- Email: [email protected]
Development of the zebrafish lateral line
Development of the zebrafish lateral line Alain Ghysen1 and Christine Dambly-Chaudie`re The lateral line system is simple (comprising six cell types), its sense organs form according to a defined and reproducible pattern, and its neurons are easily visualized. In the zebrafish, these advantages can be combined with a wealth of genetic tools, making this system ideally suited to a combined molecular, cellular and genetic analysis. Recent progress has taken advantage of these various qualities to elucidate the mechanism that drives the migration from head to tail of the sense organ precursor cells, and to approach the questions surrounding axonal guidance and target recognition. Addresses Lab. Neurogenetics, INSERM E343, cc103 Universite´ Montpellier II, Place E. Bataillon, 34095 Montpellier, France 1 e-mail: [email protected]
Current Opinion in Neurobiology 2004, 14:67–73 This review comes from a themed issue on Development Edited by Barry Dickson and Christopher A Walsh 0959-4388/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.01.012
Abbreviations ALL anterior lateral line system ngn1 neurogenin-1 PLL posterior lateral line system SDF-1 stromal-derived factor 1
Introduction The lateral line, a sensory system that is present in fish and amphibians, responds to changes in the motion of water and is involved in a large variety of behaviours, from prey detection to predator avoidance, school swimming and sexual courtship. It has disappeared in terrestrial tetrapods, with the exception of its internal counterpart, the inner ear. The lateral line comprises a set of sensory organs, the neuromasts (Figure 1a), arranged on the body surface in species-specific patterns. Neuromasts comprise a core of mechanosensory hair cells (Figure 1b), surrounded by support cells, and are innervated by sensory neurons that are localized in a cranial ganglion. The neuromasts on the head form the so-called anterior lateral line system (ALL), the ganglion of which is located between the ear and the eye, while the neuromasts on the body and tail, including those on the caudal fin, form the posterior lateral line system (PLL), its ganglion being just posterior to the ear (Figure 1c). The PLL comprises a prominent line of neuromasts that extends from head to tail along each flank and gives the system its name. www.sciencedirect.com
Cell migration in the zebrafish PLL A most unusual aspect of lateral line development is that the neuromasts are deposited by migrating primordial, which originate from cephalic placodes [1,2]. Innovative experiments on amphibians demonstrated that the PLL primordium follows a preexisting pathway, that it can follow this pathway in either direction, and that the deposition of neuromasts is due to a mechanism that is intrinsic to the primordium, rather than being a response to extrinsic, local cues [3,4]. The nature of the pathway remained elusive, however, and has been elucidated only recently, in the case of the zebrafish PLL. The lateral line system of the embryonic zebrafish is highly stereotyped [5]. The neuromasts of the PLL (Figure 2a) are deposited at regular intervals by a primordium (Figure 2c), which originates from a placode that is just posterior to the otic placode, and migrates along the horizontal myoseptum [6], all the way to the tip of the tail (Figure 2b). Recent results [7] have shown that the migration of the PLL primordium entirely depends on the interaction between the chemokine receptor CXCR4, which is present in the migrating cells (Figure 2d), and its ligand, SDF1 (stromal-derived factor 1), which is synthesized by a narrow stripe (approximately three cells wide), extending along the prospective pathway of the primordium, all the way from the first somite to the tip of the tail (Figure 2e). Morpholino inactivation of either cxcr4b or sdf1a results in a paralysis of the primordium and, consequently, in the absence of a PLL. Interestingly, in mutants where sdf1a is no longer expressed along the horizontal myoseptum, the primordium is diverted to a more ventral pathway that corresponds to another region of sdf1a expression, the pronephros (Figure 2e, arrowheads). This result confirms that SDF1 is the major (if not the only) cue that determines and guides the migration of the primordium; a conclusion that is all the more interesting when we consider the fact that the SDF1–CXCR4 interaction is involved in other long-distance migration events, such as the homing of lymphocytes (the function for which both genes were originally discovered), the migration of metastases [8] and of germ cells in both fish [9,10] and mice [11].
Downstream and upstream of migration The relative ease with which the embryonic primordium can be detected under Nomarski optics should make it feasible to laser-dissect all or part of it for subtractive hybridizations or micro-array analyses. This should allow a comprehensive analysis of the genes acting downstream Current Opinion in Neurobiology 2004, 14:67–73
68 Development
Figure 1
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Current Opinion in Neurobiology
The fish lateral line. (a) Scheme of a neuromast, illustrating the different cell types that are involved and their organization. The cupula is secreted by the support cells. The glial cells wrapping the afferent fibers have not been represented. (b) Scheme of a hair cell, illustrating its functional asymmetry, as well as its afferent and efferent innervation. The implication that excitatory efferents act on the afferent fiber rather than directly on the hair cell is plausible but not demonstrated. (c) Scheme of an adult fish, illustrating the innervation of the ALL and PLL. The size of the ear (dotted line) and central projections has been grossly exaggerated for illustration purposes. The photograph shows the organization of the ALL and PLL projections in young larvae, as seen after dextran-fluorescein (green) and dextran-rhodamine (red) labeling of head and body neuromasts, respectively [31].
