Canada Archives Canada Published Heritage Direction Du Branch Patrimoine De I'edition

Roles of Iroquois in mouse development

Niki Alizadeh Vakili

A thesis submitted in conformity with requirement for the degree of Masters of Science Graduate department of Molecular Genetics University of Toronto

395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada

Your file Votre reference ISBN: 978-0-494-38935-5 Our file Notre reference ISBN: 978-0-494-38935-5

NOTICE: AVIS: The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats.

The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission.

In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these.

While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada Roles of Iroquois in mouse development

Masters of Science, 2008 Niki Alizadeh Vakili Department of Molecular Genetics University of Toronto

Iroquois encode conserved transcriptional activators or repressors that are

important for neurogenesis, patterning and brain development. In mice, six Irx genes

show redundant expression pattern and single knock out mice are viable and fertile. In

this study, knock out ofIrx3 and Irx5 was used to decipher the functions of Iroquois in

mouse development. Irx3/Irx5 double knock out mutants die at El4.5 with defects in

various organs, such as limbs and diencephalon, confirming redundancy in Iroquois

function. In addition, I generated novel mouse models such as EnR-Irx3 line, which can

be used to conditionally overexpress the dominant repressor Irx3.1 have shown that

overexpression of EnR-Irx3 in neural progenitors lead to prenatal lethality. I have

generated a knock in epitope-tagged Irx3 useful for biochemical studies of identifying

Irx3 downstream targets and its protein partners. These mouse strains are useful for

deciphering the function of Irx proteins in mouse development.

n Abstract ii Table of contents iii List of figures iv List of abbreviations v

1. Introduction 1.1 Iroquois are organized into clusters. 1 1.2 Iroquois encode transcriptional activator or repressor that belong to TALE super class of homeodomain proteins. 2 1.3 Roles of Iroquois in Drosophila development. 4 1.4 Iroquois genes in vertebrate neural tube patterning: insights from the Chick. 5 1.5 Iroquois genes in vertebrate development: insights from the mouse. 10

2. Materials and methods 2.1 Generation of expression constructs and targeting vectors 14 2.2 Cell culture and Luciferase assay 15 2.3 Western Blot and Immunopercipitation 15 2.4 Generation of targeted ES clones 16 2.5 Sample preparation and histology 17 2.6 Mice 17 2.7 X-gal staining 17 2.8 Bone staining 18 2.9 In situ RNA hybridization 18

3. Results 3.1 Irx3 is expressed strongly in various tissues during late embryonic development and adulthood 20 3.2 Double knock out ailrx3 and Irx5 results in embryonic lethality at day 14.5 with multiple morphological defects 27 3.3 Irx3/5 DKO show proximal and hindlimb specific distal limb defects 29 3.4 Irx3/5 DKO brains have defected forebrain organization 31 3.5 Conditional knockout oiIrx3 in Irx5 background delays death until birth in double knock out embryos 34 3.6 Dominant activator, dominant repressor and HA-Irx3 fusion proteins are functionally active both in vivo and in vitro 35 3.7 Generation of targeted conditional Irx3 overexpressing mouse lines 39 3.8 Generation of epitope tagged Irx3 mouse line 42

4. Discussion and Future Directions 4.1 Dynamic expression of Irx3 at various stages and tissues suggest multiple roles during development. 47 4.2 Iroquois function redundantly during mouse development. 48 4.3 Iroquois are involved in neural crest development. 51 4.4 Iroquois play a role in limb development. 55 4.5 Iroquois are important for patterning of diencephalon. 57

5. References 60

in List of Figures

Figure 1: Organization of Iroquois genes into clusters 2 Figure 2: Roles of Iroquois in A/P patterning of neural tube 7 Figure 3: Role of Irx3 in D/V patterning of neural tube 9 Figure 4: FwW toes mutants have the entire cluster A deleted 11 Figure 5: Expression of Irx3 during embryonic development 22 Figure 6: Expression of Irx3 in adult organs 24 Figure 7: Expression of Irx3 in developing and adult brain 26 Figure 8: Morphological defects seen in Irx3/5 DKO embryos 28 Figure 9: Irx3/5 DKO mutants show defects in their heart and eyes 29 Figure 10: Limb defects in Irx3/5 DKO mutant embryos 31 Figure 11: Analysis of Irx3/5 DKO brain 33 Figure 12: Conditional deletion of Irx3 in Irx5 background 35 Figure 13: Fusion proteins of Irx3 are functionally active 38 Figure 14: Generation of Irx3 knock in ES cell lines 40 Figure 15: Generation of conditional EnR-Irx3 overexpression mouse line 42 Figure 16: Generation of epitope tagged Irx3 mouse line 44 Figure 17: Irx3-3M6H pups are comparable to their wild type littermates 46

IV List of abbreviations

ac-sc: achaete-scute A/P: anteroposterior am: araucan AVSD: artrioventricular septal defect BMPs: bone morphogenetic proteins bp: base pair caup: caupolican CNC: cardiac neural crest CNS: central nervous system DCN: deep cerebellar neurons DKO: double knock out D/V: dor so-ventral EnR: Engrailed fr: fasciculus retroflexus Ft: Fused toes Hb: habenular nucleus Irx: Iroquois homeobox gene kb: kilo basepair Iso: isthmic organizer LBWC: limb-body wall complex mirr: mirror NC: neural crest NTD: neural tube defect RA: retinoic acid Shh: sonic hedgehog SOP: sensory organ progenitor TALE: three amino acid extension loop Vp 16: viral protein 16 ZLI: zona limitans intrathalamica 1. Introduction

1.1 Iroquois are organized into clusters.

Irx genes were first identified in a screen for sensory organ development in

Drosophila. Further studies identified three genes within the locus; araucan (ara),

caupolican (caup), and mirror (mirr) (Gomez-Skarmeta et ah, 1996b; McNeill et ah,

1997). Vertebrates have more than three Irx genes that are organized into clusters in the

genome (Fig 1 A). Based on paralogs, sequence homology, and general structure of the

cluster, such as orientation and distance, a prevailing hypothesis is that the Irx clusters are

derived from duplication events during evolution (Peters et ah, 2000; Houweling et ah,

2001; Gomez-Skarmeta et ah, 2002).

Mouse and human have 6 Irx genes organized into two clusters. Cluster A

contains Irxl, Irx2, and Irx4 on mouse chromosome 13 or human chromosome 5. Irx3,

Irx5, and Irx6 belong to cluster B, positioned on mouse chromosome 8 or human

chromosome 16 (Fig. 1A) (Bosse et ah, 2000; Peters et ah, 2000; Gomez-Skarmeta et ah,

2002). Irxl and Irx2 share a similar expression pattern while Irx3 expression resembles the expression pattern of lrx5 (Cohen et ah, 2000; Houweling et ah, 2001). These Irx gene pairs are oriented in the opposite direction in the genome, making it possible that

same regulatory elements control expression of both genes. Another explanation for this

expression pattern relies on presence of conserved regulatory elements within the

clusters. Iroquois clusters are exceptionally long, with long stretches of non-coding DNA.

Preservation of these non-coding DNA is an indication of evolutionary pressure for their maintenance. Recently, highly conserved regulatory sequences have been identified in these genomic "deserts". In addition to mouse and human, these sequences are found in

1 Xenopus and zebrafish, where they have been tested for their ability to drive reporter gene expression in patterns similar to a subset of Iroquois expression (de la Calle-

Mustiens et al., 2005). In Drosophila, the regulatory sequence between araucan (ara) and caupolican (caup) regulates the expression of both genes, providing additional support for regulatory elements controlling more than one Irx genes in a cluster (Cavodeassi et al., 2001). Thus, the overlapping expression pattern of Iroquois members in various tissues can be explained by presence of shared regulatory elements that control their expression.

Drosophila Early functions: specification of ^ ^s. Iro protein: large termones —-~^

Mouse chr. 13 Human chr. 5 Late functions; Mouse chr. S specification ot Human chr. 16 pattern elements (subset of bristles, Dm chr. 3L wing veins, etc.)

Territories affected:

Fig.l. Organization of Iroquois gene into clusters. (A) Three Irx genes are organized into one cluster in Drosophila. In mice and human, six Irx genes are organized into two clusters. Also the protein structure of the Irx proteins is shown with the conserved Iro box and homeodomain. (B) Early and late roles of. Iroquois in Drosophila development in specification and patterning. HD: homeodomain, ib: Iro box.This figure was modified from Cavodeassi et al., 2001.

2 1.2. Iroquois encode transcriptional activator or repressor that belong to TALE

superclass of homeodomain proteins.

Iroquois proteins are part of the TALE (Three Amino acid Loop Extension)

superclass of homeodomain proteins. TALE homeodomains contain a stretch of three

conserved amino acids between the first and second helices. Position 50 of most

homeodomains (position 53 in TALE homeodomain) is a conserved basic amino acid

residue that plays a major role in their DNA binding. However, in the TALE

homeodomain, a small neutral amino acid occupies position 53, indicating variability in

their DNA binding ability compared to other homeodomain proteins (Burglin, et al.,

1997). In addition, Iroquois proteins are uniquely identified by presence of a stretch of thirteen amino acids in their C-terminal region, called the IRO box, whose function remains unknown (Fig. 1 A) (McNeill et al., 1997; Gomez-Skarmeta and Modolell, 2001).

Many TALE homeodomain proteins function as cofactors in combination with

other transcription factors (Burglin, 1997). For example, the Pbx family of TALE

homeodomains requires Hox proteins for their affinity in DNA binding (Mann and Chan,

1996). Drosophila studies have also illustrated the ability of Iroquois to bind DNA as

homo- or hetero-dimers with other members of their cluster (Bilioni et al., 2005). For

example, Mirror, a member ofDrosophila Iroquois proteins, can bind the fringe promoter

and repress its transcription in vivo (Bilioni et al., 2005). Additional binding sites have

been identified for ara in the achaete-scute (ac-sc) enhancer (Gomez-Skarmeta et al.,

1996; Bilioni et al., 2005). However, the consensus binding sites for Iroquois are

identified by using in vitro experiments, and therefore the physiological significance of

these sites and whether the same sites are utilized in higher organisms are still unclear.

3 Although not much is known about the binding sites of Iroquois in higher

organisms, their ability to function as a repressor or activator is well established. For

example in mice, Irx4 is shown to interact with Vitamin D receptor and retinoic acid receptor to repress expression of slow MyHC3, a gene important for chamber specific

control of gene expression, in cardiac ventricles (Wang et al., 2001). Irx5 can also repress the expression of the Kv4.2 potassium channel in cardiac myocytes by recruiting

chromatin remodeling factors to its promoter (Costantini et al., 2005). In Xenopus, Xirol

can induce expression of Otx2 through a repressor function; injection of a dominant repressor form of Xirol resulted in Otx2 expression, while the dominant activator form of

Xirol down regulated Otx2 expression (Glavic et al., 2002). Another study has shown the importance of Xirol as a transcriptional repressor in neural differentiation (Gomez-

Skarmeta et al., 2001). In zebrafish embryos, Iro proteins can function as activators to

directly induce broad expression of ngnl in dorsal ectoderm or as repressors in the ventral ectoderm to induce expression of ngnl, a proneural gene (Itoh et al., 2002).

Chimeric protein containing the Irx4 homeodomain and a strong repressor domain has the opposite effect to the wild type Irx4 cDNA when overexpressed in chick heart, revealing a potential for Irx4 as a transcriptional activator during chick heart development (Bao et al., 1999). Taken together, these experiments show that Irx proteins can function as transcriptional repressors or activators in a context dependent manner.

1.3 Roles of Iroquois in Drosophila development.

Irx genes have multiple roles during Drosophila development. They can act as pre-patterning genes, identifying large territories (Fig. IB) (Cavodeassi et al., 2001). For

4 example, am and coup are involved in the specification of notum by limiting the

formation of wing hinge (Diez del Corral et al., 1999). Later in development, the

expression of Iroquois becomes more restricted where they act as patterning genes (Fig.

IB). For example, they specify sensory organ progenitors (SOP) by controlling ac-sc

(Gomez-Skarmeta et al., 1996; Kehl et al., 1998). Loss of function mutation of Iroquois

lead to deletion of the bristles from the lateral notum leaving the medial bristles intact

(Leyns et al., 1996). In addition, Iroquois can function in cell differentiation and control

axonal projections of neurons after formation of sensory organs (Grillenzoni et al., 1998).

Iroquois genes are also expressed in the dorsal half of the developing eye and play a

major role in the localization of the equator in the developing eye (McNeill et al., 1997;

Cavodeassi et al., 1999).

