FIGURE 8 - uploaded by Annelies Schrott-Fischer
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| Overview of the crista ampullaris in the human fetal VO at W12 in (A). The DC area of the cristae (arrow) is clearly distinct from both the lower portion of sensory epithelium (SE) close to the transitional zone (arrow) and the simple cuboidal epithelium (CE) (arrow). The cells in the CE (B) is single layered and uniformly sized while the DC (C) begin to develop their basal labyrinth with various spaces separated by a basal lamina (colored red) toward the mesothelial compartments. Large lobulated nuclei (N) placed in the upper portion of the cells further characterize these cells. Most of the cytoplasmic organelles are located in the upper two-thirds of the DC's, including numerous irregular vacuoles. Deep infoldings of the lateral and basal plasmalemma divide the lower third of these cells into numerous foliate cytoplasmic compartments containing mitochondria (C). Scale bar: 20 ?m (A), 2 ?m (B), and 1 ?m (C).
Source publication
Balance orientation depends on the precise operation of the vestibular end organs and the vestibular ganglion neurons. Previous research on the assemblage of the neuronal network in the developing fetal vestibular organ has been limited to data from animal models. Insights into the molecular expression profiles and signaling moieties involved in em...
Contexts in source publication
Context 1
... calcification process was still not complete as revealed by the white spots among these crystals, which were the sites of initiation of the calcium deposition in its carbonate form. (Figure 8A) gives an overview of the sensory epithelium close to the transitional zone, dark cell area, and the simple cuboidal epithelium in a semi- circular canal. (Figure 8B) is a higher magnified view of the simple cuboidal epithelium and (Figure 8C) shows the DC area of the ampullary crest. ...
Context 2
... 8A) gives an overview of the sensory epithelium close to the transitional zone, dark cell area, and the simple cuboidal epithelium in a semi- circular canal. (Figure 8B) is a higher magnified view of the simple cuboidal epithelium and (Figure 8C) shows the DC area of the ampullary crest. The cells in the CE were uniformly shaped with a centrally placed nucleus, while those in the DC area had an irregular, laminated appearance with a large, lobulated nucleus. ...
Context 3
... 8A) gives an overview of the sensory epithelium close to the transitional zone, dark cell area, and the simple cuboidal epithelium in a semi- circular canal. (Figure 8B) is a higher magnified view of the simple cuboidal epithelium and (Figure 8C) shows the DC area of the ampullary crest. The cells in the CE were uniformly shaped with a centrally placed nucleus, while those in the DC area had an irregular, laminated appearance with a large, lobulated nucleus. ...
Citations
... The tissue preparation for paraffin embedding, the immunohistochemistry procedure and the digital examination of human cochlear sections were described in detail in our previous publications [61][62][63][64][65]. Negative controls were acquired by substituting the primary antibodies with isotype-matching immunoglobulins. ...
A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at −80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.
... The immunostaining of S100 protein is mainly related to the development of non-sensory cells such as pillars, Deiters' cells of the cochlea, hair cells of the vestibule, and their connecting nerve fibers. Also, it is present in neural crestderived cells such as Schwann cells, chondrocytes, melanocytes, and intermediate cells of the stria vascularis (Buckiová and Syka, 2009;Johnson Chacko et al., 2016). The morphological changes during the different developmental stages of the inner ear in the rabbit species are scarcely reported (Retzius, 1884), so the aim of the current work was to provide a detailed morphological source for the rabbit inner ear development histologically and immunohistochemically. ...
... 53 Vestibular development, which precedes that of the cochlea, has a more mature phenotype with distinct epithelial cell types at FW9.2. 46,54 We used differentially expressed genes and marker genes ( Figures S6A-S6D) as well as recently published single-cell datasets of the mouse inner ear 24,27,28,55,56 to annotate the populations of the integrated inner ear dataset ( Figure 3C). We identified a mesenchymal population, various epithelial populations of both cochlear and vestibular identity, hair cells, neurons, glial cells, cycling cells, endothelial cells, macrophages, and melanocytes ( Figures 3C and S6). ...
... Although the fetal immature vestibular hair cells cluster together ( Figure 5B and S6B), a distinction can be made between developing type I and type II vestibular hair cells at FW9. 54 Type I and type II vestibular hair cells can be distinguished by their shape and the type of synapse ( Figure 5D). We identified calyx-type synapses (TUBB3 + ) in D75 IEOs ( Figure 5E), which are typically associated with type I vestibular hair cells. ...
