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Early development of the cochlea of the common marmoset, a non-human primate model
Makoto Hosoya1,
Masato Fujioka1 nAff2,
Junko Okahara3,4,
Sho Yoshimatsu3,5,
Hideyuki Okano3,5 &
Hiroyuki Ozawa1
Neural Development volume 17, Article number: 6 (2022) Cite this article
Fine-tuned cochlear development is essential for hearing. Owing to the difficulty in using early human fetal samples, most of our knowledge regarding cochlear development has been obtained from rodents. However, several inter-species differences in cochlear development between rodents and humans have been reported. To bridge these differences, we investigated early otic development of a non-human primate model animal, the common marmoset (Callithrix jacchus).
We examined 20 genes involved in early cochlear development and described the critical developmental steps for morphogenesis, which have been reported to vary between rodents and marmosets.
The results revealed that several critical genes involved in prosensory epithelium specifications showed higher inter-species differences, suggesting that the molecular process for hair cell lineage acquisition in primates differs considerably from that of rodents. We also observed that the tempo of cochlear development was three times slower in the primate than in rodents.
Our data provide new insights into early cochlear development in primates and humans and imply that the procedures used for manipulating rodent cochlear sensory cells cannot be directly used for the research of primate cells due to the intrinsic inter-species differences in the cell fate determination program.
Auditory perception is the process by which mechanical sound waves are detected by the inner ear and converted into neuronal electrical impulses perceived by the brain. A coiled organ, the cochlea, plays an essential role in hearing during this sequential process in the inner ear. In the cochlea, hair cells convert mechanosensory sound waves into neural electrical pulses, which are transmitted by the synapses between the hair cells and spiral ganglion neurons and eventually reach the brain's auditory cortex [1, 2].
Fine-tuned cochlear development is essential for the acquisition of hearing. This development requires several well-controlled steps, including the formation of coiled structure, specification of sensory epithelium, differentiation of hair cells and spiral ganglion neurons, and their subsequent maturation [3, 4]. Most of our knowledge regarding cochlear development has been obtained from rodent models, specifically from mice and rats. However, several inter-species differences in cochlear development between rodents and humans have been reported. For example, the expression pattern of PRPH (peripherin), a neurofilament of the spiral ganglion neurons, differs between primates and rodents during cochlear development [5]. In addition, a human-specific pattern was reported for myelination of spiral ganglion neurons [6]. Despite these inter-species differences in cochlear development between rodents and humans, knowledge regarding cochlear development in humans is scarce because of ethical issues in obtaining human fetal tissues.
To bridge these inter-species differences between rodents and humans, the common marmoset (Callithrix jacchus), a primate model, has been exploited and investigated for cochlear development [7,8,9]. This animal model has also been used in studying deafness-related genes [10,11,12,13,14] and presbycusis [15], as well as in other areas of neuroscience [16]. These previous reports on the primate model have demonstrated the usefulness of the animal model in research on hearing.
In particular, previous reports have shown that cochlear development in this model differs from that of rodent models [7, 8]. For example, while Na–K-Cl cotransporter 1 (NKCC1) is well-known as a transporter of the stria vascularis and lateral wall, a distinct transient expression of NKCC1 in the organ of Corti during development has been observed in the common marmoset [7]. Distinct and complex changes in the expression patterns of synaptic vesicle exocytosis-related genes in hair cells during the developmental process of this primate have been reported [8]. However, neither have been reported in rodent models.
Previous embryological studies in this primate have focused only on the fetus after embryonic day 96 (E96), a relatively late stage of cochlear development [7,8,9]. Therefore, early development (before E96) of the cochlea in this animal model has not thoroughly been investigated, although several critical developmental processes are known to occur at this early stage. For example, when cochlear duct coils are formed or how sensory epithelial differentiation occurs in this animal are not well understood.
This study examined the cochlea from E70 to E92 of the common marmoset. We investigated important cochlear developmental steps, including formation of the coiled structure, specifications of the sensory epithelium, differentiation of hair cells, and development of neurons.
Cadaverous temporal bone samples of common marmosets at E70 (n = 4), E77 (n = 2), E87 (n = 4), and E92 (n = 3) were used in this study. The animal experiments were approved by the Animal Experiment Committee of Keio University (number: 11006, 08,020) and were performed in accordance with the guidelines of the National Institutes of Health and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Tissue preparation
The temporal bone was dissected and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight immediately after euthanasia. Specimens were embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan) for cross-sectioning. Seven-micrometer sections were used for immunohistochemical analysis.
After a brief wash with PBS, the sections were heated (80 °C) in 10 µM citrate buffer (pH 6) for 15 min. After another brief wash, the sections were pre-blocked for 1 h at room temperature in 10% normal serum in PBS, incubated overnight with primary antibodies at 4 °C, and then incubated with Alexa Fluor-conjugated secondary antibodies for 60 min at room temperature. Nuclei were counterstained with Hoechst 33,258.
The primary antibodies used in this study are listed in Table 1. The following secondary antibodies were used: goat anti-rabbit IgG, Alexa Fluor Plus 555 (A32732, 1:500, Invitrogen), goat anti-rabbit IgG, Alexa Fluor Plus 647 (A32733, 1:500, Invitrogen), goat anti-mouse IgG, Alexa Fluor Plus 488 (A32723, 1:500, Invitrogen), goat anti-mouse IgG1, Alexa 488 (A21121, 1:500, Invitrogen), goat anti-mouse IgG2b, Alexa 647 (A21242, 1:500, Invitrogen), goat anti-chicken IgY, Alexa Fluor 555 (A32932, 1:500, Invitrogen), donkey anti-mouse IgG, Alexa Fluor Plus 488 (A32766, 1:500, Invitrogen), donkey anti-chicken IgY, and Alexa Fluor 647 (703–605-155, 1:500, Jackson Immuno-Research).
Table 1 Primary antibodies used in this study
Early morphological development of the common marmoset cochlea
The common marmoset has a gestation period of approximately 143–150 days [17]. Previously, we have reported that cochlear duct formation was complete, with apical, middle, and basal turns, in E96 embryos of the common marmoset. In this study, we first examined morphological changes in the cochlea during early development in the common marmoset using hematoxylin–eosin staining (Fig. 1).
Histological analysis of the developing cochlea in the common marmoset using hematoxylin–eosin staining. (A) A cross-sectional view of the common marmoset cochlear duct in an E70 embryo. Cochlear duct formation had already started at E70. However, the coiled structure appeared only as a hook-like structure. (B) A cross-sectional view of the common marmoset cochlea duct in an E77 embryo. At E77, the elongation and coiling of the cochlear duct were more prominent. At this stage, the apical and basal turns can be distinguished. On the modiolar side, spiral ganglion neuron formation was observed at this stage. (C) A cross-sectional view of the common marmoset cochlea duct in an E87 embryo. At E87, the overall coiled structures of the cochlear duct were well developed and approximately two turns of the coiled structure were observed. The scala vestibuli and the scala tympani were not formed. (D) A cross-sectional view of the common marmoset cochlea duct in an E92 embryo. At E92, the coiled structure of the cochlear duct was almost completely formed. At this stage, the immature scala vestibuli and scala tympani were observed in the basal turns
In E70 embryos of the common marmoset, the cochlear duct appeared as a hook-like structure, and the coiled structure was incomplete (Fig. 1A). In E77 embryos, at least a one-turn coiled structure was observed (Fig. 1B). At this stage, a premature bony capsule of the cochlea was also observed. In E87, the cochlear duct was elongated, and a two-turn coiled structure was observed (Fig. 1C). At this stage, the structures of the cochlear bony capsules became more prominent. In E92, an almost entirely elongated coil was formed and a two-and a half-turn coiled structure was observed (Fig. 1D). Our observations indicated that the coiled structure of the common marmoset formed before E70 and its elongation continued until E92.
Development of hair cells
Next, we investigated the initial formation of hair cells by examining the expression of hair cell markers, POU4F3 (POU class 4 homeobox 3) [18] and ATOH1 (atonal bHLH transcription factor 1) [19, 20].