1.4 Iroquois genes in vertebrate neural tube patterning: insights from the Chick.

Development of the vertebrate nervous system begins by the formation of a neural

plate that rolls up to form the neural tube. Subsequently, a series of vesicles develop at the anterior end of this tube, which with the aid of various signaling centers and transcription factors become organized into the prosencephalon or forebrain, the

mesencephalon or midbrain, and the rhombencephalon or hindbrain (Fig. 2A) (Wurst and

Bally-Cuif, 2001). Wealth of information on Iroquois function is obtained from chick

experiments where they are found to play an important role in patterning and

development of the anteroposterior (A/P) axis of brain and dorso-ventral (D/V) patterning

of the neural tube.

5 One of the signaling centers in the A/P axis is the isthmic organizer (IsO)

positioned at junction of midbrain and hindbrain. Chick Irx2 expression is restricted to

rostral hindbrain and presumptive rhombomeres prior to expression of the signaling

molecule, Fg/8, in the IsO. Misexpression of Irx2 together with Fgf8 in the midbrain

leads to development of ectopic cerebellum (Fig. 2B) (Matsumoto et al., 2004).

Furthermore, the development of the ectopic cerebellum relies on transformation of Irx2

from a transcriptional repressor into a transcriptional activator by Fgf8 signaling, which

involves phosphorylation of Irx2 by the MAP kinase. Since both midbrain and hindbrain

are exposed to Fgf8, presence of Irx2 in the hindbrain appears to determine the different

response to FGF signaling and development of cerebellum in the hindbrain (Matsumoto

et al., 2004). Another signaling center important for A/P patterning of the brain is the

zona limitans intrathalamica (ZLI) that separates the thalamus from the prethalamus in

the diencephalon. One of the earliest manifestations of ZLI development is the restricted

expression of Irx3 and Six3 in posterior and anterior forebrain, respectively.

Overexpression studies demonstrated that counter repressive function of these proteins is

important for positioning of the ZLI in the diencephalon (Braun et al., 2003; Kobayashi et

al., 2002). Later in brain development, chick Irx3 is exclusively expressed posterior to the

ZLI and mediates the asymmetric response to Shh signaling secreted from the ZLI in this

region. The ectopic expression of Irx3 leads to conversion of prethalamic cells into thalamic cells in a Shh-dependent manner, indicating its role as a thalamic competence

factor (Fig. 2C) (Kiecker and Lumsden, 2004; Kobayashi et al., 2002).

6 Fig.2. Roles of Iroquois in A/P patterning of neural tube. (A) Schematic of A/P patterning of neural tube. Presence of different transcription factors across signalling centers such as ZL1 and IsO provides competence to development of different structures across these boundaries (modified from Kobayashi et al., 2002). (B) Overexpression of Irx2 along with FgfE leads to development of ectopic cerebellum (modified from Matsumoto et al., 2004). (C) Overexpression of lrx3 in forebrain leads to development of ectopic optic tectum (modified from Kobayashi et al., 2002).

7 In addition to their role in A/P patterning, Iroquois genes are known to be important in dorso-ventral (D/V) patterning of the neural tube as well. Neural progenitors

acquire identity according to their position along the D/V axis by the gradient of the Shh

concentration in the ventral region or presence of bone morphogenetic proteins (BMPs) at the dorsal end (Fig. 3A) (Roelink et al., 1995; Marti et al., 1995; Ericson et al, 1997; Lee

and Jessell, 1999). Retinoic acid (RA) can also induce an array of ventral cell types in neural explants. RA is found in the proximity of the developing spinal cord and plays a role in medial part of the spinal cord where signals from ventral and dorsal structures are weak (Novitch et al., 2003; Pierani et al., 1999). Chick lrx3 is identified as class I genes whose expression is repressed by Shh from the ventral neural tube (class II genes are induced by Shh in ventral regions). Pairs of class I and II genes have complementary

expression domain in the neural tube and act as mutual repressors to sharpen their

expression pattern. These expression patterns create a combinatorial code that defines the progenitor domains (Briscoe et al, 1999; Briscoe et al, 2001; Jessell, 2001). For

example, Irx3 is paired with Olig2, a class II gene to which it has complementary

expression pattern in the D/V axis. Overexpression of Irx3 inhibits the specification of motoneurons while inducing V2 interneuron identity (Fig. 3B) (Briscoe et al, 2000).

Although wealth of information is provided by these chick experiments, the

conclusions on roles of Iroquois in neural tube patterning relied mostly on overexpression

studies. Validation of these conclusions awaits loss of function studies in other model

organisms, such as mice.

8 0 0,5 1 2 3 4 Shh (Shh], nM

Dbxl, Dbx2, Irx3. PaxB " ;,. »• Obxi Irx3 Obx2 Dbx2, Irx3, PaxB flfl| • ||| PaxB Nkx6,1, Irx3, Pax6 19 —> ^

Nkx6.1,Pax6 pMN * MN Nkx6.1 Nkx6 1, Nkx2.2H

Class I Class I Class! Neuronal Shhl Tl Class II Fate \ Class II Class II

Fig.3. Role of Irx3 in D/V patterning of neural tube. (A) Schematic of neural tube patterning model. Presence of cross-repressing transcription factors induced by gradient of Shh controls development of neural progenitors. Modified from Jacob and Briscoe, 2003. (B) Role of Irx3 in neural tube patterning. Irx3 induces V2 neuronal fate while repressing motor neuron development. MNR2 is the marker for motor neurons, while ChxlO marks the V2 interneurons (Briscoe et al., 2000).

9 1.5 Iroquois genes in vertebrate development: insights from the mouse.

The Fused toes (Ft) mutation consists of a deletion of 1.6-Mb on chromosome 8 that disrupts the entire Iroquois cluster B in addition to three neighboring genes (Fig. 4A)

(Fts, Ftm and Fto) (Peters et al., 2002). In addition to defects in programmed cell death that lead to unsuccessful separation of digits in the limbs (Hoeven et al., 1994), various

abnormalities are observed in Irx-expressing regions in these mutants. Homozygous Ft

embryos die at midgestation exhibiting deformation of craniofacial structures, syndactyly

and Polydactyly of limbs and disorganization of the ventral spinal cord in addition to randomized embryonic turning and heart looping (Peters et al., 2002; Gotz et al, 2005).

The A/P and D/V patterning of the brain are greatly affected in these mutants. The

forebrain is expanded caudally to the cost of midbrain and the hypothalamus, a ventral

structure in the forebrain, and medial pallium of the dorsal forebrain are reduced in size

(Anselme et al., 2007). While the reduced midbrain structure is consistent with the

expression of Irx3/5 in this region, the defects in hypothalamus and medial pallium is most probably caused by reduced expression of Shh in the ventral regions (Anselme et

al., 2007). In the Ft mutants, the V2 interneuron domain is greatly reduced while the motor neurons are expanded dorsally (Fig. 4B) (Gotez et al., 2005). However, the neural tube defects are relatively subtle and V0/V1 interneuron development is not affected in these mutants. The expanded expression of Irxl (Fig 4C) suggests that expression of

other members of Iroquois genes can partially compensation for absence of cluster B

members. However, the Ft mutants have defects in the maintenance of floor plate in the

neural tube, suggesting a possibility that the defects in the Ft neural tube could be due to

floor plate defects (Gotz et al., 2005; Anselme et al., 2007). Thus the role of Irx genes in

10 ventral neural tube patterning remains unclear. In addition, other Irx unrelated genes deleted in these mutants are widely expressed in the affected organs during embryogenesis. Therefore, in order to determine the roles of Iroquois during embryonic development, analysis of Irx3, Irx5 and Irx3/5 KO mice is essential.

mn, Mmp2

to 5

Dsletlon

100 500 1000 X I 'I' i—i- —I— —i—i—i—i—H- ++• '•>'••' i"" I-

OUg2

••nt m '%?.

Fig.4. Fused toes mutants have the entire cluster A deleted. (A) The 1.6MB deletion of Fused toes mutants takes away the entire cluster A in addition to four other genes. (B) The mutat neural tubes lack V2 neurons shown by ChxlO staining in cost of expanded motor neurons, shwon by HB9 staining. (C) The mutant neural tube is dorsalized with higher and expanded expression of/a;/. This Figure was modified from Gotz et al., 2005.

Among cluster A members, single knock out of lrx4 and lrx2 has been reported previously. Consistent with the high and restricted expression of Irx4 in the developing

11 heart ventricles as early as E8.5, the Irx4-deficient mice shows abnormal expression of

atrium markers in the cardiac ventricles and reduced expression of ventricular markers,

which leads to adult onset of cardiomyopathy (Bruneau et al., 2000; Bruneau et al.,

2001). Irx2-deficient mice appear normal with no defects in their heart and brain morphology and function (Lebel et al, 2003), despite the important role of chick Irx2 in midbrain-hindbrain boundary (MHB) development.

Knock out of Irx5 results in viable and fertile animals that show defects in their heart and eye. In Irx5-deficient heart, the gradient expression of Kv4.2 is disturbed,

leading to defects in cardiac repolarization and susceptibility to inducible tachyarrythmia

(Costantini et al, 2005). In the eye, Irx5 is expressed in the developing bipolar cells and

is localized to a subset of mature cone bipolar cells in the adult retina. Lack of Irx5 leads to defects in the differentiation of Type 2 and Type 3 OFF cone bipolar cells (Cheng et

al, 2005). In the Hui laboratory, Irx3 knock out mice, which contain an insertion of the

LacZ reporter gene in the coding region, has been generated by Dr. Rui Sakuma. Irx3-

deficient mice are viable and fertile but have reduced body weight compared to their wildtype littermates. No major brain patterning defects was observed in these mutants

during early embryonic development.

Iroquois are expressed in overlapping and distinct spatio-temporal patterns during

early embryonic development. Irx3 and Irx5 share a similar domain of expression as early

as E8.0 in the central nervous system, foregut and the limbs suggesting an important role

for Iroquois in embryogenesis (Houweling et al., 2001; Cohen et al, 2000; Bosse et al.,

1997). Not much is known about the expression pattern of Irx3 during later stages of

development and adulthood. The objectives of my Masters thesis are to study the

12 expression pattern of Irx3 using the lrx3au ac line, study the potential overlapping functions of Irx3 and Irx5, and develop mouse models for future genetic and biochemical studies of Iroquois.

13 2. Materials and Methods

2.1 Generation of expression constructs and targeting vectors

In order to generate the activator and repressor forms of Irx3, the full length Irx3

expression vector, pCMV-HA-Irx3 construct, was used as a template. For the dominant

activator (Vpl6-Irx3) form of Irx3, the Vpl6 domain was digested from the pCMX-Vpl6 plasmid using the Hindlll sites and ligated into the Clal mdXhoI sites of pCMV -HA-

Irx3 using a linker oligo (oligo sequence: CGA TAA GCT TGG). The dominant

repressor (EnR-Irx3) vector was made by PCR amplification of the EnR repressor

domain from the Drosophila engrailed cDNA. Clal m&XhoI sites were introduced into the EnR domain through PCR primers (forward primer: CCA ATC GAT ACC ATG

GCC CTG GAG GAT CGC TGC and reverse primer: CAG CTC GAG GGG ATC CCA

GAG CAG ATT TCT CT). The amplified sequence was then digested and ligated into the Clal and Xhol sites of pCMV-HA-Irx3 construct and sequenced.

For generation of targeting vectors, the pIRES2-EGFP plasmid was modified by

digestion with BamHI and Bglll, and then self-ligation. The Nhel and NotI sites were then used to release the IRES-EGFP sequence. Three-piece ligation was performed to

introduce the FIA-Irx3-IRES-EGFP into the Clal and NotI sites of pBigT plasmid. The

Pad and AscI sites were used to introduce homology arms, neomycin cassette and Irx3-

IRES-EGFP from pBigT into the Rosa targeting vector reported previously (Srinivas et

al., 2001).

In order to check the function of the fusion proteins in the chick forebrain, the

coding sequences of three different forms of Irx3 from the pCMV vectors generated as

14 mentioned previously, were digested and subcloned into pCX plasmid that contains the

chick beta-actin promoter using blunt end ligation.

