The membranous labyrinth during larval development in lamprey (Lampetra planeri, Bloch, 1784)

www.elsevier.com/locate/heares

Hearing Research 201 (2005) 37–43

The membranous labyrinth during larval development inlamprey (Lampetra planeri, Bloch, 1784)

Bice Avallone a, Umberto Fascio b, Angela Senatore a, Giuseppe Balsamo a,Pier Giorgio Bianco c, Francesco Marmo a,*

a Department of Genetics, General and Molecular Biology, University of Naples ‘‘Federico II’’, Naples, Italyb CIMA, University of Milan, Milan, Italy

c Department of Zoology, University of Naples ‘‘Federico II’’, Naples, Italy

Abstract

SEM and CLSM studies were performed on the membranous labyrinth of Lampetra planeri, a threatened species of brook lam-

prey, spanning from the 1st to the 4th year of ammocoetes larval stages and on the adults. In all the examined stages, the entire

membranous labyrinth does not show any morphologic differences, but only a progressive increase in size. SEM and CLSM obser-

vations show that the ciliated chamber is lined with numerous unsensorial multiciliated cells. In the early stages, the ciliary bundles

were approximately 15 lm long, while in the late stages they reached 30 lm. In the crista sensory area, we observed two populations

of hair cells. ‘‘Type II’’ cells are peculiar for this species and show both long stereocilia decreasing in length and a long kinocilium

(10–12 lm). Two other types of ciliary bundles have been found on the sensory hair cells of the Macula communis: the first one has

both kinocilium and stereocilia about 4–5 lm long; the second shows a long kinocilium (7–10 lm in length) and short stereocilia

bundles with a gradual increase in length. In the early stages of development, the three macular areas show few and sparsely dis-

tributed hair cells. In the late developmental stages, hair cells become more numerous and densely populated.

� 2004 Published by Elsevier B.V.

Keywords: Lampetra planeri; Brook lamprey; Membranous labyrinth; Hair cells; Macula communis; Ciliated chamber

1. Introduction

In general, the Agnatha morphology of the ears dif-

fers from the one of the other vertebrate gnathostomata

0378-5955/$ - see front matter � 2004 Published by Elsevier B.V.

doi:10.1016/j.heares.2004.09.002

Abbreviations: CLSM, confocal laser scanning microscope; DAB-

CO, Diazabicyclo-octane; FITC, fluorescein isothiocyanate; MCE,

multiciliated epithelial cells; MS 222, ethyl 3-aminobenzoate; OsO4,

osmium tetroxide; PBS, phosphate buffered saline; SEM, scanning

electron microscope; TEM, transmission electron microscope; TRI-

TC, tetramethylrhodamine isothiocyanate* Corresponding author. Tel.: +39 81 2535012/+39 81 2535006; fax:

+39 81 2535000.

E-mail addresses: [email protected] (B. Avallone),

[email protected] (F. Marmo).

(de Burlet and Versteegh, 1930; Hagelin, 1974; Hosh-

ino, 1975; Lowenstein et al., 1968; Popper and Hoxter,

1987; Retzius, 1881; Thornhill, 1972). It is well known

that the inner ear of the lampreys has two semicircular

canals and a single elongate epithelium, the Macula

communis, instead of the three semicircular canals and

three distinct sensory epithelia of gnathostomes. TheM. communis is covered by a mass of calcareous oto-

liths (Carlstrom, 1963; Fermin et al., 1998; Lowenstein

et al., 1968). These crystalline spherules are held to-

gether by a fibrous ground substance (Lowenstein

et al., 1968).

A few studies about the inner ear petromyzontids

have been done. The ear of the ammocoetes larva of

Lampetra fluviatilis was studied by a light microscope

38 B. Avallone et al. / Hearing Research 201 (2005) 37–43

and a transmission electron microscope (TEM)

(Lowenstein et al., 1968; Thornhill, 1972). They have

pointed out in the sensory epithelium of cristae and

maculae two types of hair cells, called ‘‘Type I’’ or

‘‘F3’’. They are characterized by the kinocilium being

just a little longer than the adjacent stereocilia. ‘‘TypeII’’ or ‘‘F2’’ showed the kinocilium approximately 10

lm long and was straight and stiff; the stereocilia were

strikingly short in comparison with the kinocilium.

