTTGCTTTATTCTCCCA GGGAGATTGfuÂV\CTAJ\ATGTTATGAGAAGGCTGAAGGATGGTATCAATCA GTAAGTGGATG GTT GAJqATTTC CGATAATAGTTGTTAÁTT AJqJAACATCAG AAGTCTCTTCT AA ATTACAGAAC A AAGAGATGCATTTTCCACAGTCCAGCA AAGCAG

Figure 10: Four regions ("fragments") selected from the Ten-c gene promoter. Bold letters are putative franscription factor binding sites.

61 *"%*{".S ."dur"Ë Ten-Cl

TeniG2

Ten:C3

Ten-C4

Antl-ÞLX2 Ant¡-PAJ(6

Figure 1 I : ChIP assays of DLX2 or PAX6 an d, the Tenascin-C promoter. ChIp assays were performed on E13.5 mouse forebrain tissues. The striatum was used with DLX2 (left panel), while the neocortex pAX6 1ntibg_dy was used with antibody (righr fanel). Specific bands verified the interaction between transcription factors unã t*i"t r"n-C gene promoter sequences. Negative controls (negative tissue conkol and nelative antibody control) failed to precipitate chromatin. Positive controls *rr, *oi.e genomic DNA @DNA\

62 Tæn-C-t

Tæn-&.

Tæn-C3

T*n-ffi

1234 56 78 9 12 34 5 67.8 9 ChfF {er*i:FPji.ûT CÌûP {enti-OL¡rz}- reCfÈP (anti-tlLll2) re{hlP {afli-P.Â,}lû}

Figure 12: ChIP-reChIP assays. Left panel: The primary ChIP was carried out with an antibody against PAX6 (from left to right, I't to 4ü lanes), and the secondarv chlp was performed with an antibody against DLx2 (from left to right, 5th to gû ianes). rugllpTît: Tlî primary ChIP was carried out with antibody ãgainst DLxz(from left to 1" right, to 4* lanes), and the s.econdary ChIP was performed with antibody against PAX6 (from left to lgtrr, s* to 8ú lanes). cis: coretex/shiutum, No-Ab: no antibody, gDNA: genomic DNA conrrol (lane 9).

63 3.2.2. DLX2 binds to the Tenascin-C promoter invitro

To determine whether DLX? specifically binds to the Ten-Cpromoter in vitro, recombinant DLX2 proteins and radiolabeled Ten-C promoter regions generated from the

ChIP assay were used. Electrophoretic gel mobility shift assays (EMSA) showed that

DLX? binds to all4 fragments of the Ten-Cpromoter (Figure 13). Conhols incubated with unlabeled probe showed reduction of the specific band as well ("cold competition").

Moreover, the addition of specific anti-DLX2 antibodies in the experiment resulted in significant band mobility shifts ("supershift"). These experiments demonstrated that

DLXZ specifically binds to the Ten-C promoter in vitro.

64 A *r¡tbody +l rBLIQ -++ ++ Urlabeledprúe + -\. '+ Lrbeledprobe + *+ 123 45

Superslifred barË: çlü!å2

,$Sfcffic DLXZffsÞ GtlåfÐ,bard

free prdbe

îen{l & Ten{,lt

B årrfüo{¡*: .;!"+l* DUCA "*#,+,,*t Unlùelodproh jf ': t Laþlled,protrè "1.+' ! # * 123.fl.5

.superehifred Þ¡nd: s OLXZ --.Þ

$pecÏic,IlLX2trTen -t2''band::'--'-- + freepruba :Tern¡C2

65 c f,rrifroqy rLxz llrttnelâfproæ lábdËd pobe

$rperefiiftedband: o BLI(2

SpecÏic OLX2fTerÞ tl*r'B bärid 'frægmhe

Ten-f,3S Terrü3F

D nñtDq4f -:-: +iI $uË - + +.+..+ tlnlabeledpruüe ,+ l-$eledprnbe ++,t 4,.* 13345

Super*f-üedþänd: o BIJ(2 -+

Specifi c^ Il LtlS ¡lÏrn-t4 bÊnd ----)

frcæ pnibe Tl;lr.G*

66 Figure 13: EMSA of recombinarÍDLX2 and ren-cpromoter sequences.

Panel A, EMSAs show binding of recombinant DLXzto the region Ten-cl of Tenascin-C promoter containing putative homeodomain binding sites in vitro. Radiolabeled Ten-Cl oligonucleotide probes were incubated alõne Qane I, lane 6),with recombinantDLx2 protein (lanes 2-5, 7-17),with unlabeled. Ten-ClA or Ten-C1B probes (lanes 3, 8), with affinity-purifed DLX antibodies (lanes 4, 9), and,with nonspecific antibodies (lanes 5, I0).