In common marmosets, neither POU4F3 nor ATOH1 expression was observed in the E77 cochlear duct, while both of their expression was observed in the vestibular organ (Fig. 2A). In E87, expression of both POU4F3 and ATOH1 was observed in the SOX2 (SRY-box transcription factor 2) positive-prosensory domain (Fig. 2B-D). In the E87 cochlear duct, one row of inner hair cells was observed between the mid-basal and basal turns. In the basal turn, the development of outer hair cells was observed at this stage. In E92, hair cell development proceeded more apically, and both inner and outer hair cell development was observed in the middle turns (Fig. 2E-G). In contrast, no expression of hair cell markers was observed in the apical turns at this stage. These observations indicated that hair cell formation in the common marmoset started between E77 and E87 from the basal turns and progressed from the base to the apex.
Expression of POU4F3 and ATOH1 in the early developing cochlea of the common marmoset. (A) In E77, no expression of POU4F3 and ATOH1 was detected in the cochlear, while expression of POU4F3 or ATOH1 was detected in the vestibular hair cells (arrow in A). (B-D) In E87, expression of POU4F3 and ATOH1 was detected in the basal and mid-basal turn. No expression of POU4F3 or ATOH1 was detected in the apical turn. In the basal turn, expression of POU4F3 and ATOH1 was detected in the inner hair cells and one row of the outer hair cells, while in the mid-basal turn, their expression was observed only in the inner hair cells. (E–G) In E92, POU4F3 and ATOH1 expression was observed in the middle and basal turns. No expression of POU4F3 or ATOH1 was detected in the apical turns. POU4F3 and ATOH1 expression was observed in both the inner and outer hair cells in both the middle and basal turns. Nuclei were counterstained with Hoechst stain (blue). Scale bar: 200 µm in A, 50 µm in B-G. VE, vestibular organ; CO, cochlea; IHC, inner hair cells; OHC, outer hair cells
Specification of the prosensory domain in the developing common marmoset cochlea
Next, we investigated the timing of the specification of the prosensory domain in this animal using the cochlear prosensory domain markers, SOX2 [21, 22], CDKN1B (cyclin dependent kinase inhibitor 1B) [23], and JAG1 (jagged canonical Notch ligand 1) [23,24,25].
Ventral expression of SOX2 in the cochlear duct was observed in E70; however, CDKN1B and JAG1 were not observed in these SOX2-positive cells (Fig. 3A). In E77, CDKN1B and SOX2 double-positive cells were observed in the apical turns of the cochlear duct, but JAG1 expression was not observed at this stage (Fig. 3B and C). In E87, JAG1 expression was observed in the medial-ventral region of the cochlear duct (Fig. 3D). These observations indicated that the prosensory domain specification in this animal started at around E77 at the apical turn and spread to the basal turn. In E92, JAG1 expression was observed in the medial side of the SOX2-positive region in the apical turn, whereas it was broadly distributed in the SOX2-positive region in the basal turn (Fig. 3D-F). These expression patterns of SOX2 and JAG1 with apical to basal gradients were similar to those previously reported in mice [25,26,27]. In summary, as shown in the schematic diagrams in Fig. 4, CDKN1B expression in the SOX2- positive region preceded JAG1 expression in the common marmoset cochlear sensory epithelium specifications.
Sensory epithelium specifications of the cochlea of the common marmoset. (A) In E70, CDKN1B and JAG1 expressions were not observed on the floor of the cochlear duct. (B and C) In E77, CDKN1B expression was detected in the floor of the elongating cochlear duct and in the apical turns (asterisk in B). At this stage, no JAG1 expression was detected in the cochlear duct. In the apical turn, CDKN1B expression was observed in a part of the SOX2-positive regions (C). (D) In E87, JAG1 expression was observed in the cochlea. (E–G) In E92, both CDKN1B and JAG1 expression was observed in the cochlea. JAG1 expression was observed in the medial side of the SOX2-positive region in the apical turn (Arrow in E). In contrast, JAG1 expression was detected more laterally and broadly in the basal turn (Arrow in F and G). The nuclei were counterstained with Hoechst (blue). Scale bar: 50 µm. OC: organ of Corti
Schematic diagram showing expression of SOX2, CDKN1B, and JAG1. CDKN1B expressions in the SOX2-positive regions were observed at E77, while no JAG1 expression in the SOX2-positive regions was observed at this stage. At E87, both CDKN1B and JAG1 expression were observed in the SOX2-positive regions. This observation indicated that JAG1 expression followed CDKN1B expression in developing common marmoset cochlea
Early patterning of the cochlear duct of the common marmoset
Early patterning of the cochlear duct, such as the organ of Corti, stria vascularis, and Reissner's membrane, is essential for the subsequent development of each compartment of the cochlear duct. This pattern of cochlear development is known to be fine-tuned by several genes [28,29,30]. Next, we evaluated the early patterning of the cochlear duct in this animal model by examining the expression of several markers, including OTX1 (orthodenticle homeobox 1) [31] and OTX2 (orthodenticle homeobox 2) [31] as dorsal markers, PAX2 (paired box 2) [32] as lateral markers, GATA3 (GATA binding protein 3) [33, 34] and ISL1 (ISL LIM homeobox 1) [35,36,37] as ventral markers (Fig. 5 and 6).
Expression patterns of OTX1, OTX2, PAX2, GATA3, SOX2, and ISL1 in E70 cochlea. (A) GATA3 expression was observed in the ventral portion of the cochlear duct at E70, while OTX2 expression was detected in the dorsal portion. (B) ISL1 expression was observed in the ventral portion of the cochlear duct. (C) PAX2 expression was detected in the lateral portion of the cochlear duct (arrowhead). (D) SOX2 expression was observed in the ventral portion and no overlapping expression with OTX2 was detected. (E) OTX1 expression was detected in the dorsal part and no overlapping expression with GATA3 was observed. (F and G) Expression of PAX8 and DLX5 was not detected in the cochlear duct at this stage. The nuclei were counterstained with Hoechst (blue). Scale bar: 100 µm. CD: cochlear duct, VO: vestibular organ
OTX2, PAX2, GATA3, and ISL1 expression patterns in E77, E87, and E92 cochlea. (A-D) Expression patterns of E77 cochlea. GATA3 and OTX2 expression was observed in the ventral and dorsal portion of the cochlear duct at E77, respectively (A). In the cochlear duct, ISL1 and PAX2 expression was observed in the ventral (B) and lateral portion (C), respectively. OTX1 expression diminished and no expression was detected in E77 (D). (E–G) Expression patterns in E87 cochlea. GATA3 and OTX2 expression was observed in the ventral and dorsal portion of the cochlear duct, respectively (E). ISL1 expression was observed in the ventral portion of the cochlear duct (F). PAX2 expression was detected in the lateral portion of the cochlear duct and hair cells (arrow in G). (H-J) Expression patterns in E92 cochlea. GATA3 and OTX2 expression was observed in the ventral and dorsal portion of the cochlear duct, respectively (H). ISL1 expression was observed in the ventral portion of the cochlear duct (I). PAX2 expression was detected in the lateral portion of the cochlear duct and hair cells (arrow in J). The nuclei were counterstained with Hoechst (blue). Scale bar: 100 µm in A-D, 50 µm in E-J. SpGN: spiral ganglion neuron
In E70 embryo of the common marmoset, GATA3 expression was observed in the ventral portion of the cochlear duct, while OTX2 expression was detected in the dorsal portion (Fig. 5A and D). ISL1 expression was observed in the ventral portion of the cochlear duct (Fig. 5B) and PAX2 expression was detected in the lateral portion of the cochlear duct (Fig. 5C). OTX1 expression was detected in the dorsal part (Fig. 5E). At this stage, expression of DLX5 and PAX8 was not detected in the cochlear duct (Fig. 5F and G).
In E77 cochlea, GATA3 and OTX2 expression was observed in the ventral and dorsal portion of the cochlear duct (Fig. 6A). In the cochlear duct, ISL1 and PAX2 expression was observed in the ventral and lateral portion (Fig. 6B and C). OTX1 expression diminished and no expression was detected in E77. In in E87 cochlea. GATA3 and OTX2 expression was observed in the ventral and dorsal portion of the cochlear duct (Fig. 6E). ISL1 expression was observed in the ventral portion of the cochlear duct (Fig. 6F). PAX2 expression was detected in the lateral portion of the cochlear duct and hair cells (Fig. 6G). In E92 cochlea, GATA3 and OTX2 expressions were observed in the ventral and dorsal portion of the cochlear duct (Fig. 6H). ISL1 expression was observed in the ventral portion of the cochlear duct (Fig. 6I). PAX2 expression was detected in the lateral portion of the cochlear duct and hair cells (Fig. 6J).