of the CXCR4 receptor and, more generally, of the genes that control the distinctive features of migrating lateral line cells. Several genes that are specifically expressed in the primordium and/or in the neuromasts have already been described. Some of them code for transcription factors and might, therefore, play a role in determining specific properties of lateral line cells, proneural genes (discussed later), or the runxb gene [12]. Other genes that Current Opinion in Neurobiology 2004, 14:67–73
are preferentially expressed in the lateral line system code for various proteins that are involved in cell behaviour, such as cldnb [13], coding for an essential tight junction protein, or robo1 [14], coding for a transmembrane receptor that is, possibly, involved in guidance. An additional potential of the PLL is the study of the control of cell migration, arising from the continuation of www.sciencedirect.com
Development of the zebrafish lateral line Ghysen and Dambly-Chaudie`re 69
Figure 2
Cell migration in the development of the PLL. (a) The pattern of neuromasts at the end of embryogenesis. The major branch of the PLL comprises five regularly spaced neuromasts (L1-L5) and 2–3 terminal neuromasts, clustered at the tip of the tail (ter). The pattern of the ALL has been described elsewhere [5]. (b) The primary neuromasts (green dots) are deposited by a migrating primordium that follows the horizontal myoseptum over most of its course, then follows a more ventral course near the tip of the tail (dotted red line). (c) The migrating primordium, as seen under Nomarski optics. (d) Expression of cxcr4b (chemokine receptor) in the primordium. (e) Expression of sdf1a (stromal-derived factor) along the prospective pathway of the primordium, as well as along the pronephros (arrowheads).
the migration process during post-embryonic growth. In zebrafish, the embryonic primordium leads to the formation of a rudimentary PLL, comprising eight neuromasts. The PLL of the adult fish, however, comprises many more neuromasts. It has long been assumed that the transition from embryonic to adult pattern would involve a budding process, whereby, the embryonic neuromasts would form additional neuromasts. To the contrary, a detailed analysis of the post-embryonic growth of the PLL revealed that new primordia are generated and progressively fill in the gaps that are left by the embryonic primordium until all somite borders are occupied by one neuromast [15,16].
Neuromast deposition and pattern formation It is unlikely that the deposition of neuromasts is due to external cues, because a diverted primordium will continue to deposit neuromasts despite traveling through abnormal territories [4]. The idea that the deposition process is intrinsic to the primordium has been confirmed in zebrafish [17], through analyses of the variability of the normal pattern, of the effect of experimental interference, www.sciencedirect.com
and of the patterned expression of genes within the primordium. In particular, cxcr4b is down-regulated in the cells at the trailing edge (those that will be soon deposited) and is completely repressed in the proneuromasts. The proneural gene Zath1, and the neurogenic genes Notch and Delta, which are involved in the choice between hair and support cell fates, are expressed within the primordium in small groups of cells that, presumably, prefigure the deposition of neuromasts [18]; however, the mechanism that initiates the up-regulation of the proneural and neurogenic genes, and down-regulates the expression of cxcr4b, remains elusive. Although the adult PLL pattern varies greatly between teleosts, in relation to various factors such as fin position, morphology or habitat, the embryonic line appears to be amazingly conserved in fish as morphologically and phylogenetically different as the zebrafish, the turbot and the eel ([19] and unpublished observations). One mechanism of variability generation in adult patterns is the formation of additional primordia, which can vary in number and time, between species. A second mechanism Current Opinion in Neurobiology 2004, 14:67–73
70 Development
Figure 3
Afferent innervation of the PLL. (a) Afferent neurites remain in contact with the primordium from the onset of its migration. Note that several neurons already extend a central projection in the hindbrain at the level of the ear. (b) The growth cones of neurons that will end up innervating more anterior neuromasts are invariably simpler than the growth cones that will innnervate more posterior neuromasts (c).
of variability generation is the dorso-ventral migration that differentiated neuromasts can undergo; the amplitude of which depends on their position along the anteroposterior axis, thereby, defining the final shape of the PLL [15,16].
Neurons The early work of Harrison, on amphibians [1], demonstrated that the growth cones of sensory neurites accompany the primordium, thereby, establishing a physical link between the cranial ganglion and the body neuromasts. Also in the zebrafish, the primordium is accompanied by growth cones as soon as it begins to move (Figure 3a). Based on ectopic expression experiments, it has been suggested that sensory axons are guided independently of the primordium and are constrained along their course, due to the repulsive effect of two broad regions of sema3A expression, dorsal and ventral to the prospective pathway [20]. Yet, when the primordium is immobilized, as a result of the inactivation of the SDF1/ CXCR4 system, or when it is forced to follow an ectopic course in mutants, the sensory neurons invariably follow it [7], indicating that the axons are, indeed, guided by the primordium, as surmised by Harrison. Although the repulsive effect of the dorsal and ventral somites plays no role in the normal course of development, it might be a remnant of a more primitive mechanism and/or be part of a ‘back-up’ system. The reason why the growth cones follow the primordium seems to be related to the expression of HNK-1, a Current Opinion in Neurobiology 2004, 14:67–73
glycoepitope that is prominently expressed by the PLL neurons [21]; injecting the antibody in embryos results in some axons taking an extremely abnormal course [22]. The details of the interaction between neurons and primordium are not known, however, and other surface proteins, such as Tag1, might also be involved [23]. Likewise, we do not know where the neurons that innervate the neuromasts that are formed during larval life originate from, nor do we know whether the various sets of neurons that innervate the successive rounds of neuromasts remain distinct from each other – a feature that would facilitate a Fourier analysis of water movements along the fish (N Ko¨ nig, personal communication), because the final spacing between successive neuromasts will differ widely for the different rounds. The ability to use Fourier transforms would enormously increase the analytical capability of the system and would show the astonishing performances of the blind fish Astyanax fasciatus [24] to be somewhat less incredible (this fish is capable of discriminating between two surfaces covered with parallel glass rods that are, respectively, 10 and 11 mm apart!). The formation of the PLL ganglion depends on the previous expression of the proneural gene neurogenin-1 (ngn1) [25]. Interestingly, while the inactivation of ngn1 wipes out the PLL ganglion, it does not prevent the formation of neuromasts. This duality demonstrates that the neuromasts can form independently of innervation — a conclusion that has already been established in amphibians — and that ngn1 is not required for the www.sciencedirect.com
Development of the zebrafish lateral line Ghysen and Dambly-Chaudie`re 71
determination or differentiation of the hair cells. Given that Zath1 is expressed in the presumptive hair cells [18], it seems likely that, in the PLL, as in the mammalian ear, the expression of neurogenin determines the neurons, while the expression of atonal homologs determines the hair cells.
