Retinoic acid, suspended in cottonseed oil, was administered via gavage to pregnant mice (ICR strain) on day 11 (E 11) of gestation at doses of either 20, 40, or 80 mg/kg. Fetuses were examined for external malformations on day 17 (E 17). Retinoic acid treatment induced micromelia (with the elimination of several long bones at higher doses) and digital defects (ectrodactyly and syndactyly) in a dose‐dependent manner in fetuses examined on day 17. Hindlimbs were affected more than forelimbs. In another group of experiments, limbs exposed to retinoic acid treatment in utero on E 11 were cultured on E 12 and maintained for 3 days in submerged culture. Cultured limbs were examined qualitatively for digital and long bone defects, and image analysis of the area and form of bone anlagen of cultured limbs was used to quantitatively evaluate the teratogenic potential of retinoic acid. The qualitative evaluation indicated that the retinoic acid‐induced effects obtained in vivo and with pretreated, cultured limbs were essentially the same, except that the severity of regional effects changed as a result of culture. The incidence of ectrodactyly was higher with cultured limbs than with E 17 fetal limbs, but fewer cultured limbs were missing long bones. These results suggest that culturing limbs, after they have been pretreated in utero, modifies their response to a teratogen and demonstrates that the paw skeleton is extremely sensitive to teratogen treatment under these experimental conditions. Therefore, care must be exercised when attempting to compare in vivo and in vitro teratogenic data. This study also clearly demonstrates the power and usefulness of image analysis for quantitative evaluation of both the area and form of a cultured specimen such as the developing limb bud. Quantitative, image analysis of cultured limbs showed a dose‐dependent decrease in area of both fore‐ and hindlimbs. The effect was most severe in hindlimbs. In the forelimb, the paw was affected more than the long bones; as the dose increased, this disparity of effect also increased. With the hindlimb, a greater effect on the paw occurred only at 80 mg/kg. Computing the soft tissue/bone ratio illustrated that retinoic acid had a greater effect on chondrogenic tissue than on soft tissue.

Maternal administration of a single dose of retinoic acid (vitamin A acid, 100 mg/kg) on either the 11 th, 11 1/2, 12th, 12 1/2, 13th or 13 1/2 day of gestation produced phocomelia or partial phocomelia in ICR/DUB fetuses. The results depended upon the time of treatment and two gradients of effect were produced: 1) cranio-caudal gradient, since forelimb defects resulted from treatment between days 11 and 13, while similar hindlimb abnormalities were produced by administration of retinoic acid 12 to 24 hours later: 2) proximo-distal gradient, due to the heterogenous sensitivity among individual bones of the limb. In the forelimb, early treatment (11th day) produced humero-ulnar defects and later treatment (12th day) ulnoradial defects. A similar proximo-distal gradient was observed in the hindlimb. The use of teratological studies as a tool to assist morphogenetic investigation is discussed.