As shown in schematic diagrams, the expression patterns of these well-established conventional markers, which related to the early patterning of the cochlear development in the rodent models, were well conserved in the common marmoset cochlear development (Fig. 7).
Schema showing expression patterns of OTX1, OTX2, PAX2, GATA3, and ISL1. Expression patterns of the genes that control early cochlear duct patterning were investigated. In common marmoset, OTX1 expression and OTX2 expression were observed in the dorsal portion of the cochlear duct, and PAX2 expression was observed in the lateral portion of the cochlear duct and hair cells. GATA3 expression and ISL1 expression were observed in the ventral portion of the cochlear duct
Early development of the spiral ganglion neuron of the common marmoset
Appropriate differentiation of spiral ganglion neurons and their innervation into the cochlear epithelium is essential for proper acquisition of hearing ability. This developmental process of the spiral ganglion neurons is controlled by the fine tuning of expression of transcription factors such as NEUROD1 (neuronal differentiation 1) [38,39,40], POU4F1 (POU class 4 homeobox 1) [41, 42], ISL1 [34, 36], GATA3 [34, 43], and MAFB (MAF bZIP transcription factor B) [44]. Next, we investigated early development of the spiral ganglion neuron of the common marmoset.
In the common marmoset, cochleovestibular ganglion neurons were observed in the E70 cochlea (Fig. 8A-E). At this stage, the expression of TUBB3 and PRPH can be detected in cochleovestibular ganglion neurons (Fig. 8 A and D). Both NEUROD1 [38,39,40] and POU4F1 [41, 42] expression can be detected in the cochleovestibular ganglion neurons (Fig. 8B). In E70 embryo of the common marmoset, ISL1 expression was observed in the cochleovestibular ganglion neurons (Fig. 8C), whereas no expression of GATA3 and MAFB was detected (Fig. 8D and E). In the E77 cochlea (Fig. 8 F-L), spiral ganglion neurons come to be observed apparently and both NEUROD1 and POU4F1 were detected in the spiral ganglion neurons (Fig. 8G). At this stage, POU4F1 expression being apparent in most of the spiral ganglion neurons; however, NEUROD1 expression was restricted to neurons in the relatively apical turns (Fig. 8G). In contrast, RBFOX3 (RNA binding fox-1 homolog 3) expression was detected at E77 in NEUROD1-negative neurons located in the basal turns (Fig. 8H). After E77, ISL1, GATA3, and MAFB were observed in the spiral ganglion neurons (Fig. 8 J-L). At E87 (Fig. 9), although RBFOX3 expression was broader (Fig. 9B), NEUROD1 expression could not be detected in the spiral ganglion neurons (Fig. 9E and F). POU4F1 expression was reduced in the basal turns (Fig. 9F). At E92 (Fig. 10), RBFOX3 expression was detected broadly in the spiral ganglion neurons, whereas NEUROD1 and POU4F1 expression was not observed.
Cochlear neuronal development in E70 and E77. (A-E) Cochleovestibular ganglion in E70 cochlea. Expression of TUBB3, ISL1, and NEUROD1 was observed at E70 (A). Expression of POU4F1, SOX2, and PRPH was also observed in E70 cochleovestibular ganglion (B, C, and D). However, expression of MAFB and GATA3 was not detected in E70 cochleovestibular ganglion (D and E). (F-L) Spiral ganglion in E77 cochlea. Expression of TUBB3 and PRPH was observed in E77 spiral ganglion neurons (F). In E77, S100B-positive glial cells surrounding spiral ganglion neurons were observed (F). In E77, expression of NEUROD1 was observed in only an apical turn (asterisk in G), while RBFOX3 expression was observed in basal turns (H). No RBFOX3 expression was observed in the NEUROD1-positive spiral ganglion neurons (I). Expression of GATA3, ISL1, and MAFB was observed in the spiral ganglion neurons in E77 (J-L). The nuclei were counterstained with Hoechst (blue). Scale bar: 50 µm in A-E, G, and I, 100 µm in F, H, and J-L
Spiral ganglion neurons in E87. Expression of PRPH3, RBFOX3, GATA3, ISL1, and MAFB was observed in the E87 spiral ganglion neurons (A-D). POU4F1 expression was observed in all spiral ganglion neuron turns, more in apical turns than in mid-apical turns. POU4F1 expression was observed in parts of the basal turns of spiral ganglion neurons. This indicated that the expression of POU4F1 decreased in basal turns. NEUROD1 expression was not detected in E87 spiral ganglion neurons (E and F). Nuclei were counterstained with Hoechst stain (blue). Scale bar: 50 µm
Spiral ganglion neurons in E92. Expression of PRPH3, RBFOX3, GATA3, ISL1, and MAFB was observed in the E92 spiral ganglion neurons (A-D). POU4F1 expression was not observed in the spiral ganglion neurons at this stage (E). Nuclei were counterstained with Hoechst stain (blue). Scale bar: 50 µm
As shown in the schematic diagram, changes of the expression patterns of the transcription factors, which are known to be involving neuronal development in mice, were also observed during the development and maturation of spiral ganglion neurons in the development of the common marmoset cochlea (Fig. 11).
Schema showing the expression patterns of neuronal markers in early neuronal development. The expression patterns of genes involving the early development of spiral ganglion neurons were investigated. In the common marmoset, expressions of NEUROD1 and POU4F1 were observed in E77, and these expressions disappeared as development progressed. In contrast, broader RBFOX3 expression was observed after E87. GATA3 expression and MAFB expression followed ISL expressions in the spiral ganglion neuron of the common marmoset
Integration of glial cells into the spiral ganglion neuron
Glial cells surrounding the spiral ganglion neurons originate from neural crest cells and migrate to the spiral ganglion [45, 46]. After integration of the migrating glial cells into the spiral ganglion, they differentiate to form myelin [47]. Previously, we have observed myelination of the spiral ganglion neurons at E115 [8]; however, migration and integration of glial cells in this primate have not been evaluated. Hence, we investigated the development of glial cells in the spiral ganglion using SOX10 (SRY-box transcription factor 10) and S100B (S100 calcium binding protein B), which have been used for the markers of glial cells derived from migrating neural crest cells in the spiral ganglion neurons [47]. Finally, we investigated the integration of glial cells into the spiral ganglion neurons.
In the common marmoset, SOX10+ /S100B+ glial cells [47] were observed at the E70 cochleovestibular ganglion (Fig. 12 A and B). This indicated that the neural crest cells migrated into the ganglion [45, 46] before E70 in this animal. At E77 (Fig. 12 C-F), many of the glial cells were located around the spiral ganglion neurons, while several cells were positioned between the neurons. The integration of satellite glial cells was completed at E87 and E92 (Fig. 12 G-L). At E70, SOX2 expression was observed in SOX10-negative cells in the cochleovestibular ganglion; At E77, SOX2 expression in the spiral ganglion was observed only in SOX10+/S100+ glial cells.
Early development and integration of the glial cells of the cochlear spiral ganglion. (A and B) Glial cells in the E70 cochleovestibular ganglion. SOX10+/S100B+ glial cells were observed in the E70 cochleovestibular ganglion (A). Notably, the small number of SOX10−/SOX2+/S100B+ glial cells were also observed at this stage (arrowhead in B). (C-F) Glial cells in the E77 spiral ganglion. At E77, glial cells were observed surrounding the spiral ganglion neurons at this stage (arrow in E). No SOX10-positive cells were observed in the center of the spiral ganglion (asterisk in C). At this stage, S100B-positive glial cells expressed both SOX2 and SOX10 (F). (G-I) Glial cells in E87. Several SOX10-positive cells were observed between the spiral ganglion neurons (arrow in G). (J-L) Glial cells in E92. Integration of the SOX10-positive glial cells in the spiral ganglion was more prominent. The number of SOX10-positive cells observed between the spiral ganglion neurons increased (arrow in J). The nuclei were counterstained with Hoechst (blue). Scale bar: 20 µm in A, B, D, and F. 50 µm in C, E, and G-L
Development of hair cells and specification of the prosensory domain in the developing common marmoset cochlea
Development of hair cells and specification of the prosensory domain following the cochlear elongation is the one of the most essential steps in cochlear development. Our observation of the early cochlear development of the common marmoset identified the time points of these critical steps in this model animal. Combining observation in this study with our previous report, [7] hair cells developments of the common marmoset start between E77 and E87 from the basal turns and the progression reach to the apex turn at E101 (Fig. 13A). Preceding to this hair cells development, the elongation of the cochlear duct is observed until E92 (Fig. 13B).