fied (N Gompel, V Chaar and F Soubiran, personal communication). This result implies a, hitherto unsuspected, heterogeneity among similar sensory neurons, and makes it possible to envision, in vertebrates, a level of neuronal specification that is more usually associated with the development of insect sensory organs [33].
Glia
Although presently we have no idea of the mechanism that encodes the position of extension of a given PLL axon, this issue has been extensively studied in Drosophila, where the slit/robo system is used, not only to decide whether an axon will or will not cross the midline, but most importantly, to set up the distance from the midline at which the axon will branch and extend, longitudinally [34,35]. It might be that a similar mechanism is used to set up the distance from the midline in fly and fish, providing afferent axons with the opportunity to extend at various distances from the midline in an organized, programmable manner. Interestingly, it appears that most somatosensory maps use similar coordinates, with the antero-posterior axis of the body being represented along the ventro-dorsal axis of the brain, suggesting the existence of a general and highly conserved mechanism of establishing somatotopic projections.
The PLL nerve is ensheathed by glial cells. In what could have been an excessive expectation of developmental conservation between flies and vertebrates, it was thought that these glial cells might play a role in early axonal guidance [26]. However, Harrison [1] demonstrated that the neurites are towed in the wake of the primordium, thus, making this hypothesis unlikely. This issue has been convincingly settled by a set of experiments that were based on the design of fluorescent reporter lines to follow the migration of glial cells during embryogenesis [27]; they achieved the expected result that the glial cells do, indeed, migrate along the sensory axons rather than guiding them. In addition to their myelinating function, glia also play a role in maintaining the fasciculation of PLL axons [27]. The origin of the glial cells remains a disputed question, however, with some data pointing towards a neural crest origin [28], and uncaging experiments suggesting that glial cells originate from the primordium [17]. A dual origin has already been suggested for the hair cells [29]. The importance and role of the placodal versus neural crest contributions to the lateral line system is an important area that remains to be fully investigated and settled, as it might impact on our understanding of the role and evolution of the neural crest contribution to sensory systems in vertebrates.
Neural connectivity Each neuromast has dual innervation: afferent and efferent. The afferent projections of the ALL and PLL form two ribbons that extend at different levels in the hindbrain, the ALL projection being ventral to the PLL projection (Figure 1c; [30]). The PLL projection, itself, appears to be topologically organized, with neurons innervating more posterior neuromasts projecting more dorsally in the hindbrain [31]. This could reflect a capability of the sensory neurons to respond to the position of the organ they innervate by adjusting their central projection accordingly. Contrary to this interpretation, however, it was found that the central neurite extends before the peripheral neurite has reached its target organ [32]; furthermore, individual differences in growth cone morphology prefigure the position of the organ that the neuron will eventually innervate, suggesting that each neuron is somehow specified to seek an organ at a given position along the antero-posterior axis (Figures 3b,c). In agreement with this conclusion, genes that are specifically expressed in subsets of PLL neurons have been identiwww.sciencedirect.com
Efferent innervation and information processing In addition to the afferent axons, the PLL is also innervated by two sets of efferent axons that modulate its responsiveness to external stimuli [36]. One set originates from the hindbrain. This set is cholinergic and probably functions as an inhibitory feedback and/or feed-forward mechanism. The vestibular efferent nucleus of the mouse, which originates in rhombomere 4, has been shown to depend on the gene Phox2b for its differentiation, and on the gene Mash1 for its proper migration [37]. It will be interesting to determine to what extent these genetic requirements are conserved in the fish. The second efference originates from the forebrain, is dopaminergic and probably subserves an excitatory modulation, possibly related to the level of ambient light. Unfortunately, we still have no knowledge of the genetic mechanisms that allow the PLL information to be matched to other sensory information, such as vision and hearing. This matching is at the basis of our capacity to combine sensory information, corresponding to different modalities and, as such, is of paramount importance in the building of our perceptual map of the world. The experimental qualities of the PLL might provide one entry point into this challenging arena.
