Sonic Hedgehog Rescues Cranial Neural Crest from Cell Death Induced by Ethanol Exposure

Total Page:16

File Type:pdf, Size:1020Kb

Sonic Hedgehog Rescues Cranial Neural Crest from Cell Death Induced by Ethanol Exposure Sonic hedgehog rescues cranial neural crest from cell death induced by ethanol exposure Sara C. Ahlgren*, Vijaya Thakur, and Marianne Bronner-Fraser Division of Biology 139-74, California Institute of Technology, Pasadena, CA 91125 Communicated by Eric H. Davidson, California Institute of Technology, Pasadena, CA, June 14, 2002 (received for review March 14, 2002) Alcohol is a teratogen that induces a variety of abnormalities Materials and Methods including brain and facial defects [Jones, K. & Smith, D. (1973) Embryos. Chicken eggs were obtained from local sources. Eggs Lancet 2, 999-1001], with the exact nature of the deficit depending were incubated at 37°C with constant humidity. In preliminary on the time and magnitude of the dose of ethanol to which experiments, unopened eggs were injected with 200 ␮lofa developing fetuses are exposed. In addition to abnormal facial solution of 10% ethanol in Ringer’s solution, or with Ringer’s structures, ethanol-treated embryos exhibit a highly characteristic solution alone, at 26 h of incubation. This procedure has been pattern of cell death. Dying cells are observed in the premigratory previously demonstrated to result in craniofacial abnormalities and migratory neural crest cells that normally populate most facial (6). We then modified this procedure, such that eggs were structures. The observation that blocking Sonic hedgehog (Shh) opened at stage 9–10, and 20 ␮l of a 1% ethanol solution (in signaling results in similar craniofacial abnormalities prompted us Ringers) was placed between the vitelline membrane and the to examine whether there was a link between this aspect of fetal embryo. Both procedures resulted in similar cranial neural crest alcohol syndrome and loss of Shh. We demonstrate that adminis- cell death and loss of frontonasal tissues. Embryos were col- tration of ethanol to chick embryos results in a dramatic loss of Shh, lected at the desired time, and either harvested in TRIzol (Life as well as a loss of transcripts involved in Shh signaling pathways. Technologies, Grand Island, NY) for RNA isolation or fixed in In contrast, other signaling molecules examined do not demon- 4% paraformaldehyde (PFA) for either cell death analysis or in strate such dramatic changes. Furthermore, we demonstrate that situ hybridization. Whole-mount in situ hybridizations were both the ethanol-induced cranial neural crest cell death and the performed as described (11). Whole mount HNK-1 staining was associated craniofacial growth defect can be rescued by application performed by soaking fixed embryos in HNK-1 supernatant of Shh. These data suggest that craniofacial anomalies resulting (Development Studies Hybridoma Bank, Iowa City), followed by from fetal alcohol exposure are caused at least partially by loss of overnight incubation in 1:100 goat anti-mouse IgM–AP Shh and subsequent neural crest cell death. (Zymed), using BCIP (5-bromo-4-chloro-3-indolyl phosphate)͞ NBT (nitroblue tetrazolium chloride) to visualize. he secreted protein Sonic hedgehog (Shh) is essential for Tmultiple events during embryogenesis, including proper Cranial Neural Crest Cell Death. Fixed embryos were cryoprotected craniofacial development (1–4). Of the vertebrate homologues in sucrose, then embedded in gelatin and sectioned on a cryostat of the Drosophilia segment polarity gene Hedgehog, Shh has the at 10 ␮m. Sections were briefly heated to 42°C to remove the widest expression pattern in the developing head, including the gelatin, then blocked in 20% heat-inactivated goat serum in a presence of transcripts in the developing branchial arches during solution of PBSϩBSA ϩ 0.1% Triton, with all further dilutions the time of neural crest migration (5). SHH mutations in both also being made in this solution. The sections were incubated mice and humans cause significant defects in head development, overnight at 4°C in HNK-1 supernatant (1:10), followed by including distinctive facial and brain abnormalities (3, 4). Anal- anti-mouse IgM–FITC (1:500; Zymed). Finally, the sections ogously, inhibition of Shh-signaling during facial development were incubated for 10 min in 10 ␮g͞ml of the nuclear marker causes neural crest cell death and a subsequent reduction in head 4Ј,6-diamidino-2-phenylindole (DAPI) made in water, then cov- size (1). erslips were applied. The percentage of dead cranial crest cells There are several intriguing similarities in the craniofacial was determined as described (1). All statistics were performed phenotypes observed in embryos treated with ethanol and those using STATVIEW software. treated with antibodies that block Shh signaling. Both treatments result in a reduction in head size and selective apoptosis of Size Measurements. Embryos were photographed at fixed mag- cranial neural crest cells. The neural crest cell