The sensory epithelium of the otolithic maculae of

the Japanese lamprey, Entosphenus japonicus, was

examined using a scanning electron microscope

(SEM) and a TEM, (Hoshino, 1975). He showed

two distinct types of sensory hair cells: ‘‘type A’’had a bundle of 30–40 stereocilia with an organ pipe

configuration and a kinocilium generally not distinctly

discerned from amongst the longest stereocilia;

‘‘type B’’ had stereocilia of approximately uniform

heights (ca. 1 lm) and a very long and evident

kinocilium.

Popper and Hoxter (1987) used a SEM and a TEM

to investigate the inner ear of the sea lamprey, Petromy-

zon marinus. Two types of ciliary bundles were found on

the cristae as well as on the M. communis: one bundle,

‘‘F2’’, which was similar to the ‘‘F2’’ bundle of bony

fish (Popper, 1977) and ‘‘type B’’ (Hoshino, 1975),

had a long kinocilium and relatively short stereocilia.

The ‘‘F3’’ bundle which was similar to ‘‘type A’’ (Hosh-

ino, 1975) had a graded series of stereocilia and a kino-

cilium that was just longer than the longest of thestereocilia.

The present study was designed to provide a descrip-

tion of membranous labyrinth during larval develop-

ment in petromyzontids L. planeri, a threatened

species of sandy brook lamprey. This primitive verte-

brate have a larva, named ammocoetes, for a term of

four years. Each larval stage lasts for a period of one

year. In order to determine possible differences betweenammocoetes larval stages and in an attempt to elucidate

the different types of sensory cells found on the maculae,

we have carried out studies on the membranous laby-

rinth of L. planeri ammocoetes, spanning from the 1st

year to the 4th year of larval stages, and of adults, with

a SEM and a confocal laser scanning microscope

(CLSM).

2. Materials and methods

Studies were carried out on five specimens for each of

the ammocoetes larval and adult stages of L. planeri.

Specimens were collected in a brook of ‘‘Parco Nazio-

nale degli Abruzzi’’ (Italy). Animals were deeply anaes-

thetized in ethyl 3-aminobenzoate (MS 222) (Sigma, St.Louis, MO, USA), sacrificed, and the cartilaginous otic

capsule was exposed.

2.1. Confocal Laser Scanning Microscope

For each of the ammocoetes larval and adult stages,

the cartilaginous otic capsules were exposed to reveal

the membranous labyrinth. These were fixed in metha-

nol and the otoliths were removed. The membranouslabyrinth was hydrated in a graded sequence of metha-

nol to H2O and then treated with 0.25 Triton X-100

and 0.1% Tween 20 in 0.1 M PBS at pH 7.4 for 1 h.

After several rinses in 0.1 M PBS at pH 7.4, the speci-

mens were incubated overnight at 4 �C, using primary

antibodies against b-actin FITC conjugated (mono-

clonal from mouse clone 1A4; 1:500) (Sigma) and

against a-tubulin (clone B 5-1-2; 1:500) (Sigma). Afterrinsing in the same buffer (6 · 10 min), the specimens

were incubated using anti-mouse IgG TRITC (from

goat; 1:400) in 0.1 M PBS at pH 7.4 for 2 h at room tem-

perature. Both steps were carried out in a moist and

dark chamber. They were rinsed again several times in

0.1 M PBS at pH 7.4, then the specimens were mounted

on slides with diazabicyclo-octane (DABCO) (Sigma).

Preimmune sera, instead of specific antisera, were usedin the control. Finally, fluorescence observations were

carried out by a confocal microscope (Leica TCSNT)

with laser argon–krypton 7–5 mW multilines. Focal ser-

ies of horizontal planes of sections were simultaneously

monitored using the 488- and 568-nm laser line and, for

FITC, band-pass 530/30 filter and a long-pass 590 filter

for TRITC. A Leica TCSNT confocal microscope was

equipped with an AOTF filter. This filter was necessaryto minimize cross-talk during image acquisition with a

simultaneous detection of FITC/TRITC labels.

2.2. Scanning Electron Microscope

The cartilaginous otic capsules were fixed in 2.5% glu-

taraldehyde in 0.1 M PBS at pH 7.4 for 3 h at 4 �C.After microdissection of the membranous labyrinth,the specimens were rinsed in PBS and post-fixed in 1%

OsO4 in the same buffer for 1 h at 4 �C. After several

rinses in PBS, the specimens were dehydrated in a

graded sequence of ethanol–Freon mixture to 100%

Freon, then the critical point was dried. Specimens were

mounted on aluminium stubs, coated with gold for

about 30 s at a distance of 15 cm from the source, at

40 kV and 2–3 mA. The observations were carried outwith a Cambridge Stereoscan 250 MK III Microscope.