Conclusions This review has illustrated how the exceptional accessibility and definition of the PLL, together with the experimental and genetic qualities of the zebrafish, make this system ideally suited to address several important Current Opinion in Neurobiology 2004, 14:67–73
72 Development
questions in vertebrate development, from the control of cell migration and cell proliferation to the organization of axonal projections. We still do not have a comprehensive picture of the genetic program that underlies the successive steps of lateral line development, from placode determination to cell differentiation and the establishment of an accurate sensory map in the brain. Nevertheless, the convergent efforts of many laboratories, and the fact that our knowledge of PLL development has enormously increased over the past few years, suggest that such a complete understanding has now become a realistic aim. The abundance of mutations and genetic tools, which are increasingly available to the zebrafish community, will, undoubtedly, be a major asset in achieving this aim.
Acknowledgements We thank T Whitfield for excellent criticisms and J Hudspeth for careful reading of the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Raible DW, Kruse GJ: Organization of the lateral line system in embryonic zebrafish. J Comp Neurol 2000, 421:189-198.
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Metcalfe WK, Kimmel CB, Schabtach E: Anatomy of the posterior lateral line system in young larvae of the zebrafish. J Comp Neurol 1985, 233:377-389.
David N, Sape`de D, St-Etienne L, Thisse C, Thisse B, Dambly-Chaudie`re C, Rosa F, Ghysen A: Molecular basis of cell migration in the fish lateral line: role of the chemokine receptor CXCR4 and of its ligand, SDF1. Proc Natl Acad Sci USA 2002, 99:16297-16302. The authors demonstrate the role of the chemokine receptor CXCR4 and its ligand SDF1 in the migratory process that mediates the formation of the posterior lateral line system in the zebrafish embryo. They show that the receptor is expressed by the migrating cells, that the ligand is expressed by the pathway cells, and that the inactivation of either gene prevents migration.
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Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN et al.: Involvement of chemokine receptors in breast cancer metastasis. Nature 2001, 410:50-56.
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Doitsidou M, Reichman-Fried M, Stebler J, Koprunner M, Dorries J, Meyer D, Esguerra CV, Leung T, Raz E: Guidance of primordial germ cell migration by the chemokine SDF-1. Cell 2002, 111:647-659. The authors provide evidence that the presence of the CXCR4 receptor on the primordial germ cells of the zebrafish is required for their directional
Current Opinion in Neurobiology 2004, 14:67–73
migration towards the presumptive gonads. They also show that alterations of the migratory pathway correlate with alterations in the expression of SDF1 in various mutant conditions, thereby, elucidating the mechanism of this classical example of long-range, precisely controlled cell migration event. 10. Knaut H, Werz C, Geisler R, Nusslein-Volhard C: A zebrafish homologue of the chemokine receptor Cxcr4 is a germ-cell guidance receptor. Nature 2003, 421:279-282. This paper confirms the results of Doitsidou et al. [9], by studying a lossof-function mutant of the cxcr4 receptor gene in the zebrafish. They show that the primordial germ cells are still able to migrate but have lost directionality, suggesting that migration and guidance are independent processes. 11. Molyneaux KA, Zinszner H, Kunwar PS, Schaible K, Stebler J, Sunshine MJ, O’Brien W, Raz E, Littman D, Wylie C, Lehmann R: The chemokine SDF1/CXCL12 and its receptor CXCR4 regulate mouse germ cell migration and survival. Development 2003, 130:4279-4286. The authors demonstrate the implication of the SDF1/CXCR4 interaction in the migration of primordial germ cells from the endoderm towards the genital ridges, in the mouse. The accompanying movies reveal that the migrating germ cells extend long thin processes that span at least 2–4 cell diameters, such that migration guidance could depend on a contact-based mechanism, as in the case of the zebrafish lateral line [7]. 12. Kataoka H, Ochi M, Enomoto K, Yamaguchi A: Cloning and embryonic expression of the zebrafish Runt domain genes, runxa and runxb. Mech Dev 2000, 98:139-143. 13. Kollmar R, Nakamura SK, Kappler JA, Hudspeth AJ: Expression and phylogeny of claudins in vertebrate primordia. Proc Natl Acad Sci USA 2001, 98:10196-10201. 14. Lee JS, Ray R, Chien CB: Cloning and expression of three zebrafish roundabout homologs suggest roles in axon guidance and cell migration. Dev Dyn 2001, 221:216-230. 15. Ledent V: Postembryonic development of the posterior lateral line in zebrafish. Development 2002, 129:597-604. This study reports a complete description of the post-embryonic growth of the posterior lateral line in the zebrafish, and shows that three processes are at work: a progressive completion of the embryonic line by additional waves of neuromast formation, a dorso-ventral migration of differentiated nueuromasts and a budding process that transforms each organ in a ‘stitch’, a cluster of closely apposed organs. 