Development of the External Ear in the Dachs Rabbit ' DORCAS D. CRARY The Jackson Laboratory, Bar Harbor, Maine ABSTRACT Development of that part of the external ear which protects the external auditory meatus appears to be similar in all animals since the hillocks of His have been described for reptiles, amphibia, and birds as well as mammals. Disagreement exists as to the origin and fate of the various hillocks, especially tragus and antitragus. Most observations of the developing ear have begun with the formation of the hillocks at which time the tragus is situated on the mandibular arch. Because of the rapid early growth, transitional steps from the open branchial groove to this stage have not been described previously. The retarded growth of the dachs rabbit makes it possible to see intermediate steps. Here it is shown that the entire auricle arises from an intact and continuous primordium, with the tragus arising from the hyoid arch and migrating to the mandibular bar in the normal course of cell proliferation and differentiation. Variations in normal and heterozygous animals can be found in the relative rates of development of the surrounding areas. The dachs rabbit has typical features of Ichondrodystrophy. The gene responsible ( D a ) produces various effects which contribute to the understanding of basic developmental processes. These are primarily skeletal, but the external ear and its musculature are also modified (Lamb and Sawin, '63). The ear carriage is abnormal and a distinctive papilla projects free and separate from the lateral margins at its base (Sawin and Crary, '57). In the heterozygote this papilla is only partially free, being attached to the anterior margin and projecting from it in varying degrees. This structure, not manifest in the normal, has been shown by Lamb and Sawin ('63) to be the anterior cornu of the tragus. Its presence or absence has been used routinely to identify the three genotypes a t birth. In an attempt to find the earliest possible age at which the three genotypes could be identified for further histologic investigations, observations on the development of the external ear were made, aiding materially the understanding of such development. They were re-examined at 9 X , 24 hours later to verify the genotyping and to make drawings of the external appearance of the intact ear. Table 1 shows the distribution of the 345 specimens examined. Of these 81 were dachs ( D a D a ) , 178 heterozygous ( D a d a ) , and 86 normal ( d a d a ) . A test of the reliability of the typing was made for each age since identification of the genotypes at early ages was difficult. This showed no significant departure from the expected single gene segregation based either on any one age or on the pooled values . Comparative terminology used is as follows : Human (International Anatomical Nomenclature Commission, ' 5 6 ) T r a"m s Antitragus Crus helicis Anthelix Rabbit (Lamb Sawin, '63)and Anterior Posterior Anterior Posterior cornu of t r a-m s cornu of tragus helix helix Since many mammalian ears have a simpler, more straightforward form than that of the human, a simpler terminology is more readily applicable, hence anterior MATERIALS AND METHODS and posterior helix rather than crus helicis Embryos and fetuses were removed by and anthelix. In the rabbit, tragus and C!aesarean section from DA race females antitragus can be shown to be processes of at 11-23 and at 27 days postcopulation. a larger projection attached to the posteThey were placed in aceto-formol-alcohol rior helix (Lamb and Sawin, '63). The for a few minutes to distinguish the ear 1 This investigation was supported in part by PHS and its tubercles, tentatively identified for research grant C281 from the National Cancer Instltute, Public Health Service, and in part by Grant E-40 genotype, and then fixed in Bouin's fluid. from the American Cancer Society. AVAT.REC.,150: 441-448. 441 442 DORCAS D. CRARY TABLE 1 Distribution of genotypes Age postcopulation DaDa Dada 11 12 13 14 15 Subtotal 16 17 18 19 20 21 = 22 2 23 27 Totcl 1 3 2 3 1 10 5 5 5 4 3 14 20 9 6 81 1 6 5 6 2 20 1 2 12 6 10 9 13 56 37 10 5 178 dada Total 1 3 12 9 13 6 43 19 14 20 18 24 99 72 3 2 4 3 13 2 3 5 5 8 29 15 3 3 86 22 14 345 ___ 0.3333 0.0 0.1112 0.2@00 2.0000 0.6195 2.0527 0.8572 0.0 0.1112 2.2500 1.7171 4.0906 3.4555 2.4285 2.1103 No chi square value deviated .from the expected 1 : 2 : 1 ratio at the 0.05 level of significance. This sample contains some animals from normal x heterozygote matings. process of migration, comes to lie in the open end of the groove where it remains free of the crus helicis. Its anterior margin projects downward