Molecular mechanisms of hearing loss in Nager syndrome.

Nager syndrome is a rare human developmental disorder characterized by hypoplastic neural crest-derived craniofacial bones and limb defects. Mutations in SF3B4 gene, which encodes a component of the spliceosome, are a major cause for Nager. A review of the literature indicates that 45% of confirmed cases are also affected by conductive, sensorineural or mixed hearing loss. Conductive hearing loss is due to defective middle ear ossicles, which are neural crest derived, while sensorineural hearing loss typically results from defective inner ear or vestibulocochlear nerve, which are both derived from the otic placode. Animal model of Nager syndrome indicates that upon Sf3b4 knockdown cranial neural crest progenitors are depleted, which may account for the conductive hearing loss in these patients. To determine whether Sf3b4 plays a role in otic placode formation we analyzed the impact of Sf3b4 knockdown on otic development. Sf3b4-depleted Xenopus embryos exhibited reduced expression of several pan-placodal genes six1, dmrta1 and foxi4.1. We confirmed the dependence of placode genes expression on Sf3b4 function in animal cap explants expressing noggin, a BMP antagonist critical to induce placode fate in the ectoderm. Later in development, Sf3b4 morphant embryos had reduced expression of pax8, tbx2, otx2, bmp4 and wnt3a at the otic vesicle stage, and altered otic vesicle development. We propose that in addition to the neural crest, Sf3b4 is required for otic development, which may account for sensorineural hearing loss in Nager syndrome.

Initial stages of development of the submandibular gland (human embryos at 5.5-8 weeks of development).

The aim of this study was to determine the main stages of submandibular salivary gland development during the embryonic period in humans. In addition, we studied submandibular salivary gland development in rats on embryonic days 14-16 and expression in the submandibular salivary gland region with the monoclonal antibody HNK-1. Serial sections from 25 human embryos with a greatest length ranging from 10 to 31 mm (Carnegie stages 16-23; weeks 5.5-8 of development) and Wistar rats of embryonic days (E) 14-16 were analysed with light microscopy. Five stages of submandibular salivary gland development were identified. The prospective stage (1), between weeks 5.5 and early week 6, is characterized by a thickening of the epithelium of the medial paralingual groove in the floor of the mouth corresponding to the primordium of the submandibular salivary gland parenchyma. At this stage, the primordium of the parasympathetic ganglion lies below the lingual nerve. The primordium of the submandibular salivary gland parenchyma is observed in rats on E14 in the medial paralingual groove with mesenchymal cells, underlying the lingual nerve. These cells are HNK-1-positive, corresponding to the primordium of the parasympathetic ganglion. The bud stage (2), at the end of week 6 in humans and on E15 in rats, is characterized by the proliferation and invagination of the epithelial condensation, surrounded by an important condensation of the mesenchyme. The pseudoglandular stage (3) at week 6.5 is characterized by the beginning of the formation of lobes in the condensed mesenchyme. The canalicular stage (4), between week 7 and 7.5, is characterized by the appearance of a lumen in the proximal part of the submandibular duct. The innervation stage (5) occurs during week 8, with the innervation of the submandibular and interlobular ducts. Nervous branches arriving from the parasympathetic ganglion innervate the glandular parenchyma. Numerous blood vessels are observed nearby. Our results suggest that submandibular salivary gland development requires interactions among epithelium, mesenchyme, parasympathetic ganglion and blood vessels.

Developmental abnormalities of the otic capsule and inner ear following application of prolyl-hydroxylase inhibitors in chick embryos.

