Question

2. At what stage is the zebrafish embryo after 24 hours of development at 24ºC. List the structures that you can see in the embryo at this point.

3. When does the larva hatch from its chorion, and at what stage must you start feeding the larva?

4. How do you remove the chorion from an embryo?

5. What effects do low temperatures have on the development of the embryo? What effect does low temperatures have on microtubules?

6. What forms of vitamin A are known to have teratogenic effects? What forms are considered safe for developing embryos?

8. What developmental abnormalities can be caused by alcohol? Is there any recognized safe limit of alcohol that a pregnant woman can imbibe?

ANSWER ALL QUESTIONS

Answer 1

2) The zebrafish is a premier model organism yet lacks a system for assigning post-embryonic fish to developmental stages. To provide such a staging series, we describe post-embryonic changes in several traits that are visible under brightfield illumination or through vital staining and epiflourescent illumination. These include the swim bladder, median and pelvic fins, pigment pattern, scale formation, larval fin fold and skeleton. We further identify milestones for placing post-embryonic fish into discrete stages.A post-embryonic staging system is needed because changes occur in a variety of traits during this period in zebrafish and in other organisms. In the zebrafish for example, post-embryonic development entails the appearance of median adult fins and pelvic fins, increasing stratification and complexity of skin, continued skeletal development, loss of the larval fin fold, and marked changes in the gut, kidneys, and gonads, as well as the peripheral and central nervous systems. There have been some descriptions of such changes, either for the whole organism or focusing on particular traits.

ADAN EN 1-cell 0.2 h 64-cell 1k-cell 3 h B 2 h . prim-6 25 h protruding mouth 75%-epiboly 8 h 10-somite 14 h 72 h 50%-epiboly 5.3 h

The approximate generation time for Danio rerio is three months. A male must be present for ovulation and spawning to occur. Females are able to spawn at intervals of two to three days, laying hundreds of eggs in each clutch. Upon release, embryonic development begins; absent sperm, growth stops after the first few cell divisions. Fertilized eggs almost immediately become transparent, a characteristic that makes D. rerio a convenient research model species.[22]

The zebrafish embryo develops rapidly, with precursors to all major organs appearing within 36 hours of fertilization. The embryo begins as a yolk with a single enormous cell on top (see image, 0 h panel), which divides into two (0.75 h panel) and continues dividing until there are thousands of small cells (3.25 h panel). The cells then migrate down the sides of the yolk (8 h panel) and begin forming a head and tail (16 h panel). The tail then grows and separates from the body (24 h panel). The yolk shrinks over time because the fish uses it for food as it matures during the first few days (72 h panel). After a few months, the adult fish reaches reproductive maturity (bottom panel).

3)The time of hatching is not useful as a staging index for the zebrafish, in contrast to some other types of embryos, because individuals within a single developing clutch hatch sporadically during the whole third day of development (at standard temperature), and occasionally later. Whether or not an embryo has hatched, its development progresses, hour by hour, and generally individuals that have spontaneously hatched are not more developmentally advanced that ones remaining in their chorions. We arbitrarily call the creatures "embryos" until the end of the third day, and afterwards, "larvae", whether they have hatched or not.During the hatching period the embryo continues to grow at about the same rate as earlier . Morphogenesis of many of the organ rudiments is now rather complete and slows down considerably, with some notable exceptions including the gut and its associated organs. However, these endodermal structures are difficult to visualize in the living embryo because of their deep positions, and we do not consider them completely here. Much easier to see are the rapidly developing rudiments of the pectoral fins, the jaws, and the gills.

