The Vertebrate Nervous System Has Fascinated Scientists for Decades, Puzzling Over the Wonders of Consciousness, Movement and the Perception of the Outer World

Early Development of the Olfactory Placode and Early Rostrocaudal Patterning of the Caudal Neural Tube

Esther Maier

Umeå Centre for Molecular Medicine Umeå University Umeå 2009

Front cover: Upper Panel: Collage of Hes5, Msx1/2 and Lhx2 expression in the olfactory pit of a stage 22 embryo and in stage 14OP explants, and GFP-electroporated embryo, stained with HuC/D (red) and DAPI (blue). Lower Panel: Flat-mounted hindbrain of a stage 20 embryo whole mount immunohistochemistry for Tbx20 (red) and Hb9 (green) and collage of HB and SC explants expressing Tbx20, Isl1 (red) and Hb9 (green).

Copyyright © Esther Maier ISBN: 978-91-7264-804-3 ISSN: 0346-6612 New Series Number:1274 Printed by Print & Media Umeå, Sweden 2009

And the end of all our exploring Will be to arrive where we started And know the place for the first time

T.S. Eliot, “Little Gidding”

Table of contents

Abstract______7 Papers in this thesis______9 Abbreviations______10 Introduction______11-23 Organization of the nervous system………………………………………… 11 - Signalling pathways and their effectors - Terminology Development of the nervous system…………………………………………. 38 Development of the CNS……………………………………………………… 13 - Neural induction and neural plate formation - Rostrocaudal regionalization of the early CNS - Hox genes and motor neuron subtype - Cdx genes as potential mediators of Hox induction - Ventral patterning of the neural tube Development of the PNS……………………………………………………… 18 - Neural plate border specification - Neural crest and placodes The olfactory system…………………………………………………………… 19 - Development and neurogenesis - Molecular regulation Why study developmental processes in the chick?...... 23 Aims______24 Results______25-36 Project I: Rostrocaudal patterning of the caudal neuraxis………………... 25 - Cells of caudal spinal cord character are specified at the late gastrula stage - Wnt and FGF signalling is required to specify spinal cord character - Graded Wnt and FGF signalling distinguish between hindbrain and spinal cord character - Cells of caudal hindbrain character are specified at early neurulation stages - RA imparts caudal character in presumptive hindbrain and rostral character in presumptive spinal cord cells - FGF signalling distinguishes between cells of caudal hindbrain and rostral spinal cord character - Progenitor cells in the hindbrain and spinal cord have acquired

Table of contents

sufficient positional information to differentiate into dMNs and vMNs - Early Wnt signalling is required for the generation of MN subtype - Combinatorial Wnt, RA and FGF signals are sufficient to induce hindbrain and spinal cord character from neural cells - Acquisition of rostrocaudal specific Hox gene profiles determines the emergence of MN subtype Project II: Patterning and neurogenesis in the olfactory placode…………… 30 - Hes5 is the earliest neurogenic marker expressed in the olfactory placodal region in chick - Expression of neurogenesis markers in the olfactory region - Msx1/2 and Id3 are valid markers for respiratory epithelial character - Cells in the olfactory placodal area are specified as cells of both sensory epithelial and respiratory epithelial character - BMP signalling promotes the generation of respiratory epithelial character - BMP signals promote the generation of respiratory epithelial character in vivo - A switch in the competence of olfactory placodal cells to respond to BMP signals - FGF signalling is required for cells of sensory epithelial character - Manipulation of FGF signalling in vivo - Notch signalling is required to maintain progenitor cells in the olfactory placode - Hes5-positive cells are primarily affected by perturbed Notch signalling - In ovo manipulation of Notch signalling -BMP signalling affects the expression of Notch1 and Delta1 Discussion______37-51 Project I: Rostrocaudal patterning of the caudal neuraxis……………… … 37 - Wnt and FGF signalling is required for caudal neural character - Caudal spinal cord character is specified by graded Wnt and FGF signalling - Instructive FGF signalling during caudal neural development - RA signalling and caudal hindbrain development - A role for RA signalling in spinal cord development - Early Wnt signalling establishes a positional context for later RA and FGF signalling in specifying caudal hindbrain and spinal cord cells - Opposing actions of RA and FGF during caudal neural development - Rostrocaudal patterning and body axis extension - Hox gene expression in progenitor cells and the emergence of motor neuron subtype identity - Coordinated development of the CNS and PNS Project II: Patterning and neurogenesis in the olfactory placode…………… 45 - Expression of Hes5 genes in the olfactory placodal region

Table of contents

- Dynamic expression patterns of neurogenic markers in the olfactory placodal region - Olfactory placodal progenitor cells give rise to respiratory and sensory epithelial cells - BMP signals specify respiratory epithelial cells - BMP signalling is required for the differentiation of the olfactory sensory lineage - BMP signalling acts sequentially during the development of the olfactory placode - FGF signalling is required for the generation of cells of sensory epithelial character - BMP and FGF act in an opposing manner - Notch signalling is required to maintain progenitor cells - Early born neurons join the migratory mass - Antagonism between BMP and Notch signalling pathways Acknowledgements______52 References______53-64 Supplementary Figures______65-67 Papers I-IV______68

Abstract

The development of the nervous system is a complex process. Cell divisions, cell differentiation and signalling interactions must be tightly regulated. To comprehend the mature nervous system, we have to understand its assembly during development. Two main questions were addressed in this thesis: (1) how is the caudal part of the central nervous system specified and (2) how is the early development of the olfactory placode regulated? By using tissue and whole embryo assays in the chick, we identified signalling molecules involved in these processes and propose possible mechanisms for their function.

The central nervous system is regionalized along its rostrocaudal axis during development. However, the mechanisms by which cells in the caudal part of the neuraxis acquire rostrocaudal regional identity have been unresolved. We provide evidence that at gastrula stages cells in the caudal neural plate are specified as cells of caudal spinal cord character in response to Wnt and FGF signals and that cells of rostral spinal cord and caudal hindbrain character only emerge later at neurulation stages in response to retinoic acid signalling acting on previously caudalized cells. In the hindbrain and spinal cord distinct motor neuron subtypes differentiate at precise rostrocaudal positions from progenitor cells. We provide evidence that cells in the caudal neural plate have acquired sufficient positional information to differentiate into motor neurons of the correct rostrocaudal subtype.

The olfactory placode gives rise to all the structures of the peripheral olfactory system, which, in the chick consists of the olfactory nerve, the sensory epithelium, where the olfactory sensory neurons (OSN) are located and the respiratory epithelium, that produces the mucus. Several studies have addressed the role of signalling cues in the specification of OSNs but much less is known about the regulation of sensory versus respiratory patterning and the events controlling early neurogenesis in the developing olfactory placode. We show that by stage 14 the olfactory placode is specified to give rise to both cells of sensory and respiratory epithelial character. Moreover, cells of respiratory epithelial character require BMP signalling, whereas cells of sensory epithelial character require FGF signalling. We suggest a mechanism

7 Abstract in which FGF and BMP signals act in an opposing manner to regulate olfactory versus respiratory epithelial cell fate decision. BMP signalling has also been implicated in the regulation of neurogenesis in the sensory epithelium, and we show that BMP signals are required for the generation of OSNs, because in the absence of BMP signalling cells in the sensory epithelium do not mature. Independently, we also analyzed the role of Notch signalling during early olfactory development both in vitro and in vivo and provide evidence that active Notch signalling is required to prevent cells in the olfactory placode from premature differentiation.

8 Papers

Papers in this thesis

This thesis is based on the following papers and manuscripts:

I. Nordström U, Maier E, Jessell TM, Edlund T (2006). An early role for Wnt signalling in specifying neural patterns of Cdx and Hox gene expression and motor neuron subtype. PLoS Biology, 4(8) 1438-1452.

II. Maier E, Gunhaga L (2009). Dynamic expression of neurogenic markers in the developing chick olfactory epithelium. Dev Dyn, accepted

III. Maier E, von Hofsten J, Gunhaga L. Opposing activities of FGF and BMP regulate the olfactory sensory versus respiratory epithelial cell fate decision. Manuscript

IV. Maier E, von Hofsten J, Gunhaga L. Notch signalling under the negative regulatory influence of BMP activity, controls neurogenesis in the olfactory placode. Manuscript

9 Abbreviations

Abbrevations bHLH basic helix-loop-helix BMP bone morphogenetic protein BMPR BMP receptor Bra Brachyury c caudal CNS central nervous system CRABP cellular retinoic acid binding protein DiI 1,1´-dioctadecyl-3,3,3´,3´-tetramethylinocarbocyanine perchlorate Dpp decapentaplegic E embryonic day FB forebrain FGF fibroblast growth factor FGFR FGF receptor GDF growth/differentiation factor HB hindbrain IC intracellular domain INP immediate neuronal precursor mFrz8CRD mouse frizzled 8 cysteine rich domain MN motor neuron OSN olfactory sensory neuron PNS peripheral nervous system r rhombomere RA retinoic acid RAR RA receptor RALDH retinaldehyde dehydrogenase RARE RA response element RXR retinoid X receptor SC spinal cord Sfrp soluble frizzled related protein Shh sonic hedgehog Smad small mothers against dpp TAP transient amplifying cell TGFβ transforming growth factor β Wnt wingless/Int

10 Introduction

Introduction

The vertebrate nervous system has fascinated scientists for decades, puzzling over the wonders of consciousness, movement and the perception of the outer world. The complex pattern of the adult nervous system is the product of genetic instructions, tissue and cellular interactions and, later on, the interactions with the environment. Development of the nervous system begins during early embryogenesis with the induction of neural cells, then the formation of the neural plate and afterwards the establishment of the primordial nervous system of the embryo. Neurons are generated from undifferentiated precursor cells and some neurons will later migrate from their site of origin to their final position within the organism. Axonal pathways form and synapses are established, leading to the formation of the complex cellular network the nervous system represents.

Organization of the nervous system

The vertebrate nervous system has two anatomical distinct parts: the central nervous system (CNS) which consists of the brain and the spinal cord and the peripheral nervous system (PNS), which is composed of the sensory organs, and the cells and neurons of the autonomic and the enteric nervous system. Even though the two systems are anatomically distinct they still form a functional unit. All the sensory input is either generated or transferred from the PNS to the CNS. For example in the olfactory system, the sensory information is transmitted from the olfactory sensory neurons (OSN) via the olfactory bulbs to the brain, generating the impression of smell. The execution of motor commands from the brain and spinal cord is also part of the function of the PNS. The nervous system is organized along two main axes, the rostrocaudal (head-to-tail) and the dorsoventral (back-to-belly) axis, which arise during early development. The CNS develops from an initially flat sheet of cells, the neural plate. During neurulation the neural plate folds up at the margins and forms the neural tube. In the rostral neural tube, three brain vesicles form, the forebrain-, the midbrain- and the hindbrain-vesicle. In the caudal part, the neural tube gives rise to the spinal cord. These initial regions become further subdivided as development proceeds, to give rise to the adult structures of

11 Introduction the CNS. The olfactory bulb, the cerebrum, the thalamus and the hypothalamus arise from the forebrain vesicle, the midbrain from the midbrain vesicle, the cerebellum, the pons and the medulla from the hindbrain vesicle and finally the spinal cord. The spinal cord is less complex than the brain, nevertheless, the motor neurons located in the ventral area display diversity, based on muscle target and axon trajectory (Figure 2). How is such complexity created during development? External signalling cues are integrated along both axes, to provide a grid of positional information. Thus, the fate of progenitor cells in the forming nervous system depends on the position of a cell along both the rostrocaudal and the dorsoventral axis. But also the state of the responding cell plays a role, since the same external signals can produce different outcomes.

Signalling pathways and their effectors During embryogenesis only a limited number of signalling pathways control the generation of the embryo, generating the vast number of different cell types during development. The Wnt, Fibroblast Growth Factor (FGF), Bone morphogenetic proteins (BMP), Sonic hedgehog (Shh), retinoic acid (RA) and Notch-Delta signalling pathways are some of the most common and important pathways acting during development (Table 1). The same signalling pathway can act in the same cell linage at different time points; elucidating different responses in the cell and subsequently, lead to the generation of the vast number of cell types found in the adult.

pathway Receptors major intracellular effectors antagonists Wnt Frizzeled 1-10, β-Catenin, Tcf Dkk, Sfrp LRP FGF FGFR1-4 MAPK, Ras Sprouty, Pyst BMP BMPR-type-I Smad1,5,8, Smad4, p38 Smad6,7 (Alk2, 3&6) Bambi & type-II Shh Patched, Gli1-3 Gli3 smoothened RA RAR, RXR CRABP Cyp26 Notch Delta & Serrate IC of Notch, CSL

Table1: Overview over the signalling pathways and their effectors. Only pathways that were manipulated in the course of this thesis are included.

12 Introduction

Terminology It is a long way from the fertilized egg to the adult. The acquisition of cell identity during development is a multi-step process, which classically is subdivided into the following concepts: Fate: A cells contribution to the organism proper, if development goes its path. In practise, to determine the fate of an area, cell labelling techniques are used. Specification: A cell is specified to a certain fate when it keeps its fate in an environment removed from further signalling cues by surrounding tissues. Determination/Commitment: A cell is determined/committed to a certain fate if it keeps this fate even if challenged by new signalling cues. In practice, to test if a cell is determined/committed to a certain fate, a group of cells is often transplanted from its site of origin to a new position in the embryo and the further development is observed. Differentiation: Step in the maturation of progenitor cells. The cell leaves the cell cycle and start to express genes that determine its specific phenotype. (after [209])

Development of the nervous system

The nervous system originates from the ectoderm during embryonic development. The neural plate is induced during early development and will give rise to the CNS. Cells at the border between neural plate and epidermal ectoderm (the border region) generate the placodes and the neural crest, from which the structures of the PNS and some non-neural tissues/cells arise.

Development of the CNS

Neural induction and neural plate formation Originally the concept of induction was developed by Hans Spemann and Hilde Mangold around 1924, simply meaning a process by which one group of cells or tissue regulates the development of another group of cells or tissues [1]. Neural induction occurs at blastula stages [2, 3]. Epiblast cells are specified as cell of rostral neural character, and a dual role for FGF

13 Introduction signalling in neural induction in chick has been proposed. First, FGFs act to down regulate BMP mRNA expression in the medial part of the epiblast, and second FGFs play an instructive role in neural induction [4]. After induction of neural cells, these cells will autonomously start the process of neural plate formation. This process includes the apicobasal thickening of the neuroectoderm and later on during gastrula stages, convergent extension movements. Thus the neural plate gets narrower mediolaterally and elongated along its rostrocaudal extend [5].

Rostrocaudal regionalization of the early CNS How the caudal part of the nervous system arises during development is a relatively old question in the field of developmental biology. Already in 1927 Spemann proposed a model, wherein independent organizers induce tissues with different rostrocaudal identity. An alternative model was proposed by Nieuwkoop in 1952, the so called “activation-transformation”- model of caudal neural specification. In this model neural tissue is first induced by factor(s) X and then transformed to a caudal fate by factor(s) Y [6]. Since all presumptive neural cells initially are specified as cells of forebrain- like character, a Nieuwkoop-like model is more probable. Nevertheless, this still raises the question, when and how the more caudal cells of the CNS arise. After neural tissue induction and formation of the neural plate, the neural plate becomes subdivided into distinct domains along the rostrocaudal axis that later will give rise to the distinct regions of the central nervous system: the forebrain, midbrain, hindbrain and spinal cord. Fate-labelling of cells in the caudal part of the neural plate in chick embryos has shown that these cells will contribute to the caudal nervous system [7] even though these same cells are specified as rostral neural cells at stage 3 (early/mid gastrula stages). Just a few hours later at mid/late gastrula stages, cells in the neural plate have acquired specific rostrocaudal identities (Figure 1). Hence, specification of caudal neural character occurs at gastrulation stages, by signals emanating from the neighbouring tissues [7, 8]. At gastrula stages, Wnts and FGFs are expressed in the caudal part of chick, mouse, frog and fish embryos. They are therefore candidates for the caudalizing activity since their spatio-temporal expression patterns fit with a role in neural caudal specification. A previous study from our lab has shown that graded Wnt and

14 Introduction permissive FGF signalling pattern the early neural plate of the chick into the caudal forebrain, the midbrain and the rostral hindbrain [9]. Moreover, graded Wnt signalling is also involved in the rostrocaudal patterning of Xenopus and zebrafish neural tissue [10-12]. Another molecule implicated in the early rostrocaudal patterning is RA. In the chick, the RA-producing enzyme Raldh2 is expressed from stage 4 onwards in the paraxial mesoderm. Moreover, RA has an important in role in the rhombomeric patterning of the hindbrain [13], but its role in the early rostrocaudal subdivision is less well understood.