For generation of Irx3-3M6H targeting vector, oligos were used to construct the tag. Oligo sequences used are: 5'CTA GTG AAC AAA AAC TCA TCT CAG AAG

AGG ATC 3', 5'TGG AGC AGA AAC TGA TCT CAG AGG AGG ATC TCG AA 3',

5'CAA AAG CTC ATT TCT GAA GAA GAC CTG CAT 3', 5'CAT CAC CAT CAC

CAT TGA AGT 3', 5' ATC TCA ATG GTG ATG GTG ATG ATG CAG GTC TTC TT

3', 5' CAG AAA TGA GCT TTT GTT CGA GAT CCT CCT CTG AG 3', 5' ATC AGT

TTC TGC TCC AGA TCC TCT TCT 3', 5' ATC AGT TTC TGC TCC AGA TCC TCT

TCT 3', 5'GAG ATG ATG TTT TGT TCA 3'. The long arm starts at the EcoRI site upstream of the Irx3 coding sequence and is 6.5kb in length. This arm adds the 3 myc and

6 His sequence to the C-terminus of the coding region. The short arm contains the 1.3kb

sequence of the 3' end from Ncol to EcoRI site.

2.2 Cell culture and Luciferase assay.

COS cells were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. Transfection was done using Lipofectamine 2000 (Invitrogen).

Transfected cells were collected after 48 hours in luciferase harvest buffer and the

activity of the luciferase reporter previously described (Ding et al., 1999) was measured using Luciferase Reporter Assay (Promega). Data were obtained from two independent experiments, each performed in triplicate.

15 2.3 Western Blot and Immunopercipitation

E 11.5 embryos were dissected in cold PBS and flash frozen. Total cell lysates

were prepared in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM

EDTA, 1 mM EGTA, 0.1% SDS, 0.5% Doc, 1 % NP-40, 25 mM sodium pyrophosphate,

1 mM sodium orthovanadate, 10 mM NaF, 1 mM ^-glycerophosphate, and EDTA-free

complete protease inhibitor cocktail (Roche), followed by sonication. Total cell lysates

from transfected COS cells were prepared in SDS lysis buffer. For immunoprecipitation

HEPES buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH

8.0) and 1% TritonX-100 (0.5 % TritonX-100 for HEPES wash buffer) without

sonication was used for lysis of the embryos and anti-c-Myc agarose conjugated beads

(Sigma) was used for pull down of the protein complexes. Proteins were separated by 8%

SDS-PAGE and transferred to nitrocellulose for immunoblotting overnight at 4°C with an

Irx3 antibody generated by Vijitha Puviindran.

2.4 Generation of targeted ES clones

Rosa targeting vectors were linearized using Xhol enzyme site and electroporated

into W4 ES cell lines. Cells were then subjected to selection using G418. Targeted ES

clones were identified by Southern blot analysis. Positive clones were sent to Sickkids transgenic facility for blastocyst injection. Fl animals with germline transmission were

identified using PCR genotyping. The PCR primers for Rosa targeting were described

previously (Soriano, 1999). Primers used for genotyping of Irx3-3M6H line are: 5'CAA

GAA GGG GTG ATG AGA GTC GCT GGG CG 3' and 5' GGA GAG GGA ACC ACG

16 GCG AGA AAG GCC TA 3' resulting in a 323 bp wildtype band and a 439 bp mutant band.

2.5 Sample preparation and histology

Embryos and organs were fixed in 4% PFA in PBS overnight at 4°C. Dehydration was performed in series of ethanol and xylene washes. Embedded embryos in paraffin were sectioned using microtome (6um). Slides were de-waxed and re-hydrated, stained with hematoxylin for 10 minutes, and rinsed in water. Series of incubations were performed with 0.5% HC1, 70% ethanol, water, and 1% lithium carbonate. Samples were then partially dehydrated, stained with 0.5% eosin and dehydrated through ethanol and xylene series. For counter staining of the X-gal stained sections, eosin staining was performed followed by the subsequent steps.

2.6 Mice

Mice used in this study were housed in standard vented cages in conformity with the Hospital for Sick Children's animal care committee recommendations. For genotyping of the mice, ear notches or tail clips were digested in 300 ul of 50 mM NaOH at 100°C for 10 minutes. NaOH was then neutralized by adding 100 ul of 0.5 M Tris (pH

8.0). 3 ul of DNA solution was used for PCR genotyping.

17 2.7 X-gal staining

Embryos were dissected in cold PBS and fixed in 2.7 % formaldehyde, 0.02 %

NP-40 in PBS. After fixation, embryos were washed in 2 mM MgCl2, 0.02 % NP-40 in

PBS at 4°C. For whole mount X-gal staining, the embryos were incubated overnight at

room temperature with X-gal solution containing lmg/ml X-gal, 2 mM MgCl2, 0.02 %

NP-40, 5 mM K4Fe(CN)6.3H20, 5 mM K3Fe(CN)6 in PBS. The embryos were

dehydrated in methanol and cleared in methyl salicylate prior to taking picture. For

section X-gal staining, embryos were embedded in OCT compound and 12 um sections

were cut using cryostat prior to staining in X-gal solution.

2.8 Bone staining

El 8.5 mice were fixed in 80% ethanol overnight. After removing most of skin

and viscera, the mice were fixed in 95% ethanol and incubated with 15% Alcian Blue in

20% Glacial acetic acid, 80% ethanol for 24 hours at room temperature. Samples were

rinsed in 95% ethanol and stained with 7.5% Alizarin Red, 1% KOH for 24 hours.

Clearing was performed using 20% glycerol: 1% KOH over several days. Skeletons were

stored and imaged in 50% glycerol: 50% ethanol.

2.9 RNA in situ hybridization

Antisense RNA digoxigenin-dUTP-labeled riboprobes were generated from

linearized DNA plasmids; Irx3 (Cohen et al., 2000), Lhxl (Fuji et al., 1994), Ebfl (R.

Grosschedl), Otx2 (Matsuo et al, 1995), Gbx2 (G. Martin), and Dlxl (Qiu et al, 1995).

Probes were synthesized using a DIG-labeling kit (Roche) according to manufacturer's

18 instructions and precipitated in 0.1 M LiCl, 75% ethanol at -80° C over two hours. The probes were dissolved in 50% formamide and 50% H2O and stored at -80°C. In brief,

slides were rehydrated and fixed in 4% PFA. DNA was denatured in 0.2 N HC1 and neutralized with 0.1 M Triethanolamine-HCl and 20% acetic anhydride. Sections were then dehydrated and air dried prior to hybridization. Hybridization was performed with the buffer containing 50 % formamide, 5x SSC (pH 4.5), 1% SDS, 50 mg/ml heparin, and

50 mg/ml yeast t-RNA in humidified chamber (50% formamide, 5x SSC) overnight at

55°C. Samples were then washed in series of solutions: 5x SSC (at 60°C, 5'), 50%

formamide/2xSSC (at 60°C, 15'), TNE (1 M NaCl, 1 mM Tris pH 8.0, 0.5 mM EDTA at

37°C, 5'), RnaseA (0.2 mg/ml at 37°C, 45'), TNE (at 37°C, 5'), 2x SSC (twice at 55°C,

15' each), and 0.2x SSC (twice at 55°C, 15' each). Sections were then incubated with

TBST prior to blocking (Roche). Anti-DIG-AP (1:2000; Roche) was performed at room temperature for 1 hour. Slides were rinsed in TBST-Levamisole (0.2 mM-lev) twice and

incubated in APB buffer (50 mM MgCl2, 0.1 M Tris (pH 9.5), 0.1 M NaCl, 0.01 %

Tween-20, and 0.2 mM Levamisole) for 30'. BM purple reagent (Roche) was used to

develop the color in the humidified chamber overnight.

19 3. Results

3.1. Irx3 is expressed strongly in various tissues during late embryonic development and adulthood.

There is increasing evidence from experiments in chick and other model organisms that Iroquois genes are important for the development and patterning of neural tissue and other organs. In order to study the role of Irx3 in mammalian development, a mutant knock out mouse strain of Irx3, the Irx3talllMcZ, in which the exon I of Irx3 is replaced by a tauLacZ cassette followed by a stop signal, was generated by Dr. Rui

Sakuma. The absence of Irx3 does not interfere with viability or fertility of the animals, but leads to smaller body size in homozygous mutants. In this line, the expression of tauLacZ is driven by the Irx3 promoter in a pattern that recapitulates the Irx3 RNA expression and this reporter line is useful for detailed temporal expression analysis.

Dynamic expression of Iroquois during development led to analysis of Irx3 expression pattern during various stages. Irx3 has previously been shown to be expressed in the midbrain, neural tube, developing heart and prospective limb territories at E9.5

(Bosse et al., 1997; Cavodeassi et al., 2001; Bruneau et al., 2001; Christoffels et al.,

2000; Cohen et al., 2000; Houweling et al., 2001). Sections of the hindbrain showed expression in the middle neuro-epithelium region at this stage (Fig. 5A). Expression in the neural tube extended throughout the entire body and this expression was limited to the middle regions of the neural tube, excluding the most dorsal and ventral regions. The notochord was also marked by lacZ staining (Fig. 5B). Using a flatmount to look at the expression of Irx3 in the hindbrain and neural tube, weaker expression was observed in rhombomere 5 (Fig. 5C). It is thought that Ohg2 and Irx3 are cross repressor partners that

20 together form the sharp boundaries in the brain (Novitch et al., 20001). Lower expression of Irx3 in rhombomere 5 corresponds with a higher expression of Olig2 in this region and this complementary expression pattern is consistent with the cross-repressor role of Irx3 and OHg2 in neural development.

By El0.5, the expression pattern of Irx3 in the midbrain extends rostrally to cover the prethalamus in the dorsal diencephalon. The ventrolateral region of the metencephalon was marked with lacZ expression, with a gap separating the expression pattern between the midbrain and hindbrain, the latter extending through the ventral regions of the entire neural tube. At this stage, Irx3 also marked the cephalic mesoderm surrounding the optic cup (Fig. 5D) and ventricles of the developing heart (Fig. 5E). A loop-like pattern is visible in the rostral cardiac walls of ventricles. LacZ expression was also seen in the developing craniofacial nerves and the axons extending from the anterior neural tube (Fig. 5F, arrow). Irx3 expression, evident in the limb territory at E9.5 becomes specific to proximal and anterior limb buds by El0.5 in both hindlimbs and forelimbs (Fig. 5G).

Sections of the embryo at stage El3.5 revealed high expression in the mesenchyme surrounding the eye (Fig. 5H). Weaker expression was seen in the retina and no expression was visible in the lens (Fig. 51). LacZ expression was present in the maxillary facial tissues especially in the nasal region. The bronchi and the developing lung showed Irx3 expression (Fig. 5H, J). In the kidney, lacZ staining was localized to the

S-shape bodies, comma bodies and the developing collecting ducts (Fig. 5K).

21 Fig. 5. Expression of Irx3 during embryonic development. (A-B) LacZ staining on coronal sections of E9.5 embryos. Expression is seen in midbrain, notochord (arrow in B) and middle region of neural tube. (C) Flat-mount of LacZ stained neural tube. High expression is seen throughout the neural tube except at the rhombomere 5. (D) Whole mount LacZ staining of El 0.5 embryo showing high expression in hindlimbs and forelimb in addition to midbrain and and optic cup. (E) Expression oflrxS in the developing heart at EI 0.5. (F) At this stage LacZ marks the axons that are extending from the carniofacial region (G) and the anterior limbs. (H) Sagital section of E13.5 embryo. Expression is seen in (1) retina (arrow in 1) and (H) meseneymal region surrounding the eye, (J) lung (K.) and kidney. fb:fore brain, nt: neural tube, nc: notochord, r5: rhombomere 5, nib: midbrain, oc:optic cup, fl: forelimb, hi: hindlimb, ba: branchial arch, vn: heart ventricles, kid: kidney, cd: collecting duct, sb: S-shaped bodies, co: comma bodies.

22 In the adult, various organs that show embryonic Irx3 expression continue to

express Irx3. For example, the interior walls of the heart ventricles were stained with lacZ

(Fig 6A), and high LacZ expression was found in the epithelial cells of the alveolar duct

and sacs of the lungs (Fig. 6B,C). Staining of the kidneys showed Irx3 expression in both the cortex and renal pelvis though weaker in some parts of medulla (Fig. 6D). In the male reproductive organ, Irx3 was seen in the epididymal epithelium (Fig. 6E) while testis

itself did not express Irx3 (not shown). In the ovaries, expression was observed in the germinal epithelium that covers the ovaries and in late-stage developing follicles (Fig.

6H). In the skin, Irx3 expression was limited to the outer root sheet of the hair follicles

(Fig. 6F). The epithelium of esophagus, but not the stomach, was stained with lacZ (Fig.

5G). In addition, the Irx3-tauLacZ expression is also seen in other tissues, such as bones

and prostate (data not shown).

