Genetic analyses of mammalian ear development

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evelopment of the vertebrate D ear and periotic tissues is Geneticanalysesof mammalianear development characterized by a series of tissue movements and inductive interactions that promote cytodifferentiation and spatial programming of inner, middle and outer ear structures. Several recent studies have identified genes that affect these processes and, together with data from cellular and classical genetic approaches, are improving our understanding of the complexity of ear development and the possible etiology of certain hearing deficits. The membranous labyrinth (consisting of the semicircular canals with vestibular sensory transducers, and the cochlear duct with the organ of Corti) and associated VIIIth ganglion neurons are derived from the otic placode, which develops in close apposition to rhombomeres 5 and 6 of the hindbrain (Fig. 1). Transplantation and organ-culture studies have shown that close proximity to this region of the hindbrain is necessary for otic placode invagination and the early stages of otic vesicle morphogenesis x'2. These early interactions are sufficient to initiate differentiation of sensory receptor cells, but spatial organization of vestibular and auditory receptors occurs only in the continued presence of periotic mesenchyme. Furthermore, reciprocal interactions between otic epithelium and the surrounding mesenchyme are essential for aggregation and differentiation of periotic cartilage precursors, which form a protective capsule around the inner ear. The morphogenesis of middle and outer ear tissues involves interactions between neural crest cells, surface ectoderm, endoderm of the dorsal wing of the first pharyngeal pouch, and the otic vesicle. Neural crest precursors of the ear ossicles arise in the neural folds at axial levels corresponding to rhombomeres 2 and 4 (Refs 3,4). Some of these migratory cells settle at the interface of the first and second branchial arches above the first pharyngeal pouch (Fig. 2), and then shift dorsolaterally as the auditory (Eustachian) tube elongates. Crest cells are also critical to the positioning of the tympanum ~ and the subsequent morphogenesis of the outer ear. TINS, Vol. 15, No. 7, 1992

It is interesting that abnormal differentiation of auditory receptors is often associated with abnormalities in peripheral pigmentation and is observed in several syndromes in mice < 7 (e. g. splotch, piebald, lethal spotting, patch); in domesticated mammalss (e.g. dominant-white cats, Dalmations, the merle pattern in collie dogs); and in humans 9 (e.g. recessive piebaldism, X-linked pigmentary abnormalities). However, the genetic bases and times of sensory receptor degeneration vary. Homozygous splotch animals die in utero and display white spotting on the abdomen and limbs, spina bifida and severe deficits of the peripheral sensory nervous system, cardiac abnormalities, and a primary dysmorphogenesis of the membranous labyrinth, including the receptors of the inner ear. Defects in heterozygotes usually lead to lack of pigment in ventral skin only. Most of these abnormalities are attributable to widespread dysplasia of neural fold and dorsal neural tube tissues, including the focal loss of migration-competent neural crest precursors 1°. One aUelic form of splotch results from a partial deletion of the Pax-3 gene ~l, which then expresses a truncated protein product. Pax-3 is one member of a family of paired-box genes

and is normally expressed in the dorsal part of the spinal cord and hindbrain before and during the period of otic vesicle formation r). Waardenburg's syndrome (WS) is an autosomal dominant disorder that accounts for 2-3% of hereditary hearing deficits in humans and causes variable dysmorphogenesis of auditory sensory receptors ~. The syndrome includes pigment dysplasias of the iris and integument, and is frequently associated with a white blaze of skin or hair on the forelock. WS type 1 is characterized by a facial defect in which the inner corner of each eye is displaced laterally, and approximately 25c~ of these persons have hearing deficiencies. Two recent studies, one with 17 unrelated WS type 1 patients 1:~and the other with a large family having 26 affected members 11, identified gene mutations lying close to one of the helix-coding regions of the HuP2 paired homeodomain. At least three different deletions were identified in the first study, while the second reported a single basepair substitution (changing Pro to Leu). Linkage analysis and sequence data indicate that HuP2 and Pax-3 are homologous genes. While it has been suggested that Pax-3 might affect expression of the neural cell adhesion molecule (NCAM) gene 1'~, the complete

Drew M. Noden

Deptof Anatomy, Co//egeof Veterinary Medicine,Come// University,Ithaca, NY 14853,USA. Tom R. Van De Water

Deptsof Oto/aryngo/ogyand Neuroseiences,A/bert EinsteinCo//egeof Medicine, 1410 Pe/hamParkway SouthBronx, NY 10451,USA.