Figure 1: cells acquire rostrocaudal positional character at stage 4 At blastula and early gastrula stages (stage 1-3) all cells in the neural plate are specified as cells of forebrain (FB) character. By mid-gastrulation stage (stage 4) rostrocaudal positional patterning emerges and cells in the neural plate are specified as cells of FB, midbrain (MB), hindbrain (HB) and spinal cord (SC) character. (modified after Muhr 1999).

Since graded Wnt and permissive FGF signals pattern the neural plate until the level of the rostral hindbrain the questions remains how the more caudal part of the neuraxis is established. Muhr et al (1999) showed that by stage 4 the caudal most part of the chick neural plate is specified to express Hoxb8, a marker for spinal cord cells when expressed in neural tissue. But no information is given on the rostrocaudal identity of these spinal cord cells and the whereabouts of caudal hindbrain cells. Moreover, it remains unclear, how extrinsic signalling molecules, progenitor cell specification in the neural plate and the generation of different neurons along the rostrocaudal axis are connected.

15 Introduction

Hox genes and motor neuron subtype In the neural tube, interneuron subclasses are generally generated along the entire extent of the hindbrain and spinal cord, whereas motor neurons exhibit distinct features at the various levels of the caudal neuraxis. One fundamental distinction in motor neuron subtype at hindbrain and spinal cord levels is the generation of two subtypes of motor neurons that display either a dorsal exiting (dMNs) or a ventral exiting (vMNs) axon trajectory [14, 15]. Most spinal motor neurons, as well as the hypoglossal and abducens MNs of the caudal hindbrain, belong to the vMN subtype, wheras dMNs are found mainly in the hindbrain and at cervical spinal cord levels [15-17]. The further diversification in motor neuron subtype identity emerges from further subdivision of these two most basic subclasses (Figure 2, [18]). dMN NE visceral MN vMN somatic MN motor column motor pool

Figure 2: motor neuron subtype development Neuroepithelial cells (NE) generate dMNs or vMNs. Based on their target, vMNs are divided into visceral and somatic MNs. Somatic MNs innervate skeletal muscles, whereas visceral MNs innervate branchial and smooth muscles and autonomic neurons. Somatic MNs are grouped into columns that consist of several motor pools. MNs in the same column project axons to major common targets, whereas MNs located in the same pool, project axons towards a single muscle group (modified after: [19]).

The vertebrate body plan is patterned in the area approximately located caudal to the midbrain-hindbrain boundary by the expression of Hox genes [20]. In chick, the four chromosomal Hox clusters contain 39 genes and members from each cluster are expressed in either nested or overlapping domains in most tissues [210]. In the neural tube, distinct rhombomeres in the hindbrain are delineated by the nested expression of 3´Hox genes [21, 22] whereas 5´Hox genes are expressed in the spinal cord [23-27]. Positional differences of rostrocaudal identity of motor neurons correlates, at the molecular level, with the expression of different members of Hox genes along the rostrocaudal axis [28-30]. In addition, several lines of evidence indicate that Hox genes are determinants of motor neuron subtype identity in the caudal neural tube, both at hindbrain [16, 31-33] and spinal cord levels [23-25, 30, 34-39].

16 Introduction

Cdx genes as potential mediators of Hox induction Cdx genes, the vertebrate homologues of the Drosophila caudal gene [40] have been implicated as intermediators of external signalling-mediated Hox gene induction. In mouse Cdx1 null mutant embryos, Hox gene boundaries are shifted in the mesoderm [41]. Moreover, in Xenopus and chick, FGF- mediated shifts in Hox gene expression are preceded by changes in Cdx gene expression [27, 42]. In addition, RAREs and TCF/Lef binding sites have been found in the promoter region of the mouse Cdx1 gene [43, 44].

Ventral patterning of the neural tube Dorsoventral patterning of the neural tube plays an important role in generating the functional organization of the mature CNS. Together with rostrocaudal positional information a coordinate grid is established in the neural tissue. The readout of this grid is the generation of different neuronal progenitors along both axes which later will give rise to the vast array of neurons in the CNS. The mechanisms responsible for the dorsoventral patterning of the neuraxis are best described for the spinal cord, since the structural organization in the spinal cord is simpler compared to the brain. Less is known about the mechanisms which govern the dorsoventral patterning of the hindbrain and midbrain, but all over they are not considered to differ from those in the spinal cord. The mechanisms patterning the forebrain however are distinct from those acting at more caudal levels [45]. Neurons involved in the processing of the sensory information reside in the dorsal part of the spinal cord. Motor neurons and visceral neurons involved in motor output and proprioreception are located in the ventral half of the spinal cord [18, 21]. Several interneuron populations connect these two neuronal populations. During development, neurons arise from distinct progenitor domains along the dorsoventral axis, each characterized by a unique combination of transcription factors. Shh is the main patterning molecule involved in ventral specification. Shh secreted by the axial mesoderm induces the floor plate in the ventral most region of the spinal cord. The floor plate itself then starts to act as a signalling centre. Initially, Shh represses the expression of class I transcription factors in the ventral spinal cord which then allows for the induction of class II transcription factors at distinct thresholds of Shh [46].

17 Introduction

Repressive feedback-loops between class I and class II genes act to refine and maintain the progenitor domains after the initial induction. The sharp domain boundaries are thus created. However, only V3, MN and V2 neurons are lost in the Shh -/- mice, whereas V1 and V0 interneurons are still generated [47], implicating the involvement of other factors in their generation. Both FGF and RA signalling has been implicated in this process [48-50].

Development of the PNS

Neural plate border specification In chick, cells acquire neural plate border character at the late blastula stage in a BMP- and Wnt-dependent manner [51]. Early Wnt and BMP signals together block neural and induce epidermal cells [4, 52-55]. Wnt signals play no instructive role in the context of border induction but induce BMP gene expression in neural cells, thereby avoiding the simultaneous exposure of prospective border cell to Wnt and BMP signals. The subsequently later BMP signals then specify border cells [51]. At later stages Wnt signals distinguish between cells of rostral and cells of caudal border character [53, 56].

Neural crest and placodes Both the cranial placodes and the neural crest are genuine vertebrate innovations. The neural crest as well as the placodes develop as specialized areas within the ectoderm and share some common features. First both can give rise to a variety of non-epidermal cell types, including neurons. Second, during development shape changes occur, which enable these cell types to either migrate or to undergo morphogenetic movements. The neural crest is a transient population of migratory cells, which will give rise to most of the mesenchymal structures of the head but also to some of the neural cells in the cranial ganglia, and to peripheral neurons, glia and melanocytes. Wnt, BMP and FGF have been suggested to play a role in the induction of the neural crest but the exact mechanism is still unresolved. One current model suggests that BMPs and Wnts act sequentially to induce the neural crest. First BMPs are thought to induce a region of competence in the neural plate border and these cells then respond to Wnt signalling [56-59], whereas an other model claims that the Wnt and FGF pathway act in parallel, each

18 Introduction activating genes required for neural crest formation [60, 61]. A recent study however shows that BMPs have the capability to induce neural crest at late gastrula and neurulation stages in chick and Wnt only provides a positional context for this induction [53]. Cranial placodes develop from cranial ectoderm and constitute a quite heterogeneous group of structures which form as thickenings in the cranial ectoderm mostly after neural tube closure. Some of the placodes will later invaginate and/or cells originating from the placodes will migrate away [62]. The cranial placodes include in approximately rostrocaudal order: the adenohypophyseal, olfactory, lens, trigeminal and profundal placode, a series of epibranchial and hypobranchial placodes, the otic placode and a series of lateral line organ placodes. Not all of these placodes are present in all the vertebrate taxa; lateral line organ placodes for example are lost in amniotes [63]. In the rostral border, the olfactory and the lens placode in chick are specified at gastrula stages by BMP signalling. The difference in the specification of an olfactory or a lens placodal fate is that lens placodal cells have to be exposed to BMP signalling for a longer time than olfactory placodal cells [54].

The olfactory system

The sense of smell is very common in animals and is used for food search and food evaluation, localization of predators/prey and for the chemical communication between conspecific individuals, mainly in regard to mating behaviour. A large array of volatile chemical stimuli is detected via the olfactory system in terrestrial animals. The human nose for example can detect up to 400 000 different odors [64]. The peripheral olfactory system in vertebrates consists of three subsystems: (1) the main olfactory system, (2) the accessory olfactory system and (3) the septal organ. Whereas the main olfactory system is present in all terrestrial vertebrates, the accessory olfactory system is lacking in salamanders, crocodiles, birds and old world monkeys, apes and humans [65, 66]. In the main olfactory system the odorants enter the nose via the airstream and bind to the olfactory sensory neurons (OSN) in the olfactory epithelium. The OSNs project their axons in an ordered fashion to the olfactory bulb where the neuronal input from the OSNs is processed. The resulting

19 Introduction information is send to the cortical areas involved in decoding the neuronal activation patterns, into the impression of a smell. One of the fascinating features of the olfactory system is the capability of OSNs to regenerate throughout life [67, 68]. In mouse the average life span of an OSN is 90 days [69] but they may survive up to 12 months [70]. Neurogenesis in the sensory epithelium proceeds in an ordered fashion and four distinct cell populations have been implicated by both in vivo and in vitro studies: neuronal stem cells, which express among others Sox2 and Hes genes, the transient amplifying cells (TAP), that express Mash1/Cash1 and are generated through uneven division of the stem cells, the immediate neural precursor cells (INP), the progeny of the TAPs, which express Ngn and generate in some divisions the OSNs. OSNs can be characterized by the expression of Gap43, HuC/D and Lhx2 (Figure 3)[71-76].

SLPC TAP INP OSNi OSNm Figure 3: neurogenesis in the olfactory sensory lineage Stem-like progenitor cells (SLPC) divide asymmetrically to generate the transient amplifying cells (TAP) which divide and finally give rise to the immediate neural precursor cells (INP) that in their turn divide and differentiate into the immature olfactory sensory neurons (OSNi) that further develop into the mature OSN (OSNm) (after Calof 2002).

Several studies have addressed the roles for these intracellular factors in OSN generation: Hes genes are direct targets of Notch signalling [77, 78] and previous studies in the mouse indicate a dual function for Hes genes in olfactory placodal development. First, from around E10.5 in mouse Hes1 acts as a pre-patterning gene, which defines the neurogenic region in the placode and later, Hes genes negatively regulate neurogenesis in the sensory epithelial domain of the placode [79]. Mice mutant for Hes1 have an enlarged neurogenic region in the olfactory placode and later on also an increased clustering of Mash1-positive cells, suggesting a role in both limiting the neurogenic area and regulating the density of Mash1-positive precursors. Mice double mutant for Hes1 and Hes5 show a strong increase in neuronal density, especially the Ngn-positive INPs are increased, suggesting that Hes1 and Hes5 converge on INP regulation [79]. Mice mutant for Mash1 (the mouse homologue of chick Cash1) fail to generate olfactory

20 Introduction progenitors and thereby also olfactory sensory neurons, among other neurogenic defects [80, 81]. In Ngn1 null mutant mice differentiation of neural progenitors in the olfactory epithelium is blocked [81], whereas Lhx2- deficient mice display a relatively normal organization of the olfactory epithelium, but show a perturbed generation of olfactory sensory neurons [76, 82].

The cells implied in the regeneration of OSNs are not the only cells to be found in the olfactory sensory epithelium. Apart from OSNs, it is composed of glial-like sustentacular cells, which have a supporting function and of Bowman gland cells, which produce the mucus.

Development and neurogenesis During embryonic development the structures of the peripheral olfactory system arise from the olfactory placode. In the chick the peripheral olfactory system comprises the olfactory sensory epithelium, the olfactory nerve and the olfactory respiratory epithelium. As mentioned before, the olfactory placode is induced at gastrula stages by BMP signalling. By stage 14 in chick the placodal ectoderm, located at the anterior side of the head, starts to thicken. Later the placode invaginates into the head mesenchyme, thereby forming the olfactory pit from which the structures of the peripheral olfactory system start to form. The olfactory sensory epithelium differentiates from placodal tissue located deep in the invaginating pit, whereas the olfactory respiratory epithelium forms at the rim of the pit.

Molecular regulation Several signalling molecules are expressed in and around the olfactory placode/pit during development. Most of them have been implicated in the specification of the olfactory sensory neurons, especially in the mature olfactory epithelium. Less is known about the molecular mechanisms which regulate the initial subdivision of the olfactory placode into the sensory and the respiratory epithelium and the early neurogenesis in the olfactory placode.

In mouse, early addition of FGF2 to cultures of sensory epithelial cells (mostly late OE, mature) stimulates TAP division and the maintenance of stem cells, thereby affecting neurogenesis [83]. However, FGF2 mutant mice

21 Introduction show only very slight defects in developmental neurogenesis [84, 85]. FGF8 is expressed in the rim of the olfactory pit (mouse E10.5dpc) in the domain where cells divide [86]. Mice with a targeted deletion of anterior FGF8 (FGF8 flox/flox//Foxg1Cre [87]), fail to generate the sensory epithelium proper, the vomeronasal organ and the entire nasal cavity [86].

Several BMPs are expressed in the sensory epithelium [88], but BMP4 is the prevalent ligand in mice during mid/late stages of development [89]. Moreover, GDF11, another Transforming Growth Factor β (TGFβ) family member is also expressed in mouse sensory epithelium from E12.5 to adulthood [90]. In vitro studies using neuronal colony forming assays of sensory epithelium dissected from E14.5-16.5 mice showed both inhibitory and stimulatory effects of TGFβ signalling on neurogenesis [89-91]. In addition, Noggin, a BMP inhibitor, strongly inhibited the formation of neuronal colonies in the in vitro system [89]. Moreover, cell proliferation is increased in the sensory epithelium of mice mutant for GDF11 and decreased in mice lacking Follistatin, an antagonist of GDF11 [90]. In summary, TGFβ signalling has been implicated in proliferation and differentiation of cells in the sensory epithelium of the nose.

The Notch signalling pathway has been shown to regulate the status of cells in a variety of systems [92-94]. The best described role of Notch signalling during the development of the nervous system is the process of lateral inhibition. Hereby, Notch and its ligands are expressed in individual cells in a salt-and-pepper-like pattern. Cells containing high ligand levels differentiate and stimulate neighbouring cells. Activation of Notch signalling leads to the expression of Hes genes and to continuous proliferation [77, 95, 96]. Other modes of pathway function have been described. In the spinal cord stem zone, uniform and high expression of Delta1 is suggested to result in uniform Notch signalling between cells [97]. In epidermal stem cells, however, high level Delta has been associated with signalling only at the edges of the cell group [98]. It is suggested that cells expressing high levels of Notch ligands form homodimers, which act as dominant-negative inhibitors of Notch signalling [99-101]. In the mouse sensory epithelium the expression pattern of several Notch pathway components has been described, mostly at later stages of sensory epithelium development [102-104]. Differential expression of Notch1/3

22 Introduction compared to Notch2 has been implicated in the segregation of neuronal versus glial lineages in the sensory epithelium respectively [104]. At postnatal stages Notch2 is required to maintain sustentacular cell function in the adult sensory epithelium [105]. Furthermore, mice mutant for Hes1 or double mutant for Hes1/Hes5, both known target genes of Notch signalling [96], display enlarged numbers of TAPs and INPs respectively. Nevertheless, Notch function in the developing sensory epithelium has not directly been studied.