Schema showing early cochlear duct and sensory epithelium formation. (A) Schema showing differentiation of hair cells. Hair cells differentiated along the basal-to-apical axis and from inner to outer side. (B) Schema showing cochlear duct development. Cochlear duct elongation was observed between E70 and E92. CDN1B expression started from the apical turns in E77. In contrast, hair cells were observed in E87 in the basal turns. IHC, inner hair cells; OHC, outer hair cells
In the human fetus, elongation of the cochlear duct starts at 4 − 5 weeks of gestation [48, 49], and the coiled cochlea can be observed at approximately 9 − 10 weeks of gestation [48,49,50]. In humans, specification of the sensory epithelium is observed at 9 weeks of gestation [51], and the differentiation of auditory hair cells begins at 12–14 weeks of gestation [5, 52]. Contrastingly, cochlear anlage becomes evident at E10.75 in mice, the first turn of the cochlea appears at E12, and the completely coiled cochlea is observed at E17 [53]. POU4F3 expression is observed at E15.5[18] and ATOH1 is expressed in a single row of cells from the base to the mid-base of the cochlea at E14.5 [19]. Combining the results of these previous reports with our present observations, we concluded that the E70 cochlea of the common marmoset was equivalent to the human cochlea at approximately 8 weeks of gestation and the E12 mouse cochlea. Similarly, the E87 cochlea of the common marmoset corresponded to the human cochlea at approximately 12 weeks of gestation and the E15 mouse cochlea.
Specification of the prosensory domain, which is essential for the subsequent formation of the organ of Corti, is an important step in cochlear development. Reports show that this specification developed during the elongation of the cochlear duct both in mice (E12.5 − E14.5) [22, 25, 54] and humans (9 weeks of gestation) [51]. Next, we investigated the timing of the specification of the prosensory domain in this animal using the cochlear prosensory domain markers, SOX2 [21, 22], CDKN1B [23], and JAG1[23,24,25]. This investigation revealed that prosensory domain specification was started at around E77 at the apical turn and spread to the basal turn until E87 in common marmoset (Fig. 13B).
Unlike that observed in mice, CDKN1B expression was followed by JAG1 expression during the specification of the cochlear prosensory domain in the common marmoset (Fig. 3 and 4). In mice, JAG1 expression is observed as early as in E12.5 and it overlaps with the SOX2 domain [25]. CDKN1b expression is observed at around E13.5–E14.5 [23, 25]. This determination of the prosensory domain via JAG1-mediated Notch1 signal activation occurs via lateral induction [26]. It is known that conditional knockout of Jag1 in the cochlea results in downregulation of Sox2 and Cdkn1b; therefore, prior expression of JAG1 is essential for the expression of SOX2 and CDKN1B in mice [25, 55]. However, prosensory domain specification occurred in this conditional knockout mouse, indicating the possibility of compensation of JAG1 by other Notch ligands [25].
Our observation that JAG1 expression was preceded by CDKN1B or SOX2 expression suggested that CDKN1B and SOX2 expression was controlled by other factors such as other Notch ligands, and that the role of JAG1 in controlling the expression of CDKN1B or SOX2 may be less dominant in this primate but needs validation. Until now, the expression patterns of JAG1 and CDKN1B in developing human fetuses have not been reported. Thus, we cannot conclude that this inter-species difference between common marmosets and mice will also be observed between humans and mice. However, our observations suggest the possibility of primate-specific control of prosensory domain specification of the cochlea. Nonetheless, further studies are warranted in the future.
Despite the difference in initial expression patterns and the lateral induction process, expression of JAG1, CDKN1B, and SOX2 in the later process of prosensory domain specifications are well conserved between common marmoset and mice [25,26,27]. In both mice at E14.5 and the common marmoset at E87, JAG1 was expressed mainly in the Kölliker's organ. Thus, JAG1 expression showed only a slight overlap with CDKN1B expression, and most of the prosensory domain, as assessed by CDKN1B expression, was JAG1-negative. Along with cochlear development, the JAG1-positive region progressed to the lateral side. In the later stages of development, Notch signaling is believed to divert the development of the prosensory domain into hair cells via lateral inhibition [26]. This preservation of expression pattern may suggest that lateral inhibition of cochlear development is well-preserved in mammals.
For investigating the early patterning of the cochlear duct, we used OTX1 and OTX2 as dorsal markers of the developing cochlear duct, GATA3 and ISL1 as ventral markers, and PAX2 as a lateral marker.
OTX1 and OTX2 encode members of the bicoid subfamily of homeodomain-containing transcription factors, which are required for the normal development of the inner ear [31]. In mouse cochlea, co-expression of OTX1 and OTX2 can be observed in the roof of the cochlear duct until E13, whereas OTX1 expression was not detected in the cochlea at E14 [31]. In contrast, OTX2 expression was observed in the non-sensory area of the roof of the cochlear duct, corresponding to the Reissner's membrane [56]. In the common marmoset, co-expression of OTX1 and OTX2 was detected in the E70 cochlea; however, after E77, only OTX2 expression was observed in the dorsal part of the cochlear duct.
GATA3 encodes a protein that belongs to the GATA family of transcription factors. In the developing cochlea of mouse, GATA3 expression has been reported in the cochlear sensory epithelium and spiral ganglion neurons [33, 34]. In the cochlear duct of the E13.5 mouse, GATA3 expression was observed in the prosensory domain and the outer sulcus. ISL1 encodes a member of the LIM/homeodomain family of transcription factors. In the developing cochlea of mouse, ISL1 expression has also been observed in the cochlear sensory epithelium and spiral ganglion neurons [35, 36]. Reports show that ISL1 expression is dispensable for the development of the mouse cochlear prosensory region [37]. In the common marmoset, we detected the expression of both GATA3 and ISL1 in the developing cochlear duct. Expression of both was observed in the ventral cochlear sensory epithelium during the periods examined in this study.
PAX2 encodes a paired box protein, which is essential for normal inner ear morphogenesis [32]. In the developing cochlea of mouse, PAX2 expression was reported in the lateral wall of the cochlear duct and presumptive stria vascularis [32]. PAX2 expression in hair cells has also been reported in the developing cochlea of mice [43] and humans [57]. In the common marmoset, we detected the expression of PAX2 in the developing cochlear duct. Relatively broad PAX2 expression was detected in the E70 cochlea; however, PAX2 expression was restricted to the lateral wall of the cochlear duct in E77. In E87 and E97, PAX2 expression was observed on the lateral side of the cochlear epithelium and hair cells.
In addition, we examined the expression patterns of PAX8 (paired box 8) and DLX5 (distal-less homeobox 5). PAX8 encodes a paired box protein and DLX5 encodes a member of the homeobox transcription factor gene family. Both PAX8 and DLX5 are important for the specification of the otocyst and subsequent cochlear development [58, 59]. PAX8 expression has been reported in the otic placode, where it cooperates with PAX2 for inner ear morphogenesis and innervation in mice [58]. DLX5 expression has also been reported in developing otic placodes, and it is essential for the morphogenesis of the semicircular canal [59, 60]. In the common marmoset, we did not detect the expression of either PAX8 or DLX5 in the developing cochlear duct at E70, whereas they were expressed in some of the vestibular organs (Fig. 5F and G).
Although the expression patterns of genes investigated in this study were well conserved between the species, inter-species differences were observed in the expression patterns of genes related to prosensory domain specification that proceed simultaneously with this patterning.
So far, relatively large inter-species differences between rodents and primates have been reported with respect to the development of spiral ganglion neurons [5, 7]. For example, unlike in rodents [5, 61], in which PRPH expression is detected in relatively late stages of cochlear development, PRPH was expressed in spiral ganglion neurons prior to hair cell innervation in humans and common marmosets [5, 7]. In this study, we investigated the early development of spiral ganglion neurons in the common marmoset.
