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Genetic control of early neuronal development in vertebrates
Genetic control of early neuronal development
in vertebrates
Vladimir P Korzh University
of Umea, Umed, Sweden
The specification of neuronal fate startswith cell commitment and determination. These early events are accompanied by rearrangement and reshaping of presumptive neural cells. Later, the neural differentiation begins, and its course can be followed using specific molecular markers. Such events take place long before the cells acquire a typical neuronal phenotype. Primary neurons of lower vertebrates differ from secondary neurons by their size, position, timing of differentiation and length of axon. Primary neurons start to express early markers of neural differentiation at the end of gastrulation. Recent data indicate that in lower vertebrates the neural induction of primary neurons differs from the induction of secondary neurons; however, neural induction in higher vertebrates appears to be similar to the induction of secondary neurons
in lower vertebrates.
Current Opinion in Neurobiology 1994, 4:21-28
Introduction Neuronal cell fate is determined during early developmental events. Vertebrate neuronal development is characterized by the appearance of a neural plate at the end of gastrulation. This is a result of neural induction, i.e. the interaction between mesoderm and ectoderm. The next stage involves specification of the neuroblasts, which may be a result of local interactions within cell groups. These events are probably responsible for the different ways that presumptive neuroblasts react to inductive signals. Neural induction signals cause multipotential undifferentiated cells to develop into particular cell lineages. Recent studies have shown that regulatory genes and signal transduction pathways are evolutionarily conserved. In this review, I discuss the molecular mechanisms of neural induction and the recent progress in the understanding of the early stages of genetic control of neural development. Some aspects of this problem have been discussed in earlier reviews D-41.
Developmental decisions One of the major problems in developmental biology is understanding the mechanisms restricting possible cell fates or cell commitment [51. Studies of neurogenic genes in Drosophilu (reviewed in 161)and recent observations on vertebrates both show the importance of the developmental decisions taken by the neuroepidermal precursor. The genes involved in regulation of neural determination include positive and negative regulators of neurodevelopment. For example, the genes of the achaetae-scute complex (AS-O encode transcription
factors of the basic helix-loop-helix (HLH) class that exerts positive control over cellular differentiation in a variety of developmental systems 171.Although studies of the mammalian AS-C homologue 04AW1) suggest that it could be involved in neural ontogeny [81, the specific interactions remain to be defined. The prox-1 gene, a homologue of Drosophila prospero, could be one of the genes that interacts with the AMY genes. The prox-1 gene is expressed in neuroblasts where its expression pattern overlaps with that of MAW1 and partially with those of evxl (even-skipped homeobox) and en-2 (engruiled-2) Dl. Interestingly, expression patterns of the positive regulator V-MASH1 appear to be similar to that of its negative regulator, Notch [lo]. Genetic and biochemical analyses of Notch and Jd, another gene with similar function, suggest that both positive and negative developmental decisions are important in determining neural cell fate [7,11,12,13’1.
Neural induction The nature of neural-inducing signals has only been discovered recently. Sources of the signals might include the organizer (e.g. the embryonic shield in fishes, dorsal lip of blastopore in Amphibia, or Hensen’s node in birds and mammals) and the midline structures derived from the organizer, i.e. notochord and floor plate (recently reviewed in [14’1). Two routes of neural induction have been proposed: a vertical route from mesoderm to overlying ectoderm; and a horizontal route, involving planar signals through the dorsal ectoderm [151. Recent results indicate that both vertical and planar signals interact and contribute to the induction and axial patterning of the nervous system
Abbreviations ASX-achaerae-scute complex; CNS-central HNF-hepatocyte nuclear factor; HLH-helix-loop-helix;
nervous system; en-2-engrailed-t evx-even-skipped homeobox; MASH7-mammalian AS-C homologue; TGF--transforming growth factor.
0 Current Biology Ltd ISSN 0959-4388
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Development
duriog early embryogenesis [16-181. Some progress has been made in characterizing factors involved in induction events. These are encoded by genes expressed within the organizer region and midline structures, and include the following: the homeobox gene goosecoid ~9,201, involved in cell migration; members of the HNF/fork head family 121.1, which are implicated in specification of the floor plate I22.1; the Brachyury Cnt0 gene [23,24,25*1, which is necessary for organization of the notochord and development of posterior mesenchyme; genes encoding secreted factors, such as Wnt proteins [26l, whose products are thought to be involved in cell-cell interactions; and members of the LIM/homeodomain gene family, which encode transcription activation factors [27,28*] (see Fig. 1).
Primary and secondary neurons The primary and secondary neurons in adult lower vertebrates are arranged in the dorsal and ventral portions of the motor column, and they innervate different groups of muscles 1311. Early in development primary neurons form a coordinated system responsible for the escape reaction. They have large somata and long axons [32l. In zebrafish embryo, the primary neurons originate in the brain and spinal cord, and pioneer major axonal tracts [28*,33361. In embryonic spinal cord their final precise position may be adjusted to fit the segmentation of axial mesoderm [32,341. Furthermore, primary motor neurons are committed to a specific cell fate within each segmental group, 2-3 h before axonogenesis 1371. The specification of individual neurons within a segmental group may depend on the expression of the LIM/homeodomain gene family (VP Korzh, S Thor, T Edlund, unpublished data). In Amphibia, segmental organization of the central nervous system (CNS) exists very early in development [381, but after the secondary neurons appear, longitudinal columnar organization becomes more apparent 1391.
The importance of secreted factors in neural induction has long been suspected. Recent experimental evidence now supports this theory. A secreted factor, noggin, is expressed in the organizer region and can mimic the effect of the organizer 1291. Furthermore, noggin directly induces neural tissue in Xenopus embryo, thus making it the first neural inducer to be identified 130’1. Recent data indicate that other secreted molecules, such as vertebrate homologs of the Drosophila hedgehog gene product, are differentially expressed within the organizer and its midline derivatives, the notochord and floor plate. These homologs may play an important role in inducing midline derivatives of neuroectoderm (V Korzh, T Edlund, H Roelink, J Dodd, T Jesse& unpublished data; see Note added in proof).