3. Statistical analysis

Thirty images of hair cells for each stage of develop-

ment of each macula were used for quantitative analysis.

Statistical analysis was performed with a single factorialanalysis of variance (ANOVA). A P value of less than

0.01 was considered to be significant.

B. Avallone et al. / Hearing Research 201 (2005) 37–43 39

4. Results

The membranous labyrinth did not show morpho-

logic differences among all the stages examined, but only

a progressive increase in size (Fig. 1(a) and (b)). The

membranous labyrinth changed in width from the 1styear (0.32 ± 0.017 cm) to the adult stage (0.57 ± 0.014

cm) and increased in height up to 0.36 ± 0.017 cm in

the adult stage (Fig. 2).

SEM observations showed that, in all the examined

ammocoetes larval and adult stages, the ciliated cham-

ber (Fig. 1(c)) confined only to the labyrinth of the pet-

romyzontes, is lined with numerous multiciliated

Fig. 1. (a) Membranous labyrinth of ammocoetes at the 1st year of the lar

(b) Membranous labyrinth of ammocoetes at the 4th year of the larval stage.

chamber of ammocoetes at the 4th year of the larval stage, which is lined with

a basal lamina and vary in shape from cuboid to columnar; SEM micrograph.

The ciliary bundles of MCE cells are approximately 15 lm long. Microvill

(f) Ciliated chamber of ammocoetes at the 4th year of the larval stage. The

epithelial cells (MCE according to Popper and Hoxter,

1987). These cells rested directly on a basal lamina and

varied in shape from cuboid to columnar (Fig. 1(d)).

Each MCE cell showed a ciliary bundle projecting from

an apical surface. Microvilli covered the epithelium sur-

rounding the MCE cells and also laid between the ciliarybundles (Fig. 1(e) and (f)).

In the early stages, the ciliary bundles of MCE cells

were approximately 15 lm long (Fig. 1(e)), while in late

stages they reached about 30 lm (Fig. 1(f)). MCE cells

seemed unsensory, in fact CLSM observations showed

a red stain that only revealed the presence of tubulin

(Fig. 7(a)–(c)).

val stage. The arrows indicate where the ciliated chamber is located.

The arrows indicate where the ciliated chamber is located. (c) Ciliated

numerous MCE cells; SEM micrograph. (d) MCE cells rest directly on

(e) Ciliated chamber of ammocoetes at the 2nd year of the larval stage.

i cover the epithelium surrounding the MCE cells; SEM micrograph.

ciliary bundles of MCE cells reach about 30 lm; SEM micrograph.

Labyrinth's increase

0

0,2

0,4

0,6

1st year 2nd year 3rd year 4th year Adult

incr

aese

(cm

)

width

height

Fig. 2. The progressive increase of membranous labyrinth in size at

different stages of development. Values represented means ± SE.

40 B. Avallone et al. / Hearing Research 201 (2005) 37–43

Each Crista ampullaris (Fig. 3(a)) consisted of three

arms oriented along different planes of spaces (Fig.

3(b)). In the crista sensory area, we observed two dis-

tinct populations of hair cells characterized by the mor-phology of their sensory hairs. The ‘‘type I’’ cells (Fig.

3(c)) had a kinocilium (usually 4–5 lm in length), and

the kinocilial bulb was moderately large; the stereovilli

were very short (0.5–1 lm). The ‘‘type II’’ cells (Fig.

3(d)) were peculiar for this species and showed both long

stereovilli, decreasing in length (organ pipe configura-

tion) and a long kinocilium (10–12 lm), not distinctly

discerned from amongst the longest stereovilli. CLSMobservations showed that each stereovillus contained a

number of microfilaments, which basically continued

Fig. 3. (a) Crista ampullaris of ammocoetes at the 3rd year of the larval stage

along different planes of spaces. (c) Crista ampullaris ‘‘type I’’ cells show a ki

and a very short stereovilli (�0.5–1 lm); SEM micrograph. (d) ‘‘Type II

configuration) and a long kinocilium (10–12 lm), not distinctly discerned fr

into the cuticolar plate in the apical part of the hair cell

(Fig. 7(d)).