Long-term treatment with all trans-retinoic acid (RA) induces neuronal differentiation and apoptosis. However, the effect of short-term RA treatment on cell proliferation, migration and invasion of neuroblastoma cell lines (SH-SY5Y and IMR-32) remains unclear. RA induces expression of tissue-transglutaminase (TGase) and promotes migration and invasion after 24 h of treatment in SH-SY5Y cells, but not in IMR-32 cells. RA receptor (RAR) agonist (4-(E-2-[5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid) and RAR/retinoid X receptor (RXR) agonist (9-cis-RA) promote expression of TGase, migration and invasion of SH-SY5Y cells, while RXR agonist has no significant effect. RAR antagonist blocks RA effect on migration and invasion, indicating that RAR receptors are required. Retinoid receptors are expressed and activated by RA in both cell lines. However, only transient activation of RAR is observed in IMR-32 cells. These findings suggest that different responses observed in SH-SY5Y and IMR-32 cells could be due to differential activation of retinoid receptors. Overexpression of TGase has no effect on migration or invasion, while overexpression of antisense TGase blocks RA-induced migration and invasion, indicating that other molecules along with TGase mediate RA effects. In addition to the long-term effects of RA that are coupled with cell differentiation, short-term effects involve migration and invasion of neuroblastoma SH-SY5Y cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Fetal alcohol spectrum disorder (FASD) is a result of prenatal alcohol exposure, producing a wide range of defects including craniofacial, sensory, motor and cognitive deficits. Many ocular abnormalities are frequently associated with FASD including microphthalmia, optic nerve hypoplasia, and cataracts. FASD is highly prevalent in low socioeconomic populations, where it is also accompanied by higher rates of malnutrition and alcoholism. Using zebrafish as a model to study FASD retinal defects has been extremely insightful in understanding the ethanol-induced retinal defects at the cellular level. Zebrafish embryos treated with ethanol from mid-blastula transition through somitogenesis (2-24 hours post fertilization; hpf) showed defects similar to human ocular deficits including microphthalmia, optic nerve hypoplasia, and photoreceptor differentiation defects. Ethanol exposure altered critical transcription factor expression involved in retinal cell differentiation. Retinoic acid (RA) and folic acid (FA) nutrient co-supplementation rescued optic nerve and photoreceptor differentiation defects. Ethanol exposure during retinal morphogenesis stages (16-24 hpf), produced retinal defects like those seen with ethanol exposure between 2-24 hpf. Significantly, during ethanol-sensitive time window (16-24 hpf), RA co-supplementation moderately rescued these defects, whereas, FA cosupplementation showed significant rescue of optic nerve and photoreceptor differentiation. RA, but not FA, supplementation after ethanol exposure could restore ethanol-induced optic nerve and photoreceptor differentiation defects. Ethanol exposure did not affect timing of retinal cell differentiation induction, but later increased retinal cell death and proliferation. Ethanol-treated embryos showed increased retinal proliferation in the outer nuclear layer (ONL), inner nuclear layer (INL), and ciliary marginal zone (CMZ) at 48 hpf and 72 hpf. In order to identify the genesis of ethanol-induced persistent retinal defects, ethanol effects on retinal stem cell populations in the CMZ and the Müller glial cells (MGCs) were examined. Ethanol treated retinas had an expanded CMZ indicated by histology and Alcama, a retinal stem cell marker, immunolabeling, but reduced expression of rx1 and the cell cycle exit marker, cdkn1c. Ethanol treated retinas also showed reduced MGCs. At 72 hpf, ONL of ethanol exposed fish showed fewer photoreceptors expressing terminal differentiation markers. Importantly, these poorly differentiated photoreceptors co-expressed the basic helix-loop-helix (bHLH) proneural differentiation factor, neurod, indicating that ethanol exposure produced immature and undifferentiated photoreceptors. Reduced differentiation along with increased progenitor marker expression and proliferation suggest cell cycle exit failure due to ethanol exposure. These results suggested that ethanol exposure disrupted stem cell differentiation progression. Wnt, Notch and proneural gene expression regulate retinal stem cell proliferation and transition into progenitor cells. Ethanol exposure disrupted Wnt activity in the CMZ as well as Notch activity and neurod gene expression in the retina. RA and FA co-supplementation were able to rescue Wnt activity in the CMZ and rescue downstream Notch activity. To test whether the rescue of these Wnt-active cells could restore the retinal cell differentiation pathways, ethanol treated embryos were treated with Wnt agonist. This treatment could restore Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. We conclude that ethanol exposure produced persistent defects in the stem cell Wnt signaling, a critical pathway in retinal cell differentiation. Further analysis of underlying molecular mechanisms will provide insight into the embryonic origins of ethanol-induced retinal defects and potential therapeutic targets to cure this disorder.

No description available.