23 Early onset expression of Irx3 in the midbrain and hindbrain suggests a role of

Irx3 in brain development. In order to elucidate the potential functions of Irx3 in the development of these structures, we examined its expression through several developmental stages. In general, Irx3 expression becomes more restricted as the embryo develops. By E15.5, wholemount staining of the brain revealed strong Irx3 expression in the dorsal midbrain (Fig. 7A). The postnatal brain maintains the midbrain expression that covers areas from the thalamus to pons (Fig. 7B). Sections cut through P10 brain showed

24 expression in the deep cerebellar neurons (DCN) (Fig. 7C,D). This region of the cerebellum is one of the three principal neuronal subclasses that receive input from external sources, in addition to functioning as a major source of cerebellar output (Saab,

2003). One of the external sources that can stimulate DCN is the pontine nuclei that also maintains high level of Irx3 expression throughout adulthood (Fig. 7F). DCN also contains neurons that extend their axons to other midbrain regions such as superior colliculus with high level of Irx3 expression throughout embryonic development and adulthood (Fig. 7B-F). The inferior colliculus is another midbrain structure that expresses high levels of Irx3 from the postnatal stage to adulthood (Fig. 7E). A very thin lining that covers the fourth ventricle and fissures of the cerebellar cortex was stained with lacZ

(Fig. 7G). Weaker expression was observed at the Purkinje cell layer that lies between the molecular layer and internal granule cell layer in the cerebellar cortex (Fig. 7G, arrow).

In the adult brain, very strong expression was observed in the habenular nucleus (Hb) and fasciculus retroflexus (fr) (Fig. 7E,F). Maintenance of high levels of Irx3 expression in the adult brain suggests additional roles for Iroquois in brain function and behaviour.

25 •*s».

Figure 7. Expression of Irx3 in developing and adult brain. (A) Wholemount LacZ staining of E15.5 brain. High expression is limited to the midbrain. (B) Sagittal section of PO brain shows high expression in differentiating field of the inferior colliculus. Expression is also seen in the anterior pons, and regions of tegmentum. (C) Coronal section of P10 brain. High expression is seen in deep cerebellar nuclei, periaqueductal gray, and inferior colliculus.(D) Transverse section of P10 brain. Expression is seen in most regions of midbrain and fasciculus retroflexis. (E-F) Sagittal sections of adult brain. High expression is maintained in the anterior pons, superior colliculus, and tegmental area. Habenular nuclei and pontine nuclei are distinctly marked by Irx3. Weaker expression is seen in inferior colliculus. (G) High magnification of a cerebellar fissures. Expression is seen in Purkinje cells (arrow in G) and the lining of fourth ventricle and fissures of cerebellum. mb:midbrain, cb: cerebellum, dcmdeep cerebellar nuclei, fr: fasciculus retroflexis, ic: inferior colliculus, sc: superior colliculus, hn: habenular nuclei, pn: pontine nuclei.

26 3.2. Double knockout of Irx3 and Irx5 results in embryonic lethality at day 14.5 with multiple morphological defects.

Despite the high level of Irx3 expression throughout embryonic development, the

Irx3 single knock out led to viable animals with no major patterning defects. The overlapping expression of Iroquois, specifically similar domain of Irx3 and Irx5 expression (Cohen et al. 2000, Houweling et al., 2001), suggests a functional redundancy among Iroquois members. In order to overcome the problem of redundancy, the Irx3/5 double knockout mouse line was generated by Dr. Rui Sakuma. First, he introduced a new conditional deletion allele of Irx3 into the Irx5EGFP mouse ES line and screened for cis targeted clones of Irx3 and Irx5 genes (both genes are on chromosome 16). In the

Irx5 line, the Irx5 coding region has been replaced by the EGFP sequence resulting in

Irx5 knock out allele. In the presence of Cre, the Irx3 allele will be deleted leading to double knock out of Irx3/5. In order to study general roles of Irx3 and Irx5 in mouse development, Dr. Sakuma crossed the conditional double knockout line to the NLS-Cre mouse line. NLS-Cre has ubiquitous expression of Cre leading to a complete knock out of

Irx3/5. No homo2ygous mutant mice were recovered at El5.5 and growth retardation was already observed at day 14.5, suggesting embryonic lethality of Irx3/5 double knockout mice between E14.5 and E15.5.

Irx3/5 double knockout (DKO) embryos displayed several morphological phenotypes. Approximately 33% of the embryos exhibited severe exencephaly where the entire brain was exposed with abnormalities seen in the midbrain and forebrain (Fig.

8C,D). Edema was observed with 56% of the embryos with or without liver hernia (Fig.

8E-G). About 20% of double knockout embryos displayed cleft defects with missing

27 nasal processes (data not shown). In addition, some embryos show relatively normal brain structure and craniofacial features (Fig. 8H).

Figure 8. Morphological defects seen in \rx3/5 double knockout embryos. (A) Front view and (B) side view of a wild type El 4,5 embryo. (C) Front and (D) side view of double mutant littermate embryo shown in A-B with exencepahly. The double mutants show a range of phenotypes including edema seen in E and F, liver hernia seen in F and G or look normal as seen in H.

Tissues with high expression of Iroquois were harvested at E14.5 and sectioned for examination. Severe defects were observed in the heart of DKO embryos. The right ventricle was enlarged relative to the wild type (Fig. 9A,B). Transverse sections of the body at the level of the heart revealed artrioventricular septal defect (AVSD) (Fig. 9D, arrow) and double ventricle outlet defect (data not shown). Malformed ventricle walls lead to abnormal division between the two chambers. Furthermore, the chest wall was significantly thinner relative to the wild type (Fig. 9F, arrow).

At first glance, Irx3/5 DKO mutants did not appear to develop eyes (Fig. 9H,J).

However, sagittal sections of the head indicated that the eyes indeed formed but were not

28 correctly positioned (Fig. 9L). Pups of the mutant genotype had eyes positioned under a thick mesenchymal tissue and were only visible by sectioning deeper into the head (Fig.

9H-L).

3.3. Irx3/5 DKO show proximal and hindlimb specific distal limb defects

The limbs showed highly specific expression of Irx3 in the anterior proximal regions as early as El0.5 (Fig. 10A). Consistent with this expression pattern, lrx3/5 DKO mutants lack scapula and hip girdle, the major bones in the shoulder and hip respectively.

29 In addition, Irx3 DKO pups lacked digit I from the hindlimbs and approximately 15% did not have digit II (Fig. 10B). Interestingly, this phenotype is hindlimb-specific and forelimbs are predominantly normal with the exception of few embryos that exhibited

Polydactyly. Since Irx3 and Irx5 are not expressed in the distal limbs, this phenotype suggests an unexpected role for Iroquois in limb patterning. Due to lethality of homozygous mutants at El4.5, we used conditional deletion of Irx3 in Irx5 mutant background. In order to delete lrx3 gene, transgenic mouse lines that express Cre in

specific tissues can be used. We crossed the conditional double mutant line to Prxl-Cre

(Logan et al., 2002) to specifically delete Irx3 in the limb mesenchyme at about E10.5.

Strikingly, bone staining of these conditional Irx3/5 mutants revealed no defects in the

distal limb developments although the development of scapula and hip girdles is affected

similarly to the Irx3/5 DKO mice (Fig. IOC). This observation revealed a very early

function for Iroquois in the formation of distal hindlimb elements (prior to El 0.5).

30 A B

lrx3/5 DKO

*/ ^¥r:

wt lrx3/5 DKO

Figure 10. Limb defects in Irx3/5 double knockout mutant embryos. (A) The forelimbs look similar to that of wild type littermates while the hindlimbs are missing digit I and sometimes digit II. (B) Bone staining shows lack of scapula in forelimbs and digit I, tibia and hip bones in hindlimbs. (C) Bone staining of conditional knock out of Irx3 in lrx5 kock out background does not show the distal hindlimb specific phenotype while detects in hip girdles and scapula are still observed.

3.4. lrxS/5 DKO brains have defected forebrain organization.

To further chracterize the brain defects of Irx3/5 DKO, we performed RNA in situ hybridization on El4.5 mutant brains, using markers that identify various structures of

31 the developing brain (Fig. 11 A). We used non-exencephalic embryos to avoid secondary structural defects caused by exencephaly. The fr (fasciculus retroflexus) tract, which is the boundary of Irx3 expression in the diencephalon, is a structure that separates the pretectum from the thalamus. The markers that identify the pretectum are Ebfl and LhxJ.

In the DKO mutants, while no change was observed for Ebfl expression, Lhxl expression was missing from the rostal pretectum (Fig. 1 IB). Furthermore, the expression of Gbx2, a thalamus marker, expanded caudally. Other markers such as Otx2, a marker for midbrain,

and Dlxl, a marker for the prethalamus and GE, did not show significant difference from the wild type brain. Based on these observations, we concluded that lack of lrx3 and Irx5 expression specifically affects the development of structures across the fr tract,

subsequently leading to the expansion of the thalamus at the cost of the rostal pretectum.

32 Dlx1

B wt /rx3/5 DKO

***^i«>$!

CM O" /"

<*r Pit* ^ SI % ui •* *m » ^¥ " X

'* V

X A

I #

Figure ll. Analysis of lrx3/5 double mutant brain. (A) Diagram of the In Sim markers that are expressed in various structures of the brain and used in B. (B) In Situ hybridization on sagital sections of El4.5 heads with no exencephaly. The double mutant shows reduced Lhxl and expanded Gbx2 expression. PT: pre-tecturn, TH: thalamus; PTh: pre-thalamus.

33 3.5. Conditional deletion of Irx3 in Irx5 knockout background delays death until birth in double knockout embryos.

Most structures in the brain do not develop until later stages of embryonic development or until after birth, hence the death of Irx3/5 double knockout embryos at day 15.5 curtail detailed studies on brain development. To by-pass this limitation, we performed a neural specific knock out of Irx3/5 by crossing the Irx3/5 conditional double knockout line to the Nestin-Cre mouse line (Isaka et al., 1999) (Fig. 12A). This line expresses Cre in the neural progenitors as early as El0.5. The conditional deletion ofIrx3 and Irx5 leads to lethality within few hours after birth (Fig. 12B). Sagittal sections through the newborn brain of the conditional mutants did not reveal any severe morphological defects (Fig. 12C). Future experiments using RNA in situ markers would be required to identify more specifically the defects in the structures across the fr tract (as explained in 3.5).

34 B

Irx3 Irx5

1 in presence of ere

wt lrx3/5 CDKO;Nestin-cre

S\3

"J' vr

Figure 12. Conditional deletion of Irx3 in Irx5 background. (A) Irx3 can be conditionally deleted in presence of ere. (B) The conditional deletion of lrx3 in neural tissue leads to lethality at birth. (C) The mutant brains at birth do not show any severe morphological defects.

3.6. Dominant activator, dominant repressor and HA-Irx3 fusion proteins are functional both in vivo and in vitro.

Previous studies in chick and Xenopus have shown that Iroquois can function as either a transcriptional activator or repressor in a context dependent manner (Glavic et al,

2002; Gomez-Skarmeta et al., 2001; Bao et al., 1999; Matsumoto et al., 2004; Kiecker and Lumsden, 2004; Kobayashi et al., 2002). In order to determine which form of Irx3 is important during embryogenesis, I generated three different forms ofIrx3 to be used in overexpression studies. The Irx3 coding sequence was fused to an HA tag to generate a

35 wild-type form of Irx3 (HA-Irx3). To make a repressor form of Irx3, its coding sequence was fused to a 207 amino acids repressor domain from the Drosophila Engrailed protein

(EnR-Irx3). The activator form of Irx3 was generated by fusing the Irx3 coding region to the Vpl6 activator domain (Vpl6-Irx3) (Fig. 13 A). Expression of the three constructs were verified by transient transfection in cultured cells. Lysates from the transfected cells were run on an SDS-PAGE gel and probed with the Irx3 antibody generated in our lab.

The HA-Irx3 protein appeared as an 85 kD-sized band. Vpl6-Irx3 and EnR-Irx3 ran as slightly larger bands (90 and 95 kD respectively) as expected due to presence of the extra amino acids in the repressor and activator domains. A plasmid with a pCMV promoter lacking a coding sequence served as a negative control (Fig. 13B).

To check the activity of the fusion proteins, I used a luciferase assay system designed in the laboratory of Dr. Pfaff (Lee et al., 2004). The reporter plasmid included

TK, a strong ubiquitous promoter, driving the luciferase expression and a 2.5 kb distal region of Hb9 gene upstream of the TK promoter (Fig. 13C). Expression of Irx3 has been shown to significantly reduce the activity of this reporter (Lee et al., 2004). This result was reproducible by HA-Irx3, which was used as the wild-type Irx3 control in our experiments, leading to eight-fold repression of the luciferase activity. Using EnR-Irx3, an even stronger repression was obtained and the luciferase activity became negligible.

