The Mouse Nervous System

Table of Contents

Cover image

Front Matter

Copyright

Dedication

List of Contributors

Preface

Acknowledgments

Chapter 1. Molecular Regionalization of the Developing Neural Tube

Chapter 2. Gene Targeting

Chapter 3. Genetic Neuroanatomy

Chapter 4. Neocortex

Chapter 5. Hippocampus

Chapter 6. Piriform Cortex and Amygdala

Chapter 7. Subpallial Structures

Chapter 8. Hypothalamus

Chapter 9. Diencephalon

Chapter 10. Midbrain

Chapter 11. Cerebellum

Chapter 12. Hindbrain

Chapter 13. Spinal Cord

Chapter 14. Vascular Supply

Chapter 15. Magnetic Resonance Imaging of the Mouse Brain

Chapter 16. Motor Nuclei of the Cranial Nerves

Chapter 17. Visceral Motoneurons

Chapter 18. Neurosecretory Nuclei of the Hypothalamus and Preoptic Area

Chapter 19. Motor Cortex

Chapter 20. Hypothalamic Goal-directed Behavior – Ingestive, Reproductive and Defensive

Chapter 21. The Somatosensory System

Chapter 22. Gustatory

Chapter 23. Pain

Chapter 24. Auditory System

Chapter 25. Visual System

Chapter 26. The Olfactory System

Chapter 27. Vestibular System

Chapter 28. The Basal Forebrain Cholinergic Projection System in Mice

Chapter 29. An Introduction to the Neurobiology of Emotions and Social Behavior

Chapter 30. Prefrontal Cortex

Chapter 31. Overview of Mouse Models for Psychiatric and Neurologic Disorders

Chapter 32. Mouse Models of Mental Illness and Neurological Disease

Chapter 33. The Neuroanatomy of Addictive Processes

Index

Front Matter

The Mouse Nervous System

Edited by

C harles W atson

Faculty of Health Sciences, Curtin University, Perth, Australia, and Neuroscience Research Australia, Sydney, Australia

G eorge P axinos

Neuroscience Research Australia, Sydney, Australia and University of New South Wales, Sydney, Australia

L uis P uelles

Department of Human Anatomy, University of Murcia, Spain

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

Academic Press is an imprint of Elsevier

Copyright

Academic Press is an imprint of Elsevier

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First edition 2012

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Dedication

To Mario Capecchi, whose discovery of gene targeting has made the mouse the center-piece of neuroscience research.

List of Contributors

EDITORS

Charles Watson

Faculty of Health Sciences, Curtin University, Perth, Australia and Neuroscience Research Australia, Sydney, Australia

George Paxinos

Neuroscience Research Australia, Sydney, Australia and University of New South Wales, Sydney, Australia

Luis Puelles

Department of Human Anatomy, University of Murcia, Spain

CONTRIBUTORS

Antonio Abellan

Laboratory of Brain Development and Evolution, Department of Experimental Medicine, Faculty of Medicine, University of Lleida, Institute of Biomedical Research of Lleida, Lleida, Catalonia, Spain

Manisha Aggarwal

Johns Hopkins University School of Medicine, Department of Radiology, Division of NMR, Baltimore, MD, USA

Ken Ashwell

Department of Anatomy, School of Medical Sciences, The University of New South Wales, NSW, Australia

Sylvia Bardet

Unité de Génétique Moléculaire Animale-INRA UMR 1061, University of Limoges, Limoges, France

Alan M. Brichta

School of Biomedical Sciences and Pharmacy, Hunter Medical Research Institute, University of Newcastle, Callaghan, NSW, Australia

Robert John Callister

School of Biomedical Sciences & Pharmacy, University of Newcastle and Hunter Medical Research Institute, Newcastle, NSW, Australia

Newton Sabino Canteras

Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

Mario Capecchi

Department of Human Genetics, The University of Utah, Salt Lake City, UT, USA

Marie-Françoise Chesselet

Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

Yogita Chudasama

Department of Psychology, McGill University, Montreal, QC, Canada

Trevor A. Day

School of Biomedical Sciences & Pharmacy, and the Centre for Brain & Mental Health Research, University of Newcastle and the Hunter Medical Research Institute, Newcastle, Australia

Diego Echevarria

Instituto de Neurociencias, UMH-CSIC, San Juan, Alicante, Spain

Jose-Luis Ferran

University of Murcia, Faculty of Medicine, Department of Human Anatomy, Murcia, Spain

Keith B.J. Franklin

Department of Psychology, McGill University, Montreal, QC, Canada

YuHong Fu

Neuroscience Research Australia, Sydney, Australia

Nicholas R. Franich

Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

Lorise C. Gahring

SLC-VA GRECC and the University of Utah School of Medicine, Salt Lake City, UT, USA

Brett Anthony Graham

School of Biomedical Sciences & Pharmacy, University of Newcastle and Hunter Medical Research Institute, Newcastle, NSW, Australia

Nicolás Gutiérrez-Castellanos

Laboratori de Neuroanatomia Funcional Comparada, Universitat de València. Departments of Functional Biology and Cell Biology, Burjassot, Spain

Erika Gyengesi

Prince of Wales Medical Research Institute and The University of New South Wales, Sydney, NSW, Australia

Miriam A. Hickey

Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

Daniel P. Holschneider

Departments of Psychiatry and the Behavioral Sciences, Neurology, Cell & Neurobiology, Keck School of Medicine, and Department of Biomedical Engineering, Viterbi School of Engineering, Los Angeles, CA, USA

Phil Jobling

School of Biomedical Sciences and Pharmacy, University of Newcastle, NSW, Australia

Alexandra Joyner

Developmental Biology Program, Sloan-Kettering Institute, New York, NY, USA

Jon H. Kaas

Department of Psychology, Vanderbilt University, Nashville, TN, USA

Matthew Kirkcaldie

School of Medicine, University of Tasmania, Hobart, Tasmania, Australia

Enrique Lanuza

Laboratori de Neuroanatomia Funcional Comparada, Universitat de València, Department of Cell Biology, Burjassot, Spain

Rebecca Lim

School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia

Mark D. Lindner

Lindner Preclinical Consulting, LLC, Cheshire, CT, USA

Manuel S. Malmierca

Auditory Neurophysiology Unit, Institute for Neuroscience of Castilla y León and Faculty of Medicine, University of Salamanca, Salamanca, Spain