Why study developmental processes in the chick?

To understand the development of organisms in general it is not sufficient to study the developmental processes in just one species. In the comparison of data stemming from different model organisms, the general principles of developmental biology emerge. Moreover, different model organisms are especially suited to address specific question. In the chick, large embryo size and the flat morphology of the early embryonic stages together with comparatively easy obtainable embryos make it a good model for both in vitro and in vivo studies. From an evolutionary perspective, birds have an intermediate position: cladistically closely related to reptiles they are endothermic, resembling in physiology more to mammals. Thus, studying the developmental biology of chick may give insights into questions that concern evolutionary versus physiological constraints [65; 66].

23 Aims

Aims

The aims of this thesis are to understand when and how cells in the caudal neural plate are specified and how patterning and neurogenesis in the olfactory placode are controlled.

Specific aims are:

• To determine the character of caudal neural plate cells at gastrula stages.

• To determine when cells in the caudal neural plate acquire caudal hindbrain and rostral spinal cord character.

• To determine the signalling molecules involved in the specification of caudal hindbrain and rostral and caudal spinal cord character.

• To define early markers for the respiratory epithelium and for the sensory epithelial cell types in chick.

• To determine how the olfactory placode is patterned into respiratory and sensory epithelial domains.

• To define signals involved in early neurogenesis in the olfactory placode.

24 Results

Results

Project I: Rostrocaudal patterning of the caudal neuraxis

Cells of caudal spinal cord character are specified at the late gastrula stage Earlier studies in our lab have shown that cells of rostral hindbrain and spinal cord character have both been specified at gastrula stages (stage 4)[7]. To further investigate the axial identity of the spinal cord cells and to analyze whether cells in the caudal neural plate also are specified as cells of caudal hindbrain character, we examined the expression of region-specific Hox genes in caudal neural plate explants. By stage 17 neural progenitor cells in rhombomere 7 and 8 of the caudal hindbrain are characterized by the expression of Hoxb4, cells in the rostral spinal cord (somites 5-19) by the expression of Hoxb4 together with Hoxb8 and cells in the caudal spinal cord by the expression of Hoxb4, Hoxb8 and Hoxc9 together (somites 19-tail). Stage 4 caudal neural plate explants (4C), grown in vitro for 48hrs, generated cells of rostral hindbrain character and of caudal spinal cord character, in distinct domains of the explant. Only a few cells of rostral spinal cord character were generated but no cells of caudal hindbrain character were present. Thus, at mid-gastrula stage, cells in the caudal neural plate are mainly specified as cells of rostral hindbrain or caudal spinal cord character.

Wnt and FGF signalling is required to specify spinal cord character What are the extrinsic signalling cues involved in the specification of spinal cord cells? Earlier studies in our lab have shown that convergent Wnt and FGF signalling is required and sufficient for the induction of cells of rostral hindbrain character at gastrula stages. To investigate if Wnt and FGF signalling play a role in the induction of cells of spinal cord character, we cultured 4C explants in the presence of Frz8-CRD-IgG, an inhibitor of Wnt signalling or in the presence of SU5402, an inhibitor of FGF signalling. In both cases, the generation of cells of caudal and rostral spinal cord character, as well as the generation of cells of rostral hindbrain character was blocked and instead cells of rostral forebrain character were generated. Thus, both Wnt and FGF signalling are required for the generation of spinal cord character.

25 Results

Graded Wnt and FGF signalling distinguishes between hindbrain and spinal cord character If both rostral hindbrain and spinal cord cells are dependent on Wnt and FGF signalling for their initial specification, how is the difference between these domains established? To investigate this further, we DiI-marked 4C explants to keep track of their initial rostrocaudal orientation in the embryo during the culture period. The part of the explant located caudally and close to the primitive streak generated cells of caudal spinal cord character, whereas the rostral-medial part of the explant generated cells of rostral hindbrain character. The distribution of several Wnt and FGF ligands in gastrula stage embryos suggested that the difference between rostral hindbrain and caudal spinal cord character might be imposed by differences in the level/duration of exposure to Wnt and/or FGF signals. To investigate this issue, we exposed 4C explants to Wnt and FGF alone or in combination. Neither Wnt nor FGF signalling alone had any effect on the specification state of 4C explants. Wnt and FGF signalling in combination however, suppressed the generation of cells of rostral hindbrain character and most cells acquired caudal spinal cord character. This suggested, together with the fact that cells of caudal spinal cord character are located at lateral-caudal positions in the neural plate, that Wnt and FGF signals from the primitive streak and the caudal neural plate promote the specification of cells of caudal spinal cord character.

Cells of caudal hindbrain character are specified at early neurulation stages But when are cells of caudal hindbrain and rostral spinal cord character specified? To address this question we isolated caudal neural plate explants from stages 5-8 embryos. Prior to stage 8, the caudal neural plate is either specified as rostral or caudal spinal cord, but no cells of caudal hindbrain character can be isolated. By stage 8 however, explants isolated rostral to the node generated cells of caudal hindbrain character, explants isolated at the level of the node generated cells of rostral spinal cord character and explants located caudal to the node generated cells of caudal spinal cord character. Hence, by stage 8, cells in the caudal neural plate are specified, in distinct domains, as cells of rostral and caudal hindbrain and of rostral and caudal spinal cord character.

26 Results

RA imparts caudal character in presumptive hindbrain and rostral character in presumptive spinal cord cells What are the extrinsic signalling cues establishing the caudal hindbrain and the rostral spinal cord? Since Wnt signalling contributes to the distinction between cells of rostral hindbrain and cells of caudal spinal cord character at gastrula stages, we first examined whether exposure to Wnt was required for the further distinction of cells of prospective rostral and caudal hindbrain and spinal cord character. Blocking Wnt signalling in vitro in stage 8 caudal neural plate explant did not influence the rostrocaudal character in these explants, indicating that prolonged Wnt signalling is not required for the generation of these subdomains. Thus, by stage 8 rostral hindbrain and caudal spinal cord subdomains have become independent of further Wnt signalling. Several lines of evidence [106-109] indicate a role for RA signalling in the differentiation of cells of caudal hindbrain character. Expression of the RA synthesizing enzyme Raldh2 in the paraxial mesoderm adjacent to the caudal neural plate starts at stage 4+ in the chick. Between stages 5 to 8 Raldh2 expression in the paraxial mesoderm increases. Since RA is synthesised at the right time and place, we asked whether RA signalling is involved in the specification of caudal hindbrain and rostral spinal cord cells by acting on cells that already have acquired an initial caudal identity. To test this, we exposed 4C explants to RA. Under these conditions the generation of rostral hindbrain and of caudal spinal cord cells was blocked and instead cells of caudal hindbrain and rostral spinal cord character were generated. Caudal hindbrain cells emerged in the rostral domain of the explant, which would otherwise generate rostral hindbrain cells, and rostral spinal cord cells emerged in the caudal domain of the explant normally generating caudal spinal cord cells. Thus, these results further strengthen the notion that RA can induce caudal hindbrain character in rostral hindbrain cells [16, 108-110] and provide evidence that rostral spinal cord cells are induced in cells being previously exposed to the caudalizing signals Wnt and FGF.

FGF signalling distinguishes between cells of caudal hindbrain and rostral spinal cord character Both caudal hindbrain and rostral spinal cord cells depend on RA signalling for their induction at approximately the same time in development. How then, is the difference between these two regions of the neuraxis established?

27 Results

Several studies have shown that FGF signalling can promote the expression of Hox genes characteristic for the spinal cord [10, 24, 27, 42] and that FGFs can oppose RA signalling during patterning of Hox gene expression in the neural tube [24, 27]. Moreover, at stage 8, FGF8 is expressed in the node and in the primitive streak, close to the cells specified as cells of rostral spinal cord character. To investigate whether FGF signalling plays a role in the discrimination between cells of caudal hindbrain and cells of rostral spinal cord character, we exposed stage 4C explants to RA and FGF in combination. No cells of caudal hindbrain character were generated; instead all the cells were of rostral spinal cord character. Thus, FGF signalling promotes the generation of rostral spinal cord cells at the expense of caudal hindbrain cells.

Progenitor cells in the hindbrain and spinal cord have acquired sufficient positional information to differentiate into dMNs and vMNs The early patterning of progenitor cells along the rostrocaudal and dorsoventral axes of the neural tube is a prerequisite for the later generation of post-mitotic neurons. Within the ventral hindbrain and spinal cord various motor neuron subtypes differentiate at different rostrocaudal levels from progenitor cell populations in response to Shh [17, 18, 21, 111]. Therefore, we tested whether prospective hindbrain and spinal cord cells have acquired sufficient positional information at gastrula stages to differentiate into MNs of the correct rostrocaudal subtype in response to Shh-N, the N-terminal domain of the Shh protein, which has been shown to exhibit MN inducing activity [112, 113]. 4C explants, which normally display a dorsal phenotype, were exposed to Shh-N after 28hrs of culture for an additional 38hrs, Under these conditions dMNs and vMNs were generated in different regions of the explant and most of the vMNs expressed Hoxc9 protein, indicative of a caudal (thoracic) character. Hence, by gastrula stages, cells in the caudal neural plate have acquired sufficient positional information to permit them to differentiate into the correct MN subtypes in responses to Shh activity.

Early Wnt signalling is required for the generation of MN subtype Next we wanted to test if Wnt signalling in prospective hindbrain and spinal cord cells is required for the generation of dMNs and vMNs. To explore this, we exposed stage 4C explants to Frz8CRD-IgG for 28h and Shh-N for an additional 38h. No dMNs and vMNs characteristic of the hindbrain and

28 Results spinal cord were generated under these culture conditions and instead the cells acquired the characteristics of ventral forebrain MNs. Thus, Wnt signalling is required for the generation of dMNs and vMNs.

Combinatorial Wnt, RA and FGF signals are sufficient to induce hindbrain and spinal cord character from neural cells To further test the sufficiency of the signals implied in hindbrain and spinal cord patterning, we investigated if Wnt, RA and FGF signals alone or in combination are sufficient to reconstruct expression profiles characteristic of hindbrain and spinal cord cells in rostral forebrain cells, which have not been exposed to any caudalizing signal in ovo. Wnt, FGF and RA alone or FGF and RA in combination did not illicit any change in rostral identity in rostral forebrain cells in accordance with previous studies [7, 9]. Wnt and FGF in combination induced a Hox gene profile characteristic for cells of caudal spinal cord character, whereas Wnt and RA in combination induced a Hox gene profile characteristic for cells of caudal hindbrain character and a combination of Wnt, FGF and RA induced a Hox gene profile characteristic for cells of rostral spinal cord character. Thus, Wnt signalling in combination with FGF and/or RA signalling is sufficient to reconstruct Hox gene profiles characteristic of cells of caudal hindbrain and rostral and caudal spinal cord character form rostral forebrain cells.

Acquisition of rostrocaudal specific Hox gene profiles determines the emergence of MN subtypes Hox gene combinations are informative markers of rostrocaudal positional identity of progenitor cells in the hindbrain and spinal cord [26]. Moreover, Hox proteins have been suggested to be determinants of MN subtype [17, 26, 114]. Therefore we addressed whether Hox gene expression profiles expressed by both stage 8 neural explants and induced by Wnt, FGF and/or RA in isolated rostral forebrain cells, predict the later generation of dMNs and vMNs. In both cases, exposure to Shh-N leads to the generation of dMNs and vMNs of the regional identity predicted by the Hox gene profile. Thus, Wnt together with FGF and/or RA is sufficient to impart Hox gene profiles that prefigure the emergence of position specific MN subtypes.

29 Results

Project II: Patterning and neurogenesis in the olfactory placode

Hes5 is the earliest neurogenic marker expressed in the olfactory placodal region in chick In chick there are three members of the Hes5 family, Hes5-1, Hes5-2 and Hes5-3 [115]. Since the expression patterns of the three genes during the development of the olfactory placodal region are rather similar, we focused on the pattern of Hes5-1 and hereafter call it Hes5. Interestingly, we found that Hes5 expression can be detected in the olfactory region already at stage 10, indicating earlier onset of expression than previously reported in mouse [79]. Moreover, the uniform expression of Hes5 is indicative of a role in pre- patterning the placodal ectoderm.

Expression of neurogenesis markers in the olfactory region To better understand the early events governing neurogenesis in the early olfactory area, we analyzed the expression of Hes5 (stem-like cells), Cash1 (TAPs), Ngn1 (INPs) and Gap43, Lhx2 and HuC/D which are all expressed by OSNs [73, 116]. Expression of Cash1, Ngn1, Gap43 and HuC/D can be detected from stage 14, when the olfactory placode becomes visible as an ectodermal thickening by the side of the head, whereas Lhx2 can first be detected by stage 17. At these early stages the majority of Gap43-, HuC/D- and Lhx2-positive cells are migrating away from the olfactory placode towards the brain and only a few cells positive for these markers are detected in the placode. By stage 22 the olfactory placode has started to invaginate and all of the analyzed markers (Hes5, Cash1, Ngn1, Gap43, Lhx2 and HuC/D) are expressed in the olfactory pit, but in distinct anterior-to-posterior and inferior-to-superior patterns. By stage 34 (~E8) first signs of stratification can be observed in the sensory epithelium. Cells expressing HuC/D, Lhx2 and Gap43 are located in basal positions in the sensory epithelium, whereas Hes5 expressing cells are located more apically.

Msx1/2 and Id3 are valid markers for respiratory epithelial character During development both the respiratory and the sensory epithelium of the nose arise from the olfactory placode, but how the emergence of these two distinct epitheliums is regulated during development remains elusive. Several transcription factors expressed in the sensory epithelium have been

30 Results described, but early markers for the respiratory epithelium are poorly defined. To gain a better understanding of the subdivision of the olfactory pit and the location from which respiratory epithelial cells emerge in vivo, we performed homotopic grafting experiments at E3.5. Small explants of the medial-posterior rim were dissected out from E3.5 quail embryos and grafted in ovo into a chick E3.5 embryo. Chick embryos were allowed to develop to E5, when the sensory and respiratory epithelium can be morphologically distinguished [117]. The site of integration of the crafted quail cells was determined by immunohistochemistry with the quail specific antibody QPCN. In the majority of the embryos, the quail derived cells were observed in the respiratory epithelium, providing evidence that precursor cells of the respiratory epithelium are located at the rim of the olfactory pit at E3.5. Next we analyzed the expression of several candidate molecules and among others, the expression patterns of the transcription factors Msx1/2 and Id3 at stages E3.5 and E5. At E3.5 Msx1/2 and Id3 are expressed in the medial- posterior rim and in the posterior part of the olfactory pit and at E5 expression is restricted to the morphologically thinner prospective respiratory epithelial region. Thus, we concluded that Msx1/2 and Id3 are expressed in the respiratory region of the developing nose and are valid molecular markers for cells of respiratory epithelial character.

Cells in the olfactory placodal area are specified as cells of both sensory epithelial and respiratory epithelial character By stage 14 the olfactory placodes become visible as ectodermal thickenings on both sides of the head. Ectodermal explants isolated from the presumptive placodal region of stage 14 chick embryos (14OP explants) and cultured in vitro for ~35h (approximately corresponding to a stage 22 chick embryo) generated Hes5+, Ngn1+, HuC/D+ and Lhx2+ cells, characteristic of the sensory epithelium and Id3+, Msx1/2+ cells characteristic of the respiratory epithelium in distinct parts of the explants. Thus, at stage 14, progenitor cells in the olfactory placodal region are specified as cells of sensory epithelial and respiratory epithelial character.

