The aim of this study was to assess sensitivity of early life stage zebrafish to cadmium (Cd). Embryos and larva were exposed to Cd before the formation of skeletal elements. Exposure times were selected to determine whether Cd perturbed developmental processes that lead to skeletal deformities, and to characterize the types of skeletal deformities that occurred. Embryo/larva were exposed to 3μM, 10μM, 30μM, 100μM, 300μM, and 1000μM Cd at 12-36 hours post fertilization (hpf), 36-60hpf, 60-84hpf, and 144-168hpf in one series of experiments. This experiment was conducted at circumneutral pH. A second series of experiments with these same Cd concentrations were also conducted at pH 6 with embryo/larva exposed at 12-36hpf, 36-60hpf, and 60-84hpf. The fish that survived the exposures were raised for 40-60 days whereupon the surviving fish were overdosed with MS222, and X-rayed. The later early life stage fish were more sensitive to Cd toxicity; the chorion appeared to protect the earlier exposed embryos (zebrafish hatch 48-72hpf). Embryo/larva were more resistant to toxicity from dissolved Cd (pH 6) than particulate Cd (circumneutral pH); absence of functional gills during early life stages perhaps explained resistance to dissolved Cd. Notochord lesions (typically lethal within two weeks) occurred when embryos were exposed 12-36hpf. There was no evidence for sensitivity of early life stage zebrafish to Cd induced skeletal deformities that occurred within 40-60 days of Cd exposures. / Graduation date: 2004

<p>The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. While these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a, and H+-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role for notochord vacuoles in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.</p> / Dissertation

Back pain is a highly prevalent condition whose pathogenesis is associated with intervertebral disc (IVD) degeneration. Degeneration is driven by abnormal cell biology, particularly within the IVD’s inner core, the nucleus pulposus (NP). In recent years, there has been an ever-increasing search for cell-based therapies aimed at correcting the cell biology and thus repairing/regenerating the degenerate IVD. The success of these novel therapies, however, requires a thorough understanding of IVD development and of the phenotype of its cells. The embryonic, foetal and juvenile NP is populated by large vacuolated notochordal cells that with skeletal maturity are replaced by smaller NP cells. Since notochordal cells have been shown to display protective and anabolic roles in the IVD their loss in humans has often been suggested to initiate the degenerative process. As such, a detailed understanding of notochordal cells and their regulatory pathways may help identify factors involved in IVD homeostasis and aid the development of novel cell-based therapies targeting IVD degeneration. The study of human notochordal cells has, however, been hindered by ethical, logistical and technical difficulties in obtaining suitable samples and, as such, the human notochordal cell phenotype is, to date, unknown, constituting a major limitation in the field. The work presented here was conducted with the objective of developing a methodology to isolate human developing notochordal cells (NP progenitors) from adjacent sclerotomal cells (annulus fibrosus and vertebral body progenitors), to characterise the notochordal cell phenotype and identify potential factors involved in notochordal cell biology. Initially, human embryonic and foetal spines were characterised to assess their suitability as a source of notochordal cells and to identify a notochord-specific marker that could be used to isolate notochordal cells for microarray studies. The human developing spine contained large vacuolated notochordal cells in all stages analysed (3.5-18 weeks post-conception (WPC)) that specifically expressed KRT8, KRT18 and KRT19 at all stages and CD24 between 5.5-18 WPC. KRT18 and CD24 were independently used to label notochordal cells (7.5-14 weeks post-conception) and separate them from sclerotomal cells. Methodologies were developed to allow extraction of RNA of sufficient quality for microarray analysis from fixed, permeabilised (in the case of KRT18) and/or, labelled and sorted cells (CD24). Microarray analysis identified and real-time qPCR and, for some markers, immunohistochemistry, validated GRB14, SLC19A1, FGF10, ADORA3, TBXA2R, CDH6, ANPEP, CD69, CD24, RTN1, PRPH, MAP1B, ISL1 and CLDN1 as human notochordal cell markers. Ingenuity pathway analysis was performed to investigate the pathways/networks and upstream regulators and downstream effectors of notochordal cells. Inhibition of inflammation and angiogenesis were identified as relevant to notochordal cell biology, function and, possibly, to the known protective and anabolic role notochordal cells display in the IVD. Notochordal marker gene expression was identified in adult NP tissue, and negatively correlated with degeneration. Proteins encoded by ADORA3 and MAP1B were expressed by a proportion of adult NP cells, suggesting the presence of notochord-derived cells in the adult NP.Importantly, this is the first study to detail a methodology and successfully isolate human notochordal cells. Such methodology has the potential to be used to culture and investigate the biology of viable human notochordal cells (CD24+ve). Future studies aimed at developing cell-based therapies for IVD degeneration could also use these identified markers to assess appropriate stem cell differentiation to notochordal cells.