&pax-b)
-t Wnt- I+
This evolutionary tendency is more obvious in higher vertebrates. In the chick, segmental organization of the CNS is preserved in the brain but disappears in the spinal cord where CNS columnar organization becomes established as soon as neurulation comes to an end [401. In mammals, this process results in subdivision of the motor column into discrete motor pools 1411.
en-2
Neuroectoderm
pik?lla “is
0 O
c.) Signal
precursors
O
cl.
Somite
hial
,
>
II Bracn:rury
muscle
Danforth’s short tail
pioneers
n
0
v
0 1994 Current ODinion m Neurobiolorv
Fig. 1. General scheme of neural induction in vertebrates. Recent analysis of mutants of floor plate (FP) and notochord have shown that the induction of primary neurons of lower vertebrates could be dependent on inductive signals from cell predecessors of midline structures. This type of inductive signalling disappears in higher vertebrates, where neurodifferentiation starts only after neural tube closure. cyc-7, ntl - zebrafish mutations; Danforth’s short tail, Brachyury, Pintail, truncate - murine mutations; NP - notoplate. Based on [2,19,20,21 ?? ,22*,23,24,25*,29,43*,44,45,48,49,5Ooj; S Dietrich, P Gruss, F Schubert, personal communication.
Genetic control of early neuronal development in vertebrates Korzh
The difference in the organization of primary and secondary neurons within the spinal cord might be predetermined by the difference in molecular mechanisms involved in establishing anterior-posterior patterning or in the competence of primary and secondary neurons to respond to specific signals during early neuronal induction and/or specification.
Early events in neurodifferentiation Recent progress in understanding neural induction in zebrafish using early markers of neural differentiation have shown that early events of neural differentiation, such as cell commitment, cell rearrangement, neural induction and expression of the first neural markers, take place within 2-3 h (Fig. 2). For example, cells of the hypoblast, which participate in the formation of mesoderm and entoderm, become committed to a specific fate and position at the mid-gastrula stage (8 hours post fertilization) 142.1. Cell-labelling experiments have shown that immediately after commitment, intensive sorting of neural and ectodermal progenitors completely separates cells with neurogenic and epidermogenic capacities 143’1. The induction of LIM/homeodomain proteins, recognized by anti-Isl-1 antibody, takes place at the end of gastrulation in motor neurons located along the midline
(a)
The neural differentiation in chick initially follows the same trend. Cells become committed and patterns of neuroepithelium are arranged before an inductive interaction with the notochord. Neural induction takes place very early during stages 2-4 147,481. Inductive signals from the notochord may, therefore, act on the dividing neural plate cells to confer the potential to generate the neurons. The selection of the particular cell fate and appearance of corresponding markers in individual cells is postponed until stage I5 1491 and could then be influenced by other factors 150’1. The chick motor neurons start to express 1~1-1 before any other known molecular markers, immediately after the final division of motor neuron progenitors. At that time point cell progenitors are located close to ventricular zone 1491. In addition to inductive signals, the notochord and the floor plate provide trophic signals,
Hox cluster,
Gtx, Wnt, D/x, Pax, Ems, hedgehog
and Rohon-Beard cells located at the lateral edges of the neural plate. This occurs at the very beginning of morphological differentiation of the midline structures 128’1. Almost simultaneously, a subset of interneurons starts to express puxCzf-b) 144,451.Regulatory interactions between pax&f-b), Wnt-1 and en-2 suggest that functional relations between these genes are conserved during evolution from Drosophila to teleosts, and perhaps even further 1461. Secondary motor neurons start to express 1~1-1after formation of a spinal cord 128.1.
Pax, Wnt, 5’
hedgehog
3’
I
Anterior 4
Cross section 1
(b)
b Posterior
Cross section 2
4
a
Dorsal
I
Cross section 1
Ventral Cross section 2
0
1994 Current Opinion in Neurobiology
Fig. 2. Early stages of neurodifferentiation in zebrafish. The first markers of neurodevelopment start to appear in zebrafish embryo at the end of gastrulation, before yolk plaque closure. The regionalization of the neuroectoderm could be predetermined as a result of expression of several gene families. (a) Top view on the neuroectoderm of whole-mount immunostained embryos. (b) Cross settions of such embryos. ACC - anterior group of cells; Trg - trigeminal ganglia; PMN - primary motor neurons; IN - interneurons; RB - Rohon-Beard neurons. Based on [29,43*,52*,53]; VP Korzh, unpublished observations.
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promoting either cell division or cell survival in neural plate explants. These could be peptide growth factors, such as acidic and basic FGF (fibroblast growth factor) and TGF-fl2 (transforming growth factor-p2), enhancing proliferation within neural plate in vitro 151'1. The specification of neuronal classes in the spinal cord depends on anterior-posterior and dorsal-ventral regionalization of the spinal cord, and might be established due to the expression of extracellular matrix components 152’1, Hox code 153,541, dorsalin-1, Pax-3, Pax-6, and Pa-7 [55’,56’,571 (Fig. 3).
Analysis of midline
lower vertebrates
In zebrafish, several mutants lacking midline structures have been identified. cyc-1 mutants lack floor plate and develop cyclopia due to the lack of the ventral cells in the forebrain [21. One study of cyc1 mutants shows nearly normal development of all motor neurons, i.e. both primary and secondary ones L62.1. In another study, a reduction of ventral GABAergic Kolmer-Aghuhr neurons, located close to the floor plate, has been reported [63*1. The ventral axonal pathway is disorganized in cyc-1 mutants, implying that floor plate is important for the pathfinding of motor neuron axons. Interestingly, the cyc-1 phenotype, in general, is similar to the Xenopus phenotype that arises after reduction of the quantity of the organizer during late blastula WI, illustrating ‘community’ effect during induction of dorsal mesoderm [651. Another tebrafish mutation disrupting development of midline structures, ntl, produces mutants lacking a differentiated notochord. Nevertheless, the presence of the differentiated notochord in this mutant is not a prerequisite for the induction of floor plate and neurons. Perhaps cell predecessors of the notochord supply the inductive signals f25.1.
mutants
Higher vertebrates
Molecular analyses in higher vertebrates have shown that the lack of midline structures leads to dramatic changes in the organization of ventral spinal cord and specific classes of neurons in murine mutants or chick embryos deprived of their notochord. The floor plate and motor neurons are missing after removal of notochord in chick embryo 149,581. In contrast, dorsal spinal cord is less affected by close opposition of transplanted notochord, which cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest derived dorsal root ganglia 1591.