BACKGROUND: Naturally hypoxic conditions in amniote embryos play important roles in normal development. We previously showed that a hypoxic condition is required to produce a sufficient amount of neural crest cells (NCCs) during embryogenesis and that promoting a hypoxic response by prolyl-hydroxylase (PHD) inhibitors increases NCCs. Given that PHD inhibitors are considered as a potential treatment for anemia and ischemic diseases, we investigated the phenotypic effect of PHD inhibitors on embryonic development. METHODS: Chick embryos were administered with PHD inhibitors prior to the induction of NCCs on day 1.5. Three main events relating to hypoxia, NCCs induction, vasculogenesis and chondrogenesis, were examined. RESULTS: PHD inhibitors caused an increase of Sox10-positive NCCs in vivo. Vasculogenesis was promoted temporarily, although rapid vasculogenesis diminished the effect by day 5 in cephalic and pharyngeal regions. Studies on chondrogenesis at day 7 showed advanced development of the otic capsule, a cartilaginous structure encapsulating the inner ear. Analysis by X-ray micro-computed-tomography (µCT) revealed smaller otic capsule, suggesting premature differentiation. This in turn, deformed the developing semicircular canals within it. Other skeletal structures such as the palate and jaw were unaffected. The localized effect on the otic capsule was considered a result of the multiple effects from the hypoxic responses, increased NCCs and promoted chondrogenesis. CONCLUSION: Given the wide range of clinical applications being considered for PHD inhibitors, this study provides crucial information to caution and guide use of PHD inhibitors when treating women of childbearing age.

Early Fetal Development of the Otic and Pterygopalatine Ganglia with Special Reference to the Topographical Relationship with the Developing Sphenoid Bone.

The otic and pterygopalatine ganglia are located close to the greater wing (alisphenoid) of the sphenoid bone and many researchers have noted nerves connecting these ganglia in human embryos. The greater wing (alisphenoid) arises from the cartilaginous ala temporalis independently of the lesser wing, but no topographical changes between this cartilage and nerve elements have been demonstrated. We examined histological sections of 20 human embryos and fetuses from 6 to 15 weeks of development (WD). At 6 WD, the ala temporalis, the alar process and ganglia were all identified as a single, undifferentiated cell mass. Subsequently, the two ganglia became identifiable, but were continuous on the superior side of the initial ala temporalis. The temporal, superior spine of the ala temporalis was surrounded by the part that connected the ganglia. At 7 WD, the superior spine of the ala temporalis was reduced in size and the continuity of these ganglia was lost. At this point, a secondarily-formed communicating branch between the ganglia, the nervus sphenoidalis was first identifiable. At 9 WD, the ala temporalis and the alar process had clearly become cartilages, and the anterior end of the otic ganglion was separated from the ala temporalis. The nervus sphenoidalis became longer. At 15 WD, the otic and pterygopalatine ganglia were clear separated from the alisphenoid, which consisted of the cartilaginous ala temporalis and membranous bone. Consequently, the separation between the otic and pterygopalatine ganglia seemed to be due to the developing ala temporalis. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Submandibular parasympathetic gangliogenesis requires sprouty-dependent Wnt signals from epithelial progenitors.

Parasympathetic innervation is critical for submandibular gland (SMG) development and regeneration. Parasympathetic ganglia (PSG) are derived from Schwann cell precursors that migrate along nerves, differentiate into neurons, and coalesce within their target tissue to form ganglia. However, signals that initiate gangliogenesis after the precursors differentiate into neurons are unknown. We found that deleting negative regulators of FGF signaling, Sprouty1 and Sprouty2 (Spry1/2DKO), resulted in a striking loss of gangliogenesis, innervation, and keratin 5-positive (K5+) epithelial progenitors in the SMG. Here we identify Wnts produced by K5+ progenitors in the SMG as key mediators of gangliogenesis. Wnt signaling increases survival and proliferation of PSG neurons, and inhibiting Wnt signaling disrupts gangliogenesis and organ innervation. Activating Wnt signaling and reducing FGF gene dosage rescues gangliogenesis and innervation in both the Spry1/2DKO SMG and pancreas. Thus, K5+ progenitors produce Wnt signals to establish the PSG-epithelial communication required for organ innervation and progenitor cell maintenance.

Prox1 identifies proliferating neuroblasts and nascent neurons during neurogenesis in sympathetic ganglia.

