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Patterns of Epithelial Cell Death during Early Development of the Human Inner Ear Juan J. Represa, Jose A. Moro, Francisco Pastor, Angel Gato and Enrique Barbosa Ann Otol Rhinol Laryngol 1990 99: 482 DOI: 10.1177/000348949009900613 The online version of this article can be found at: http://aor.sagepub.com/content/99/6/482

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Ann 0101 Rhinal Laryngol99: 1990

PATTERNS OF EPITHELIAL CELL DEATH DURING EARLY DEVELOPMENT OF THE HUMAN INNER EAR JUAN J. REPRESA, MD, PHD BROOKLYN, NEW YORK

JOSE A. MORO, MD, PHD

ANGEL GATO, MD

V ALLADOLID, SPAIN

V ALLADOLID, SPAIN

FRANCISCO PASTOR, MD

ENRIQUE BARBOSA, MD, PHD

V ALLADOLID, SPAIN

VALLADOLID, SPAIN

A light microscopic study of cell death in a developmental series of otic primordia from 23 human embryos (Carnegie stages 9 to 14) was completed. Degenerated cells were noted predominantly in the placode (stages 9 and 10), cup (stages 11 and 12), and otocyst (stages 13 and 14). A systematic camera lucida study of the appearance and topography of degenerating epithelial cells showed four different areas of cell death in the otic primordia that related to 1) invagination and detachment of the otic anlage, 2) early histogenesis of the statoacoustic ganglion, and 3) development of the endolymphatic duct. The possible role of cell death in the morphogenesis of the inner ear related to morphogenetic movements is discussed. KEY WORDS -

cell death, human embryo, inner ear, morphogenesis.

INTRODUCTION

bryology, Complutense University, Madrid, Spain. The material was classified according to the Carnegie stages devised by O'Rahilly," as summarized in the Table. The age of each embryo was estimated from the developmental stage according to the data of Olivier and Pineau. 27

Embryonic development encompasses not only the process of differentiation and growth but also cell death. Cell death is known to be involved in both normal and pathologic development. A detailed knowledge of the distribution and cytologic characteristics of the areas of em bryonic cell death is important for understanding normal and abnormal development.":"

HUMAN EMBRYOS USED IN PRESENT STUDY

Carnegie Stages'

Programmed cell death has been shown to be active in the regression of transient embryonic tissues and in those parts of the embryo undergoing morphogenetic movements such as folding, separation, and confluence of anlagen.":" These morphogenetic movements playa role in the formation of the inner ear of humans and other vertebrates during early development. 10-18

9 (2) 10 (3)

11 (3)

Human Embryo Designation GV CV-2 Ca Rm CANO OY-6

Ve-7 Be.

Areas of cell death have been reported in different primordia of the human embryo'r", however, their occurrence in the epithelial folds of the inner ear has received little attention. We previously have shown that programmed cell death occurs during early morphogenesis of the otic and olfactory plates of chick and mouse embryos.23-26 The aim of the present work was to study areas of cell death that occur during the early development of the human inner ear and to document their location, morphology, and evolution in order to assess their importance in this developing system.

12 (3)

13 (3)

14 (3)

MATERIALS AND METHODS

Ceo Ro FAUS-5 C-l GV-3 Pt-13 00-1 JD-l JD-2 Pt-8

OY-4 GV-5 Mu-3 VG 00-2

Pairs oj Somites

Length (mm)

3 6

2.0

9 12 14 16

2.4

20 20 24 26

27 30

3.0 4.0 4.5 4.8 4.9

5.0 5.3

5.0 6.0

7.0 6.4

7.0 7.0

Parentheses indicate number of otic primordia studied for obtaining cell counts.

This paper is based on the examination of human embryos from the Department of Anatomy and Em-

'Devised by O·Rahilly."

From the Department of Morphology, Faculty of Medicine, University of Valladolid, Valladolid, Spain. This work was partially funded by grants from the Spanier Ministry of Education and Science (04-8847) and the Fondo de Investigaciones Sanitarias (FISS 89/0301). REPRINTS - Juan J. Represa, MD, PhD, Dept of Anatomy and Cell Biology, Box 5, SUNY, Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203-2098.

