J. Embryol. exp. Morph., Vol. 14, Part 3, pp. 265-272, December 1965
Printed in Great Britain
Experimental studies on the columella-capsular
interrelationship in the turtle Chelydra serpentina
by M. J. TOERIEN 1
From the Department of Anatomy, University of Stellenbosch
THE reciprocal effect of two neighbouring structures of different origins on each
other during development is of considerable interest to the embryologist.
Such a relationship exists between the columella auris and the auditory capsule
in the vicinity of the fenestra ovalis. This particular relationship, in addition,
embraces an aspect of fundamental importance to the morphologist viz. the
possible derivation of part of the columella from the capsule.
The vast literature pertaining to this latter aspect and particularly in regard
to the development of the human stapes, was recently reviewed in great detail
by Strickland, Hanson & Anson (1962). References to literature related to other
vertebrates are found in e.g. van der Klaauw (1924), Versluys (1936), and Werner (1960). An earlier paper (Toerien, 1963) deals with the rather specialized
problem of Amphibian stapes development
The results of certain extirpation procsduivS on early embryos of the turtle
{Chelydra serpentina) throw interesting h^ .. on the cclumella-capsule interrelationship. The planning of the operations was based on the following
earlier observations:
1. The visceral skeleton is almost entirely derived from neural crest cells and
consequently the removal of the cranial neural folds will lead to the absence
of the visceral arch skeleton (Horstadius, 1950; Mangold, 1961; Hammond
& Yntema, 1964). Since the stapes is part of the visceral skeleton, its absence
was also anticipated.
2. Removal of the otic placode or otocyst inhibits the development of the
auditory capsule in most vertebrates (see Yntema, 1955; Benoit, 1957;
Toerien, 1965).
MATERIAL AND METHODS
The eggs were obtained from turtles collected in the vicinity of Syracuse N.Y.
and from the Lemberger Co. of Oshkosh, Wisconsin. The recovery of the eggs,
their preparation for the operation and the subsequent treatment of the embryos
are described by Yntema (1964).
1
Author's address: Department of Anatomy, University of Stellenbosch, Stellenbosch,
S. Africa.
266
M. J. TOERIEN
The following operations were carried out by means of glass needles and
sharpened watchmaker's forceps:
1. Unilateral extirpation of the otic placode at somite Stages 12-20 (Toerien,
1965).
2. Bilateral removal of the neural folds between the second somite and the
optic enlargement (somite stages 4-6).
3. Bilateral removal of the neural folds anterior to the optic enlargement but
excluding the anterior (transverse) fold (somite Stages 4-6).
The eggs were incubated at 30° C for a period of 30 days. At this temperature,
30 days constitutes about half the total incubation time for normal eggs.
The average shell lengths of the specimens when sacrificed are given in Table 1.
Serial sections, 16 /x in thickness, were prepared of all the heads and graphic reconstructions made of the chondrocrania.
TABLE 1
Extirpation of
otic placode
No. of specimens
Specimens selected for study
Average shell length
when sacrificed
Extirpation of
anterior
cranial
neural fold
Extirpation of
posterior
cranial
neuralfold
14
7
13-7mm.
16
5
12-7
RESULTS AND DISCUSSION
The following results bear on the present problem.
(a) Absence of almost the entire auditory capsule and a deficient footplate of
the columella follow the removal of the placode on that side (Toerien,
1965).
(b) One of the most conspicuous results of the removal of the anterior cranial
neural folds is the absence of the cartilaginous elements of the mandicular
visceral arch. In addition to Meckel's cartilage and the quadrate, the
development of the tympanic membrane, which is supported by the latter
structure, is also inhibited.
(c) Extirpation of the posterior cranial neural folds lead to the suppression of
the development of the cartilaginous derivatives of the hyoid visceral arch,
including the columella auris.
Although the changes occurring in the chondrocrania of the operated animals
are largely consistent for the different experiments, minor variations are found.
Cartilaginous structures representing remnants of the ear capsule (Toerien,
1965) or parts of the visceral arch skeleton are not always completely suppressed.