Neurogenesis in embryonic sympathetic ganglia involves neuroblasts that resume proliferation following neuronal differentiation. As cell cycle exit is not associated with neuronal differentiation, the identity of proliferating neuroblasts is incompletely understood. Here, we use sympathetic ganglia of chick embryos to define the timing of neurogenesis and neuroblast identity focusing on the expression and function of the transcription factor Prox1. We show that a large fraction of neuroblasts has initially withdrawn from the cell cycle at embryonic day 3 (E3), which is reflected by a high proportion of p27(+)/Islet1(+) neuroblasts (63%) and low numbers of EdU(+)/Islet1(+) cells (12%). The proportion of proliferating Islet1(+) neuroblasts, identified by EdU pulse labeling and by the absence of the postmitotic marker p27 increases to reach maximal levels at E5, when virtually all neuroblasts are in the cell cycle (95%). Subsequently, the proportion of EdU-labeled and p27(-) neuroblasts is reduced to reach low levels at E11. Interestingly, the expression of the transcription factor Prox1 is restricted to the neuronal lineage, that is, Sox10(+)/Phox2b(+) neuron progenitors, proliferating p27(-)/Islet1(+) neuroblasts and nascent neurons but is rapidly lost in postmitotic neurons. In vitro and in vivo knockdown and overexpression experiments demonstrate effects of Prox1 in the support of neuroblast proliferation and survival. Taken together, these results define the neurogenesis period in the chick paravertebral sympathetic ganglia including an initial cell cycle withdrawal and identify Prox1 as a marker and regulator of proliferating sympathetic neuroblasts.

Signaling via the transcriptionally regulated activin receptor 2B is a novel mediator of neuronal cell death during chicken ciliary ganglion development.

The TGF-ß ligand superfamily members activin A and BMP control important aspects of embryonic neuronal development and differentiation. Both are known to bind to activin receptor subtypes IIA (ActRIIA) and IIB, while in the avian ciliary ganglion (CG), so far only ActRIIA-expression has been described. We show that the expression of ACVR2B, coding for the ActRIIB, is tightly regulated during CG development and the knockdown of ACVR2B expression leads to a deregulation in the execution of neuronal apoptosis and therefore affects ontogenetic programmed cell death in vivo. While the differentiation of choroid neurons was impeded in the knockdown, pointing toward a reduction in activin A-mediated neural differentiation signaling, naturally occurring neuronal cell death in the CG was not prevented by follistatin treatment. Systemic injections of the BMP antagonist noggin, on the other hand, reduced the number of apoptotic neurons to a similar extent as ACVR2B knockdown. We therefore propose a novel pathway in the regulation of CG neuron ontogenetic programmed cell death, which could be mediated by BMP and signals via the ActRIIB.

RARß regulates neuronal cell death and differentiation in the avian ciliary ganglion.

Programmed cell death during chicken ciliary ganglion (CG) development is mostly discussed as an extrinsically regulated process, guided either by the establishment of a functional balance between preganglionic and postganglionic activity or the availability of target-derived neurotrophic factors. We found that the expression of the gene coding for the nuclear retinoic acid receptor ß (RARB) is transiently upregulated prior to and during the execution phase of cell death in the CG. Using retroviral vectors, the expression of RARB was knocked down during embryonic development in ovo. The knockdown led to a significant increase in CG neuron number after the cell death phase. BrdU injections and active caspase-3 staining revealed that this increase in neuron number was due to an inhibition of apoptosis during the normal cell death phase. Furthermore, apoptotic neuron numbers were significantly increased at a stage when cell death is normally completed. While the cholinergic phenotype of the neurons remained unchanged after RARB knockdown, the expression of the proneural gene Cash1 was increased, but somatostatin-like immunoreactivity, a hallmark of the mature choroid neuron population, was decreased. Taken together, these results point toward a delay in neuronal differentiation as well as cell death. The availability of nuclear retinoic acid receptor ß (RARß) and RARß-induced transcription of genes could therefore be a new intrinsic cue for the maturation of CG neurons and their predisposition to undergo cell death.

Fgf-signaling-dependent Sox9a and Atoh1a regulate otic neural development in zebrafish.

Fibroblast growth factors (Fgfs) play important roles in developmental processes of the inner ear, including the ontogeny of the statoacoustic ganglia (SAG) and hair cells. However, the detailed genetic mechanism(s) underlying Fgf/Fgfr-dependent otic neural development remains elusive. Using conditional genetic approaches and inhibitory small molecules, we have revealed that Fgfr-PI3K/Akt signaling is mainly responsible for zebrafish SAG development and have determined that Sox9a and Atoh1a act downstream of Fgfr-Akt signaling to specify and/or maintain the otic neuron fate during the early segmentation stage. Sox9a and Atoh1a coregulate numerous downstream factors identified through our ChIP-seq analyses, including Tlx2 and Eya2. Fgfr-Erk1/2 signaling contributes to ultricular hair cell development during a critical period between 9 and 15 hours postfertilization. Our work reveals that a genetic network of the previously known sensory determinant Atoh1 and the neural crest determinant Sox9 plays critical roles in SAG development. These newly uncovered roles for Atoh1and Sox9 in zebrafish otic development may be relevant to study in other species.

