Induction of the Isthmic Organizer and Specification of the Neural Plate Border
Cédric Patthey
Umeå Center for Molecular Medicine Umeå University, Umeå Sweden 2008
1 Front Cover: Photomontage showing how neural, neural crest and epidermal cells are already specified in the blastula stage chick embryo. Explants were taken as indicated in the ectoderm, cultured for 28 hr and 8µm sections were stained for Sox1 (red), HNK-1 (Green) or Cytokeratins (Blue).
Copyright © Cédric Patthey ISBN: 978-91-7264-603-2 ISSN: 0346-6612 New Series No: 1197 Printed by Print & Media Umeå, Sweden, 2008
2
To Annika
“Let us seek with the desire to find, and find with the desire to seek still more.” Saint Augustine
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Table of contents
Abstract...... 6 Papers in this thesis...... 8 Abbreviations ...... 9 Introduction...... 10 Signaling factors and cell fate specification...... 11 Why focus on the nervous system?...... 13 Description of the nervous system...... 13 Generation of cell diversity in the nervous system ...... 14 The Isthmic Organizer...... 16 The neural plate border ...... 17 Specification of placodal and NC cells ...... 19 Aims...... 21 Methodology ...... 22 Results ...... 23 Convergent Wnt and FGF signaling at the gastrula stage induce the formation of the Isthmic Organizer (Paper I)...... 23 Early development of the central and peripheral nervous systems is coordinated by Wnt and BMP signals (Paper II) ...... 26 Wnt-regulated temporal control of BMP exposure directs the choice between neural plate border and epidermal fate (Paper III)...... 29 Discussion...... 35 A reasonable approach to study early vertebrate development...... 35 The IsO is induced by convergent Wnt and FGF signals ...... 36 Source of IsO-Inducing signals...... 38 Distinct time windows for IsO induction and maintenance...... 39 Mechanism of IsO establishment...... 39 Refinement of Wnt1 and Fgf8 expression domains ...... 42 Context of neural plate border induction ...... 44 Temporal mechanism of Border induction ...... 45 A novel patterning mechanism ...... 47 The BMP gradient model...... 48 Duration of BMP/Wnt signaling...... 49 Intracellular integration of BMP and Wnt signals ...... 50
4 Temporal Shift in response to BMP and Wnt signals...... 51 Border cells derive from neural cells ...... 53 OLP cells derive from cells initially specified as NC cells...... 53 Distinct roles of BMP and Wnt signals in NC cell specification...... 55 Role of FGF signals in border induction...... 56 Common origin of NC and placodal cells...... 57 Induction of other placodes...... 58 Are all NC cells induced simultaneously by the same mechanism?...... 59 Coordination of CNS and PNS development...... 60 Conclusions ...... 62 Acknowledgements...... 63 References...... 65 Supplementary Figures...... 74
5 Abstract
The vertebrate nervous system is extremely complex and contains a wide diversity of cell types. The formation of a functional nervous system requires the differential specification of progenitor cells at the right time and place.
The generation of many different types of neurons along the rostro-caudal axis of the CNS begins with the initial specification of a few progenitor domains. This initial coarse pattern is refined by so-called secondary organizers arising at boundaries between these domains. The Isthmic Organizer (IsO) is a secondary organizer located at the boundary between the midbrain and the hindbrain. Although the function and maintenance of the IsO are well understood, the processes underlying its initial specification have remained elusive. In the present work we provide evidence that convergent Wnt and FGF signals initiate the specification of the IsO during late gastrulation as part of the neural caudalization process.
The initial step in the generation of the nervous system is the division of the embryonic ectoderm into three cell populations: neural cells giving rise to the CNS, neural plate border cells giving rise to the peripheral nervous system, and epidermal cells giving rise to the outer layer of the skin. While the choice between neural and epidermal fate has been well studied, the mechanism by which neural plate border cells are generated is less well understood. At rostral levels of the neuraxis, the neural plate border gives rise to the olfactory and lens placodes, thickenings of the surface ectoderm from which sensory organs are derived. More caudally, the neural plate border generates neural crest cells, a transient population that migrates extensively and contributes to neurons and glia of the peripheral nervous system. How the early patterning of the central and peripheral nervous systems are coordinated has remained poorly understood. Here we show that the generation of neural plate border cells is initiated at the late blastula stage and involves two phases. During the first phase, neural plate border cells are exposed to Wnt signals in the absence of BMP signals. Simultaneous exposure to Wnt and BMP signals at this early stage leads to
6 epidermal induction. Wnt signals induce expression of Bmp4, thereby regulating the sequential exposure of cells to Wnt and BMP signals. During the second phase, at the late gastrula stage, BMP signals play an instructive role to specify neural plate border cells of either placodal or neural crest character depending on the status of Wnt signaling. At this stage, Wnt signals promote caudal character simultaneously in the neural plate border and in the neural ectoderm. Thus, the choice between epidermal and neural plate border specification is mediated by an interplay of Wnt and BMP signals that represents a novel mechanism involving temporal control of BMP activity by Wnt signals. Moreover, the early development of the central and peripheral nervous systems are coordinated by simultaneous caudalization by Wnt signals.
7 Papers in this thesis
I. Olander S, Nordstrom U, Patthey C, Edlund T (2006). Convergent Wnt and FGF signaling at the gastrula stage induce the formation of the Isthmic Organizer. Mechanisms of Development 123, 166-176.
II. Patthey C, Gunhaga L, Edlund T (2008). Early development of the central and peripheral nervous systems is coordinated by Wnt and BMP signals. PLoS ONE 3(2) e1625.
III. Patthey C, Edlund T, Gunhaga L (2008). Wnt-regulated temporal control of BMP exposure directs the choice between neural plate border and epidermal fate. Submitted
8 Abbreviations
B: Border BF-1: Brain factor 1 BMP: Bone morphogenetic protein C: Caudal CB: Caudal Border CNS: Central nervous system En1/2: Engrailed 1/2 FB: Forebrain FGF: Fibroblast growth factor HB: Hindbrain IsO: Isthmic Organizer Ker: Cytokeratins L: Lateral M: Medial MB: Midbrain mFrz8CRD: mouse Frizzled 8 cysteine rich domain MHB: Midbrain-hindbrain boundary OLP: Olfactory and lens placodes PNS: Peripheral nervous system R: Rostral RB: Rostral Border RT-PCR: Reverse transcription-polymerase chain reaction Sfrp: Soluble Frizzled-related protein Shh: Sonic hedgehog TGFβ: Transforming growth factor β Wnt: Wingless/Int
9 Introduction
Introduction
The human body is composed of approximately 1014 cells. Each cell is found at a correct position, exhibits appropriate structural and biochemical properties, and is coordinated within the whole organism. Particularly impressive is the nervous system which gives us the ability to sense and process information from our surroundings and to respond in an appropriate manner, a process involving elaborate cognitive functions including memory, feelings and thinking. But the most marvelous is that all of this complexity derives, at each and every generation, from the development of a single cell: the fertilized zygote.
During the journey that is the development from egg to adult, a great number of cells and cell types are produced. Animal development proceeds by the unfolding of a genetic program. All of the information needed to build the organism is contained within the fertilized egg and deployed step by step, the consequences of each developmental event becoming causes for later increases in complexity.
Although biological diversity is almost infinite in the animal kingdom, animals can be classified in groups sharing basic morphology (phyla). This body plan, as it is called, is established during early development, involving common mechanisms that appear to be strongly conserved across species. Thus, studying early development in any chordate model organism is relevant to understanding how the body plan is laid down – and broadly applicable to humans.
There are two main questions in developmental biology: 1. How is cell diversity achieved? What are the mechanisms and signals that direct a cell to acquire a particular character rather than another? 2. How are tissues patterned? How are cells generated at the right place and in the right temporal order? The work described here addresses these two questions in specific systems.
10 Introduction Signaling factors and cell fate specification Initially, cells share a common undifferentiated state and have the capacity to become any one of many cell types (a property called pluripotency). As development proceeds, groups of cells acquire distinct characteristics, although they may remain plastic and retain multiple potentialities. Groups are divided into subgroups of progenitor cells with restricted potentialities until they become committed to differentiate into a particular cell type.
Diversification of cell types involves differential gene expression. Progenitor cells are specified by expression of particular sets of transcription factors. In animals with regulative development, gene expression is largely under the control of secreted extracellular signals. Historically, the first step toward understanding early development was to identify these factors. It has now become apparent that the generation of many types of progenitor cells is determined by the activity of only a few families of signaling factors: TGFβ/BMP, Wnt, FGF, Retinoids, Hedgehog and Notch.
Elucidating the expression patterns of the above-mentioned signaling molecules has proven useful for investigating how distinct progenitors are specified. However, expression patterns, though informative, are not conclusive. One reason for this is that the diffusion range of signaling factors is largely unknown and interactions with inhibitors make it difficult to deduce the signaling activity of a given morphogen at a particular location. Moreover, the presence of signaling proteins and activation of the cognate signaling pathways cannot be excluded based on expression patterns, because levels of transcripts under the threshold of detection by in situ hybridization could be sufficient to yield significant levels of signals. Thus, there is now a need to map the effects of signaling factors in space and time in order to determine how it might affect the generation of cell diversity.
A central question is how a relatively small number of signaling pathways can specify a much larger number of different cell types. The effects of a particular signal on a cellular substrate may depend on the concentration of the ligand and/or the time of exposure1-6. Signals can also act in a combinatorial manner7-10, although the significance of this is only beginning
11 Introduction to be understood. The response to a particular signal may depend on the presence or absence of other signals. Finally, signaling molecules are reused at different times and places to mediate various fate specification events. Depending on timing and the cell population on which they are acting, signals can operate in a synergistic or antagonistic manner. Generally, the developmental history of a cell determines its response to a particular signal or combination of signals, which means that signaling factors can act in a sequential manner. For a thorough understanding of cell diversification in early development, it is not sufficient to identify the signals involved in the specification of particular cell types. It is also desirable to determine in what sequence signaling factors act upon the cells. In consequence, the key to understanding cell lineage specification lies in the study of how combinations of signaling factors affect cell fate over time.
The acquisition of cell identity proceeds by consecutive steps. Because development is stereotyped, the fate of cells at a defined time and location is determined even before the cells have been instructed to follow that path of differentiation. Fate maps describe the position of cells that will later contribute to particular tissues. The process by which a cell is actually instructed to adopt a particular character is called specification. The specification state of a cell refers to the character this very cell will acquire if isolated and maintained in neutral conditions. In organisms with regulative development, specification usually occurs in response to continued exposure to extracellular signals and is therefore referred to as induction. After a period of ongoing signaling activity, cells become independent of the signals that induced them, while still able to change character in response to alternative signals. As development proceeds, cells become committed as cells of a particular type and lose the ability to change fate in response to extracellular signal. Finally, cells acquire their definitive biochemical and physiological characteristics, a process known as differentiation. Differentiation is almost invariably coupled to cell cycle exit.
12 Introduction Why focus on the nervous system? The debilitating effects of diseases affecting the nervous system testify how essential its proper functioning is to our well-being. Our ability to selectively take in and integrate information from our surroundings and elaborate appropriate behavioral responses is dependent on a functional nervous system. Even our feelings and thoughts have a biological basis in the nervous system, although the source of these is poorly understood. Thus, the nervous system is a particularly important area of study. We can attain a better understanding of how the nervous system functions by determining how it is assembled during embryogenesis. An obligatory first step in that direction is to describe the mechanisms leading to the generation of neural progenitor cells at appropriate positions within the nascent brain, spinal cord and peripheral organs. In addition, deeper knowledge of neural development should make it possible to produce specific types of neural cells in vitro, which could possibly be used to develop therapeutic strategies for treating dysfunctions of the nervous system. The nervous system is also an appropriate system for studying general principles of pattern formation: many markers are available and early patterning of the nervous system occurs in the morphologically simple ectodermal germ layer.
Description of the nervous system In all vertebrates, the nervous system is divided into the central nervous system (CNS) and the peripheral nervous system (PNS). The PNS is composed of sensory cells sensing external and internal physical stimuli, sensory neurons and glia conducting impulses to the CNS, as well as cells of the autonomic and enteric nervous systems. Cells in the PNS are found throughout the body and at its surface. The CNS is composed of interneurons, motoneurons, and glia. It receives and integrates inputs from the PNS and elaborates a response to maintain homeostasis and control appropriate behaviors. Cells in the CNS are gathered into a continuous internal structure comprising the brain and spinal cord.
In the embryo, the developing CNS is subdivided along the rostro-caudal (antero-posterior or head to tail) axis into five primitive regions: telencephalon, diencephalon, midbrain, hindbrain and spinal cord. These
13 Introduction different regions will generate distinct types of neurons with specific physiologies and connectivities. Similarly, in the developing PNS, several types of cells are generated at specific positions along the rostro-caudal axis11.
Generation of cell diversity in the nervous system The entire nervous system is derived from the ectoderm, which also gives rise to the epidermis. The very first step in the generation of the nervous system is the segregation of neural plate cells from epidermal cells, which give rise to the CNS and the skin, respectively. This process, commonly called neural induction, occurs at the early blastula stage, before the onset of gastrulation12. The molecular nature and interactions of signals operating during the neural induction process are well established12. FGF signals act to specify neural cells and BMP signals specify epidermal cells. To make the system bistable, BMP and FGF act in a double-negative reciprocal feedback loop, by repressing each other’s expression. In addition, Wnt signals regulate the choice between neural and epidermal fates by blocking the response of epiblast cells to FGF signals, thereby permitting BMP signals to induce an epidermal fate. Finally, at later stages, BMP antagonists secreted by the dorsal/axial mesoderm help to maintain and stabilize neural character13.
The PNS derives from the border between the neural plate and the epidermal ectoderm (Figure 1). Both the time at, and the mechanism by, which neural plate border cells are segregated from neural plate cells and epidermal cells have remained unknown.
14 Introduction
Figure 1: Ectodermal cell types. Schematic illustrations of chick embryos at the late gastrula stage (stage 4) representing the prospective neural plate (a), the neural plate border (b) and the epidermal ectoderm (c).