Transfection of Vpl6-Irx3 did not show any significant reduction in luciferase activity

(Fig 13D). Together, these results confirmed the transcriptional activities of the Irx3 fusion proteins in vitro.

To check the activity of the fusion proteins in vivo, we overexpressed the three forms of Irx3 into the chick brain. The laboratory of Dr. Shimamura had previously

36 shown that overexpression of Irx3 in the chick forebrain induces ectopic expression of the En2 protein, which is normally expressed in the midbrain-hindbrain boundary region

(Kobayashi et al., 2002). They have also shown that the induction of En2 relies on the repressor activity of Irx3. To use the chick system, I subcloned the three forms of Irx3 into pCX, a promoter that induces high expression in chicks. In collaboration with the

laboratory of Dr. Shimamura, the three plamids were electroprated into the chick forebrain. Overexpression of HA-Irx3 and EnR-Irx3 led to the induction of En2 in the forebrain in the experimental side. No induction of En2 was observed on the control side

of the brain with electropration of GFP plasmid alone (Fig 13E). Since the expression of

En2 relies on repressor function of Irx3, this assay could not be used to examine the

activity of Vpl6-Irx3. However, Irx3 is known to function as a cross-repressor with Six3 in the formation of ZLI (explained in 1.4) Therefore if a dominant activator form of Irx3

is expressed at the ZLI boundary, the expression of genes in ZLI such as Six3 and Shh is

expected to be repressed, leading to disruption of ZLI formation. Indeed, eletroporation of Vpl6-Irx3 disturbed ZLI development as shown by the absence of Shh expression

(Fig. 13E). Together, these results illustrated that the Irx3 fusion proteins are functional

in vivo.

37 B 3<

Repressor effects of Irx3 on luciferase activity M l 4

» 600000 ' ^B : : T *> 500000 . ^^B " J -500000 ' ^B< •g 300000 .' ^H : •i | 200000 :'.' ~^B V I 100000 ^H ^^ •

jiCMV HA tf*3 construct

EP:HA-lrx3 control side .^ /

% fc£n2 •- ...KM ' EP:Vpl6-lrx3 ttmTiotside t «/*) r<- iShh / j#hh EP:EnR-lrx3 control side

En2 En2

Figure 13. Fusion proteins ofIrx3 are functionally active. (A) Schematic of the wild type (lrx3 fused with HA tag), dominant activator (Irx3 fused with Vpl6 activator domain) and dominant repressor (lrx3 fused with engrailed repressor domain) forms of Irx3. (B) Western blot with Irx3 antibody showing expression of the three forms of Irx3 in the COS1 cell line. (C) Schematic of the luciferase reporter plasmid used in D. Wild type Irx3 can repress expression of luciferase reporter from the ubiquitous promoter. (D) HA-Irx3 and EnR-lrx3 maintain their repressor function while Vpl6-lrx3 does not repress the luciferase expression. (E) Electroporation of the three fusion proteins in the chick forebrain confirms their functional activity in vivo. HA-Irx3 and EnR-Irx3 can induce En2 while Vpl6-lrx3 can disturb ZLI development (refer to the text for more details).

38 3.7. Generation of targeted conditional Irx3 overexpressing mouse lines

In order to induce overexpression of Irx3, we used the Rosa targeting vector for

the generation of our mouse lines. Rosa26 is a ubiquitous gene that is not essential for

mouse development, as homozygous mutants do not show any defects (Srinivas et al.,

2001). We introduced the Irx3 sequence downstream of a stop signal flanked by LoxP

sites into the Rosa26 locus. In the presence of Cre, the stop signal will be removed and

Irx3 will be expressed under the control of the endogenous Rosa promoter (Fig. 14A).

I introduced HA-Irx3, EnR-Irx3 and Vpl6-Irx3 into the Rosa targeting vector and

electroporated them into mouse embryonic stem cells. Approximately 100 drug-resistant

clones were selected for each targeting experiment. All of these clones were tested for

correct integration into the Rosa locus by Southern blot hybridization. Two different

probes for both the 5' and 3' ends were used for each targeting vector to confirm the

homologous recombination events. For the 5' probe, the band resulting from the wildtype

locus is llkb. The targeting vector introduces an EcoRV enzyme site in the Rosa locus that leads to the formation of a 3.8kb band upon correct integration. The 3' probe

detected a 9.5kb wild type band. The mutant band size varies depending on the size of the

introduced sequence. The EnR-Irx3 and Vpl6-Irx3 integrated constructs produce slightly

larger mutant bands since their coding sequence is larger than the HA-Irx3. Between

eight to thirteen correctly targeted ES clones were identified for each targeting vector

using both probes. Southern blot hybridization experiments showing a few of these

clones are presented in Figure 14B.

39 / pBigT Pad -| ^ PGK-Neo _' Stop p|-»- fpifr- AscI

Targeting vector PROSA26PA Rosa26 genomic sequence |~| PSK-DTA j Pacl/Ascl Genomic EcoRV •V y\. EcoRV Ftasa26 locus •• ' '" ' ' ' '-" •' " . ".'.: —H

H EcoRV Targeted Rosa 26 C.„BU locus cc. EcoRV -| • PGK-Neo Stop M 'r*3 bpA

In presence of Cre Targeted locus EcoRV EcoRV after ere- ' • "! H • j S" bpA mediated excision

HA-lrx3 Vp16-lrx3 EnR-lrx3

5' probe llkb

m- 4* 3.8kb

3' probe 11.5-12 kb "m % 9.5 kb

Figure 14. Generation of lrx3 knock in ES cell lines. (A) The targeting design For introduction of three different forms of Irx3 shown in 8A into Rosa locus. 5' and 3' probe used in B are also shown, (B) Southern results showing positive ES clones for all three lines. EcoRV enzyme site was used for both 5'and 3'probe. With the 5' probe the llkb band is produced from the wild type locus and the 3.8kb band is produced from the correctly targeted Rosa locus. With the 3' probe the 9.5kb band is produced by the wild type locus while the correctly targeted locus will produce a band from 11.5 to 12kb depending on the form of Irx3 introduced into the Rosa locus.

40 Correctly targeted clones were sent for blastocyst injections using C57BL/6 donor

host embryos. Male chimeras were mated to C57BL/6 females and resulting brown coat-

coloured Fl progeny were PCR genotyped for the presence of the constructs. The HA-

Irx3 ES clones generated chimeras with very little contribution which did not produce

any pups. Germline transmission was obtained from the EnR-Irx3 construct and we are

currently in the process of crossing the Vpl6-Irx3 chimeras (high chimerism) to C57BL/6

females.

In order to overexpress the EnR-Irx3 in the neural tissue, the Rosa-EnR-Irx3 mouse line was crossed to Nestin-Cre. Presence of Cre-recombinase in neural tissue leads to the deletion of the stop signal from upstream of the EnR-Irx3 sequence, and the

subsequent expression of the dominant repressor form of Irx3 in the neural tissue. To verify the efficiency of Cre excision, we performed in situ hybridization experiments using the Irx3 probe on E14.5 brain tissue. Detection of a signal in the forebrain, where

Irx3 is normally not expressed, indicates efficient overexpression of Irx3 from the Rosa

locus (Fig. 15A).

To determine the effects of misexpression of EnR-Irx3 in the forebrain, we

obtained sagittal sections of E14.5 brain tissue. Histology of these sections did not reveal any severe morphological defect (Fig. 15B). Results were similar at El8.5 (Fig. 15C). To

check viability of these mice, litters were genotyped at birth. No Rosa-EnR-Irx3; Nestin-

Cre mice were found in these litters indicating that overexpression of the repressor form

of Irx3 in the entire neural tissue leads to perinatal lethality. While the cause of lethality remains to be determined, the overexpression of EnR-Irx3 in the CNS appears to be

incompatible with life.

41 3.8. Generation of epitope tagged Irx3 mouse line

In order to identify Irx3 interacting partners and downstream targets, an epitope- tagged Irx3 mouse line was generated. Protein tags containing 3-myc and 6-his motifs were introduced at the 3' end of the protein (Irx3-3M6H) to facilitate the purification of the protein complexes with use of high quality antibodies. The epitope-tagged Irx3 was subcloned into the pCX chick promoter and sent to the laboratory of Dr. Shimamura where the tagged protein was electroporated into the chick forebrain. The induction of the

42 En2 protein in the forebrain (similar to the levels of wildtype Irx3) verified the functional activity of Irx3-3M6H construct in vivo. A vector, which generates a similar C-terminus tag was constructed for gene targeting. This vector introduced an EcoRV enzyme site along with the neomycin selection cassette (Fig. 16A). Similar to those described above, drug resistant ES cells were selected and correct targeting was confirmed using Southern blot hybridization. The 3' probe detected the 19 kb wild type band and a 4.4 kb mutant band (resulting from the EcoRV site introduced by the targeting vector). The 5' probe detected a 19 kb wild type band and 15.6 kb mutant band (Fig. 16B). Several positive clones were confirmed by using both probes. We were able to successfully obtain germline pups by blastocyst injection from one clone (Fig. 17A).

43 19 HJ

Wild-type ™,/ '•' • ,W HI RV allele Y/~~ : M t I,,,

5' |Hiit,K:; X *\i\

Targeting vcct4ir t ' "

l?*tt

Targeted "* >f ~]~4'W7WU'«f<- alkie *^

> •- lovi' B 5' probe 3' probe

15.6kb *.«#«*•*» 19kb 15kb

if *:• •• 4.4kb

Figure 16. Generation of epitope tagged Irx3 mouse line. (A) The targeting stragedy for introduction of 3Myc and 6His tag at the 3'end of Irx3 coding region. The 5' and 3' probe used in B are also shown. (B) Southern blot analysis confirming correctly targeted ES clones. With the 5' probe the 15.6kb band is produced from the wild type locus and the 15kb band is produced from the correctly targeted locus. The 19kb band produced by 3' probe is from the wild type locus and the 4.4 kb band is produced from the targeted locus.

In order to verify expression of the epitope-tagged Irx3, embryo lysates were collected from wildtype, heterozygous and homozygous embryos at El 1.5. Lysates were run on SDS-PAGE gel and the membrane was probed with Irx3 antibody. In the heterozygous lysate, both wildtype Irx3 and Irx3-3M6H were detected with similar

intensity. Mice homozygous for Irx3-3M6H showed only one band at the expected size.

44 Using the myc antibody, a weaker band was seen in the heterozygous embryo lysate and

strong band was observed with the homozygous preparation (Fig. 17B).

In order to confirm that the tagged Irx3 are functionally similar to the wildtype

Irx3, we compared the phenotype of Irx3tag/" mice to Irx3_/" mice. Irx3"~ mice are smaller

and slimmer in comparison to wild type mice (unpublished data). However, Irx3tag/" mice

resemble their wild type littermates in body size and weight (Fig. 17C) indicating that the tagged form of Irx3 can functionally replace wild type Irx3. Next, we used this mouse

line for immunoprecipitation (IP) experiments. We were able to IP the tagged protein

efficiently from homozygous and heterozygous embryo lysates using myc beads and the

myc antibody (Fig. 17D). Therefore, this line will provide a useful tool for biochemical

studies.

45 A B

Irx3-3M6H Irx3 ab Irx3

Irx3-3M6H myc ab

3 3 3

Irx3tag/- Irx3-3M6H 34.8g Irx3 I 3 3

Figure 17. Irx3-3M6H pups are comparable to their wild type Httermates. (A) Genotyping of tagged Irx3 showing germline transmission. (B) Western blot analysis of tagged and wild type Jrx3 from El 1.5 whole embryo lysates. (C) Animals with one copy of tagged Irx3 and another knock out copy (tauLacZ) are identical to their wild type Httermates in size and body weight. (D) Western blot with Irx3 antibody on myc IPs from wild type and homozygous embryo lysates.

46 4. Discussion

4.1. Dynamic expression of Irx3 at various stages and tissues suggest multiple roles during development.

Our expression analysis has revealed that Irx3 continues to be highly expressed in various tissue and organs during later stages of development and adulthood, suggesting that Iroquois genes may play additional roles in the development and function of various organs. For example, the expression in cephalic mesoderm surrounding the optic cup at

El 3.5 suggests a role in the development of outer eye layers including the eye muscle and lachrymal glands that are derived from this region (Bosse et al., 1997). Our analysis revealed expression of Irx3 in the cardiac conduction system of the adult heart ventricles.