Fig. 1. Scanning electron micrograph of an 18-somite mouse embryo (day 9 of gestation) fractured in the transverse plane, showing the otic cup (OC), rhombencephalon (R) and pharynx (Ph). The mesenchymal cells around the otic cup, dorsal aorta (DA) and aortic arch (a) are of both neural crest and mesodermal origin. CV represents the cranial cardinal vein. Scale bar, 100 Ibm. © 1992, ElsevierSciencePublishersLtd, (UK)

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function of Pax-3 is unknown. Certainly, the different modes of activity (recessive or incomplete dominant in mice, dominant in humans) and the more severe brain and spinal cord defects seen in homozygous splotch mice must make one wary of extrapolating from the two systems. Furthermore, this discrepancy is exacerHindbrain - bated since so little is known about Neural crest how the distribution or differenParaxial mes tiation of melanocytes and dysplasia of auditory receptors are causally Rhombomerc linked 16, or about how otic epiPlacodes thelia promote the differentiation of pigment cells derived from the neural crest. Pharyngeal Lc An alternative approach to Branchial a rc h e s . ~ . , , , . / ~ - ~ / ~ assessing the genetic basis of ear morphogenesis is presented in reports by Lufkin et al. 17 and Chisaka et al. 18 Both groups used homologous recombination (gene targeting) to generate lines of Fig. 2. The changing spatial relations between the otic vesicle and nearby transgenic mice that are unable to mesenchymal and epithelial structures. Neural crest cells (dark grey tint) make normal H o x - l . 6 gene prodmoving towards the pharyngeal area are deflected by the invaginating otic ucts. Normally this gene is excup. Later, paraxial mesoderm tissue (stippled) expands laterally to surround pressed along the caudal and middle the otic vesicle. Dorsal tissues progressively shift rostrally, while ventral tissues regions of the hindbrain before the recede caudally. These movements bring the first pharyngeal pouch and onset of otic placode formation; the associated neural crest cells of the first and second arches into close contact with the ventrally expanding cochlear primordium, and establish new spatial signal recedes caudally following relations between middle and inner ear structures. In the mouse, motor neuron otic vesicle formation. Lufkin et al. pools are located in rhombomere (r) rl-r3 (nerve V), r4-r6 (nerve VII), r5 used a nucleotide construct that (nerve VI), r6 (nerve IX) and r7-r8 (nerve X). disrupted a control region and part of an amino-terminal coding region of exon 1, while Chisaka et al. used TABLE I. Comparison of anatomic defects in two Hox-l.6 gene deletion experiments a smaller construct that deleted Feature Lufkin et al. 17 Chisaka et al. TM the homeodomain region of exon 2. Disruption of Hox-1.6 expression Brain results in defects of the inner ear Hindbrain closure Delayed Normal and also of both motor and sensory Rhombomeres Normal Poorly delineated neurons associated with cranial Motor nuclei VII, IX, X Reduced or absent Reduced or absent nerves VI[, VIII, IX and X Superior olivary complex (Data not available) Absent (Table I). Homozygotes die during Inner ear the perinatal period. In the study Otic vesicle (9--9.5 days) (Data not available) Reduced; displaced rostrally and by Lufkin et al. 17 the membranous laterally labyrinth fails to show regional Membranous labyrinth No regional specification Cochlear duct reduced in size specification, but middle and outer Sensory receptors Occasional macular zone Several foci present ear tissues are normal. Hindbrain Vestibular ganglion Reduced Reduced neural fold closure is delayed, but Auditory ganglion Absent Reduced once closed, the hindbrain exhibits Perilymphatic spaces Reduced Present its normal rhombomeric organization. In contrast, in the report by Middle and outer ear Auditory tube Present Present Chisaka et al. 18, development of Tympanic membrane Present Absent the cochlear duct is abnormal, and Ossicles Present Absent the middle ear cavity, ossicles, and Ear canal Present Distorted outer ear tissues are severely Pinna Present Hypoplastic hypoplastic or absent. Embryonic rhombomeres are indistinct, and Peripheral nerves and ganglia the otic vesicle and adjacent perTrigeminal nerves and ganglia Normal Normal ipheral ganglia and nerves are Nerve roots VII, VIII, IX, X Absent Absent shifted rostrally so that ganglia VII Proximal ganglia VII, IX, X Absent Reduced; shifted rostrally (left and V are often contiguous. more than right) Distal ganglia IX, X Present Present; position uncertain The tissues that are abnormal or absent in these studies do not 'Present' implies that the structures have formed and are qualitatively, although not necessarilyquantitatively, share a common embryonic origin, normal. 236