The hair cells were surrounded by supporting cells;

their apical surface was covered by microvilli. The pres-

ence of two types of hair cell has been confirmed by

CLSM analyses that have also indicated that both typesof cells were present in the early stages of larval develop-

ment (Fig. 7(e)), while in the ammocoete at the 4th year

of the larval and adult stages the ‘‘type II’’ cells were

prevalently present (Fig. 7(f)).

SEM observations revealed that the M. communis

occupied the ventro-medial floor of the labyrinth and

comprised three well defined macular areas lying in dif-

ferent planes of space: the anterior horizontal macula,the vertical macula, and the posterior horizontal macula

(Fig. 4) corresponding to saccule, utricle, and lagena

maculae (in agreement with Lowenstein et al., 1968).

In the literature, the nomenclature of the otolithic mac-

ulae in lamprey was based on a topographical view of

the lamprey labyrinth. We have also distinguished on

the ground of the resemblance between the types of cells

observed in the three macular areas and those that wehave found in numerous groups of vertebrate.

SEM observations, proved by CLSM images, pointed

out that in the three macular areas, in early stages (1st

and 2nd year larval stage) there were very few and

spaced sensory cells. Moreover, these were separated

from one another by numerous supporting cells (Fig.

5(b), (e), and (h)). In the following larval stages, these

; SEM micrograph. (b) Crista ampullaris consists of three arms oriented

nocilium (usually 4–5 lm in length), a moderately large kinocilial bulb,

’’ cells show both long stereovilli, decreasing in length (organ pipe

om amongst the longest stereovilli; SEM micrograph.

Fig. 4. Drawing of the three maculae in the M. communis.

B. Avallone et al. / Hearing Research 201 (2005) 37–43 41

sensory cells progressively increased and, in the

ammocoete at the 4th year larval and adult stages an ex-

panse of sensory cells in which each sensory cell was sur-

rounded by supporting cells was present (Figs. 5(c), (f)and (i); 7(g)–(i)).

Fig. 5. (a) Hair cells of macula lagenae with a bundle of stereovilli and a kino

from amongst the stereovilli. (b) Macula lagenae: In ammocoete at the 2nd y

are separated from one another by numerous supporting cells. (c) Macula lag

sensory cells in which each sensory cell is surrounded by supporting cells is

kinocilium (approximately 7–10 lm long) and very short stereovilli of graded

stage, there are very few and spaced sensory cells and these are separated from

small hair bundles, probably immature (arrows). (f) Macula sacculi: The adu

cells of macula utriculi are characterized by a long kinocilium (7–10 lm in

(h) Macula utriculi: Hair cells of ammocoetes at the 1st year of the larval sta

(arrows). (i) Macula utriculi: In the ammocoete at the 4th larval stage an

supporting cells is present; SEM micrographs.

Fig. 6 shows the mean number of hair cells for each

stage of development and for each macula ± SD. The

significance of differences was evaluated at P < 0.01

(ANOVA).

In the posterior horizontal macula (macula lagenae)

cells were present (Figs. 5(a) and 7(g)) with a bundleof stereovilli and a kinocilium (usually 4–5 lm in length)

generally not distinctly discerned from amongst the ster-

eovilli. This type of sensory cell seemed peculiar for this

species. In the anterior horizontal (macula sacculi) and

in the vertical macula (macula utriculi) the population

of hair cells (Figs. 5(d) and (g) and 7(h) and (i)) ap-

peared to be characterized by a long and evident kinocil-

ium (approximately 7–10 lm long) and stereovilli, very

cilium (usually 4–5 lm in length) are generally not distinctly discerned

ear of the larval stage, there are few and spaced sensory cells and these

enae: In the ammocoete at the 4th year of the larval stage an expanse of

present. (d) Hair cells of macula sacculi are characterized by a long

heights. (e) Macula sacculi: In ammocoete at the 2nd year of the larval

one another by numerous supporting cells. There are present cells with

lt hair cells appear to be close to one another and numerous. (g) Hair

length) and short stereovilli bundles with a gradual increase in length.

ge. There are cells with small hair bundles present, probably immature

expanse of sensory cells in which each sensory cell is surrounded by

Fig. 7. Antibodies against b-actin (green color), antibodies against a-tubulinlarval stage. (b) Ciliated chamber of ammocoetes at the 2nd year of the larva

(c) Ciliated chamber of ammocoetes at the 4th year of the larval stage. The c

crista. Stereovilli contain a number of microfilaments, which basically contin

at the 2nd year of the larval stage. Both ‘‘type I’’ (arrow head) and ‘‘type II’’ c

adult. The ‘‘type II’’ cells are prevalently present. (g) Macula lagenae of adult

present. (h) Macula sacculi of ammocoetes at the 4th year of the larval stage

heights present. (i) Macula utriculi of adult. There are cells with a long kino

increase in length present.