Neurodevelopment. Parasympathetic neurons originate from nerve-associated peripheral glial progenitors.

The peripheral autonomic nervous system reaches far throughout the body and includes neurons of diverse functions, such as sympathetic and parasympathetic. We show that the parasympathetic system in mice--including trunk ganglia and the cranial ciliary, pterygopalatine, lingual, submandibular, and otic ganglia--arise from glial cells in nerves, not neural crest cells. The parasympathetic fate is induced in nerve-associated Schwann cell precursors at distal peripheral sites. We used multicolor Cre-reporter lineage tracing to show that most of these neurons arise from bi-potent progenitors that generate both glia and neurons. This nerve origin places cellular elements for generating parasympathetic neurons in diverse tissues and organs, which may enable wiring of the developing parasympathetic nervous system.

Neurodevelopment. Parasympathetic ganglia derive from Schwann cell precursors.

Neural crest cells migrate extensively and give rise to most of the peripheral nervous system, including sympathetic, parasympathetic, enteric, and dorsal root ganglia. We studied how parasympathetic ganglia form close to visceral organs and what their precursors are. We find that many cranial nerve-associated crest cells coexpress the pan-autonomic determinant Paired-like homeodomain 2b (Phox2b) together with markers of Schwann cell precursors. Some give rise to Schwann cells after down-regulation of PHOX2b. Others form parasympathetic ganglia after being guided to the site of ganglion formation by the nerves that carry preganglionic fibers, a parsimonious way of wiring the pathway. Thus, cranial Schwann cell precursors are the source of parasympathetic neurons during normal development.

Differential effects of RET and TRKB on axonal branching and survival of parasympathetic neurons.

Interactions between neurons and their targets of innervation influence many aspects of neural development. To examine how synaptic activity interacts with neurotrophic signaling, we determined the effects of blocking neuromuscular transmission on survival and axonal outgrowth of ciliary neurons from the embryonic chicken ciliary ganglion. Ciliary neurons undergo a period of cell loss due to programmed cell death between embryonic Days (E) 8 and 14 and they innervate the striated muscle of the iris. The nicotinic antagonist d-tubocurarine (dTC) induces an increase in branching measured by counting neurofilament-positive voxels (NF-VU) in the iris between E14-17 while reducing ciliary neuron survival. Blocking ganglionic transmission with dihyro-ß-erythroidin and α-methyllycacontine does not mimic dTC. At E8, many trophic factors stimulate neurite outgrowth and branching of neurons placed in cell culture; however, at E13, only GDNF stimulates branching selectively in cultured ciliary neurons. The GDNF-induced branching at E13 could be inhibited by BDNF. Blocking ret signaling in vivo with a dominant negative (dn)ret decreases survival of ciliary and choroid neurons at E14 and prevents dTC induced increases in NF-VU in the iris at E17. Blocking TRKB signaling with dn TRKB increases NF-VU in the iris at E17 and decreases neuronal survival at E17, but not at E14. Thus, RET promotes survival during programmed cell death in the ciliary ganglion and contributes to promoting branching when synaptic transmission is blocked while TRKB inhibits branching and promotes maintenance of neuronal survival. These studies highlight the multifunctional nature of trophic molecule function during neuronal development.

The Sympathetic and Parasympathetic Contributions to the Cardiac Plexus: a Fetal Study / Contribuciones Simpáticas y Parasimpáticas al Plexo Cardíaco: Estudio Fetal

