Growth and cellular patterning during fetal human inner ear development studied by a correlative imaging approach

Abstract

Background: Progressive transformation of the otic placode into the functional inner ear during gestational development in humans leads to the acquisition of hearing perception via the cochlea and balance and spatial orientation via the vestibular organ.

Results: Using a correlative approach involving micro-computerized tomography (micro-CT), transmission electron microscopy and histological techniques we were able to examine both the morphological and cellular changes associated with human inner ear development. Such an evaluation allowed for the examination of 3D geometry with high spatial and temporal resolution. In concert with gestational progression and growth of the cochlear duct, an increase in the distance between some of the Crista ampullaris is evident in all the specimens examined from GW12 to GW36. A parallel increase in the distances between the macular organs - fetal utricle and saccule - is also evident across the gestational stages examined. The distances between both the utricle and saccule to the three cristae ampullares also increased across the stages examined. A gradient in hair cell differentiation is apparent from apex to base of the fetal cochlea even at GW14.

Conclusion: We present structural information on human inner ear development across multiple levels of biological organization, including gross-morphology of the inner ear, cellular and subcellular details of hearing and vestibular organs, as well as ultrastructural details in the developing sensory epithelia. This enabled the gathering of detailed information regarding morphometric changes as well in realizing the complex developmental patterns of the human inner ear. We were able to quantify the volumetric and linear aspects of selected gestational inner ear specimens enabling a better understanding of the cellular changes across the fetal gestational timeline. Moreover, these data could serve as a reference for better understanding disorders that arise during inner ear development.

Fig. 1

Growth of the cochlear duct/ scala media between the gestational weeks 11 to 36. 3D images are manually segmented fetal human temporal bones rendered using Amira 6.4® software. Substantial increase in the endolymphatic fluid volume and length of the scala media were apparent in the segmented datasets. Since all specimens were randomly oriented during scanning all the left datasets (GW14, GW19, GW23 & GW36) were mirrored so that all the specimens appear to be that of a right inner ear

Fig. 2

Change in the length of the scala media between the gestational weeks 11 to 36. The growth of the endolymph filled scala media estimated via centerline analysis using Amira 6.4®. A substantial increase in scala media length measured from apex to base occurs between the gestational age 11 (youngest specimen examined) and gestational age 36 (oldest specimen examined), while strongest increase in analyzed stages occurs between GW 11 and GW19

Fig. 3

Change in the volume of the scala media between the gestational weeks 11 to 36. The increase in volume of the endolymph filled scala media estimated via material statistics module using Amira 6.4®. A substantial increase in scala media volume measured occurs between the gestational age 11 (youngest specimen examined) and gestational age 36 (oldest specimen examined), while strongest increase in analyzed stages occurs between GW 14 and GW23

Fig. 4

Change in volume/length ratio of the scala media between the gestational weeks 11 to 36. The increase in the volume/length ratio of the scala media parallels the increase in the volumes of the scala media of individual fetal specimens. A substantial increase in this ratio occurs between the gestational ages 11 (youngest specimen examined) and gestational age 23

Fig. 5

Growth of the endolymph between the gestational weeks 11 to 36. 3D rendered images obtained from manually segmented fetal human temporal bones using Amira 6.4® software. Substantial increase in the endolymphatic fluid spaces was visually apparent in the segmented datasets. Since all the specimens were randomly oriented during scanning all the left datasets (GW14, GW19, GW23 & GW36) were mirrored so that all the specimens appear to be that of a right inner ear

Fig. 6

Variation in the distances between the vestibular end organs during the gestational weeks 11 to 36. Trend lines added based on linear regression. a Change in distance between the developing cristae ampullares. b Change in distance between the developing macula organs. c Change in distance between the developing macula of the utricle and the three individual cristae ampullares. d Change in distances amongst the developing macula of the saccule and the three individual Cristae ampullares between the fetal gestational weeks 11 to 36