NEUROD1, a member of the neuroD family of basic helix-loop-helix (bHLH) transcription factors, is also known as one of the proneural genes responsible for the development of neuroectodermal progenitor cells. NeuroD expression has been reported in the developing mouse cochlea [39], and it is essential for neuronal differentiation of spiral ganglion neurons [38, 39]. In mice, NeuroD expression was detected in the spiral ganglion in E9.5, and its expression was observed until E13.5 [40]. POU4F1 encodes a member of the POU-IV class of neural transcription factors, POU4F1, also known as brain-specific homeobox/POU domain protein 3A (BRN3A). POU4F1 regulates soma size, target field innervation, and axon pathfinding of spiral ganglion neurons [41]. In mice, POU4F1 expression was observed as early as E9.5, and it was downregulated in the spiral ganglion neurons in E17.5 [42]. RBFOX3 encodes a member of the RNA-binding FOX protein family, which is involved in the regulation of alternative splicing of pre-mRNA. RBFOX3 is also known as the neuronal nuclei (NeuN) antigen, which has been widely used as a marker for post-mitotic neurons [62, 63]. RBFOX3 expression has been reported in post-mitotic mature spiral ganglion neurons after E11.5 in mice [42]. The sequential changes in the expression of NEUROD1, POU4F1, and RBFOX3 during cochlear development are well conserved between mice and common marmosets.
Studies have shown that ISL and GATA3 are essential for the proper development of spiral ganglion neurons and sensory epithelium [34]. In the mouse cochlea, Isl1 expression was observed in the inner ear neuronal lineage from embryonic day E8.5 to E9.5, similar to the expression of NeuroD [36]. In contrast, at E9.5, GATA3 expression was detected only in the otic epithelium, and no expression was detected in the neuronal lineage [43]. GATA3 expression was first observed in the selected neuronal lineage at E12.5, which increased all over the spiral ganglion neurons after E13.5 [34]. Previous studies have shown that GATA3 is essential not for differentiation but for the survival of spiral ganglion neurons [34]. MAFB encodes MAFB, a basic leucine zipper (bZIP) transcription factor. MAFB acts downstream of GATA3 to control the differentiation of spiral ganglion neurons and drive the differentiation of the large post-synaptic density characteristic of the ribbon synapse [44]. The sequential expression patterns of ISL1, GATA3, and MAFB were conserved between mice and marmosets.
Finally, we compared the timing of integration of glial cells into the spiral ganglion neuron between the species. In mouse cochlea, SOX10-positive glial cells originating from neural crest cells were observed in the spiral ganglion neurons at E12.75. At this stage, the SOX10-positive glial cells wrapped the spiral ganglion neurons [64]. and also expressed SOX2. As development progressed, the SOX10-expressing cells were integrated throughout the spiral ganglion at E17.5 [64].
The satellite glial cells of the developing cochlea of the human fetus express S100B, which is a member of the S100 family of proteins containing two EF-hand calcium-binding motifs [5]. In humans, both SOX10- and S100B-positive glial cells can be observed in 9 weeks gestation fetuses [47], which integrate into the spiral ganglion neurons and envelope them between 12 and 14 weeks of gestation in humans [47].
In common marmoset, the integration of satellite glial cells was observed at E87 (approximately 12 weeks of gestation in human and the E15 mouse cochlea). Our observation indicated that glial cell integration of the common marmoset is more similar to human, and this integration was observed before hair cell differentiation in the primate. This timing was relatively earlier than that in mice [64].
Inter-species difference of cochlear development between rodents and primates
The time course of early cochlear development in the common marmoset is summarized in Fig. 14. The essential developmental steps of early cochlear development include elongation of the cochlear duct, specification of the sensory epithelium, and differentiation of hair cells. This schema indicates the temporal relationship between the crucial steps.
Schematic diagrams showing early cochlear development. The essential developmental steps of early cochlear development in the common marmoset investigated in this study are indicated
This study confirmed that most of the developmental steps of the common marmoset were conserved in humans or mice. However, several inter-species differences were observed as summarized in the schema (Fig. 15). Two of the most prominent inter-species differences observed in this study were the tempo of the cochlear development, and expression patterns of the genes associated with the sensory epithelium specifications.
Schematic diagrams showing early cochlear development comparing common marmoset, mouse, and human. This study confirmed that most of the early cochlear developmental steps of the common marmoset were conserved in humans or mice. Gene expression patterns in hair cells development and neural development were well-conserved between the species. However, several inter-species differences were also observed. Expression patterns of the genes associated with the sensory epithelium specifications (CDKN1B and JAG1) showed more significant inter-species differences between the common marmoset and mouse. In marmosets, the progression of differentiation of hair cells from basal to apical turn is about three times slower than in mice
So far, although the inter-species differences in the tempos of cochlear development have been underestimated, they may be essential for organogenesis from pluripotent stem cells or organ regeneration [65]. In this study, it was observed that cochlear elongation (from E70 to E92) takes about three weeks, and differentiation of hair cells from basal to apical turn takes about two weeks (from E87 to E101). In contrast, in mice, it has been reported that cochlear elongation took about one week (from E10.75 to E17), [53] and hair cells differentiation needed four days (from E13.5 to E17.5) [19]. This comparison indicates that cochlear development is approximately three times slower in the common marmoset than in mice. Overall, the tempo of cochlear development of the common marmoset was more similar to that of humans than that of mice. In general, the body pattern formation of an embryo, known as segmentation, progresses more slowly in humans than in mice [66, 67]. This process is governed by an oscillating biochemical process known as the segmentation clock [68]. Recently, the inter-species difference in the segmentation clock has been investigated. Reports have shown that this inter-species difference in the tempo of the segmentation clock is caused by the difference in the rates of biochemical reactions; the synthesis and degradation of the critical molecules are two or three times slower in humans than in mice [66]. Our observation indicates that a similar phenomenon that explains the inter-species differences in the tempos of the segmentation clock may also underlie cochlear development.
The inter-species differences in the expression patterns of prosensory domain-specific genes are another prominent feature of this study. The molecular mechanism of prosensory domain specification and subsequent hair cell differentiation is of considerable interest in the field of hearing research, as regeneration of hair cells retracing the developmental steps may be utilized for designing therapy for hearing loss [69, 70]. Our data imply that the procedures used for manipulating rodent cochlear sensory cells cannot be directly used to research primate cells due to the intrinsic inter-species differences in the cell fate determination program. More detailed studies are needed on the species-specific mechanisms that determine sensory cell fate during cochlear development in primates.
We determined the time course of the essential developmental stages of early cochlear development in the common marmoset, a non-human primate model. In addition, we clarified the similarities and differences in the expression patterns of developmental proteins in the cochlea between previously established rodent models and the primate model. Our data will be helpful for studying early cochlear development in primates and human.
All data generated or analysed during this study are included in this published article.
PRPH:
Peripherin
Na-K-Cl cotransporter 1
POU4F3:
POU class 4 homeobox 3
ATOH1:
Atonal bHLH transcription factor 1
SOX2:
SRY-box transcription factor 2
CDKN1B:
Cyclin dependent kinase inhibitor 1B
JAG1:
Jagged canonical Notch ligand 1
OTX1:
Orthodenticle homeobox 1
PAX2:
Paired box 2
GATA3:
GATA binding protein 3
ISL1:
ISL LIM homeobox 1
DLX5:
Distal-less homeobox 5
NEUROD1:
Neuronal differentiation 1
RBFOX3:
RNA binding fox-1 homolog 3
MAFB:
MAF bZIP transcription factor B
SOX10:
SRY-box transcription factor 10
S100B:
S100 calcium binding protein B
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We thank Saki Ninomiya for providing technical support.
MH was supported by a grant from the Japanese government, MEXT KAKENHI (Grant-in-Aid for Scientific Research (B) 20H03836, Grant-in-Aid for Challenging Research (Exploratory) 21K19581), the Keio Medical Association and Keio University Medical Science Fund, the Society for Promotion of International Oto-Rhino-Laryngology (SPIO), the Kanae Foundation for the Promotion of Medical Science, the Takeda Science Foundation, and Keio Gijuku Academic Development Funds. This study was also partially supported by grants to MF from the Japanese government MEXT KAKENHI (Grant-in-Aid for Scientific Research (A) 18H04065, 19H05473, 21H04839 ), Keio Gijuku Academic Development Funds, the foundation of Mitsubishi Tanabe, and the Takeda Science Foundation. This study was also partially supported by a grant for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) JP21dm0207001.