Salvador Martínez

Instituto de Neurociencias, UMH-CSIC, San Juan, Alicante, Spain

Fernando Martínez-García

Laboratori de Neuroanatomia Funcional Comparada, Universitat de València. Department of Functional Biology, Burjassot, Spain

Margaret Martinez-de-la-Torre

University of Murcia, Faculty of Medicine, Department of Human Anatomy, Murcia, Spain

Robert A. McArthur

McArthur and Associates GmbH, Basel, Switzerland

Loreta Medina

Laboratory of Brain Development and Evolution, Department of Experimental Medicine, Faculty of Medicine, University of Lleida, Institute of Biomedical Research of Lleida, Lleida, Catalonia, Spain

Vera Medvedeva

Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

Susumu Mori

Johns Hopkins University School of Medicine, Department of Radiology, Division of NMR, Baltimore, MD, USA

Amparo Novejarque

Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, London, UK

Anthony N. van den Pol

Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA

Eduardo Puelles

Alicante Institute for Neuroscience, UMH-CSIC, San Juan, Alicante, Spain

Yue Qi

Neuroscience Research Australia, Randwick, NSW, Australia

Scott W. Rogers

SLC-VA GRECC and the University of Utah School of Medicine, Salt Lake City, UT, USA

John L.R. Rubenstein

Nina Ireland Laboratory of Developmental Neurobiology, Center for Neurobiology and Psychiatry, University of California at San Francisco, San Francisco, CA, USA

David K. Ryugo

The Garvan Institute of Medical Research Darlinghurst, NSW, Australia

Oscar U. Scremin

VA Greater Los Angeles Healthcare System, Research Department and David Geffen School of Medicine at UCLA, Physiology Department, Los Angeles, CA, USA

Gulgun Sengul

Department of Anatomy, Ege University School of Medicine, Izmir, Turkey

Roy V. Sillitoe

Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, USA

Iwona Stepniewska

Department of Psychology, Vanderbilt University, Nashville, TN, USA

Anamaria Sudarov

Developmental Biology Program, Sloan-Kettering Institute, New York, NY, USA. Present address: Laboratory of Neurogenetics and Development, Weill Medical College of Cornell University, New York, NY, USA

Petr Tvrdik

Department of Human Genetics, The University of Utah, Salt Lake City, UT, USA

F. Rohan Walker

School of Biomedical Sciences & Pharmacy, and the Centre for Brain & Mental Health Research, University of Newcastle and the Hunter Medical Research Institute, Newcastle, Australia

Mark Whitehead

UCSD School of Medicine, Department of Surgery, Division of Anatomy, La Jolla, CA, USA

Menno Witter

Kavli Institute for Systems Neuroscience, Centre for the Biology of Memory, Department of Neuroscience, Norwegian University of Science and Technology NTNU, Trondheim, Norway. Department of Anatomy & Neurosciences, VU University Medical Center, Amsterdam, The Netherlands

Nicole A. Young

Department of Psychology, Vanderbilt University, Nashville, TN, USA

Laszlo Zaborszky

Center for Molecular and Behavioral Neuroscience, Rutgers, The State University of New Jersey, Newark, USA

Jiangyang Zhang

Johns Hopkins University School of Medicine, Department of Radiology, Division of NMR, Baltimore, MD, USA

Preface

When we announced our intention to put this book together, some of our colleagues questioned the need for a large book dedicated to the mouse nervous system. They pointed out that Elsevier already publishes a comprehensive book on the rat ( The Rat Nervous System by Paxinos) and suggested that a book devoted to the mouse would be something of a luxury. We persisted with our plan for three reasons.

The first reason is the different readership. The development of gene targeting in mice has led to an explosion of research on the mouse brain, but the overwhelming majority of the researchers do not have a strong background in neuroanatomy. We have produced a book that does not assume a systematic knowledge of the structure of the mouse brain and spinal cord. We believe that many of our readers need an introductory guide to their region of interest. We do not wish to patronize our audience, but we are acutely aware of the limited number of experienced anatomists in laboratories devoted to molecular genetics. We admire the extraordinary contribution made by molecular biologists to neuroscience and we hope to enhance the quality of their work by giving them access to the neuroanatomical information they need.

The second reason is the need for a book that presents the new data on the role of gene expression during development of the mouse brain. Over the past twenty years, many traditional views on the organization of the nervous system have been either discarded or substantially reworked because of thousands of studies on gene expression during development using gene-targeting technologies. The chapters on the hypothalamus, diencephalon, and midbrain are prime examples of the need to change views on the organization of the brain, based on gene expression during development.

The third justification for this book is that mice are not small rats. The mouse brain has unique features that must be seriously considered in any rigorous study. While there is a wealth of information on the morphology, neurochemistry and connections of cell groups in the rat nervous system, there is a limited range of information on areas of the mouse brain. A feature of this book is the presence of many anatomical illustrations of the mouse brain and spinal cord that will be easy for beginners in the field to interpret.

Charles Watson

George Paxinos

Luis Puelles

Acknowledgments

We thank the chapter authors who have made this book a substantial contribution to the neuroscience library. In a complex project of this size, there is inevitable frustration from the early contributors who have been forced to wait for the laggards to complete the book. We appreciate their understanding and their patience.

We thank the Elsevier Academic Press editorial staff, who took this book from concept to reality; at first Johannes Menzel, and later Mica Haley and Melissa Turner.

Charles Watson is particularly grateful to his outstanding team of research assistants, Lucy Adams, Megan Harrison, Aine O'Brien, and Tiza Chipungu, who created illustrations, carried out literature searches, and edited chapters.

We wish to acknowledge support from an NHMRC Australia Fellowship to G Paxinos (Grant # 568605) in the preparation of Chapter 9, Chapter 10, Chapter 11, Chapter 12, Chapter 13, Chapter 16, Chapter 18, Chapter 21 and Chapter 25.

Chapter 1. Molecular Regionalization of the Developing Neural Tube

Salvador Martínez, Eduardo Puelles, Luis Puelles and Diego Echevarria

Instituto de Neurociencias, UMH-CSIC, San Juan, Alicante, Spain.