BMP signalling promotes the generation of respiratory epithelial character At stage 14 BMP4 is expressed in the proximity of the posterior olfactory placodal area and Id and Msx genes are target genes of BMP signalling [118- 120]. Therefore, we asked whether BMP signalling could play a role in

31 Results sensory versus respiratory patterning of the olfactory placode. To address this issue we cultured 14OP explants in the presence of Noggin, an antagonist of BMP signalling [121]. In the presence of Noggin, the generation of Id3+ and Msx1/2+ respiratory epithelial cells was blocked. Moreover, the expression of Ngn1, Lhx2 and HuC/D was downregulated or blocked in explants exposed to Noggin and most cells expressed Hes5, characteristic of neuronal progenitor cells in the sensory epithelium. Thus, at stage 14 BMP signalling is required for both the generation of respiratory epithelial cells and the differentiation of progenitor cells in the sensory epithelium. To investigate whether BMP signalling is sufficient to promote the generation of cells of respiratory epithelial character, we exposed 14OP explants to BMP4 (3nM) for ~35hours. In the presence of additional BMP signals, the generation of Hes5+ and Ngn1+ cells was blocked and the expression of HuC/D+ and Lhx2+ cells was greatly reduced and instead most cells expressed Msx1/2 and Id3, a marker profile characteristic of cells of respiratory epithelial character. Thus, at stage 14, additional BMP signalling suppresses the generation of sensory epithelial cells and promotes the induction of respiratory epithelial cells.

BMP signals promote the generation of respiratory epithelial cells in vivo To further examine the role of BMP signals in the patterning of the olfactory placode, the status of BMP signalling was manipulated in vivo. Stage 12 chick embryos were electroporated with 1//a control GFP-vector 2//a Noggin-containing vector [122] or 3//a vector containing a constitutively active Alk6 receptor (caAlk6, [123]) and grown in ovo to ~stage 22. Embryos electroporated with the control GFP-vector displayed normal phenotypes. In Noggin-electroporated embryos, the expression of Msx1/2 and Id3 was reduced and Hes5 was upregulated in the same area in the olfactory pit on the electroporated side of the embryo. In caAlk6- eletroporated embryos, Hes5 expression was reduced and expression of Msx1/2 and of Id3 was upregulated. Moreover, expression of HuC/D was reduced in the electroporated region. These results further strengthen the notion that BMP signalling is required for the generation of cells of respiratory epithelial character and that sensory epithelial character is inhibited by BMP signalling.

32 Results

A switch in the competence of sensory epithelial cells to respond to BMP signals Next we analyzed whether the ability of BMP signalling to induce respiratory character in cells of sensory epithelial character persist at later stages of olfactory placode development. The BMP activator construct caAlk6 was electroporated into stage 17-21 embryos. In stage 17-20 embryos electroporated with the caAlk6 construct, Msx1/2 and Id3 expression was up-regulated (18/20), but expression of Hes5 remained unaffected (20/20). In embryos electroporated with caAlk6 slightly later, at stage 21, no change in the expression of Msx1/2, Id3, Hes5 and HuC/D was detected (3/5). Thus, the ability of BMP signalling to induce respiratory epithelial character in sensory epithelial cells declines around stage 21.

FGF signalling is required for cells of sensory epithelial character FGF signalling has been implicated in the development of the olfactory placode [86] and in the regulation of neurogenesis within the sensory epithelium [83]. To determine whether FGF signalling plays a role in the specification of sensory versus respiratoy epithelial character, we exposed 14OP explants to SU5402 (5-10nM), an inhibitor of FGF signalling and cultured them for ~35h. Under these conditions, the generation of Hes5+, Ngn1+, HuC/D+ and Lhx2+ cells of sensory epithelial character was blocked and most cells expressed Msx1/2 and Id3, characteristic of respiratory epithelial cells. Thus, FGF signalling is required for the generation of sensory epithelial cells and in the absence of FGF signalling, sensory epithelial cells acquire respiratory epithelial character. Next we asked whether levels of FGF activity affect the differential generation of sensory and respiratory epithelial cells in the olfactory placode. We cultured 14OP explants in the presence of FGF4 (200ng) or FGF8 (300ng). Under these conditions, no change in the generation of Hes5+, Ngn1+, HuC/D+, Lhx2+ sensory epithelial cells could be observed. Moreover, Msx1/2+ and Id3+ respiratory epithelial cells are only non-significantly decreased, indicating that additional FGF exposure does not affect the differential generation of sensory and respiratory epithelial cells. In addition, no shift in the distribution of cells in the neurogenic lineage was observed; hence we conclude that FGF signalling does not regulate neurogenesis under these conditions

33 Results

Manipulation of FGF signalling in vivo In order to examine the role of FGF signals in the patterning of the olfactory placode in vivo, stage 12 chick embryos were electroporated in ovo with 1//a control GFP-vector 2//a truncated FGF-ReceptorIII-containing vector (FGFRIII-ΔC, [124]), which inhibits FGF signalling in electroporated cells or 3//a vector containing FGF8b [125] and grown in ovo to ~stage 22. Embryos with GFP expression in the olfactory region were selected for further analysis. In agreement with our explant results, activation of the FGF pathway resulted in a mild reduction of Msx1/2 and Id3 expression. Inhibition of FGF activity reduced or blocked the expression of Hes5 and HuC/D and increased the expression of Msx1/2 and Id3 in the electroporated area compared to the non-electroporated control side. In the most severely affected embryos the invagination of the olfactory placode was blocked. Variation in the severity of the phenotype displayed by embryos electroporated with FGFRIII-ΔC, correlated clearly with electroporation efficiency, as the amount of transferred GFP/FGF3c-∆C was consistent with the reduction in levels of Hes5 and HuC/D expression. Thus, FGF signalling in vivo is required for the generation or maintenance of cells of sensory epithelial character and for the formation of the nasal cavity. In summary our results provide evidence that FGF signals are required for the generation of sensory epithelial cells and for proper invagination of the olfactory placode, whereas BMP signals promote respiratory epithelial character

Notch signalling is required to maintain progenitor cells in the olfactory placode Notch signalling has been implicated in several regions of the nervous system, where it acts to maintain neuronal progenitors or to drive glial differentiation [126, 127]. This, together with the expression patterns of several members of the Notch signalling pathway during early stages of placodal development ([102] and own data) prompted us to analyze the role of Notch signalling in the generation of OSNs. We exposed stage 14OP explants to DAPT, an inhibitor of Notch signaling. DAPT blocks the γ- secretase enzyme that cleaves the intracellular domain of Notch after ligand binding, which normally translocates into the nucleus and activates target genes [95]. Stage 14OP explants exposed to DAPT for 48h generated many Gap43-positive and HuC/D-positive cells, but no cells expressing Hes5,

34 Results

Cash1 or Ngn1 could be detected. Thus, in the absence of active Notch signalling most cells acquire OSN character.

Hes5-positive cells are primarly affected by perturbed Notch-signalling To explore which cells are primarily affected by the inhibition of Notch signalling, we cultured stage 14OP explants in the presence of DAPT for 7h, 15h and 24h. After 7h most cells express Cash1 and the generation of Hes5+ cells is reduced, by 15h, most cells express Ngn1 and a light expression of Gap43 can be detected and by 24h most cells express Ngn1, Gap43 and HuC/D. Thus, the first cells affected by the absence of Notch signalling are the Hes5-expressing cells. Subsequently Cash1 and Ngn1 expression is lost in olfactory placodal cells and instead the cells acquire characteristics of OSNs. Thus, Notch signalling is required to maintain Hes5-expressing cells in the olfactory placodal area. In the absence of Notch signalling Hes5 progenitor cells enter the differentiation pathway prematurely.

In ovo manipulation of Notch signalling Next we tested the role of Notch signals in intact embryos. We electroporated the olfactory placodal region of stage 14 embryos with 1//a GFP-control vector 2//a dominant-negative mastermind-like (MAMLI) containing vector, to inactivate Notch signalling [128] or 3//a constitutively- active Notch receptor, to activate the pathway [129], and cultured the embryos in ovo to ~stage 22. Control embryos displayed no defects. In embryos electroporated with the dnMAMLI construct Ngn1, Gap43 and HuC/D expression was slightly increased. Most of the Gap43/HuC/D- positive electroporated neurons were detected in the migratory mass. Embryos electroporated with the constitutively-active Notch receptor generated a decrease in Ngn1, Gap43 and HuC/D expressing cells in the forming olfactory pit. Moreover, the number of Gap43/HuC/D-positive cells is reduced in the migratory mass. Quantification of all HuC/D-GFP-double positive cells in the migratory mass of control electroporated, dnMAMLI electroporated and constitutively-active Notch receptor electroporated embryos, revealed that HuC/D-positive cells were increased in dnMAMLI electroporated embryos and decreased in constitutively-active Notch electroporated embryos. Taken together, our results indicate, that a decrease in Notch signalling in vivo leads to an increase of neuron differentiation,

35 Results whereas an increase in Notch signalling leads to a decrease in neuron differentiation.

BMP signalling affects the expression of Notch1 and Delta1 Next, we wanted to elucidate if BMP signalling affects Notch pathway members in olfactory placodal cells. Since Notch1 and Delta1 is the receptor-ligand pair prominently expressed in the placode already at early stages, we focused on these two. Stage 14OP explants cultured for 24h express Notch1 and Delta1 in a salt-and-pepper pattern and Hes5 in some cells. In stage 14OP explants cultured together with BMP4 (5nM), the expression of Notch1, Delta1 and Hes5 is blocked. Explants cultured in the presence of Noggin (200µl) showed decreased numbers of Notch1+ cells, and uniform high expression of Delta1 and Hes5 throughout the explant. Thus, the effect of BMP signalling on neuronal differentiation in the olfactory placode is, at least in part mediated by the effect BMP signals excerpt on Notch pathway members.

36 Discussion

Discussion

Project I: Rostrocaudal patterning of the caudal neuraxis

In summary our data proposes the following model of rostrocaudal specification in the caudal neural tube: At gastrula stages prospective caudal neural cells are exposed to Wnt signals derived from the emerging paraxial mesoderm, the neural plate and epiblast cells, and to FGF signals emerging from the primitive streak. Both Wnt and FGF act upon caudal neural plate cells and specify those cells as either cells of rostral hindbrain or of caudal spinal cord character. Slightly later during development, RA produced by Raldh2 in the caudal paraxial mesoderm and newly formed somites will act upon cells that already have acquired an initial caudal identity. RA signalling induces caudal hindbrain identity in cells of rostral hindbrain character and rostral spinal cord character in cells of caudal spinal cord character. In caudal spinal cord cells, RA suppresses the expression of Hoxc9. FGFs expressed by Hensen´s node and in the primitive streak at neurulation stages, promote the generation of rostral spinal cord cells instead of caudal hindbrain cells in the presence of RA signalling. Moreover FGF signals also maintain caudal spinal cord cells, possibly by opposing RA signalling. In addition to this, we provide evidence that the early interaction of Wnt, FGF and RA signalling establishes Hox gene profiles in progenitor cells, which anticipate the later generation of distinct rostrocaudal motor neuron subtypes.

Wnt and FGF signalling are required for caudal neural character Wnt signalling has been implicated in the generation of caudal neural cells in several animal systems. For example, zebrafish embryos mutant for wnt8 lack trunk and tail structures, implicating that Wnt signalling is required for the generation and/or patterning of the mesoderm and the neurectoderm [130]. In Xenopus a direct caudalizing role for Wnt signals in patterning the neurectoderm has been suggested [11]. FGF signalling has also been implicated in the generation of mesodermal and neural cells and a role in specifying caudal neural character by FGF signals has been suggested [131- 134]. Previous results in our lab have shown that cells of caudal forebrain, midbrain and rostral hindbrain character are specified by graded Wnt

37 Discussion signalling, whereby the most caudal cells require the highest levels of Wnt signalling [9]. In line with this, Wnt inhibitors are expressed in the rostral neuroectoderm and head mesenchyme maintaining cells of rostral forebrain character [135, 136]. Low level FGF signalling has been shown to have a permissive function in this context and varying the levels of FGF signalling did not influence the caudal identity of the cells in this study [9]. Cells of spinal cord character are specified at the same developmental stages as cells of caudal forebrain, midbrain and rostral hindbrain character [7]. Our results indicate that at gastrula stages both Wnt and FGF signalling is required for the acquisition of caudal spinal cord character. At gastrula stages several Wnt family members are expressed in the primitive streak, the paraxial mesoderm and the neuroectoderm at levels caudal to the node [136- 138]. Moreover, FGFs are expressed in the primitive streak [24, 139, 140]. The expression pattern of Wnt and FGF genes during gastrula stages indicates that cells in the caudal neural plate are exposed to Wnt and FGF signals at higher levels or for a longer time.

Caudal spinal cord character is specified by graded Wnt and FGF signalling Previous results provide evidence that, in the presence of FGF, graded Wnt signalling specifies progressively more caudal character at gastrula stages [9]. Our results indicate that cells of caudal spinal cord character require higher levels of both Wnt and FGF signals at gastrula stages. Prospective forebrain cells exposed to low level FGF and Wnt generated cells of rostral hindbrain and caudal spinal cord character in distinct domains of the explant (Suppl Figure 1). Increasing the levels of FGF in this context resulted in the generation of cells of caudal spinal cord character in the entire explant, indicating that in the presence of high level Wnt signalling, graded FGF signalling might be sufficient to distinguish between cells of rostral hindbrain and caudal spinal cord character. Nevertheless, in caudal neural plate cells only simultaneous exposure to both Wnt and FGF, leads to the acquisition of caudal spinal cord character. Thus, in the caudal neural plate graded activities of both Wnt and FGF signalling distinguish between rostral hindbrain and caudal spinal cord character.

38 Discussion

Instructive FGF signalling during caudal neural development At the time when caudalization of the neural plate occurs several other patterning events take place in the developing embryo. In the neural plate border, the placodal and neural crest derivates of the peripheral nervous system are specified from neural plate cells [53]. Interestingly, high level Wnt signalling (higher than the amount used in Paper I) leads to a loss of neural character in gastrula stage rostral forebrain cells and BMP signalling specifies gastrula stage neural plate cells as cells of peripheral neural character. Wnt signalling in this context has been shown to up-regulate BMP expression [51]. Since FGF signalling is required for the specification of caudal neural cells, we speculated that in part the permissive role might be to inhibit BMP signals. FGF signalling has been shown to exert inhibitory effects on BMP signalling by phosphorylating the linker region of Smad1 [141]. Exposure of stage 4 rostral forebrain cells to Wnt and the BMP inhibitor Noggin is sufficient to induce spinal cord cells, indicative that FGF signalling is required to inhibit BMP signals [53, 54]. Nevertheless, exposure of rostral forebrain cells to Wnt, Noggin and SU simultaneously blocked the induction of spinal cord (Suppl Figure 2), suggesting that FGF signalling has additional, instructive roles in the induction of caudal neural plate tissue.