Irradiation of Xenopus fertilized eggs by ultraviolet light produces notochord-less embryos. Analysis of CNS differentiation has shown that lack of the notochord results in the arrest of floor plate. The number of secondary neurons is dramatically reduced, whereas primary neurons are less affected 1661.
Analysis of mutants has shown that Danforth’s short tail mutants in mice lack notochord caudally, resulting in the arrest of development of floor plate and motor neurons 1601. Experimental removal of notochord or the absence of notochord in notochord-less mutants of mouse, such as Brachyury curtailed (Tc), Danforth’s short tail (Sd), Pintail (Pt) and truncate (tc), leads to ectopic ventral spread of expression of molecular markers normally mapped more dorsally ([55*,56*,611; S Dietridh, F Schubert, P Gruss, personal communication).
??zf
(pax-b)
B 1994 Current Op~mon I” Neurobiology
These observations show that neural induction in lower vertebrates takes place very early in development, before the morphological differentiation of midline structures (Fig. 4). Cell precursors of midline structures are probably responsible for the signalling of neural induction in this case. Primary neuron differentiation may be less dependent on influences from the organizer or differentiated midline structures than dif-
IPax3
.
Pax8
ElPax6
0
Pax2
IDPax
0
Pax5
Fig. 3. Neurodifferentiation in the spinal cord. The specification of cell types in the spinal cord could be dependent on inductive signalling from two poles of the spinal cord: dorsal plate (dorsally); and floor plate and notochord (ventrally). The interaction of these signals could determine the distribution of expression patterns of other cell fate determinants, like Pax genes. AP-alar plate; BP-basal plate; FP-floor plate; IN-interneurons; PMN-primary motor neurons; RB-Rohon-Beard cells; RProof plate; SL-suicus limitans; SMNsecondary motor neurons. Based on [22*, 29,42.,43*,44,54,55.,56*].
Genetic control of early neuronal development in vertebrates Korzh
Fertilization
0 I
Gastrulation
Neural tube formation
Neurulation
A
/
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Neural induction
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Commitment
-1
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--I
Competence Primary neurons . Neurodifferentiation
Secondary neurons I
3 1994 Current Opinion in Neurobiilogy
ferentiation of secondary neurons. First, primary neurons might be saved in ultraviolet-treated Xenopus embryos owing to the different sensitivity of the signalling pathway involved in inductive events leading to an appearance of primary and secondary neurons. Second, development of primary neurons might depend more on maternally derived factors. Interestingly, CNS determination in Xenopus can occur even without cell division, and the phenotype of cells arising after differentiation is more like primary neurons 1671. Primary neurons are also preserved in the neurodegenerative zebrafish mutant ned-1, in which secondary neurons die [68]. Currently, several laboratories are using saturation mutagenesis on zebrafish so as to be able to mutate all, or at least most, of the zebrafish genes (e.g. [691>. We will be in a better position to tackle the problem of primary neuron induction when more mutants are identified.
Conclusion Higher vertebrates do not have primary neurons. In lower vertebrates some of the primary neurons disappear during development. This could be attributed to the development of complex reactions based on processes such as the vision pathway, which substitute primitive avoidance reactions that involve primary neurons. During evolution, segmental organization of the CNS characteristic for primary neurons has been substituted by a columnar one, characteristic for secondary neurons. Finally, in higher vertebrates primary neurons and segmental organization of CNS in the spinal cord disappear completely. This could be related to changes in developmental programs, including elongation of embryogenesis and emergence of the parental guardianship over their progeny.
??
Fig. 4. Early events of neurodifferentiation of primary and secondary neurons. The early events of neurodifferentiation, such as neural induction, cell commitment, rearrangement and reshaping, take place at the end of gastrulation. The appearance of specific neural markers in primary and secondary nyurons occurs in two stages. Primary neurons start to express 1~1-1,a marker for neurodifferentiation, at the end of gastrulation before neural tube formation. This event is postponed in secondary neurons until after neural tube formation.
Recent studies have shown that the organizer and derived midline structures are the source of neural induction. Lack of notochord or floor plate leads to dorsalization of the spinal cord and impaired development of ventral CNS. In particular, defects of midline structures have minor effects on lower vertebrate primary neuron development, which can be accounted for by inductive signals from cell-predecessors of differentiated midline structures. Comparative analysis of embryos of lower and higher vertebrates lacking midline structures shows an apparent difference in the mechanism of neural induction of primary and secondary neurons. Primary neurons start to express 1~1-1 early, while they are still in the neuroepithelium, perhaps because of the early inductive signals from cell predecessors of the midline structures. The neurons of higher vertebrates also become committed to neuronal fate early, but the appearance of specific molecular markers is postponed until after formation of the spinal cord. Further studies of molecular mechanisms involved in neural differentiation may account for the difference in induction between primary and secondary neurons. It is clear that mutations of all genes known to participate in emission, transmission, reception and processing of neural induction signal? will be instrumental in understanding the details of the molecular apparatus involved. Once these are clear, we may be able to understand why higher vertebrates have no primary neurons. The analysis of neural induction mechanisms indicates that the evolution of developmental programs can be traced not only phylogenetically but also by using molecular genetics and experimental embryology. During early evolution of vertebrates the neural tube might be induced by signals of cell predecessors of midline structures.
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Development
Note added in proof Recently, vertebrate homologues of the Drosophila segment polarity gene hedgehog have been cloned and found to be expressed in regions implicated in polarizing activity, such as the ZPA (zone of polarizing activity) in the limb and the notochord/floor plate. Functional evidence is emerging that vertebrate hedgehog gene products, which are secreted proteins, act as signals for antero-posterior patterning in the limb and dorso-ventral patterning in the neural tube [X1-73].