The cardiac plexus is formed by sympathetic nerves originating from the superior, middle, inferior cervical or cervicothoracic ganglia as well as from the first to the fifth thoracic ganglia. Furthermore, the vagus nerve and its counterpart, the recurrent laryngeal nerve supply the cardiac plexus with parasympathetic cardiac nerves. This investigation aimed to review and record the medial contributions of the cervical ganglia, first to fifth thoracic ganglia and medial contributions of the vagus and recurrent laryngeal nerves to the cardiac plexus. The study involved bilateral micro-dissection of forty cadaveric fetal specimens (n=80). The origins of sympathetic contributions to the cardiac plexus were described as either ganglionic, inter-ganglionic or from both the ganglion and the inter-ganglionic sympathetic chain. The number of cervical sympathetic ganglia varied from two to five in this study; the superior cervical ganglion was constant while the middle cervical, vertebral, inferior cervical or cervicothoracic ganglia were variable. The prevalence of cardiac nerves were as follows: superior cervical cardiac nerve (95%); middle cervical cardiac nerve (73%); vertebral cardiac nerve (41%); inferior cervical cardiac nerve (21%) and cervicothoracic cardiac nerve (24%). This investigation records the thoracic caudal limit of the thoracic sympathetic contributions to the cardiac plexus as the T5 ganglion. The findings of this study highlight the importance of understanding the medial sympathetic contributions and their variations to the cardiac plexus as this may assist surgeons during minimal access surgical procedures, sympathectomies, pericardiectomies and in the management of diseases like Raynaud's Phenomenon and angina pectoris.

El plexo cardíaco está formado por los nervios simpáticos procedentes de los ganglios cervicales superior, medio e inferior o cervicotorácico, así como los ganglios torácicos desde el primero al quinto. Por otra parte, el nervio vago y su contraparte, el nervio laríngeo recurrente suministra al plexo cardíaco nervios cardíacos parasimpático. Esta investigación tuvo como objetivo revisar y registrar las contribuciones mediales de los ganglios cervicales, ganglios torácicos del primero al quinto ganglios y contribuciones mediales de los nervios laríngeos recurrentes y vagos en el plexo cardíaco. Se realizó la micro-disección bilateral de cuarenta especímenes cadavéricos fetales (n = 80). Los orígenes de las contribuciones simpáticas hacia el plexo cardíaco se describen de forma independiente como ganglionar o inter-ganglionar, o desde ambos ganglios y la cadena simpática interganglionar. El número de ganglios simpáticos cervicales varió de dos a cinco; el ganglio cervical superior fue constante, mientras que los ganglios medio-cervical, vertebral, cervical inferior o cervicotorácico fueron variables. La prevalencia de los nervios cardíacos fueron: nervio cardíaco cervical superior (95%); nervio cardíaco cervical medio (73%); nervio cardiaco vertebral (41%); nervio cardíaco cervical inferior (21%) y nervio cardíaco cervicotorácico (24% ). La investigación registró el límite torácico caudal de las contribuciones torácicas simpáticos al plexo cardíaco como el ganglio T5. Los resultados de este estudio muestran la importancia de comprender las contribuciones simpáticas mediales y sus variaciones en el plexo cardíaco, ya que podrían ayudar a los cirujanos durante los procedimientos quirúrgicos mínimanente invasivos, simpatectomías, pericardiectomías y en el manejo de enfermedades como el fenómeno de Raynaud y la angina de pecho.

Development of cardiac parasympathetic neurons, glial cells, and regional cholinergic innervation of the mouse heart.

Very little is known about the development of cardiac parasympathetic ganglia and cholinergic innervation of the mouse heart. Accordingly, we evaluated the growth of cholinergic neurons and nerve fibers in mouse hearts from embryonic day 18.5 (E18.5) through postnatal day 21(P21). Cholinergic perikarya and varicose nerve fibers were identified in paraffin sections immunostained for the vesicular acetylcholine transporter (VAChT). Satellite cells and Schwann cells in adjacent sections were identified by immunostaining for S100ß calcium binding protein (S100) and brain-fatty acid binding protein (B-FABP). We found that cardiac ganglia had formed in close association to the atria and cholinergic innervation of the atrioventricular junction had already begun by E18.5. However, most cholinergic innervation of the heart, including the sinoatrial node, developed postnatally (P0.5-P21) along with a doubling of the cross-sectional area of cholinergic perikarya. Satellite cells were present throughout neonatal cardiac ganglia and expressed primarily B-FABP. As they became more mature at P21, satellite cells stained strongly for both B-FABP and S100. Satellite cells appeared to surround most cardiac parasympathetic neurons, even in neonatal hearts. Mature Schwann cells, identified by morphology and strong staining for S100, were already present at E18.5 in atrial regions that receive cholinergic innervation at later developmental times. The abundance and distribution of S100-positive Schwann cells increased postnatally along with nerve density. While S100 staining of cardiac Schwann cells was maintained in P21 and older mice, Schwann cells did not show B-FABP staining at these times. Parallel development of satellite cells and cholinergic perikarya in the cardiac ganglia and the increase in abundance of Schwann cells and varicose cholinergic nerve fibers in the atria suggest that neuronal-glial interactions could be important for development of the parasympathetic nervous system in the heart.