and in later stages becomes parallel to the posterior margin OBSERVATIONS (figs. 5-12). Thus it becomes the distincAs in all mammals the external ear of tive papilla of the dachs and as such is the rabbit is a modification of the first identifiable as (the anterior cornu of) the branchial groove with additions from the tragus (Lamb and Sawin, '63). In the mandibular and hyoid arches (Arey, '54). heterozygote where the cleft is somewhat At 11 days postcopulati6n (fig. 1 ) the narrower, usually only the posterior rnargroove is open ventrally in both dachs and gin of the tragus projects into the groove heterozygous individuals and the diagnos- although variation may be almost contintic projections are just becoming visible. uous between dachs and normal. The skin Arising from the hyoid arch is a rather fold covering the crus helicis bulges out large appendage in the process of migra- to cover it and creates a ridge which protion around the open end of the groove. jects laterally where it covers the anterior The heterozygote is slightly more ad- margin. In normal animals the tragus lies vanced than the dachs and by comparing in proximity to the crus helicis and is DaDa figures 1 and 2 , Dada figure 2 , and eventually almost completely obscured by dada figure 1, in that order, the progress of the skin which overlies it. The ridge prothis migration can be readily seen. In the duced in the skin fold projects upwards. normal the migration is nearly complete, This distinguishing feature separates the and by 13 days (fig. 3 ) it is complete in three genotypes at all ages once migration all three genotypes. The groove is closed of the tragus is completed (figs. 3-12), and appears as an elongated slit with but it is somewhat less prominent after small tubercles on each side. The width birth when the ear reopens and begins to of this slit varies with genotype, being assume its ultimate adult conformation. widest in the dachs, narrowest in normals, This ridge is particularly useful in typing and intermediate in heterozygotes. Varia- early developmental stages where the tion in development makes some speci- tragus is still prominent in normal animens identifiable only by the width of the mals. As the auricle grows, the ventral groove. Because of the relatively broad portions of the ear are suppressed and cleft in the dachs, the appendage, in the certain of the features seen in early develterms anterior and posterior cornu of the tragus thus seem more apt. In using this terminology, Lamb and Sawin followed that of Meinertz ( ' 3 5 ) . DEVELOPMENT O F RABBIT EXTERNAL EAR opment become less noticeable. Thus, in the normal of 16 days (fig. 6 ) the tragus is quite visible, but at birth it is difficult to discern and in the adult it is almost never seen. The auricular cartilage is first detectable around 12 days (fig. 2) as a slight projection of the posterior dorsal edge of the lbranchial groove. It projects out over the groove (figs. 2-5) and by 16 days (fig. 6 ) covers it completely. As it grows, the helices elongate and the ear opening narI*OWS until it closes completely. It does not open again until after birth. At around 21-22 days (fig. 11) the ear folds back revealing the now closed external auditory meatus and assuming a position along the back of the head where it remains until after birth when it is erected in normal and heterozygous animals. Because of the abnormalities of skull (Sawin, Ranlett, and Crary, '59) and musculature (Lamb and Sawin, '63) the dachs ear never attains the erect position but projects dorsolaterally (Crary and Sawin, '52). DISCUSSION AND CONCLUSIONS That the external ear develops from hillocks (His, 1885, cited by Streeter, '22) or ridges (Hammar, '02, cited by Streeter, '22) arising from the hyoid and mandibular arches early in embryonic development is a generally accepted fact. It is interesting to note that these hillocks have been described for mammals with very different types of ears (Gradenigo, 1888; Schwalbe, 1897, '16; Schmidt, '02, all cited by Streeter, '22) and also for reptiles, amphibia, and birds, which never acquire a distinct auricle (Schwalbe, 1891, cited by Streeter, '22). This would seem to indicate that the auricle, per se, is an independent functional adaptation and that the development of that part of the external ear which protects the entrance to the external auditory meatus is probably very similar in all animals. However, considerable disagreement exists as to the ultimate fate of thle various hillocks or to the origin of the definitive parts of the fully formed ear. The greatest confusion appears to exist in the origin of the tragus and antitragus. His (1885, cited by Streeter, '22) in his classic description, which has dominated subsequent literature, attributed the origin 443 of the tragus to the mandibular arch and the antitragus to the hyoid. Streeter ('22) believed that too much emphasis had been placed on the importance of the hillocks which are transitory and incidental rather than fundamental to the development of the auricle. They are due to closely packed masses of mesenchymal cells beneath actively proliferating ectoderm. The ectoderm of the hyoid arch shows more marked changes than that of the mandibular bar and resembles that of the early stages of limb buds. Streeter felt that the auricle arises from an intact and continuous primordium which becomes elaborated into its final form by the ordinary processes of differentiation. Although he makes the statement that it is possible that the auricle is entirely of hyoid origin, the mandibular elements being products of cells which have migrated into this region from the hyoid bar, he was unable to demonstrate it with his material and continued to describe the tragus as of mandibular origin. Wood-Jones and Wen ('34) concluded that the tragus was derived from the mandibular arch, but that the remainder of the external ear was of hyoid origin. Boas ('l2), in a comprehensive study of the comparative anatomy of the adult mammalian ear and its cartilage, perfected a method by which the cartilage could be flattened out for ease of comparison. In so doing, he demonstrated that the tragus is a part of the posterior border of the ear cartilage which is rolled so that the proximal portion lies over the anterior edge. He pointed out the strong similarity of all mammalian ears and discussed how the individual parts vary from one form to another, but he made no attempt to trace their origins. His reclassification of parts into anterons, posterons, and notches, while perhaps facilitating his comparisons, serves only to confuse the picture. More recently, Lamb and Sawin ('63) prepared ear cartilages of dachs and normal rabbits, again showing that the auricular cartilage is nearly continuous but with the margins unconnected anteriorly. The tragus is continuous for most of its length with the posterior helix (anthelix) and lies just posterior to the anterior helix (crus helicis). In these preparations, the papilla-like structure of the 444 DORCAS D. CRARY Stages in the development of the external ear in dachs ( D a D a ) , heterozygous ( D a d a ) , and normal ( d a d a ) rabbits. Fig. 1 Eleven days postcopulation. Note projection (tragus) arising from the hyoid arch which is in process of migration around the open end of the gill cleft in DaDa and Dada animals. Fig. 2 Twelve days. The tragus is still migrating around the gill cleft, Note beginning of auricular cartilage. Fig. 3 Thirteen days. Migration of tragus is complete in all three genotypes. Note difference in width of branchial groove. Figs. 4, 5 Fourteen and fifteen days. Note projection of auricular cartilage over ear opening. Fig. 6 Sixteen days. Auricular cartilage (dotted lines) now covers ear opening completely. 445 DEVELOPMENT O F RABBIT EXTERNAL EAR AC, Auricular cartilage Ah, Anthelix At, Antitragus Abbreviations BG, 1st branchial groove CH, Crus helicis HA, Hyoid arch 11, Incisura intertragica MA, Mandibular arch T, Tragus Whole embryos and heads from Minot and Taylor ('05) x 2.5. Figs. 7-10 Seventeen-twenty days. Note angle of anterior margin of tragus in DaDa animals and of the ridge produced by skin fold covering it in heterozygotes (Dada) and normals (dada). Figs. 11, 12 Twenty-two and twenty-three days. The auricular cartilage has folded back along the head revealing the closed external auditory meatus. 446 DORCAS D. CRARY dachs proves to be the superior anterior cornu of the tragus (tragus) which in normal rabbits develops in contact with the anterior helix (crus helicis) where it is not apparent beneath the skin. This suggests that the tragus is, therefore, of hyoid rather than of mandibular origin. Most observations of the developing ear have begun with the time of formation of the auricular hillocks. Because of the rapidity of the early development of this region, the steps in the transition from the open branchial groove to this stage have not been described previously and confusion concerning the origin of certain of the parts of the ear has resulted. I n the dachs rabbit growth in general is retarded, making it possible to observe intermediate steps in development not heretofore demonstrated. The projection seen i n the earliest embryos, later identifiable as the tragus, becomes the distinctive diagnostic papilla of the