death observed nifications, which changed at different ages and with different after blocking Shh signaling in the chick (1) is similar to that head positions. The digital photographs were analyzed using the observed after ethanol exposure in a number of species including measuring tool available in PHOTOSHOP software (Adobe Sys- mouse, chick, and quail, both in vivo and in vitro (6–10). tems, Mountain View, CA). However, the mechanisms by which alcohol affects selective target tissues remain unknown. Hybridoma Treatments. Stage 9–10 chick embryos were injected In this study, we investigate the impact of embryonic exposure with a function-blocking anti-Shh antibody (Development Stud- to ethanol on Shh signaling during the critical period of early ies Hybridoma Bank) as described (1). cranial neural crest migration. After ethanol exposure, we noted a reduction in the mRNA levels of Shh both by in situ hybrid- RT-PCR. RNA was pooled from 4–6 embryos per experiment and ization and reverse transcriptase (RT)-PCR, as well as a reduc- treated to remove genomic DNA, then cDNA was synthesized tion in the mRNA for a number of genes in the Shh signaling using SuperscriptII. Alternatively, RNA was collected from the cascade. We further demonstrate that cranial neural crest cells heads of 6–7 embryos per condition. The relative amounts of can be rescued from ethanol-induced cell death, and that the cDNA in each matched set was normalized for the presence of associated decrease in size of the frontonasal mass can be prevented by application of exogenous Shh. We have thus identified a molecular deficit associated with embryonic expo- Abbreviations: Shh, Sonic hedgehog; RT, reverse transcriptase; DAPI, 4Ј,6-diamidino-2- sure to ethanol that in turn represents a link to the etiology of phenylindole. craniofacial defects. *To whom reprint requests should be addressed. E-mail: [email protected]. 10476–10481 ͉ PNAS ͉ August 6, 2002 ͉ vol. 99 ͉ no. 16 www.pnas.org͞cgi͞doi͞10.1073͞pnas.162356199 Downloaded by guest on October 2, 2021 Fig. 2. There is increased cranial neural crest cell death after ethanol Fig. 1. Ethanol treatment results in a reduction in the size of craniofacial tissues. treatment. Embryos were treated with ethanol at stage 9–10, collected 1 day (a and b) Embryos collected approximately 24 h after ethanol treatment (stage later, fixed, sectioned, and the sections stained for the neural crest marker 9–10) show distinct differences in craniofacial growth. The measurements for the HNK-1 (in green) and the nuclear marker DAPI (in blue). (a and b) A section frontonasal mass are indicated by the bar in each figure; control, 0.42 mm; taken from the optic cup (oc) region of a control embryo contains HNK-1- ethanol, 0.33 mm (see Table 1). (c–f) HNK-1 whole-mount analysis of embryos Ϸ1 positive neural crest cells (nc). (c and d) A section taken from the same region day after ethanol treatment demonstrates a reduction in the HNK-1-positive of an ethanol-treated embryo also contains HNK-1-positive neural crest cells. cranial neural crest. Streams of neural crest are indicated with arrows, and the ec, ectoderm; di, diencephalon. (e–eЈЈ). Magnified views of the area outlined region around the otic vesicle (ov) is enlarged in d and e. Although crest is clearly in a. A single pyknotic nucleus is present (arrow). This nucleus has an HNK-1- present in all appropriate regions, the intensity of staining appears reduced in the positive membrane (eЈ and eЈЈ). (f–fЈЈ). Magnified views of the area outlined whole-mount. HNK-1 staining of non-neural tissue, including the heart (h) ap- in c. Arrows point to three examples of pyknotic nuclei in f, all of which have pears unchanged. (f) The trunk region of ethanol-treated embryos did not HNK-1-positive membranes (fЈ). (g) Analysis of the pyknotic cells demonstrates display any neural crest abnormalities when stained with HNK-1. (g) Ethanol- that exposure to ethanol resulted in a dramatic increase in the percentage of treated (stage 9–10) embryos collected 3 days after treatment show distinct size the cranial neural crest cells (HNK-1-positive) that appear pyknotic, whereas differences of the frontonasal mass (Table 1). no change in the survival of the non-neural crest mesenchyme (HNK-1- negative) was observed. HNK-1-positive: control n ϭ 44; ethanol n ϭ 48. *, Unpaired t test, P Ͻ 0.0001. HNK-1-negative: control n ϭ 44; ethanol n ϭ 39, the ubiquitous gene GAPDH (12), using a Molecular Dynamics P not significant. (h) Merged image of DAPI and HNK-1 from the region PhosphorImager and IMAGEQUANT software (Molecular Dy- marked ‘‘h’’ in c, a region that contains almost no neural crest cells, and namics). GAPDH was used as a loading control for each PCR demonstrates no pyknotic nuclei. reaction. By the amounts of GAPDH and by visual assessment of 18S RNA, there did not appear to be a significant alteration in cShh, cFgf-8, or control constructs. The resulting supernatant the amount of RNA that was obtained from ethanol-treated versus control embryos.