Acknowledgements I thank T Edlund, J Eisen and D Milton for critical reading of the manuscript. I am indebted to C Kimmel, J Eisen, T Jessell, M Westemeld and P GNSS, and many members of his lab, for sending preprints. I am a permanent member of the Koltsov Institute of Developmental Biology, Moscow, Russia.
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of the Zebra&h
in zebrafish was studied in detail using high resolution histology and cell labelling. During neurulation, cells with different developmental capabilities within the neuroectoderm became resolved into territories. This is due to intensive cell migration, when cells with neurogenic and epidermogenic capacities separate from each other, in some cases moving a distance of up to about 400 pm within 1-2 h. This causes three areas of thickening in the neural plate, a medial and two lateral ones, corresponding to the neural folds in higher vertebrates. This establishes that neurulation in zebrafish is very similar to neurulation in higher vertebrates. &?UNkitiOn
44.
KRAUSSS, JOHANSENT, KORZH V, FJOSEA: Expression of the ZebraIish Paired Box Gene Pax(zf-b) During Early Embryogenesis. Lkwelopmenf 1992, 113:1193_1206.
45.
MIKKOLA I, FJOSE A, KLJWADAJ, WILSON S, GUDDAL PH, KRAUSS S: The Paired Domain Containing Nuclear Factor Zpax-2 is Expressed in Specific Commissural Interneurons in Zebrafish Embryos. J Neurobiol 1992, 23:933-946.
46.
KRAUSS S, MADEN M, HOLDER N, WIL-SONS: Zebrafish pa@] is Involved in the Formation of the Midbrain-Hindbrain Boundary. Nature 1992, 360:87-89.
47.
ALVAREZI, SCHOENWOLF G: Patterns
48.
STOREYK, CROSSLEYJ, DE ROBERTISE, NORRISW, SXRN C:
of Neuroepithelial Cell Rearrangement During Avian Neurulation arc Determined Prior to Notochordal Inductive Interactions. Deu Biol 1990, 143:7&92.
Neural
Induction
and Regionalisation
of the Chick
Embryo.
Development 1992, 114:72%741. 49.
in the Brain of Zebralish
J Neurosct 1990, 10:1892-1905.
Embryo
ERIC~ON J,
THOR S, EDLUND T, JESSELL T, YAMADA T: Early Stages of Motor Neuron Differentiation Revealed by Expression of Homeobox Gene Islet-l. Science 1992, 256:1555-1560.
YAMADA T, PFAFF S, EDLUND T, J&SELL T: Control of Cell Pattern in the Neural Tube: Motor Neuron Induction by Diffusible Factors from Notochord and Floor Plate. Cell 1993, 73:673-686. Neuronal differentiation in the spinal cord on different levels along the dorsal-ventral axis was studied using explants from ventral, dorsal and intermediate parts of the chick spinal cord. Specification of cell fate begins at the neural plate stage. Determination of the ventral cell fate depends on diffusible signals from the notochord and the floor plate. These signals act directly on the neural plate cells and are sufficient to induce motor neuron differentiation. The floor plate acquires motor neuron-inducing activity after the notochord, but at this stage many neural plate cells are still competent to generate motor neurons, and the signalling from the floor plate could be involved in generating motor neurons as well. 50. .
27
28
Development PWCZEK M, JF..%ELL T, DODD J: Induction of Floor Plate Differentiation by Contact-Dependent, Homeogenic Sials. &uelopment 1993, 117:205-218. Methods of transplantation and culturing of various regions of the neuroectoderm in tirro were used to evaluate the role of midline StNCmRS, in particular the notochord, during neural induction. Specification of cell fate and the dorso-ventral patterning of cell types begin at the neural plate stage. The notochord provides the initial source of signals responsible for both the differentiation of the floor plate and motor neurons. Induction of the floor plate is dependent on the contact with the notochord. The rostra1 region of the neural plate that gives rise to the forebrain is refractory to the floor plate induction. 51. .
A, SAN&J: Retrovirally Intro duced Antisence Integrin RNA Inhibits Neuroblast Migration fn nf.0. Nertrofl 1992, 9:1117-1131. The method of retrovirus-mediated gene transfer was used for the antisense RNA knockout of fit-integrin in migrating neuroblasts of the chick tectum. These cells failed to migrate and accumulated in the ventricular zone showing integrin involvement in the migratory process. 52. .
53.
GALILEOD, MAJORSJ, Ho~wrrz
HUNT P, WHITING J, NONCHEV S, SHAM M, MARSHALL H, GRACHAM A, COOK M, ALLEMANN R, RIGBY P, GUI.ISANO M, ET AL.: The Branchial Hox Code and Its Implications for Gene Regulation, Patterning of the Nervous System and Head Evolution. Lkuelopment 1991, Suppl 2:63-78.
54.
KESSEL M, Gauss P: Homeotic Transformations of Murine Vertebrae and Concomitant Alteration of Hox Codes Induced by Retinoic Acid. Cell 1991, 67:89-104.
of Cell Pattern in the Neural Tube: Regulation of Cell Differentiation by Dorsalin-I, a Novel TGF Family Member. Cell 1993, 73687-702. A novel member of the TGF gene family, dot~-alin-l, was cloned in chick. It appears to be expressed selectively in the dorsal spinal cord due to restrictive signalling from the notochord. Experiments in tiifro have shown that dorsalinpromotes differentiation of cells with neural crest-like properties and inhibits induction of motor neurons acting as a dorsalizing factor. This study establishes the importance of dorsal signalling for the overall organization of the neural tube along the dorsal-ventral axis and the specification of dorsal neurons. 55. .
60.
BOVOLENTA P, DODD J: Perturbation of Neuronal Diierentiation and Axon Guidance in the Spinal Cord of Mouse Embryos Lacking a Floor Plate: Analysis of Danforth’s Short Tail Mutation. Development 1991, 113:625-639.
61.
PHELPS D, DRESSLERG: Aberrant forth’s Short Tail (Sd) Mutants.