Importantly, Irx3 knock out mice show prolonged ventricle contraction suggesting that

Irx3 plays a role in the adult ventricular conduction function (Dr. B. Bruneau, unpublished communication).

Brain development, which is a complex process that involves differentiation, migration and bundle formation of axons, relies on different signaling molecules including Shh and Fgf8, axon guidance cues and transcription factors. The dynamic expression of Irx3 in the midbrain and hindbrain suggest a potential role for Irx3 in various processes during brain development. Irx3 expression marks the DCN and various structures that are connected to cerebellar structures such as pontine nuclei and superior colliculus. These structures together form a neuronal circuit that connects the dorsal midbrain to the ventral brain and spinal cord. In addition, superior colliculus, is important for visual processing and eye movement (Saab, 2003). Superior colliculus is the counter part of optic tectum in the chick (Nagy, 2006) and overexpression studies in chick

47 embryos have shown that Irx3 is important for the development of this structure

(Kobayashi, 2002). Inferior colliculus, another midbrain structure with high expression of

Irx3, functions in auditory processing (Tongjaroenbuangam, 2006). Fasciculus retroflexus (fr) tract is a tight bundle of axons that extend from the neurons of the habenular nucleus (Klemm, 2004) and both structures show a high level of Irx3 expression during adulthood. Hb is known to play a role in control of behaviour, learning and memory (Klemm, 2004; Kelly and Lecourtier, 2007). Thus, it will be interesting to investigate whether Irx3 knock out mice manifest any defects in behaviour, learning and/or memory.

4.2. Iroquois function redundantly during mouse development.

Iroquois homologs in Drosophila and Xenopus have been shown to play important roles in patterning and neurogenesis (Gomez-Skarmeta et al., 1996, McNeill et al, 1997, Netter et al, 1998, Grillenzoni et al, 1998, Kehl et al., 1998, Bellefroid et al,

1998, Gomez-Skamreta et al., 1998). Previously, the generation of single knockout mutants of Irx2, Irx4 and Irx5 have been reported (Bruneau et al., 2001; Cheng et al.

2005; Costantini et al, 2005; Lebel et al., 2003). These animals are all viable and fertile

tautacZ with no major patterning defects. The irx3 ijne used in the expression studies presented above represents a null allele of Irx3 (Irx3 protein can not be detected in the homozygous embryos, data not shown). Consistent with the functional redundancy of

Iroquois genes, knockout of Irx3 also results in viable and fertile animals. In addition to being highly expressed in the neural tube (as discussed above), Irx3 has the earliest onset of expression, detected at E6.5 (Houweling et al., 2001). However, no major patterning

48 defects were found in homozygous knockout embryos at these early stages of

development (unpublished data). The presence of six Iroquois genes that show

overlapping expression from early onset suggests the possibility of functional

compensation from other Irx genes in absence of one (Houweling et al., 2001, Cohen et

al., 2000, Bosse et al., 1997). Expression patterns of Irx3 resemble those of Irx5 as early

as E8.0 (Cohen et al., 2000, Houweling et al., 2001), hence it is highly possible that the

lack of patterning phenotype in Irx3 KO is due to compensation by Irx5. Additional

support for redundancy among Iroquois is provided from studies with other model

organisms, where the absence of one Iroquois does not generate a strong phenotype. For

instance, in Drosophila the identification of Iroquois relied on a mutation that affected two of the Irx genes, ara and caup, as mutation in only one of the genes has very mild or

no visible phenotype (Gomez-Skarmeta et al., 1996, Leyns et al., 1996). In zebrafish,

knock down of two Iroquois, Irxlb and Irx7, was required to result in a MHB patterning

defect, which was not observed in single knock down embryos (Itoh et al., 2002).

In order to overcome the redundancy of Iroquois and elucidate their roles in

patterning and development, we took two approaches: (1) double knockout of Irx3/5 and

(2) the generation of Irx3 overexpression lines. The double knockout of Irx3 and Irx5

leads to embryonic lethality at day 14.5-15.5 with defects in various tissues and organs.

Therefore, compared to single knock out of Irx3 or Irx5, inactivation of both genes

significantly reduces viability, confirming the functional redundancy among Iroquois.

The defects seen in various organs of the double knockout embryos is consistent with the

redundancy between Irx3 and Irx5 since these organs show expression of both genes

during development. Furthermore, other Irx genes are still expressed in these organs and

49 could to some degree compensate for lack of the Irx3 and Irx5 and resulting in a less

severe phenotype. For example, in addition to lrx3 and Irx5, Irx4 and Irx6 are expressed

in the heart ventricles at high levels. It is possible that more severe heart defects will be

observed upon deletion of additional Irx genes. Knock out of Irx4 or Irx2, both of which have some overlapping expression with Irx3/5 expression domain, are available. It is possible to cross the double mutant mouse to these single knock out lines to generate

triple knock out embryos. Of our interest would be examination of the neural tube and

hindbrain in Irx2; Irx3/5 triple knock out embryos, since all three genes are highly

expressed at MHB and the P2 level of ventral neural tube.

Overexpression studies in chick have revealed important insights into the roles of

Irx genes in neural development, such as the current model of neural tube patterning and

brain development that involves the cross-repressor function of transcription factors

(Briscoe et al., 2000; Matsumoto et al., 2004; Kobayashi et al., 2002). These experiments,

though very informative, have their limitations. The culture time is limited and long-term

effects of overexpression throughout development cannot be determined. In addition, these experiments are not highly feasible in the mouse model due to limitations on

accessibility of the embryos and their culturing difficulties. In order to study the effects

of Irx3 overexpression in the mouse, we used gene targeting to generate mice that can

conditionally overexpress Irx3 from the ubiquitous Rosa locus. Surprisingly, neural

specific overexpression of EnR-Irx3 at El0.5 did not result in severe morphological

defects in the brain. It is possible that the overexpression at earlier stage of development

is required to disturb brain development. Further, marker gene analysis of the midbrain

structures is required to reveal if any molecular defects are seen in the EnR-Irx3

50 overexpressing brain. It is also possible that Irx3 functions as an activator in the midbrain and overexpression of the repressor form can not induce development of midbrain structures in the forebrain. Future experiments with the Vpl6-Irx3 are required to elucidate which form of Irx3 is important for the development of midbrain structures. In conclusion, we have shown that this line can be used to drive overexpression of Irx3 and it is possible to use other Cre lines to target expression in various tissues. For example,

Emx2-cre can be used to induce overexpression specifically in the forebrain (Kimura et al., 2005), Emxl-Cre to drive the cerebral cortex and hippocampus expression (Guo et al., 2000), and the alpha-myosin heavy chain-Cre to induce heart-specific expression

(Agah et al, 1997).

4.3 Iroquois are involved in neural crest development.

Consistent with high expression of Irx3/5 in various tissues, Irx3/5 double mutants showed defects in the development of multiple organs. Noticeably, prevalence of phenotypes in the organs that are neural crest derived suggests a role for Iroquois in neural crest development.

The high occurrence of Irx3/5 DKO mutants with liver hernia indicated abnormal closure of the abdominal wall. This abnormality is not limited to the abdominal region as transverse sections at the heart level also shows thinning of the anterior ventral wall.

Furthermore, skeletal staining of El4.5 embryos revealed defective development of the sternum (data not shown). Thus, Iroquois is involved in ventral midline closure, a defect seen in various human syndromes. Recently, genetic studies and animal models were employed to understand the syndromes associated with body wall closure. Malformations

51 concerning ventral body wall development are observed in 1 in 2000 human births that often leads to perinatal death (Brewer et al. 2004). Recent evidence suggests that genetic

changes are associated with multiple syndromes characterized with body wall defects.

For example, Beckwith-Wiedemann Syndrome (BWS), the most recognized disease associated with abdominal wall defect, is caused by improper imprinting of several genes

(Brewer et al., 2004). Rieger Syndrome caused by mutations in Pitx2, results in eye, craniofacial and cardiac abnormalities in addition to ventral body wall defects (Semina et

al., 1996, Gage et al., 1997). Body wall defects often occur with other major

developmental abnormalities such as limb defects, exencephaly or facial clefts as seen in limb-body wall complex (LBWC) (Brewer et al., 2004). Remarkably, our Irx3/5 DKO mutants exhibit all of the defects associated with LBWC. In addition to the eye phenorype, the craniofacial and heart defects seen in our Irx3/5 DKO resemble the mouse model of the Rieger Syndrome, the Pitx2 knockout (with the exclusion of left right

asymmetry defect seen in Pitx2 KO) (Lin et al., 1999; Kitamura et al., 1999). We propose that our Irx3/5 DKO mouse line will provide a useful tool for understanding the

developmental process of body wall closure and its associated defects.

In addition to body wall closure defect of the Irx3/5 DKO mice, exencephaly is

another phenotype associated with congenital conditions. Exencephaly often indicates the presence of neural tube defect (NTD), where closure of the neural tube fails in the

developing brain (Greene and Copp, 2005). The closure of the neural tube occurs relatively early during development and is completed by E9.5-10 (Copp et al. 2003). In

Irx3/5 DKO mice, the neural tube defect was not observed at El0.5 (data not shown), and

exencephaly only becomes apparent by El 1.5. Therefore, the neural tube closure defect

52 may not be the primary cause of exencephaly in Irx3/5 DKO mutants. Alternatively, the phenotype can be caused by defects in the cephalic ectoderm where Irx3 is highly expressed.

In addition to exencephaly, the craniofacial structures of the mutants are affected.

Initial fate mapping experiments have shown that rostral neural crest (NC) cells that have the ability to form membrane bones, such as the skull, migrate from the cephalic ectoderm (Couly et al., 1993, Douarin et al, 2007). It has been shown that no facial structures develop, if this population of the NC cells are removed (Couly et al., 2002,

Creuzet et al., 2002). The specification, migration, survival and fate determination of the cranial NC cells play an important role in craniofacial development (Chai and Maxson,

2006). Together, these observations suggest that craniofacial malformations in Irx3/5 mutants are probably caused by defects in the cranial neural crest development.

NC cells arise at the boundary between the neural plate and the epidermis. They are pluripotent cells that migrate from their origin and differentiate into various tissues from cartilage and skeletal structures to pigment cells and peripheral nerves. Any defect interfering with the induction, migration, or differentiation of neural NC cells would lead to abnormalities that affect various structures derived from these cells (Wada, 2001).

Consistent with our hypothesis on the importance of Iroquois on neural crest function, various tissues that are derived from NC cells are affected in Irx3/5 DKO mutants.

Afferent neurons and glial cells of the cranial and dorsal root ganglia are derived from the neural crest (Barlow et al., 2002, Cordes 2001). One of the defects seen in mouse models of NC development, such as Msxl/2 mutants, is mispatterned cranial nerves (Ishii et al.,

2005). In Irx3/5 mutants, cranial nerve III is missing, while cranial nerve IV is

53 defasciculated and misguided, and the trigeminal nerve (V) is hypoplastic. Other mutant mice defective in NC development, such as knockout mice of Msxl/2 and AP-2 also show defects very similar to those of IrxS/5 mice (Zhang et ah, 1996, Schorle et al., 1996,

Ishii et al., 2005). In addition to the nerve defects as observed above, Msxl/2 double knockout embryos display a ventral body wall closure phenotype. AP-2 knockout mice exhibit sternal defects in addition to incomplete closure of the abdominal wall similar to those observed in lrx3/5 DKO mice (Zhang et al., 1996, Schorle et al, 1996).

Lastly, the phenotype seen in the heart of Irx3/5 mutants can also be caused by from neural crest defect. The cardiac neural crest (CNC) cells migrate from the lower hindbrain (between the otic placode and fourth somite) towards the heart to provide mesenchymal cells to the heart and the great arteries (Snider et al, 2007). The CNC cells can migrate to the artrioventricular cushions and surround the conduction system

(Poelmann and Gittenberger-de Groot, 1999). The AVSD seen in Irx3/5 mutants is caused by failure in ventricular separation most probably due to incomplete cushion development. Interestingly, the conduction system of the adult mice is affected in Irx3 single knockout (unpublished data). Therefore, it is possible that the defects observed during embryonic development or in the adult heart are caused by the neural crest defects.

Consistent with these observations, studies in other model organisms have shown a role for Iroquois in neural crest development. In Xenopus, overexpression of Xirol led to the enlargement of neural crest territory while blocking its activity inhibited expression of neural crest markers (Glavic et al., 2004). In zebrafish, irol and iro7 have been shown

54 to be essential for neural crest formation (Itoh et al., 2002). Our studies revealed for the first time the role of Iroquois in mouse neural crest development.