Outline

Introduction 2

Morphogenesis of the Neural Tube 3

Dorso-Ventral (DV) Patterning in the Neural Tube 6

Ventralization 6

Dorsalization 8

Antero-Posterior (AP) Patterning in the Neural Tube 9

Secondary Organizers Control Molecular Regionalization 11

Diencephalic Regionalization: How the Diencephalon Creates its Structural Complexity 12

The Diencephalic Transcriptome Map Supports the Prosomeric Model 13

Acknowledgments 14

List of Abbreviations 15

Introduction

Brain function originates in the precise integration of activities of different neural structures. The peculiar richness of the brain’s integrative output is dependent upon the development of multiple anatomical subregions, each having cytological and functional specificity. This requires stringent developmental patterning in space and time of the molecular and cellular processes that build up the hypercomplex structure of the brain. We have come to realize that a delicate balance between spatio-temporal sequences of gene expression regulates normal brain development, forming a complex causal network. Since space and time appear inextricably linked in developmental genoarchitectonics, we review in this chapter the cartography of genes which are known to represent key factors for brain development in the neural tube, focusing mainly on those that code for morphogenetic information: signaling molecules and transcription factors.

The neuroepithelium of the neural plate and tube contains the progenitors for the neural cells – neurons and glia – that will jointly form operative brain structures. Cells at different initial domains of these primordia receive multiple morphogenetic positional signals, encoded by the graded distribution of signaling molecules produced at restricted sites within or outside the developmental field. These signals act upon specific receptors expressed in the neuroepithelial cells, and regulate at each locus the expression of a specific set of transcription factors controlling proliferation, neurogenesis and cellular differentiation, and, eventually, the emergence of connections and functional properties. These molecular codes progress from initial dynamic states to more stable or definitive states, characterizing the ‘molecular identity’ of the locally produced cell populations. The topology of the differential possible genomic interpretations of these graded signals – the catalogue of different possible interactive genomic readings of the sets of gradiental signals – creates the sets of presumptive regional subdivisions of the brain wall, a progressive process called regionalization.

We will focus our chapter on the mechanisms which act early in development, from early neural tube to midgestation stages in mouse embryos, that is, between 7.5 and 14.5 days of embryonic development (E7.5–E14.5). In the E14.5 mouse embryo, most of the varieties of neurons composing the central nervous system have been generated, and have migrated from the germinative neuroepithelium into the mantle layer. However, at this embryonic stage important and complex migratory processes that shape the future brain have not yet initiated. Therefore, the specific genetic signature of each neuronal population in the mantle layer can be easily correlated to that of their progenitors in the neuroepithelial ventricular zone. At this stage, gene expression patterns (which are publicly accessible at several sites, such as the Eurexpress web page: www.eurexpress.org, or the Allen Developing Mouse Brain Atlas web page: www.developingmouse.brain-atlas.org) may reveal transient or permanent readouts of positional information, established at the level of either epithelial (progenitors) cells or mantle (neural differentiated) cells. The progressive stabilization and/or further regionalization of such patterns bespeaks of the underlying differential specification of cellular fates. Thus, further longitudinal analysis of such gene expressions will allow us to correlate temporo-spatial patterns of selected genes with the study of precursor proliferation versus generation, migration and differentiation of neuronal cell types.

Morphogenesis of the Neural Tube

Neurulation is a fundamental process of embryogenesis that culminates in the formation of the neural tube after progressive infolding of the early neural plate. The bending of the neural plate involves the formation of hinge regions and lines where the neural tube contacts surrounding tissues (for review see Colas and Schoenwolf, 2001). Elevation of the neural folds establishes a trough-like space called the neural groove, which becomes the lumen of the primitive neural tube after closure of the neural groove. The neural tube closes as the paired neural folds are brought together at the dorsal midline, establishing the roof plate of the neural tube (Fig. 1.1 A–C and 1.2 A–C; Copp et al., 2003). In addition, the neural folds will generate the specialized neuromesenchymal cells of the neural crest, at the origin of neural populations of the peripheral nervous system, jointly with some ectodermal cephalic placodes.

The early neural tube is, in most vertebrates, a rather straight, elongated structure. However, even before its posterior portion has fully formed via neurulation, the most anterior portion of the neural tube is undergoing drastic changes. In this region, the tube balloons into three primary vesicles: the forebrain (prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon) (Fig. 1.1 A–C). By the time the posterior end of the neural tube closes, secondary bulges – the optic vesicles – have extended laterally from each side of the developing forebrain. At this early stage of development (the so-called three-vesicle stage), the long axis of the neural tube bends considerably, as already incipiently observed at late neural plate stages, but more markedly occurring after neurulation, leading to the development of the cephalic and cervical ventral flexures of the neural tube (Fig. 1.1 C, D). Subsequently, the prosencephalon becomes subdivided into the anterior secondary prosencephalon (telencephalon and hypothalamus) and the more caudal diencephalon proper (Fig. 1.1 D).

The discovery that putative regulatory genes are expressed in regionally restricted patterns in the developing forebrain has provided new tools for defining neural histogenetic domains and their boundaries at higher resolution than was possible before with methods based on differentiation traits. Based on gene expression patterns together with morphological information, two main models have been used to interpret neural plate and tube regionalization: a topographic columnar model, largely aimed at saving the classic concept of sulcal division of the diencephalon into the 4 longitudinal columnar zones of Herrick (1910) (Alvarez-Bolado et al., 1995) and a topological segmental model known as the prosomeric model, constructed upon evidence of serial transversal neural tube divisions sitting across the primary longitudinal zones of His, 1893a and His, 1893b. The latter is more consistent with emergent morphological, molecular and experimental data, which cannot be satisfactorily rationalized in terms of the zones of Herrick (Fig. 1.1 D; see below, and also the chapters on Hypothalamus and Diencephalon, this volume).

The topologic prosomeric paradigm proposes that the embryonic forebrain is molecularly articulated into a neuromeric framework – a segmental Bauplan – that is subdivided into a grid-like pattern of differentially developing longitudinal (columnar) and transverse (segmental) histogenetic domains. This would be a result of early evolutionary genomic channelization of this fundamental Cartesian organization of the neural primordium in chordates, irrespective of the substantial varying secondary morphogenetic deformations which are characteristic of different vertebrate lineages (Fig. 1.1 D; revised in Puelles, 1995). The prosomeric model has been successfully applied to understand the molecular regionalization of the neural tube in almost all vertebrate species, in what amounts to an important validation of its assumptions. As a result, the number of longitudinal neural zones, the number of transversal neural segments, and their main subdivisions seem to be constant in all vertebrate brains, allowing easy topological comparisons part-to-part across species, and the extrapolation of causal mechanisms from one form to another. The model explicitly emphasizes shared patterns, but can as easily be used to study differential developmental patterns, or pathological patterns (mutant and other pathological phenotypes).