The position of progenitor cells along the rostro-caudal and dorso-ventral (back to belly) axes determines the combination of signaling factors they are exposed to, which in turn dictates cellular identity. After neural cells have been specified during the process of neural induction, the CNS is subdivided into rostro-caudal domains containing cells expressing particular combinations of transcription factors14. As proposed by Nieuwkoop in his Activation-Transformation model15, all cells of the neural plate are initially specified (activation) as cells of rostral (anterior) forebrain character. Cells of the midbrain, hindbrain and spinal cord are then caudalized (transformation) and cells of the forebrain maintain a rostral character. In most species studied, caudal (posterior) character is imparted by signals emanating from the blastopore (the streak in amniotes) and the mesoderm that derives from it. The closer cells are to the source of the signal, the more caudalized they will become. In chick, graded levels of Wnt signals together with permissive FGF signals specify caudal forebrain, midbrain, rostral hindbrain and caudal spinal cord during the early phase of caudalization at the late gastrula stage3. Antagonists of Wnt signals, such as Dickkopf-1 and Sfrp, are active in the forebrain region to prevent caudalization16. Later, at the neural plate stage, retinoic acid produced in the paraxial mesoderm enters the scene to specify cells of caudal hindbrain and rostral spinal cord character in interplay with FGF signals10.
15 Introduction The Isthmic Organizer The initial rostro-caudal patterning that occurs during late gastrulation subdivides the neural plate into a few gross domains. However, dozens of different cell types are generated at different levels of the CNS. The signals that direct the cells to alternative pathways of differentiation emanate from local sources called secondary organizers. Secondary organizers appear at boundaries: the Isthmic Organizer (IsO) is found at the boundary between the midbrain and the hindbrain, the zona limitans intrathalamica at the boundary between the rostral forebrain and caudal forebrain, and the anterior neural ridge at the rostral edge of the CNS.
The IsO is one of the best-described secondary organizers. Located at the midbrain-hindbrain boundary (MHB), the IsO can be defined as a group of cells secreting the signaling factors FGF8 and Wnt1. Other Wnt and FGF family members are also expressed in the IsO. FGF signals emanating from the IsO are important for the specification of dopaminergic and serotonergic cells as well as polarity in the tectum and cerebellum17.
The IsO is characterized by a complex pattern of gene expression. Early during caudalization, the neuro-ectoderm is divided into rostral and caudal regions expressing the transcription factors Otx2 and Gbx2, respectively18. In the HH stage 10 embryo19, the Otx2-Gbx2 boundary is well established and sharp and defines the MHB. At that stage, Otx2 is expressed in the entire forebrain and midbrain and Gbx2 is expressed in the rostral hindbrain (rhombomere r1). Expression of the transcription factors Pax2 and Engrailed-1/2 (En1/2) in the midbrain and rostral hindbrain broadly define the MHB-region. The IsO appears as a thin band of Wnt1-expressing cells in the Otx2+ domain and an adjacent thin band of Fgf8-expressing cells in the Gbx2+ domain (Figure 2).
16 Introduction
Figure 2: Expression pattern of genes characteristic of the IsO. Schematic image of a 10 somite (stage 10) chick embryo showing expression of genes characteristic of the Isthmic Organizer (IsO). The IsO is defined by the cells expressing Wnt1 and Fgf8 and is located at the midbrain-hindbrain boundary (MHB). Otx2 is expressed in the forebrain (FB) and midbrain (MB). Gbx2 is expressed in the rostral hindbrain (HB). Pax2 and En1/2 are expressed in a region that spans the MHB.
The refinement and maintenance of the IsO is under the control of intricate genetic interactions between the transcription factors En1/2, Pax2, Otx2, Gbx2, and the secreted molecules Wnt1 and FGF8. However, the identity of the extracellular signals that lead to initial induction of Fgf8 and Wnt1- expressing cells at the IsO has remained unidentified.
The neural plate border The PNS has a dual embryonic origin from the neural crest (NC) and the cranial placodes. Both arise at the junction between the neural and the epidermal ectoderm, the neural plate border (from here referred to simply as Border). The neural crest is a cell population that appears in the Border region at the level of the mid-diencephalon and caudally (epichordally), but not at rostral forebrain levels (prechordally). NC cells gives rise to a vast array of cell types, including neurons and glia from the PNS, smooth muscle cells, osteoblasts and chondroblasts, endocrine cells of the adrenal medulla, and pigmented cells of the dermis. NC progenitors delaminate from the ectoderm and migrate extensively to colonize target organs. Cranial
17 Introduction placodes are thickenings of the non-neural ectoderm contributing cells to sensory organs and cranial nerve ganglia in the head. Adjacent to the forebrain, the hypophyseal (pituitary), olfactory and lens placodes are formed. More caudally, in the hindbrain region, the trigeminal, otic (statoacoustic) and epibranchial placodes are generated lateral to the domain where NC cells are produced (Figure 3).
Figure 3: Neural crest and placodes. Schematic drawing of the head region of a 4-somites (stage 8) embryo showing the location of prospective cranial placodes (grey) and the neural crest (black), based on diverse fate maps. The neural fold does not produce NC cells at forebrain level.
Neural character is specified by FGF signals in the absence of Wnt and BMP signals, while it is firmly established that epidermal character is specified by convergent Wnt and BMP signaling12,20. However, BMP and Wnt signals are also involved in the generation of NC cells. Thus, how ectodermal cells become Border cells in response to Wnt and BMP signals without acquiring epidermal character has remained a mystery. It has been suggested that both placodal cells and NC cells are induced by intermediate BMP levels21-23, but at present there is no conclusive evidence that intermediate levels of BMP signaling are sufficient to induce a Border.
18 Introduction Specification of placodal and NC cells At the late gastrula stage, Border cells at forebrain levels are specified as olfactory and lens placodal (OLP) cells, while more caudal Border cells are specified as NC cells1,24. Around that stage, transcription factors of the Pax3/7 and Zic families start to be expressed medially in the ectoderm while Msx1/2 and Dlx3/5 are expressed laterally. The Border is believed to be specified by the overlapping expression of these transcription factors25. Yet, our understanding of the mechanism by which extracellular signals instruct ectodermal cells to become Border cells has remained rudimentary.
The neural crest represents a classical example of embryonic induction. That is, NC formation is thought to result as the consequence of secreted signals from the adjacent epidermis and/or paraxial mesoderm, because these tissues are sufficient to promote the generation of NC cells when ectopically located or in vitro. The mechanism by which cells from the Border region are instructed to become NC cells is known to involve Wnt and BMP signals26.
The first evidence that BMP signaling is involved in NC specification was the demonstration that BMP4 is sufficient to re-specify spinal cord neural cells as NC cells27,28. The role of BMP signals in the choice between neural and epidermal character being firmly established, it was natural to suggest that the NC located in-between would be induced by intermediate levels of BMP signaling, and this theory has been supported by animal cap experiments in the frog Xenopus laevis and analysis of zebrafish mutants21- 23,29,30.
Both gain- and loss-of-function experiments in Xenopus and zebrafish pointed to a role of Wnt signaling in NC specification (reviewed in ref. 31 and 32) However, it has been debated whether Wnt acts directly in NC induction or via caudalization of the neuro-ectoderm33,34. In amniotes, it has been claimed that Wnt1 can induce NC cells in the chick spinal cord35. Others have suggested that Wnt is involved in the choice between placodal and NC paths of differentiation36-38.
19 Introduction In summary, reduction of BMP or Wnt signaling leads to defects in NC formation, and overexpression of either signal results in ectopic induction of NC markers. However, the specific roles of Wnt and BMP signals in NC specification have remained ill-defined.
20 Aims
Aims This thesis studies the way in which signaling factors of the Wnt, BMP and FGF families interact to differentially specify cell types in the embryonic ectoderm.
The specific aims of this thesis are:
• To determine at what stage the Isthmic Organizer is induced
• To define the signals responsible for the initial induction of the Isthmic Organizer
• To determine at what stage the neural plate border is initially specified
• To determine how Wnt and BMP signals are integrated to differentially specify neural plate border and epidermal cells
• To determine the specific roles of BMP and Wnt signals in the generation of NC and placodal cells
21 Methodology
Methodology
Embryonic development is an intricate process and involves numerous interactions. When manipulating embryos in vivo, cross-regulatory events between different cell types can influence results. Moreover, the potential for redundancy makes genetic studies difficult to interpret.
Signaling factors of the same family are often reused during development. A signal can be interpreted differently by the same cell even within a relatively short period of development. It follows that an appropriate time resolution is required to study how signals interact to specify cell fates and to get meaningful insight into the numerous developmental mechanisms involving timing.
In the present work, to circumvent such difficulties, ex vivo explant assays of cell fate specification in chick were designed. The strength of the method stems from the fact that a piece of tissue can be cleanly dissected and isolated with the exclusion of non-desired cells types. In such a simplified system, indirect effects can be ruled out. Tools are available that reliably suppress signaling pathways regardless of genetic redundancy. The activity of the signaling pathways can be controlled by incubation in the presence of defined concentrations of (natural) activators or inhibitors of signaling pathways. Such an agonistic or antagonistic pressure usually leads to a clear-cut response, which is not often the case in vivo. Furthermore, signals and antagonists can be tested in various combinations and at graded concentrations or durations of exposure. Most importantly, signaling pathways can be activated/inhibited almost instantly at precise stages of development and at defined time during culture. Contrary to other approaches, the biological time is considered: the embryos are staged by morphology. Finally, the fate acquired by the cells can be accurately monitored by analyzing the expression of a collection of markers on consecutive sections by immunohistochemistry and/or in situ hybridization.
22 Results
Results
Convergent Wnt and FGF signaling at the gastrula stage induce the formation of the Isthmic Organizer (Paper I)
The IsO is induced at the late gastrula stage By the late gastrula stage, the neural plate of chick embryos is divided into regions of forebrain, midbrain, hindbrain and spinal cord precursors3,39. Later, during somitogenesis, this coarse pattern is refined by signals emanating from the IsO appearing at the boundary between the midbrain and hindbrain. However, it is not known at what stage the IsO itself is induced. Expression of genes characteristic of the MHB region such as Pax2, En1/2, Wnt1 and Fgf8 is initiated almost synchronously during early somitogenesis, indicating that the inducing process must occur at that stage, or earlier. At stage 4, the midbrain and hindbrain primordia are specified and the ectoderm is already divided into an Otx2 expressing region and a Gbx2-expressing region18. Moreover, explants spanning the whole extent of the rostro-caudal axis generate a fine pattern of neural cells including sharply segregated midbrain and rostral hindbrain, and even distinct rhombomeres 3 and 540. This prompted us to assess whether the IsO is already specified at the late gastrula stage. Because the formation of the fine pattern of cells characteristic of the IsO may require long range planar signals, we first isolated large explants that span most of the rostro-caudal extent of the stage 4 neural plate. Cultured to the equivalent of a stage 10 embryo, such explants generated two thin bands of Fgf8 and Wnt1- expressing cells, centered in a region of Pax2+ and En1/2+ cells - a profile characteristic of the IsO. In contrast, explants isolated at stage 3+ or earlier generated only cells of forebrain character.
To test whether long range signals are required for the formation of the IsO, small explants restricted to the prospective MHB region were isolated and cultured. Such explants generated a region of Otx2+/Wnt1+ cells and an adjacent region of Gbx2+/Fgf8+ cells. Pax2+ and En1/2+ cells spanned these domains. Thus, although the Wnt1 and Fgf8 domains became somewhat broader in small explants than in larger explants, the complex pattern of
23 Results gene expression characteristic of the IsO was generated in MHB explants. In conclusion, the prospective MHB region has got sufficient positional information by stage 4 to produce the IsO independently of signals from non-neural sources or long range signals within the neural plate. Thus, the IsO is induced at stage 4, the late gastrula stage.
IsO induction requires Wnt and FGF signals Previous results have shown that convergent Wnt and FGF signals mediate the acquisition of midbrain and hindbrain character in the caudal neural plate3. To test whether the same signals are required for IsO induction, we cultured stage 4 MHB explants in the presence of mFrz8CRD, a dominant negative soluble form of the Wnt receptor Frizzled41, or in the presence of SU5402, a pharmacological inhibitor of FGF receptor activation42. As expected, when either Wnt or FGF signals were blocked, cells acquired forebrain character and no cells expressed IsO markers. In order to define better the period, during which FGF and Wnt are required, we aimed to test when the formation of the pattern of gene expression characteristic of the IsO becomes independent of Wnt and FGF signals. MHB explants isolated at stage 6 and exposed to mFrz8CRD or SU5402 still generated an IsO, including Wnt1+ and Fgf8+ cells. Thus, Wnt and FGF signals are required for the initial specification of the IsO and IsO induction is ongoing between stage 3+ and 5.
Convergent Wnt and FGF signals are sufficient to produce an IsO Our results strongly suggest that long range signals within the neural plate or signals from non-neural sources are not required for IsO specification after the MHB region has been caudalized. Are the caudalizing Wnt and FGF signals sufficient to induce a complete IsO, or are other signals also required? To address this question, we isolated non-caudalized cells from the prospective forebrain and exposed them to Wnt and FGF signals in combination. Stage 4 forebrain explants cultured in the presence of Wnt3A (~100ng/ml) and FGF4 (15ng/ml) generated Wnt1+/Otx2+ cells in the core of the explant and Fgf8+/Gbx2+ cells at the periphery, while Pax2 and En1/2 where expressed throughout the explant. Thus, Wnt3A and FGF4 in
24 Results combination are sufficient to reconstruct the complex pattern of gene expression characteristic of the IsO.
The IsO was discovered for its ability to ectopically induce structures characteristic of the MHB when grafted into the forebrain-midbrain region43. To address whether the IsO-like structure generated in vitro by exposure to FGF and Wnt signals possessed organizing activity, we isolated explants in the prospective forebrain of stage 4 quails and cultured them in the presence of Wnt3A (~100ng/ml) and FGF4 (15ng/ml) for 24 hours in order to allow the generation of the pattern of cells characteristic of the IsO. These explants were then recovered and grafted into the caudal forebrain of stage 10 chick embryos. The chimera was cultured in ovo until stage 17 and the expression of MHB markers was analyzed by immunohistochemistry. Explants cultured with Wnt3A and FGF4 induced expression of En1/2 and Pax2 in adjacent host tissue, but explants cultured alone or with FGF4 only did not. These results imply that convergent Wnt and FGF signals are sufficient to induce a complete and functional IsO in non-caudalized cells.
25 Results
Early development of the central and peripheral nervous systems is coordinated by Wnt and BMP signals (Paper II)
NC specification is ongoing at the late gastrula stage Recent results have provided evidence that OLP cells are being specified during the late gastrula stage and that the specification of NC cells is also ongoing at the same stage1,24. In the stage 10 embryo, NC cells are defined by expression of Snail2 (previously known as Slug) and/or HNK1. Sox1 is expressed exclusively in definitive neural cells of the neural tube and cytokeratins (Ker) are expressed in the epidermal ectoderm and cranial placodes (Table 1a). At stage 17, cells of the olfactory placode co-expresses Raldh3 and Ker, while cells of the lens placode co-express δ-crystallin and Ker (Table 1b). In order to study the signals involved in early development of the Border, we cultured explants from the rostral Border (RB) and caudal Border (CB). The identity of the cells was examined by assessing expression of Sox1, Snail2, HNK-1, Ker after 22 hr of culture (equivalent to stage 10) and/or Raldh3, δ-crystallin and Ker after 42 hr of culture (equivalent to stage 17).