Student Number : 9502128Y - MSc dissertation - School of Anatomical Sciences - Faculty of Science / Retinoic acid, the active metabolite of Vitamin A is known to play a major role in embryonic growth and differentiation during development. It has been shown that either excess or deficiency of retinoic acid during embryogenesis can be teratogenic. In order to study the teratogenic effects of retinoic acid, the aim of the present study was therefore to investigate the effect of all-trans retinoic acid on the migration and fate of neural crest cells in vitro and in vivo. In addition, the study investigated the effect of retinoic acid on the cytoskeletal elements of neural crest cells and on Rac and Rho, two members of the Rho family of GTPases. The neural tubes containing neural crest cells of quail embryos were removed at cranial levels and cultured on fibronectin as a substrate. The neural tubes were cultured in either Dulbecco’s minimal essential medium (DMEM) or in DMEM+Dimethylsulphoxide (DMSO) as controls. In order to test the effect of retinoic acid, the neural tubes were cultured in 10-5M all-trans retinoic acid (RA) which was reconstituted in DMSO. The distance of migration of the cultured quail neural crest cells was measured and compared between the controls and the experimentals. To study the effect of RA on the cell actin cytoskeleton in vitro, cultured neural crest cells were stained with rhodamine phalloidin. In addition, following 24 hours of culture, the quail neural crest cells were brought into suspension and micro-injected into 36 hour-old chick hosts. While the migration of neural crest cells was extensive in the control cultures in vitro, migration was inhibited in the retinoic acid-treated neural crest cells. In addition, retinoic-acid treated neural crest cells showed pigmentation and neuronal processes earlier than did the control neural crest cells. Retinoic acid-treated neural crest cells showed a disarray of the cytoskeletal elements as they were devoid of stress fibres and focal adhesions. In addition, retinoic acid appears to decrease the expression of Rac and Rho of cultured quail neural crest cells. Following micro-injection of cultured control and RA-treated quail neural crest into the cranial region of chick hosts, the control cells populated the beak area, whereas the retinoic acid-treated quail neural crest cells migrated to the retina of the eye, a region they normally do not populate. These results suggest that retinoic acid disturbs the migration of neural crest cells. It appears to do this by affecting the cytoskeletal elements of neural crest cells and the genes that are involved in forming these elements.

Pattern formation ensures that tissues, organs, and structures develop in the correct place and orientation within the body. Patterning processes are at the heart of morphogenesis yet remain poorly understood due to their complexity. The sea urchin larval skeleton provides a simple model to study skeletal patterning, where the skeleton-producing primary mesenchyme cells (PMCs) receive patterning cues from the overlying ectoderm. The normal skeletal patterning process requires the PMCs to migrate within the blastocoel to specific positions. While ectodermal and endodermal signals regulate PMC positioning and differentiation, additional signals act to regulate biomineralization per se in the PMCs. However, the distinction between these effects is not well understood and new efforts have been made to identify these patterning and biomineralization cues that regulate sea urchin skeletal development. Understanding the mechanism by which PMCs interpret and transduce patterning cues into a migratory bias and/or positional information will provide insight into tissue patterning and developmental plasticity both in sea urchins and, more broadly, in deuterostomes. Ethanol is a known vertebrate teratogen that causes craniofacial defects as a component of fetal alcohol syndrome. Perturbations to retinoic acid biosynthesis and the Hedgehog signaling pathway are thought to be causal for the fetal alcohol syndrome phenotype in vertebrates. We used the sea urchin embryo to gain evolutionary insight into how ethanol affects embryonic development in a basal deuterostome animal. We found that ethanol specifically perturbs skeletal patterning. When sea urchin embryos are exposed to ethanol, they exhibit conspicuously delayed development, and broad skeletal patterning defects that are potentially analogous to fetal alcohol syndrome associated facial patterning defects in vertebrates and humans. PMC transplantation experiments demonstrated that ethanol-induced defects are not specific to the PMCs, and instead reflect the perturbation of patterning cues. We also found that the expression of both patterning cues and PMC-specific genes was delayed by ethanol exposure. Surprisingly, our results indicate that retinoic acid and Hedgehog pathways are not functionally relevant for the teratogenic effects of ethanol in the larval skeletal patterning process, indicating a lack of evolutionary conservation of these pathways in ethanol-mediated teratogenesis among deuterostomes. Temporal transcriptome analysis revealed significant impacts of ethanol on signaling and metabolic gene expression and a disruption in the timing of expression for sea urchin specification gene regulatory network (GRN) genes. Surprisingly, multiple circuits with the GRN exhibit precocious expression while others are delayed. Taken together, our results suggest that the skeletal patterning perturbations in ethanol-treated sea urchin embryos arise from a loss of temporal synchrony within and between the instructive and responsive tissues during pattern formation.