482

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Represa et al, Cell Death in Otocysts

483

Fig 1. Schematic drawings taken from cross sections of otic primordia in human embryos. Black dots show location of dead cells and phagocytes. Age in postovulatory days is plotted on abscissa and embryonic length in millimeters on ordinate. Rectangles indicate Carnegie stages. (Drawings are not to scale.)

E E

z

4

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C) 3 Z W ...J

STAGE-9

20

21

22

23

26

25

AGE

28

29

30

31

32

IN DAYS

Twenty-three human embryos (Carnegie stages 9 to 14) were studied. The embryos were sectioned and stained for light microscopy. Seventeen specimens were stained with hematoxylin and eosin and the remaining six specimens with Mallory's or van Cieson's stain. Necrotic cells were observed under oil immersion with a 100/1.32 objective microscope and were identified on the basis of nuclear morphology using the criteria described by Clucksmann. 1 Serial sections of otic primordia were drawn with use of a camera lucida at x25, and the sites of cell degeneration were marked. Degenerating cells were counted in all sections. Serial and total counts of pyknotic figures of each stage are represented as averages of three otic primordia of different embryos, except for stage 9, in which both otic primordia were studied from the same embryo. Although no corrections were made to allow for splitting of degeneration sites between adjacent sections, the numbers obtained were comparable among different embryos. RESULTS

Early development of the human inner ear can be divided into stages based on the shape of the otic primordium. Initially, cephalic ectoderm forms a placode; subsequent invagination gives rise to a cup that closes, detaches from the surface, and forms a cyst. This simple pear-shaped otocyst in later development will be expanded into the membranous labyrinth. The studies reported here reveal that intense necrotic activity takes place during the developmental period extending from the placode stage to the otocyst stage of inner ear development. The pattern and number of necrotic sites throughout this period of otic morphogenesis are summarized in Fig 1. Otic Placode (Stage 9). The otic primordium of the stage 9 embryo is represented by a thickening of

the cephalic ectoderm adjacent to the hindbrain, which has become a three- or four-layered pseudostratified epithelium. The otic invagination has not yet developed. Cell degeneration is limited to a few pyknotic figures (18 ± 5 necrotic cells per rudiment) that are localized randomly in the border between the otic epithelium and the marginal cephalic ectoderm (Fig 2A). Early Otic Cup (Stages 10 and 11). The area of programmed cell death is detected in the otic epithelium at the beginning of its invagination (stage 10) as a single zone of cellular necrosis (97 ± 22 necrotic cells per rudiment) located at the edge of the lateral and caudal folds of the invaginating otic cup; medial and cranial portions remain free of cell degeneration. The appearance of the otic anlage at the beginning of stage 11 (Fig 2B) is similar to that of stage 10. The distribution and number of degenerating cells (119 ± 29 necrotic cells per primordium) have not changed significantly, and the otic epithelium maintains a steady rate of programmed cell death. Concurrently, several mitotic figures are present, homogeneously scattered around the lumen of the otic epithelium (data not shown). Late Otic Cup (Stages 11 and 12). During the end of stage 11 and most of stage 12, further invagination of the otic primordium transforms the cup into a vesicle. This vesicle remains open until the end of stage 12 and maintains communication with the exterior (amniotic cavity) through the otic pore. There is a second and significant change in the morphology of the otic rudiment during stages 11 and 12. Some of the epithelial cells of the ventral wall of the cup migrate across the basement membrane into the underlying ventral periotic mesenchyme and give rise to the primordium of the stato-

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Represa et al, Cell Death in Otocysts

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Fig 2. A) Micrograph of frontal section through external edge of otic placode in later stage 9 embryo (H & E, original x400). Few degenerating cells are present at border (arrowheads) between otic epithelium and cephalic ectoderm. B) Transverse section of stage II embryo through anterior portion of external edge of otic cup (H & E, original xl,OOO). Some degenerating cells and associated cellular debris (arrows) are present in epithelial layer. C) Transverse section through otic cup of human embryo at stage 12 (Mallory's stain, original xI60). Arrow shows location of programmed cell death in otic primordia at this developmental stage. D) Lowpower view of stage 12 otocyst (ov) that appears closed but remains connected to cephalic ectoderm (ce) (H & E, original xlOO). E) Higher-power view of otocyst shown in D (H & E, original xl,OOO). Note many dead or dying cells (arrows) located in zone of fusion and closure.