Inner ear disorders are among the most common congenital abnormalities; however, current tissue culture models lack the cell type diversity to study these disorders and normal otic development. Here, we demonstrate the robustness of human pluripotent stem cell-derived inner ear organoids (IEOs) and evaluate cell type heterogeneity by single-cell transcriptomics. To validate our findings, we construct a single-cell atlas of human fetal and adult inner ear tissue. Our study identifies various cell types in the IEOs including periotic mesenchyme, type I and type II vestibular hair cells, and developing vestibular and cochlear epithelium. Many genes linked to congenital inner ear dysfunction are confirmed to be expressed in these cell types. Additional cell-cell communication analysis within IEOs and fetal tissue highlights the role of endothelial cells on the developing sensory epithelium. These findings provide insights into this organoid model and its potential applications in studying inner ear development and disorders.
... The first major sensory system to come online during fetal development is the vestibular system Bradley and Mistretta (1975); Johnson Chacko et al. (2016)). Vestibular input has a significant influence in early movement development and is directly connected to the the existence of primitive reflexes (Brandt and Dieterich, 2015;Grzywniak, 2016) and head and neck movement (Lovell, 2021). ...
The prefrontal cortex is included in a neuronal system that includes the basal ganglia, the thalamus, and the cerebellum. Most of the higher and more complex motor, cognitive, and emotional behavioral functions are thought to be found primarily in the frontal lobes. Insufficient connectivity between the medial prefrontal cortex (mPFC) and other regions of the brain that are distant from each other involved in top-down information pro- cessing rely on the global integration of data from multiple input sources and enhance low-level perception processes (bottom-up information processing). The reduced deactivation in mPFC and in the rest of the Default Network during global task processing is consistent with the integrative modulatory role served by the mPFC. We stress the importance of understanding the degree to which sensory and movement anomalies in individuals with autism spectrum disorder (ASD) can contribute to social impairment. Further investigation on the neurobiological basis of sensory symptoms and its relationship to other clinical features found in ASD is required Treatment perhaps should not be first behaviorally-based but rather based on facilitating sensory motor development
... Twenty-Six human embryos and foetuses (GW09 x2, GW10 x3, GW11x2, GW12x3, GW13 x3, GW14 x2, GW15 x2, GW16 x3, GW17 x3, GW18 x3 and GW19 x1 as biological/technical replicates were done via sectioning) were used for this study. Tissue preparation, immunohistochemistry and fluorescencebased immunohistochemistry procedure on human cochlear section was described in previous publications (29)(30)(31)(32). ...
Background
Human inner ear contains macrophages whose functional role in early development is yet unclear. Recent studies describe inner ear macrophages act as effector cells of the innate immune system and are often activated following acoustic trauma or exposure to ototoxic drugs. Few or limited literature describing the role of macrophages during inner ear development and organogenesis.
Material and Methods
We performed a study combining immunohistochemistry and immunofluorescence using antibodies against IBA1, CX3CL1, CD168, CD68, CD45 and CollagenIV. Immune staining and quantification was performed on human embryonic inner ear sections from gestational week 09 to 17.
Results
The study showed IBA1 and CD45 positive cells in the mesenchymal tissue at GW 09 to GW17. No IBA1 positive macrophages were detected in the sensory epithelium of the cochlea and vestibulum. Fractalkine (CX3CL1) signalling was initiated GW10 and parallel chemotactic attraction and migration of macrophages into the inner ear. Macrophages also migrated into the spiral ganglion, cochlear nerve, and peripheral nerve fibers and tissue-expressing CX3CL1. The mesenchymal tissue at all gestational weeks expressed CD163 and CD68.
Conclusion
Expressions of markers for resident and non-resident macrophages (IBA1, CD45, CD68, and CD163) were identified in the human fetal inner ear. We speculate that these cells play a role for the development of human inner ear tissue including shaping of the gracile structures.
... Nuclear antigen Ki-67 is lacking in resting cells (G0 phase) and hence exclusively positive in the nuclei of proliferating cells [24] . In addition, Johnson Chacko et al. [45] stated that Ki-67 expression is restricted to the undifferentiated cells, whereas the differentiated cells are negative for this marker. In the current work, significant reduction in the ki-67 expression level was detected in the apparently normal newly formed spindle muscle in PRP treated group. ...
... The inner ear develops from the otic placode, a thickening of non-neural ectoderm (NNE) forming in the cranial portion of the embryo at the level of rhombomeres V and VI. 10,11 The otic placode receives signals from the surrounding mesenchyme and the developing neural tube including Wnt 12,13 and Fgf signaling [14][15][16] [25][26][27][28][29][30][31][32] have been recently published. 65,66 Direct reprogramming has recently emerged as a potential complementary strategy. ...