62. HATTA K: Role of the Floor Plate in Axonal Patterning in . the Zebrafish CNS. Neuron 1992, 9:629-642. This study of the zebra&h mutant cyc-1, which lacks the floor plate, demonstrates the importance of the floor plate in normal axonal pathfmding. The preservation of neurons within the spinal cord where the development of all spinal motor neurOns is nearly normal is particularly noteworthy. The induction and proper positioning of motor neurons may not require a differentiated floor plate and could depend on the presence of the notochord or undifferentiated signalling cell predecessors of the floor plate. 63. .
BEHNHAHM R, P~I’EL C, WILSON S, KUWADA J: Axonal Trajectories and Distribution of GABAergic Spinal Neurons in Wildtype and Mutant Zebrafish Lacking Floor Plate Cells. J Comp Neural 1992, 326~265272. Analysis of the neural differentiation in cyc-1 mutants, lacking the floor plate, detects reduction of ventral GABA-positive neurons in the spinal cord and more severe defects in the overall organization of anterior brain. It also shows disturbances in axonal trajectories of some interneurons suggesting that floor plate cells provide one of the several guidance cues for axonal outgrowth and play a role in cellular differentiation.
64.
STEWART R, GERHART J: The Anterior Extent of Dorsal Development of the Xenopus Embryonic Axis Depends on the Quantity of Organiser in the Late Blastula. Drvelopment 1990, 109:363-372.
65.
GUKDON J, TILLERE, ROBERTSJ, KATO K: A Community
BASLER K, EDLUND T, JESSELLT, YAMADA T: Control
in Muscle
CHALEPAKISG, STOYKOVAA, WIJNHOLDSJ, TREMBLAYP, GHUSS P: Pax: Gene Regulators in the Developing Nervous System. J Newohol 1993, in press.
58.
YAMADAT, PLACZEKM, TANAKA H, DODD J, JESELL T: Control of Cell Pattern in the Developing Nervous System. Cell 1991, 664:635-647.
59.
ARIINGER K, BHONNEH-FRASEW M: Notochord
Suppress Formation of Neural Crest rons. Lkvelopment 1992, 116:877-886.
Grafts Do Not or Commissural Neu-
Cztrr
Effect
Biol 1993, 3:1-11.
CLARKE J, HOLDER N, SOFFE S, STORM-MATHISENJ: Neuroanatomical and Functional Analysis of Neural Tube Formation in Notochordless Embryos; Laterality of the Spinal Cord is Lost. Dewlopment 1991, 112:499-516.
67.
HARRIS W, HARTENS~INV: Neuronal Determination Without Cell Division in Xenopus Embryos. Nerrron 1991, 6:499-515.
68.
GRUNWALD D, KIMMEL C, WESTERFIELD M, WAI.KEK C, STREISINGER G: A Neural Degeneration Mutation that Spares Primary Neurons in the Zcbrakish. L&v Biol 1988, 126:11%128.
69.
MULINS M, NOSSLEIN-VOLHARD C: Mutational
.
57.
Development.
66.
56.
GOULDING M, LUMSDEN A, Gauss P: Signals from the Notochord and Floor Plate Regulate the Region-Specific Expression of Two Pax Genes in the Developing Spinal Cord. Lkvelq.nnent1993, 117:1001-1016. Members of a Pax gene family are expressed in a regionalized way in the spinal cord. Chick homologs of par-3 and panG were cloned, and fn sitar hybridization, along with the transplantation of the notochord, were used to study the effect of the notochord on the distribution of pa3c expression during early development. The results were interpreted in favor of the model where two signals control the regional expression of par-3 in the spinal cord. The dorsal signal activates plrr-3, and the ventral signal, dependent on the notochord, represses p-3 expression. parGexpression maps the intermediate region of the spinal cord only, perhaps due to negative effect of the notochord on pu.x-G expression in the ventral region of the spinal cord.
Expression of Pax-2 in DanDa, Bloll993, 157:251-258.
Studying Embryonic Pattern Formation Cirrr Opin Genet Lkv 1993, 3648-654. 70.
Approaches to in the Zebrafish.
RIDDLE R, JOHNSON R, LAUFERE, TABIN C: Sonic Hedgethe Polarising Activity of the ZPA. CeN 1993, 75:1401-1416.
hog Mediates
71.
ECHELAHD Y, EPSTEIND, ST-JACQUESB, SHEN L, MOHLERJ, of MCMAHON J, MCMAHON A: Sonic Hedgehog, a Member
a Family of Putative Signalling Molecules, Is Implicated in the Regulation of CNS Polarity. CelI 1993, 75:1417-1430. 72.
KRAUSS S, CONCORDF~J-P, INCHAM P: A Functionally Conserved Homolog of the Drosopbfka Segment Polarity Gene &h is Expressed in Tissues with Polarizing Activity in Zebrafish Embryos. Cell 1993, 75:1431-1444.
73.
ROELINKH, AUGSBURGER A, HEEMSKERK J, KORZHV, NORLINS, RUIZ I ALTABAA, TANABEY, PLACZEKM, EDLUNDT, JE.WI.I.T, DODD J: Floor Plate and Motor Neuron Induction by vhb
I, a Vertebrate Homolog of hedgehog Notochord. Cell 1994, in press.
V Korzh, Depanment S-90187, Sweden.
of Microbiology,
University
Expressed
of Urn&,
by the
Urn&
in vertebrates
Vladimir P Korzh University
of Umea, Umed, Sweden
The specification of neuronal fate startswith cell commitment and determination. These early events are accompanied by rearrangement and reshaping of presumptive neural cells. Later, the neural differentiation begins, and its course can be followed using specific molecular markers. Such events take place long before the cells acquire a typical neuronal phenotype. Primary neurons of lower vertebrates differ from secondary neurons by their size, position, timing of differentiation and length of axon. Primary neurons start to express early markers of neural differentiation at the end of gastrulation. Recent data indicate that in lower vertebrates the neural induction of primary neurons differs from the induction of secondary neurons; however, neural induction in higher vertebrates appears to be similar to the induction of secondary neurons
in lower vertebrates.