16. Sape`de D, Gompel N, Dambly-Chaudie`re C, Ghysen A: Cell migration in the post-embryonic development of the fish lateral line. Development 2002, 129:605-615. The authors demonstrate the presence of an additional primordium that migrates and forms new neuromasts during larval life, indicative of a reiteration of the embryonic process. They also show that differentiated neuromasts can migrate through the epidermis. The data indicate that modulations of these two processes are largely responsible for the large variety in lateral line patterns across fish species. 17. Gompel N, Cubedo N, Thisse C, Thisse B, Dambly-Chaudie`re C, Ghysen A: Pattern formation in the lateral line of zebrafish. Mech Dev 2001, 105:69-77. 18. Itoh M, Chitnis AB: Expression of proneural and neurogenic genes in the zebrafish lateral line primordium correlates with selection of hair cell fate in neuromasts. Mech Dev 2001, 102:263-266. 19. Okamura A, Oka HP, Yamada Y, Utoh T, Mikawa N, Horie N, Tanaka S: Development of lateral line organs in leptocephali of the freshwater eel Anguilla japonica (teleostei, Anguilliformes). J Morphol 2002, 254:81-91. Despite being closest to the primitive teleosts, eels have been refractory to developmental studies, due to their peculiar breeding behaviour. This paper reports a method to generate eel embryos, and gives a first account of how their lateral line develops. 20. Shoji W, Yee CS, Kuwada JY: Zebrafish semaphorin Z1a collapses specific growth cones and alters their pathways in vivo. Development 1998, 125:1275-1283. 21. Metcalfe WK, Myers PZ, Trevarrow B, Bass MB, Kimmel CB: Primary neurons that express the L2/HNK-1 carbohydrate during early development in the zebrafish. Development 1990, 110:491-504. www.sciencedirect.com
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22. Becker T, Becker CG, Schachner M, Bernhardt RR: Antibody to the HNK-1 glycoepitope affects fasciculation and axonal pathfinding in the developing posterior lateral line nerve of embryonic zebrafish. Mech Dev 2001, 109:37-49. 23. Warren JT Jr, Chandrasekhar A, Kanki JP, Rangarajan R, Furley AJ, Kuwada JY: Molecular cloning and developmental expression of a zebrafish axonal glycoprotein similar to TAG-1. Mech Dev 1999, 80:197-201. 24. Hassan ES: Hydrodynamic imaging of the surroundings by the latral line of the blind cave fish Anoptichtys jordani. In The Mechanosensory Lateral Line. Edited by Coombs S, Go¨ rner P, Mu¨ nz H. New York: Springer Verlag; 1989:217-227. 25. Andermann P, Ungos J, Raible DW: Neurogenin 1 defines zebrafish cranial sensory ganglia precursors. Dev Biol 2002, 251:45-58. The authors report that the expression of neurogenin-1 delineates the lateral line (and other cranial) placodes, and prefigures the expression of another bHLH (basic helix–loop–helix) factor, NeuroD. They also show that the inactivation of neurogenin-1 prevents the formation of the sensory neurons, but does not affect the associated glial cells or the neuromasts. 26. Chien CB, Piotrowski T: How the lateral line gets its glia. Trends Neurosci 2002, 25:544-546. 27. Gilmour DT, Maischein HM, Nusslein-Volhard C: Migration and function of a glial subtype in the vertebrate peripheral nervous system. Neuron 2002, 34:577-588. This paper demonstrates that the glial cells that ensheath the peripheral axons of the lateral line sensory neurons are guided along the growing axons, and will follow them along ectopic courses in mutant conditions. Thus, glial cells are dispensable for axon elongation, but they are required to maintain proper fasciculation of the lateral line nerve. 28. Kelsh RN, Dutton K, Medlin J, Eisen JS: Expression of zebrafish fkd6 in neural crest-derived glia. Mech Dev 2000, 93:161-164.
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29. Collazo A, Fraser SE, Mabee PM: A dual embryonic origin for vertebrate mechano-receptors. Science 1994, 264:426-430. 30. McCormick CA: Central lateral line mechanosensory pathways in bony fish. In The Mechanosensory Lateral Line. Edited by Coombs S, Go¨ rner P, Mu¨ nz H. New York: Springer Verlag; 1989:341-364. 31. Alexandre D, Ghysen A: Somatotopy of the lateral line projection in larval zebrafish. Proc Natl Acad Sci USA 1999, 96:7558-7562. 32. Gompel N, Dambly-Chaudie`re C, Ghysen A: Neuronal differences prefigure somatotopy in the zebrafish lateral line. Development 2001, 128:387-393. 33. Ghysen A, Dambly-Chaudie`re C: A genetic programme for neuronal connectivity. Trends Genet 2000, 16:221-226. 34. Rajagopalan S, Vivancos V, Nicolas E, Dickson BJ: Selecting a longitudinal pathway: robo receptors specify the lateral position of axons in the Drosophila CNS. Cell 2000, 103:1033-1045. 35. Zlatic M, Landgraf M, Bate M: Genetic specification of axonal arbors: atonal regulates robo3 to position terminal branches in the Drosophila nervous system. Neuron 2003, 37:41-51. In the fly CNS, the terminal branches of sensory axons extend along specific fascicles within the longitudinal connectives. The authors show that the choice of a particular fascicle depends on the same mechanism that earlier led to the establishment of this fascicle at this position. 36. Bricaud O, Chaar V, Dambly-Chaudie`re C, Ghysen A: Early efferent innervation of the zebrafish lateral line. J Comp Neurol 2001, 434:253-261. 37. Tiveron MC, Pattyn A, Hirsch MR, Brunet JF: Role of Phox2b and Mash1 in the generation of the vestibular efferent nucleus. Dev Biol 2003, 260:46-57.