4.4 Iroquois play a role in limb development.

Both Irx3 and Irx5 are expressed in the prospective limb territory as early as E9.0

(Cohen et al., 2005). Limb development starts at the onset of Iroquois expression and the early expression of Irx genes suggests a potential role in limb patterning. Consistent with the expression pattern of Irx3 and Irx5, the development of scapula and hip was found to be severely defective in Irx3/5 DKO mutants. Signaling pathways important for distal limb patterning have little or no effect on the formation of proximal skeletons and many of the studied mutants with limb phenotype have normal girdle skeleton, indicating distinct mechanism controlling proximal and distal limb development (Kuijper et al.,

2004;,Capdevila and Belmonte, 2001). Interestingly, another TALE homeobox protein,

Pbxl, is nuclear in proximal limb bud and cytoplasmic in the distal regions. Expression of Meisl/2 (Pbx shuttling proteins) and Hox genes, which contain Pbx dimerization motifs, is restricted to the proximal regions. Together, these observations suggest that nuclear localization of Pbx with the aid of Meis allows it to function as a cofactor with

Hox proteins in proximal limb development. Pbxl -deficient mice display defects in proximal skeletal elements confirming the importance of Meis/Pbx/HOX interaction in proximal limb development (Capdevila and Belmonte, 2001). Similar to Pbx proteins,

Iroquois contain a TALE-homeodomain and can potentially function as cofactors for other homeodomain containing proteins. Among the few genes that have been identified to be important for proximal limb development is the homeobox-containing gene, Emx2.

55 Emx2 mutants lack the scapula blade and ilium (Pellegrini et al., 2001), a phenotype highly similar to Irx3/5 mutant limbs. Chick experiments have shown that Emx2 expression is induced by retinoic acid. While Emx2 is an essential gene for the development of proximal limb elements, its expression itself is not sufficient to program proximal limb development (Prols et al., 2004), suggesting that Emx2 may require additional factors in specifying the development of proximal limb elements. Interestingly, we have shown that Irx3 and Irx5 are induced in P19 carcinoma cell lines upon differentiation with retinoic acid (unpublished data). Therefore, the facts that the lrxS/5 and Emx2 mutant mice display similar phenotypes and that they are regulated by the same signaling pathway (RA) suggest that Iroquois may function as co-factor with Emx2 during proximal limb development. It is possible that the cofactor function of Iroquois is not limited to Emx2. They can possibly interact with other proximal patterning genes, such as Hoxc6. However elucidation of this role of Iroquois awaits further experiments.

One approach will be to use our epitope-tagged mice to identify protein partners of Irx3 in the proximal limb using co-immunoprecipitation and mass spectrometry experiments.

Surprisingly, the distal hindlimbs of the Irx3/5 are affected though the expression of Irx3/5 is proximally restricted. While the loss of digit I and occasionally digit II could be a secondary result of cell death in the proximal region, an intriguing possibility is that, after specification of anterior cells during early limb development, limb progenitor cells migrate from the proximal region to form the more distal elements. Consistent with this hypothesis, conditional deletion of Irx3 at El0.5 in the Irx5 mutant background did not result in a distal limb phenotype, indicating a role for Irx3 prior to E10.5. To determine the time point that Irx3 is required for specification of distal limb elements, we can

56 employ tamoxifen inducible-Cre mouse line to examine the temporal effect of Irx3 deletion.

4.5. Iroquois are important for patterning of diencephalon.

Patterning of the diencephalon is thought to rely on the subdivision of the anteroposterior axis into segments that express specific transcription factors. In addition to gene expression, the subdivision relies on morphology and cell lineage restriction

(Puelles et al. 1993, Puelles et al. 2003, Larsen et al. 2001). The prosomeric model of brain development divides the diencephalon into three prosomeres, pi to p3 (Fig. 18).

The major structure developed in pi is the pretectum while the thalamus and prethalamus are formed in p2 and p3 respectively. The boundary that separates p2 from p3 is the zona limitans intrathalamica (ZLI), which restricts cell mixing between p2 and p3. The sharp expression of transcription factors across the boundaries is thought to be important for the development of the structures within a boundary. While separation of p2 from p3 by the

ZLI is well established, not much is known about the segmentation of pi and p2. Despite the distinct gene expression pattern and tissue morphology between the pretectum and thalamus, in one study true segmentation of pl/p2 was challenged and presence of a boundary that restricts cell mixing between pi and p2 was questioned (Larsen et al.,

2001). The rostral expression of Irx3 and Irx5 is limited to the developing fr tract that separates the pretectum from the thalamus. The expansion of thalamus into pretectum in the Irx3/Irx5 mutants suggests that the development of segments restricting development of thalamus in the p2 region are defective. However, the double mutant embryos die before complete development of brain structures including the fr tract. Since it is possible

57 to bypass lethality till birth by using a conditional deletion of Irx3 in neural tissue, injection of labeled dye (Dil) can be used to visualize the fr tract right after birth. At this stage, the fr tract is completely formed and in the wild type embryos the dye can mark the tight bundle of axons from habenula to the ventral regions. If Irx3/5 indeed play a role in the development of fr, we expect to see severe de-fasciculation or absence of the fr tract in the conditional mutants. In addition to supporting the prosomeric model of brain development and presence of a boundary that separates pi from p2, currently the Irx3/5

DKO mutant appears the only mouse model available for studying the development of

Hb and fr although few zebrafish strains have been used in these structures.

Boundary formation in the CNS is thought to be controlled by cross-repressing transcription factors that allow the formation of a sharp line of expression across boundaries. Previous experiments in chick have shown that Irx3 and Six3 are cross- repressing partners involved in the development of the ZLI (Braun et al. 2003). However, in mice, the rostral limit of Irxl expression expands to the ZLI while the rostral limit of

Irx3/5 expression is the fr tract (posterior to ZLI). It has been shown that fez/fezl genes are Irxl cross-repressing partners in ZLI development (Hirata et al. 2006). Thus, it would be of interest to determine the cross-repressing partners for Irx3/5 in fr tract development.

For this purpose, we would need to identify genes whose expression are rostral to Irx3/5 expression and whether their expression is altered in Irx3/5 DKO brain. In addition to

£7x3, Olig2, Emx2, and Pax6 are potential candidates since they display complementary expression pattern to that of Irx3/5. In a microarray study of SimV'' embryos, Irx3 expression was found to be up-regulated and expanded in the hypothalamus of the SimT'

58 mice suggesting that Siml would be another candidate partner of Irx3/5 (Caqueret et al.,

2006).

In conclusion, while increasing evidence from overexpression studies suggest multiple roles for Iroquois in development of various tissues, single knock out Irx members in mice do not cause severe patterning defects. Irx3/5 DKO studies revealed important patterning functions of two highly redundant genes, Irx3 and Irx5, indicating that lack of phenotype in single knock out mice is due to functional compensation by other members of Iroquois. In order to understand the mechanism of Iroquois function during mammalian development, identification of their molecular targets and protein partners is required and the reagent mice generated in this study will provide useful tools for this purpose.

59 5. References:

Agah, R, Frenkel, P., French, B., Michael, L., Overbeek, P. and Schneider, M. (1997). Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. J Clin Invest 100, 169-79.

Anselme, L, Laclef, C, Lanaud, M., Rtither, U. and Schneider-Maunoury, S. (2007). Defects in brain patterning and head morphogenesis in the mouse mutant Fused toes. Dev Biol 304, 208-20.

Bao, Z., Bruneau, B., Seidman, J., Seidman, C. and Cepko, C. (1999). Regulation of chamber-specific gene expression in the developing heart by Irx4. Science 283, 1161-4. Barlow, L. (2002). Cranial nerve development: placodal neurons ride the crest. Curr Biol 12,R171-3.

Bellefroid, E., Kobbe, A., Grass, P., Pieler, T., Gurdon, J. and Papalopulu, N. (1998). Xiro3 encodes a Xenopus homolog of the Drosophila Iroquois genes and functions in neural specification. EMBO J11, 191-203.

Bilioni, A., Craig, G., Hill, C. and McNeill, H. (2005). Iroquois transcription factors recognize a unique motif to mediate transcriptional repression in vivo. Proc Natl Acad Sci USA 102, 14671-6.

Bosse, A., Stoykova, A., Nieselt-Struwe, K., Chowdhury, K., Copeland, N., Jenkins, N. and Grass, P. (2000). Identification of a novel mouse Iroquois homeobox gene, Irx5, and chromosomal localisation of all members of the mouse Iroquois gene family. Dev Dyn 218, 160-74.

Bosse, A., Ziilch, A., Becker, M., Torres, M., Gomez-Skarmeta, J., Modolell, J. and Grass, P. (1997). Identification of the vertebrate Iroquois homeobox gene family with overlapping expression during early development of the nervous system. Mech Dev 69, 169-81.

Braun, M., Etheridge, A., Bernard, A., Robertson, C. and Roelink, H. (2003). Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain. Development 130, 5579-87.

Brewer, S. and Williams, T. (2004). Finally, a sense of closure? Animal models of human ventral body wall defects. Bioessays 26, 1307-21.

Briscoe, J., Chen, Y., Jessell, T. and Struhl, G. (2001). A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube. Mol Cell 7, 1279-91.

60 Briscoe, J. and Ericson, J. (1999). The specification of neuronal identity by graded Sonic Hedgehog signalling. Semin Cell Dev Biol 10, 353-62.

Briscoe, J. and Ericson, J. (2001). Specification of neuronal fates in the ventral neural tube. Curr Opin Neurobiol 11, 43-9.

Briscoe, J., Pierani, A., Jessell, T. and Ericson, J. (2000). A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101, 435-45.

Bruneau, B., Bao, Z., Fatkin, D., Xavier-Neto, J., Georgakopoulos, D., Maguire, C, Berul, C, Kass, D., Kuroski-de Bold, M., de Bold, A. et al. (2001). Cardiomyopathy in Irx4-deficient mice is preceded by abnormal ventricular gene expression. Mol Cell Biol 21, 1730-6.

Bruneau, B., Bao, Z., Tanaka, M., Schott, X, Izumo, S., Cepko, C, Seidman, J. and Seidman, C. (2000). Cardiac expression of the ventricle-specific homeobox gene Irx4 is modulated by Nkx2-5 and dHand. Dev Biol 217, 266-77.

Burglin, T. (1997). Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals. Nucleic Acids Res 25, 4173-80.

Capdevila, J. and Izpisua Belmonte, J. (2001). Patterning mechanisms controlling vertebrate limb development. Annu Rev Cell Dev Biol 17, 87-132.

Caqueret, A., Boucher, F. and Michaud, J. (2006). Laminar organization of the early developing anterior hypothalamus. Dev Biol 298, 95-106.

Chai, Y. and Maxson, R. J. (2006). Recent advances in craniofacial morphogenesis. Dev Dyn 235, 2353-75.

Cheng, C, Chow, R., Lebel, M., Sakuma, R., Cheung, H., Thanabalasingham, V., Zhang, X., Bruneau, B., Birch, D., Hui, C. et al. (2005). The Iroquois homeobox gene, Irx5, is required for retinal cone bipolar cell development. Dev Biol 287, 48-60.

Copp, A., Greene, N. and Murdoch, J. (2003a). Dishevelled: linking convergent extension with neural tube closure. Trends Neurosci 26, 453-5.

Copp, A., Greene, N. and Murdoch, J. (2003b). The genetic basis of mammalian neurulation. Nat Rev Genet 4, 784-93.

Cordes, S. (2001). Molecular genetics of cranial nerve development in mouse. Nat Rev Neurosci 2, 611-23.

61 Couly, G., Coltey, P. and Le Douarin, N. (1993). The triple origin of skull in higher vertebrates: a study in quail-chick chimeras. Development 117, 409-29.

Couly, G., Creuzet, S., Bennaceur, S., Vincent, C. and Le Douarin, N. (2002). Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head. Development 129, 1061-73.

Creuzet, S., Couly, G., Vincent, C. and Le Douarin, N. (2002). Negative effect of Hox gene expression on the development of the neural crest-derived facial skeleton. Development 129, 4301-13. de la Calle-Mustienes, E., Feijoo, C, Manzanares, M., Tena, J., Rodriguez-Seguel, E., Letizia, A., Allende, M. and Gomez-Skarmeta, J. (2005). A functional survey of the enhancer activity of conserved non-coding sequences from vertebrate Iroquois cluster gene deserts. Genome Res 15, 1061-72.

Diez del Corral, R., Aroca, P., G mez-Skarmeta, J., Cavodeassi, F. and Modolell, J. (1999). The Iroquois homeodomain proteins are required to specify body wall identity in Drosophila. Genes Dev 13, 1754-61.