The longitudinal boundaries segregate columns (longitudinal zones of His, 1893a and His, 1893b) of cells with similar properties. These are specified at neural tube stages by interactive dorso-ventral (DV) patterning mechanisms, which result from the earlier latero-medial patterning mechanisms of the neural plate (FIGURE 1.1 and FIGURE 1.2). Dorsalization, mediated by dorsally-originated diffused signals, tends to pattern typical dorsal neural derivatives, while ventralization, mediated by signals diffusing from the axial mesoderm (notochord) and the neural floor plate, tends to pattern ventral neural fates. These important signaling interactions, globally known as DV patterning, give rise to four fundamental longitudinal zones, which, listed from ventral to dorsal, are the floor plate, basal plate, alar plate and roof plate (FIGURE 1.1 and FIGURE 1.2). All neural segments share this basic DV pattern, irrespective of their antero-posterior position. This common or repeated basic pattern is what is meant when the neural segments are described as metameric.

Transverse molecular boundaries (each with diverse causes, depending on their sites), which can be traced along the entire DV dimension of the brain wall (from roof to floor), subdivide it into a constant antero-posterior set of segments (neuromeres). As a result of the primary differential genetic specification, each of these fields subsequently independently regulates the proliferative dynamics of its neuroepithelium (proliferative maxima at their centers and minima at the interneuromeric boundaries), often leading to transient external bulging of their lateral walls. This bulging was the secondary morphogenetic phenomenon recorded by pioneering 19th century neuroembryologists as the appearance of neuromeres (e.g., Orr, 1887). Lack of understanding of their secondary and transient nature led to much discussion about the number and duration in time of true neural segments, apart from the exclusion of artifactual analogues. In the prosencephalon, 6 prosomeres were first postulated (p1–6; Puelles and Rubenstein, 1993), with subsequent simplification to p1–p3 in the diencephalon, plus an incomplete bipartition of the secondary prosencephalon, Puelles and Rubenstein, 2003; Fig.1.1 D). See the chapter on Hypothalamus (this text) for recent developments on rostral forebrain neuromeres. In the rhombencephalon, the segments are termed rhombomeres (r1–r11, counting the isthmus as r0; Fig.1.1 D), of which only r2–r6 are definable by their distinct bulging (Marin and Puelles, 1995), and the r0–r1 and r7–r11 units were called pseudorhombomeres (Cambronero and Puelles, 2000), or, as we now prefer, cryptorhombomeres, because their clear cut molecular and fate boundaries do not overtly give rise to separate bulges (Marin et al., 2008). The mesencephalon, which clearly generates different structures along the AP axis (at least four in its alar plate: tectal gray, superior and inferior collicula and preisthmus), was classically divided into two mesomeres (m1, m2). However, the expected partial gene expression domains distinguishing such AP sub-regions were not easily found (e.g., none recorded after exploration of 1281 midbrain genes in the Eurexpress database). Therefore, it has been common in the literature until quite recently to regard the midbrain as a segmentally undivided vesicle (Fig. 1.1 D; Puelles et al., 2004). However, a report by Hidalgo-Sanchez et al. (2005) first identified the preisthmus as a histologically distinct caudal midbrain transverse domain intercalated between the inferior colliculus and the isthmic domain, which expresses Otx2 (thus clearly belonging to the midbrain; Figs. 1.3 C, E) and also Pax2, the latter selectively (not present in the rest of the midbrain after stabilization of its earlier ampler dynamic pattern), but does not express isthmic markers such as Fgf8. This has led to the introduction of the m1/m2 mesomere subdivisions in the reference atlases of the public database Allen Developing Mouse Brain Atlas, where additional molecular evidence of the distinct (though small) m2 mesomere can be found.

The prosomeric model has unveiled the morphological significance of numerous gene expression patterns in the forebrain, suggesting the existence of additional molecular subdivisions of the main AP and DV regions, representing histogenetically differentially-specified domains of neural precursors with characteristic nuclear derivatives in the adult brain (Garcia-Lopez and Martinez, 2010 and Gimeno and Martinez, 2007; Ferran et al., 2007 1 and Ferran et al., 2009 1; Puelles, 1995, Puelles and Martinez, 2004, Puelles, 2007, Rubenstein et al., 1994 28 and Shimamura et al., 1995; and recently revised in Puelles and Rubenstein, 2003). Examination of this molecular-structural association under the new concept of neural genoarchitectonics (Ferran et al., 2009) has shown how the topologically patterned expression of particular genes is directly related to specific morphogenetic and cytogenetic developments.

Dorso-Ventral (DV) Patterning in the Neural Tube

The differential molecular fate specification of intersecting longitudinal and transversal regions within the neuroepithelium involves patterning along the medio-lateral (ML) and antero-posterior (AP) dimensions of the neural plate. The ML patterning in the neural plate is topologically equivalent to DV patterning in the neural tube.

Ventralization

Our knowledge of ventral patterning is strongly based on recent results obtained about the spinal cord, which derives from the posterior (caudal) parts of the neural plate. Molecules produced by adjacent non-neural tissues first specify the diverse DV regional identities. Ventral signaling is represented by Sonic Hedgehog ( Shh), which is expressed at the axial mesendoderm and codes for a secretable protein morphogen, SHH (Chiang et al., 1996 3, Echelard et al., 1993, Ericson et al., 1996 15, Hynes et al., 1995 and Roelink et al., 1994 25, 1995; Shimamura and Rubenstein, 1997; Tanabe et al., 1995; for review see Tanabe and Jessel, 1996). SHH is first secreted by the notochord, and soon the expression of its gene is homeogenetically induced in the overlying median neural plate (Fig. 1.1 B), conferring to this region a floor plate identity and the ability to function in patterning of the basal and alar plates as a secondary source of the diffusing SHH morphogen. Because the notochord primarily ends rostrally under the prospective mamillary pouch, subsequently the floor plate markers also end at this neighborhood (Figs. 1.1 B, C; 1.2A, B, D, F). This singularity causes the left and right basal and alar plates to meet rostrally at the hypothalamic median plane between the end of the floor plate and the end of the roof plate (the latter represented by the terminal ridge; asterisk in Figs.1.1 A, B, C; roof plate colored in yellow, basal plate in green, and floor plate in pink, in Fig. 1.1 C; the rest is alar plate). This extended roof-alar-basal-floor rostral end of the neural tube wall has been identified as the terminal wall (see chapter on Hypothalamus, present volume).