Current Opinion in Neurobiology 2004, 14:67–73
Current Opinion in Neurobiology 2004, 14:67–73 This review comes from a themed issue on Development Edited by Barry Dickson and Christopher A Walsh 0959-4388/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.01.012
Abbreviations ALL anterior lateral line system ngn1 neurogenin-1 PLL posterior lateral line system SDF-1 stromal-derived factor 1
Introduction The lateral line, a sensory system that is present in fish and amphibians, responds to changes in the motion of water and is involved in a large variety of behaviours, from prey detection to predator avoidance, school swimming and sexual courtship. It has disappeared in terrestrial tetrapods, with the exception of its internal counterpart, the inner ear. The lateral line comprises a set of sensory organs, the neuromasts (Figure 1a), arranged on the body surface in species-specific patterns. Neuromasts comprise a core of mechanosensory hair cells (Figure 1b), surrounded by support cells, and are innervated by sensory neurons that are localized in a cranial ganglion. The neuromasts on the head form the so-called anterior lateral line system (ALL), the ganglion of which is located between the ear and the eye, while the neuromasts on the body and tail, including those on the caudal fin, form the posterior lateral line system (PLL), its ganglion being just posterior to the ear (Figure 1c). The PLL comprises a prominent line of neuromasts that extends from head to tail along each flank and gives the system its name. www.sciencedirect.com
Cell migration in the zebrafish PLL A most unusual aspect of lateral line development is that the neuromasts are deposited by migrating primordial, which originate from cephalic placodes [1,2]. Innovative experiments on amphibians demonstrated that the PLL primordium follows a preexisting pathway, that it can follow this pathway in either direction, and that the deposition of neuromasts is due to a mechanism that is intrinsic to the primordium, rather than being a response to extrinsic, local cues [3,4]. The nature of the pathway remained elusive, however, and has been elucidated only recently, in the case of the zebrafish PLL. The lateral line system of the embryonic zebrafish is highly stereotyped [5]. The neuromasts of the PLL (Figure 2a) are deposited at regular intervals by a primordium (Figure 2c), which originates from a placode that is just posterior to the otic placode, and migrates along the horizontal myoseptum [6], all the way to the tip of the tail (Figure 2b). Recent results [7] have shown that the migration of the PLL primordium entirely depends on the interaction between the chemokine receptor CXCR4, which is present in the migrating cells (Figure 2d), and its ligand, SDF1 (stromal-derived factor 1), which is synthesized by a narrow stripe (approximately three cells wide), extending along the prospective pathway of the primordium, all the way from the first somite to the tip of the tail (Figure 2e). Morpholino inactivation of either cxcr4b or sdf1a results in a paralysis of the primordium and, consequently, in the absence of a PLL. Interestingly, in mutants where sdf1a is no longer expressed along the horizontal myoseptum, the primordium is diverted to a more ventral pathway that corresponds to another region of sdf1a expression, the pronephros (Figure 2e, arrowheads). This result confirms that SDF1 is the major (if not the only) cue that determines and guides the migration of the primordium; a conclusion that is all the more interesting when we consider the fact that the SDF1–CXCR4 interaction is involved in other long-distance migration events, such as the homing of lymphocytes (the function for which both genes were originally discovered), the migration of metastases [8] and of germ cells in both fish [9,10] and mice [11].
Downstream and upstream of migration The relative ease with which the embryonic primordium can be detected under Nomarski optics should make it feasible to laser-dissect all or part of it for subtractive hybridizations or micro-array analyses. This should allow a comprehensive analysis of the genes acting downstream Current Opinion in Neurobiology 2004, 14:67–73
68 Development
Figure 1
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(b)
Kinocilium
Cupula +
–
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Hair cell Mantle cell Inhibitory efferent
Afferent fibers
Excitatory efferent
Nucleus
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(c)
Current Opinion in Neurobiology
The fish lateral line. (a) Scheme of a neuromast, illustrating the different cell types that are involved and their organization. The cupula is secreted by the support cells. The glial cells wrapping the afferent fibers have not been represented. (b) Scheme of a hair cell, illustrating its functional asymmetry, as well as its afferent and efferent innervation. The implication that excitatory efferents act on the afferent fiber rather than directly on the hair cell is plausible but not demonstrated. (c) Scheme of an adult fish, illustrating the innervation of the ALL and PLL. The size of the ear (dotted line) and central projections has been grossly exaggerated for illustration purposes. The photograph shows the organization of the ALL and PLL projections in young larvae, as seen after dextran-fluorescein (green) and dextran-rhodamine (red) labeling of head and body neuromasts, respectively [31].
of the CXCR4 receptor and, more generally, of the genes that control the distinctive features of migrating lateral line cells. Several genes that are specifically expressed in the primordium and/or in the neuromasts have already been described. Some of them code for transcription factors and might, therefore, play a role in determining specific properties of lateral line cells, proneural genes (discussed later), or the runxb gene [12]. Other genes that Current Opinion in Neurobiology 2004, 14:67–73
are preferentially expressed in the lateral line system code for various proteins that are involved in cell behaviour, such as cldnb [13], coding for an essential tight junction protein, or robo1 [14], coding for a transmembrane receptor that is, possibly, involved in guidance. An additional potential of the PLL is the study of the control of cell migration, arising from the continuation of www.sciencedirect.com
Development of the zebrafish lateral line Ghysen and Dambly-Chaudie`re 69
Figure 2
Cell migration in the development of the PLL. (a) The pattern of neuromasts at the end of embryogenesis. The major branch of the PLL comprises five regularly spaced neuromasts (L1-L5) and 2–3 terminal neuromasts, clustered at the tip of the tail (ter). The pattern of the ALL has been described elsewhere [5]. (b) The primary neuromasts (green dots) are deposited by a migrating primordium that follows the horizontal myoseptum over most of its course, then follows a more ventral course near the tip of the tail (dotted red line). (c) The migrating primordium, as seen under Nomarski optics. (d) Expression of cxcr4b (chemokine receptor) in the primordium. (e) Expression of sdf1a (stromal-derived factor) along the prospective pathway of the primordium, as well as along the pronephros (arrowheads).