RA signalling and caudal hindbrain development At neurulation stages (stage 8) cells of caudal hindbrain character start to be specified, a time point at which strong expression of Raldh2 can be detected in the paraxial mesoderm underlying the caudal neural plate [142-144]. Our results indicate that RA induces caudal hindbrain character in cells with an initial caudal identity. This resembles the phenotype observed in embryos exposed to exogenous RA, which display an expansion of the caudal hindbrain at the expense of the rostral hindbrain [108, 110, 145]. Moreover, embryos with depleted RA signalling show an inverse phenotype, displaying expanded rostral hindbrain territories at the expense of caudal hindbrain territories [108, 109, 145, 146], a defect that can be rescued by administration of RA [109, 147]. CYP26 enzymes are members of the cytochrome oxidase p450 family and catabolize RA [148-150]. Expression of Cyp26 family members in mouse, Xenopus, chick and zebrafish has been reported in the rostral hindbrain [151- 159]. Moreover, loss of Cyp26 in anterior hindbrain territories causes the extension of posterior hindbrain territories associated with a mis-

39 Discussion specification of rostral hindbrain territories [10, 151, 160, 161], further strengthening the notion that caudal hindbrain character is specified by RA signals acting on cells initially specified as cells of rostral hindbrain character by Wnt and FGF signals. Moreover, since caudal hindbrain cells are not specified until stage 8, whereas rostral spinal cord cells emerge already at stages 5-7 this suggest that caudal hindbrain cells require RA at higher levels/for a longer time. Interestingly in 4C explants exposed to high levels of RA, caudal hindbrain cells emerged in the rostral domain of the explant, which would otherwise generate rostral hindbrain cells and rostral spinal cord cells emerged in the caudal domain of the explant normally generating caudal spinal cord cells (Figure 4). Moreover, administering low levels of RA to 4C explants still generated rostral spinal cord character in the caudal spinal cord region, but rostral hindbrain character was maintained, further arguing that caudal hindbrain cell require higher levels of RA signalling compared to rostral spinal cord cells.

Figure 4: RA respecifies caudal neural plate cells (A) Schematic picture of a stage 4 embryo. The expression of members of the Wnt and FGF signalling pathway is summarized. The black dotted line indicates the neural plate. The grey dotted line indicates the area depicted in B. (B) Schematic drawing of a stage 4C explant. The caudal-medial part is specified as caudal spinal cord (cSC), the rostral-lateral part of the explant is specified as rostral hindbrain (rHB). (C) Stage 4C explant exposed to RA, caudal hindbrain (cHB) character will emerge in the rHB domain and rostral spinal cord (rSC) character will emerge in the cSC domain, whereas exposure to RA and FGF together results in the emergence of rSC character in the entire explants and exposure to Wnt and FGF results in the emergence of cSC character in the entire explant (modified after U. Nordström).

40 Discussion

A role for RA signalling in spinal cord development Cells specified as cells of rostral spinal cord character can be isolated in low numbers already in stage 4 embryos and between stages 5-7 cells in the caudal neural plate are predominantly specified as cells of rostral spinal cord character. The spatial emergence of cells of rostral spinal cord character correlates with the expression of Raldh2 in the paraxial mesoderm close to the caudal neural plate. Our results clearly implicate RA signalling in the specification of cells of rostral spinal cord character by suppressing Hoxc9 expression in cells of caudal spinal cord character. Interestingly, at stages 5-7, when the caudal neural plate is mainly specified as rostral spinal cord, Raldh2 expressing mesoderm underlies almost the entire caudal neural plate [142]. By stage 8, Raldh2 expression is restricted to the paraxial mesoderm located at the level of/just rostral to the node and our results show that at this stage the caudal neural plate caudal to the node is specified as caudal spinal cord [143, 144]. These findings are in line with previously reported results, which have suggested that the expression of Hox genes characteristic of the rostral spinal cord is promoted by RA signals derived from the paraxial mesoderm [7, 10, 24, 27, 162]. Expression of Raldh2 persists in the somatic mesoderm adjacent to the forming neural tube, and grafting experiments have indicated that spinal cord tissue at brachial and thoracic neural tube levels is still divertible in its fate until the time of neural tube closure [29]. Interestingly, also at these later stages Raldh2 expression remains excluded from caudal neural tube domains [144], further supporting a role for RA signalling in the distinction of rostral versus caudal spinal cord phenotypes.

Early Wnt signalling establishes a positional context for later RA and FGF signalling in specifying caudal hindbrain and spinal cord cells We provide evidence that by stage 8 RA and FGF signalling imposes positional identity on hindbrain and spinal cord progenitor cells. The role of RA and FGF signalling in early hindbrain and spinal cord regionalization has previously been studied. RA signalling promotes Hox gene expression characteristic of caudal hindbrain and rostral spinal cord levels [7, 10, 24, 27, 107, 109, 162] and FGF signals derived from the regressing primitive streak promote the expression of more caudal Hox genes in a concentration dependent manner [24, 25]. Our results extend these findings by establishing that early Wnt signals provide the positional context for the later actions of

41 Discussion

RA and FGF signals in rostrocaudal regional specification. Neither RA nor FGF signals alone or in combination are sufficient to induce caudal neural cells in prospective forebrain cells. Only in concert with Wnt signalling are caudal neural cells induced in prospective forebrain cells by RA and/or FGF.

Opposing actions of RA and FGF during caudal neural development Opposing activities of RA and FGF pathways have been implicated in patterning the caudal part of the body. RA signalling has been shown to downregulate FGF8 expression and FGF signalling to repress Raldh2 expression [163, 164]. Our results show that blocking FGF signalling in stage 8 CN explants blocks the generation of Hoxc9-positive cells and instead cells express Hoxb4 and Hoxb8 a Hox gene profile characteristic of cells of rostral spinal cord character (data not shown), the same shift in specification is observed in the caudal-medial domain of 4C explants exposed to RA. This shift from caudal to rostral spinal cord in 4C explants can be reversed by adding high levels of FGF together with the same amount of RA to the explants. In addition in the rostral-lateral domain of the 4C explants exposed to RA and FGF together, FGF promotes the emergence of rostral spinal cord cells, instead of caudal hindbrain cells (Figure 4C). Thus our results further strengthen the idea that RA and FGF signalling acts in an opposing manner during regionalisation of the caudal neuraxis at neurulation stages on cells primed by the previous exposure to Wnt signalling.

Rostrocaudal patterning and body axis extension Rostrocaudal patterning of the neuraxis is tightly linked with body axis extension. In higher vertebrates, rostrocaudal patterning of neural tissue proceeds in a two step process [165]. In the rostral part, from the forebrain to the rostral hindbrain, rostrocaudal patterning can be viewed as a simple subdivision of the pre-existing neural plate into caudal forebrain, midbrain and rostral hindbrain domains by graded Wnt and permissive FGF signalling [9, 11]. In the caudal neural plate, the common view is that cells in the stem zone produce neural structures de novo while the stem zone regresses in concert with the regressing node [97, 164, 166]. Wnt, FGF and RA signals have all been implicated in the regulation of stem zone maintenance and exit [97, 164, 166, 167]. FGF-dependent Notch signalling has been shown to maintain cells in an undifferentiated state in the stem zone [97], whereas RA signalling drives differentiation [164]. In view of these studies our findings

42 Discussion could be reinterpreted. The emergence of caudal neural structures in a sequential pattern could be viewed as a progressive assignment of rostrocaudal identity onto cell populations exiting the stem zone at distinct time points. Nevertheless, our results provide evidence that cells in the caudal neural plate do not emerge in an ordered “caudal hindbrain first- caudal spinal cord last” fashion. In addition, our observations suggest that at late gastrula to early neurula stages, RA signalling specifies cells of caudal hindbrain and rostral spinal cord character and is not merely driving progenitor cells to differentiate. Moreover between stages 5-7 cells in the caudal neural plate are predominantly specified as cells of rostral spinal cord character and expression of Raldh2 in the mesoderm underlies the entire neural plate [142], suggesting that at these early stages the entire neural plate is exposed to RA signalling. Moreover at later developmental stages the expression of Raldh2 protein is no longer detectable in the presomitic mesoderm at around stage HH14 (when the 20th somite forms). In addition, caudally located somites do not contain Raldh2 protein in their ventral portions, arguing against a requirement for RA in the differentiation of ventral neural cell types in the caudal neural tube [144]. These findings suggest a difference in RA exposure between rostral and caudal spinal cord cells in the embryo.

Hox gene expression in progenitor cells and the emergence of motor neuron subtype identity In the hindbrain and spinal cord neural progenitor cells located at distinct rostrocaudal levels in the ventral neural tube differentiate into two major neuronal subtypes: dMNs and vMNs [15, 168]. Our findings indicate that the exposure of neural plate cells to Wnt signalling during gastrula stages is required for the emergence of dMNs and vMNs in the hindbrain and spinal cord. This extends previous studies that established a role for RA, FGF and GDF11 signals in the generation of spatially distinct MNs in the caudal neural tube [24, 25, 169]. At hindbrain and spinal cord levels Hox gene profiles correlate with the rostrocaudal identity of neural progenitor cells [28, 29]. Moreover, recent studies have suggested instructive roles for Hox genes in motor neuron subtype specification. In the hindbrain, Hoxa3 is expressed in rhombomeres (r) caudal to the r4/r5 boundary and misexpression at rostral hindbrain levels leads to the ectopic generation of somatic motor neurons [16] and the combined action of Hoxa3 and Hoxb3 is required for the

43 Discussion specification of somatic progenitor cells in r5 [170]. Moreover, the nested expression of Hoxb1 in r4 is required for the correct specification of branchiomotor neurons [32, 33, 171, 172]. At spinal cord levels, Hoxc8 is required for the specification of brachial motor neurons [35, 36] and members of the Hox10 gene group are required for the proper specification of the lumbosacral region of the spinal cord [23, 30, 37-39]. Thus, Hox gene profiles expressed in progenitor cells are major determinants of MN identity. Our findings indicate that Wnt, FGF and/or RA are sufficient to induce Hox gene profiles in prospective forebrain cells, thereby reconstructing neuronal progenitor cells that in response to Shh signalling differentiate in MNs of the predicated spatial subtype. Cdx genes have been implicated as mediators between external signalling cues and Hox gene induction in vertebrates. In the caudal neural plate Cdx genes are transiently expressed [173-175]. Both in Xenopus and chick, FGF induced changes in Hox gene expression are preceded by changes in Cdx gene expression [27, 42]. In zebrafish, cdx1 and cdx4 have been implicated in mediating the responsiveness to FGF and RA signalling [176]. Moreover, both RARE and Tcf/Lef binding sites have been found in the mouse Cdx1 promoter region [43, 44]. Our results indicate that by stages 6-8 prospective spinal cord cells express Cdx genes and that the interplay of Wnt, RA and FGF signalling establishes Cdx gene profiles indicative of spinal cord identity. Thus our results provide evidence for a link between gastrula stage Wnt, FGF and RA signalling, Cdx and Hox gene profiles expressed by progenitor cells and the emergence of dMNs and vMNs at hindbrain and spinal cord levels.

Coordinated development of the CNS and PNS For the nervous system to function properly, the different classes of neurons generated by the CNS and PNS precursors along the rostrocaudal axis have to establish connections in an appropriate manner. At gastrula stages Wnt signalling is both required for the caudalization of neural plate and neural plate border cells, giving rise to CNS and PNS precursors respectively ([9, 53], Paper I). Thus, Wnt signalling provides a means to spatially coordinate the early development of the central and the peripheral nervous system. Since Hox genes are expressed in neural crest cells as well as in neural cells [27, 177, 178], it would be interesting to see if RA and FGF dependent

44 Discussion mechanism are involved in the patterning of caudal neural crest tissue arising at spinal cord levels.

Project II: Patterning and neurogenesis in the olfactory placode

In summary, we show that the olfactory placode becomes subdivided into sensory and respiratory epithelial domains by a mechanism involving FGF and BMP signalling. BMP signals induce cells of respiratory epithelial character, whereas FGF signals are required to generate cells of sensory epithelial character. Moreover, we show that later during development at stage 21, cells in the sensory epithelial domain no longer respond to elevated levels of BMP signalling by fate shifting to respiratory epithelial character. In addition, we show that neurogenesis in the olfactory placode is regulated by Notch signalling. In the absence of active Notch signalling, cells prematurely differentiate into OSNs, whereas overactivation of the Notch signalling pathway maintains progenitor cells and suppresses the generation of OSNs.

Expression of Hes5 genes in the olfactory placodal region We analyzed the expression patterns of the bHLH repressor gene Hes5 in the olfactory placodal region. We found that uniform expression of Hes5 genes starts around stage 10 in the olfactory placodal region. Previous studies describe a dual role for Hes genes in the development of the olfactory sensory epithelium in mice: first, Hes genes act as pre-patterning genes in the olfactory placode and define the neurogenic region and second, at later developmental stages Hes genes play a role in regulating the number of progenitor cells emerging in the neurogenic domain of the olfactory placode by negatively regulating neurogenesis [79]. Thus, the early uniform expression detected around stage 10 indicates that Hes5 might be involved in the early patterning of the developing olfactory placode, possibly by delineating the presumptive neurogenic region in the placodal ectoderm. Moreover since Hes5 is expressed before any other marker of the neurogenic lineage, it seems to be the earliest marker associated with neuronal determination in the olfactory placodal region in chick.

45 Discussion

Dynamic expression patterns of neurogenic markers in the olfactory placodal region We analyzed the expression patterns of Hes5 (Hes5-1), Cash1, Ngn1, Lhx2 and HuC/D from stage 14 to stage 34 in the olfactory placode and olfactory sensory epithelium. Our results have the following implications: (1) By stage 14, Cash1, Ngn1 and HuC/D are expressed in the olfactory placode. This is much earlier than previously reported in the mouse [80, 81, 179], but this difference is most likely due to a lack of expression analyzes in the mouse rather than species differences. (2) From stage 22 neurogenesis proceeds along distinct axes in the olfactory pit. Differentiated cells are located preferentially at medial-superior positions within the pit. This highlights the importance to indicate which area of the pit is shown in a given section since the expression patterns can vary greatly along the anterior-posterior extent of the olfactory pit. (3) Stratification in the chick olfactory sensory epithelium becomes apparent at stage 34, which is later compared to the development of the mouse olfactory sensory epithelium [76, 116].

Olfactory placodal progenitor cells give rise to respiratory and sensory epithelial cells Our results provide evidence, that by stage 14 cells in the olfactory placodal region are specified as cells of respiratory and sensory epithelial character. This is consistent with previous descriptive studies, stating that the olfactory placode give rise to both sensory and respiratory epithelial cells [117]. Due to a lack of molecular markers, the molecular mechanism by which cells in the olfactory placode become subdivided into sensory and respiratory epithelial cells has remained unresolved.

BMP signals specify respiratory epithelial cells Our results provide evidence, that BMP signalling is required for the generation of cells of respiratory epithelial character. Moreover, elevated BMP signalling is sufficient to induce cells of respiratory epithelial character from cells of sensory epithelial character. Expression of BMP4 can be detected already at stage 10 in the presumptive olfactory placodal region and persists in the caudal rim of the olfactory pit. At stage 22 the expression pattern of BMP4 correlates spatially with the localization of respiratory epithelial cells in the olfactory pit (Suppl Figure 3). Several studies have shown that BMP signalling can block neural and promote epidermal

46 Discussion character at early developmental stages [180-182]. Interestingly, prospective epidermal cells are specified to express Msx1/2 [4] raising the possibility that a similar molecular cassette is employed during patterning of the olfactory placode. In the olfactory region, previous results have shown that BMP signalling exerts a negative effect on neurogenesis in a neuronal colony forming assay (OE tissue isolated from E14.5-15.5 mice), but no increase in non-neural colonies was observed [91]. In a similar assay Noggin blocked the formation of neuronal colonies, but again no change in non-neuronal colonies could be observed [89]. The effects of BMP and Noggin on neurogenesis are in line with our observation. In addition, we find that both increasing and abolishing BMP signalling in our study had an effect on the non-neuronal cells of respiratory epithelial character. Since the character of the non-neuronal colonies was not addressed by Shou et al in either study, it is difficult to draw a conclusion. The earlier developmental time point of our analysis may account for some of the observed differences.