Current Opinion in Neurobiology 1994, 4:21-28
Introduction Neuronal cell fate is determined during early developmental events. Vertebrate neuronal development is characterized by the appearance of a neural plate at the end of gastrulation. This is a result of neural induction, i.e. the interaction between mesoderm and ectoderm. The next stage involves specification of the neuroblasts, which may be a result of local interactions within cell groups. These events are probably responsible for the different ways that presumptive neuroblasts react to inductive signals. Neural induction signals cause multipotential undifferentiated cells to develop into particular cell lineages. Recent studies have shown that regulatory genes and signal transduction pathways are evolutionarily conserved. In this review, I discuss the molecular mechanisms of neural induction and the recent progress in the understanding of the early stages of genetic control of neural development. Some aspects of this problem have been discussed in earlier reviews D-41.
Developmental decisions One of the major problems in developmental biology is understanding the mechanisms restricting possible cell fates or cell commitment [51. Studies of neurogenic genes in Drosophilu (reviewed in 161)and recent observations on vertebrates both show the importance of the developmental decisions taken by the neuroepidermal precursor. The genes involved in regulation of neural determination include positive and negative regulators of neurodevelopment. For example, the genes of the achaetae-scute complex (AS-O encode transcription
factors of the basic helix-loop-helix (HLH) class that exerts positive control over cellular differentiation in a variety of developmental systems 171.Although studies of the mammalian AS-C homologue 04AW1) suggest that it could be involved in neural ontogeny [81, the specific interactions remain to be defined. The prox-1 gene, a homologue of Drosophila prospero, could be one of the genes that interacts with the AMY genes. The prox-1 gene is expressed in neuroblasts where its expression pattern overlaps with that of MAW1 and partially with those of evxl (even-skipped homeobox) and en-2 (engruiled-2) Dl. Interestingly, expression patterns of the positive regulator V-MASH1 appear to be similar to that of its negative regulator, Notch [lo]. Genetic and biochemical analyses of Notch and Jd, another gene with similar function, suggest that both positive and negative developmental decisions are important in determining neural cell fate [7,11,12,13’1.
Neural induction The nature of neural-inducing signals has only been discovered recently. Sources of the signals might include the organizer (e.g. the embryonic shield in fishes, dorsal lip of blastopore in Amphibia, or Hensen’s node in birds and mammals) and the midline structures derived from the organizer, i.e. notochord and floor plate (recently reviewed in [14’1). Two routes of neural induction have been proposed: a vertical route from mesoderm to overlying ectoderm; and a horizontal route, involving planar signals through the dorsal ectoderm [151. Recent results indicate that both vertical and planar signals interact and contribute to the induction and axial patterning of the nervous system
Abbreviations ASX-achaerae-scute complex; CNS-central HNF-hepatocyte nuclear factor; HLH-helix-loop-helix;
nervous system; en-2-engrailed-t evx-even-skipped homeobox; MASH7-mammalian AS-C homologue; TGF--transforming growth factor.
0 Current Biology Ltd ISSN 0959-4388
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Development
duriog early embryogenesis [16-181. Some progress has been made in characterizing factors involved in induction events. These are encoded by genes expressed within the organizer region and midline structures, and include the following: the homeobox gene goosecoid ~9,201, involved in cell migration; members of the HNF/fork head family 121.1, which are implicated in specification of the floor plate I22.1; the Brachyury Cnt0 gene [23,24,25*1, which is necessary for organization of the notochord and development of posterior mesenchyme; genes encoding secreted factors, such as Wnt proteins [26l, whose products are thought to be involved in cell-cell interactions; and members of the LIM/homeodomain gene family, which encode transcription activation factors [27,28*] (see Fig. 1).
Primary and secondary neurons The primary and secondary neurons in adult lower vertebrates are arranged in the dorsal and ventral portions of the motor column, and they innervate different groups of muscles 1311. Early in development primary neurons form a coordinated system responsible for the escape reaction. They have large somata and long axons [32l. In zebrafish embryo, the primary neurons originate in the brain and spinal cord, and pioneer major axonal tracts [28*,33361. In embryonic spinal cord their final precise position may be adjusted to fit the segmentation of axial mesoderm [32,341. Furthermore, primary motor neurons are committed to a specific cell fate within each segmental group, 2-3 h before axonogenesis 1371. The specification of individual neurons within a segmental group may depend on the expression of the LIM/homeodomain gene family (VP Korzh, S Thor, T Edlund, unpublished data). In Amphibia, segmental organization of the central nervous system (CNS) exists very early in development [381, but after the secondary neurons appear, longitudinal columnar organization becomes more apparent 1391.
The importance of secreted factors in neural induction has long been suspected. Recent experimental evidence now supports this theory. A secreted factor, noggin, is expressed in the organizer region and can mimic the effect of the organizer 1291. Furthermore, noggin directly induces neural tissue in Xenopus embryo, thus making it the first neural inducer to be identified 130’1. Recent data indicate that other secreted molecules, such as vertebrate homologs of the Drosophila hedgehog gene product, are differentially expressed within the organizer and its midline derivatives, the notochord and floor plate. These homologs may play an important role in inducing midline derivatives of neuroectoderm (V Korzh, T Edlund, H Roelink, J Dodd, T Jesse& unpublished data; see Note added in proof).
&pax-b)
-t Wnt- I+
This evolutionary tendency is more obvious in higher vertebrates. In the chick, segmental organization of the CNS is preserved in the brain but disappears in the spinal cord where CNS columnar organization becomes established as soon as neurulation comes to an end [401. In mammals, this process results in subdivision of the motor column into discrete motor pools 1411.
en-2
Neuroectoderm
pik?lla “is
0 O
c.) Signal
precursors
O
cl.