Panel B, EMSAs show binding of recombinant DLX2 to the region Ten-C2 of Tenascin-C promoter containing putative homeodomain binding sites in vitro. Radiolabeled Ten-C2 oligonucleotide probes were incubated alõne Qane I), with recombinantDLx2 protein (tane 2-5), with affinity-purified DLX antib oiies (lanes 3), with unlabeled Ten-C2'probes (lane 5). A nonspecific antibody was used in laie 4.

Panel c, EMSAs show binding of recombinant DLX2 to the region Ten_c3 of Tenascin-C promoter containing homeodomain binding sites in vitro.Radiolabeled Ten-C3 oligonucleotide probes were incubated alone (lane I, Iane 6),with recombinant DLx2 protein (lanes 2-5, T-r0), with unrabeled, Ten-c3A or-?B probes (lanes J g), with affinity-purifed DLX antibodies (lanes 4, 9), andwith nonspr.ifi. antiùodies ('lanes 5, r 0).

Panel D, EMSAs show binding of recombinant DLX2 to the region Ten_c4 of Tenascín-C promoter containing homeodomain binding sites in vitro.Radiolabeled Ten-C4 oligonucleotide probes were incubated alone (lane l),with unlabeled. Ten-C4 probes (lanes -1) with recombinantDLx2 protein (lanes 2-5),withaffinity-purified DLX antibodies (lane 4), and with a nonspecific antibody (lane 5).

Gel shifts, denoting specif,rc binding of DLX2 to DNA, are indicated with solid arrows. Srrpershifts with specific DLX antibodies are indicate ãby broken a/.rows;I, nonspecific polyclonal antibody.

67 3.3. DLX? and PAX6 transfection promotes Tenøscin-C promoter expression fn vitro

To show the functional consequence of binding of DLX2 and.PAX6 to the Ten-C promoter, luciferase reporter gene assays were performed. Luciferase assays were performed in IIEK293 cell lines. There was a low level of expression of DLX2, but no

PAX6 expression in IIEK 293 cell lines (data not shown). Luciferase assays were carried out using Dlx2 and Pax6 constructs and either the full-len glh Tenascin-C promoter or smaller regions of the Tenascin-cpromoter charactenzed by chlp and EMSA.

Co-transfection with either DLX2 or PAX6 expression constructs and the full-length

Ten-C promoter resulted in a significant increase in luciferase activity compared to controls (2.4*0.2-fold for DLx2 and 1 .5+0.2 -fold for pAX6, p<0.01; Figure 14), suggesting that both DLX2 and PAX6 proteins can up-regulate the expression of the

Ten-C promoter in vitro- Co-transfection with both DLX2 and pAX6 with the full-length

Ten-C promoter showed a 3'1+0.5-fold (p<0.05) increase of luciferase activity, greater upregulation than for either hanscription factor alone.

68 '5e o o U' (g L E pGl3-Basic P2o J I 'Ten-C-F J þo õ1ìo lJ-

pcDNA3 DLX2 PAX6 DLX2+PAX6

Figure l4: Luciferase reporter assays with the Tenascin-C full-length promoter. Luciferase reporter gene assays followed transient co-transfections with the Tenascin-C full-length promoter inserted into a pGL3-L-uciferase reporter, in the absence o, pr.rrrrr. of DLX2 expression in FIEK293 cells. DLX2 and PAX6 both up-regulate the of the Tenascin-C full-le^ngth promoter. pGL3-Ten- C-F: Tenasi¡" ð r¡l-l*gth"*pr.rrio' prorrot", inserted to a pGL3-Luciferase reporter vector.