Only those specimens in which the structures operated for are completely or
Columella-capsular interrelationship
267
almost completely absent from otherwise fairly normal skulls, were selected for
detailed study. The results reported above derive from these specimens (Table 1).
They allow the study of the following aspects of cranial development:
1. The configuration of the stapes in the absence of the two structures with
which it is most intimately connected viz. the tympanic membrane and the
auditory capsule;
2. The development of the fenestra ovalis in the absence of the columella and
3. The possible contribution of capsular material to the formation of the
columella footplate.
The Columella Amis
In the normal embryo of 14 mm. shell length, the columella is entirely cartilaginous. It is dumb-bell shaped with a slender, gently curved rod, connecting
the oval footplate with a lateral disc. (Dohrer, 1919). A ventral process (interhyal process, Bender, 1911, Kunkel, 1912b) of varying length extends ventrally,
posteriorly and medially from the ventral rim of the lateral plate. Posterior to
this process the plate is excavated from behind (Text-fig. 1).
Aud . cap.
• Fen.
oval.
• Col. ft pi.
Proc. metot.
Col. latpl.
Proc . interh.
1mm.
TEXT-FIG. 1. Graphic reconstruction of lateral view of part of normal auditory capsule of
embryo of Chelydra serpentina Columella auris incompletely drawn. Aud. cap., auditory
capsule; Fen. oval., fenestra ovalis; Col. ftp!., columella auris footplate; Col. laptl., columella auris lateral plate; Proc. interh., interhyal process; Proc. metot., metotic process
(parotic crest).
Medial aspect.
The development of the columella was studied in several chelonians and the
possible capsular origin of its footplate received much attention. According to
Smith (1914) and Shaner (1926) the columella in Chrysemys marginata is
268
M. J. TOERIEN
chondrified from two centres. The precartilaginous mesenchyme is, however, laid
down in one piece which is also continuous with that of the interhyal and hyoid
cornu (Smith, 1914). At a later stage the interhyal process (remnant of the interhyal) joins Meckel's cartilage or becomes connected to it by means of a dense
connective tissue string, (Emys lutaria; Kunkel, 19126).
The view of these authors as to the non capsular origin of the entire columella
is in contrast to that of earlier workers, Parker (1880, Chelone virides), Noack
(1907) and Fuchs (1907, 1915 Emys europaea) who claimed a capsular origin
for (at least) the footplate of the columella. At first glance the experimental
evidence seems to support this earlier point of view since the medial end—footplate—is always deficient after suppression of the cartilaginous ear capsule.
Aud. cap.
Col. latpl.
Proc. interh.
Imm.
2. Graphic reconstruction of ventral view of auditory capsule and columella auris
of Chelydra serpentina. Both structures are virtually unaffected by the absence of a quadrate
and tympanic membrane which was induced experimentally. Abbreviations as in Text-fig. 1.
TEXT-FIG.
This is in conformity with the results of similar experiments on other tetrapods.
(Luther, 1927, Rana, Bombinator; Violette, 1930, Rana; Reagan, 1915, 1917
chicken and Toerien, 1963, Ambystoma).
The columellar rod in the experimental animals is complete. Since the medial
half of the columella, i.e. the footplate and half the rod, is developed from one
chondrification centre it seems clear that it is the footplate of the columella that
is affected by the absence of the ear capsule and not the entire medial chondrofication. A variable number of footplate cells tend to be present around the medial
end of the columella. These two observations, together with the fact that a
separate chondrification centre in the capsular wall representing the footplate
was not found by any investigator, indicate that the footplate as such cannot be
regarded as part of the cartilaginous ear capsule.
The process whereby cartilage cells surrounding the fenestra ovalis can 'dedifferentiate' and then join the rudimentary stapes footplate as described for man
Columella-capsular interrelationship
269
by Bast & Anson (1949) and Hanson et al. (1962) cannot be verified by the
present experiments and it is also possible that the capsule merely induces the
full development of the footplate as in the amphibians that were investigated experimentally (Toerien, 1963).
Lateral Aspect
The absence of a tympanic membrane and quadrate, following extirpation of
the anterior neural folds, does not seem to have much effect on the development
of the lateral plate of the columella. The ventral (interhyal) process, however,
appears to be displaced more dorsally (Text-fig. 2).