Masato Fujioka
Present address: Department of Molecular Genetics, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0374, Japan
Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, 160-8582, Japan
Makoto Hosoya, Masato Fujioka & Hiroyuki Ozawa
Laboratory for Marmoset Neural Architecture, Center for Brain Science, RIKEN, 2-1 Hirosawa Wako, Saitama, 351-0193, Japan
Junko Okahara, Sho Yoshimatsu & Hideyuki Okano
Department of Marmoset Biology and Medicine, Central Institute for Experimental Animals, 3-25-12 Tonomachi Kawasaki-ku Kawasaki, Kanagawa, 210-0821, Japan
Junko Okahara
Department of Physiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, 160-8582, Japan
Sho Yoshimatsu & Hideyuki Okano
Makoto Hosoya
Sho Yoshimatsu
Hideyuki Okano
Hiroyuki Ozawa
M.H., M.F., J. O., S.Y., H.Okano, and H.Ozawa, conceived and designed the experiments. M.H. and M.F. wrote the manuscript. M.H. performed most of the experiments and analyzed the data. All authors read and approved the final version of the manuscript.
Correspondence to Masato Fujioka.
The animal experiments were approved by the Animal Experiment Committee of Keio University (number: 11006, 08020) and were performed in accordance with the guidelines of the National Institutes of Health and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
H. Okano is a founding scientist, and a paid scientific advisory board member of San Bio Co., Ltd. MH and MF were founding scientists of Otolink Inc.
Hosoya, M., Fujioka, M., Okahara, J. et al. Early development of the cochlea of the common marmoset, a non-human primate model. Neural Dev 17, 6 (2022). https://doi.org/10.1186/s13064-022-00162-8
Sensory epithelium
Cochlear development


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Nonprofit work is essential and effective. It is not simple or straightforward. With numerous stakeholders, a considerable number of regulations and complex cultural norms, nonprofits operate within a unique and often challenging framework.
Successful Nonprofits navigate this path by being clear in their capacity, intentional in their work, and entrepreneurial in their outlook. Sustainable Strategic Planning will help you achieve this operational model.
Businesses can meet their corporate social responsibility goals by outsourcing to nonprofits embedded in the desired impact field. Shifting from a one-off sponsorship mindset to long-term, intentional partnerships sets up win-win scenarios for both parties and exponentially increases the impact to the social field. Again, clear goals and an entrepreneurial outlook are fundamental to success.
Anne Willson works with nonprofit organizations and businesses that partner with nonprofits to strengthen their social impact. With a grounded, real-world perspective gained through twenty-seven years in the nonprofit sector as well as in small business and with a public university, Anne addresses core issues relevant to compelling community engagement: strategic planning focused on impact, organizational integrity, identifying and leveraging social assets, and convening multiple constituents and interests to work within a collaborative framework. In addition, she works with nonprofit organizations on identifying and leveraging assets for earned income streams. Anne lives in Cary, NC.
Anne's artwork may be found at annewillsonart.com.


A renowned Indian-American cardiologist Suresh Gadasalli, who performed the world's first simultaneous hybrid revascularisation in 2005, was killed by his friend and business associate who then committed suicide in Odessa, Texas according to police.
Dr Gadasalli, 53, was killed by his patient and business associate Ayyasamy Thangam at the 'Healthy Heart Centre' on Thursday, according to a release issued by the Odessa Police Department.
The 60-year old then closed the door of the room the two men were in, and committed suicide.According to corporal Steve LeSueur, the two men were close friends and business associates.
Dr Gadasalli's brother-in-law said that the two families knew each other for a decade, and that the last rites will be held in Odessa likely on Wednesday.
LeSueur on Friday declined to release a possible motive for the killings and the police had no information on why Thangam was being treated.
Dr. Gadasalli was born in Bangalore and studied the Jawaharlal Nehru Medical College in Belgaum, Karnataka, before enrolling in the Internal Medicine and Cardiology at the University of Wisconsin Medical School and the Sinai Samaritan Centre in Milwaukee.
He was recruited by Medical Centre Hospital in Odessa, Texas in 1994.
The Healthy Heart Centre profile mentions that Dr Gadasalli was recognised as a leading physician of the world by the International Association of Cardiologists.
He was named a "super doctor" by Texas Monthly in 2008.
In 2005, he performed the world's first simultaneous hybrid revascularisation, which involved two major procedures - coronary artery bypass and stent replacement, using a robotic system known as the Da Vinci Surgical System.
Gadasalli was previously investigated in a federal court case on a currency structuring charge.
The FBI began their investigation into the Healthy Heart Center in June of 2012.
He was accused of violating federal law in structuring transactions to avoid reporting requirements, according to previous reports.
According to court records, Gadasalli's case was closed on January 22, 2014.


At this level, students will work in various ways, independently, in pairs or in small groups on oral or written activities, developing their autonomy.
More advanced texts and works of youth literature will be studied and will sustain the vocabulary enrichment. More advanced grammar rules will be taught and the emphasis will be on more complex verb tenses.
Having acquired those skills, students will be encouraged to work on longer, more structured written expression and will practice writing their essays. Creativity in written expression will be strongly encouraged.
Promote independent thinking and work.


Organizations around the world are expanding increasingly demanding of ways to harness the power of the Internet to promote their product and services. Among one of the most substantial and also advantageous means of utilizing the Internet to own website traffic, leads as well as sales is through the web application development services available within a web development company. Actually, considering the launch of an online organization without factoring the benefits of web application development solutions is one of the fastest and also simplest methods to lose out on some genuinely incredible possibilities.
Web application development services usually drop under the web development umbrella, which likewise has the tendency to consist of web content development, web design, shopping development and also other associated services. The services that lay underneath that umbrella are most frequently offered by professionals and also experts who concentrate on that specific location. Just as you would certainly employ an expert concentrating on web design to build you a site, you would do the same for one offering web application development solutions.
In choosing a development company, customers have to take into consideration more than just the level of proficiency. There are several factors to consider consisting of the needs of the particular project in addition to the spending plan of the customer. While the procedure of discovering the best web application development services for your job can show long term as well as engaged, a terrific location to begin is by referencing portfolios to check previous job carried out by the provider.
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Posts in yorktown
Penniman, The Canary Girls and The Spanish Flu
April 20th, 2020 Posted by analytics History, Local History, Trish Thomas, williamsburg, yorktown No Comment yet
On land that is now occupied by Cheatham Annex, about six miles from Colonial Williamsburg, there was once a thriving little village known as Penniman. This village was originally founded in 1916 by the Dupont Company to make dynamite, but World War I changed their plan and the sole industry in Penniman became the manufacture of TNT.
The wages being paid were so high that people from all over came to work at Penniman's factories, yet local farmers found it hard to find laborers to work in the fields. The village had electricity, sewer, water and hard roads, all at a time when pigs roamed free on muddy Duke of Gloucester Street. The population grew to about 20,000 with 10,000 of them working in the plant.
Because so many men were in the military, women were the obvious choice for factory workers, encouraged to step out of their traditional roles and work at the plant to support "The Boys at the Front."
Stuff one for the Kaiser! became a recruiting slogan, and the women came. Their job was to load powdered TNT into artillery shells. The TNT was as finely powdered as talcum, and it didn't take long for it to poison the women. TNT poisoning turned their skin and nails dark yellow and turned their lips purple. The women were soon called "The Canary Girls." The skin and nail discoloration weren't the only effects; their bone marrow and their livers were damaged. The women suffered dizziness and nausea but the worst effect was that their immune systems were compromised.
The name Canary Girls was not a compliment or a term of endearment; these women were shunned by the people in the village and by the other workers. The Canary Girls were not allowed to sit with other people in the cafeteria; they were "socially distanced" from everyone else.
In spite of the poisoning the work went on, the village boomed, townspeople had to wait in long lines for a seat at one of the local restaurants, the bank, and the post office. Penniman was the most modern town in rural Virginia.
It all came to a halt in 1918 when the Spanish Flu came to the Peninsula — and to Penniman.
Because of the pandemic, public gatherings became illegal, schools were closed to children and reopened as hospitals, and public funerals were outlawed. People were told to wear a gauze mask whenever they went outside.
On October 13, 1918 a reporter at the Daily Press newspaper on the Peninsula wrote, "A Williamsburg undertaker had to requisition a truck to haul bodies from Penniman this morning… There is a scarcity of coffins here, the dealers having in hand only a small stock prior to the grip of epidemic."
By October 18, 1918, around 6,000 Virginians had died of the Spanish Flu. On that day another reporter wrote that the undertakers in the Williamsburg area were being kept very busy, and that "baggage cars are always full of caskets."
Locals later recalled seeing coffins stacked to the ceiling at the rail depot. So many died that the plant ran out of burial space, forcing the bu
rial of many at a local undertaker's farm. There is no record of exactly how many died at Penniman; the only reference I have found was in a newspaper where a local reporter said the number he saw was "so large as to be unthinkable."