69 Since the Tenascin-C fail-length promoter was activated by either DLX2 (2.4+0.2 fold) or PAX6 (1.5+ 0.2 fold) co{ransfection, the question of interest was whether any special region of the Tenascin-C promoter was regulated by DLX2 or PAX6. Luciferase assays following co-transfection of each fragment of the Tenascin-Cpromoter with DLX2 or

PAX6 were performed. We revealed that DLX2 co-transfection also increased the expression of each fragment of Ten-C (1.39t0.24-fold for Ten-Cl, l.l2+0.09-fold for

Ten-c2, r.65+0.12-fold for Ten-C3, 1.28+0.lg-fold for Ten-c4), while pAX6 only increased the expression of one fragment of Ten-C, named Ten-C3 (1.33+0.11-fold)

(Figure 15).

To further investigate the increase of expression of the Ten-C3 promoter region in vitro, mutated Ten-C3 constructs were examined using luciferase assays (Figure 16). There are three putative homeobox domain-binding sites in Ten-C3, each mutant of which was generated by site-directed mutagenesis. The three mutants are named Ten-C3-Ml,

Ten-C3-MZ, and Ten-C3-M3. We found that the expression of the Ten-C3-M2 mutant is compromised when transfected with either DLIçZ or PAX6. This result indicates that this second'candidate homeodomain binding site is critical for up-regulation in vítro,and the third site is also likely to be involved. The first site is not necessary, as the expression of the Ten-C3-Ml mutant is not significant affected in vitro.

70 * ï '=è 2.5 o li3' o) _T m pGL3-Basic U) ll * * g r pGL3-TenCf g 1.5 TW () f, tr pGL3-TenC1 J T .l I pGL3-TenC2 ol rr# w õ T Bs pGL3-TenC3 o LL 0.5 W. IW n pGL3-TenC4 W,,'* pcDNA3 DLX2 PAX6

Figure 15: Luciferase reporter assays with four selected fragments of the Tenascin-C promoter' HEK293 cells were transfected wúhTenascin-C promoter expression vectors and either DLx2 or PAX6 expression vector. DLX2was foìrnd to increàse the expression of each of the four fragments of Ten-cpromoter, while pAX6 onl¡, i¡"r.uses the *: expression of fragment Ten-c3. n:3. p<0.01; Tencf: Ten-c dtt_tength.

7t 3

.à ,.s o

c) Ø Lõ o 1.5 'õ f J o 1 ! O n" LL "'"

0 pcDNA3

Figure 16: Luciferase reporter assays with the mutated Ten-Cpromoter region, Ten-C3. HEI<293 cells were co-transfected with DLX2 or PAX6 and wild type Ten'-Cí qin green; or mutated Ten-c3 (Ten-c3-Mi in light yellow, Ten-c3-M2 in dark yellow, aìd Ten-C3-M3 in blue)' The second site (M2) is critical for up-regulation, and tie third site is likely to be involved in the regulation. n:3. *, p<0.05, y, p<0.01.

72 3,4. DLX2 interacts with PAX6

Given the preceding results, there is a possibility thatDLX2 directly interacts with pAX6.

To test this possibility, the HEK293 cell line was tansfected with a Dlx2expression plasmid accompanied by Pax6 expression plasmid, and then subjected to a co-immunoprecipitation (co-IP) experiment. We found thatDLx}bound top,\X6 in vitro @igtxe 17, upperpanel). To further test the interaction between DLX2 andpAXl in vivo, cell lysates of forebrain embryo extract (of cortex/striatum border) was immunoprecipitated with DLX2 antibody, and PAX6 antibody was used in Western blotting to assess whether PAX6 was accompanied by DLx2 in the immunoprecipitates.

As shown in Figure 17 (lower panel), DLX2 formed protein-protein complexes with

PAX6 in both embryonic cortex/striatum as well as pancreas. Direct western blotting of cofical extracts performed with PAX6 antibody are used as a positive control (lane 6). A negative control is included using beads conjugated to an irrelevant antibody @at IgG, lanel). Another control performed was an IP without any antibody at alland western blotting with PAX6 antibody (tane 3).