the migration process during post-embryonic growth. In zebrafish, the embryonic primordium leads to the formation of a rudimentary PLL, comprising eight neuromasts. The PLL of the adult fish, however, comprises many more neuromasts. It has long been assumed that the transition from embryonic to adult pattern would involve a budding process, whereby, the embryonic neuromasts would form additional neuromasts. To the contrary, a detailed analysis of the post-embryonic growth of the PLL revealed that new primordia are generated and progressively fill in the gaps that are left by the embryonic primordium until all somite borders are occupied by one neuromast [15,16].
Neuromast deposition and pattern formation It is unlikely that the deposition of neuromasts is due to external cues, because a diverted primordium will continue to deposit neuromasts despite traveling through abnormal territories [4]. The idea that the deposition process is intrinsic to the primordium has been confirmed in zebrafish [17], through analyses of the variability of the normal pattern, of the effect of experimental interference, www.sciencedirect.com
and of the patterned expression of genes within the primordium. In particular, cxcr4b is down-regulated in the cells at the trailing edge (those that will be soon deposited) and is completely repressed in the proneuromasts. The proneural gene Zath1, and the neurogenic genes Notch and Delta, which are involved in the choice between hair and support cell fates, are expressed within the primordium in small groups of cells that, presumably, prefigure the deposition of neuromasts [18]; however, the mechanism that initiates the up-regulation of the proneural and neurogenic genes, and down-regulates the expression of cxcr4b, remains elusive. Although the adult PLL pattern varies greatly between teleosts, in relation to various factors such as fin position, morphology or habitat, the embryonic line appears to be amazingly conserved in fish as morphologically and phylogenetically different as the zebrafish, the turbot and the eel ([19] and unpublished observations). One mechanism of variability generation in adult patterns is the formation of additional primordia, which can vary in number and time, between species. A second mechanism Current Opinion in Neurobiology 2004, 14:67–73
70 Development
Figure 3
Afferent innervation of the PLL. (a) Afferent neurites remain in contact with the primordium from the onset of its migration. Note that several neurons already extend a central projection in the hindbrain at the level of the ear. (b) The growth cones of neurons that will end up innervating more anterior neuromasts are invariably simpler than the growth cones that will innnervate more posterior neuromasts (c).
of variability generation is the dorso-ventral migration that differentiated neuromasts can undergo; the amplitude of which depends on their position along the anteroposterior axis, thereby, defining the final shape of the PLL [15,16].
Neurons The early work of Harrison, on amphibians [1], demonstrated that the growth cones of sensory neurites accompany the primordium, thereby, establishing a physical link between the cranial ganglion and the body neuromasts. Also in the zebrafish, the primordium is accompanied by growth cones as soon as it begins to move (Figure 3a). Based on ectopic expression experiments, it has been suggested that sensory axons are guided independently of the primordium and are constrained along their course, due to the repulsive effect of two broad regions of sema3A expression, dorsal and ventral to the prospective pathway [20]. Yet, when the primordium is immobilized, as a result of the inactivation of the SDF1/ CXCR4 system, or when it is forced to follow an ectopic course in mutants, the sensory neurons invariably follow it [7], indicating that the axons are, indeed, guided by the primordium, as surmised by Harrison. Although the repulsive effect of the dorsal and ventral somites plays no role in the normal course of development, it might be a remnant of a more primitive mechanism and/or be part of a ‘back-up’ system. The reason why the growth cones follow the primordium seems to be related to the expression of HNK-1, a Current Opinion in Neurobiology 2004, 14:67–73
glycoepitope that is prominently expressed by the PLL neurons [21]; injecting the antibody in embryos results in some axons taking an extremely abnormal course [22]. The details of the interaction between neurons and primordium are not known, however, and other surface proteins, such as Tag1, might also be involved [23]. Likewise, we do not know where the neurons that innervate the neuromasts that are formed during larval life originate from, nor do we know whether the various sets of neurons that innervate the successive rounds of neuromasts remain distinct from each other – a feature that would facilitate a Fourier analysis of water movements along the fish (N Ko¨ nig, personal communication), because the final spacing between successive neuromasts will differ widely for the different rounds. The ability to use Fourier transforms would enormously increase the analytical capability of the system and would show the astonishing performances of the blind fish Astyanax fasciatus [24] to be somewhat less incredible (this fish is capable of discriminating between two surfaces covered with parallel glass rods that are, respectively, 10 and 11 mm apart!). The formation of the PLL ganglion depends on the previous expression of the proneural gene neurogenin-1 (ngn1) [25]. Interestingly, while the inactivation of ngn1 wipes out the PLL ganglion, it does not prevent the formation of neuromasts. This duality demonstrates that the neuromasts can form independently of innervation — a conclusion that has already been established in amphibians — and that ngn1 is not required for the www.sciencedirect.com
Development of the zebrafish lateral line Ghysen and Dambly-Chaudie`re 71
determination or differentiation of the hair cells. Given that Zath1 is expressed in the presumptive hair cells [18], it seems likely that, in the PLL, as in the mammalian ear, the expression of neurogenin determines the neurons, while the expression of atonal homologs determines the hair cells.
fied (N Gompel, V Chaar and F Soubiran, personal communication). This result implies a, hitherto unsuspected, heterogeneity among similar sensory neurons, and makes it possible to envision, in vertebrates, a level of neuronal specification that is more usually associated with the development of insect sensory organs [33].