The Auditory Capsule
The fenestra ovalis is found between the capsular cartilage superiorly and
anteriorly, the basal plate ventrally and the 'stapes inferior' ventro-posteriorly.
It was shown elsewhere (Toerien, 1965) that the 'stapes inferior' of Kunkel
(1912a) is not of capsular origin and therefore not homologous with the amphibian operculum. The fenestra is oval shaped with its long axis directed anteroposteriorly. The footplate of the columella fits closely into this opening (Textfig. 1).
-Aud./cap.
Proc. metat.
1 mm.
3. Graphic reconstruction of lateral view of auditory capsule in embryo of Chelydra
serpentina in which the development of a columella auris is experimentally suppressed.
H. cart, rem., cartilaginous remnants of hyoid branchial arch. Other abbreviations as in
Text-fig. 1.
TEXT-FIG.
270
M. J. TOERIEN
Fairly normal auditory capsules were found in a number of specimens of
which the posterior neural folds were removed. This was also found in Ambystoma by Horstadius & Sellman (1946). In the absence of a columella the
fenestra ovalis has the appearance of an irregular fissure (Text-fig. 3). A separate
(loose) footplate was not found in the absence of a columella. The irregularity
of the fenestra ovalis is apparently not caused by the effect of the removal of the
neural fold on the development of the capsule. In some specimens, where the
capsule is less complete but a columella present, the fenestra is normal. However,
the fenestra is sometimes entirely absent in the small vesicular capsules.
SUMMARY
1. As in the chicken and amphibia the presence of the auditory capsule in the
turtle is necessary for the full development of the medial end of the columella
auris.
2. The experimental evidence supports the more recent views obtained from
the study of normal series of chelonians that the footplate is not developed as a
separate entity from capsular cartilage.
3. The columella is virtually unaffected by the absence of the quadrate and
tympanic membrane.
4. The development of a normal fenestra ovalis appears to be dependant on
the presence of a columella.
RESUME
Recherches experimentales sur les relations mutuelles entre la capsule auditive
et la columelle chez la tortue Chelydra serpentina.
1. Comme chez le poulet et les Amphibiens, la presence de la capsule auditive
est necessaire, chez la tortue, au plein developpement de l'extremite mediane de
la columelle.
2. Les resultats experimentaux appuient les considerations les plus recentes
tirees de l'etude de series de Cheloniens, selon lesquelles la plaque stapediale ne
se developpe pas en tant qu'entite separee a partir du cartilage capsulaire.
3. L'absence du carre et de la membrane tympanique n'affecte virtuellement
pas la columelle.
4. Le developpement d'une fenetre ovale normale apparait comme dependant
de la presence de la columelle.
ACKNOWLEDGEMENTS
The experiments on which these results are based were carried out in the Department of
Anatomy, State University of New York, Upstate Medical College at Syracuse. I am most
grateful to Professor Chester L. Yntema who not only suggested the problem to me but supervised and assisted in its execution while I was a guest in his laboratory as International Postdoctoral Research Fellow of the National Institutes of Health.
Columella-capsular interrelationship
271
REFERENCES
BAST, T. H. & ANSON, B. J. (1949) The Temporal Bone and Ear. Springfield: Charles C. Thomas.
BENDER, O. (1911). t)ber Herkunft und Entwickelung der Columella auris bei Testudo graeca
Anat.Anz. 40, 161-77.
BENOIT, J. (1957). Sur un exemple de cascade d'inductions dans l'organogenese des vertebres:
la genese de l'oreille interne. Ann. Biol. Paris 33, 385-412.
DOHRER, J. (1919). Die Entwicklung der Paukentasche bei Chelydra serpentina. Morph. Jb.
50, 103-12.
FUCHS, H. (1907). Uber die Entwicklung des Operculums der Urodelen und des Distelidiums
(Columella auris) einiger Reptilien. Verh. anat. Ges. Jena. (Wiirzburg) 21, 8-34.
FUCHS, H. (1915). Uber den Bau unt Entwicklung des Schadels der Chelone imbricata.