73 1 2 3

* f -È Ë iÐ f, I g s # g û' 4tt Püü4: ffi 1W

B Lane 1 2 34 Crrb¡r#r¡üfñ + + + Fancgæ:11ç4tO :+ Êt lgg Rt-antFttrl : + -"+ $ende. + + ++ ti,Fwtl

P*t(Ê ¡+ ,

Figure 17: Co-IP of DLX2 and PAX6. Co-IP was performed with ÍIEI{¿13 cell lysates and embryo extracts. A. Invitro co-IP experiment in HEK293 cells. pcDNA3-DLX2 and poDNA3-PAX6 were co-transfected in HEK293 cells and then cell lysates were immunoprecipitated using rabbit anti-DLX 2 antibody. The immunoprecipitates were examined by westem blotting using an anti-PAX6 antibody. An IP control is shown in lane 2' B' In vivo co-IP experiment with E13.5 tissue lysates from cortex/striatum and pancreas. Cell lysates from tissues were incubated with anti-DLX2 antibody for Ip. For western blotting of immunoprecipitates, anti-PAX6 antibody was used. Negative controls were performed without antibodies. Positive controls were results from onf western blotting experiments, for both corte>r/striatum border and pancreas (Figure lourtesy of Shunzhen Zhang).

74 4. Discussion

We have shown that PAX6 binds tothe promoter of Tenascin-C,probably up-regulating

its expression in the developing forebrain. We demonstrated that pax6 is necessary for

the expression of Tenascin-C at the pallio-subpallial boundary. Althou gh Dlx2 is not

necessary for the expression of Tenascin-C,DLXZ protein still binds to the same region pAX6 of the Tenascin-C promoter to which binds, indicating DLx2is part of a

kanscriptional complex that binds to the Tenasctn-Cpromoter. It is notable thatDLX2

forms protein-protein complexes with PAX6, which is the first report of such an

interaction.

4.1. Pax6 is essential for the expression of Tenascin_C

In the developing cortex, Pax6 is expressed in the progenitors of the radial glial that

perform a dual function as precursor cells and as guides for migrating neurons, part of the functional tole of Paxó in vertebrate forebrain development (Gotz et al., 199g; Simpson

and Price, 2002)- Stoykova (Stoykova et al., 1997) first reported that pax6 is critical for

the expression of Tenascin-C RNA, as Tenascin-C expression is abolished at the

cortex/striatum border in the forebrains of Pax6 homozygous mutant mice. This research

was performed 3ts-labelled using anti-sense RNA probe s. Tenascin-C is highty expressed

at the PSB and in cortical VZ cells (Götz et al.,1997). In the Pax6 mutant, the expression

of Ten-C is absent. However, it is interesting to note that the express ion of Ten-C is not altered in the YZ of the medial (hippocampal) telencephalic wall or in other regions

(mesencephalon, cerebellum) of the Pax6 mutant brain, which suggests that there are other factors that are expressed at PSB that may be involved in regulating TEN-C

75 expression along wíth Pax6.ln our experiments, a polyclonal TEN-C antibody was

utilized in immunohistochemistry and immunofluorescence experiments to show the

alteration of expression of TEN-C in mutant mouse embryonic forebrain. Our results

confirmed that TEN-C expression was significantly compromis ed, in Paxlhomozygous

mutant mice, but not in Pax6 heterozygotes. However, the expression of TEN-C is still

detected in DlxI /2 double knock-out mice, which suggests that DlxI /2 do not play a key

role in regulating the expression of TEN-C.

Recent research (von Holst et a1.,2007) has demonstrated that there are 20 different

TEN-C isoforms presenting in neural stem/progenitor cells, and the expression of short

and long isoforms of TEN-C was differentially regulated by transient overexpression of

Pax6 in neurospheres. The larger TEN-C isoforms contained four, five, and six additional

alternatively spliced fibronectin type III domains, and were up-regulate dby Pax6, while

the smaller TEN-C isoforms that did not contain any or just contained one additional

domain were down-regulated by Pax6. However, the total mRNA level of Ten-C and the

composition of TEN-C isoforms remained unchanged. Some large TEN-C isoforms were

lost in sey (small eye) mutant forebrain tissues. These findings indicated Pax6 canbe a

modulator of the spliceosome. Compared to previous studies, our f,rndings provide new

insights regarding the regulation of expression of TEN-C. It is also possible that this

differential regulation of TEN-C isoforms is also regulated by Dlx2 as well.