Stage 4 CB explants generated NC cells but no placodal cells. In contrast, RB explants generated OLP progenitor cells but no NC cells. The generation of OLP or NC cells occurred in the absence of mesodermal, neural or epidermal cells (data not shown). Thus, NC cells are specified in the caudal, but not rostral Border at the late gastrula stage. a. Stage 10 b. Stage 17
Sox1 Sox1 Sox2 Snail2 HNK-1 Ker Neural + + - - - -cryst -cryst NC - - + + - Raldh3 δ Ker Epidermal - - - - + olfactory + - + Placodal - + - - + Lens - + + Table 1: Marker profiles for ectodermal cell types.
26 Results Both BMP and Wnt signals are required for NC specification BMP signals have been suggested to induce both placodal (olfactory and lens) cells rostrally and NC cells caudally. Wnt, on the other hand is a well known caudalizing signal, but it has also been suggested to contribute to NC induction. In order to gain insight into the respective roles of BMP and Wnt signals in rostro-caudal patterning of the Border and NC induction, we analyzed the fate adopted by prospective NC cells when exposed to inhibitors of the BMP and Wnt pathways. When BMP signaling was suppressed by application of Noggin (~25ng/ml), a specific antagonist of BMP signals44, the generation of NC cells was blocked and the cells acquired a caudal neural character instead, as shown by co-expression of Sox1 and Pax2 (Supplementary Figure 1). When Wnt signals were suppressed by mFrz8CRD, a soluble form of the Frizzled receptor, the generation of NC cells was also blocked, but this time cells acquired olfactory and lens placodal character.
Wnt signals caudalize the Border The finding that prospective NC cells acquire OLP character when Wnt signals are suppressed indicates that Wnt signaling caudalizes the Border. To test this hypothesis, we exposed stage 4 RB to Wnt3A (~50ng/ml) in order to activate Wnt signaling. In such conditions, the generation of OLP cells was blocked and NC cells were generated instead. Thus, Wnt signals are both required and sufficient to promote the acquisition of NC character at the expense of OLP character, i.e. for caudalization of the Border.
BMP signals instruct caudal cells to become NC instead of neural ectoderm Our BMP loss-of-function experiments suggest that BMP signals are responsible for induction of NC at the expense of caudal neural tissue. However, it has been proposed in previous studies that both Wnt and BMP signals can induce NC cells ectopically in caudal neural ectoderm35,45. Therefore, we tested the ability of Wnt and BMP signals to induce NC cells in the caudal neural plate of stage 4 embryos. In stage 4 explants exposed to Wnt3A (50-200ng/ml), nuclear β-catenin was increased, confirming activation of the canonical Wnt pathway (Supplementary Figure 2). However, no NC cells were generated at any concentration tested and the
27 Results cells maintained a neural state. In contrast, BMP4 (5ng/ml) blocked the generation of neural cells and induced NC cells. Similar results were obtained in caudal (C) neural explants isolated at intermediate dorso-ventral levels of the spinal cord at stage 10 (Supplementary Figure 3). Thus, BMP signals, but not Wnt signals, are required and sufficient to induce NC cells at the expense of neural cells at caudal levels of the neuraxis.
BMP signals block neural character and Wnt signals caudalize the ectoderm Previous studies have provided evidence that BMP signals induce OLP cells at forebrain levels1 and our present data indicate that BMP signals induce NC cells at more caudal levels, whilst Wnt signals caudalize the Border. Together, these results suggest that BMP signaling blocks neural character and induces Border character, while Wnt signals impart the rostro-caudal context for this induction. To test this idea, we investigated the ability of Wnt and BMP signals alone or in combination to induce NC cells. When stage 4 rostral (R) explants, isolated at the level of the prospective forebrain (equivalent to FB explants in Paper I), were cultured in the presence of Wnt3A (~100ng/ml), the generation of neural cells was blocked and NC cells were induced. Wnt signals have previously been shown to lie upstream of BMP signals in specification of epidermal at the expense of neural character at the early blastula stage20. We therefore examined whether Wnt signals induce expression of Bmp genes. Indeed, Bmp2 and Bmp4 were up- regulated 2-3 fold in stage 4 R explants treated with Wnt3A (~100ng/ml) compared to control explants. To examine whether the induction of NC cells by Wnt3A requires BMP signals, we cultured stage 4 R explants in the presence of Wnt3A (~100ng/ml) and Noggin (25-50ng/ml). Under such conditions, Noggin blocked the generation of NC cells and neural cells were generated. Wnt3A, however, acted as a caudalizing factor, as previously described, and caudal neural cells of midbrain-hindbrain character were generated. Thus, Wnt signals caudalize forebrain cells and have the ability to independently promote BMP signals, which in turn induces NC cells.
28 Results NC cells become independent of BMP and Wnt signals by the head fold stage Neural cells at midbrain and rostral hindbrain levels acquire a caudal character between the late gastrula stage and the head fold stage3,39,46. To define the time window of Border caudalization, we sought to determine the stage at which NC specification becomes independent of BMP and Wnt signals. CB explants isolated at stage 6, but not before, still generated NC cells when exposed to Noggin or to mFrz8CRD. Thus, the generation of NC cells becomes independent of further BMP and Wnt signals by the head fold stage.
Together, the results provide evidence that the caudalization of the Border occurs at late gastrula stage under the control of Wnt signals, and the induction of placodal or NC cells occurs at the same stage and is dependent on BMP signals.
Wnt-regulated temporal control of BMP exposure directs the choice between neural plate border and epidermal fate (Paper III).
Border cells are specified at the late blastula stage During the late gastrula stage, OLP cells are induced by BMP signals and NC cell induction involves Wnt and BMP signals. However, induction of epidermal ectoderm is ongoing at blastula stages and is also mediated by BMP and Wnt signals. What is the patterning mechanism leading to differential induction of the Border and epidermal ectoderm by the same signaling molecules? As a first step toward answering this question, we sought to determine the time at which Border induction is initiated. Explants were isolated in the medial (M), Border (B) and lateral (L) regions of the chick epiblast at early and late blastula stages, and the fate adopted by the cells was monitored by analysis of gene expression after 28 hr in culture, corresponding to stage 10, using immunohistochemistry. Expression of Sox1 and Sox2 is characteristic of neural cells, Snail2 and HNK1 mark NC cells, co-expression of Sox2 and Ker marks placodal cells and Ker alone is
29 Results characteristic of epidermal cells (Table 1). EK stage XI47 B explants mainly generated neural or epidermal cells, or a mixture of both, but rarely NC cells and never placodal cells. In contrast, stage 2 B explants reliably generated a vast majority of NC cells. NC cells were generated to the same extant at all rostro-caudal levels of the Border, but no placodal cells could be isolated at that stage. Thus, Border cells are first specified at the late blastula stage, stage 2 in chick, and Border cells are initially specified as NC cells irrespective of their position along the rostro-caudal axis.
BMP and Wnt signals are required for Border induction At stage 4, BMP signals induce OLP cells in the absence of Wnt signals and NC cells in the context of Wnt signaling (see Paper II). To investigate the roles of BMP and Wnt signals in the initial induction of the Border, we analyzed the effects of suppressing BMP or Wnt signals on cell fate specification in prospective Border cells at stage 2. As expected, Noggin (~25ng/ml) blocked the generation of NC cells in stage 2 B explants and promoted the generation of neural cells. The Wnt antagonist mFrz8CRD also blocked the generation of NC cells in stage 2 B explants. However, unlike at stage 4, cells did not acquire placodal but rather neural character, when Wnt signals were suppressed. Thus, both Wnt and BMP signals are required to inhibit neural fate and induce the generation of Border cells.
Wnt signals promote the generation of Border cells by a BMP dependent mechanism Previous results have provided evidence that Wnt and BMP together induce epidermal ectoderm at the early blastula stage20, and the present data provide evidence that both Wnt and BMP signals are required for the generation of Border cells. To gain insight into how Wnt and BMP signals interact during Border induction, we first analyzed the character adopted by prospective neural cells of stage 2 embryos when exposed to Wnt or BMP signals. Stage 2 M explants cultured in the presence of BMP4 (5ng/ml) generated epidermal cells, but no NC or placodal cells at any concentration tested. In contrast, Wnt3A (~100ng/ml) induced massively the generation of NC cells in stage 2 M explants. Within a 10-fold range of concentrations, Wnt3A induced NC cells but never epidermal cells (data not shown). To determine whether the induction of Border cells by Wnt signals is dependent
30 Results on BMP signals, we cultured stage 2 M explants in the simultaneous presence of Wnt3A (~100ng/ml) and Noggin (~25ng/ml). In such conditions, the cells maintained a neural character and Noggin blocked the generation of NC cells. In addition, the cells lost forebrain character and acquired more caudal, hindbrain character (Supplementary Figure 4). To address whether Wnt signals may support NC cell formation by inducing Bmp expression, we analyzed mRNA levels by quantitative RT-PCR in stage 2 M explants cultured together with Wnt3A (~100ng/ml) for 10 hr. Bmp4 expression was upregulated 2.4-fold in explants exposed to Wnt3A, compared to explants cultured alone. Conversely, mFrz8CRD downregulated Bmp4 1.7-fold in stage 2 B explants. Moreover, Bmp4 levels were 1.8-fold higher in stage 2 B explants compared to stage 2 M explants. Together, these results indicate that Wnt signals induce BMP activity, which in turn blocks neural and induces Border character.
Simultaneous exposure of epiblast cells to BMP and Wnt signals induces epidermal ectoderm The above results suggest that Wnt induces (caudal) Border cells in a BMP dependent manner, while exposure to BMP signals leads to induction of epidermal cells instead of Border cells. To test whether the combination of BMP and Wnt induces Border cells, we exposed stage 2 M explants to BMP4 (2-5ng/ml) and Wnt3A (20-100ng/ml) simultaneously. Surprisingly, in such conditions, epidermal cells but no NC or placodal cells were generated. This finding points to the idea that simultaneous exposure to Wnt and BMP signals from stage 2 onwards leads to epidermal specification, which in turn implies that Wnt signals are present in stage 2 M explants to which BMP4 was added. Supporting the idea that cells in stage 2 M explants experience Wnt signaling, such explants cultured until a stage corresponding to Stage 14 embryos generated Brain factor-1 (BF-1)+/Pax6+ cells characteristic of dorsal telencephalon, a cell population previously shown to be Wnt-dependent9. Moreover, stage 2 M explants cultured together with mFrz8CRD generated BF-1+/Nkx2.1+ cells characteristic of ventral telencephalon (Supplementary Figure 5). Thus, Wnt signaling is active in prospective neural cells isolated at stage 2, but at levels lower than required to promote formation of Border cells. Together, these results
31 Results provide evidence that simultaneous exposure to BMP and Wnt signals at stage 2 induces epidermal instead of Border cells.
To determine whether treatment with a BMP agonist fails to induce Border cells because of the simultaneous presence of Wnt signals, we exposed stage 2 M explants to BMP4 (5ng/ml) and mFrz8CRD. Under these conditions, the generation of neural cells was blocked, and Sox2+, Ker+ placodal cells but no epidermal cells were generated. Prolonged culture yielded Raldh3+ olfactory, and δ-crystallin+ lens, placodal cells. Thus, in the absence of Wnt activity, BMP signals are sufficient to block the acquisition of neural character and induce (rostral) Border cells instead of epidermal cells. These results also provide evidence that Wnt signals do not play an instructive role in the specification of Border cells, raising the possibility that the role of Wnt signals in the initial specification of Border cells is to provide temporal control of exposure to BMP signals to avoid simultaneous early exposure of epiblast cells to the two classes of signals.
Border cells initially have a neural character To gain insight into the timing of exposure to Wnt and BMP signals in the Border, we examined the character of cells specified as Border cells after 8h of culture (corresponding in time to late gastrula stage). Stage 2 B explants cultured alone or stage 2 M explants cultured in the presence of Wnt3A (~100ng/ml) for 8h generated cells expressing the early neural markers Sox2 and Otx2 to the same extent as stage 2 M explants cultured alone. In contrast, BMP4 (5ng/ml) inhibited Sox2 and Otx2 expression in stage 2 B explants (Supplementary Figure 6). These results show that the Border is derived from cells that initially exhibit neural character. Moreover, since BMP but not Wnt signals are sufficient to block neural character, these results suggest that Border cells are initially exposed to Wnt but not to BMP signals.
Wnt signals are required during the early but not late phase of Border induction The results described above raise the possibility that the temporal order of exposure to Wnt and BMP signals is key to the differential specification of epidermal and Border cells. To assess this issue directly, we examined the
32 Results requirements for Wnt and BMP signals over different time windows of the induction process. In stage 2 B explants exposed to mFrz8CRD only for the first 10 hr of culture (out of 28 hr), the generation of NC cells was blocked and the cells acquired a neural character. In contrast, when mFrz8CRD was added after 10 hr of culture (and thus present only during the last 18 hr), cells acquired placodal instead of neural character. When cultured to a stage corresponding to HH 14, such explants generated Raldh3+ olfactory and δ- crystallin+ lens placodal cells (data not shown). Thus, Wnt signals are required for the early but not late phase of Border induction. In agreement with the data presented in Paper II, Wnt signals are required during the late phase to specify NC cells at the expense of olfactory and lens cells.
Border induction requires late BMP activity Stage 2 B explants exposed to Noggin (~25ng/ml) during the first 10 hr of culture (out of 28 hr) generated NC cells to the same extant as explants cultured alone, and no neural cells appeared. In contrast, when Noggin was added after 10 hr of culture (and thus present only during the last 18 hr), the generation of NC cells was blocked and virtually all cells acquired a neural character. Thus, the early phase of Border induction is BMP-independent, and BMP signals are required during the late phase. To confirm that the early Wnt-dependent phase requires absence of BMP signaling, we exposed stage 2 B explants to BMP signals either starting at the time of isolation or after 10 hr in culture. When BMP4 (5ng/ml) was added directly to stage 2 B explants, the generation of NC cells was inhibited and epidermal cells were generated instead. In contrast, when BMP4 was added after 10 hr of culture to the same final concentration, stage 2 B explants generated NC cells to the same extent as control explants. These temporally controlled loss-of- function experiments provide evidence that Border induction involves an early phase that requires Wnt signals in the absence of BMP signals, followed by a later phase requiring BMP signals in the presence or absence of Wnt signals.