Recommended publications
  • Fetal Blood Flow and Genetic Mutations in Conotruncal Congenital Heart Disease
    Journal of Cardiovascular Development and Disease Review Fetal Blood Flow and Genetic Mutations in Conotruncal Congenital Heart Disease Laura A. Dyer 1 and Sandra Rugonyi 2,* 1 Department of Biology, University of Portland, Portland, OR 97203, USA; [email protected] 2 Department of Biomedical Engineering, Oregon Health & Science University, Portland, OR 97239, USA * Correspondence: [email protected] Abstract: In congenital heart disease, the presence of structural defects affects blood flow in the heart and circulation. However, because the fetal circulation bypasses the lungs, fetuses with cyanotic heart defects can survive in utero but need prompt intervention to survive after birth. Tetralogy of Fallot and persistent truncus arteriosus are two of the most significant conotruncal heart defects. In both defects, blood access to the lungs is restricted or non-existent, and babies with these critical conditions need intervention right after birth. While there are known genetic mutations that lead to these critical heart defects, early perturbations in blood flow can independently lead to critical heart defects. In this paper, we start by comparing the fetal circulation with the neonatal and adult circulation, and reviewing how altered fetal blood flow can be used as a diagnostic tool to plan interventions. We then look at known factors that lead to tetralogy of Fallot and persistent truncus arteriosus: namely early perturbations in blood flow and mutations within VEGF-related pathways. The interplay between physical and genetic factors means that any one alteration can cause significant disruptions during development and underscore our need to better understand the effects of both blood flow and flow-responsive genes.
    [Show full text]
  • Dlx3b/4B Is Required for Early-Born but Not Later-Forming Sensory Hair Cells
    © 2017. Published by The Company of Biologists Ltd | Biology Open (2017) 6, 1270-1278 doi:10.1242/bio.026211 RESEARCH ARTICLE Dlx3b/4b is required for early-born but not later-forming sensory hair cells during zebrafish inner ear development Simone Schwarzer, Sandra Spieß, Michael Brand and Stefan Hans* ABSTRACT complex arrangement of mechanosensory hair cells, nonsensory Morpholino-mediated knockdown has shown that the homeodomain supporting cells and sensory neurons (Barald and Kelley, 2004; Raft transcription factors Dlx3b and Dlx4b are essential for proper and Groves, 2015; Whitfield, 2015). Inner ear formation is a induction of the otic-epibranchial progenitor domain (OEPD), as multistep process initiated by the establishment of the preplacodal well as subsequent formation of sensory hair cells in the developing region, a zone of ectoderm running around the anterior border of the zebrafish inner ear. However, increasing use of reverse genetic neural plate containing precursors for all sensory placodes (Streit, approaches has revealed poor correlation between morpholino- 2007). The preplacodal region is further specified into a common induced and mutant phenotypes. Using CRISPR/Cas9-mediated otic-epibranchial progenitor domain (OEPD) that in zebrafish also mutagenesis, we generated a defined deletion eliminating the entire contains the progenitors of the anterior lateral line ganglion (Chen open reading frames of dlx3b and dlx4b (dlx3b/4b) and investigated a and Streit, 2013; McCarroll et al., 2012; Hans et al., 2013). potential phenotypic
    [Show full text]
  • Stages of Embryonic Development of the Zebrafish
    DEVELOPMENTAL DYNAMICS 2032553’10 (1995) Stages of Embryonic Development of the Zebrafish CHARLES B. KIMMEL, WILLIAM W. BALLARD, SETH R. KIMMEL, BONNIE ULLMANN, AND THOMAS F. SCHILLING Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403-1254 (C.B.K., S.R.K., B.U., T.F.S.); Department of Biology, Dartmouth College, Hanover, NH 03755 (W.W.B.) ABSTRACT We describe a series of stages for Segmentation Period (10-24 h) 274 development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad peri- Pharyngula Period (24-48 h) 285 ods of embryogenesis-the zygote, cleavage, blas- Hatching Period (48-72 h) 298 tula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the Early Larval Period 303 changing spectrum of major developmental pro- Acknowledgments 303 