Fig. 6. The number of hair cells at different stages of development for

each macula. Values represented means ± SD. Significance of differ-

ences was evaluated at P < 0.01 (ANOVA).

42 B. Avallone et al. / Hearing Research 201 (2005) 37–43

short in comparison with the kinocilium, of graded

heights; were similar to ‘‘type B’’ or ‘‘II’’ according to

other authors (Hoshino, 1975; Lowenstein et al.,

1968). Sometimes, cells were present with small hair

bundles, probably immature (Avallone et al., 2003;

Bhave et al., 1995) (Fig. 5(e) and (h)).The CLSM observations showed no fluorescence in

all negative controls.

5. Discussion

The present study provides the first CLSM descrip-

tion of the ear�s sensory epithelia of brook lamprey.CLSM observations show a red stain level of cilia that

(red color). (a) Ciliated chamber of ammocoetes at the 1st year of the

l stage. The red stain of MCE cells only reveals the presence of tubulin.

iliary bundles of MCE cells reach about 30 lm. (d) Sensory area of the

ue into the cuticolar plate. (e) Sensory area of the crista of ammocoetes

ells (arrow) of hair cells are present. (f) Sensory area of the crista of the

. There are cells with both kinocilium and stereovilli about 4–5 lm long

. There are cells with a long kinocilium and short stereovilli of graded

cilium (7–10 lm in length) and short stereovilli bundles with a gradual

B. Avallone et al. / Hearing Research 201 (2005) 37–43 43

only reveals the presence of tubulin, so the MCE cells

are epithelial. SEM observations point out that their ci-

lia bundles have projected into the otic chamber with the

same orientation. According to Lowenstein et al. (1968),

MCE cells keep up vigorous vortices of endolymph with

a constant orientation. Although the ciliated chambersare in open communication with the other labyrinth

spaces, de Burlet and Versteegh (1930) stated that the

endolimph vortices do not transgress beyond the ciliated

chambers. The fluid motion might function as part of a

vestibular system in lieu of a semicircular canal (de Bur-

let and Versteegh, 1930; Mygind, 1948). However, in

hagfish (Myxiniformes) MCE cells are not present

though they only have one semicircular canal (Jørgensenet al., 1998; Lowenstain and Thornhill, 1970; Retzius,

1881). In spite of this, the single canal systems of hagfish

respond to stimuli in all planes (Lowenstain and Thorn-

hill, 1970). It is possible to believe that the flow of endo-

lymph with a constant orientation might avoid the

displacement of the numerous small otoconia from M.

communis. This function is probably necessary because

of the primitive organization of lamprey�s M. comunis.In all the L. planeri stages examined, the whole mem-

branous labyrinth does not show morphologic differ-

ences. It must be borne in mind that the ammocoetes

at the 1st larval stages are one year old and, therefore,

it is logical to think that the two semicircular canals with

the ampullae and the M. communis are already present.

During larval development, there is only a progressive in-

crease in size of membranous labyrinth. In SEM obser-vations, the presence of cells with small hair bundles,

probably still immature or new, as well as the progressive

increase of sensory cell numbers indicate that, during

development, there is, simultaneously, both a growth

and an acquisition of maturity of the sensory area.

According to Hardisty (1979), Jarvik (1980) and Ro-

mer (1966), petromyzontids and gnathostomes separated

from a common ancestor sometime in the early silurianperiod. In the inner ear, the presence of cells with com-

mon features in the lamprey and in numerous groups

of vertebrate suggests convergent evolution. We, who

are in agreement with Popper and Hoxter (1987), may

say that the common ancestor probably already had an

inner ear with otoliths as well as sensory cells with a char-

acteristic orientation pattern, and so its inner ear did not

differ greatly from those in modern vertebrates.Furthermore, in this species of lamprey we have ob-

served a peculiar variety of sensory cell types. It is pos-

sible to state that the observed cytoarchitectonic type

corresponds to a common model for all the petromyzon-

tes, even if the size and the ratio between kinocilia and

stereovilli in sensory cells may show variations.

Acknowledgements

SEM studies were carried out at the Centro di Servi-

zio di Microscopia Elettronica (CISME) of the Univer-

sity of Naples Federico II. The authors thank the staff

for their assistance.

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