In this study, p-chlorophenylalanine (pCPA), an inhibitor of tryptophan hydroxylase (the rate limiting enzyme of serotonin synthesis), was used to reduce serotonin (5HT) levels during early development in zebrafish embryos. One day old dechorionated embryos were treated with 25μM pCPA for 24h and subsequently rescued. Immunohistological studies using a 5HT antibody confirmed that 5HT neurons in the brain and spinal cord were depleted of transmitter by 2days post fertilization (dpf). Twenty four hours after pCPA exposure embryos were unable to burst swim and were nearly paralyzed. Movement began to improve at 4dpf, and by 7dpf, larvae exhibited swimming activity. Rescued larvae continued to grow in rostrocaudal length over 5days post-rescue, but their length was always 16-21% below controls. Surprisingly, both groups displayed the same number of myotomes. To examine whether hypertonicity of myotomes in treated embryos played a role in their shorter rostrocaudal lengths, 1dpf embryos were exposed to a combination of 25μM pCPA and 0.6mM of the sodium channel blocker ethyl 3-aminobenzoate methanesulfonate (MS-222). After a 24hour exposure, the embryos exhibited the same rostrocaudal length as control embryos suggesting that myotome hypertonicity plays a major role in the decreased axial length of the treated larvae. In addition, pCPA treated 2dpf embryos exhibited abnormal notochordal morphology that persisted throughout recovery. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the relative levels of the serotonin 1A receptor (5HT 1A) transcript and the serotonin transporter (SERT) transcript in the brain and spinal cord of control and treated embryos. Transcripts were present in both brain and spinal cord as early as 1dpf and reached maximal concentrations by 3dpf. Embryos treated with pCPA demonstrated a decrease in the concentration of 5HT 1A transcript in both brain and spinal cord. While SERT transcript levels remained unaffected in brain, they were decreased in spinal cord. Five days subsequent to pCPA rescue, 5HT 1A transcript concentrations remained decreased in brain while SERT transcript levels were elevated in both regions. These findings suggest that reduction of 5HT during early zebrafish development may have an adverse effect on body length, notochordal morphology, locomotor behavior, and serotonin message-related expression.

Pastaruoju metu tyrimams dažniausiai naudojami žinduoliai, kadangi rezultatai šiek tiek atspindi žmogaus organizme vykstančius procesus. Šis darbas parengtas analizuojant besivystančias paukščio embriono sistemas: tai būdas, leidžiantis sutaupyti lėšų bei dėka trumpo embrioninio vystymosi laikotarpio suteikiantis galimybę operatyviam rezultatų gavimui. Iki šiol literatūroje neradome duomenų apie pirmojo raumens susitraukimo ypatybes, o šį procesą įtakojančios nervų sistemos vystymąsi apibūdina keletas hipotezių. Taigi darbo tikslas buvo išsiaiškinti pirmojo susitraukiančiojo raumens atsiradimo laiką, jo morfologiją (1) ir išanalizuoti ankstyvajame embriogenezės laikotarpyje kontraktilų aparatą įtakojančios nervų sistemos vystymosi ypatumus (2). Tyrimams naudojome kalakuto ir vištos embrionus – kadangi skirtingų porūšių skirtumai mums nebuvo svarbūs, palyginimas tarp minėtų paukščių embrionų neatliktas. Embrionai buvo stebimi in vivo, tyrimams in vitro atlikti buvo naudojami šie molekulinės biologijos metodai: paruoštų pjūvių dažymas histo- ir imunohistochemiškai, polimerazės grandininės reakcijos, ląstelių kultūrų auginimas ir Western blotas. Pirmasis aktyvus raumens susitraukimas kalakuto embrione atsiranda 19-22 Hamburgerio Hamiltono stadijoje, t.y., praėjus 116 val. nuo inkubacijos pradžios: porinis raumuo sudarytas iš keturių susiliejusių miomerų, yra 1 mm ilgio ir 0,1 mm pločio – šis iš dviejų ląstelių tipų sudarytas audinys atsakingas už pirmuosius aktyvius embriono... [toliau žr. visą tekstą] / The purpose of the study was to characterize the first contraction of an isolated muscle in turkey embryo and to determine whether at early stages of chicken brain development during neurogenesis, cells from the astrocytic lineage are present in relevant amounts, where they are located in the neural tube, and to what extend brain regional differences exist. MATERIAL AND METHODS. From the 3rd day of incubation on until the 6th day the embryos were continuously watched through a cellophane window in the eggshell. For histology the embryos were fixed in Bouin's fluid, then completely cut in serial sections of 5 microm thickness and stained according to Masson-Goldner's trichrome procedure plus resorcin-fuchsin. For gene expression analysis markers for stem cells, neurons and astrocytes were used. Immunohistochemistry was made against SMI 312 and GFAP antibodies and Western blot against GFAP as well. RESULTS. A paired muscle 1 mm long and 0.1 mm broad, derived by fusion of the four occipital myomeres, is responsible for the first individual contraction. The contraction produces a stretching in the neck region. The muscle named M. occipitalis primordialis consists of four end-to-end connected groups of mononucleated muscle cells. The muscle contains two types of cells according to the cell nuclei. The elastic rod-shaped notochord represents an endoskeleton. Immediately after contraction it brings the body of the embryo back into its former shape. We also demonstrate that specific... [to full text]

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