cesses that occur during the first 3 days after fer- tilization, and we review some of what is known Glossary 303 about morphogenesis and other significant events that occur during each of the periods. Stages sub- References 309 divide the periods. Stages are named, not num- INTRODUCTION bered as in most other series, providing for flexi- A staging series is a tool that provides accuracy in bility and continued evolution of the staging series developmental studies. This is because different em- as we learn more about development in this spe- bryos, even together within a single clutch, develop at cies. The stages, and their names, are based on slightly different rates. We have seen asynchrony ap- morphological features, generally readily identi- pearing in the development of zebrafish, Danio fied by examination of the live embryo with the (Brachydanio) rerio, embryos fertilized simultaneously dissecting stereomicroscope.
    [Show full text]
  • Brain-Derived Neurotrophic Factor and Neurotrophin 3 Mrnas in The
    Proc. Nail. Acad. Sci. USA Vol. 89, pp. 9915-9919, October 1992 Developmental Biology Brain-derived neurotrophic factor and neurotrophin 3 mRNAs in the peripheral target fields of developing inner ear ganglia ULLA PIRVOLA*, JUKKA YLIKOSKI*t, JAAN PALGIt§, EERO LEHTONEN*, URMAS ARUMAE§, AND MART SAARMAt§ *Department of Pathology, University of Helsinki, SF-00290 Helsinki, Finland; *Institute of Biotechnology, University of Helsinki, SF 00380 Helsinki, Finland; and §Department of Molecular Genetics, Institute of Chemical Physics and Biophysics, Estonian Academy of Sciences, 200026 Tallinn, Estonia Communicated by N. Le Douarin, July 1, 1992 ABSTRACT In situ hybridization was used to study the site tyrosine kinase receptors likewise bind neurotrophins with and timing of the expression of nerve growth factor (NGF), low and high affinity (21-23). The individual neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin 3 exert similar functional effects on overlapping but also on (NT-3), and neurotrophin 5 (NT-5) mRNAs in the developing distinct neuronal populations (24). In addition, there is evi- inner ear of the rat. In the sensory epithelia, the levels of NGF dence that they have regulatory roles in non-neuronal tissues and NT-5 mRNAs were below the detection limit. NT-3 and as well (25, 26). In principle, all neurotrophins could regulate BDNF mRNAs were expressed in the otic vesicle in overlapping the development of the inner ear. We have used in situ but also in distinct regions. Later in development, NT-3 hybridization to determine the spatiotemporal expression pat- transcripts were localized to the differentiating sensory and tern of NGF, BDNF, NT-3, and NT-5 mRNAs in the cochlea supporting cells of the auditory organ and vestibular maculae.
    [Show full text]
  • Neurosensory Development in the Zebrafish Inner Ear
    NEUROSENSORY DEVELOPMENT IN THE ZEBRAFISH INNER EAR A Dissertation by SHRUTI VEMARAJU Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2011 Major Subject: Biology NEUROSENSORY DEVELOPMENT IN THE ZEBRAFISH INNER EAR A Dissertation by SHRUTI VEMARAJU Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Bruce B. Riley Committee Members, Mark J. Zoran Brian D. Perkins Rajesh C. Miranda Head of Department Uel Jackson McMahan December 2011 Major Subject: Biology iii ABSTRACT Neurosensory Development in the Zebrafish Inner Ear. (December 2011) Shruti Vemaraju, B.Tech., Guru Gobind Singh Indraprastha University Chair of Advisory Committee: Dr. Bruce B. Riley The vertebrate inner ear is a complex structure responsible for hearing and balance. The inner ear houses sensory epithelia composed of mechanosensory hair cells and non-sensory support cells. Hair cells synapse with neurons of the VIIIth cranial ganglion, the statoacoustic ganglion (SAG), and transmit sensory information to the hindbrain. This dissertation focuses on the development and regulation of both sensory and neuronal cell populations. The sensory epithelium is established by the basic helix- loop-helix transcription factor Atoh1. Misexpression of atoh1a in zebrafish results in induction of ectopic sensory epithelia albeit in limited regions of the inner ear. We show that sensory competence of the inner ear can be enhanced by co-activation of fgf8/3 or sox2, genes that normally act in concert with atoh1a.