BMP signals are required for the differentiation of the olfactory sensory lineage BMP signalling has been implicated in the regulation of neurogenesis during development of the cerebellum, hippocampus and the neural crest [129, 183- 185]. In the olfactory sensory epithelium, in vitro studies using neuronal colony forming assays of sensory epithelium dissected from E14.5-16.5 mice showed both inhibitory and stimulatory effects of TGFβ signalling on neurogenesis in sensory epithelium cultures [89-91]. BMP2,4 and 7 reduced the number of proliferating progenitors and inhibited the production of OSNs by a mechanism involving the degradation of Mash1 protein [91]. In contrast, at lower concentrations, BMP4, but not BMP7 could also stimulate neurogenesis in the same assay, by promoting the survival of newly generated OSNs. Moreover, Noggin strongly inhibited the formation of neuronal colonies in the in vitro system [89]. In the same assay, GDF11 inhibited neurogenesis by arresting INP proliferation through induction of p27KIP. Moreover, cell proliferation of INPs is increased in the OE of mice mutant for GDF11 and decreased in mice lacking Follistatin, an antagonist of GDF11 [90]. Our results indicate, that BMP signalling is required for the progression of cells in the olfactory sensory lineage, since inhibition of BMP activity inhibits TAP, INP and OSN generation and cells instead acquire progenitor-like character. Moreover, pSmad1/5/8 expressing cells can be detected in a scattered pattern in the neurogenic region of stage 22 olfactory

47 Discussion pits (data not shown), indicating that BMP signalling is active in those cells. Thus, our results provide evidence, that BMP signalling is required for neuron production in the olfactory sensory lineage.

BMP signalling acts sequentially during the development of the olfactory placode At gastrula stages in the chick, the olfactory and the lens placode are specified by BMP signals and sustained BMP activity at neural fold stages promotes the acquisition of a lens placodal fate [54]. Around stage 14 BMP signalling is required to specify cells of respiratory epithelial character. Later in development, starting around stage 21, the cells of sensory epithelial character in the olfactory placode respond to BMP signals no longer by the acquisition of a respiratory epithelial fate but maintain their sensory epithelial character. In summary, our data show that the competence to respond to BMP signalling in olfactory placodal cells changes during a relatively short time period.

FGF signalling is required for the generation of sensory epithelial character Starting around stage 8, several FGFs are expressed in the anterior part of the embryo and later expression of FGF8 can be detected in the olfactory placode/pit [86, 139]. Our results indicate that FGF signalling is required for the generation of cells of sensory epithelial character both in in vitro and in vivo, and that cells of sensory epithelial character in the absence of FGF signalling acquire respiratory epithelial character. Consistent with this, FGF signalling is required for the acquisition of pro-neural gene expression in the otic placode [186]. Previous results, obtained in olfactory sensory epithelium in vitro cultures, indicate that FGF signalling exerts stimulatory effects on neurogenesis [83]. Moreover, mice lacking the anterior expression domain of FGF8, initiate establishment of the sensory lineage at E10.5, probably due to compensatory effects of FGF3 and FGF10 [187]. At later stages (E12.5) however, development does not proceed further, possible because FGF8 is required to maintain and produce neural stem and progenitor cells in the olfactory sensory lineage [86]. We have shown that blocking FGF signalling in stage 14OP explants results in a reduction in size, but both, the relative and the total amount of Msx1/2-positive cells is increased in these explants, arguing that FGF is not merely required to sustain sensory epithelial cells as

48 Discussion suggested by the mouse data. Nevertheless, it can not be excluded that some sensory epithelial progenitor cells are dependent on FGF for their survival.

BMP and FGF act in an opposing manner Our results indicate that FGF and BMP signalling act in an opponent manner in the regulation of sensory versus respiratory epithelial fate specification. One possible role for FGF might be to restrict BMP signalling, since inhibition of FGF signalling leads to the emergence of respiratory epithelial character only in the presence of endogenous BMP signals. Inhibition of FGF might increase the activity of BMP signals or lower the total amount needed for the induction of cells of respiratory epithelial character. Opposing activities of FGF and BMP signals have been described in several systems. A recent study reported that a balance of FGF and BMP signals is required for the generation of anterior placodal progenitor cells in part by preventing the generation of epidermal and neural cells, respectively [54]. Moreover, it has been suggested that tooth development and the progression of chondrocytes are controlled by antagonistic BMP and FGF signalling [188-191]. During the development of the cerebellum, FGF8 emanating from the isthmus is thought to influence the balance between Notch and BMP by phosphorylating Smad1 in the linker region, which leads to the deactivation of Smad1 [129, 192]. Moreover during development of the forebrain and the limb bud BMP4 signals have been shown to repress FGF8 expression [193- 195]. Additionally, in the developing forebrain FGF activity restricts the expansion of BMP4 expression via Foxg1, a transcriptional repressor [196- 198]. Several studies indicate that the primary role for Foxg1 is to maintain neuronal progenitor cells [199-201]. In the olfactory placode of mice Foxg1 expression is widespread (E10.5) but becomes restricted to the ventral regions later on [202]. Storm et al suggest a Foxg1-survival pathway, which is proposed to be especially sensitive to FGF-induced inhibitors, resulting in cell death when endogenous FGF levels are further raised, an observation that is contrary to our results. Taken together, our results provide evidence that FGF and BMP signals act in an opponent manner in cells of the olfactory placode to regulate olfactory versus respiratory epithelial cell fate determination.

49 Discussion

Notch signalling is required to maintain progenitor cells Diverse roles for Notch signalling have been described in the nervous system. Notch signalling can both inhibit and promote neurogenesis and regulate cell type diversification [203-205]. Our results indicate that Notch signalling in the olfactory placode is required to maintain Hes5-positive progenitor cells. Inhibition of Notch signalling in vitro and in vivo leads to increased neurogenesis. Interestingly, in our in vitro assay, the entire progenitor pool is depleted when Notch signalling is blocked. At late stages of olfactory sensory epithelium development, Notch signalling has been implicated in the segregation of OSN versus glial cell types [104]. Moreover, Notch mediated lateral inhibition has been implicated in regulating the density of neuronal progenitor cells in the developing sensory epithelium [96].

Early born neurons join the migratory mass Our results have shown, that in embryos with perturbed Notch signalling, most HuC/D/GFP-double-positive cells leave the placode and migrate toward the forebrain as part of the migratory mass. This is in line with other descriptive in vivo studies that have shown that between stages 14 and 17 most of the differentiated neurons leave the placode ([74] EM unpub observation). Little is known about the fate of these early migrating neuronal cells arising in the olfactory placode. This wave of migratory cells raises the possibility that in the olfactory placode, like in the CNS [129] several cell types arise at different time points from the same progenitor population and that in the olfactory placode the emergence of functional OSNs in the sensory epithelium might occur at later stages of development.

Antagonism between BMP and Notch signalling pathways Our results indicate that inhibition of BMP signalling in the olfactory placodal region is required for the generation of neuronal cells, since stage 14OP explants cultured in the presence of the BMP-inhibitor Noggin express Hes5 in most of the cells. Hes5 is a direct target gene of the Notch signalling pathway [77, 206, 207] and Notch signalling is required to maintain Hes5- positive progenitor cells in the olfactory placodal region, raising the possibility, that BMP signalling influences the status of Notch signalling. Indeed, elevated levels of BMP signalling blocked expression of Delta1 and Notch1 and promotes respiratory epithelial character. In line with this Notch

50 Discussion activity in the otic placode is required to exclude expression of non-neural genes form the pro-neural territory [128]. Moreover, we show that inhibition of BMP signalling lead to increased uniform expression of Delta1 and down- regulation of Notch1. A similar uniform high Delta1 expression domain has been reported in the spinal cord stem zone, where it functions in progenitor maintenance [97]. Previous studies have reported both synergistic and antagonistic effects of the two pathways. In neural crest stem cells, BMP signals promote neurogenesis, whereas Notch is dominant over BMP signalling and promotes gliogenesis instead [183]. Moreover in the rhombic lip, Notch signalling has been shown to regulate the responsiveness of progenitor cells to BMP signals. Notch activity inhibits the responsiveness to BMP signals, thereby maintaining progenitor cells [129]. In addition, in epithelial cell migration, BMP signals promote migration, whereas Notch signalling inhibits migration [208]. In summary, our results indicate that in the olfactory placode BMP and Notch signalling act antagonistically in the sensory epithelium.

51 Acknowledgements

Acknowledgements

First, I like to thank Thomas, my supervisor during the work on the rostrocaudal project, for giving me the possibility to join his lab and the support throughout the years.

I like to thank Lena, my supervisor during the olfactory project, for all the support and enthusiasm, and for giving me experimental freedom.

Ullis, thanks for sharing the rostrocaudal project with me; I learned a lot from you during the early years, and miss having you around, in the lab and dinner-wise.

Jonas, thanks for all the work you put into the electroporations, it’s really nice to have some in vivo support! Moreover your presence kept my Swedish practiced and we could share being “old”.

Helena, thanks for all the assistance during the years, CM, organization, and lately the Westerns. And for all the non-lab related talks and activities.

Former members of group TE: Ferran, Sanna, Matthew, Anna, My and Cédric for creating a really great atmosphere in the lab and for all the non-lab fun we had, I miss you.

Members of group LG: Tanushree, Hanna and Vijay for discussions, work- and otherwise, and during the month of March, generously donating eggs.

Angelica, Christopher and Daniela, who all did some project or other related to olfactory development, thanks.

Tom Jessell contributed with ideas and writing skills to the rostrocaudal project.

PA, Kristina, Brigitta and Clara, I honestly don´t know how I would have managed without you.

All the people at UCMM for making it the great workplace it is.

52 References

References

1. Spemann, H.M., H., Uber Induktion von Embryonanlagen durch Implantation artfremder Organisatoren. Roux. Arch. Entw. Mech. Organ., 1924(100): p. 599- 638. 2. Streit, A., et al., Initiation of neural induction by FGF signalling before gastrulation. Nature, 2000. 406(6791): p. 74-8. 3. Wilson, S.I., et al., An early requirement for FGF signalling in the acquisition of neural cell fate in the chick embryo. Curr Biol, 2000. 10(8): p. 421-429. 4. Wilson, S., et al., The status of Wnt signalling regulates neural and epidermal fates in the chick embryo. Nature, 2001. 411(6835): p. 325-30. 5. Colas, J.F. and G.C. Schoenwolf, Towards a cellular and molecular understanding of neurulation. Dev Dyn, 2001. 221(2): p. 117-45. 6. Nieuwkoop, P.D., et al., Activation and organisation of the central nervous system in amphibians. J Exp Zool, 1952(120): p. 1-108. 7. Muhr, J., et al., Convergent inductive signals specify midbrain, hindbrain, and spinal cord identity in gastrula stage chick embryos. Neuron, 1999. 23(4): p. 689- 702. 8. Muhr, J., T.M. Jessell, and T. Edlund, Assignment of early caudal identity to neural plate cells by a signal from caudal paraxial mesoderm. Neuron, 1997. 19(3): p. 487-502. 9. Nordstrom, U., T.M. Jessell, and T. Edlund, Progressive induction of caudal neural character by graded Wnt signaling. Nat Neurosci, 2002. 5(6): p. 525-32. 10. Kudoh, T., S.W. Wilson, and I.B. Dawid, Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development, 2002. 129(18): p. 4335- 46. 11. Kiecker, C. and C. Niehrs, A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus. Development, 2001. 128(21): p. 4189-201. 12. Kiecker, C. and C. Niehrs, The role of prechordal mesendoderm in neural patterning. Curr Opin Neurobiol, 2001. 11(1): p. 27-33. 13. Niederreither, K., et al., Retinoic acid synthesis and hindbrain patterning in the mouse embryo. Development, 2000. 127(1): p. 75-85. 14. Landmesser, L.T., The acquisition of motoneuron subtype identity and motor circuit formation. Int J Dev Neurosci, 2001. 19(2): p. 175-82. 15. Sharma, K., et al., LIM homeodomain factors Lhx3 and Lhx4 assign subtype identities for motor neurons. Cell, 1998. 95(6): p. 817-28. 16. Guidato, S., F. Prin, and S. Guthrie, Somatic motoneurone specification in the hindbrain: the influence of somite-derived signals, retinoic acid and Hoxa3. Development, 2003. 130(13): p. 2981-96. 17. Guthrie, S., Neuronal development: putting motor neurons in their place. Curr Biol, 2004. 14(4): p. R166-8. 18. Jessell, T.M., Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat Rev Genet, 2000. 1(1): p. 20-9.

53 References

19. Song, M.R. and S.L. Pfaff, Hox genes: the instructors working at motor pools. Cell, 2005. 123(3): p. 363-5. 20. Krumlauf, R., Hox genes in vertebrate development. Cell, 1994. 78(2): p. 191-201. 21. Lumsden, A. and R. Krumlauf, Patterning the vertebrate neuraxis. Science, 1996. 274(5290): p. 1109-15. 22. Trainor, P.A. and R. Krumlauf, Hox genes, neural crest cells and branchial arch patterning. Curr Opin Cell Biol, 2001. 13(6): p. 698-705. 23. Shah, V., E. Drill, and C. Lance-Jones, Ectopic expression of Hoxd10 in thoracic spinal segments induces motoneurons with a lumbosacral molecular profile and axon projections to the limb. Dev Dyn, 2004. 231(1): p. 43-56. 24. Liu, J.P., E. Laufer, and T.M. Jessell, Assigning the Positional Identity of Spinal Motor Neurons. Rostrocaudal Patterning of Hox-c Expression by FGFs, Gdf11, and Retinoids. Neuron, 2001. 32(6): p. 997-1012. 25. Dasen, J.S., J.P. Liu, and T.M. Jessell, Motor neuron columnar fate imposed by sequential phases of Hox-c activity. Nature, 2003. 425(6961): p. 926-33. 26. Carpenter, E.M., Hox genes and spinal cord development. Dev Neurosci, 2002. 24(1): p. 24-34. 27. Bel-Vialar, S., N. Itasaki, and R. Krumlauf, Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups. Development, 2002. 129(22): p. 5103-15. 28. Belting, H.G., C.S. Shashikant, and F.H. Ruddle, Multiple phases of expression and regulation of mouse Hoxc8 during early embryogenesis. J Exp Zool, 1998. 282(1- 2): p. 196-222. 29. Ensini, M., et al., The control of rostrocaudal pattern in the developing spinal cord: specification of motor neuron subtype identity is initiated by signals from paraxial mesoderm. Development, 1998. 125(6): p. 969-82. 30. Lance-Jones, C., et al., Hoxd10 induction and regionalization in the developing lumbosacral spinal cord. Development, 2001. 128(12): p. 2255-68. 31. Hale, M.E., et al., Hox gene misexpression and cell-specific lesions reveal functionality of homeotically transformed neurons. J Neurosci, 2004. 24(12): p. 3070-6. 32. Jungbluth, S., E. Bell, and A. Lumsden, Specification of distinct motor neuron identities by the singular activities of individual Hox genes. Development, 1999. 126(12): p. 2751-8. 33. Studer, M., et al., Altered segmental identity and abnormal migration of motor neurons in mice lacking Hoxb-1. Nature, 1996. 384(6610): p. 630-4. 34. Wu, Y., et al., Hoxc10 and Hoxd10 regulate mouse columnar, divisional and motor pool identity of lumbar motoneurons. Development, 2008. 135(1): p. 171-82. 35. Vermot, J., et al., Retinaldehyde dehydrogenase 2 and Hoxc8 are required in the murine brachial spinal cord for the specification of Lim1+ motoneurons and the correct distribution of Islet1+ motoneurons. Development, 2005. 132(7): p. 1611- 21. 36. Tiret, L., et al., Increased apoptosis of motoneurons and altered somatotopic maps in the brachial spinal cord of Hoxc-8-deficient mice. Development, 1998. 125(2): p. 279-91.