Hearing loss is the most widely spread sensory disorder in our society. In the majority of cases, it is caused by the loss or malfunctioning of cells in the cochlea: the mechanosensory hair cells, which act as primary sound receptors, and the connecting auditory neurons of the spiral ganglion, which relay the signal to upper brain centers. In contrast to other vertebrates, where damage to the hearing organ can be repaired through the activity of resident cells, acting as tissue progenitors, in mammals, sensory cell damage or loss is irreversible. The understanding of gene and cellular functions, through analysis of different animal models, has helped to identify causes of disease and possible targets for hearing restoration. Translation of these findings to novel therapeutics is, however, hindered by the lack of cellular assays, based on human sensory cells, to evaluate the conservation of molecular pathways across species and the efficacy of novel therapeutic strategies. In the last decade, stem cell technologies enabled to generate human sensory cell types in vitro, providing novel tools to study human inner ear biology, model disease, and validate therapeutics. This review focuses specifically on two technologies: directed differentiation of pluripotent stem cells and direct reprogramming of somatic cell types to sensory hair cells and neurons. Recent development in the field are discussed as well as how these tools could be implemented to become routinely adopted experimental models for hearing research.
... The earliest time point for fully adult-like vestibular hair cells immune stained for myosin VIIa was in the cristae ampullaris at GW9. We previously observed positive immune staining for glutamine synthetase (Johnson Chacko et al. 2016), a metabolic regulator of glutamate in the transitional cells of the vestibular end organs. Lineage-specific expression of glutamine synthetase in luminal cells of breast epithelia enables cellular survival (Kung et al. 2011). ...
... GATA3 expression could signal auditory specification via multiple gene expression cascades, one of which could be the associated transcriptional factor MAFB. We observed MAFB expression in the fetal cochlear (Pechriggl et al. 2015) and vestibular ganglia (Johnson Chacko et al. 2016) between the gestational weeks 8 and 12. MAFB was previously reported to be part of a GATA3-MAFB transcriptional network directing postsynaptic differentiation in auditory synapses (Yu et al. 2013), an indication of its role in SG terminal differentiation. The timely expression of the transcription factors GATA3 and SOX2 observed here with MAFB (Johnson Chacko et al. 2016;Pechriggl et al. 2015) suggests the existence of a cochlear prosensory gene-signaling cascade. ...
... We observed MAFB expression in the fetal cochlear (Pechriggl et al. 2015) and vestibular ganglia (Johnson Chacko et al. 2016) between the gestational weeks 8 and 12. MAFB was previously reported to be part of a GATA3-MAFB transcriptional network directing postsynaptic differentiation in auditory synapses (Yu et al. 2013), an indication of its role in SG terminal differentiation. The timely expression of the transcription factors GATA3 and SOX2 observed here with MAFB (Johnson Chacko et al. 2016;Pechriggl et al. 2015) suggests the existence of a cochlear prosensory gene-signaling cascade. This signaling cascade could be similar to the cooperative interaction of SOX2, SIX1, and EYA1 proteins in guiding ATOH1mediated hair cell specification (Ahmed et al. 2012). ...
Expression patterns of transcription factors leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), transforming growth factor-β-activated kinase-1 (TAK1), SRY (sex-determining region Y)-box 2 (SOX2), and GATA binding protein 3 (GATA3) in the developing human fetal inner ear were studied between the gestation weeks 9 and 12. Further development of cochlear apex between gestational weeks 11 and 16 (GW11 and GW16) was examined using transmission electron microscopy. LGR5 was evident in the apical poles of the sensory epithelium of the cochlear duct and the vestibular end organs at GW11. Immunostaining was limited to hair cells of the organ of Corti by GW12. TAK1 was immune positive in inner hair cells of the organ of Corti by GW12 and colocalized with p75 neurotrophic receptor expression. Expression for SOX2 was confined primarily to the supporting cells of utricle at the earliest stage examined at GW9. Intense expression for GATA3 was presented in the cochlear sensory epithelium and spiral ganglia at GW9. Expression of GATA3 was present along the midline of both the utricle and saccule in the zone corresponding to the striolar reversal zone where the hair cell phenotype switches from type I to type II. The spatiotemporal gradient of the development of the organ of Corti was also evident with the apex of the cochlea forming by GW16. It seems that highly specific staining patterns of several transcriptions factors are critical in guiding the genesis of the inner ear over development. Our findings suggest that the spatiotemporal gradient in cochlear development extends at least until gestational week 16.