Glia
Although presently we have no idea of the mechanism that encodes the position of extension of a given PLL axon, this issue has been extensively studied in Drosophila, where the slit/robo system is used, not only to decide whether an axon will or will not cross the midline, but most importantly, to set up the distance from the midline at which the axon will branch and extend, longitudinally [34,35]. It might be that a similar mechanism is used to set up the distance from the midline in fly and fish, providing afferent axons with the opportunity to extend at various distances from the midline in an organized, programmable manner. Interestingly, it appears that most somatosensory maps use similar coordinates, with the antero-posterior axis of the body being represented along the ventro-dorsal axis of the brain, suggesting the existence of a general and highly conserved mechanism of establishing somatotopic projections.
The PLL nerve is ensheathed by glial cells. In what could have been an excessive expectation of developmental conservation between flies and vertebrates, it was thought that these glial cells might play a role in early axonal guidance [26]. However, Harrison [1] demonstrated that the neurites are towed in the wake of the primordium, thus, making this hypothesis unlikely. This issue has been convincingly settled by a set of experiments that were based on the design of fluorescent reporter lines to follow the migration of glial cells during embryogenesis [27]; they achieved the expected result that the glial cells do, indeed, migrate along the sensory axons rather than guiding them. In addition to their myelinating function, glia also play a role in maintaining the fasciculation of PLL axons [27]. The origin of the glial cells remains a disputed question, however, with some data pointing towards a neural crest origin [28], and uncaging experiments suggesting that glial cells originate from the primordium [17]. A dual origin has already been suggested for the hair cells [29]. The importance and role of the placodal versus neural crest contributions to the lateral line system is an important area that remains to be fully investigated and settled, as it might impact on our understanding of the role and evolution of the neural crest contribution to sensory systems in vertebrates.
Neural connectivity Each neuromast has dual innervation: afferent and efferent. The afferent projections of the ALL and PLL form two ribbons that extend at different levels in the hindbrain, the ALL projection being ventral to the PLL projection (Figure 1c; [30]). The PLL projection, itself, appears to be topologically organized, with neurons innervating more posterior neuromasts projecting more dorsally in the hindbrain [31]. This could reflect a capability of the sensory neurons to respond to the position of the organ they innervate by adjusting their central projection accordingly. Contrary to this interpretation, however, it was found that the central neurite extends before the peripheral neurite has reached its target organ [32]; furthermore, individual differences in growth cone morphology prefigure the position of the organ that the neuron will eventually innervate, suggesting that each neuron is somehow specified to seek an organ at a given position along the antero-posterior axis (Figures 3b,c). In agreement with this conclusion, genes that are specifically expressed in subsets of PLL neurons have been identiwww.sciencedirect.com
Efferent innervation and information processing In addition to the afferent axons, the PLL is also innervated by two sets of efferent axons that modulate its responsiveness to external stimuli [36]. One set originates from the hindbrain. This set is cholinergic and probably functions as an inhibitory feedback and/or feed-forward mechanism. The vestibular efferent nucleus of the mouse, which originates in rhombomere 4, has been shown to depend on the gene Phox2b for its differentiation, and on the gene Mash1 for its proper migration [37]. It will be interesting to determine to what extent these genetic requirements are conserved in the fish. The second efference originates from the forebrain, is dopaminergic and probably subserves an excitatory modulation, possibly related to the level of ambient light. Unfortunately, we still have no knowledge of the genetic mechanisms that allow the PLL information to be matched to other sensory information, such as vision and hearing. This matching is at the basis of our capacity to combine sensory information, corresponding to different modalities and, as such, is of paramount importance in the building of our perceptual map of the world. The experimental qualities of the PLL might provide one entry point into this challenging arena.
Conclusions This review has illustrated how the exceptional accessibility and definition of the PLL, together with the experimental and genetic qualities of the zebrafish, make this system ideally suited to address several important Current Opinion in Neurobiology 2004, 14:67–73
72 Development
questions in vertebrate development, from the control of cell migration and cell proliferation to the organization of axonal projections. We still do not have a comprehensive picture of the genetic program that underlies the successive steps of lateral line development, from placode determination to cell differentiation and the establishment of an accurate sensory map in the brain. Nevertheless, the convergent efforts of many laboratories, and the fact that our knowledge of PLL development has enormously increased over the past few years, suggest that such a complete understanding has now become a realistic aim. The abundance of mutations and genetic tools, which are increasingly available to the zebrafish community, will, undoubtedly, be a major asset in achieving this aim.
Acknowledgements We thank T Whitfield for excellent criticisms and J Hudspeth for careful reading of the manuscript.
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