Together, our results provide evidence that during the induction of Border cells Wnt signals do not play an instructive role but act by inducing Bmp expression in prospective Border cells. The Wnt-regulated induction of Bmp expression avoids simultaneous exposure of prospective Border cells to
33 Results BMP and Wnt signals, allowing BMP signals to induce Border cells instead of epidermal cells. The main data underlying this model are summarized in Figure 4.
Figure 4: Summary of data. a. Character adopted by the cells of stage 2 medial (M, red box) or Border (B, green box) explants exposed to various combinations of Wnt3A, BMP4, Noggin (α-BMP) and mFrz8CRD (α-Wnt). b. Character adopted by the cells of stage 4 rostral neural (R, red box), caudal neural (C, yellow box), rostral Border (RB, purple box) or caudal border (CB, green box) explants exposed to Wnt3A, BMP4, Noggin (α-BMP) or mFrz8CRD (α-Wnt). See text for details. All data from Paper I and II except the one marked with * from ref. 1.
34 Discussion
Discussion
A reasonable approach to study early vertebrate development The emergence of the enormous diversity of cell types during vertebrate nervous system development relies on the ordered generation of progenitor cells in the early embryo. Surprisingly, most cell lineage decisions studied thus far have been shown to depend on only a handful of signals. Whilst new genes that have a role in cell fate specification are continually being identified, most are involved in either the modulation or transduction of known signaling pathways. Indeed, signals are reused in several systems, at different stages of development, and even in different places at the same time or repeatedly on the same cell lineage. Whereas the signaling factors are well-established, the way in which signals act in a series of causative inductive events is largely unknown. The present challenge to understand development is not so much in finding unknown factors but rather to discover how the known factors are deployed and integrated, in specific combinations and in specific sequences to promote particular developmental lineages.
Since the embryo develops in a highly integrated manner, different parts of the embryo are constantly influencing each other. This implies that manipulation of a signal in vivo potentially impinges on tissues other than the one being studied, rendering the results difficult to interpret.
For these reasons, a method is needed that allows the experimenter i) To test the action of signals on a cell population directly, ii) To manipulate several signals at the same time, and iii) To study cells that are accurately defined, with sufficient temporal and spatial resolution. Explants culture in chick combines all of these advantages. The ability to isolate cells at precise developmental stages and to manipulate signaling activities at desired time points during culture, makes chick explants a very valuable model to study the role of timing in cell fate specification. The data generated using explants can be used to design specific experiments in intact embryos in order to validate the results.
35 Discussion Signaling factors belonging to the same family often activate a common downstream transducer. For example, Wnts stimulate β-catenin nuclear localization, BMPs activate Smad1/5/8 phosphorylation and FGFs activate the Ras-Erk pathway. Accordingly, different ligands in a same family show similar activity in several assays. Protostomes have a relatively small number of genes in each family of signaling factors (5-6 Wnts in flies48, 2 Fgfs in nematodes49, 3 Bmps in flies), but still have a rather complex body plan, arguing against the idea that paralogous factors have specific intracellular effects. Moreover, experiments have shown examples in which family members are largely interchangeable. What is the purpose then, of such great apparent redundancy in each signaling family? One possibility is the importance of activation at different thresholds due to differential affinity of receptors and/or co-receptors for the activating ligand. Furthermore, distinct ligands of the same family may act on cells with specific competence due to their expression at a distinct time and place. Several members in each family of signals have presumably evolved because paralogs are differentially regulated (due to differences in cis- regulatory sequence), which allows the signal to be deployed at various times and in various places. A logical consequence of this is that the choice of a particular ligand in a family of signaling factors that is used in gain-of- function experiments has little consequence. Wnt3A, BMP4 and FGF4 can thus be used as tools to activate the cognate signaling pathways. Analysis of gene expression patterns can give a hint to what ligands might be active in the embryo.
Additionally, signaling pathways can be reliably suppressed in vitro by the use of inhibitors that block all signals within a family. Hence, redundancy is not likely to be a problem in the explant system.
The IsO is induced by convergent Wnt and FGF signals As for other caudal neural cells, the progenitors of the MHB region are first specified as neural cells with a forebrain character during blastula stages. This rostral specification persists until mid-gastrulation, when caudalizing signals are secreted by the elongating streak and the mesoderm that derives from it. Later, at the late gastrula stage, neural cells have acquired sufficient
36 Discussion positional information to adopt a cell lineage specific to one of the main subdivisions of the neuraxis: forebrain, midbrain, hindbrain or spinal cord. In chick, formation of this primitive rostro-caudal pattern takes only a few hours. Expression of Gbx2 at the late gastrula stage in the caudal neural plate is one of the earliest molecular readouts of caudalization.
Our results show that the complex pattern of gene expression characteristic of the IsO is specified in the MHB ectoderm as soon as midbrain and hindbrain cells are specified. In other words, no explants could be isolated that generated midbrain and hindbrain cells without also generating Wnt1+ and Fgf8+ IsO-like cells. This strongly supports the idea that the IsO is induced during initial neural caudalization and by the same signals that specify midbrain and hindbrain character: Wnt and FGF. The idea that IsO induction is an integral part of the neural caudalization process has been put forward by Wnt8 gain- and loss-of-function experiments in zebrafish embryos50. Moreover, our results show that the generation of the IsO has become independent of caudalizing signals by stage 6, the neural fold stage. Thus, the IsO is induced much earlier than the onset of expression of IsO- specific genes.
The specification of midbrain and hindbrain character is under the control of convergent Wnt and FGF signals3,39. In this context, hindbrain cells require a ~2-fold higher Wnt concentration than midbrain cells. When FGF or Wnt signals are suppressed, prospective midbrain and hindbrain cells acquire a rostral forebrain character. Accordingly, we found that cells from the MHB region adopt a forebrain fate when Wnt or FGF signals are suppressed, and as expected, none of these forebrain cells expressed IsO-specific markers in the presence of SU5402 or mFrz8CRD. Our gain-of-function experiments provide evidence that simultaneous exposure to Wnt and FGF signals is sufficient to reconstruct a functional IsO. This confirms that no other exogenous signals are required for IsO induction.
It is somewhat astonishing that the finely organized pattern of gene expression characteristic of the IsO can be generated by the combination of two signals in an apparently homogenous forebrain cell population. Presumably, a peripheral-high to medial-low gradient of Wnt signaling is
37 Discussion generated in the explants, which leads to the formation of an IsO-like structure. The fact that a complex pattern can be generated in stage 4 forebrain explants exposed to Wnt and FGF highlights the regulative nature of rostro-caudal patterning. If the symmetry is broken, a self-regulative patterning process is triggered and the whole set of cell types will be generated in proper proportions and at appropriate places.
Source of IsO-Inducing signals What is the source of the IsO-inducing signals? As the embryo develops, the position of the MHB region relative to mesodermal and endodermal structures varies due to ongoing movements of gastrulation. At the time of initial induction, the cells that will contribute to the IsO are found next to the rostral part of the streak/node, overlying the ingressing mesoderm. Around that stage, several Wnt and FGF ligands are expressed in the streak and/or node51-54. In addition, Wnt11 is expressed in ingressed paraxial mesoderm and Wnt8c is expressed in the caudal ectoderm/epiblast. As a consequence of the regression of the node, Wnt8c expression is down- regulated in a rostral to caudal wave in such a way that progressively more caudal cells are exposed to Wnt signals for a gradually increasing duration52,53. Analysis of Wnt/β-catenin reporter mice have revealed that Wnt signaling is activated in the MHB region around the late gastrula stage55. Since caudalization is dependent on the canonical Wnt pathway56, Wnt3A and Wnt8c are likely to be involved in IsO induction.
Liguori et al have reported that in mice lacking Cripto, a co-receptor for the TGFβ super-family ligand Nodal, no mesoderm ingresses but a basic rostro- caudal pattern is generated in the neuro-ectoderm, including an essentially normal IsO57. It should be noted that a source of Wnt signals might exist in the proximal part of the Cripto-/- embryos, where Brachyury and Fgf8 are expressed58. These results suggest that ingressed mesoderm does not contain positional information, but rather that a localized source of caudalizing signals is sufficient to determine the rostrocaudal orientation. Wnt signals induce Wnt8C expression in the ectoderm/epiblast3, which may in turn drive caudalization by planar signals. Although the Drosophila Wnt ligand Wg has been shown to act directly and at long-range59, Wnts have generally a
38 Discussion limited capacity to diffuse freely60. Hence, the Wnt morphogen gradient active during caudalization of the neuro-ectoderm is likely to be established by a relay mechanism rather than by diffusion from a local source. It has also been suggested that signals from the notochord are important for IsO specification61,62. However, IsO formation does not require any non- ectodermal signals after stage 4, since explants isolated at that stage can generate an IsO, and the notochord first appears at stage 5. It follows that the notochord is not required for IsO formation, in agreement with the notion that the notochord is required for dorso-ventral patterning but not for caudalization of the neuro-ectoderm57,63,64.
Distinct time windows for IsO induction and maintenance Several lines of research have established that Wnt1, Wnt10b, Wnt3a and Fgf8 are required not only for the function but also for maintenance of the IsO65-68. In zebrafish and/or mouse mutants lacking one or several of those genes, all IsO markers are initially expressed before they are lost around or after the 12 somite stage (equivalent to stage 11 in chick).
Our loss-of-function experiments provide evidence that Wnt and FGF signals are required for IsO induction during late gastrula stages, between stage 3+ and 5, but not at stage 6. Thus, expression of IsO-specific genes requires Wnt and FGF during two separate periods: from stage 3+ to stage 5 and from stage 11 onwards. In this respect it is interesting that the induction and the maintenance of Pax2 are regulated by different enhancers69,70. Between these two periods, there is a time window from stage 6 to stage 11, during which expression of IsO-specific genes is independent of the signals that initially induced it. Since we cultivated stage 4 MHB explants to the equivalent of stage 10, the blockade of IsO markers by Wnt and FGF antagonists reflects a requirement for the initial induction and not for the maintenance of the IsO.
Mechanism of IsO establishment The data presented above show that IsO induction occurs at the late gastrula stage (stage 3+ to 5) as part of the Wnt-mediated caudalization process.
39 Discussion Expression of IsO-specific markers such as En1/2, Pax2, Wnt1 and Fgf8 is initiated nearly synchronously at the onset of somitogenesis (stage 7+). Each one of these transcription factors/secreted signals is capable of promoting the expression of the others in the MHB region62,71-73. Functional studies in chick and mouse have established that Pax2 lies upstream in the genetic network that controls Fgf8 expression, while Wnt1 and En1 are induced independently74. Yet, the steps linking the initial induction of the IsO to the later restricted expression of Wnt1 and Fgf8 remain unknown.
By the time IsO-specific genes are expressed, Wnt and FGF signals are no longer required for IsO induction. This time gap between exposure to the inducing signals and expression of IsO-specific markers might at first sight seem incongruent. However, a delay between the perception of the signal and expression of specific marker proteins is a rule more than an exception1,9,10,39,75. It is not known whether this is due to slow kinetics of transcription and translation or a sign of intermediate steps in the process.
The factor(s) lying upstream of Pax2 are not known. A conserved regulatory sequence required for initiation of Pax2 expression in the MHB has been identified69,70. The murine transcription factor Oct3/4 binds this sequence and its zebrafish ortholog Pou2 is involved in Pax2 induction76. Pou2 expression, however, is not specific to the MHB region. Other genes have been shown to be specifically expressed in the MHB region prior to somitogenesis, such as the Hes-related genes Her5 in zebrafish and XHR1 in Xenopus77,78. The early expression during the time window of IsO induction makes Her5 and XHR1 good candidates as direct targets of Wnt/FGF signals. However, the signals and factors upstream of Her5 and XHR1 have not yet been reported. No ortholog of Her5 has been identified in amniotes.
Because Otx2 and Gbx2 expression domains overlap slightly during gastrulation, it has been proposed that an interaction between these transcription factors is responsible for IsO specification79,80. This intriguing proposition would imply that IsO specification is due to induction of Gbx2 by caudalizing signals at a lower threshold than that required to repress Otx2 expression. However, this hypothesis has been discredited since the Gbx2
40 Discussion and Otx2 knock-out mice have shown that an interaction between the two transcription factors is not required for IsO specification81.
Comparison of gene expression in several phyla led to the hypothesis of the tripartite brain: the neuraxis of the bilaterian ancestor was divided into a rostral domain expressing Otx/Otd, an intermediate domain expressing Pax2/5/8 and a caudal domain expressing Hox genes82. Such a pattern could be established by induction of the three territories at different concentrations of a caudalizing signal. In vertebrates, however, the situation is different since the Pax2 and Otx2 expression domains overlap. There is now substantial evidence that induction of Fgf8 is under the control of Pax2 and that the Otx2/Gbx2 interface decides the position of the IsO within the Pax2+ domain31,74,81.
Figure 5: Induction and positioning of the IsO by Wnt signaling. Pax2 is induced by levels of Wnt activity between an upper and a lower threshold. Otx2 and Gbx2 are repressed and induced, respectively, by levels of Wnt activity over an intermediate threshold. Pax2 induces Fgf8 expression in cell expressing Gbx2.
41 Discussion Regardless if Pax2 induction by caudalizing signals is direct or indirect, the following model is conceivable (Figure 5): Pax2 is induced at a concentration of Wnt signals comprised between a lower and an upper threshold. Otx2 and Gbx2 are repressed and induced, respectively, when Wnt concentration reaches a threshold half-way between the two Pax2 thresholds. Cross repression between Otx2 and Gbx2 refines the boundary83, which will determine the position of the IsO within the Pax2+ domain.
Refinement of Wnt1 and Fgf8 expression domains One of the most striking features of the IsO is the restricted expression of Wnt1 and Fgf8 in two adjacent thin domains. During normal development, Wnt1 transcripts are found initially dorsally in the midbrain of the stage 8 (3 somites) embryo. At stage 9, Wnt1 is expressed in almost the entire midbrain. A few hours later, Wnt1 expression is restricted to the caudal- most part of the midbrain. After stage 10, the Wnt1 and Fgf8 expression domains are progressively refined and segregated84,85. Our results showing that the stage 4 neuro-ectoderm has acquired sufficient positional information to reconstitute this fine pattern raise the question of how Wnt1 and Fgf8 are restricted to a 2-3 cell diameter band. A first hint is given by the fact that the Wnt1 stripe is thinner in explants extending from the forebrain to the spinal cord than in smaller explants restricted to the MHB region (MHB explants). This suggests the presence of a forebrain-derived signal repressing Wnt1 expression in the adjacent midbrain. However, the IsO pattern is eventually refined in MHB explants cultured for an additional 24 hours (data not shown). An alternative explanation would be that Wnt1 expression requires FGF8 activity for maintenance and vice-versa. Indeed, cells at the boundary are distinguished from others in the compartment by proximity to the adjacent compartment. The possibility of a cross dependence between FGF8 and Wnt1 has received support from several studies65,67,68 and is inline with the observation that all explants generating an IsO-like pattern contain Wnt1+ and Fgf8+ cells in approximately constant proportions. This “both-or-none” phenomenon could also reflect a common progenitor for Wnt1 and FGF8 cells. In that scenario, prospective Wnt1+ and Fgf8+ cells would be produced in a salt and pepper pattern before Wnt1 and Fgf8 transcripts are expressed, and then segregated by cell sorting.