TINS, Vol. 15, No. 7, 1992

and experimental lesions to any one of the components (e.g. otic vesicle, neural crest, placodes) would not produce the array of primary morphogenetic defects observed. However, the progenitors of these tissues are normally situated close together in the middle hindbrain region (beside and beneath rhombomeres 4-7), and their morphogenesis is known to be dependent on sequential interactions. The absence of neuroblasts derived from the neural crest would mimic the isolation of placode-derived sensory neurons and the absence of dorsal roots ~'~, which would secondarily disrupt normal morphogenesis of motor nucleff°. However, given that most jaw, hyoid and branchial arch structures are normal in these animals, it is difficult to ascribe the missing middle ear ossicles and the outer ear hypoplasia seen by Chisaka et al. ~ to a primary lesion of the neural crest, unless only specific subpopulations of crest cells were affected. A particularly interesting feature of the Hox-1.6 disruption produced by Chisaka et al.l~ is the rostral shift in otic and adjacent peripheral ganglionic tissues. This alteration in spatial relations may disrupt interactions of otic, periotic and neural crest cells with the hindbrain. Treatment of mouse ~ and

Xenopus '~'~ embryos with retinoic

acid causes a similar rostral shift of hindbrain-level axial and paraxial structures, including the otic vesicle, but later morphogenesis of ear tissues has not been examined in these animals. The different phenotypes of homozygotes in these two partial gene deletion studies are enigmatic. In the absence of direct assays for the presence and integrity of H o x - l . 6 gene products, it can only be assumed that the constructs used for gene targeting produced different functional deletions. Defining how such differences then generate inner ear or middle and outer ear dysmorphologies may be particularly illuminating. Furthermore, it is not known whether the H o x - l . 6 deletions also included sequences whose products regulate the activity of other genes in the Hox family. It is likely that clarification of these critical attributes will reveal subtle but important aspects of the genetic control of ear morphogenesis.

Selected references 1 Noden, D. M. and Van De Water, T. R. (1986)in The Biology of Change in Otolaryngology (Ruben, R. J., Van De Water, T. R. and Rubel, E. W., eds), pp. 15-46, Elsevier 2 Van De Water, T. R. and Represa, J. (1991) Ann. NY Acad. Sci. 630, 116-128