4.2. Dk2 and, Pax6 transcription factors bind to the same region of the Tenascin-C

promoter

'We showed that both DLX2 and PAX6 bind to the Tenascín-C promoter ín vivo and in

76 vitro,using ChIP and EMSA experiments. When these experiments were initiated, the

fust step was to review the sequence of the Tenascin-C promoter. We found that there are

several putative transcription factors binding sites (Table 6, Figure 10). A recent

publication regarding interaction between regulators and the human Ten-C promoter was

reported (Ghaùrekar and Trojanowska, 2008). A ChIP assay was carried out to show that

endogenous GATA-6 binds to the Ten-C promoter in vivo.In addition, a GATA-6

response element was located at position - 467 to - 460 bp of the Ten-C promoter,

verified by mutational analysis (Ghatnekar and rrojanowska, 200g).

Table 6: Putative paired and homeodomain binding sites are distributed in the Ten-C promoter

Number Homeodomain Paired domain Paired-homeodomain Name binding site bindins site Binding site Ten-Cl 2 I Ten-C? 4 Ten-C3 J Ten-C4 J I

As mentioned previously, Tenascin-C is induced or repressed by multiple factors that

include avaiety of growth factors, c¡rtokines, integrins and mechanical forces (Jones and

Jones, 2000a; Jones and Jones, 2000b). For instance, fetal bovine serum and transforming

growth factor-81 (TGF-Pl) have both been revealed to variabty regulate Ten-C. The

mwine EVX-I protein was also shown to be an activator of the Ten-Cpromoter.

Moreover, the response element was mapped to an 89 bp region of the Ten-C proximal promoter. The Tenascin-C promoter also contains two important DNA regulatory motifs: an NF-xB binding site, and a TRE/AP-I motif (Jones et al., lggz).Maoy activators of

Ten-C gene expression have been identified; however, it remains unknown whether these

77 factors act directly or indirectly on the Ten-Cpromoter. DLX2 and PAX6 bind to the

same region of Ten-C promoter, which suggests the hypothesis that DLX} and PAX6

may compete with each other for binding to the Ten-C promoter. It will be of interest to

examjne whether these two transcription factors act together as part of a regulatory

complex. It has been well reported that homeodomain proteins can interact with other

factors. For example, it was shown that GATA-4 interacts with Ntø2.5 in the heart to

regulate expression of the atrial natriuretic factor and cardiac actin promoter (Saadane et

aI.,1999; Sepulveda et al., 1998);

4.3. PAX6 and DLX2 interact physically and function in the forebrain development

It was interesting to find that PAX6 and DLX2 proteins interact in vivo and. ín vitro, as

revealed by co-immunoprecipitation experimentS. PAX6 interacts with other factors, such

as transcription factor AP-2. The expression of AP-2 overlaps with that of pax6 in the

eye and the brain. AP-2 has a significant role in eye development as well, as Ap-2 mutant

mice show eye defects similar to the Pax6heterozygote (West-Mays et al., lggg).

Interactions between Pax6 andAP-2 have been verified by transfection and gel shift

experiments; Pax6binds to AP-2 through its C-terminal domain. Cooperative interactions

between Pax6 and AP-2 are necessary for normal corneal epithelial development and

maintenance (Sivak et al., 2004). Other studies have found evidence of interactions

between Pax6 and cooperative partner proteins in vertebrate development. pAX6 also

interacts with the transcription factor sox2, and the complex of pAX6 and soX2

activates the expression of y-crystallin gene during lens placode development (Kamachi et a1.,2001). Also in the lens, Pax6 and form a complex and participate in lens development (Cveki ef al., !999). In the pancreas, Pax6 hasbeen

78 revealed to interact with Maf and -2 transcription factors to cooperatively induce the

expression of the glucagon gene (Hussain and Habener, 1999; Planque et al., 2001). Here we have provided evidence showing that PAX6 andDLX2 interact in the developing

forebrain. To our knowledge, our observation is the first to propose a mechanism for the regulation of a specific ECM gene by PAX6, and also to suggest an interaction between

PAX6 and DLX2. Taken together, all of these studies indicate a mechanism by which

PAX6 protein controls the expression of an ECM protein through interactions with other proteins, including the transcription facto r DLX2.