42 Discussion
Ye et al74 provide a detailed model of the interactions leading to restricted Wnt1 and Fgf8 expression: Wnt1 expression requires Otx2 cell- autonomously and is therefore expressed in the midbrain but not in the hindbrain. Fgf8 is induced by Pax2 in the hindbrain but not in the midbrain because Otx2 prevents the ability of Pax2 to induce Fgf8. Induction of Fgf8 by Pax2 requires Wnt1 activity and is as a result restricted to the anterior- most cells in rhombomere r1. The questions of how Pax2 is induced, what induces Wnt1 in the first place and what restricts the anterior border of Wnt1 remain open.
43 Discussion
Context of neural plate border induction The embryonic ectoderm gives rise to three cell populations: the neural cells in the medial (dorsal in anamniotes) region, epidermal cells in the lateral (ventral in anamniotes) region and Border cells in between. The Border gives rise to placodal and NC cells, which together form the precursors of the PNS. The secreted signals involved in the specification of NC and OLP cells have been identified: BMP and FGF play important roles in the specification of placodal cells and BMP, Wnt and FGF are instrumental in the specification of NC cells1,24,37. However, the same families of signals are also involved in the choice between neural and epidermal cells, raising the question of when and how the Border is segregated from the neural and epidermal moieties.
The choice between neural and epidermal lineages occurs very early during development, already at the early blastula stage. Our results provide evidence that cells specified as Border cells can be found reliably only later, at the late blastula stage. The BMP signals responsible for Border specification are required until the headfold stage, stage 5-6 in chick. Until that stage, epiblast/ectodermal cells exhibit extensive plasticity and can change fate if exposed to a new combination of signals. Thus, specification of Border cells is ongoing during gastrulation, from stage 2 to stage 5.
The source of the signal(s) that induce the Border is not known. Epiblast cells from the Border region of stage 2 embryos have received sufficient positional information to reproducibly generate Border cells when isolated. At that stage, no mesoderm or definitive endoderm has ingressed yet86,87 and the only possible sources of signals in proximity are the underlying hypoblast/endoblast and the adjacent epiblast. If the hypoblast/endoblast is not required, long range planar signals are likely to be involved until stage 2, since explants isolated in the same region at the early blastula stage generate mostly neural or epidermal cells. From stage 2 onwards, long range signals are not required and Border cells can develop independently of epidermal or neural cells.
44 Discussion Temporal mechanism of Border induction Together, the results presented in this thesis suggest a model of Border induction that can be divided into two distinct temporal phases (Figure 6):
The first phase is initiated at the late blastula stage, when epiblast cells in the Border region have got sufficient positional information to become Border cells if isolated from the rest of the embryo. From stage 2 to stage 4, prospective Border cells are exposed to Wnt signals, which promotes BMP activation, at least in part, by promoting Bmp4 transcription. During this first phase, which lasts approximately 10 hours, BMP signals are not required. Rather, an absence of BMP is required to avoid induction of epidermal ectoderm by the combined action of Wnt and BMP signals. In the presence of Wnt and absence of BMP signals, prospective Border cells maintain a neural character until the late gastrula stage. How the Wnt signaling pathway is initially activated in the prospective Border is not known. Because Wnts are expressed and active at the periphery of the blastula20,88, Wnt activity is probably readily regulated in the Border. Since Wnt signals do not diffuse far60, it is likely that Wnt expression is induced in the prospective Border itself. Alternatively, an increase in Wnt activity could be the result of dilution or decay of a Wnt inhibitor.
The second phase of Border induction is initiated around stage 4, when the specification of Border cells starts to require BMP signals. During this phase, BMP signals block the acquisition of neural character and promote the acquisition of either OLP or NC fate. The expression pattern of Bmp genes fits with this model: The lateral-most epiblast of the blastula embryo expresses Bmp4 and Bmp712,89, while Bmp4 and BMP2 expression is upregulated in the Border at the late gastrula stage89,90, in accordance with the view that the prospective epidermal ectoderm is exposed to BMP signals before the Border. At caudal levels, BMP induces NC character in the context of continued exposure to Wnt signals. At rostral forebrain levels, where Wnt signals are inhibited, BMP signals induce placodal cells. Border induction comes to an end when the specification of NC and OLP cells has become independent of BMP signals, by stage 6.
45 Discussion
46 Discussion
Figure 6: Model of Border cells specification. (a) Role of timing in Border versus epidermal induction. Border cell types are induced by sequential exposure to Wnt and BMP signals. Short duration of Wnt signaling allows induction of OLP cells whereas continued Wnt signaling promotes NC induction. Simultaneous and ongoing exposure to a combination of Wnt and BMP signals promotes epidermal induction. (b) Model of Wnt and BMP activity in the epidermal and Border regions at blastula to gastrula stages. Neural and epidermal fate choice is initiated at the early blastula stage in a Wnt and BMP dependent manner. Wnt signals are activated in the prospective Border in the absence of BMP signals around the late blastula stage. This in turn leads to a BMP-dependent induction of Border cells around the late gastrula stage. Active suppression of Wnt signals by Wnt antagonists (α-Wnt) in the rostral Border yields OLP induction and continued Wnt signaling in the caudal Border leads to NC induction.
A novel patterning mechanism The model described above represents a novel patterning mechanism whereby the three main cell types of the ectoderm (neural, Border and epidermal) are specified by temporal combinations of two signals. Neural cells are generated in the absence/at low levels of Wnt and BMP, Border cells are induced by sequential activation of the Wnt and BMP pathways, and epidermal cells are induced by simultaneous exposure to Wnt and BMP signals. The effect of Wnt through BMP requires extracellular BMP protein, since it can be blocked by an extracellular antagonist. Also, the induction of BMP activity is correlated with induction of Bmp4 expression. The period of time separating initiation of exposure to Wnt signals from exposure to BMP signals is crucial to Border specification, because cells exposed to both Wnt and BMP directly acquire epidermal fate due to cooperation between the signals. Because activation of Wnt signaling in epiblast cells results in deferred activation of BMP signaling, cells exposed to Wnt only will be automatically exposed to BMP at a later time when the response to the combination of Wnt and BMP has changed.
About 10 hours elapse between the initiation of Border cell specification and activity of the effectors, namely BMP signals. Several molecular mechanisms can be suggested that would explain why BMP activity is induced by Wnt only after a period of time. Wnt signaling might be integrated over time until a threshold required for BMP induction is reached. In addition, several steps might be required, for example a cascade
47 Discussion of induction of transcription factors. Finally, production of BMP proteins could take time and/or be regulated even though induction of Bmp mRNA might be fast. Temporally regulated degradation of a BMP inhibitor represents another possible mechanism.
The BMP gradient model A morphogen gradient is a widely used concept to explain pattern formation in the developing embryo. According to the French Flag model91, gene expression is restricted spatially to contiguous domains in response to different concentrations of the morphogen. There are many examples of a graded response to increasing concentrations of a secreted signal. Some examples are: Shh in the ventral spinal cord92, BMP in the dorsal spinal cord93, Wnt in caudalization of the neural plate3,56, retinoic acid in rostro- caudal patterning of the hindbrain94, and BMP and Wnt in imaginal discs of the fruit fly95. Mainstream opinions in the field of NC induction consider that epidermal ectoderm is specified by high levels of BMP/Smad signaling and neural ectoderm by low or absent BMP/Smad signaling, while NC cells are specified at an intermediate level of BMP signaling. This theory is mainly supported by studies involving titration of BMP antagonists in Xenopus animal cap explants and by examination of zebrafish mutants21- 23,29,30. Some of the most conclusive evidence for the existence of a BMP gradient comes from a study in zebrafish which reports a graded distribution of phosphorylated Smad along the dorso-ventral axis of the blastula and gastrula embryos96.
In contrast, titration of BMP antagonists in smaller Xenopus animal cap explants did not yield a significant number of NC cells, unless other factors, such as Wnt or FGF, were added30,36. Streit and Stern97 also refuted the BMP gradient model based on the observation that Bmp4 expression is higher in the Border than in the epidermis at neural fold stages, although this does not exclude a gradient of BMP activity earlier during the time of Border induction. A current consensus is that, although an intermediate concentration of BMP signaling might be required for NC and placodal specification, it is not sufficient. Accordingly, we found that exogenous BMP4 induced epidermal cells at all concentrations tested, but never Border
48 Discussion cells, in blastula stage prospective neural plate around the time these cell types are specified. Thus, the French Flag model of morphogen action cannot by itself account for the specification of neural, NC/placodal and epidermal cells in the ectoderm.
Graded BMP signals can induce OLP and epidermal cells in stage 4 forebrain explants, however. Likewise, BMP signals induce NC and epidermal cells at different concentrations in stage 4 caudal neural explants. Moreover, although stage 4 Border cells tolerate exogenous BMP4 at a concentration that would induce epidermis at stage 2, higher concentrations of BMP4 induced epidermal cells in Border explants at stage 4 (unpublished results). Thus, although the initiation of Border induction involves temporally controlled exposure to BMP signals rather than a BMP gradient, graded levels of BMP can still influence the Border versus epidermal fate choice. It is conceivable that Border and epidermal fates are indeed decided by intermediate and high levels of Smad1/5/8 phosphorylation, respectively. In that scenario, exposure to BMP from early stages on results in high activation, even at low BMP concentrations, while Wnt-induced delayed exposure to BMP reliably yields moderate activation of the pathway. In summary, intermediate concentrations of BMP ligands are not sufficient to induce Border cells, but our model involving temporal control of BMP signaling is compatible with the hypothesis that levels of BMP signaling ultimately determine ectodermal cell fates.
Duration of BMP/Wnt signaling Several studies have shown ectopic induction of NC cells and, more rarely, placodal cells by BMP inhibition21,22,36,98. The interpretation of this data depends critically on what signaling factors epidermal cells have been exposed to by the time BMP is inhibited. In the prospective epidermis, BMP inhibition at late blastula stage leads to neural specification (Supplementary Figure 7 and Ref. 96 and 99). In contrast, inhibition at late gastrula stage leads to ectopic generation of NC cells96,99. This difference in response after inhibition at different stages indicates that BMP activity between the blastula and the gastrula stage is sufficient to block neural fate and specify Border cell type. Ongoing, combined activities of Wnt and BMP are
49 Discussion required for epidermal differentiation. These data are consistent with a model in which Wnt and BMP together specify epidermal ectoderm, provided that the duration of combined exposure is sufficient.
Wnt inhibition in prospective epidermis results in specification of rostral Border, placodal precursor cells, whether it is started at late blastula or late gastrula stage (Supplementary Figure 7 and L.Gunhaga, personal communication). These results are in accordance with the idea that epidermal ectoderm is normally exposed to both Wnt and BMP signals, and the remaining BMP induces Border cells when Wnt is inhibited. In the latter scenario, prospective epidermal cells have not been exposed to the combination of Wnt and BMP long enough by the late gastrula stage in order to follow an epidermal path of differentiation. Altogether, the possibility of Border induction by BMP and Wnt inhibition at the late gastrula stage indicates that Border cells can be specified by BMP signals in the presence of Wnt, provided that the Wnt-BMP combination is interrupted before the cells are committed to an epidermal fate.
Whilst in some instances, duration of signal is responsible for cell fate specification1,4, there are several examples were longer duration of signaling and higher concentration of signals can achieve the same effect: caudalization of the neural plate55 and dorso-ventral patterning of the spinal cord100. Dessaud et al have reported that graded concentrations of Shh determine the duration rather than intensity of downstream activation of the transducer Gli3100. Thus, increased concentration means prolonged signaling and not increased signal intensity. The idea that the duration of combined Wnt-BMP determines Border or epidermal fate choice is consistent with the observed effect of manipulations suppressing Wnt or BMP signals at the late gastrula stage. Our model whereby Wnt-mediated temporal control of BMP signals specifies Border cells is also compatible with the hypothesis of cell fate specification by the duration of BMP/Wnt signaling.
Intracellular integration of BMP and Wnt signals As discussed above, early simultaneous activation of Wnt and BMP pathways might induce epidermal cells because of higher levels, or
50 Discussion increased duration, of intracellular signaling as compared to sequential exposure to Wnt and BMP. A possible molecular explanation for the cooperation between Wnt and BMP is provided by a study from Fuentealba et al that reports positive effects of Wnt signaling on Smad1 stability, which results in prolonged duration of Smad activity in response to BMP signals101. Another possible level of integration is the convergence of Wnt/β-catenin and Smad signaling on a common cis-regulatory unit, as it is the case in Vent, Vox and Tbx6 activation during mesoderm patterning in zebrafish7,102. Similarly, it has been shown in vitro that Lef1/β-catenin and Smad4/Smad1 interact on the Msx2 promoter, explaining the synergy between these two pathways in Msx2 induction103.
Temporal Shift in response to BMP and Wnt signals Our data provide evidence that BMP signals play an instructive role in the induction of the Border. However, neural cells exposed to BMP at the late blastula stage adopt an epidermal fate. In contrast, neural cells exposed to BMP signals at the late gastrula stage become NC or OLP cells. Here I discuss possible reasons for this difference.
Border cells can be induced by BMP exposure at stage 2, provided that Wnt signals are inhibited at the same time. Thus, time is not a determinant, but rather the simultaneous exposure to both signals at that early stage. BMP induces epidermal cells instead of Border cells in blastula medial explants because of the simultaneous presence of Wnt signals. Wnt signals are present in medial cells at that stage, as witnessed by the Wnt-dependent acquisition of dorsal telencephalic character in neural cells isolated and cultured alone.
At stage 2, a combination of BMP and Wnt signals induces epidermal ectoderm. However, about 10 hr later at stage 4, Wnt and BMP signals induce NC cells in the caudal Border region. Since the caudalizing effect of Wnt can be blocked by an extracellular antagonist, the ligand must be present, the receptor must be activated and the signal is probably transduced through β-catenin. Likewise, the Border-inducing effect of BMP signals can be blocked by Noggin, which implies that the ligand is present and the
51 Discussion receptor activated. Thus, the same combination of Wnt and BMP signals can now specify caudal Border cells of NC character.