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acoustic ganglion. While this is occurring in the region of the otic epithelium, where cells of this epithelium are migrating away to form the eighth sensory cranial ganglion, the thickness of the epithelium increases considerably, the cells are clearly disorganized, and the basal lamina seems to be discontinuous (Fig 3D). At the same time several mitotic figures can be seen among ganglionic and otic cells. Cellular degeneration in this later cup stage is localized in two distinct areas. The first zone (which started during the previous stage) is in the lateral and caudal folds of the otic cup (Fig 2C), where it forms an extensive area of cell death (190±37 pyknotic sites per otic cup) at the end of stage 11. By stage 12 this region of necrosis encircles the otic pore (243 ± 34 pyknotic sites per otic cup).

The second zone of cell death is limited to the ventral otic epithelium overlying the area of the developing primordium of the statoacoustic ganglion. This new locus of cell death, the ventral otic cup pyknotic zone, makes its first appearance at the end of stage 11 (57 ± 14 pyknotic sites per otic cup) as a single wave of degeneration that increases considerably during stage 12 (141 ± 32 pyknotic sites per primordium) and continues as a band of pyknotic cells in the most ventral wall of the otic cup and in the statoacoustic ganglion primordium, where cellular degeneration is scattered. Early Otocyst (Stages 12 and 13). During the end of stage 12, growth of the otic walls brings the edges of the otic pore into close apposition and eventually they fuse. But the newly closed otocyst remains connected to the surface ectoderm via a small epithelial stalk (Fig 2D). At stage 13 the insertion of the otic

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Represa et at, Cell Death in Otocysts

485

Fig 3. A) Low-power view of stage 13 otocyst (ov) already detached from cephalic ectoderm (ce) and appearing embedded in cephalic mesenchyme close to neural tube (nt) (Mallory's stain, original xlOO). Note zone on lateral wall of otocyst displaying remains of connecting stalk (between arrowheads). B) Higher-power view of remains of connecting stalk in A (Mallory's stain, original x1,OOO). Arrowheads - dead cells at closure point and connecting stalk of otocyst. C) Frontal section through otocysts at early otic vesicle stage (stages 12 and 13) showing morphology of periotic head region during this period (H & E, original x25). nt - neural tube, ov - otocyst, sag - statoacoustic ganglion, tg - trigeminal ganglion. D) Higher magnification of ventral wall of otocyst (ov) and statoacoustic ganglion (sag) at stage 12 (H & E, original x250). Cell degeneration and epithelial disorganization are found in this region of otocyst's wall. E) Higher-power view of wall of otocyst at stage 12 (H & E, original xl,OOO). Note number of dead or dying cells (arrowheads) located in ventral epithelium of otocyst next to otic ganglion.

stalk on the otocyst is interrupted as this vesicle finally detaches from the cephalic ectoderm and becomes a closed cyst surrounded by cephalic mesenchyme (Fig 3A). Histologic sections reveal the otocyst to be a simple pear-shaped vesicle with significant differences in the thickness of its walls. The dorsal portion of the otocyst thins into a simple cuboidal epithelium, while the ventral portion remains multilayered. The statoacoustic ganglion has already formed (Fig 3C) . At stage 12, the closure of the otic pore is accompanied by cell death. A wide necrotic area appears in this zone of epithelial fusion (286 ± 64 pyknotic cells per otocyst), overlapping and extending the cell death area in the otic folds that accompanied the process of invagination during the previous stages. At the present stage, most necrotic cells appear to be located in the epithelial stalk, the adjacent cephalic ectoderm, and the superficial otic epithelium (Fig 2D,E). Some dying cells were also found in the space between the ectoderm and the epithelium of the otocyst's wall.