However, it is still unclear whether the patterning of the ventral forebrain is regulated by mechanisms which are distinct from those that occur in more posterior regions. Anterior to the notochord, a median mesendodermal tissue named the prechordal plate underlies the rostral part of the neural plate. (However, we must be aware that, if the notochord defines the axis, then essentially nothing can be ‘anterior’ to it; the morphologic classification of the prechordal plate as ‘axial’ is therefore controversial. An added difficulty is that the prechordal tissue is not fixed – its cells move progressively towards the terminal ridge or rostral neuropore.) Several lines of molecular and genetic evidence now suggest that the ‘ventralization’ of the forebrain involves molecular mechanisms which are similar to those demonstrated in more posterior neural regions, but nevertheless are regulated by the combined influences of the notochord and the prechordal plate. In fact, analysis of mice lacking a functional Shh gene has demonstrated that SHH is essential for the ventral patterning of the entire central nervous system (CNS; Chiang et al., 1996). Moreover, Shimamura and Rubenstein (1997), using chick neural plate explants, directly showed that prechordal plate tissue (and associated dorsal foregut) induces ventral fates and represses dorsal properties in prosencephalic explants. Furthermore, they showed that the prechordal plate functions alone in the initial specification of the ventral prosencephalon. Note, however, that the concept of ‘ventral forebrain’ does not distinguish between patterning of floor plate tissue, as opposed to basal plate tissue (both being considered ‘ventral’).

Hence, the prechordal plate and notochord may have parallel roles in neural plate ventralization and longitudinal neural patterning. There are data that suggest that such collaboration extends all the way back to the midbrain tegmentum (Garcia-Calero et al., 2008). However, the two inducers also exhibit distinct molecular properties. For instance, though both inducing tissues express Shh, the two show some differential markers (e.g., goosecoid only in prechordal plate), which may endow them with specific inductive abilities (Placzek et al., 1993). The related idea of prechordal and epichordal parts of the neuroepithelium (Puelles et al., 1987; Puelles and Rubenstein, 2003 and Swanson, 2003) has been tentatively connected with such differences (Vieira et al., 2006). Due to the molecular and structural complexity of the rostral forebrain, definition of the anterior pole of the basal plate in the hypothalamus is still a controversial matter in the literature (some authors actually see it within the ‘basal’ telencephalon). However, gene mapping results seem to reinforce the prosomeric model concept represented in Fig. 1.1 C, where the rostral basal plate expands dorsoventrally in breadth within the hypothalamus, encompassing the entire mamillary and tuberal regions up to the retrochiasmatic area. Complementarily, the alar plate also extends into the hypothalamic rostral forebrain, encompassing the classic anterior, chiasmatic, and supraoptic parts of the hypothalamus, plus the eye vesicle and the whole telencephalon (Fig. 1.1 C). This molecular analysis is largely consistent with the pioneering one of His, 1893a and His, 1893b, but now requires us to develop more precise models of hypothalamic topological DV regionalization, in order to understand causally the multiple structural subdivisions of these large basal and alar domains (Puelles and Rubenstein, 2003; Puelles et al., 2004; Shimogori et al., 2010; see chapter on Hypothalamus, this volume).

Dorsalization

Regarding the dorsal signals that specify alar territories, gain-of-function experiments and gene expression data have shown that some dorsal signals are coded by members of the TGF-β superfamily, such as BMP4 and BMP7, or members of the Wnt family, which are produced by the non-neural ectoderm next to the neural plate folds, later fused over the roof plate, and by the roof plate itself (Basler et al., 1993; Dickinson et al., 1995; Liem et al., 1995 22 and Shimamura and Rubenstein, 1997). The interface between the neural and non-neural ectoderm is separately specified as neural crest up to mid-diencephalic levels. This primordium will generate free migratory neural crest cells that variously disperse into the embryonic body after the closing of the neural folds (FIGURE 1.1 and FIGURE 1.2). BMPs and Wnts are expressed in the roof plate, causing diffusion of dorsal signals that, together with ventral signals (mainly the SHH signal), establish a combined molecular code of positional information along the DV axis in the neural tube (Basler et al., 1993; Dickinson et al., 1995; Lee and Jessell, 1999; Liem et al., 1995; Shimamura and Rubenstein, 1997). Recently, FGF8, the product of the Fgf8 gene, has been identified as a required factor for the normal development of the diencephalic alar plate, by interacting locally with Wnt1 expression in the roof plate (Martinez-Ferre and Martinez, 2009).

The basic common result of this process of DV regionalization, as it operates throughout the length of the neural tube, is the specification of four basic longitudinal zones in its neuroepithelial wall that correspond to: floor, basal, alar and roof plates. In addition to signaling genes, like Shh expressed in the floor (across spinal cord and hindbrain) and also in the basal plate of the midbrain+forebrain tagma (FIGURE 1.1 and FIGURE 1.2), or Wnt1, expressed widely in the roof plate (Figs. 1.2 A–C), several transcription factors have been observed to be specifically expressed in the floor and basal plate of the brain ( Foxa1 and Lmbx1;Figs. 1.2 D–G) or in the alar plate ( Zic1 and Lhx2;Figs. 1.2 H, I).

There are no important outstanding controversies between different models of longitudinal zonation in the neural tube, if we consider that neuroanatomists still conceiving an end of the floor and basal plate of the forebrain within the telencephalon (e.g., Altman and Bayer, 1995; 200…; Alvarez-Bolado et al., 1995 and Swanson, 1992, 2003) have not adduced so far any molecular or experimental evidence in support of their position, nor seem able to explain in a simpler way the sort of molecular data illustrated in this chapter and other cited sources (revised in Puelles and Rubenstein, 2003 and Puelles at al., 2007). Sometimes authors try to wed together the incompatible columnar and prosomeric models (with the apparent aquiescence of reviewers and journal editors), generating ambiguous and misleading interpretations, as observed recently in the important work of Shimogori et al. (2010), where the hypothalamic basal plate is presented according to the prosomeric model (without mentioning it), whereas the hypothalamic and prethalamic alar plate are inconsistently described according to the columnar model (not mentioned, either). This weak morphologic rationale led, among other latent pitfalls, to the conceptually dangerous definition of a ‘diagonal hypothalamic band’, presumably as a compromise between the ‘conciliated’ models. This band is strictly longitudinal in the prosomeric model, and strictly meaningless within the columnar model (since it does neither predict, nor explain it).