One can envision several intracellular pathways: one that is activated by the combination of BMP and Wnt and leads to epidermal specification. Another one activated by Wnt that drives caudalization. A third one activated by BMP signals that directs Border induction. At stage 2, the epidermal- inducing pathway is activated by the combination of BMP and Wnt. At stage 4, however, when Border cells have been exposed to Wnt in the absence of BMP, the epidermal-inducing pathway is blocked intracellularly and the cells can acquire caudal and Border character in the presence of both BMP and Wnt signals. Put in a more anthropomorphic way, the cells become deaf to the combination of Wnt and BMP, while still listening to Wnt and BMP signals separately. A Border can be induced in the context of Wnt signaling when the pathway transducing the epidermal-inducing response is inactivated, which presumably occurs in the Border between stage 2 and stage 4. A molecular explanation for this change is yet to be found. It has recently been suggested that expression of different Lef/TCF genes might confer stage-specific and cell-specific responses to Wnt signals104, providing an explanation why Wnt signals promote epidermal specification at blastula stages and caudalization at gastrula stages. It is worthy to note in this respect that Wnt and FGF signals act in an antagonistic manner at blastula stages but cooperate during caudalization.
The fact that cells are exposed to Wnt but not BMP during the first phase of Border induction suggests that Wnt signaling itself might be responsible for the change in the way cells respond to the combination of Wnt and BMP signals. If this is the case, Wnt as an initial inducer of BMP ensures that the Wnt-BMP combination will not induce epidermal ectoderm at later stages. In summary, the shift in competence of Border cells between blastula and gastrula stages represents a loss of the ability to induce epidermal fate in response to the Wnt/BMP combination.
52 Discussion Border cells derive from neural cells The name of the neural plate border implies that it is a subregion of the neural plate. This assertion appears evident when considering the position of premigratory NC cells in the neural tube. However, the idea has been challenged by the observation that NC cells can apparently be derived from either epidermal or neural ectoderm in fate mapping, grafting or recombination experiments105,106. Our results provide evidence that Border cells derive from cells initially exhibiting neural character. Despite being specified as non-neural cells at stage 2, explanted prospective Border cells express transiently the neural markers Sox2 and Otx2 during the first few hours of culture. Sox2 and Otx2 are eventually down-regulated as cells acquire NC character, presumably in response to BMP signals. Supporting our conclusion, Sox2 mRNA can be detected transiently in the prospective neural crest region in chick embryos107,108
According to our model, Border specification is initiated by exposure of neural cells to Wnt signals. In agreement with the model, HH stage 2 medial cells exposed to Wnt3A develop into NC cells. In contrast EK stage XI medial cells exposed to Wnt3A acquire an epidermal character20. Since increasing Wnt3A concentration was not sufficient to induce epidermal cells in stage 2 neural cells, this difference is likely to reflect an intrinsic difference in the responding cells between early and late blastula stages. It is tempting to speculate that this “maturation” involves exposure to FGF signals, since medial cells in the blastula embryo acquire neural character in response to FGF signals. Hypothetically, the response of cells to FGF signals cannot be blocked by Wnt at the late blastula stage as it is the case at early blastula stages.
OLP cells derive from cells initially specified as NC cells Nieuwkoop’s Activation-Transformation model for neural caudalization has received increasing support109. Cells of the prospective CNS are first specified as rostral neural cells and later re-specified as caudal cells in response to Wnt and FGF signals from the streak and nascent mesoderm. Accordingly, we found that cells in the neural plate of late blastula stage embryos are specified as BF-1+ telencephalic cells. It has been suggested
53 Discussion that, similarly to what is observed in the neural plate, the Border is initially specified as rostral placodal cells before being caudalized by Wnt, FGF and retinoic acid signals36,98. However, no cells specified as OLP cells could be found in stage 2 or stage 3 embryos (present study and ref. 1) and RB explants generated NC cells at those stages. In contrast, stage 4 RB explants generated OLP cells but no NC cells. These results provide evidence that Border cells are initially specified as NC cells and rostral Border cells are re-specified as OLP in response to signaling inputs from other tissues. The later conclusion is supported by the observation in Xenopus that Snail1 and Pax3, two genes expressed in NC but not placodal cells, are transiently expressed in the rostral neural fold38.
Our results reveal that Wnt signals are required and sufficient to switch between OLP and NC fate at stage 4. Prospective Border cells isolated at stage 2 and specified as NC cells can be re-specified as OLP cells by exogenous Wnt antagonists. At stage 4, the Wnt antagonists Dickkopf-1 and Crescent are expressed in the hypoblast and rostral definitive endoderm underlying the forebrain and OLP regions53. A picture is thus emerging from these data, whereby prospective Border cells are first specified as NC cells and re-specified as OLP cells around stage 4 by Wnt antagonists. From stage 5 on, Dickkopf-1 is expressed in the head mesoderm, which might be important to maintain rostral placodal cells in a Wnt-free region. Favoring the later idea, repression of NC fate in Xenopus rostral Border requires secretion of Dickkopf-1 by the head mesoderm38.
The apparent discrepancy in the state of caudalization of neural and Border cells at the late blastula stage can be explained by a higher Wnt activity in the Border, presumably because of proximity to the marginal zone. Since Wnt signals need to be actively precluded from the rostral Border for OLP specification, it makes sense that rostral Border cells are specified as NC cells before ingression of the mesodermal/endodermal source of Wnt inhibitors.
At first sight, it may seem surprising that during Border induction, BMP activity is induced by Wnt and not by another signal, since the cooperation of both classes of signals induce epidermal ectoderm. NC cells are specified
54 Discussion by convergence of Wnt and BMP signals, whereas OLP cells are specified by BMP alone. With Wnt as an inducer of BMP, NC cells are specified in the context of persistent Wnt signaling. In contrast, exposure to Wnt antagonists specifically at forebrain level allows OLP induction in the absence of Wnt signals. Thus, a mechanism involving induction of BMP by Wnt provides an opportunity for a spatial and temporal coordination of forebrain and OLP specification by Wnt antagonists.
Distinct roles of BMP and Wnt signals in NC cell specification Both BMP and Wnt are involved in NC induction. We have been able to distinguish the specific roles of the two classes of signals in the process: Wnt signals caudalize the Border and BMP signals induce NC character in the caudalized context. The fact that graded Wnt signaling caudalizes the neural plate, together with the finding that BMP signals induce rostral Border (OLP) cells, strongly suggests that the role of Wnt is general caudalization of the ectoderm, while the role of BMP is specification of Border cell types.
Acquisition of cell fate is the result of activation of genes characteristic for that particular cell type and repression of genes characteristic of other cell types. Clearly, Wnt signals are both required and sufficient to restrict the formation of OLP cells, whilst BMP signals are both required and sufficient to block the generation of neural cells. Which pathway activates the NC- specific genes is less clear. An indication is highlighted by the fact that BMP4 can still induce the generation of NC cells after stage 6 when the caudalization process is terminated, even when Wnt signals are suppressed simultaneously (unpublished data). Thus, NC cells can be induced in the absence of extracellular Wnt signals, provided that the cells have been caudalized previously. It follows that BMP, but not Wnt signals are instructive in induction of NC character. This conclusion is supported by studies that examined activation of the Snail2 promoter by BMP and Wnt signaling: although the promoter contains functional binding sites for both Lef/TCF and Smad, the Wnt-reporter TOP-EGFP was not activated in the neural fold prior to Snail2 induction110,111. In addition, we found that the ability of Wnt signals to induce NC cells in the forebrain anlage is
55 Discussion dependent on BMP ligands and that the caudalizing activity of Wnt and the Border-inducing activity of BMP can be separated.
It has been proposed that BMP signals induce NC formation via induction of Wnt signaling. Indeed, BMP (probably BMP4) regulates Wnt1 expression in the neural fold, which in turn promotes emigration of NC cells112. This process should be distinguished from NC induction, since Wnt1 expression is initiated after NC specification has become independent of Wnt and BMP signals. Moreover, mice lacking Wnt1 and Wnt3A still generate NC cells113. Epistatic experiments indicate that BMP is upstream of Wnt signaling during Olig3+ interneuron specification114. However, Wnt cannot be downstream of BMP in NC induction, since Wnt signals can not induce NC cells ectopically in caudal neural cells, where BMP signals can.
According to our data, Wnt signaling lies upstream of BMP signals during the initial phase of Border induction. During the later phase, however, Wnt and BMP seem to converge to specify NC cells. At this stage, Wnt and BMP signals have distinct roles and do not act in sequence. Thus, both classes of signals do more than inducing expression of the other. Consistently with this view, neither Wnt nor BMP can substitute for one another in ectopic NC induction. Cross regulatory interactions between BMPs and Wnts in the prospective NC at gastrula and neurula stages have yet to be investigated.
Role of FGF signals in border induction The way in which FGF and Wnt signals are integrated varies dramatically between blastula and gastrula stages. At the early blastula stage, Wnt signals block the response of epiblast cells to FGF signals. Wnt signaling at that early stage regulates the ability of FGF to promote neural fate. At the late gastrula stage, in contrast, Wnt and FGF signals cooperate to mediate caudalization of the neuro-ectoderm. Although FGF has been involved in Border specification97, the specific role of FGF signals in Border induction has not yet been reported.
56 Discussion In contrast to what happens at the early blastula stage, Wnt signaling does not repress neural character by itself at stage 2. This observation suggests that Wnt and FGF do not act in an antagonistic manner at that stage. The differential specification of epidermal and Border cells is also likely to depend on the way in which FGF signaling is integrated with BMP and Wnt signals. Indeed, FGF is required to prevent acquisition of epidermal ectoderm both in rostral and caudal Border cells at stage 4 (Ref. 1 and Supplementary Figure 8).
Common origin of NC and placodal cells Neural crest and placodes share the property of contributing cells to the PNS. This raises the question of the existence of a common “PNS progenitor” from which both placodal and NC cells would derive. Is there a “Border state” shared by placodal and NC progenitors? As discussed recently115, gene expression, mechanisms of migration, mechanisms of differentiation and cell-types indicate that placodal and NC cells are fundamentally different and have little in common in terms of their development. Based on these arguments and evolutionary considerations, the existence of a Border state seems unlikely. If there was a Border state, one would expect that cells exist at a certain stage that are specified as Border cells but have not received enough positional information to become either NC or OLP. No such cell could be found, indicating that OLP and NC cells are specified directly as different cell types. Furthermore, among all the known NC and placodal markers, no gene has been identified that is expressed in both NC and placodal cells but not in the epidermis.
Our results suggest that OLP and NC cells are both induced by BMP signals during the late gastrula stage and that Wnt signals act at the same stage to determine OLP versus NC fate. It remains unknown whether Wnt starts to exert caudalizing activity before BMP is active, or if BMP first induces cells in a Border state, which Wnt will caudalize. Our temporally controlled loss- of-function experiments in stage 2 B explants indicate that BMP signals are not required during the first 10 hr of culture, corresponding to the time elapsed between stage 2 and stage 4. At stage 4, when BMP signals start to be required, OLP and NC cells are already differentially specified. Thus, the
57 Discussion temporal requirements for BMP and Wnt signals suggest that the two classes of signals act at approximately the same time to induce the Border and caudalize it, respectively. However, since OLP and NC specific markers are expressed only after stage 6, it is difficult to determine whether Border cells have a caudal or rostral character at the time non-neural character is first acquired.
Induction of other placodes Our data provide evidence that OLP cells are generated by early exposure to Wnt signals followed by Wnt-induced exposure to BMP signals in the context of suppressed Wnt activity. What about the other placodes?
The hypophyseal placode is arguably derived from cells first specified as OLP cells. At the late gastrula stage, cells of the prospective hypophyseal placode, situated immediately rostral to the neural plate and rostro-medial to the olfactory and lens primordia, develop an olfactory or lens placodal character when cultured in isolation (L. Gunhaga, personal communication). Moreover, Hedgehog signals seem to be required to promote hypophyseal character at the expense of OLP character (L. Gunhaga, personal communication). Consistent with this hypothesis, chicken and zebrafish mutants with defects in Shh signaling have increased formation of lens cells116-118. Thus, it seems that the hypophyseal, olfactory and lens placodes are part of a same subfamily: the rostral placodes that are induced in the Border by BMP in the absence of Wnt.
The trigeminal, otic and epibranchial placodes are probably induced later, laterally to the NC domain, perhaps by another mechanism. It is not known whether these placodes are derived from Border cells that are specified as NC at stage 4 or from adjacent cells, specified as epidermal ectoderm at stage 4. Ker+ cells isolated directly adjacent to the NC territory at stage 4 did not express Sox2 or Pax2 (unpublished data).
Located at a relatively caudal and lateral position, the trigeminal, otic and epibranchial placodes are likely to be exposed to Wnt signals, raising the question of how these cells avoid NC specification. Wnt inhibitors like
58 Discussion Cerberus originating from the underlying intermediate mesoderm might play a role as suggested by Litsiou et al (Ref. 37).
Are all NC cells induced simultaneously by the same mechanism? There appears to be a maturation gradient along the rostro-caudal axis of vertebrate embryos. Events such as the folding of the neural tube and initiation of NC cell migration are progressive from rostral to caudal levels. Tucker et al.96 provided evidence in zebrafish that the time window of BMP requirement in NC induction progresses temporally from rostral to caudal. Unexpectedly, it appears that NC cells are induced simultaneously at all levels from forebrain to spinal cord in chick. We found that Border induction is initiated at all rostro-caudal levels tested at stage 2. NC cells at spinal cord level become independent of BMP and Wnt signals at the same time as NC cells at midbrain level, namely between stage 5 and stage 6 (unpublished data). It cannot be excluded, however, that the requirements for BMP signals vary within this time range.
Similarly to short germ-band insects, vertebrates are patterned in a two step process119. From forebrain to hindbrain levels, a rostro-caudal pattern is generated directly and simultaneously on an already present neural anlage. In the trunk, neural tissue is produced as cells leave a spatially limited “proliferation zone” and generate more and more caudal structures. More neural and NC cells are believed to be induced “de novo” during this process. Whether this involves another mechanism of Border induction is not known. Nevertheless, our results show that BMP, but not Wnt, signals can induce NC cells in trunk neural tissue, consistent with what is observed more rostrally. In the neural plate, Hoxb9+ caudal spinal cord cells are specified at the same time as more rostral cell types, namely at the late gastrula stage10. This points to a simultaneous specification of caudal cell types irrespective of the rostro-caudal level, at least from midbrain to brachial levels. Yet BMP retains the ability to induce NC cells in the spinal cord at stage 10 (Ref. 28 and 45 and present study). Thus, more NC cells might be induced in the dorsal neural tube in response to BMP signals emanating from the dorsal midline and/or adjacent epidermal ectoderm.