    [Show full text]
  • 20. Placodes and Sensory Development
    PLACODES AND 20. SENSORY DEVELOPMENT Letty Moss-Salentijn DDS, PhD Dr. Edwin S. Robinson Professor of Dentistry (in Anatomy and Cell Biology) E-mail: [email protected] READING ASSIGNMENT: Larsen 3rd Edition Chapter 12, Part 2. pp.379-389; Part 3. pp.390- 396; Chapter 13, pp.430-432 SUMMARY A series of ectodermal thickenings or placodes develop in the cephalic region at the periphery of the neural plate. Placodes are central to the development of the cranial sensory systems in vertebrates and are among the innovations that appeared in the early evolution of vertebrates. There are placodes for the three organs of special sense: olfactory, optic (lens) and otic placodes, and (epibranchial) placodes that give rise to the distal cells of the sensory ganglia of cranial nerves V, VII, IX and X. Placodes (with the possible exception of the olfactory placodes) form under the influence of surrounding cranial tissues. They do not appear to require the presence of neural crest. The mesoderm in the prechordal plate plays a significant role in the initial development of the placodes for the organs of special sense, while the pharyngeal pouch endoderm plays that role in the development of the epibranchial placodes. The development of the organs of special sense is described briefly. LEARNING OBJECTIVES You should be able to: a. Give a definition of placodes and describe their evolutionary significance. b. Name the different types of placodes, their locations in the developing embryo and their developmental fates. c. Discuss the early development of the placodes and some of the possible factors that feature in their development.
    [Show full text]
  • AMBLYSTOMA PUNCTATUM Stage of the Embryo at The. Time of Operation
    238 ZO6LOG Y: R. G. HARRISON PROC. N. A. S. the other for some cells and in the opposite manner for certain others, seem to be explainable best by regarding the concentrated phase of a melanophore to be dependent upon the activity of one type of nerve; and the dispersed phase, upon the activity of another type of nervous connection. Abramowitz, A. A., "Regeneration of Chromatophore Nerves," Proc. Nat. Acad. Sci., 21, 137-141 (1935). Mills, S. M., "The Double Innervation of Fish Melanophores," Jour. Exp. Zool., 64, 231-244 (1932). Smith, D. C., "The Action of Certain Autonomic Drugs upon the Pigmentary Re- sponses of Fundulus," Ibid., 58, 423-453 (1931). Wyman, L. C., "Blood and Nerve as Controlling Agents in the Movements of Melano- phores," Ibid., 39, 73-132 (1924). RELATIONS OF SYMMETRY IN THE DEVELOPING EAR OF AMBLYSTOMA PUNCTATUM By Ross G. HARRISON OSBORN ZOOLOGICAL LABORATORY, YALE UNIVBRSITY Read before the Academy, November 19, 1935 The present investigation is an attempt to study the changes underlying embryonic differentiation by means of transplantation experiments in which the orientation of the grafted organ rudiment is varied. The ear was chosen for the purpose, as was the fore limb in a previous study, be- cause it is a paired organ which in its final stage is asymmetric and is the mirror image of the corresponding structure on the opposite side. It has been found that there are definite relations between the orientation of the graft and the character of the resulting organ, particularly in regard to its asymmetry, and that these relations change in regular order with the stage of the embryo at the.