Just after the otocyst has detached from the cephalic ectoderm (stage 13), the necrotic area is similar to that found in the preceding stage (Fig 3A,B), in which all degeneration sites are concentrated at the vicinity of the closing point in the otic epithelial wall, in the cephalic ectoderm (245 ± 67 pyknotic cells per otocyst), or in between these two epithelia. By the end of stage 13 the amount of cellular necrosis is notably reduced (88 ± 33) in the above-mentioned locations, and by stage 14 the amount of pyknosis at these sites is insignificant. The ventral pyknotic zone follows a similar time course during these early otocyst stages. It intensifies while the statoacoustic ganglion is forming and reaches its maximum (187 ± 26) by the end of stage 12 (Fig 3D,E). Pyknosis then diminishes along this ventral wall (74 ± 26 pyknotic cells per otocyst), and by the end of stage 13 no cellular degeneration is detected in the ventral wall of the otocyst.

Late Otocyst (Stages 13 and 14). From the end of stage 13 onward, a complex series of epithelial

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Fig 4. A) Micrograph of right otocyst (ov) of stage 14 embryo (H & E, original xlOO). Emerging endolymphatic duct (ed) is observed in form of invagination of dorsal wall. Zone between arrowheads (Anson's fold) displays normally occurring cellular degeneration. nt - neural tube, cd - cochlear duct. B) Higher-power view of Anson's fold shows localization of dead and dying cells (arrowheads) (H & E, original xl,OOO).

movements begins to transform the otocyst into the intricate structure of the mature membranous labyrinth. By the end of stage 13 or at the beginning of stage 14, the otocyst has begun its morphogenesis. From the medial-dorsal face of the otocyst a hollow tube, the endolymphatic duct, has grown out as a prolongation of the otic cavity. In the middle of stage 14 this endolymphatic duct has elongated and projects dorsally (Fig 4A). At this developmental stage an epithelial fold (Anson's fold) can be distinguished that serves as a marker to define the insertion of the lateral aspect of the endolymphatic duct on the dorsal wall of the otocyst (Fig 4A). At the site of Anson's fold a small band of cell death is observed (48 ± 11 necrotic sites per otocyst) that surrounds the insertion of the endolymphatic appendage from the outer (early stage 14) to the inner walls (later stage 14) of the endolymphatic duct (Fig 4B). At the end of stage 14 the rudiments of the semicircular canals and the coiled cochlear tube start to bulge out from the otocyst. In all these remaining portions of the otic epithelium we found no pyknotic cells. DISCUSSION

In the human embryo, programmed cell death takes place during the formation of the otocyst. Cell death seems to be a constant feature in this early development of the otic primordium. Programmed cell death shows a typical morphologic pattern in all embryos at the same stage and seems to relate to invagination and detachment of the otic anlage, early histogenesis of the statoacoustic ganglion, and growth of the endolymphatic duct. Our findings suggest four distinct temporospatial patterns of cell death during early development of the inner ear (Fig 1).

An early phase extends from the time of otic placode formation (stage 9) to the period of placodal folding (stages 10 and 11). During this developmental period, necrotic cells and cellular debris are found aggregated at the edges of the otic placode simultaneously with the formation of the otic folds. The present investigation in the human inner ear thus confirms the observation that cell death is present during and correlates with bending of several ectodermal rudiments such as the optic placodes during the formation of the lens" or the neural plate.!" The role of cell death during these morphogenetic movements remains mostly a matter of conjecture. Our observations suggest that cell death occurs simultaneously with invagination movements and proliferative activity in the otic epithelium, which has been shown in the rat and other mammals to have a higher mitotic index than cephalic ectoderm. The loss of a number of cells in restricted areas of rapidly growing tissue may have profound effects on its morphologic development, permitting lateral cell displacement into adjoining regions and therefore leading to the process of folding.s'" Furthermore, examination of the degeneration pattern in the embryonic human inner ear reveals that pyknotic cells are concentrated in those portions of the otic primordium in which invagination is more pronounced. Cell death at the edges of the otic placode may be interpreted as morphogenetic degeneration, according to the terminology of Saunders and Fallon, 5 that might be involved in the invagination and folding of the otic placode and playa mechanical role as has been described for the formation of several embryonic primordia. Nevertheless, as has been reported for these other primordia, other mechanisms are concurrently involved in the invagination of the