59 Discussion Beside the placode/NC dichotomy, several cell types from the PNS arise at specific rostro-caudal levels within the NC pool. A code of transcription factors (Hox genes, Krox-20, Teashirt, etc…) is present in the NC in a pattern similar to that observed in the adjacent neuroepithelium11, and NC cells exhibit diverse migratory behaviors. It has been widely debated whether the lineage potentiality of NC cells is restricted or not prior to migration. Either way, one can reasonably assume that secreted signals in the prospective NC act to endow cells at different rostro-caudal levels with specific lineage restriction and fate specific characteristics. It will be interesting to see in the future whether the profiles of gene expression observed at different rostro-caudal levels are instructed by the same signals that pattern the adjacent neural plate (graded Wnt during gastrulation, retinoic acid and FGF at headfold stage) , and if these signals are sufficient to specify the migratory behavior and the fate of the cells.
Coordination of CNS and PNS development Different classes of neurons are generated along the rostro-caudal axis of the CNS and PNS. These cells need to establish connections with each other in order to convey sensory information accurately. Thus, it is logical that particular progenitor cells have to be coordinately induced at correct positions in both systems.
At the late gastrula stage, Wnt signals specify different cell types at different rostro-caudal levels. This caudalization by Wnt is simultaneous in the neural plate and in the Border, between stage 4 and 6. It can be speculated that this coordination ensures that OLP cells are generated at forebrain level only, as olfactory neurons must make connections only with the rostral forebrain. Similarly, caudalization by a single signal presumably ensures that NC cells are generated at levels of the CNS where they should make connections. A note of caution to this theory is that NC cells migrate extensively.
During the time Wnt activity determines the rostro-caudal identity of ectodermal cells, BMP signals induce simultaneously OLP and NC progenitors in the rostral and caudal Border regions, respectively. The simultaneous induction of rostral and caudal Border cell types by BMP
60 Discussion signals, together with the simultaneous caudalization of the neural plate and the Border, provides a coordinated control of CNS and PNS early development.
Several patterning events have to occur simultaneously at very early stages when the embryo is small enough to facilitate the production of stable morphogen gradients and regulative systems.
61 Conclusions
Conclusions
• The induction of the Isthmic Organizer occurs at the late gastrula stage
• The Isthmic Organizer is induced by convergence of the caudalizing signals Wnt and FGF
• Induction of the Border is initiated at the late blastula stage
• Specification of Border cells is mediated by a novel patterning mechanism whereby Wnt signals control sequential activation of the Wnt and BMP pathways
• During late gastrulation BMP signals specify placodal cells or NC cells depending on the activity of the caudalizing factor Wnt
62 Acknowledgements
Acknowledgements
I owe my gratitude to all the people who have contributed to the realization of this thesis.
To Thomas Edlund for giving me the opportunity to join his lab. Working under your supervision has been enriching and inspiring.
To Lena Gunhaga, my supervisor during the last run, for being helpful, encouraging and opened to all spontaneous discussions.
Sanna Olander contributed heavily to the IsO project.
Many thanks to Helena Alstermark for excellent technical assistance over the years.
Former lab members Ullis, Matthew, Anna, My and Ferran made group TE the best working place I could dream of. I miss you a lot.
I thank members of group LG: Esther, Daniela, Tanushree, Jonas and Hanna for technical help and discussions.
PA, Kristina, Clara and the cleaning staff have been like guardian angels and made my time much easier.
I am indebted to all the people at UCMM for daily help and friendship. I am in particular grateful to Stefan for teaching me RT-PCR.
Sincere thanks to Jeremy Kenaghan and Jon Gilthorpe for their efforts to improve the English language of the manuscript.
I extend my gratitude to my parents for caring about my dreams and helping me making them come true.
63 Acknowledgements I thank sincerely my beloved future wife Annika. The list of all your contributions to my PhD and to this thesis is long, ranging from encouragements to discussions about my projects.
Last but not least, thanks to my son Linus for bringing me back down to earth by being a happy baby when I come home.
64 Supplementary Figures
References
1. Sjodal, M., Edlund, T. & Gunhaga, L. Time of exposure to BMP signals plays a key role in the specification of the olfactory and lens placodes ex vivo. Dev Cell 13, 141-9 (2007). 2. Pages, F. & Kerridge, S. Morphogen gradients. A question of time or concentration? Trends Genet 16, 40-4 (2000). 3. Nordstrom, U., Jessell, T. M. & Edlund, T. Progressive induction of caudal neural character by graded Wnt signaling. Nat Neurosci 5, 525- 32 (2002). 4. Harfe, B. D. et al. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118, 517-28 (2004). 5. Dyson, S. & Gurdon, J. B. The interpretation of position in a morphogen gradient as revealed by occupancy of activin receptors. Cell 93, 557-68 (1998). 6. Briscoe, J., Chen, Y., Jessell, T. M. & Struhl, G. A hedgehog- insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube. Mol Cell 7, 1279-91 (2001). 7. Szeto, D. P. & Kimelman, D. Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm formation. Development 131, 3751-60 (2004). 8. Kudoh, T., Concha, M. L., Houart, C., Dawid, I. B. & Wilson, S. W. Combinatorial Fgf and Bmp signalling patterns the gastrula ectoderm into prospective neural and epidermal domains. Development 131, 3581-92 (2004). 9. Gunhaga, L. et al. Specification of dorsal telencephalic character by sequential Wnt and FGF signaling. Nat Neurosci 6, 701-7 (2003). 10. Nordstrom, U., Maier, E., Jessell, T. M. & Edlund, T. An early role for WNT signaling in specifying neural patterns of Cdx and Hox gene expression and motor neuron subtype identity. PLoS Biol 4, e252 (2006). 11. Trainor, P. A. & Krumlauf, R. Patterning the cranial neural crest: hindbrain segmentation and Hox gene plasticity. Nat Rev Neurosci 1, 116-24 (2000).
65 Supplementary Figures 12. Wilson, S. I., Graziano, E., Harland, R., Jessell, T. M. & Edlund, T. An early requirement for FGF signalling in the acquisition of neural cell fate in the chick embryo. Curr Biol 10, 421-9 (2000). 13. Linker, C. & Stern, C. D. Neural induction requires BMP inhibition only as a late step, and involves signals other than FGF and Wnt antagonists. Development 131, 5671-81 (2004). 14. Lumsden, A. & Krumlauf, R. Patterning the vertebrate neuraxis. Science 274, 1109-15 (1996). 15. Nieuwkoop, P. D. N., G. V. Neural activation and transformation in explants of competent ectoderm under the influence of fragments of anterior notochord in Urodeles. J. Embryol. Exp. Morph. 2, 175-193 (1954). 16. Yamaguchi, T. P. Heads or tails: Wnts and anterior-posterior patterning. Curr Biol 11, R713-24 (2001). 17. Wurst, W. & Bally-Cuif, L. Neural plate patterning: upstream and downstream of the isthmic organizer. Nat Rev Neurosci 2, 99-108 (2001). 18. Hidalgo-Sanchez, M., Millet, S., Bloch-Gallego, E. & Alvarado- Mallart, R. M. Specification of the meso-isthmo-cerebellar region: the Otx2/Gbx2 boundary. Brain Res Brain Res Rev 49, 134-49 (2005). 19. Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92 (1951). 20. Wilson, S. I. et al. The status of Wnt signalling regulates neural and epidermal fates in the chick embryo. Nature 411, 325-30 (2001). 21. Brugmann, S. A., Pandur, P. D., Kenyon, K. L., Pignoni, F. & Moody, S. A. Six1 promotes a placodal fate within the lateral neurogenic ectoderm by functioning as both a transcriptional activator and repressor. Development 131, 5871-81 (2004). 22. Marchant, L., Linker, C., Ruiz, P., Guerrero, N. & Mayor, R. The inductive properties of mesoderm suggest that the neural crest cells are specified by a BMP gradient. Dev Biol 198, 319-29 (1998). 23. Nguyen, V. H. et al. Ventral and lateral regions of the zebrafish gastrula, including the neural crest progenitors, are established by a bmp2b/swirl pathway of genes. Dev Biol 199, 93-110 (1998). 24. Basch, M. L., Bronner-Fraser, M. & Garcia-Castro, M. I. Specification of the neural crest occurs during gastrulation and requires Pax7. Nature 441, 218-22 (2006). 25. Sauka-Spengler, T. & Bronner-Fraser, M. A gene regulatory network orchestrates neural crest formation. Nat Rev Mol Cell Biol 9, 557-68 (2008).
66 Supplementary Figures 26. Basch, M. L. & Bronner-Fraser, M. Neural crest inducing signals. Adv Exp Med Biol 589, 24-31 (2006). 27. Basler, K., Edlund, T., Jessell, T. M. & Yamada, T. Control of cell pattern in the neural tube: regulation of cell differentiation by dorsalin-1, a novel TGF beta family member. Cell 73, 687-702 (1993). 28. Liem, K. F., Jr., Tremml, G., Roelink, H. & Jessell, T. M. Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell 82, 969-79 (1995). 29. Barth, K. A. et al. Bmp activity establishes a gradient of positional information throughout the entire neural plate. Development 126, 4977-87 (1999). 30. LaBonne, C. & Bronner-Fraser, M. Neural crest induction in Xenopus: evidence for a two-signal model. Development 125, 2403-14 (1998). 31. Yanfeng, W., Saint-Jeannet, J. P. & Klein, P. S. Wnt-frizzled signaling in the induction and differentiation of the neural crest. Bioessays 25, 317-25 (2003). 32. Schmidt, C. & Patel, K. Wnts and the neural crest. Anat Embryol (Berl) 209, 349-55 (2005). 33. Lewis, J. L. et al. Reiterated Wnt signaling during zebrafish neural crest development. Development 131, 1299-308 (2004). 34. Wu, J., Yang, J. & Klein, P. S. Neural crest induction by the canonical Wnt pathway can be dissociated from anterior-posterior neural patterning in Xenopus. Dev Biol 279, 220-32 (2005). 35. Garcia-Castro, M. I., Marcelle, C. & Bronner-Fraser, M. Ectodermal Wnt function as a neural crest inducer. Science 297, 848-51 (2002). 36. Villanueva, S., Glavic, A., Ruiz, P. & Mayor, R. Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction. Dev Biol 241, 289-301 (2002). 37. Litsiou, A., Hanson, S. & Streit, A. A balance of FGF, BMP and WNT signalling positions the future placode territory in the head. Development 132, 4051-62 (2005). 38. Carmona-Fontaine, C., Acuna, G., Ellwanger, K., Niehrs, C. & Mayor, R. Neural crests are actively precluded from the anterior neural fold by a novel inhibitory mechanism dependent on Dickkopf1 secreted by the prechordal mesoderm. Dev Biol 309, 208-21 (2007). 39. Muhr, J., Graziano, E., Wilson, S., Jessell, T. M. & Edlund, T. Convergent inductive signals specify midbrain, hindbrain, and spinal cord identity in gastrula stage chick embryos. Neuron 23, 689-702 (1999).
67 Supplementary Figures 40. Fiske, B. Wnt signals lead cells down the caudal path. Nat Neurosci 5, 510 (2002). 41. Hsieh, J. C., Rattner, A., Smallwood, P. M. & Nathans, J. Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein. Proc Natl Acad Sci U S A 96, 3546-51 (1999). 42. Mohammadi, M. et al. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 276, 955-60 (1997). 43. Martinez, S., Wassef, M. & Alvarado-Mallart, R. M. Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en. Neuron 6, 971-81 (1991). 44. Lamb, T. M. et al. Neural induction by the secreted polypeptide noggin. Science 262, 713-8 (1993). 45. Taneyhill, L. A. & Bronner-Fraser, M. Dynamic alterations in gene expression after Wnt-mediated induction of avian neural crest. Mol Biol Cell 16, 5283-93 (2005). 46. Olander, S., Nordstrom, U., Patthey, C. & Edlund, T. Convergent Wnt and FGF signaling at the gastrula stage induce the formation of the isthmic organizer. Mech Dev 123, 166-76 (2006). 47. Eyal-Giladi, H. & Kochav, S. From cleavage to primitive streak formation: acomplementary normal table and a new look at the first stages of the development of thechick. I. General morphology. Dev Biol 49, 321-337 (1976). 48. Prud'homme, B., Lartillot, N., Balavoine, G., Adoutte, A. & Vervoort, M. Phylogenetic analysis of the Wnt gene family. Insights from lophotrochozoan members. Curr Biol 12, 1395 (2002). 49. Birnbaum, D., Popovici, C. & Roubin, R. A pair as a minimum: the two fibroblast growth factors of the nematode Caenorhabditis elegans. Dev Dyn 232, 247-55 (2005). 50. Rhinn, M., Lun, K., Luz, M., Werner, M. & Brand, M. Positioning of the midbrain-hindbrain boundary organizer through global posteriorization of the neuroectoderm mediated by Wnt8 signaling. Development 132, 1261-72 (2005). 51. Karabagli, H., Karabagli, P., Ladher, R. K. & Schoenwolf, G. C. Comparison of the expression patterns of several fibroblast growth factors during chick gastrulation and neurulation. Anat Embryol (Berl) 205, 365-70 (2002).