The hypothalamus is therefore still a subject of controversy today. The interpretation of hypothalamic regional topology still suffers frequent revisions. This is either due to the difficulty of interpreting genetic patterns in the absence of a clear understanding of the theoretical assumptions and postulates of each available model, or as a result of legitimate theoretical advances in the formulation of such models. For instance, earlier versions of the prosomeric model described a prechordal floor component in the hypothalamus (Puelles and Rubenstein, 2003), but new data on gene expression and some experimental results (Sanchez-Arrones et al., 2009) have suggested that the floor plate is strictly epichordal and ends at the mamillary region, thus requiring that median infundibular and median eminence structures previously held to form a prechordal floor should now be reclassified as basal plate territories (Bardet et al., 2008; L. Puelles personal observations; see chapter on Hypothalamus, this volume, and Allen, Developing Mouse Brain Atlas).

Additional food for thought and reflection on alternative models is obtained by looking at gene expression patterns mapped for instance in the Euroexpress database at E14.4, which suggest that the mamillary region shares basal plate markers with the diencephalic basal plate areas, while infundibular and tuberal hypothalamic areas instead express some genes otherwise present in the alar plate, such as Lhx2 (Fig. 1.2 I) and Dlx2 (Fig. 1.6 B). Many markers are expressed in complex patterns both in the alar and basal plates. In contrast, Zic1 maps within the alar plate and does not appear within the rest of the hypothalamus (Fig. 1.2 H). This observation fits together with Shh and Nkx2.1 expression (Figs. 1.2 A, B) within the standard prosomeric model. These partly contradictory observations illustrate clearly enough that no single gene marker can be truly correlated with a neuromorphologic concept, such as ‘basal plate’. Many markers need to be contemplated within a clearly formulated working model, and accruing evidence of various sorts (not only gene mappings, but also experimental embryologic data, mutant phenotypes, gain- and loss-of-function studies, in vitro assays) should point out at some point whether the model is solid, or needs either development, or should be abandoned altogether for a better option.

We should attend to explanatory simplicity, which a good model allows, as well as its pregnancy, that is, its capacity to suggest interesting new ideas to be tested. After all, morphologic models are just mental instruments for helping us to think, and should never be regarded as representing the Truth. Therefore, we should look forward to further descriptive and experimental data needed to more accurately approach a causal model of hypothalamic regionalization.

Antero-Posterior (AP) Patterning in the Neural Tube

AP patterning is the process that leads to the generation of distinct transverse domains at different axial positions. There is evidence that AP patterning begins during early gastrulation in the neural plate. Vertical signals spread from underlying tissues (mesoderm and endoderm) to the overlying dorsal ectoderm, and planar signals from the organizer (Hensen’s node) and from non-neural ectoderm, acting through the plane of the ectodermal epithelium, contribute to the specification of AP regional differences (for review see Doniach, 1993 and Puelles et al., 2005; also Ruiz i Altaba, 1994). The initial AP pattern is induced by the combined action of two signals produced by the axial endomesoderm.

The first signal induces anterior neural fate, that is: forebrain and midbrain. While the candidate signal is the protein Cerberus, Lim1 and Otx2 transcription factors are key molecules to localize the corresponding organizer function in the endomesoderm and its effects on the neuroectoderm. Loss-of-function mutants result in mouse embryos which lack forebrain and midbrain, suggesting that Lim1 and Otx2 have a role in early AP patterning (Acampora et al., 1995, Ang et al., 1996 and Matsuo et al., 1995 1; Shawlot and Behringer, 1995). Lim1 is expressed in the primitive streak (notochordal primordium) and prechordal mesoderm. Because expression is not detected in the neural plate, the lack of forebrain and midbrain in Lim1 mutants is evidence for an essential role of this mesoderm in anterior CNS development (Shawlot and Behringer, 1995). Understanding the mechanisms underlying the Otx2 phenotype is more difficult, due to the dynamics of its expression pattern (FIGURE 1.1, FIGURE 1.2 and FIGURE 1.3) and to the complexity of its molecular interactions.

Subsequently, graded secondary signals progressively posteriorize the anteriorized neural plate, allowing hindbrain and spinal cord development. Candidate signals for this posteriorizing activity include retinoic acid (Blumberg et al., 1997, Durston et al., 1989 13 and Papalopulu et al., 1991; Ruiz i Altaba A, 1991 and Ruiz i Altaba A, 1991), basic Fgfs (FGF2; Cox and Hemmati-Brivanlou, 1995, Hemmati-Brivanlou and Melton, 1997, Kelly and Melton, 1995 and Lamb and Harland, 1995) and Wnt signaling (Elkouby et al., 2010). These molecules regulate the local expression of Hox-family genes. In mammals, this gene family comprises 39 closely related genes coding for homeodomain transcription factors, organized in 4 homologous clusters (A, B, C and D) (Pearson et al., 2005). The Hox genes have sharply defined anterior expression boundaries, but their posterior boundaries are typically less clear and largely overlap with the expression of more posterior Hox genes (Hooiveld and Morgan, 1999 and Marín et al., 2008 15).

Furthermore, mesendodermal tissues underlying the anterior neural plate could also regulate differentially regional patterns of genes, as in the case of Otx2 and Engrailed (En1) within the rostral brain (FIGURE 1.1, FIGURE 1.2 and FIGURE 1.3; Ang and Rossant, 1993 and Ang et al., 1994). Recently we have demonstrated the requirement of prechordal mesoderm for the development of normal regionalization of the ventral forebrain and midbrain in chick embryos (Garcia-Calero et al., 2008).