79 5. Conclusion and Future Directions

In conclusion, this study has demonstrated that the homeobox genes Pax6 and Dlx2 play an important role in vertebrate forebrain development. Pax6 and Dlx2 are expressed in the pallium (neocortex) and subpallium (ganglionic eminences), respectively in the developing forebrain, overlapping at the pallio-subpallial boundary. Interestingly.

TEN-C, an adhesion protein of the extracellular matrix, is specifically expressed at this boundary. TEN-C protein expression is completely abolished in the telencephalon of the

Pax6 homozygous null mouse embryo, whereas it is unaffected in the Dlxl/Dlx2 do:u;ble knockout mouse. Yentralization of the domain of Dlx2 expression from the subpallium of telencephalon to the pallium is also revealed in fhe Pax6homo,rygous null mouse embryo. Moreover, Ien-C has been identif,red as a direct transcriptional target of PAX6.

Both DLX2 and PAX6 bind to regions of the Ten-C promoter in vitro and. ¡n vivo and, transactivate expression of a Ten-C reporter gene constru ct in vitro.It is notable that both

DLX2 and PAX6 bind together to a small region of the Ten-C promoter invivo and.

DLX? and PAX6 form protein-protein complexes in vitro and in situ (Figxe l7). In the three models shown in Figure 18, model C is more plausible, in which PAX6 interacts with DLX2 and the PAX6-DLX2 complex bind to Ten-C promoter via PAX6. Model ,.C" is hypothesized to explain how PAX6 and DLX2 may interact at the Ten-Cpromoter because Ten-C expression is significantly reduced at the pallial-subpallial boundary in the

Pax6 mutant, while unaffected at this boundary in the Dlxl/Dk2 double knockout mutant.

Hence, Pax6butnot Dlx2 is necessary for expression of TEN-C at the PSB. Furthernore, the Ten-C3 promoter region identified from the ChIP-re-ChIP assays appears to be specifically activated by PAX6 but not DLX2 in vitro. So, Pax6 is both necessary and

80 sufficient for Ten-C expression in the developing forebrain, while Dlx2 is only suffrcient

fot Ten-C expression in vitro. The schematic diagram of Ten-C promoter has been updated to include the binding site for PAX6 at nt -1863 (Figure 19). Loss of pax6- mediated activation of Ten-C may contribute to the compromise of the pallio-subpallial boundary in the developing forebrain.

Although these studies have enriched our understanding of the role of Pax6 and, Dlx2 jn vertebrate forebrain development, important questions remain. It is unclear why DLX2 transactivates Ten-C expression in vitro yet loss of Dlxl/DIx2 function does not affect

Ten-C expression at the pallio-subpallium boundary in vivo.It is possible that pAX6 and

DLX2 compete for binding to the Ten-Cpromoter. To test this possibility, a time course of PAX6 arñDLX2loading on the Ten-C promoter should be explored. It will be exciting to examine the phenotypes of Pax6/Dtxl/Dtx2 triple knock out mice, which will provide further evidence regarding the cooperation of Pax6 and, Dtxl/2 during development. The critical domains forDLX2lPAX6 interaction remain unknown. A series of Pax6 arñ Dlx2 deletion constructs removing putative functional domains could be generated in order to narrow down the region of PAX6 that binds to DLX2. Hence, further characteization of the interactions between these transcription factors and their target genes will improve ow understanding of forebrain development and help elucidate the mechani sms underlying human neuro developmental disorders.

81 t I I

r*

Figure 18: Models for PAX6 andDLX2 to the Ten-c promoter. A. pAX6 and DLx2 form a protein-protein complex, and DLx2 may bind to the Ten-c gene promoter directly. B. Both PAx6 and DLX2 bind to the Ten-C g"n. pro-ãt"r. c. PAX6-DLX2 complexes bind via pAX6 to the Ten-c gene promoter.

-ttË¡ -260 ¿3t -2m -fû0 8r

Par6+fllÉ 0T3 *to+H TDE .ilCT HF,,t Ée{ {CIh21 Egr.{ ih,lF,ltE lÊøZl {R:xlf}l

Fieure 19: updated schematic diagram of transcription factor binding sites gene promorer. pAX6-DLx2comptex yit3 !\? Ten,c The binding site for the via PAX6 is added at position -1863. p, pAX6; D, DLX2.

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