Somite
hial
,
>
II Bracn:rury
muscle
Danforth’s short tail
pioneers
n
0
v
0 1994 Current ODinion m Neurobiolorv
Fig. 1. General scheme of neural induction in vertebrates. Recent analysis of mutants of floor plate (FP) and notochord have shown that the induction of primary neurons of lower vertebrates could be dependent on inductive signals from cell predecessors of midline structures. This type of inductive signalling disappears in higher vertebrates, where neurodifferentiation starts only after neural tube closure. cyc-7, ntl - zebrafish mutations; Danforth’s short tail, Brachyury, Pintail, truncate - murine mutations; NP - notoplate. Based on [2,19,20,21 ?? ,22*,23,24,25*,29,43*,44,45,48,49,5Ooj; S Dietrich, P Gruss, F Schubert, personal communication.
Genetic control of early neuronal development in vertebrates Korzh
The difference in the organization of primary and secondary neurons within the spinal cord might be predetermined by the difference in molecular mechanisms involved in establishing anterior-posterior patterning or in the competence of primary and secondary neurons to respond to specific signals during early neuronal induction and/or specification.
Early events in neurodifferentiation Recent progress in understanding neural induction in zebrafish using early markers of neural differentiation have shown that early events of neural differentiation, such as cell commitment, cell rearrangement, neural induction and expression of the first neural markers, take place within 2-3 h (Fig. 2). For example, cells of the hypoblast, which participate in the formation of mesoderm and entoderm, become committed to a specific fate and position at the mid-gastrula stage (8 hours post fertilization) 142.1. Cell-labelling experiments have shown that immediately after commitment, intensive sorting of neural and ectodermal progenitors completely separates cells with neurogenic and epidermogenic capacities 143’1. The induction of LIM/homeodomain proteins, recognized by anti-Isl-1 antibody, takes place at the end of gastrulation in motor neurons located along the midline
(a)
The neural differentiation in chick initially follows the same trend. Cells become committed and patterns of neuroepithelium are arranged before an inductive interaction with the notochord. Neural induction takes place very early during stages 2-4 147,481. Inductive signals from the notochord may, therefore, act on the dividing neural plate cells to confer the potential to generate the neurons. The selection of the particular cell fate and appearance of corresponding markers in individual cells is postponed until stage I5 1491 and could then be influenced by other factors 150’1. The chick motor neurons start to express 1~1-1 before any other known molecular markers, immediately after the final division of motor neuron progenitors. At that time point cell progenitors are located close to ventricular zone 1491. In addition to inductive signals, the notochord and the floor plate provide trophic signals,
Hox cluster,
Gtx, Wnt, D/x, Pax, Ems, hedgehog
and Rohon-Beard cells located at the lateral edges of the neural plate. This occurs at the very beginning of morphological differentiation of the midline structures 128’1. Almost simultaneously, a subset of interneurons starts to express puxCzf-b) 144,451.Regulatory interactions between pax&f-b), Wnt-1 and en-2 suggest that functional relations between these genes are conserved during evolution from Drosophila to teleosts, and perhaps even further 1461. Secondary motor neurons start to express 1~1-1after formation of a spinal cord 128.1.
Pax, Wnt, 5’
hedgehog
3’
I
Anterior 4
Cross section 1
(b)
b Posterior
Cross section 2
4
a
Dorsal
I
Cross section 1
Ventral Cross section 2
0
1994 Current Opinion in Neurobiology
Fig. 2. Early stages of neurodifferentiation in zebrafish. The first markers of neurodevelopment start to appear in zebrafish embryo at the end of gastrulation, before yolk plaque closure. The regionalization of the neuroectoderm could be predetermined as a result of expression of several gene families. (a) Top view on the neuroectoderm of whole-mount immunostained embryos. (b) Cross settions of such embryos. ACC - anterior group of cells; Trg - trigeminal ganglia; PMN - primary motor neurons; IN - interneurons; RB - Rohon-Beard neurons. Based on [29,43*,52*,53]; VP Korzh, unpublished observations.
23
24
DeveioDment
promoting either cell division or cell survival in neural plate explants. These could be peptide growth factors, such as acidic and basic FGF (fibroblast growth factor) and TGF-fl2 (transforming growth factor-p2), enhancing proliferation within neural plate in vitro 151'1. The specification of neuronal classes in the spinal cord depends on anterior-posterior and dorsal-ventral regionalization of the spinal cord, and might be established due to the expression of extracellular matrix components 152’1, Hox code 153,541, dorsalin-1, Pax-3, Pax-6, and Pa-7 [55’,56’,571 (Fig. 3).
Analysis of midline
lower vertebrates
In zebrafish, several mutants lacking midline structures have been identified. cyc-1 mutants lack floor plate and develop cyclopia due to the lack of the ventral cells in the forebrain [21. One study of cyc1 mutants shows nearly normal development of all motor neurons, i.e. both primary and secondary ones L62.1. In another study, a reduction of ventral GABAergic Kolmer-Aghuhr neurons, located close to the floor plate, has been reported [63*1. The ventral axonal pathway is disorganized in cyc-1 mutants, implying that floor plate is important for the pathfinding of motor neuron axons. Interestingly, the cyc-1 phenotype, in general, is similar to the Xenopus phenotype that arises after reduction of the quantity of the organizer during late blastula WI, illustrating ‘community’ effect during induction of dorsal mesoderm [651. Another tebrafish mutation disrupting development of midline structures, ntl, produces mutants lacking a differentiated notochord. Nevertheless, the presence of the differentiated notochord in this mutant is not a prerequisite for the induction of floor plate and neurons. Perhaps cell predecessors of the notochord supply the inductive signals f25.1.
mutants
Higher vertebrates
Molecular analyses in higher vertebrates have shown that the lack of midline structures leads to dramatic changes in the organization of ventral spinal cord and specific classes of neurons in murine mutants or chick embryos deprived of their notochord. The floor plate and motor neurons are missing after removal of notochord in chick embryo 149,581. In contrast, dorsal spinal cord is less affected by close opposition of transplanted notochord, which cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest derived dorsal root ganglia 1591.
Irradiation of Xenopus fertilized eggs by ultraviolet light produces notochord-less embryos. Analysis of CNS differentiation has shown that lack of the notochord results in the arrest of floor plate. The number of secondary neurons is dramatically reduced, whereas primary neurons are less affected 1661.