... Likewise, it is well known that pax-8 expression is found in many normal and neoplastic tissues, including epididymis, rete testis, fallopian tumor and ovary, pancreatic islets and lymph nodes, while commonly found in bladder adenocarcinoma, seminoma, endometrial adenocarcinoma, ovarian serous carcinoma, renal cell carcinoma, oncocytoma, neuroendocrine tumors, parathyroid carcinoma and papillary, follicular and medullary thyroid carcinoma [43][44][45][46][47][48][49]. During embryogenesis, pax-2 and pax-8 transcription factors play a redundant role by together regulating inner ear development, associated with otic placode induction, where they are required to maintain otic differentiation [50,51]. Both pax-2 and pax-8 are noted in hair cell development of the vestibular end organ (otic progenitor cells) during gestational weeks 8-12 [51,52]. ...
... During embryogenesis, pax-2 and pax-8 transcription factors play a redundant role by together regulating inner ear development, associated with otic placode induction, where they are required to maintain otic differentiation [50,51]. Both pax-2 and pax-8 are noted in hair cell development of the vestibular end organ (otic progenitor cells) during gestational weeks 8-12 [51,52]. Thus, in the neoplastic setting of a tumor arising from this zone in later life, it is perhaps an explanation for pax-8 expression, and why it is so strongly expressed in ELSTs, as seen in 85% of the tumors studied. ...
Endolymphatic sac tumors (ELSTs) are rare, slowly growing temporal bone neoplasms which show a high association with von Hippel-Lindau (VHL) syndrome. The immunohistochemistry evaluation of these papillary-cystic neoplasms frequently raises the differential diagnosis with renal cell carcinoma, among other metastatic neoplasms, whether in VHL patients or not. A cohort of 26 patients with ELSTs were evaluated for histologic features, immunohistochemistry findings, and association with VHL. Standard immunohistochemistry evaluation was performed. Sixteen females and 10 males ranging in age from 10 to 69 years (mean 44; VHL mean: 32) at initial presentation, comprised the cohort of patients. Most (86%) experienced hearing changes or inner ear symptoms (vertigo, dizziness), with an average duration of symptoms for 39 months (range 2–240 months). The tumors were an average of 2.9 cm (range 0.4–8 cm), with 14 left, 11 right sided and one bilateral tumor. Nine patients had documented VHL, with 3 patients having a concurrent or subsequent clear cell renal cell carcinoma. Patients were followed an average of 6.2 years (available in 24 patients): 19 alive without disease, 7.5 years; 2 dead without disease, 1.2 years; and 3 alive with disease, 3.1 years. The neoplastic cells show the following immunohistochemistry findings: AE1/AE3, EMA, CK7, CAIX, GLUT1, VEGF: 100% of cases tested were positive; pax-8: 85% of cases positive; CD10 and RCC: 0% of cases reactive. Based on this cohort of 26 patients with ELST, 9 of whom had VHL, the strong pax-8 and CAIX should be used in conjunction with negative CD10 and RCC to help exclude a metastatic renal cell carcinoma. As CAIX is an enzyme overexpressed in hypoxia and hypoxia inducible factor is what VHL protein regulates, this is an expected, although previously unreported finding. Whether part of VHL or not, VHL mutations may be a somatic rather than germline finding in the tumors, a possible further explanation for the CAIX reaction.
... Prime among these cellular patterning genes are Maf B (V-maf musculo-aponeurotic fibrosarcoma oncogene homolog B) and Pax family genes including Pax2, Pax6, and Pax8. Besides these patterning genes, other genes specific for the neurosensory epithelia are also functional at this time point including peripherin and Class III β-Tubulin [7] whose expression are guided by neurotrophins and their factors [8]. Neurosensory cell fate specification [9] is driven by these and other factors and thus leads to the growth of a well-designed sensory organ capable of auditory and vestibular cognizance. ...
... using immune staining's were able to visualize the development of the cochlear hair cells between GW 10 to 12 [8,14]. We were also able to localize otic differentiation using transcription factors like Pax2 & Pax8 and chart the development of vestibular innervation using antibodies specific Beta III tubulin and peripherin during the same time period [7]. These and other morphological changes causes a volumetric increase in geometric size some of which are accompanied by the displacement of the sensory structures. ...
... The cellular growth in the vestibulum is much more advanced towards this time point as implicit from the well-developed utricular hair cells, their stereocilia and surrounding supporting cells. These findings support previously described data of the fetal utricle at GW11 [7] and is an indicator of the early maturation of the vestibular organ. The support structures like the protocalyx, stereocilia and the kinocilia are an indicator of the early onset of the vestibular functionality. ...
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.