As previously introduced, the protein that apparently regulates the basic AP patterning process is named Cerberus. The expression of Cerberus appears in a broad anterior domain flanking the expression of Chordin and Lim1 in the prechordal plate. This pattern may specify the anterior axial mesendoderm and the prosencephalic field of the neural plate. When Cerberus was ectopically expressed in Xenopus embryos, it induced nearly complete head structures (Bouwmeester et al., 1996). Other genes, such as Noggin, Follistatin, Cripto and Chordin also induce anterior neural tissues, but these genes may not be essential for AP patterning (Hemmati-Brivanlou et al., 1994; Lamb and Harland, 1995 and Liguori et al., 2003 15 and 2009; for review see Doniach, 1993).

Regionalization along the antero-posterior axis of the neural plate and tube generates transverse blocks of neuroepithelium with distinct molecular identities (combinations of genes), which in their turn may modulate specific competence to respond to given inductive signals (Ericson et al., 1995, Hynes et al., 1995, Shimamura and Rubenstein, 1997 and Simon et al., 1995 1). This phenomenon is clearly illustrated by the local effects of the SHH signal. This gene is expressed along the entire AP extent of the prechordal plate and the notochord (Fig. 1.1). Whereas SHH directly induces the expression of some genes (e.g. Shh, HNF3β , Nkx2.2) in all AP regions of the median neural plate or ventral neural tube, other downstream genes are induced only at particular sectors along the AP axis. For instance, Nkx2.1 is expressed only in the prosencephalic neural plate (largely prospective basal hypothalamus and prethalamus), whereas Nkx6.1 is expressed in more posterior locations (caudal basal diencephalon, midbrain and hindbrain; Fig. 1.4 A; Qiu et al., 1998). The SHH signal also induces a continuous column of Plp/dm20-expressing oligodendroglial progenitors along the spinal cord. Nevertheless, although Shh is expressed homogenously in the prosencephalic basal plate, only discontinuous clusters of this oligodendroglial column have been identified in the parabasal plate of chick embryos, in anterior mesencephalon and prosomeres 1 and 2 (Garcia-Lopez and Martinez, 2010 and Perez Villegas et al., 1999), suggesting heterogeneous competence states of the neuroepithelial cells at different segmental territories. FGF8 is another example of an inductive signal that generates differential molecular responses at different axial levels. The FGF8 signal is required for cell survival and regionalization in dorsal telencephalon and mid-hindbrain territories (Chi et al., 2003; Storm et al., 2006), but acts through the induction of different genes in prosencephalic versus mesencephalic domains: rostrodorsally (telencephalic field) it induces FoxG1 (Bf1), whereas caudally it induces En2 (Figs. 1.4 B, C; Shimamura and Rubenstein, 1997).

Secondary Organizers Control Molecular Regionalization

Regionalization of the anterior neural plate appears to result from the superposition of multiple, distinct, patterning mechanisms. So far we have described how initial phenomena of AP patterning create transverse zones, each with a distinct histogenetic competence; concomittant signaling phenomena graded along the ML axis generate longitudinally aligned domains, which later represent the DV longitudinal zones of the neural tube. The intersecting combination of DV and AP patterning accordingly generates a grid-like organization of distinct brain progenitor primordia with characteristic complex molecular identities, with shared and unshared gene markers. Shared determinants will cause general properties along the relevant dimension, whereas unshared ones will probably cause distinctive features. Therefore, neural progenitors in the epithelium will establish their proliferation and differentiation programs under the networked control of multiple positional information signals: the resulting Cartesian map is a representation of the two main topological dimensions along which the transmitted molecular signals diffuse. All the rest of CNS development is an epiphenomenon of these early decisions on cell molecular identity.

Distinct neural and glial identities are acquired by neuroepithelial derivatives through progressive restriction of early histogenetic potential, under the influence of local environmental signals. Evidence for morphogenetic control sites at specific locations in the neural tube has led to the concept of secondary organizers, which regulate the identities and regional polarity of neighboring neuroepithelial regions (FIGURE 1.1, FIGURE 1.2, FIGURE 1.3, FIGURE 1.4 and FIGURE 1.5; for review see Vieira et al., 2010). These organizers, acting in a second step, and with more restricted spatial ranges, after those that operate throughout the embryo during gastrulation, usually develop as specialized neuroepithelial patches or lines within the previously broadly regionalized neuroectoderm, usually at given genetic boundaries; frequently cells expressing different transcription factors are juxtaposed at the secondary organizers (Broccoli et al., 1999 9, Garda et al., 2001, Katahira et al., 2000 1, Li and Joyner, 2001, Martinez-Barbera et al., 2001 and Millet et al., 1999 9). Their function is to release one or more diffusible morphogens with a particular direction and range, establishing gradients of their products that can be interpreted in terms of differential regulation of genomic transcription by competent cells within the action range. Subsequently, their activity refines local neural identities along the AP or DV axes (Fig. 1.5 A–C; Figdor and Stern, 1993 17, Joyner et al., 2000, Meinhardt, 1983, Rubenstein et al., 1998 and Wassef and Joyner, 1997).

Three regions in the neural plate and tube have been identified as putative secondary organizers (Fig. 1.5 C, D): the anterior (terminal) neural ridge (ANR) at the open neural plate, which gets later incorporated into the roof of the secondary prosencephalon, the zona limitans intrathalamica (ZLI) in the diencephalon and the isthmic organizer (IsO) at the rhombencephalic-mesencephalic junction.

Whereas abundant literature has been published regarding the molecular and cellular processes underlying IsO activity and resulting midbrain-hindbrain regionalization (recently revised in Vieira et al., 2010), less is known about diencephalic regionalization and ZLI activity, and very little about ANR regulation of telencephalic compartmentalization. Therefore, we will just review the most recent data about diencephalic regionalization, which is a hot field within developmental neurobiology at present.