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Represa et al, Cell Death in Otocysts

otic placode, including interactions with the extracellular matrix or the action of cytoplasmic structures within the otic epithelial cells - for instance, contraction of an apical terminal bar network of microfilaments." At stages 12 and 13, a new area of cell death appears during the closure of the otic vesicle by fusion of the epithelial cells that form the otic pore and during its detachment from the cephalic ectoderm. This area of cell degeneration seems to be required to obliterate the lumen of the otic pore and to separate the otocyst from the surface ectoderm. Previous reports have already associated cell death with the process of epithelial fusion. Localized cell degeneration has been shown to be present during closure of the neural tube;"':" during fusion of both the palatal shelves" and facial prominences," and in detachment of the lens from the surface ectoderm of the head. Previous work" reported localized cell degeneration in the connecting stalk of the otocyst of the rat embryo and suggested that detachment of the otic primordium from the cephalic ectoderm occurs via genetically controlled cell death. Our results in the human embryo are in agreement with such a hypothesis. In summary, the morphogenesis of the human inner ear between stages 9 and 12 progresses through a complicated but orderly maze of linked epithelial cell movements. Cell death, invagination, vesicle formation, and ectodermal separation appear to be associated in a temporal and spatial relationship. Qualitatively, these results resemble those described for other embryonic anlagen, suggesting that programmed cell death in the developing inner ear may playa similar role. There is a third area of cell degeneration that occurs throughout the period of the statoacoustic ganglion formation (stages 11 and 13). This area of cell death is found in the medial-ventral wall of the otocyst overlying the developing statoacoustic ganglion. An in vitro fate map of the mouse otocysr" showed that the medial-ventral wall of the otic vesicle gives rise to the neuroblasts of the statoacoustic ganglion. The neurons that compose this cranial ganglion in the chick embryo have been shown to be

487

primarily of placodal origin, with a minimal contribution of neural crest cells forming a few vestibular neurons.P':'" Furthermore, the times of terminal mitosis documented for these otocyst-derived neurons and the time of greatest cell division activity in the otic epithelium 34.35 coincide with the period of cell degeneration in the ventral-medial wall of the otocyst. Therefore, the third cell death area observed in the ventral otic epithelium might represent histogenetic degeneration" related to neuroblast proliferation, migration, and differentiation in the formation of the statoacoustic ganglion. Perhaps some of the cells remaining in the ventral wall of the otocyst lack trophic signals for migration and differentiation and consequently degenerate, forming the ventral pyknotic zone of the otic primordium. The role of cell death in neuronal development has been chiefly considered in terms of target-dependent cell degeneration. 37-39 Since our results have documented the presence of degenerating cells at a time well before axonal or dendritic outgrowth and synaptic junctions occur in the developing inner ear, 40 this causal relationship can be excluded in the present case. Finally, the fourth area of cell death described here corresponds to the development of the endolymphatic duct and sac anlage from the dorsal wall of the otocyst. It previously has been shown in other vertebrates that the growth of the endolymphatic primordia takes place by cellular reorganization rather than proliferation and that there is a low mitotic activity in the endolymphatic wall, while proliferation is increased in the rest of the otic epithelium.18.25.26 The degenerating and necrotic cells in the endolymphatic duct next to Anson's fold may represent a barrier between two cell populations that are known to have different mitotic patterns. 25.26.41 The fact that no cell degeneration could be found in the early developing semicircular canals or cochlear duct suggests that cell death does not participate in their formation at this early developmental period of human inner ear organogenesis. However, it might be possible that cell death is present at older stages in the above-mentioned locations, as has been reported in rat embryo inner ears.:"

ACKNOWLEDGMENTS - We thank the members of the Department of Anatomy and Embryology, Complutense University. Madrid, Spain, for advice during the examination of the human embryos. We also thank Professor Giraldez (Valladolid, Spain) and Professor John Lewis (New York. NY) for critical reading of the manuscript.

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Patterns of epithelial cell death during early development of the human inner ear.

A light microscopic study of cell death in a developmental series of otic primordia from 23 human embryos (Carnegie stages 9 to 14) was completed. Deg...
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