    [Show full text]
  • Unraveling the Genetic and Developmental Mysteries of 22Q11 Deletion Syndrome
    Review TRENDS in Molecular Medicine Vol.9 No.9 September 2003 383 Unraveling the genetic and developmental mysteries of 22q11 deletion syndrome Hiroyuki Yamagishi1 and Deepak Srivastava2 1Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo 160-8582, Japan 2Departments of Pediatrics and Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Rm NA8.124, Dallas, TX 75390-9148, USA Birth defects occur in nearly 5% of all live births and are susceptibilities that predispose individuals to a variety the major cause of infant mortality and morbidity. of birth defects. Here, we review the recent advances that Despite the recent progress in molecular and develop- have begun to reveal the human genetic, developmental mental biology, the underlying genetic etiology of most biology and molecular biology underpinnings of this congenital anomalies remains unknown. Heterozygous common syndrome. deletion of the 22q11.2 locus results in the most com- mon human genetic deletion syndrome, known as Clinical features of 22q11DS DiGeorge syndrome, and has served as an entry to The clinical findings associated with del22q11 are highly understanding the basis for numerous congenital heart variable. Approximately 75% of patients with 22q11DS are and craniofacial anomalies, among many other defects. born with congenital heart defects (CHD), mainly of the Extensive human genetic analyses, mouse modeling cardiac outflow tract and aortic arch. Other common and studies of developmental molecular cascades features of 22q11DS include a characteristic facial involved in 22q11 deletion syndrome are revealing com- appearance, immunodeficiency from thymic hypoplasia, plex networks of signaling and transcriptional events velopharyngeal dysfunction with or without cleft palate, that are essential for normal embryonic development.
    [Show full text]
  • Neural Development in the Otic Vesicle Husniye Kantarci, Yunzi Gou, Bruce B Riley*
    RESEARCH ARTICLE The Warburg Effect and lactate signaling augment Fgf-MAPK to promote sensory- neural development in the otic vesicle Husniye Kantarci, Yunzi Gou, Bruce B Riley* Biology Department, Texas A&M University, College Station, United States Abstract Recent studies indicate that many developing tissues modify glycolysis to favor lactate synthesis (Agathocleous et al., 2012; Bulusu et al., 2017; Gu et al., 2016; Oginuma et al., 2017; Sa´ et al., 2017; Wang et al., 2014; Zheng et al., 2016), but how this promotes development is unclear. Using forward and reverse genetics in zebrafish, we show that disrupting the glycolytic gene phosphoglycerate kinase-1 (pgk1) impairs Fgf-dependent development of hair cells and neurons in the otic vesicle and other neurons in the CNS/PNS. Fgf-MAPK signaling underperforms in pgk1- / - mutants even when Fgf is transiently overexpressed. Wild-type embryos treated with drugs that block synthesis or secretion of lactate mimic the pgk1- / - phenotype, whereas pgk1- / - mutants are rescued by treatment with exogenous lactate. Lactate treatment of wild-type embryos elevates expression of Etv5b/Erm even when Fgf signaling is blocked. However, lactate’s ability to stimulate neurogenesis is reversed by blocking MAPK. Thus, lactate raises basal levels of MAPK and Etv5b (a critical effector of the Fgf pathway), rendering cells more responsive to dynamic changes in Fgf signaling required by many developing tissues. Introduction Development of the paired sensory organs of the head relies on critical contributions from cranial *For correspondence: placodes. Of the cranial placodes, the developmental complexity of the otic placode is especially [email protected] remarkable for producing the entire inner ear, with its convoluted epithelial labyrinth and rich cell type diversity.
    [Show full text]
  • In Vitro Study of Morphological Changes of the Cultured Otocyst Isolated from the Chick Embryo
    Int. J. Morphol., 35(1):208-211, 2017. In vitro Study of Morphological Changes of the Cultured Otocyst Isolated from the Chick Embryo Estudio in vitro de los Cambios Morfológicos del Otocisto Cultivado Aislado del Embrión de Pollo Sittipon Intarapat; Thanasup Gonmanee & Charoensri Thonabulsombat INTARAPAT, S.; GONMANEE, T. & THONABULSOMBAT C. In vitro study of morphological changes of the cultured otocyst isolated from the chick embryo. Int. J. Morphol., 35(1):208-211, 2017. SUMMARY: The aim of this study was to observe morphological changes of the cultured otocysts isolated from various stages of the chick embryo. Isolated otocysts were dissected from embryonic day, E2.5-4.5 of incubation (HH stage 16-26) according to stages of developing inner ear. Morphology of the chick otocyst exhibited an ovoid shape. The width and height of the otocyst were 0.2 mm and 0.3 mm, respectively. Elongation of a tube-like structure, the endolymphatic duct, was found at the dorsal aspect of the otocyst. The cultured otocyst is lined by the otic epithelium and surrounding periotic mesenchymal cells started to migrate outwards the lateral aspect of such epithelium. Notably, the acoustic-vestibular ganglion (AVG) was observed at the ventrolateral aspect of the otocyst. Appearance of AVG in vitro can be applied for studying chemical-induced ototoxicity and sensorineural hearing loss. It was concluded that the organ- cultured otocyst of the chick embryo could be used as a model to study sensory organ development of avian inner ear. KEY WORDS: Chick embryo; Inner ear; Otocyst; Otic development. INTRODUCTION Chicken has become a favorable model in study auditory organ regeneration and differentiation (Li et developmental biology and stem cell research (Stern, 2005; al., 2003b; Oshima et al., 2010; Ouji et al., 2012).