54 References

37. Lin, A.W. and E.M. Carpenter, Hoxa10 and Hoxd10 coordinately regulate lumbar motor neuron patterning. J Neurobiol, 2003. 56(4): p. 328-37. 38. de la Cruz, C.C., et al., Targeted disruption of Hoxd9 and Hoxd10 alters locomotor behavior, vertebral identity, and peripheral nervous system development. Dev Biol, 1999. 216(2): p. 595-610. 39. Carpenter, E.M., et al., Targeted disruption of Hoxd-10 affects mouse hindlimb development. Development, 1997. 124(22): p. 4505-14. 40. Lohnes, D., The Cdx1 homeodomain protein: an integrator of posterior signaling in the mouse. Bioessays, 2003. 25(10): p. 971-80. 41. Subramanian, V., B.I. Meyer, and P. Gruss, Disruption of the murine homeobox gene Cdx1 affects axial skeletal identities by altering the mesodermal expression domains of Hox genes. Cell, 1995. 83(4): p. 641-53. 42. Pownall, M.E., et al., eFGF, Xcad3 and Hox genes form a molecular pathway that establishes the anteroposterior axis in Xenopus. Development, 1996. 122(12): p. 3881-92. 43. Houle, M., et al., Retinoic acid regulation of Cdx1: an indirect mechanism for retinoids and vertebral specification. Mol Cell Biol, 2000. 20(17): p. 6579-86. 44. Prinos, P., et al., Multiple pathways governing Cdx1 expression during murine development. Dev Biol, 2001. 239(2): p. 257-69. 45. Lupo, G., W.A. Harris, and K.E. Lewis, Mechanisms of ventral patterning in the vertebrate nervous system. Nat Rev Neurosci, 2006. 7(2): p. 103-14. 46. Briscoe, J. and J. Ericson, The specification of neuronal identity by graded Sonic Hedgehog signalling. Semin Cell Dev Biol, 1999. 10(3): p. 353-62. 47. Pierani, A., et al., A sonic hedgehog-independent, retinoid-activated pathway of neurogenesis in the ventral spinal cord. Cell, 1999. 97(7): p. 903-15. 48. Gabay, L., et al., Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron, 2003. 40(3): p. 485-99. 49. Novitch, B.G., et al., A requirement for retinoic acid-mediated transcriptional activation in ventral neural patterning and motor neuron specification. Neuron, 2003. 40(1): p. 81-95. 50. Wilson, L., et al., Retinoic acid and the control of dorsoventral patterning in the avian spinal cord. Dev Biol, 2004. 269(2): p. 433-46. 51. Patthey, C., T. Edlund, and L. Gunhaga, Wnt-regulated temporal control of BMP exposure directs the choice between neural plate border and epidermal fate. Development, 2009. 136(1): p. 73-83. 52. Basch, M.L. and M. Bronner-Fraser, Neural crest inducing signals. Adv Exp Med Biol, 2006. 589: p. 24-31. 53. Patthey, C., L. Gunhaga, and T. Edlund, Early development of the central and peripheral nervous systems is coordinated by Wnt and BMP signals. PLoS ONE, 2008. 3(2): p. e1625. 54. Sjodal, M., T. Edlund, and L. Gunhaga, Time of exposure to BMP signals plays a key role in the specification of the olfactory and lens placodes ex vivo. Dev Cell, 2007. 13(1): p. 141-9.

55 References

55. Villanueva, S., et al., Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction. Dev Biol, 2002. 241(2): p. 289-301. 56. Litsiou, A., S. Hanson, and A. Streit, A balance of FGF, BMP and WNT signalling positions the future placode territory in the head. Development, 2005. 132(18): p. 4051-62. 57. Bastidas, F., J. De Calisto, and R. Mayor, Identification of neural crest competence territory: role of Wnt signaling. Dev Dyn, 2004. 229(1): p. 109-17. 58. Knecht, A.K. and M. Bronner-Fraser, Induction of the neural crest: a multigene process. Nat Rev Genet, 2002. 3(6): p. 453-61. 59. Ragland, J.W. and D.W. Raible, Signals derived from the underlying mesoderm are dispensable for zebrafish neural crest induction. Dev Biol, 2004. 276(1): p. 16-30. 60. LaBonne, C. and M. Bronner-Fraser, Neural crest induction in Xenopus: evidence for a two-signal model. Development, 1998. 125(13): p. 2403-14. 61. Raible, D.W., Development of the neural crest: achieving specificity in regulatory pathways. Curr Opin Cell Biol, 2006. 18(6): p. 698-703. 62. Baker, C.V. and M. Bronner-Fraser, Vertebrate cranial placodes I. Embryonic induction. Dev Biol, 2001. 232(1): p. 1-61. 63. Schlosser, G., Induction and specification of cranial placodes. Dev Biol, 2006. 294(2): p. 303-51. 64. Mori, K., et al., Maps of odorant molecular features in the Mammalian olfactory bulb. Physiol Rev, 2006. 86(2): p. 409-33. 65. Meisami, E. and K.P. Bhatnagar, Structure and diversity in mammalian accessory olfactory bulb. Microsc Res Tech, 1998. 43(6): p. 476-99. 66. Bhatnagar, K.P. and E. Meisami, Vomeronasal organ in bats and primates: extremes of structural variability and its phylogenetic implications. Microsc Res Tech, 1998. 43(6): p. 465-75. 67. Graziadei, P.P. and G.A. Graziadei, Neurogenesis and neuron regeneration in the olfactory system of mammals. I. Morphological aspects of differentiation and structural organization of the olfactory sensory neurons. J Neurocytol, 1979. 8(1): p. 1-18. 68. Graziadei, P.P. and M. Okano, Neuronal degeneration and regeneration in the olfactory epithelium of pigeon following transection of the first cranial nerve. Acta Anat (Basel), 1979. 104(2): p. 220-36. 69. Mackay-Sim, A. and P.W. Kittel, On the Life Span of Olfactory Receptor Neurons. Eur J Neurosci, 1991. 3(3): p. 209-215. 70. Hinds, J.W., P.L. Hinds, and N.A. McNelly, An autoradiographic study of the mouse olfactory epithelium: evidence for long-lived receptors. Anat Rec, 1984. 210(2): p. 375-83. 71. Baizer, L., et al., Chicken growth-associated protein (GAP)-43: primary structure and regulated expression of mRNA during embryogenesis. Brain Res Mol Brain Res, 1990. 7(1): p. 61-8. 72. Beites, C.L., et al., Identification and molecular regulation of neural stem cells in the olfactory epithelium. Exp Cell Res, 2005. 306(2): p. 309-16. 73. Calof, A.L., et al., Progenitor cells of the olfactory receptor neuron lineage. Microsc Res Tech, 2002. 58(3): p. 176-88.

56 References

74. Fornaro, M., et al., HuC/D confocal imaging points to olfactory migratory cells as the first cell population that expresses a post-mitotic neuronal phenotype in the chick embryo. Neuroscience, 2003. 122(1): p. 123-8. 75. Kawauchi, S., et al., Molecular signals regulating proliferation of stem and progenitor cells in mouse olfactory epithelium. Dev Neurosci, 2004. 26(2-4): p. 166-80. 76. Kolterud, A., et al., The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity. Development, 2004. 131(21): p. 5319-26. 77. Basak, O. and V. Taylor, Identification of self-replicating multipotent progenitors in the embryonic nervous system by high Notch activity and Hes5 expression. Eur J Neurosci, 2007. 25(4): p. 1006-22. 78. Jarriault, S., et al., Delta-1 activation of notch-1 signaling results in HES-1 transactivation. Mol Cell Biol, 1998. 18(12): p. 7423-31. 79. Cau, E., et al., Hes genes regulate sequential stages of neurogenesis in the olfactory epithelium. Development, 2000. 127(11): p. 2323-32. 80. Murray, R.C., et al., Widespread defects in the primary olfactory pathway caused by loss of Mash1 function. J Neurosci, 2003. 23(5): p. 1769-80. 81. Cau, E., S. Casarosa, and F. Guillemot, Mash1 and Ngn1 control distinct steps of determination and differentiation in the olfactory sensory neuron lineage. Development, 2002. 129(8): p. 1871-80. 82. Hirota, J. and P. Mombaerts, The LIM-homeodomain protein Lhx2 is required for complete development of mouse olfactory sensory neurons. Proc Natl Acad Sci U S A, 2004. 101(23): p. 8751-5. 83. DeHamer, M.K., et al., Genesis of olfactory receptor neurons in vitro: regulation of progenitor cell divisions by fibroblast growth factors. Neuron, 1994. 13(5): p. 1083-97. 84. Dono, R., et al., Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice. Embo J, 1998. 17(15): p. 4213-25. 85. Ortega, S., et al., Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2. Proc Natl Acad Sci U S A, 1998. 95(10): p. 5672-7. 86. Kawauchi, S., et al., Fgf8 expression defines a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the mouse. Development, 2005. 132(23): p. 5211-23. 87. Meyers, E.N., M. Lewandoski, and G.R. Martin, An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat Genet, 1998. 18(2): p. 136-41. 88. Peretto, P., et al., BMP mRNA and protein expression in the developing mouse olfactory system. J Comp Neurol, 2002. 451(3): p. 267-78. 89. Shou, J., et al., Opposing effects of bone morphogenetic proteins on neuron production and survival in the olfactory receptor neuron lineage. Development, 2000. 127(24): p. 5403-13. 90. Wu, H.H., et al., Autoregulation of neurogenesis by GDF11. Neuron, 2003. 37(2): p. 197-207.

57 References

91. Shou, J., P.C. Rim, and A.L. Calof, BMPs inhibit neurogenesis by a mechanism involving degradation of a transcription factor. Nat Neurosci, 1999. 2(4): p. 339- 45. 92. Lewis, J., Notch signalling and the control of cell fate choices in vertebrates. Semin Cell Dev Biol, 1998. 9(6): p. 583-9. 93. Lai, E.C., Notch signaling: control of cell communication and cell fate. Development, 2004. 131(5): p. 965-73. 94. Artavanis-Tsakonas, S., M.D. Rand, and R.J. Lake, Notch signaling: cell fate control and signal integration in development. Science, 1999. 284(5415): p. 770-6. 95. Bray, S.J., Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol, 2006. 7(9): p. 678-89. 96. Iso, T., L. Kedes, and Y. Hamamori, HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol, 2003. 194(3): p. 237-55. 97. Akai, J., P.A. Halley, and K.G. Storey, FGF-dependent Notch signaling maintains the spinal cord stem zone. Genes Dev, 2005. 19(23): p. 2877-87. 98. Lowell, S., et al., Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters. Curr Biol, 2000. 10(9): p. 491- 500. 99. Klueg, K.M. and M.A. Muskavitch, Ligand-receptor interactions and trans- endocytosis of Delta, Serrate and Notch: members of the Notch signalling pathway in Drosophila. J Cell Sci, 1999. 112 ( Pt 19): p. 3289-97. 100. Sakamoto, K., et al., The nephroblastoma overexpressed gene (NOV/ccn3) protein associates with Notch1 extracellular domain and inhibits myoblast differentiation via Notch signaling pathway. J Biol Chem, 2002. 277(33): p. 29399-405. 101. Sakamoto, K., et al., Intracellular cell-autonomous association of Notch and its ligands: a novel mechanism of Notch signal modification. Dev Biol, 2002. 241(2): p. 313-26. 102. Schwarting, G.A., T. Gridley, and T.R. Henion, Notch1 expression and ligand interactions in progenitor cells of the mouse olfactory epithelium. J Mol Histol, 2007. 38(6): p. 543-53. 103. Orita, Y., et al., Expression of Notch1 and Hes5 in the developing olfactory epithelium. Acta Otolaryngol, 2006. 126(5): p. 498-502. 104. Carson, C., B. Murdoch, and A.J. Roskams, Notch 2 and Notch 1/3 segregate to neuronal and glial lineages of the developing olfactory epithelium. Dev Dyn, 2006. 235(6): p. 1678-88. 105. Rodriguez, S., et al., Notch2 is required for maintaining sustentacular cell function in the adult mouse main olfactory epithelium. Dev Biol, 2008. 314(1): p. 40-58. 106. Begemann, G. and A. Meyer, Hindbrain patterning revisited: timing and effects of retinoic acid signalling. Bioessays, 2001. 23(11): p. 981-6. 107. Kolm, P.J., V. Apekin, and H. Sive, Xenopus hindbrain patterning requires retinoid signaling. Dev Biol, 1997. 192(1): p. 1-16. 108. Maden, M., Retinoid signalling in the development of the central nervous system. Nat Rev Neurosci, 2002. 3(11): p. 843-53. 109. Niederreither, K., et al., Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. Nat Genet, 1999. 21(4): p. 444-8.

58 References

110. Dupe, V. and A. Lumsden, Hindbrain patterning involves graded responses to retinoic acid signalling. Development, 2001. 128(12): p. 2199-208. 111. Briscoe, J., et al., Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling. Nature, 1999. 398(6728): p. 622-7. 112. Porter, J.A., et al., The product of hedgehog autoproteolytic cleavage active in local and long-range signalling. Nature, 1995. 374(6520): p. 363-6. 113. Roelink, H., et al., Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell, 1995. 81(3): p. 445-55. 114. Briscoe, J. and D.G. Wilkinson, Establishing neuronal circuitry: Hox genes make the connection. Genes Dev, 2004. 18(14): p. 1643-8. 115. Fior, R. and D. Henrique, A novel hes5/hes6 circuitry of negative regulation controls Notch activity during neurogenesis. Dev Biol, 2005. 281(2): p. 318-33. 116. Murdoch, B. and A.J. Roskams, Olfactory epithelium progenitors: insights from transgenic mice and in vitro biology. J Mol Histol, 2007. 38(6): p. 581-99. 117. Croucher, S.J. and C. Tickle, Characterization of epithelial domains in the nasal passages of chick embryos: spatial and temporal mapping of a range of extracellular matrix and cell surface molecules during development of the nasal placode. Development, 1989. 106(3): p. 493-509. 118. Hollnagel, A., et al., Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. J Biol Chem, 1999. 274(28): p. 19838-45. 119. Hussein, S.M., E.K. Duff, and C. Sirard, Smad4 and beta-catenin co-activators functionally interact with lymphoid-enhancing factor to regulate graded expression of Msx2. J Biol Chem, 2003. 278(49): p. 48805-14. 120. Tucker, A.S., A. Al Khamis, and P.T. Sharpe, Interactions between Bmp-4 and Msx-1 act to restrict gene expression to odontogenic mesenchyme. Dev Dyn, 1998. 212(4): p. 533-9. 121. Lamb, T.M., et al., Neural induction by the secreted polypeptide noggin. Science, 1993. 262(5134): p. 713-8. 122. Timmer, J.R., C. Wang, and L. Niswander, BMP signaling patterns the dorsal and intermediate neural tube via regulation of homeobox and helix-loop-helix transcription factors. Development, 2002. 129(10): p. 2459-72. 123. James, R.G. and T.M. Schultheiss, Bmp signaling promotes intermediate mesoderm gene expression in a dose-dependent, cell-autonomous and translation-dependent manner. Dev Biol, 2005. 288(1): p. 113-25. 124. Hasegawa, H., et al., Laminar patterning in the developing neocortex by temporally coordinated fibroblast growth factor signaling. J Neurosci, 2004. 24(40): p. 8711- 9. 125. Delfini, M.C., et al., Control of the segmentation process by graded MAPK/ERK activation in the chick embryo. Proc Natl Acad Sci U S A, 2005. 102(32): p. 11343- 8. 126. Louvi, A. and S. Artavanis-Tsakonas, Notch signalling in vertebrate neural development. Nat Rev Neurosci, 2006. 7(2): p. 93-102. 127. Yoon, K. and N. Gaiano, Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nat Neurosci, 2005. 8(6): p. 709-15.