Fig. 7

Overview of the developing fetal inner ear from GW9 to GW15 along the basal turn. a Toluidine blue stained semi-thin section of a fetal human cochlea at GW9 showing the basal turn. Accumulation of epithelial cells is evident at this stage, but no differentiation in the supporting cells or hair cells is evident. b Toluidine blue stained semi-thin section of a fetal human cochlea at GW11 showing the basal turn. At this gestational age, supporting cells (arrows) show distinct morphological characteristics. c Toluidine blue stained semi-thin section of a fetal human cochlea at GW12 with the stria vascularis (asterisk) and supporting cells (arrows) evident. d Toluidine blue stained semi-thin section of a fetal human cochlea at GW13 with tunnel of Corti development starting. The developing outer and inner hair cells are also apparent (arrow) in the histological section. e Toluidine blue stained semi-thin section of a fetal human cochlea at GW14. The opening of the tunnel of Corti (arrow) is evident at GW14 with the outer and inner hair cells lining the developing OC being apparent (arrowheads). f Toluidine blue stained semi-thin section of a fetal human cochlea at GW15 the tunnel of Corti is enlarged (arrowhead). The cytoarchitecture of the stria vascularis (arrow) is well-developed. Melanocytes invading the basilar membrane in the future stria vascularis are also visible (arrowhead). Scale bars = 50 μm

Fig. 8

Correlative microscopy of the fetal human utricle at GW13. a Semi-thin section of the utricle at GW13. Hair cell formation clearly can be seen (arrows). Scale bar = 100 μm. b Transmission electron microscopy of the utricle at GW13. This figure demonstrates the hair cells (HC) with the cuticular plate, the stereocilia and the kinocilium (arrows). SC = supporting cells. Scale bar = 20 μm

Fig. 9

Correlative imaging of the human fetal cochlea at GW14 illustrating the basal to apical gradient in the developmental progression of the organ of Corti (oc) along the cochlear duct. a Mid-modiolar microCT section through the whole cochlea at GW14 showing basal turn (bt), middle turn (mt) and apical turn (at) of the cochlear duct. b Corresponding mid-modiolar semi-thin section of the same specimen. The opening of the tunnel of Corti in the basal turn is well visible (see also Fig. 7e), while in the apical turn the tunnel of Corti has yet to form. Along the middle turn, the opening of the tunnel of Corti is very small. Furthermore, the developing spiral ganglion (spg) and nerve fibers (nf) can be seen. c Transmission electron microscopy of the same specimen confirmed that in the apical turn the tunnel of Corti has not yet formed. d Volume rendering of the microCT scan showing the exact position of the registered semi-thin section depicted in Fig. 9b. Scale bar (a-b) 500 μm (c) 5 μm and (d) 1 mm

Fig. 10

Semi-thin section of the developing OC. (a) At GW16, the organ of Corti has four rows of outer hair cells (OHC), inner hair cells (arrowhead) and the cytoarchitecture of the Stria vascularis (Stv) is well developed. b The spiral ganglion has a lone satellite glial cell (b arrow) encapsulating it at this stage. c At GW21, the organ of Corti (c) is well formed and functional with outer hair cells (OHC) and inner hair cells (arrowheads) visible. Three to four satellite glial cells (d open arrows) encapsulating the spiral ganglion cells (d arrows) is evident at this stage. Scale bars = 50 μm

Fig. 11

Scanning electron micrographs of a human cochlea at gestational week 16: a A mid-modiolar cut exposes the fluid compartments Scala media (SM), Scala tympani (ST) and Scala vestibuli (ST) that are all fully developed. Cartilaginous tissue is colored blue and borders the Modiolus (M). b Shows a detailed view of Kölliker’s organ of the middle turn with inner hair cells colored red and outer hair cells colored green. Note the primary cilium that is present in all supporting cells of the sensory epithelium! The basilar membrane is coated with a thick, spongiform layer of mesothelial cells. c The higher magnified view depicts the interior of the evolving tunnel of Corti. Medial efferent fibers span the gap between pillar cells as tunnel crossing fibers (TCF) to provide efferent innervation at the basal pole of outer hair cells (OHC, green). Inner hair cell (IHC) red. d The apical end of an inner hair cell is presented: The terminal web of the cuticular plate (colored yellow) is surrounded by long microvilli. Stereocilia (colored red) show typical arrangement similar to adult vestibular hair cell formation. A kinocilium (colored green) at the distal aspect of the apical hair cell pole is still present, its bulging insertion indicates the position of the basal body underneath. Scale bars (a) 4 mm, (b-c) 40 μm and (d) 4 μm