Voeltzkow Reise Ostafrik. Stuttg. 5, 1-320.
HAMMOND, W. S. & YNTEMA, C. L. (1964). Depletions of pharyngeal arch cartilages following
extirpation of cranial neural crest in chick embryos. Ada. Anat. 56, 21-34.
HANSON, J. R., ANSON, B. J. & STRICKLAND, E. M. (1962). Branchial sources of the auditory
ossicles in man. Arche. Otolar. 76, 200-15.
S. (1950). The Neural Crest. London: Oxford University Press.
S. & SELLMAN, S. (1946). Experimented Untersuchungen iiber die Determination des knorpeligen Kopfskelettes bei Urodelen. Nova Acta R. Soc. Scient. upsal.
13/8,1-170.
KUNKEL, B. W. (1912a). On a double fenestral structure in Emys. Anat. Rec. 6, 267-80.
KUNKEL, B. W. (19126). The development of the skull of Emys lutaria. /. Morph. 23,693-780.
LUTHER, A. (1927). Entwicklungsmechanische Untersuchungen am Labyrinth einiger Anuren.
Commentat. biol. 2,1-4$.
MANGOLD, O. (1961). Molchlarven ohne Zentralnervensystem und ohne Ectomesoderm.
Arch. EntwMech. Org. 152, 725-69.
NOACK, H. (1907). Uber die Entwicklung des Mittelohres von Emys eropaea nebst Bemerkungen zur Neurologie dieser Schildkrote. Arch, microsk. Anat. EntwMech. 69, 457-90
(Vide Smith).
PARKER, W. K. (1880). Report on the development of the green turtle (Chelone viridis).
Report on scientific results of the Voyage ofH. M.S." Challenger" during the years 1875-76.
Zoology, 1. (Vide Smith)
REAGAN, F. P. (1915). A genetic interpretation of the stapes, based on a study of avian embryos
in which the development of the cartilaginous otic capsules has been experimentally
inhibited. Anat. Rec. 9, 114-5.
REAGAN, F. P. (1917). The role of the auditory sensory epithelium in the formation of the
stapedial plate. /. exp. Zoo I. 23, 85-108.
SHANER, R. F. (1926). The development of the skull of the turtle, with remarks on fossil
reptile skulls. Anat. Rec. 32, 343-67.
SMITH, L. W. (1914). The origin and Development of the columella auris in Chrysemys
marginata. Anat. Anz. 46, 547-60.
HORSTADIUS,
HORSTADIUS,
STRICKLAND, E. M., HANSON, J. R. & ANSON, B. J. (1962). Branchial Sources of Auditory
Ossicles in Man. Archs. Otolar. 16,100-122.
M. J. (1963). Experimental studies on the Origin of the Cartilage of the Auditory
Capsule and Columella in Ambystoma. J. Embryol. exp. morph. 11,459-73.
TOERIEN, M. J. (1965). An experimental approach to the development of the ear capsule in the
Turtle, Chelydra serpentina. J. Embryol. exp. Morph. 13,141-9.
VAN DER KLAAUW, J. C. (1924). Bau und Entwicklung der Gehorknochelchen. Ergebn. Anat.
EntwGesch. 25, 565-622.
VERSLUYS, J. (1936). Kranium und Visceralskelett der Sauropsiden H.B. vergl. Anat. Wirb. 4,
699-808 (ed. Bolk, Goppert, Kallius, Lubosch) Urban & Schwarzenberg, Berlin.
VIOLETTE, H. N. (1931). Experiments upon the middle ear of Rana. Thesis, Yale University.
TOERIEN,
272
M. J. TOERIEN
C. F. (1960). Das Gehororgan der Wirbeltiere unddes Mencken. Leipsig: Georg
Thieme.
YNTEMA, C. L. (1955). Ear and Nose. In Analysis of Development (ed. Willier, Weiss and Hamburger). Philadelphia and London: Saunders.
YNTEMA, C. L. (1964). Procurement and use of the turtle embryo for experimental procedures.
Anat. Rec. 149, 577-83.
WERNER,
{Manuscript received 10th June 1965)