68 Supplementary Figures 52. Hume, C. R. & Dodd, J. Cwnt-8C: a novel Wnt gene with a potential role in primitive streak formation and hindbrain organization. Development 119, 1147-60 (1993). 53. Chapman, S. C., Brown, R., Lees, L., Schoenwolf, G. C. & Lumsden, A. Expression analysis of chick Wnt and frizzled genes and selected inhibitors in early chick patterning. Dev Dyn 229, 668-76 (2004). 54. Crossley, P. H. & Martin, G. R. The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121, 439-51 (1995). 55. Nordstrom, U. Early Rostrocaudal Patterning of the CNS. Thesis (2005). 56. Kiecker, C. & Niehrs, C. A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus. Development 128, 4189-201 (2001). 57. Liguori, G. L. et al. Anterior neural plate regionalization in cripto null mutant mouse embryos in the absence of node and primitive streak. Dev Biol 264, 537-49 (2003). 58. Ding, J. et al. Cripto is required for correct orientation of the anterior- posterior axis in the mouse embryo. Nature 395, 702-7 (1998). 59. Strigini, M. & Cohen, S. M. Wingless gradient formation in the Drosophila wing. Curr Biol 10, 293-300 (2000). 60. Christian, J. L. BMP, Wnt and Hedgehog signals: how far can they go? Curr Opin Cell Biol 12, 244-9 (2000). 61. Ang, S. L. & Rossant, J. Anterior mesendoderm induces mouse Engrailed genes in explant cultures. Development 118, 139-49 (1993). 62. Shamim, H. et al. Sequential roles for Fgf4, En1 and Fgf8 in specification and regionalisation of the midbrain. Development 126, 945-59 (1999). 63. Fekany-Lee, K., Gonzalez, E., Miller-Bertoglio, V. & Solnica-Krezel, L. The homeobox gene bozozok promotes anterior neuroectoderm formation in zebrafish through negative regulation of BMP2/4 and Wnt pathways. Development 127, 2333-45 (2000). 64. Klingensmith, J., Ang, S. L., Bachiller, D. & Rossant, J. Neural induction and patterning in the mouse in the absence of the node and its derivatives. Dev Biol 216, 535-49 (1999). 65. Reifers, F. et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125, 2381-95 (1998). 66. McMahon, A. P., Joyner, A. L., Bradley, A. & McMahon, J. A. The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from
69 Supplementary Figures stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell 69, 581-95 (1992). 67. Lee, S. M., Danielian, P. S., Fritzsch, B. & McMahon, A. P. Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain. Development 124, 959-69 (1997). 68. Buckles, G. R., Thorpe, C. J., Ramel, M. C. & Lekven, A. C. Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation. Mech Dev 121, 437-47 (2004). 69. Picker, A., Scholpp, S., Bohli, H., Takeda, H. & Brand, M. A novel positive transcriptional feedback loop in midbrain-hindbrain boundary development is revealed through analysis of the zebrafish pax2.1 promoter in transgenic lines. Development 129, 3227-39 (2002). 70. Pfeffer, P. L., Payer, B., Reim, G., di Magliano, M. P. & Busslinger, M. The activation and maintenance of Pax2 expression at the mid- hindbrain boundary is controlled by separate enhancers. Development 129, 307-18 (2002). 71. Liu, A., Losos, K. & Joyner, A. L. FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate. Development 126, 4827-38 (1999). 72. Okafuji, T., Funahashi, J. & Nakamura, H. Roles of Pax-2 in initiation of the chick tectal development. Brain Res Dev Brain Res 116, 41-9 (1999). 73. Crossley, P. H., Martinez, S. & Martin, G. R. Midbrain development induced by FGF8 in the chick embryo. Nature 380, 66-8 (1996). 74. Ye, W. et al. Distinct regulators control the expression of the mid- hindbrain organizer signal FGF8. Nat Neurosci 4, 1175-81 (2001). 75. Marklund, M. et al. Retinoic acid signalling specifies intermediate character in the developing telencephalon. Development 131, 4323-32 (2004). 76. Reim, G. & Brand, M. Spiel-ohne-grenzen/pou2 mediates regional competence to respond to Fgf8 during zebrafish early neural development. Development 129, 917-33 (2002). 77. Shinga, J., Itoh, M., Shiokawa, K., Taira, S. & Taira, M. Early patterning of the prospective midbrain-hindbrain boundary by the HES-related gene XHR1 in Xenopus embryos. Mech Dev 109, 225-39 (2001). 78. Müller, M., Weizsäcker, E. & Campos-Ortega, J. A. Transcription of a zebrafish gene of the hairy-Enhancer of split family delineates the
70 Supplementary Figures midbrain anlage in the neural plate. Dev. Genes Evol. 206, 153 -160 (1996). 79. Garda, A. L., Echevarria, D. & Martinez, S. Neuroepithelial co- expression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer. Mech Dev 101, 111-8 (2001). 80. Katahira, T. et al. Interaction between Otx2 and Gbx2 defines the organizing center for the optic tectum. Mech Dev 91, 43-52 (2000). 81. Li, J. Y. & Joyner, A. L. Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression. Development 128, 4979-91 (2001). 82. Reichert, H. A tripartite organization of the urbilaterian brain: developmental genetic evidence from Drosophila. Brain Res Bull 66, 491-4 (2005). 83. Simeone, A. Positioning the isthmic organizer where Otx2 and Gbx2meet. Trends Genet 16, 237-40 (2000). 84. Hidalgo-Sanchez, M., Millet, S., Simeone, A. & Alvarado-Mallart, R. M. Comparative analysis of Otx2, Gbx2, Pax2, Fgf8 and Wnt1 gene expressions during the formation of the chick midbrain/hindbrain domain. Mech Dev 81, 175-8 (1999). 85. Hollyday, M., McMahon, J. A. & McMahon, A. P. Wnt expression patterns in chick embryo nervous system. Mech Dev 52, 9-25 (1995). 86. Psychoyos, D. & Stern, C. D. Fates and migratory routes of primitive streak cells in the chick embryo. Development 122, 1523-34 (1996). 87. Kimura, W., Yasugi, S., Stern, C. D. & Fukuda, K. Fate and plasticity of the endoderm in the early chick embryo. Dev Biol 289, 283-95 (2006). 88. Roeser, T., Stein, S. & Kessel, M. Nuclear beta-catenin and the development of bilateral symmetry in normal and LiCl-exposed chick embryos. Development 126, 2955-65 (1999). 89. Chapman, S. C., Schubert, F. R., Schoenwolf, G. C. & Lumsden, A. Analysis of spatial and temporal gene expression patterns in blastula and gastrula stage chick embryos. Dev Biol 245, 187-99 (2002). 90. Faure, S., de Santa Barbara, P., Roberts, D. J. & Whitman, M. Endogenous patterns of BMP signaling during early chick development. Dev Biol 244, 44-65 (2002). 91. Wolpert, L. Positional information and the spatial pattern of cellular differentiation. J Theor Biol 25, 1-47 (1969). 92. Poh, A. et al. Patterning of the vertebrate ventral spinal cord. Int J Dev Biol 46, 597-608 (2002).
71 Supplementary Figures 93. Mizutani, C. M., Meyer, N., Roelink, H. & Bier, E. Threshold- dependent BMP-mediated repression: a model for a conserved mechanism that patterns the neuroectoderm. PLoS Biol 4, e313 (2006). 94. Dupe, V. & Lumsden, A. Hindbrain patterning involves graded responses to retinoic acid signalling. Development 128, 2199-208 (2001). 95. Neumann, C. & Cohen, S. Morphogens and pattern formation. Bioessays 19, 721-9 (1997). 96. Tucker, J. A., Mintzer, K. A. & Mullins, M. C. The BMP signaling gradient patterns dorsoventral tissues in a temporally progressive manner along the anteroposterior axis. Dev Cell 14, 108-19 (2008). 97. Streit, A. & Stern, C. D. Establishment and maintenance of the border of the neural plate in the chick: involvement of FGF and BMP activity. Mech Dev 82, 51-66 (1999). 98. Aybar, M. J., Glavic, A. & Mayor, R. Extracellular signals, cell interactions and transcription factors involved in the induction of the neural crest cells. Biol Res 35, 267-75 (2002). 99. Wawersik, S., Evola, C. & Whitman, M. Conditional BMP inhibition in Xenopus reveals stage-specific roles for BMPs in neural and neural crest induction. Dev Biol 277, 425-42 (2005). 100. Dessaud, E. et al. Interpretation of the sonic hedgehog morphogen gradient by a temporal adaptation mechanism. Nature 450, 717-20 (2007). 101. Fuentealba, L. C. et al. Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal. Cell 131, 980-93 (2007). 102. Ramel, M. C. & Lekven, A. C. Repression of the vertebrate organizer by Wnt8 is mediated by Vent and Vox. Development 131, 3991-4000 (2004). 103. Hussein, S. M., Duff, E. K. & Sirard, C. Smad4 and beta-catenin co- activators functionally interact with lymphoid-enhancing factor to regulate graded expression of Msx2. J Biol Chem 278, 48805-14 (2003). 104. Heeg-Truesdell, E. & LaBonne, C. Neural induction in Xenopus requires inhibition of Wnt-beta-catenin signaling. Dev Biol 298, 71-86 (2006). 105. Bronner-Fraser, M. Origin of the avian neural crest. Stem Cells 13, 640-6 (1995). 106. Moury, J. D. & Jacobson, A. G. The origins of neural crest cells in the axolotl. Dev Biol 141, 243-53 (1990).
72 Supplementary Figures 107. Wakamatsu, Y., Endo, Y., Osumi, N. & Weston, J. A. Multiple roles of Sox2, an HMG-box transcription factor in avian neural crest development. Dev Dyn 229, 74-86 (2004). 108. Rex, M. et al. Dynamic expression of chicken Sox2 and Sox3 genes in ectoderm induced to form neural tissue. Dev Dyn 209, 323-32 (1997). 109. Stern, C. D. Initial patterning of the central nervous system: how many organizers? Nat Rev Neurosci 2, 92-8 (2001). 110. Sakai, D. et al. Regulation of Slug transcription in embryonic ectoderm by beta-catenin-Lef/Tcf and BMP-Smad signaling. Dev Growth Differ 47, 471-82 (2005). 111. Vallin, J. et al. Cloning and characterization of three Xenopus slug promoters reveal direct regulation by Lef/beta-catenin signaling. J Biol Chem 276, 30350-8 (2001). 112. Burstyn-Cohen, T., Stanleigh, J., Sela-Donenfeld, D. & Kalcheim, C. Canonical Wnt activity regulates trunk neural crest delamination linking BMP/noggin signaling with G1/S transition. Development 131, 5327-39 (2004). 113. Dickinson, M. E., Selleck, M. A., McMahon, A. P. & Bronner-Fraser, M. Dorsalization of the neural tube by the non-neural ectoderm. Development 121, 2099-106 (1995). 114. Zechner, D. et al. Bmp and Wnt/beta-catenin signals control expression of the transcription factor Olig3 and the specification of spinal cord neurons. Dev Biol 303, 181-90 (2007). 115. Schlosser, G. Do vertebrate neural crest and cranial placodes have a common evolutionary origin? Bioessays 30, 659-72 (2008). 116. Kondoh, H. et al. Zebrafish mutations in Gli-mediated hedgehog signaling lead to lens transdifferentiation from the adenohypophysis anlage. Mech Dev 96, 165-74 (2000). 117. Buxton, P. et al. Craniofacial development in the talpid3 chicken mutant. Differentiation 72, 348-62 (2004). 118. Davey, M. G. et al. The chicken talpid3 gene encodes a novel protein essential for Hedgehog signaling. Genes Dev 20, 1365-77 (2006). 119. Stern, C. D. et al. Head-tail patterning of the vertebrate embryo: one, two or many unresolved problems? Int J Dev Biol 50, 3-15 (2006).
73 Supplementary Figures Supplementary Figures
Supplementary Figure 1: In the Absence of BMP activity prospective NC cells acquire a midbrain-hindbrain character. (A,B) Consecutive sections showing expression of Pax2 and En1/2 in explants cultured for 22 hr. (A) Stage 4 CB explants cultured alone (n=20) generated no Pax2+ or En1/2+ cells. (B) Stage 4 CB explants cultured in the presence of Noggin (n=20) generated Pax2+ and En1/2+ cells, characteristic of the midbrain-hindbrain. Scale bar, 100 µm.
74 Supplementary Figures
Supplementary Figure 2: In the presence of Wnt3A β-catenin nuclear staining is increased in prospective midbrain-hindbrain cells. (A,B) Consecutive sections showing expression of β-catenin in explants cultured for 6 hr. (A) Stage 4 C explants cultured alone (n=9) generated no or only a few cells with positive nuclear β-catenin staining. (B) Stage 4 C explants cultured together with Wnt3A (n=9) generated an increased number of cells with positive nuclear β-catenin staining (white arrowheads). Scale bar, 100 µm.
75 Supplementary Figures
Supplementary Figure 3: BMP4 but not Wnt3A induces neural crest in spinal cord cells under different culture conditions. (A-C) Consecutive sections showing expression of molecular markers in explants cultured for 24 hr. (A,B) Stage 10 C explants cultured alone (n=20) or together with Wnt3A (~100 ng/ml) (n=20) generated Sox1+ cells, but no Snail2+ or HNK-+ cells. (C) Stage 10 C explants cultured together with
76 Supplementary Figures BMP4 (20ng/ml) (n=20) generated Snail2+ and HNK-1+ cells, but no Sox1+ cells. Scale bar, 100 µm.
Supplementary Figure 4: In the absence of BMP activity Wnt signals induce caudal character in prospective neural cells. (A,B) Consecutive sections showing expression of molecular markers in explants cultured for 28 hr. (A) Stage 2 M explants (n=9) generated Sox1+, Otx2+ cells, but no Gbx2+ cells. (B) Stage 2 M explants cultured together with Wnt3A (~100 ng/ml) and Noggin (~25 ng/ml) (n=14) generated Sox1+ and Gbx2+ cells, but no Otx2+ cells. Scale bar, 100 µm.
77 Supplementary Figures
Supplementary Figure 5: At the late blastula stage prospective neural cells are specified as dorsal forebrain cells in response to low levels of Wnt activity. (A,B) Consecutive sections showing expression of molecular markers in explants cultured for 48 hr. (A) Stage 2 M explants (n>30) generated BF1+, Pax6+ cells, but no Nkx2.1+ cells. (B) Stage 2 M explants cultured together with mFrz8CRD (n=20) generated BF1+, Nkx2.1+ cells, but no Pax6+ cells. Scale bar, 100 µm.
78 Supplementary Figures
Supplementary Figure 6: Prospective Border cells exhibit neural character during the Wnt-dependent early phase of specification. (A-D) Consecutive sections showing expression of molecular markers in explants cultured for 8 hr. (A) Stage 2 M explants (n=8) generated Sox2+ and Otx2+ cells. (B) Stage 2 M explants cultured together with Wnt3A (~100 ng/ml) (n=8) generated Sox2+ and Otx2+ cells. (C) Stage 2 B explants (n=8) generated Sox2+ and Otx2+ cells. (D) Stage 2 M explants cultured together with BMP4 (5 ng/ml) (n=8) generated no Sox2+ or Otx2+ cells. Scale bar, 100 µm.
79 Supplementary Figures
Supplementary Figure 7 has been removed for reasons of copyright. The figure is available in the printed edition.
80 Supplementary Figures
Supplementary Figure 8: FGF signals are required in Border cells at the late blastula stage (A-C) Consecutive sections showing expression of molecular markers in explants cultured for 28 hr. (A) Stage 2 B explants (n>30) generated Snail2+ and HNK-1+ cells, but no or only a few Sox1+, Sox2+ or Ker+ cells. (B) Stage 2 L explants exposed to a dominant negative FGF receptor (FGFR4, 5 µg/ml) (n>30) generated Ker+ cells, but no Sox1+, Sox2+, Snail2+ or HNK- 1+ cells. Scale bar, 100 µm.
81