dachs because of the reduced rate of growth and development which not only slows the migration of the tragus but keeps the groove relatively broad. Thus the papilla, i n itself, is not a n abnormal structure, but its abnormal exposure is entirely the result of retarded growth. The tragus normally remains free of the crus helicis in some species of mammals, notably the camel (Camelus bactrianus) and the wild pig of Sumatra (Sus) (Boas, '12). Study of the development of the dachs ear, especially when compared with that of heterozygotes and normals, shows that the entire auricle, which in cartilage preparations of adults appears as a single continuum, arises from a n intact and continuous primordium, as Streeter suggested, with the tragus (anterior cornu) arising from the hyoid arch and migrating to the mandibular bar i n the normal course of cell proliferation and differentiation. Variations in expression of this region in both heterozygous and normal animals presumably can be found in the original width of the branchial groove and the relative rates of development of the surrounding areas. LITERATURE CITED Arey, L. B. 1954 Developmental Anatomy, 6th ed. W. B. Saunders Company, Philadelphia. Boas, J. E. V. 1912 Ohrknorpel und ausseres Ohr der Saugetiere. Kopenhagen. 266 pp, 25 plates. Crary, D. D., and P. B. Sawin 1952 A second recessive achondroplasia in the domestic rabbit. J. Heredity, 4 3 : 255-259. Gradenigo, G. 1888 Die Formentwickelung der Ohrmuschel mit Riicksicht auf die Morphologie und Teratologie derselben. Centralbl. f . d. med. Wissensch. 26:82-86, 113-117. Cited by G. L. Strceter, Development of the auricle in the human embryo. Carnegie Inst. Wash. Pub. 277. Contrib. Embryol., 1 4 : 111-138, 1922. Hammar, J. A. 1902 Studien iiber die Entwickklung des Vorderdarms und einiger angrenzenden Organe. I Abtheilung : Allgemeine Morphologie der Schlundspalten beim Menschen. Entwicklung des Mittelohrraumes und des ausseren Gehorganges. Arch. f. mikr. Anat., 59: 471-628. Cited by G. L. Streeter, Development of the auricle in the human embryo. Carnegie Inst. Wash. Pub. 277. Contrib. Embryol., 14: 111-138, 1922. His, W. 1885 Die Formentwichelung des ausseren Ohres. Anatoinie menschlicher Embryonen. Part 111, 211-221. Cited by G. L. Streeter, Development of the auricle i n the human embryo. Carnegie Inst. Wash. Pub. 277. Contrib. Embryol., 14: 111-138, 1922. International Anatomical Nomenclature Commission 1956 Nomina Anatoniica. The Willizms & Wilkins Co., Baltimore. Lamb, N. P.7 and P. B. Sawin 1963 Morphogenetic studies of the rabbit. XXXIII. Cartilages and muscles of the external ear as affected by the dachs gene ( D a ) . Am. J. Anat., 1 1 3 : 365-388. Meinertz, T. 1935 Die Hautmuskulatur der Saugetiere. Yahrb. Morph. u. Mikrosk. Anat. Abt. I. Gegenbauers Morph. Yahrb., 75: 15-51. Minot, C. S., and E. Taylor 1905 Normal plates of the development of the rabbit (Lepus cuniculus, L ) , In: Normentafeln zur Entwicklungsgeschichte der Wirbelthiere V. F. Keibel, Jena. Sawin, P. B., and D. D. Crary 1957 Morphogenetic studies of the rabbit. XVII. Disproportionate adult size induced by the DA gene. Genetics, 42: 72-91. Sawin, P. B., Mary Ranlett and D. D. Crary 1959 Morphogenetic studies of the rabbit. XXV. The spheno-occipital synchondrosis of the dachs (chondrodystrophy) rabbit. Am. J. Anat., 105: 257-280. Schmidt, J. 0. L. 1902 Vergleichend-anatoinische Untersuchuiigeii iiber die Ohrmuschel verschiedener Saugetiere. Berlin. Cited by G . L. Streeter, Dcvelopment of the auricle in the human embryo. Carnegie Inst. Wash. Pub. 277. Contrib. Embryol., 14: 111-138, 1922. Schwalbe, G. 1891 iiber Auricularhocker bei Reptilien; ein Beitrag zur Phylogenie des ausseren Ohres. Anat. Anz. 6: 43-53. Cited by G . L. Streeter, Development of the auricle i n the human embryo. Carnegie Inst. Wash. Pub. 277. Contrib. Embryol., 14: 111-138, 1922. 1897 Das aussere Ohr. In: Handbuch der Anatomie des Menschen. K. H. von Bardeleben. Vol. 5, part 2: 125-131. G. Fischer, - DEVELOPMENT O F RABBIT EXTERNAL EAR Jena. Cited by G. L. Streeter, Development of the auricle in the human embryo. Carnegie Inst. Wash. Pub. 277. Contrib. Embryol., 14: 111-138, 1922. 1916 Beitrage ZUI Kenntnis des ausseren Ohres der Primaten. Zeitschr. f. Morphol. u. Anthropol. 19: 545-668. Cited by G. L. Streeter, Development of the auricle i n the 447 human embryo. Carnegie Inst. Wash. Pub. 277. Contrib. Embryol., 14: 111-138, 1922. Streeter, G. L. 1922 Development of the auricle in the human embryo. Carnegie Inst. Wash. Pub. 277. Contrib. Embryol., 14: 111-138. Wood-Jones, F., and I-C. Wen 1934 The development of the external ear. J. Anat., 68: 525-533.