59 References

128. Abello, G., et al., Early regionalization of the otic placode and its regulation by the Notch signaling pathway. Mech Dev, 2007. 124(7-8): p. 631-45. 129. Machold, R.P., D.J. Kittell, and G.J. Fishell, Antagonism between Notch and bone morphogenetic protein receptor signaling regulates neurogenesis in the cerebellar rhombic lip. Neural Dev, 2007. 2: p. 5. 130. Lekven, A.C., et al., Zebrafish wnt8 encodes two wnt8 proteins on a bicistronic transcript and is required for mesoderm and neurectoderm patterning. Dev Cell, 2001. 1(1): p. 103-14. 131. Alvarez, I.S., M. Araujo, and M.A. Nieto, Neural induction in whole chick embryo cultures by FGF. Dev Biol, 1998. 199(1): p. 42-54. 132. Cox, W.G. and A. Hemmati-Brivanlou, Caudalization of neural fate by tissue recombination and bFGF. Development, 1995. 121(12): p. 4349-58. 133. Storey, K.G., et al., Early posterior neural tissue is induced by FGF in the chick embryo. Development, 1998. 125(3): p. 473-84. 134. Sun, X., et al., Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev, 1999. 13(14): p. 1834-46. 135. Niehrs, C., Head in the WNT: the molecular nature of Spemann's head organizer. Trends Genet, 1999. 15(8): p. 314-9. 136. Yamaguchi, T.P., Heads or tails: Wnts and anterior-posterior patterning. Curr Biol, 2001. 11(17): p. R713-24. 137. Chapman, S.C., et al., Expression analysis of chick Wnt and frizzled genes and selected inhibitors in early chick patterning. Dev Dyn, 2004. 229(3): p. 668-76. 138. Hume, C.R. and J. Dodd, Cwnt-8C: a novel Wnt gene with a potential role in primitive streak formation and hindbrain organization. Development, 1993. 119(4): p. 1147-60. 139. Karabagli, H., et al., Comparison of the expression patterns of several fibroblast growth factors during chick gastrulation and neurulation. Anat Embryol (Berl), 2002. 205(5-6): p. 365-70. 140. Crossley, P.H. and G.R. Martin, The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development, 1995. 121(2): p. 439-51. 141. Pera, E.M., et al., Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction. Genes Dev, 2003. 17(24): p. 3023-8. 142. Swindell, E.C., et al., Complementary domains of retinoic acid production and degradation in the early chick embryo. Dev Biol, 1999. 216(1): p. 282-96. 143. Hochgreb, T., et al., A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field. Development, 2003. 130(22): p. 5363-74. 144. Berggren, K., et al., Differential distribution of retinoic acid synthesis in the chicken embryo as determined by immunolocalization of the retinoic acid synthetic enzyme, RALDH-2. Dev Biol, 1999. 210(2): p. 288-304. 145. Guidato, S., C. Barrett, and S. Guthrie, eds. Patterning of motor neurons by retinoic acid in the chick embryo hindbrain in vitro. Mol Cell Neurosci. Vol. 23. 2003. 81- 95.

60 References

146. White, J.C., et al., Vitamin A deficiency results in the dose-dependent acquisition of anterior character and shortening of the caudal hindbrain of the rat embryo. Dev Biol, 2000. 220(2): p. 263-84. 147. Gale, E., M. Zile, and M. Maden, Hindbrain respecification in the retinoid- deficient quail. Mech Dev, 1999. 89(1-2): p. 43-54. 148. White, J.A., et al., Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase. J Biol Chem, 1996. 271(47): p. 29922-7. 149. Ray, W.J., et al., CYP26, a novel mammalian cytochrome P450, is induced by retinoic acid and defines a new family. J Biol Chem, 1997. 272(30): p. 18702-8. 150. Fujii, H., et al., Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos. Embo J, 1997. 16(14): p. 4163-73. 151. Abu-Abed, S., et al., The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures. Genes Dev, 2001. 15(2): p. 226-40. 152. MacLean, G., et al., Cloning of a novel retinoic-acid metabolizing cytochrome P450, Cyp26B1, and comparative expression analysis with Cyp26A1 during early murine development. Mech Dev, 2001. 107(1-2): p. 195-201. 153. Tahayato, A., P. Dolle, and M. Petkovich, Cyp26C1 encodes a novel retinoic acid- metabolizing enzyme expressed in the hindbrain, inner ear, first branchial arch and tooth buds during murine development. Gene Expr Patterns, 2003. 3(4): p. 449-54. 154. Hollemann, T., et al., Regionalized metabolic activity establishes boundaries of retinoic acid signalling. Embo J, 1998. 17(24): p. 7361-72. 155. de Roos, K., et al., Expression of retinoic acid 4-hydroxylase (CYP26) during mouse and Xenopus laevis embryogenesis. Mech Dev, 1999. 82(1-2): p. 205-11. 156. Chen, Y., et al., Increased XRALDH2 activity has a posteriorizing effect on the central nervous system of Xenopus embryos. Mech Dev, 2001. 101(1-2): p. 91-103. 157. Blentic, A., E. Gale, and M. Maden, Retinoic acid signalling centres in the avian embryo identified by sites of expression of synthesising and catabolising enzymes. Dev Dyn, 2003. 227(1): p. 114-27. 158. Reijntjes, S., E. Gale, and M. Maden, Generating gradients of retinoic acid in the chick embryo: Cyp26C1 expression and a comparative analysis of the Cyp26 enzymes. Dev Dyn, 2004. 230(3): p. 509-17. 159. Dobbs-McAuliffe, B., Q. Zhao, and E. Linney, Feedback mechanisms regulate retinoic acid production and degradation in the zebrafish embryo. Mech Dev, 2004. 121(4): p. 339-50. 160. Emoto, Y., et al., Retinoic acid-metabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal cord in zebrafish. Dev Biol, 2005. 278(2): p. 415-27. 161. Sakai, Y., et al., The retinoic acid-inactivating enzyme CYP26 is essential for establishing an uneven distribution of retinoic acid along the anterio-posterior axis within the mouse embryo. Genes Dev, 2001. 15(2): p. 213-25. 162. Grandel, H., et al., Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud. Development, 2002. 129(12): p. 2851-65.

61 References

163. Diez del Corral, R., et al., Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension. Neuron, 2003. 40(1): p. 65-79. 164. Diez del Corral, R. and K.G. Storey, Opposing FGF and retinoid pathways: a signalling switch that controls differentiation and patterning onset in the extending vertebrate body axis. Bioessays, 2004. 26(8): p. 857-69. 165. Stern, C.D., et al., Head-tail patterning of the vertebrate embryo: one, two or many unresolved problems? Int J Dev Biol, 2006. 50(1): p. 3-15. 166. Olivera-Martinez, I. and K.G. Storey, Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension. Development, 2007. 134(11): p. 2125-35. 167. Delfino-Machin, M., et al., Specification and maintenance of the spinal cord stem zone. Development, 2005. 132(19): p. 4273-83. 168. Ericson, J., et al., Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell, 1997. 90(1): p. 169-80. 169. Sockanathan, S., T. Perlmann, and T.M. Jessell, Retinoid receptor signaling in postmitotic motor neurons regulates rostrocaudal positional identity and axonal projection pattern. Neuron, 2003. 40(1): p. 97-111. 170. Gaufo, G.O., K.R. Thomas, and M.R. Capecchi, Hox3 genes coordinate mechanisms of genetic suppression and activation in the generation of branchial and somatic motoneurons. Development, 2003. 130(21): p. 5191-201. 171. Bell, E., R.J. Wingate, and A. Lumsden, Homeotic transformation of rhombomere identity after localized Hoxb1 misexpression. Science, 1999. 284(5423): p. 2168- 71. 172. Gavalas, A., et al., Neuronal defects in the hindbrain of Hoxa1, Hoxb1 and Hoxb2 mutants reflect regulatory interactions among these Hox genes. Development, 2003. 130(23): p. 5663-79. 173. Deschamps, J., et al., Initiation, establishment and maintenance of Hox gene expression patterns in the mouse. Int J Dev Biol, 1999. 43(7): p. 635-50. 174. Marom, K., E. Shapira, and A. Fainsod, The chicken caudal genes establish an anterior-posterior gradient by partially overlapping temporal and spatial patterns of expression. Mech Dev, 1997. 64(1-2): p. 41-52. 175. Shimizu, T., et al., Interaction of Wnt and caudal-related genes in zebrafish posterior body formation. Dev Biol, 2005. 279(1): p. 125-41. 176. Shimizu, T., Y.K. Bae, and M. Hibi, Cdx-Hox code controls competence for responding to Fgfs and retinoic acid in zebrafish neural tissue. Development, 2006. 133(23): p. 4709-19. 177. Trainor, P.A. and R. Krumlauf, Patterning the cranial neural crest: hindbrain segmentation and Hox gene plasticity. Nat Rev Neurosci, 2000. 1(2): p. 116-24. 178. Creuzet, S., G. Couly, and N.M. Le Douarin, Patterning the neural crest derivatives during development of the vertebrate head: insights from avian studies. J Anat, 2005. 207(5): p. 447-59. 179. Cau, E., et al., Mash1 activates a cascade of bHLH regulators in olfactory neuron progenitors. Development, 1997. 124(8): p. 1611-21.

62 References

180. De Robertis, E.M. and H. Kuroda, Dorsal-Ventral Patterning and Neural Induction in Xenopus Embryos. Annu Rev Cell Dev Biol, 2004. 181. Londin, E.R., J. Niemiec, and H.I. Sirotkin, Chordin, FGF signaling, and mesodermal factors cooperate in zebrafish neural induction. Dev Biol, 2005. 279(1): p. 1-19. 182. Wilson, S.I. and T. Edlund, Neural induction: toward a unifying mechanism. Nat Neurosci, 2001. 4 Supp 1: p. 1161-8. 183. Morrison, S.J., et al., Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell, 2000. 101(5): p. 499-510. 184. Lemaire, L. and M. Kessel, Gastrulation and homeobox genes in chick embryos. Mech Dev, 1997. 67(1): p. 3-16. 185. Bonaguidi, M.A., et al., Noggin expands neural stem cells in the adult hippocampus. J Neurosci, 2008. 28(37): p. 9194-204. 186. Alsina, B., et al., FGF signaling is required for determination of otic neuroblasts in the chick embryo. Dev Biol, 2004. 267(1): p. 119-34. 187. Chen, B., E.H. Kim, and P.X. Xu, Initiation of olfactory placode development and neurogenesis is blocked in mice lacking both Six1 and Six4. Dev Biol, 2009. 326(1): p. 75-85. 188. Yoon, B.S., et al., BMPs regulate multiple aspects of growth-plate chondrogenesis through opposing actions on FGF pathways. Development, 2006. 133(23): p. 4667-78. 189. Rice, R., I. Thesleff, and D.P. Rice, Regulation of Twist, Snail, and Id1 is conserved between the developing murine palate and tooth. Dev Dyn, 2005. 234(1): p. 28-35. 190. Neubuser, A., et al., Antagonistic interactions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell, 1997. 90(2): p. 247-55. 191. Minina, E., et al., Interaction of FGF, Ihh/Pthlh, and BMP signaling integrates chondrocyte proliferation and hypertrophic differentiation. Dev Cell, 2002. 3(3): p. 439-49. 192. Eivers, E., L.C. Fuentealba, and E.M. De Robertis, Integrating positional information at the level of Smad1/5/8. Curr Opin Genet Dev, 2008. 18(4): p. 304- 10. 193. Dahn, R.D. and J.F. Fallon, Limbiting outgrowth: BMPs as negative regulators in limb development. Bioessays, 1999. 21(9): p. 721-5. 194. Ohkubo, Y., C. Chiang, and J.L. Rubenstein, Coordinate regulation and synergistic actions of BMP4, SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles. Neuroscience, 2002. 111(1): p. 1-17. 195. Zuniga, A., et al., Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds. Nature, 1999. 401(6753): p. 598-602. 196. Storm, E.E., J.L. Rubenstein, and G.R. Martin, Dosage of Fgf8 determines whether cell survival is positively or negatively regulated in the developing forebrain. Proc Natl Acad Sci U S A, 2003. 100(4): p. 1757-62.

63 References

197. Seoane, J., et al., Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell, 2004. 117(2): p. 211-23. 198. Dou, C., et al., BF-1 interferes with transforming growth factor beta signaling by associating with Smad partners. Mol Cell Biol, 2000. 20(17): p. 6201-11. 199. Bourguignon, C., J. Li, and N. Papalopulu, XBF-1, a winged helix transcription factor with dual activity, has a role in positioning neurogenesis in Xenopus competent ectoderm. Development, 1998. 125(24): p. 4889-900. 200. Hanashima, C., et al., Foxg1 suppresses early cortical cell fate. Science, 2004. 303(5654): p. 56-9. 201. Xuan, S., et al., Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres. Neuron, 1995. 14(6): p. 1141-52. 202. Duggan, C.D., et al., Foxg1 is required for development of the vertebrate olfactory system. J Neurosci, 2008. 28(20): p. 5229-39. 203. Baker, N.E., Notch signaling in the nervous system. Pieces still missing from the puzzle. Bioessays, 2000. 22(3): p. 264-73. 204. Corbin, J.G., et al., Regulation of neural progenitor cell development in the nervous system. J Neurochem, 2008. 106(6): p. 2272-87. 205. Wheeler, S.R., S.B. Stagg, and S.T. Crews, Multiple Notch signaling events control Drosophila CNS midline neurogenesis, gliogenesis and neuronal identity. Development, 2008. 135(18): p. 3071-9. 206. Lutolf, S., et al., Notch1 is required for neuronal and glial differentiation in the cerebellum. Development, 2002. 129(2): p. 373-85. 207. de la Pompa, J.L., et al., Conservation of the Notch signalling pathway in mammalian neurogenesis. Development, 1997. 124(6): p. 1139-48. 208. Itoh, F., et al., Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells. Embo J, 2004. 23(3): p. 541-51. 209. Wolpert, L., et al., Entwicklungsbiologie. 1st edition, Spektrum akademischer Verlag, 1999. 210. Gilbert, SF., Developmental Biology. 6th edition, Sinauer, 2000.

64 Supplementary Figures

Supplementary Figures

Suppl Fig 1:

Suppl Figure 1: Graded FGF signalling is sufficient to impose progressively more caudal character in the presence of high levels of Wnt signalling (A) Schematic graph of a stage 4 embryo, red box indicates the explant region in the presumptive forebrain. (B-D) Sox2 was used as a general neural marker (B) Control stage 4 FB explants generated Sox2+/Otx2+ cells (C) Stage 4 FB explants cultured in the presence of FGF (15ng/ml) and Wnt3A (100µl/ml) generated Krox20+ cells and Hoxb4+/Hoxb8+/Hoxc9+ cells. (D) Stage 4 FB explants cultured in the presence of FGF (60ng/ml) and Wnt3A (100µl/ml) generated predominantly Hoxb4+/Hoxb8+/Hoxc9+ cells and only few Krox20+ cells. (Figure previously published in “Early Rostrocaudal patterning of the CNS” by U. Nordström, 2005)

65 Supplementary Figures

Suppl Fig 2:

Suppl Figure 2: FGF signalling is required to impose caudal neural character in the presence of Noggin (A) Schematic graph of a stage 4 embryo, red box indicates the explant region in the presumptive forebrain. (B-D) Sox2 was used as a general neural marker. (B) Control stage 4 FB explants generated Sox2+/Otx2+ cells (C) Stage 4 FB explants exposed to Wnt (100µl/ml) and Noggin (200µl/ml) generated Hoxb8+ cells. (D) Stage 4 FB explants exposed to Wnt (100µl/ml), Noggin (200µl/ml) and SU5402 (5µM) generated Sox2+/Otx2+ cells.

66 Supplementary Figures

Suppl Fig 3:

Suppl Figure 3: Expression of BMP4, Id3 and Msx1/2 in a stage 22 embryo. Schematic graph of a stage 22 embryo, indicating the plane of section. (A) DAPI stained overview of a section, the boxed area indicates the olfactory pit, blown-up in the panels B- D. (B) Expression of BMP4 can be detected at medio-posterior positions in the rim of the olfactory pit. (C) Expression of Id3 can be detected in the rim of the olfactory pit and is absent in the anterior and the superior region. (D) Expression of Msx1/2 can be detected in the rim of the olfactory pit and is absent in anterior and superior positions.