    [Show full text]
  • Wnt-Dependent Regulation of Inner Ear Morphogenesis Is Balanced by the Opposing and Supporting Roles of Shh
    Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh Martin M. Riccomagno,1,2 Shinji Takada,3 and Douglas J. Epstein1,4 1Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA; 2Institute of Cell Biology and Neuroscience, University of Buenos Aires School of Medicine, 1121 Buenos Aires, Argentina; 3Okazaki Institute for Integrative Biosciences, National Institutes of Natural Sciences Okazaki 444 8787, Japan The inner ear is partitioned along its dorsal/ventral axis into vestibular and auditory organs, respectively. Gene expression studies suggest that this subdivision occurs within the otic vesicle, the tissue from which all inner ear structures are derived. While the specification of ventral otic fates is dependent on Shh secreted from the notochord, the nature of the signal responsible for dorsal otic development has not been described. In this study, we demonstrate that Wnt signaling is active in dorsal regions of the otic vesicle, where it functions to regulate the expression of genes (Dlx5/6 and Gbx2) necessary for vestibular morphogenesis. We further show that the source of Wnt impacting on dorsal otic development emanates from the dorsal hindbrain, and identify Wnt1 and Wnt3a as the specific ligands required for this function. The restriction of Wnt target genes to the dorsal otocyst is also influenced by Shh. Thus, a balance between Wnt and Shh signaling activities is key in distinguishing between vestibular and auditory cell types. [Keywords: Wnt1/3a; Shh; Dlx5; otic vesicle; vestibulum; semicircular canals; hindbrain] Received February 9, 2005; revised version accepted May 13, 2005.
    [Show full text]
  • Origin of Acoustic–Vestibular Ganglionic Neuroblasts in Chick Embryos and Their Sensory Connections
    Brain Structure and Function (2019) 224:2757–2774 https://doi.org/10.1007/s00429-019-01934-5 ORIGINAL ARTICLE Origin of acoustic–vestibular ganglionic neuroblasts in chick embryos and their sensory connections Luis Óscar Sánchez‑Guardado1 · Luis Puelles2,3 · Matías Hidalgo‑Sánchez1 Received: 7 February 2019 / Accepted: 31 July 2019 / Published online: 8 August 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract The inner ear is a complex three-dimensional sensory structure with auditory and vestibular functions. It originates from the otic placode, which generates the sensory elements of the membranous labyrinth and all the ganglionic neuronal precursors. Neuroblast specifcation is the frst cell diferentiation event. In the chick, it takes place over a long embryonic period from the early otic cup stage to at least stage HH25. The diferentiating ganglionic neurons attain a precise innervation pattern with sensory patches, a process presumably governed by a network of dendritic guidance cues which vary with the local micro-environment. To study the otic neurogenesis and topographically-ordered innervation pattern in birds, a quail–chick chimaeric graft technique was used in accordance with a previously determined fate-map of the otic placode. Each type of graft containing the presumptive domain of topologically-arranged placodal sensory areas was shown to generate neuroblasts. The diferentiated grafted neuroblasts established dendritic contacts with a variety of sensory patches. These results strongly suggest that, rather than reverse-pathfnding, the relevant role in otic dendritic process guidance is played by long-range difusing molecules. Keywords Developing inner ear · Otic placode · Neuroblast · Sensory patch · Maculae · Cristae · Otic innervation Introduction otic epithelium itself.
    [Show full text]