3 Noden, D. M. (1975) Dev. Biol. 42, 106-130 4 Lumsden, A., Sprawson, N. and Graham, A. (1991 ) Development 113, 1281-1291 5 Noden, D. M. (1983) Dev. BioL 96, 144-165 6 Deol, M. S. (1970) Proc. R. Soc. London Set. B 175, 201-217 7 Steel, K. P. (1991)Ann. NYAcad. Sci. 630, 68-79 8 Noden, D. M. and de Lahunta, A. (1985) The Embryology of Domestic Species: Developmental Mechanisms and Malformations, Williams and Wilkins 9 Beighton, P. eta/. (1991) Ann. NY Acad. Sci. 630, 152-166 10 Moase, C. E. and Trasler, D. G. (1990) Teratology 42, 171-182 11 Epstein, D. J., Vekemans, M. and Gros, P. (1991) Ceil 67, 767-774 12 Goulding, M. D. etaL (1991) EMBOJ. 10, 1135-1147 13 Tassabehji, M. et aL (1992) Nature 355, 635-636 14 Baldwin, C. T., Hoth, C. F., Amos, J. A., da-Silva, E. O. and Milunsky, A. (1992) Nature 355, 637-638 15 Moase, C. E. and Trasler, D. G. (1991) Development 113, 1049-1058 16 Steel, K. P. and Barkway, C. (1989) Development 107, 453-463 17 Lufkin, T., Dierich, A., Lemeur, M., Mark, M. and Chambon, P. (1991) Cell 66, 1105-1119 18 Chisaka, O., Musci, T. and Capecchi, M. R. (1992) Nature 355, 516-520 19 Hamburger, V. (1961) J. Exp. Zoo/. 148, 91-124 20 Heaton, M. B. and Moody, S. A. (1980) J. Comp. NeuroL 189, 61-99 21 Morris-Kay, G. M., Murphy, P., Hill, R. E. and Davidson, D. R. (1991) EMBO J. 10, 2985-2995 22 Papalopulu, N. et al. (1991) Development 113, 1145-1158

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Acknowledgements Drs ChrisWnghtand BrigidHogan generouslyprovided many helpful suggestionsdunng thepreparationof th~s discussion.

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SantiagoRamony Cajalandmethodsin neurohistology Javier DeFelipe and Edward G. Jones

Controversy, misunderstanding or uninformed opinion abound over the extent to which the great Spanish neurohisto/ogist, Santiago Ramony Caja/, specified his staining methods in his analytical papers, the methods by which he analysed and presented his data, and the microscopes available to him. In this paper, we have attempted to outline the information on thesepoints that we have been able to obtain from a detailed examination of his writings and a study of the evidence remaining in the Caja/Museum in Madrid. The fortuitous combination of genius and the advent of a new research technique has commonly had dramatic consequences for the advancement of scientific knowledge. The genius of Santiago Ramdn y Cajal was compounded of profound biological insight, skilful exploitation of existing techniques and invention of new ones, consummate artistry and an enormous capacity for sustained hard work. In 1887, he took up a neurohistological staining method which, although introduced by Camillo Golgi in 1873 (Refs 1,2), had received scant

TINS, Vol. 15, No. 7, 1992

attention or unsuccessful application outside Golgi's Italian laboratory. The result was an unparalleled success and by 1891 Ram6n y Cajal* had published some 45 major papers 3 in which the foundations of the Neuron Doctrine, formally enunciated by Waldeyer 4 in the autumn of that year and which forms the underpinnings of all neuroscience~ were firmly laid. But genius in analytical science requires tools in order to extract, systematize and interpret data. Although there are many who would credit Ram6n y Cajal with being such a strong swimmer in the tide of biological science that he could figuratively walk on its waters, even he needed aids to facilitate his work at the microscope. In the present account we hope to provide some insights into his methods and to resolve certain controversies and misinterpretations that have arisen regarding them.

). DeFehpeis at the Instituto Caja/,Ave Dr Arce37, 28002 Madrid, Spareand E. 5. Jonesis at the Dept of Anatomyand Neurobiology, Universityof California, Irvine, CA 92717, USA.

* It is TINS' stylistic policy to refer to Santiago Ramdn y Cajal by the full version of his surname, rather than by the shorter 'Cajal'.

© 1992.ElsevierSciencePublishersLtd,(UK)

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