No description available.

No description available.

Metal-induced autoimmunity is an experimental model of environmentally induced autoimmune syndrome. Subtoxic doses of heavy metals administered to genetically susceptible mice resulted in the production of highly specific IgG antinucleolar antibodies (ANoA) accompanied by lymphoproliferation and serum increases in IgG1 and IgE. In this study, the induction of tolerance to mercuric chloride (HgCl2)-induced autoimmunity by pre-exposure to the low-dose mercury was reported. The ultimate mechanisms through which the immune system obtains the tolerance to a low dose of heavy metals remain unknown. The previous experiment showed that CD4+CD25+ regulatory T cells (Tregs) contributed to the maintenance of immunological self-tolerance and to the prevention of autoimmune diseases. The tolerized mice had a higher percentage of Tregs and ICOS+ regulatory T cells than the nontolerized mice. ICOS (Inducible T-cell COStimulator) is a costimulatory receptor homologous to CD28 and CTLA-4. The expression of ICOS occurs on activated T cells and is dependent upon TCR and CD28 signals. The anti-ICOS blockade restored the ability of tolerized mice to produce elevated amounts of IgG1, IgE and anti-nucleolar antibodies. The ICOS expression on Tregs and Teff cells increased after the mercury challenge. Mice that received anti-ICOS had a low percentage of Tregs and showed an increased production of several cytokines. Taken together, these results suggested that Tregs maintained the immune tolerance in response to chemical challenges. The ICOS pathway is important for the differentiation of Tregs and blocking this pathway could prevent peripheral tolerance to the low dose mercury. The results also showed the splenocytes from the tolerized group produced a higher amount of IL-10 than the nontolerized group. This promoted us to study the role of IL-10 in tolerance induction. To validate the role of Interleukin-10 in tolerance maintenance, the tolerized mice were treated with blocking anti-IL-10 and anti-IL-10 receptor mAb. Those tolerized mice treated with IL-10 blocking antibodies produced a higher amount of serum IgG1, IgE and anti-nucleolar antibodies compared with the group treated with control antibodies. This suggested IL-10 is critical in maintaining the peripheral tolerance in a mercury treated mouse model. All Trans retinoic acid (ATRA) is the metabolically active derivative of vitamin A and functions as potent regulator of gene expression. Vitamin A and retinoic acid play vital roles in the homeostatic control of the immune system because vitamin A-deficient individuals are incapable of controlling bacterial, viral, and protozoan diseases. In this study, the role of ATRA was reported in the first time in mercuric chloride (HgCl2)-induced autoimmunity by feeding the ASW mice ATRA. The results showed that retinoid acid exacerbated the mercury-induced autoimmunity. To study whether retinoic acid plays a role in low dose mercury induced tolerance, three groups of tolerized ASW mice were treated with either ATRA, mercury or both. The results showed retinoic acid could break the low-dose mercury-induced tolerance. The splenocytes from the group that received both mercury and ATRA treatment produced much more IL-2 and IFN-γ. This group had the lowest percentage of IL-10+ cells. The group that received ATRA had a lower percentage of early apoptosis cells. To further study the mechanism, a PCR array that included 84 genes, involved in T cell and B cell activation, was used to study four groups of mice treated with mercury, ATRA, both or neither. 10 candidate genes were selected and analyzed by Real-Time PCR to validate the PCR array results. Further study is needed to characterize the expression and the role of molecules that are upregulated by mercury and ATRA. / Microbiology and Immunology