Reconsideration of the autonomic cranial ganglia: an immunohistochemical study of mid-term human fetuses.

The cranial parasympathetic ganglia have been reported to paradoxically contain the sympathetic nerve marker, tyrosine hydroxylase (TH), in addition to neurons expressing parasympathetic markers such as vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS). However, the distribution of these molecules in the cranial ganglia of human fetuses has not yet been examined. Using paraffin sections from 10 mid-term human fetuses (12-15 weeks), we performed immunohistochemistry for TH, VIP, and nNOS in the parasympathetic ciliary, pterygopalatine, otic, and submandibular ganglia, and for comparison, the sensory inferior vagal ganglion. The ciliary and submandibular ganglia contained abundant TH-positive neurons. In the former, TH-positive neurons were much more numerous than nNOS-positive neurons, whereas in the latter, nNOS immunoreactivity was extremely strong. No or a few cells in the pterygopalatine, otic, and inferior vagal ganglia expressed TH. Ciliary TH neurons appeared to compensate for classically described sympathetic fibers arising from the superior cervical ganglion, whereas in the submandibular ganglion, nNOS-positive neurons as well as TH neurons might innervate the lingual artery in addition to the salivary glands. Significant individual variations in the density of all these markers suggested differences in sensitivity to medicine affecting autonomic nerve function. Consequently, in the human cranial autonomic ganglia, it appears that there is no simple dichotomy between sympathetic and parasympathetic function.

Differential contribution of Neurog1 and Neurog2 on the formation of cranial ganglia along the anterior-posterior axis.

BACKGROUND: The neural crest (NC) and placode are transient neurogenic cell populations that give rise to cranial ganglia of the vertebrate head. The formation of the anterior NC- and placode-derived ganglia has been shown to depend on the single activity of either Neurog1 or Neurog2. The requirement of the more posterior cranial ganglia on Neurog1 and Neurog2 is unknown. RESULTS: Here we show that the formation of the NC-derived parasympathetic otic ganglia and placode-derived visceral sensory petrosal and nodose ganglia are dependent on the redundant activities of Neurog1 and Neurog2. Tamoxifen-inducible Cre lineage labeling of Neurog1 and Neurog2 show a dynamic spatiotemporal expression profile in both NC and epibranchial placode that correlates with the phenotypes of the Neurog-mutant embryos. CONCLUSION: Our data, together with previous studies, suggest that the formation of cranial ganglia along the anterior-posterior axis is dependent on the dynamic spatiotemporal activities of Neurog1 and/or Neurog2 in both NC and epibranchial placode.

F-spondin regulates neuronal survival through activation of disabled-1 in the chicken ciliary ganglion.

The extracellular membrane-associated protein F-spondin has been implicated in cell-matrix and cell-cell adhesion and plays an important role in axonal pathfinding. We report here that F-spondin is expressed in non-neuronal cells in the embryonic chicken ciliary ganglion (CG) and robustly promotes survival of cultured CG neurons. Using deletion constructs of F-spondin we found that the amino-terminal Reelin/Spondin domain cooperates with thrombospondin type 1 repeat (TSR) 6, a functional TGFß-activation domain. In ovo treatment with blocking antibodies raised against the Reelin/Spondin domain or the TSR-domains caused increased apoptosis of CG neurons during the phase of programmed cell death and loss of about 30% of the neurons compared to controls. The Reelin/Spondin domain receptor - APP and its downstream signalling molecule disabled-1 are expressed in CG neurons. F-spondin induced rapid phosphorylation of disabled-1. Moreover, both blocking the central APP domain and interference with disabled-1 signalling disrupted the survival promoting effect of F-spondin. Taken together, our data suggest that F-spondin can promote neuron survival by a mechanism involving the Reelin/Spondin and the TSR domains.