Analysis of mutants has shown that Danforth’s short tail mutants in mice lack notochord caudally, resulting in the arrest of development of floor plate and motor neurons 1601. Experimental removal of notochord or the absence of notochord in notochord-less mutants of mouse, such as Brachyury curtailed (Tc), Danforth’s short tail (Sd), Pintail (Pt) and truncate (tc), leads to ectopic ventral spread of expression of molecular markers normally mapped more dorsally ([55*,56*,611; S Dietridh, F Schubert, P Gruss, personal communication).
??zf
(pax-b)
B 1994 Current Op~mon I” Neurobiology
These observations show that neural induction in lower vertebrates takes place very early in development, before the morphological differentiation of midline structures (Fig. 4). Cell precursors of midline structures are probably responsible for the signalling of neural induction in this case. Primary neuron differentiation may be less dependent on influences from the organizer or differentiated midline structures than dif-
IPax3
.
Pax8
ElPax6
0
Pax2
IDPax
0
Pax5
Fig. 3. Neurodifferentiation in the spinal cord. The specification of cell types in the spinal cord could be dependent on inductive signalling from two poles of the spinal cord: dorsal plate (dorsally); and floor plate and notochord (ventrally). The interaction of these signals could determine the distribution of expression patterns of other cell fate determinants, like Pax genes. AP-alar plate; BP-basal plate; FP-floor plate; IN-interneurons; PMN-primary motor neurons; RB-Rohon-Beard cells; RProof plate; SL-suicus limitans; SMNsecondary motor neurons. Based on [22*, 29,42.,43*,44,54,55.,56*].
Genetic control of early neuronal development in vertebrates Korzh
Fertilization
0 I
Gastrulation
Neural tube formation
Neurulation
A
/
I
00
Neural induction
I
Commitment
-1
Cell rearrangement
--I
Competence Primary neurons . Neurodifferentiation
Secondary neurons I
3 1994 Current Opinion in Neurobiilogy
ferentiation of secondary neurons. First, primary neurons might be saved in ultraviolet-treated Xenopus embryos owing to the different sensitivity of the signalling pathway involved in inductive events leading to an appearance of primary and secondary neurons. Second, development of primary neurons might depend more on maternally derived factors. Interestingly, CNS determination in Xenopus can occur even without cell division, and the phenotype of cells arising after differentiation is more like primary neurons 1671. Primary neurons are also preserved in the neurodegenerative zebrafish mutant ned-1, in which secondary neurons die [68]. Currently, several laboratories are using saturation mutagenesis on zebrafish so as to be able to mutate all, or at least most, of the zebrafish genes (e.g. [691>. We will be in a better position to tackle the problem of primary neuron induction when more mutants are identified.
Conclusion Higher vertebrates do not have primary neurons. In lower vertebrates some of the primary neurons disappear during development. This could be attributed to the development of complex reactions based on processes such as the vision pathway, which substitute primitive avoidance reactions that involve primary neurons. During evolution, segmental organization of the CNS characteristic for primary neurons has been substituted by a columnar one, characteristic for secondary neurons. Finally, in higher vertebrates primary neurons and segmental organization of CNS in the spinal cord disappear completely. This could be related to changes in developmental programs, including elongation of embryogenesis and emergence of the parental guardianship over their progeny.
??
Fig. 4. Early events of neurodifferentiation of primary and secondary neurons. The early events of neurodifferentiation, such as neural induction, cell commitment, rearrangement and reshaping, take place at the end of gastrulation. The appearance of specific neural markers in primary and secondary nyurons occurs in two stages. Primary neurons start to express 1~1-1,a marker for neurodifferentiation, at the end of gastrulation before neural tube formation. This event is postponed in secondary neurons until after neural tube formation.
Recent studies have shown that the organizer and derived midline structures are the source of neural induction. Lack of notochord or floor plate leads to dorsalization of the spinal cord and impaired development of ventral CNS. In particular, defects of midline structures have minor effects on lower vertebrate primary neuron development, which can be accounted for by inductive signals from cell-predecessors of differentiated midline structures. Comparative analysis of embryos of lower and higher vertebrates lacking midline structures shows an apparent difference in the mechanism of neural induction of primary and secondary neurons. Primary neurons start to express 1~1-1 early, while they are still in the neuroepithelium, perhaps because of the early inductive signals from cell predecessors of the midline structures. The neurons of higher vertebrates also become committed to neuronal fate early, but the appearance of specific molecular markers is postponed until after formation of the spinal cord. Further studies of molecular mechanisms involved in neural differentiation may account for the difference in induction between primary and secondary neurons. It is clear that mutations of all genes known to participate in emission, transmission, reception and processing of neural induction signal? will be instrumental in understanding the details of the molecular apparatus involved. Once these are clear, we may be able to understand why higher vertebrates have no primary neurons. The analysis of neural induction mechanisms indicates that the evolution of developmental programs can be traced not only phylogenetically but also by using molecular genetics and experimental embryology. During early evolution of vertebrates the neural tube might be induced by signals of cell predecessors of midline structures.
25
26
Development
Note added in proof Recently, vertebrate homologues of the Drosophila segment polarity gene hedgehog have been cloned and found to be expressed in regions implicated in polarizing activity, such as the ZPA (zone of polarizing activity) in the limb and the notochord/floor plate. Functional evidence is emerging that vertebrate hedgehog gene products, which are secreted proteins, act as signals for antero-posterior patterning in the limb and dorso-ventral patterning in the neural tube [X1-73].
Acknowledgements I thank T Edlund, J Eisen and D Milton for critical reading of the manuscript. I am indebted to C Kimmel, J Eisen, T Jessell, M Westemeld and P GNSS, and many members of his lab, for sending preprints. I am a permanent member of the Koltsov Institute of Developmental Biology, Moscow, Russia.
Papers of particular interest, published review, have been highlighted as: - . of special interest .. of outstanding interest
within
SPEMANN
Haven:
the annual
period
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V Korzh, Depanment S-90187, Sweden.
of Microbiology,
University
Expressed
of Urn&,
by the
Urn&