World War l ended November 11, 1918, the TNT plant at Penninman closed and the majority of employees still living were packed on trains and sent to their respective home towns. The employees who remained immediately started to dismantle the town. Equipment was sold as salvage; houses were moved or torn down.
By 1920 the village of Penniman was gone, disappeared as quickly as it had appeared, a victim of war, manmade calamity and widespread fatal disease.
Patricia Thomas, Williamsburg Walking Tours
Tags: canary girls, penniman tnt, spanish flu, williamsburg walking tours
Revolutionary War: Victory At Yorktown
August 2nd, 2017 Posted by admin revolutionary war, victory, yorktown No Comment yet
Past A Turning Point
For years General Washington had a strategy of survival. His mantra was that even if you couldn't win against the British in open battle, he could choose not to lose. What I mean by this is as long as the British armies could not openly defeat and decimate the Colonial Army, they could continue to fight on and on in a war of attrition. He knew the longer he could prolong the fighting, the greater the chance of victory as the British army would run low on energy, manpower, supplies and just a general will to win. In the end he was right. It did get to that point as he was able to recruit Allies to help the developing nation and overwhelm a weary British force.
September marks that turning point in the paths of the Colonial American colonies and the British Empire. And it was culminated with a brilliant strategic move by General Washington and General Rochambeau of France to trap the British at Yorktown. Wedging them between the Continental Army on the land and the French Armada by sea forced them to surrender as they became overwhelmed by artillery. The battle alone is a huge strategic victory over well trained, well disciplined forces that would govern a huge number of countries in the world all through the 19th century.
But it was something much bigger than that. We had delivered a titanic blow to the British Empire as they began their rise to Colonial Expansion worldwide. They would come to dominate the 19th century, with interests in Africa Asia and even North America.
It would certainly be a blow that the British were not going to forget anytime soon. In fact another war would break out in 1812 as the British actually invaded young America. Naturally, we resisted and were victors in this conflict as well. Truly the saying that freedom isn't free is accurate.
Years of struggle
Victory at Yorktown was not an easy one. The area stands as a monument to the eventual triumph of the colonies. But the years leading up to this outcome were marked by struggle and doubt. There was no guarantee that it would be an overwhelming victory like in Yorktown.
In fact when you see Colonial Williamsburg you see a beautiful microcosm of a gorgeous Colonial day. You don't see the day in and day out struggle that many of these colonists faced in wartime.
Our tours are designed to give you real history. History is not neat and orderly. It is a mark of struggle, sacrifice and is often brutal. Our tours give you the real dimensions of history and history is not gentile. This is a contrast from "historical marketing" or the reshaping of history to fit what certain groups want which you may have experienced elsewhere.
REVOLUTIONARY WAR – SEPTEMBER 1781
Your Bibliography: Revolutionarywararchives.org. (2017). Revolutionary War – September 1781. [online] Available at: http://www.revolutionarywararchives.org/49september1781.html[Accessed 21 Jul. 2017].
Tags: revolutionary war, september, struggle, williamsburg


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Johns Hopkins Medicine: Research
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Dada, Maqbool, Ph.D.
Dr. Maqbool Dada has a joint appointment in anesthesiology and critical care medicine at the Johns Hopkins University School of Medicine.Dr. Dada received his B.S. from the University of California at Berkeley. He earned his Ph.D. from the Massachuse...
Dadabhai, Alia Simjee, M.D.
Dr. Alia Dadabhai is an assistant professor of medicine at the Johns Hopkins University School of Medicine. Her areas of clinical expertise include hepatology and liver transplant evaluation. Her areas of clinical interest are: Viral hepatitis; ...
Dagnelie, Gislin, M.S., Ph.D.
Gislin Dagnelie, Ph.D., is an associate professor of ophthalmology at the Johns Hopkins University School of Medicine and the associate director of the Lions Vision Research and Rehabilitation Center at the Wilmer Eye Institute. His wo...
Dahoud, Wissam, M.D.
Dalal, Sonia Parimal, M.D.
Dalesio, Nicholas Michael, M.D., M.P.H.
Dr. Nicholas Michael Dalesio is an assistant professor of anesthesiology and critical care medicine and otolaryngology-head and neck surgery at the Johns Hopkins University School of Medicine. His area of clinical expertise is pediatric anesthesiolog...
D'Alessio, Franco Rafael, M.D.
Dr. D'Alessio focuses on caring for critically ill patients with particular interest in acute respiratory failure and patients with sepsis. His translational focus is in identifying changes in the immune cell landscape in pulmonary and critical...
Dalton, William Brian, M.D., Ph.D.
Dr. Dalton is an Assistant Professor of Oncology at The Johns Hopkins University School of Medicine. He is active in the Division of Hematology Malignancies, Leukemia Program at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins.&nb...
Damarla, Mahendra, M.D.
Dr. Damarla is a specialist in Pulmonary and Critical Care Medicine. He is an expert in the diagnosis and management of all forms of acute respiratory failure, including asthma, pneumonia, C.O.P.D., neuromuscular diseases, and the acute respiratory d...
Damico, Rachel Lynn, M.D., Ph.D.
Dr. Damico received her medical degree and doctoral degree in Molecular and Cellular biology from the University of Pennsylvania in 2000. She completed her residency in the Osler Internal Medicine training program at Johns Hopkins and continued on as...
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Is It Time for a Tablet?
This brings me to the oh-so-shiny-and-new tablet computers. Are they worth the money? Yes. Can you deduct the expense? At least part of it, for sure.
For the uninitiated, I should note that tablet computers have been around for years. Microsoft introduced a tablet version of Windows in 2001. I recall holding one of those early tablets, a thick slab of plastic that seemed like an overly weighty laptop with the keyboard ripped away—very unappealing. With the new iPad 2 and its elegant touch-screen interface and thousands of work and play apps, Apple has reconceived the entire platform. The company has (or will soon have) competition in this arena from BlackBerry, Motorola, Samsung, Dell, and Archos. But so far, none of their devices is as light or as thin as the 1.3-pound iPad 2. Without debating the merits of other tablets in this brief space (I do like several of them), I have two main reasons for my fondness for the iPad 2: the price—starting at $499, it's cheaper than most of the others—and the fact that some of its apps seem to be tailor-made for actors.
Like the iMovie app. For a mere $4.99, you can edit your reel and put it online using the iPad and its built-in Wi-Fi. No other tablet maker is offering anything that competes with this.
You can purchase or rent thousands of films and TV shows through the iTunes Store. You can also stream them from Netflix, at zero additional cost to members with plans starting at $8.99 per month. The iPad also makes an excellent e-book reader, with thousands of titles available for download. Play publisher Samuel French recently announced that it's actively pursuing the release of plays in digital format, something I think a lot of actors will welcome.
The iPad 2 is also a great PDF reader. This may sound mundane, but it's highly useful for show folk. In the last year, I was asked to participate in four play or screenplay readings, and in every case, the director or writer emailed me the script in PDF format and asked that I print a copy and bring it to rehearsal. At roughly 100 pages per script, I went through nearly a ream of paper and endless amounts of pricey black ink. I couldn't help but notice that one of the other actors showed up to one reading with his iPad in hand, from which he read the script. No paper, no ink, just a tiny carbon footprint. Color me leafy green.
I think it's reasonable to deduct some of the expense of your tablet on your tax return, as many of the device's uses are justifiably work-related. But how much? If you already deduct a percentage of your home Internet expenses and new computer purchases, a tablet easily fits into these categories. I'd have a chat with your accountant to determine the exact percentage to deduct.
By the way, refurbished original iPads—which lack the front- and rear-facing cameras of the iPad 2—are being offered on Apple's website and elsewhere (along with a one-year warranty, just as if they were new) at some nicely discounted prices.


The book of self-tanning is long and complex. You would think it should be easy, right? Lay out in sun, glow like a goddess on the French Riviera. But alas, the mission to gain a golden skin tone is as complex as finding a six-figure job in the middle of a recession.
Women drop hundreds of dollars a month to lie in the coffin-like tanning beds that threaten your physical health. Spray on tans like Mystic Tan are favored by wannabe-starlets like Paris Hilton, but skin can often turn out looking a bit more like a glass of Tang rather than gold. And while the budget-tanning method of lying out in the sun is wallet-friendly, the risks of burns and skin cancer are getting greater every day. So put down the SPF 600, and follow my journey to glow without risking my health, my wallet, and my common sense.