Ding, Q., Fukami, S., Meng, X., Nishizaki, Y., Zhang, X., Sasaki, H., Dlugosz, A., Nakafuku, M. and Hui, C. (1999). Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of Glil. Curr Biol 9, 1119-22.

Ericson, J., Rash bass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T. and Briscoe, J. (1997). Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90, 169-80.

Fujii, T., Pichel, J., Taira, M., Toyama, R., Dawid, I. and Westphal, H. (1994). Expression patterns of the murine LIM class homeobox gene liml in the developing brain and excretory system. Dev Dyn 199, 73-83.

Gage, P. and Camper, S. (1997). Pituitary homeobox 2, a novel member of the bicoid- related family of homeobox genes, is a potential regulator of anterior structure formation. Hum Mol Genet 6, 457-64.

Glavic, A., Gomez-Skarmeta, J. and Mayor, R. (2002). The homeoprotein Xirol is required for midbrain-hindbrain boundary formation. Development 129, 1609-21.

Glavic, A., Silva, F., Aybar, M., Bastidas, F. and Mayor, R. (2004). Interplay between Notch signaling and the homeoprotein Xirol is required for neural crest induction in Xenopus embryos. Development 131, 347-59.

Gomez-Skarmeta, J., Diez del Corral, R., de la Calle-Mustienes, E., Ferre-Marco, D. and Modolell, J. (1996). Araucan and caupolican, two members of the novel iroquois

62 complex, encode homeoproteins that control proneurai and vein-forming genes. Cell 85, 95-105.

Greene, N. and Copp, A. (2005). Mouse models of neural tube defects: investigating preventive mechanisms. Am J Med Genet C Semin Med Genet 135, 31-41.

Grillenzoni, N., van Helden, J., Dambly-Chaudiere, C. and Ghysen, A. (1998). The iroquois complex controls the somatotopy of Drosophila notum mechanosensory projections. Development 125, 3563-9.

Guo, H., Hong, S., Jin, X., Chen, R., Avasthi, P., Tu, Y., Ivanco, T. and Li, Y. (2000). Specificity and efficiency of Cre-mediated recombination in Emxl-Cre knock-in mice. Biochem Biophys Res Commun 273, 661-5.

Gomez-Skarmeta, J., de La Calle-Mustienes, E. and Modolell, J. (2001). The Wnt- activated Xirol gene encodes a repressor that is essential for neural development and downregulates Bmp4. Development 128, 551-60.

Gomez-Skarmeta, J., Glavic, A., de la Calle-Mustienes, E., Modolell, J. and Mayor, R. (1998). Xiro, a Xenopus homolog of the Drosophila Iroquois complex genes, controls development at the neural plate. EMBO J17, 181-90.

Gomez-Skarmeta, J. and Modolell, J. (2002). Iroquois genes: genomic organization and function in vertebrate neural development. Curr Opin Genet Dev 12, 403-8.

Gotz, K., Briscoe, J. and Riither, U. (2005). Homozygous Ft embryos are affected in floor plate maintenance and ventral neural tube patterning. Dev Dyn 233, 623-30.

Hirata, T., Nakazawa, M., Muraoka, O., Nakayama, R., Suda, Y. and Hibi, M. (2006). Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions. Development 133, 3993-4004.

Isaka, F., Ishibashi, M., Taki, W., Hashimoto, N., Nakanishi, S. and Kageyama, R. (1999). Ectopic expression of the bHLH gene Mathl disturbs neural development. Eur J Neurosci 11, 2582-8.

Ishii, M., Han, J., Yen, H., Sucov, H., Chai, Y. and Maxson, R. J. (2005). Combined deficiencies of Msxl and Msx2 cause impaired patterning and survival of the cranial neural crest. Development 132, 4937-50.

Itoh, M., Kudoh, T., Dedekian, M., Kim, C. and Chitnis, A. (2002). A role for irol and iro7 in the establishment of an anteroposterior compartment of the ectoderm adjacent to the midbrain-hindbrain boundary. Development 129, 2317-27.

Kehl, B., Cho, K. and Choi, K. (1998). mirror, a Drosophila homeobox gene in the Iroquois complex, is required for sensory organ and alula formation. Development 125,

63 1217-27.

Kiecker, C. and Lumsden, A. (2004). Hedgehog signaling from the ZLI regulates diencephalic regional identity. Nat Neurosci 7, 1242-9.

Kimura, J., Suda, Y., Kurokawa, D., Hossain, Z., Nakamura, M., Takahashi, M., Hara, A. and Aizawa, S. (2005). Emx2 and Pax6 function in cooperation with Otx2 and Otxl to develop caudal forebrain primordium that includes future archipallium. J Neurosci 25, 5097-108.

Kitamura, K., Miura, H., Miyagawa-Tomita, S., Yanazawa, M., Katoh-Fukui, Y., Suzuki, R., Ohuchi, H., Suehiro, A., Motegi, Y., Nakahara, Y. et al. (1999). Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism. Development 126, 5749-58.

Klemm, W. (2004). Habenular and interpeduncularis nuclei: shared components in multiple-function networks. Med Sci Monit 10, RA261-73.

Kobayashi, D., Kobayashi, M., Matsumoto, K., Ogura, T., Nakafuku, M. and Shimamura, K. (2002). Early subdivisions in the neural plate define distinct competence for inductive signals. Development 129, 83-93.

Kuijper, S., Beverdam, A., Kroon, C, Brouwer, A., Candille, S., Barsh, G. and Meijlink, F. (2005). Genetics of shoulder girdle formation: roles of Tbxl5 and aristaless- like genes. Development 132, 1601-10.

Larsen, C, Zeltser, L. and Lumsden, A. (2001). Boundary formation and compartition in the avian diencephalon. J Neurosci 21, 4699-711.

Le Douarin, N., Brito, J. and Creuzet, S. (2007). Role of the neural crest in face and brain development. Brain Res Rev 55, 237-47.

Lecourtier, L. and Kelly, P. (2007). A conductor hidden in the orchestra? Role of the habenular complex in monoamine transmission and cognition. Neurosci Biobehav Rev 31, 658-72.

Lee, K. and Jessell, T. (1999). The specification of dorsal cell fates in the vertebrate central nervous system. Annu Rev Neurosci 22, 261-94.

Lee, S., Jurata, L., Funahashi, J., Ruiz, E. and Pfaff, S. (2004). Analysis of embryonic motoneuron gene regulation: derepression of general activators function in concert with enhancer factors. Development 131, 3295-306.

Lin, C, Kioussi, C, O'Connell, S., Briata, P., Szeto, D., Liu, F., Izpisua-Belmonte, J. and Rosenfeld, M. (1999). Pitx2 regulates lung asymmetry, cardiac positioning and

64 pituitary and tooth morphogenesis. Nature 401, 279-82.

Logan, M., Martin, J., Nagy, A., Lobe, C, Olson, E. and Tabin, C. (2002). Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis 33, 77-80.

Mann, R. and Chan, S. (1996). Extra specificity from extradenticle: the partnership between HOX and PBX/EXD homeodomain proteins. Trends Genet 12, 258-62.

Marti, E., Bumcrot, D., Takada, R. and McMahon, A. (1995). Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants. Nature 375, 322-5.

Matsuo, L, Kuratani, S., Kimura, C, Takeda, N. and Aizawa, S. (1995). Mouse Otx2 functions in the formation and patterning of rostral head. Genes Dev 9,2646-58.

Nagy, A., Kruse, W., Rottmann, S., Dannenberg, S. and Hoffmann, K. (2006). Somatosensory-motor neuronal activity in the superior colliculus of the primate. Neuron 52, 525-34.

Netter, S., Fauvarque, M., Diez del Corral, R., Dura, J. and Coen, D. (1998). white+ transgene insertions presenting a dorsal/ventral pattern define a single cluster of homeobox genes that is silenced by the poly comb-group proteins in Drosophila melanogaster. Genetics 149, 257-75.

Novitch, B., Wichterle, H., Jessell, T. and Sockanathan, S. (2003). A requirement for retinoic acid-mediated transcriptional activation in ventral neural patterning and motor neuron specification. Neuron 40, 81-95.

Pellegrini, M., Pantano, S., Fumi, M., Lucchini, F. and Forabosco, A. (2001). Agenesis of the scapula in Emx2 homozygous mutants. Dev Biol 232, 149-56.

Peters, T., Ausmeier, K., Dildrop, R. and Riither, U. (2002). The mouse Fused toes (Ft) mutation is the result of a 1.6-Mb deletion including the entire Iroquois B gene cluster. Mamm Genome 13, 186-8.

Pierani, A., Brenner-Morton, S., Chiang, C. and Jessell, T. (1999). A sonic hedgehog- independent, retinoid-activated pathway of neurogenesis in the ventral spinal cord. Cell 97, 903-15.

Poelmann, R. and Gittenberger-de Groot, A. (1999). A subpopulation of apoptosis- prone cardiac neural crest cells targets to the venous pole: multiple functions in heart development? Dev Biol 207, 271-86.

65 Prols, F., Ehehalt, F., Rodriguez-Niedenfiihr, M., He, L., Huang, R and Christ, B. (2004). The role of Emx2 during scapula formation. Dev Biol 275, 315-24.

Puelles, L. and Rubenstein, J. (1993). Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosci 16, 472-9.

Puelles, L. and Rubenstein, J. (2003). Forebrain gene expression domains and the evolving prosomeric model. Trends Neurosci 26, 469-76.

Qiu, M., Bulfone, A., Martinez, S., Meneses, J., Shimamura, K., Pedersen, R. and Rubenstein, J. (1995). Null mutation of Dlx-2 results in abnormal morphogenesis of proximal first and second branchial arch derivatives and abnormal differentiation in the forebrain. Genes Dev 9, 2523-38.

Roelink, H., Porter, J., Chiang, C, Tanabe, Y., Chang, D., Beachy, P. and Jessell, T. (1995). Floor plate and motor neuron induction by different concentrations of the amino- terminal cleavage product of sonic hedgehog autoproteolysis. Cell 81, 445-55.

Saab, C. and Willis, W. (2003). The cerebellum: organization, functions and its role in nociception. Brain Res Brain Res Rev 42, 85-95.

Schorle, H., Meier, P., Buchert, M., Jaenisch, R. and Mitchell, P. (1996). Transcription factor AP-2 essential for cranial closure and craniofacial development. Nature 381, 235-8.

Semina, E., Reiter, R., Leysens, N., Alward, W., Small, K., Datson, N., Siegel- Bartelt, J., Bierke-Nelson, D., Bitoun, P., Zabel, B. et al. (1996). Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome. Nat Genet 14, 392-9.

Snider, P., Olaopa, M., Firulli, A. and Conway, S. (2007). Cardiovascular development and the colonizing cardiac neural crest lineage. ScientificWorldJournal 7, 1090-113. Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21, 70-1.

Srinivas, S., Watanabe, T., Lin, C, William, C, Tanabe, Y., Jessell, T. and Costantini, F. (2001). Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1, 4.

Tongjaroenbuangam, W., Jongkamonwiwat, N., Phansuwan-Pujito, P., Casalotti, S., Forge, A., Dodson, H. and Govitrapong, P. (2006). Relationship of opioid receptors with GABAergic neurons in the rat inferior colliculus. Eur J Neurosci 24, 1987-94.

Tronche, F., Kellendonk, C, Kretz, O., Gass, P., Anlag, K., Orban, P., Bock, R.,

66 Klein, R. and Schiitz, G. (1999). Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat Genet 23, 99-103. van der Hoeven, F., Schimmang, T., Volkmann, A., Mattei, M., Kyewski, B. and Riither, U. (1994). Programmed cell death is affected in the novel mouse mutant Fused toes (Ft). Development 120, 2601-7.

Wada, H. (2001). Origin and evolution of the neural crest: a hypothetical reconstruction of its evolutionary history. Dev Growth Differ 43, 509-20.

Wang, G., Nikovits, W. J., Bao, Z. and Stockdale, F. (2001). Irx4 forms an inhibitory complex with the vitamin D and retinoic X receptors to regulate cardiac chamber-specific slow MyHC3 expression. J Biol Chem 276, 28835-41.

Wurst, W. and Bally-Cuif, L. (2001). Neural plate patterning: upstream and downstream of the isthmic organizer. Nat Rev Neurosci 2, 99-108.

Zhang, H., Catron, K. and Abate-Shen, C. (1996). A role for the Msx-1 homeodomain in transcriptional regulation: residues in the N-terminal arm mediate TATA binding protein interaction and transcriptional repression. Proc Natl Acad Sci USA 93, 1764-9.