Diencephalic Regionalization: How the Diencephalon Creates its Structural Complexity

The diencephalon is a central area of the developing brain that generates the thalamic/prethalamic nuclear complex and the pretectum in its alar plate, whereas it generates corresponding diencephalic tegmental structures in the basal plate (FIGURE 1.1, FIGURE 1.2 and FIGURE 1.3 E). In the prosomeric model, the diencephalon is subdivided into three transversal territories (prosomeres 1–3) each displaying a characteristic molecular identity and fate (Bulfone et al., 1993, Puelles, 1995 and Puelles and Rubenstein, 1993; Puelles et al., 1987; Rubenstein et al., 1994 28 and Rubenstein et al., 1998). The respective alar territories are better understood anatomically, whereas our knowledge of the correlative tegmental units suffers from the fact that historic prevalence of the columnar forebrain model has tended to relegate them to the spurious category of ‘rostral midbrain tegmentum’, which is obviously unsupported by the molecular data we have now (see, e.g., FIGURE 1.1, FIGURE 1.2, FIGURE 1.3, FIGURE 1.4, FIGURE 1.5 and FIGURE 1.6 B, N). The atlases of Swanson, 1992 and Altman and Bayer, 1995 and Deng (2008) still illustrate that obsolete viewpoint, whereas the Neuroexpress and Allen Developing Mouse Brain Atlas databases illustrate the novel scenario also sketched here.

In the mature brain, these diencephalic transversal units contain the following anatomic complexes in their alar plates: the pretectum (PT) (in prosomere 1; p1), the thalamus (Th) and epithalamus (ET) (in prosomere 2; p2), the prethalamus (PTh) and the prethalamic eminentia (PThE) (in prosomere 3; p3; the ‘prethalamus’ term was first proposed in its present usage by Puelles and Rubenstein, 2003 and its use has spread widely; neuroanatomic literature contains some other usages with somewhat different meanings). In their basal plates the diencephalic prosomeres produce structures that are classified as pretectal tegmentum (p1Tg), prerubral tegmentum (p2Tg) and prethalamic tegmentum (p3Tg or Forel’s field) (Fig. 1.3 E). Fate mapping studies using experimental embryology (Garcia-Lopez et al., 2007, 2009) and genoarchitectonics (persistent gene expressions in derivatives; Delaunay et al., 2009 1, Ferran et al., 2007 1, Ferran et al., 2009 1 and Garcia-Lopez and Martinez, 2010), have confirmed these lineage relations. Detailed molecular characterization and delimitation of the mouse prethalamus as compared with the hypothalamus should be studied in the works of Puelles and Rubenstein (2003) and Shimogori et al. (2010) (see also chapters on Hypothalamus and Diencephalon, present volume).

The zona limitans intrathalamica (ZLI) is a transversal ventricular ridge in the diencephalic neural tube, corresponding to a surface transverse constriction, or furrow, between p2 and p3 (Figdor and Stern, 1993; Garcia-Lopez et al., 2007; Larsen et al., 2001 1, Puelles et al., 1987 8 and Rendahl, 1924), thus separating the anterior (p3) and posterior (p2 and p1) diencephalic regions (FIGURE 1.1, FIGURE 1.2 and FIGURE 1.3, 1.5D, 1.6A,B; Puelles and Rubenstein, 2003). This intrathalamic limit progressively appears in a ventrodorsal sequence in the early neural tube, and exhibits an unique pattern of molecular expression conserved among all studied vertebrates, which suggests an important role for this area as a secondary morphogenetic organizer in diencephalic histogenesis (Fig. 1. 6 A; Kiecker and Lumsden, 2004, Vieira and Martinez, 2006 and Vieira et al., 2010 for review see: Echevarria et al., 2003).

Planar AP interaction between differentially prespecified neuroepithelia may trigger the molecular mechanisms that regulate the positioning and specification of the ZLI (FIGURE 1.1, FIGURE 1.2, FIGURE 1.3, FIGURE 1.4 and FIGURE 1.5; Garcia-Calero et al., 2006; Kobayashi et al., 2002; Vieira et al., 2005). Such planar phenomena have been postulated to induce the conditions in the alar plate that permit the ‘ectopic’ expression of Shh in the ZLI (its expression domain being otherwise restricted to the basal or floor plates), and also would activate the morphogenetic (signaling) properties of this organizer. SHH signal released from the ZLI diffuses rostrally into p3 and caudally into p2 and p1; it therefore can regulate graded AP regionalization and cell fate of the different diencephalic prosomeres, through the control of specific gene expressions (Figs. 1. 5 A,C, 1.6A; Ferran et al., 2007, 2009; Kobayashi et al., 2002; Vieira et al., 2005). The pattern of Shh expression in the ZLI thus starts by being limited to the diencephalic basal plate and then extends dorsally into the presumptive ZLI epithelium, by a process of homeogenetic induction (Echelard et al., 1993; Echevarria et al, 2003; Shimamura et al., 1995 and Vieira and Martinez, 2006). The importance of Shh expression in the ZLI is supported by the fact that mice which bear a mutant Shh show defects in early development, with an important reduction in size of the diencephalic vesicle (Chiang et al., 1996; Ishibashi and McMahon, 2002).

Cellular identity in the diencephalon may be under the control of genes whose expression is regulated by signaling cascades activated by ZLI-derived morphogens (Fig. 6 A, B). Some examples of this are:

1) cells nested within the ZLI Shh expression domain express the transcription factors Sim1 and Pitx2 (Fig.1.6 B; Fan et al., 1999; Kitamura et al., 1997).

2) Nkx2.2 and Fgf15 are expressed both rostrally and caudally in shell domains next to the ZLI, (Gimeno et al., 2007; Martinez-de-la-Torre et al., 2002; Price et al., 1992; Shimamura et al., 1995).

3) The Gbx2 transcription factor is expressed caudal to the ZLI core and caudal shell –see selective labeling of this ZLI shell in Fig. 1.6 E – and serves as a marker for the thalamus, excluding the epithalamus (Figs. 1.6 B, N; Martinez-de-la-Torre et al., 2002).

4) Dlx2 and Nkx2.1 are expressed in the alar and basal plate, respectively, just rostral to the ZLI (Gonzalez et al., 2002).

5) The dorsal tip of Shh expression in the ZLI is flanked by Wnt1 caudally and Fgf8 rostrally (Martinez-Ferre and Martinez, 2009).

Recent experimental data from our group has demonstrated that SHH signals from both the ZLI and the basal plate play an important role in the molecular patterning of the diencephalic alar plate (Vieira et al., 2006). Basal plate signals probably play an initial role in the specification of longitudinal territories in the thalamic area (ventro-dorsal patterning), while subsequent development of the ZLI represents an additional source of morphogenetic signals that superimpose graded antero-posterior information upon the thalamic epithelium. The combined effect of these two types of information possibly contributes to the complexity of thalamic molecular regionalization and as a consequence, to its complex