Tanning beds are the Holy Grail for proud Tanorexics. The phrase "GTL" (which stands for gym, tan, laundry) has become a national phenomenon thanks to the cast of Jersey Shore. But with the severe risks of skin cancer combined with skyrocketing prices, many people are looking to go in a different direction. We've seen spray on tanning systems but the results are often a little more…radioactive than planned.
In lieu of getting skin cancer, or looking like an Oompa Loompa, we tested a boatload of sunless tanners so you wouldn't have to.
I first tried Fake Bake: Sunless and Skinny with major reservations. I could only imagine preteens on the Jersey Shore slathering it on under their denim cutoffs. But would it be creepy for a full-fledge adult to use it? After building a darker base on my pale skin with drug store go-to, Jergens Natural Glow, I was hoping for something a little more lively. I slathered on Fake Bake with great enthusiasm, and ignored the pair of rubber gloves that come with the bottle. My immediate reaction was "Wow, this stuff really works!" and then, "Crap! I should have worn the gloves!" My hands looked quite dirty, despite scrubbing after application. The smell is what you'd imagine from self-tanner, but luckily your skin doesn't maintain the smell after application. After a few days of applying Sunless and Skinny, I loved the natural glow. In addition to its tanning abilities, Sunless and Skinny seeks to eliminate the visibility of cellulite and smooth skin. I wasn't expecting my cellulite to diminish instantly, but the golden tone does have a slimming effect. My skin's glow made me feel like a bronze goddess, except for the splotches of brown under my arms and on the upper thigh.
As one of the frontrunners in modern cosmetics, I was eager to test Lancome's Flash Bronzing Leg Gel. After showering and exfoliating, I applied Flash Bronzer all over my legs. Satisfied with the result, I decided to use it on my arms and chest. For a few days, I basked in the glory of my golden tan. I couldn't believe how easy it was. "Your skin tone is BEAUTIFUL!" a friend said to me after viewing a party photo of me. I began to plot out buying bottles of Flash Bronzer after my sample ran out. And then the honeymoon period ended. While visiting my parents in Connecticut, a family friend grabbed a glance of my neck. "Um….Do you have leprosy or something?" Okay, that wasn't quite the look I was going for. But after a few days of laying low and not applying the bronzer, it had begun to fade into a splotchy mess which my mother described as "looking like dirt." After some major exfoliating thanks to coarse sugar, soap, and a washcloth, I was able to get rid of most of the left over tanner. If you're willing to undergo a few days of splotchy skin after a few fabulous ones, Lancôme's sunless tanners are absolutely worth your money.
If you've cultivated a fake tan like I have, you're eager to protect it. Sol Survivor is an SPF 30 spray that provides protection if you're lying in the sun. While it acts as a traditional sunscreen in the face of UVA and UVB rays, it won't diminish your carefully cultivated Fake Bake. Like other sunscreens, it needs to be reapplied after swimming of drying off, but its easy to apply spray bottle is quick and painless. But whether you're just frolicking in the sun or looking to protect your tan, remember that no golden glow is worth risking your health. Even if you're looking to get seriously tan, protect your skin by slathering on some form of sunscreen before you fry like bacon. Skin cancer isn't attractive at any age.
For more information about Fake Bake: Sunless and Skinny and Sol Survivor, visit www.fakebake.com. For more information about Lancôme Flash Bronzer, visit www.lancome.com.


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2 injured, 1 arrested in Sandwich crash
Geoff Spillane
SANDWICH — A Forestdale man was arrested at Falmouth Hospital after a car crashed into a utility pole on Snake Pond Road on Sunday night, injuring him and the vehicle's driver.
The two occupants of the vehicle, who both had a strong odor of alcohol on their breath, were taken to the hospital for treatment, according to a police report.
The car snapped the utility pole, Sandwich fire Capt. Robert Black said.
The passenger in the vehicle, Zachary Fisher, 26, was charged with assault on a family or household member and assault and battery with a dangerous weapon, the report says. He pleaded not guilty to both charges Monday in Barnstable District Court.
The woman driving the vehicle said she and Fisher were arguing and he wanted to get out of the car and repeatedly opened the door while the vehicle was moving, the report says.
The woman said Fisher grabbed the steering wheel, pulling the vehicle to the left, causing the car to cross over the double yellow line and into an ongoing lane of traffic, eventually going off the road and hitting a fence, a mailbox and the utility pole, the report says.
Fisher told officers that the vehicle began to drift to the left while the woman was driving, and he grabbed the steering wheel in an attempt to pull the vehicle back into the lane, the report says.
Fisher is scheduled to return to court April 19 for a pretrial hearing.
— Staff Writer Christine Legere contributed to this report. Follow Geoff Spilane on Twitter: @GSpillaneCCT.


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3 Top Restaurant Industry Trends to Watch Post-COVID
by Sarah Schmidt, on June 24, 2021
The COVID-19 pandemic took a heavy toll on the restaurant industry. The National Restaurant Association called 2020 the most challenging year for the industry in its history. More than 110,000 eating and drinking establishments closed in the U.S. last year, and 2.5 million restaurant industry jobs were wiped out.
However, the devastating downturn experienced in 2020 and 2021 is anticipated to be short-term. Global foodservice revenues are projected to rise 3.7% per year to reach $4.4 trillion by 2024, according to forecasts from The Freedonia Group, a leading market research firm and division of MarketResearch.com.
1. Global Foodservice Market Primed for Growth
Freedonia identified a range of factors that will boost long-term restaurant market growth:
Continued adoption of advanced technologies to streamline operations, enhance customer experiences, and increase sales
Rising popularity of online food delivery and the expansion of delivery networks, including cloud kitchens (also known as ghost kitchens or virtual kitchens)
Healthy and sustainable eating trends driving higher-value sales in mature markets
Strong investment in the global expansion of multinational chains
Growing incomes in developing markets widening the local consumer base for restaurants
2. A Burst of Digital Adoption and Tech Innovation
Due to COVID, old analog systems had to be quickly transformed as restaurants pivoted to online, contactless ordering. The intense challenges of the pandemic and the cutthroat competition within the foodservice industry will continue to accelerate innovation and force restaurants to rely more on digital technology.
A common way to leverage technology is through the use of proprietary mobile apps that help brands to offer personalized incentives and foster repeat business. Foodservice brands are also expected to invest in automated and robotic technologies such as self-service kiosks, cashierless technologies, and restaurant robots to improve efficiency.
Burger King and McDonald's employ a range of technologies including data analytics, geofencing, and mobile apps to improve customer experiences, reduce labor costs, and optimize upsells.
3. Ethical Considerations and Sustainability Remain Key
Technology is not the only way restaurants can boost revenues. Offering higher-quality menu items and ethically sourced ingredients is another popular strategy.
Particularly in mature markets, consumers are increasingly looking for foodservice options that minimize environmental impacts and align with their healthy eating lifestyles. The rising influence of generation Z and millennial consumers is also expected to reinforce the importance of corporate social responsibility and sustainability.
As a result, more and more brands are focusing on:
Healthy, high-quality menu items
Diet-friendly options such as keto, vegan, or vegetarian
Responsibly sourced and certified ingredients, including non-GMO and plant-based
Sustainable business practices to reduce waste
Chipotle, Panera Bread, and Starbucks are all examples of this trend; their menus emphasize high ingredient quality through ethical sourcing such as cage-free, pasture-raised, and antibiotic-free livestock. Subway also centers its branding on health and wellness with the "Eat Fresh" tagline.
Where to Learn More
Need more restaurant industry statistics and analysis for 2020, 2021, and beyond? Read The Freedonia Group's in-depth report Global Foodservice with COVID-19 Market Impact Analysis, which covers full-service restaurants, quick-service and fast-casual restaurants, other eating and drinking places (such as bakeries, bars/pubs, coffee shops, and sandwich shops and cafes), and other foodservice establishments (such as airline foodservice, catering services, and hotel foodservice).
If you're looking for a report with a different scope or focus area, search MarketResearch.com's vast collection of restaurants market research from leading research firms.
About the author: Sarah Schmidt is a Managing Editor at MarketResearch.com, a leading provider of global market intelligence products and services.
Topics: Food & Beverage Industry Insights The Freedonia Group
10 Global Industries That Will Boom in the Next 5 Years
How to Conduct an Effective Voice of Market Study
7 Ways to Identify New Market Opportunities in a Changing World
Top 10 Reasons Why You Need Market Research