4)To establish an inexpensive and highly reproducible method of removing chorions from about 1600 embryos at a time at 4 hpf, a Belly Dancer shaker was modified to accommodate a custom-machined, anodized aluminum shaker plate that holds 4 glass Petri dish bottoms (100 × 15 mm; VWR, Radnor, PA) and attached water delivery tubing, stainless steel nozzles, and a drain port . The internal workings of the Belly Dancer were modified with a small pump and a parametric motion control.The front panel was modified with an LED display and push button control. The on-board pump supplied rinse water from an external heated (28 °C) carboy via the tubing and nozzles to each glass dish at the appropriate time. The movement of the shaker was controlled by the same system to deliver pulsed agitation or gentle swirling, precisely when needed, to dislodge partially hydrolyzed chorions. The only manually performed steps were the addition of a pronase aliquot to commence digestion and pressing of the start button. No other steps were necessary to operate the device. The pronase digestion of the chorion was performed at 4 hpf. Approximately 2000 zebrafish Tropical 5D strain embryos were received from the Sinnhuber Aquatic Research Laboratory’s mass spawning facility in a 135-mm plastic dish and quickly cleaned by removing all dead, unfertilized, or obviously abnormal embryos with an aspirator, a 5- to 10-min process for a trained technician. Approximately 400 to 500 embryos were placed in each of the 4 glass dishes in 25 mL of FW with 50 μL of 50 mg/mL pronase for 6.5 min while the dechorionator platform constantly agitated. The pronase was then flushed away by gently overflowing the dish with the pumped-in FW for 10 min with 45-s agitation cycles separated by 15 s while still. The total volume of fish water consumed was about 1 L. After the pronase and rinse phases, the embryos were incubated for 20 min at 28 °C, agitated once more to dislodge any remaining chorions, and rinsed again to remove the dislodged chorion.

5)A series of experiments was conducted to estimate phenotypic correlations between incubation characteristics, and to evaluate the effects of cold stress and genotype during incubation on chick weight, egg weight loss, hatching time, and embryonic mortality. Eggs were cooled at 18 or 24 C, for 12, 24, 36, 48, or 72 h beginning on Day 8, 12, 14, 16, or 18 of incubation. Other eggs were cooled intermittently for 6 h every 48 h or 12 h every 96 h. A control group in each experiment was not cold stressed. Results indicated a low and negative correlation between hatching time and chick weight, and a low and positive correlation between hatching time and weight loss from transfer to hatching when variability due to egg weight was removed. Chick weights at hatching were lower in chicks from cooled eggs than those of chicks from eggs incubated under normal temperature. The chicks from cooled eggs were more susceptible to dehydration during holding in the hatcher. Incubation times were delayed approximately as long as the times of embryonic cooling. Embryonic mortality was significantly increased under continuous (single period) cold stress, but not under intermittent cooling (6 h every 48 h). Significant genotype by environment interactions were found in the response of embryos of various strains to cold stress. Exposure for 36 h or longer had detrimental effects on chick weight and embryo viability, but these effects were modified by interactions among the factors involved. The results indicated that embryos from cooled eggs lose more weight during incubation and that the neonatal chicks are more susceptible to dehydration during holding time, and have a longer incubation period, and a greater embryonic mortality.

Microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.

6)Retinoids, the synthetic derivates of vitamin A, have a key role on cellular differentiation and developmental tissue specificity. Their effects are mediated by nuclear receptors which transactivate homeobox genes. They are teratogenic to animals and they all induce similar malformations dependent on the dose and the duration of exposure. This is a review of the teratogenic effects of vitamin A and its synthetic derivates--isotretinoin, acitretine and topical retinoids--in humans. High dose vitamin A have a potent teratogenic effect and are therefore contra-indicated during pregnancy. Isotretinoin is responsible for a syndrome including malformations of the central nervous system, heart and thymus, together with craniofacial defects. The incidence rate is high and comparable to thalidomide (ie, 25%). This high teratogenic potency justifies a strict limitation of such a prescription in women susceptible to become pregnant. Acitretine, which replaces etretinate because of its long half life of 120 days, might also be teratogenic in humans. In addition, it may be back transformed into etretinate, thus contraindicating pregnancy for 2 years after withdrawal. Finally, despite a low percutaneous resorption, available data on the use of retinoids as topicals are limited and their use during pregnancy is therefore not recommended. Although they are efficient in skin diseases, the use of retinoids in women of the child bearing age is very limited because of their potent teratogenic effect.

8)According to the Centers for Disease Control and Prevention (CDC), birth defects occur in one of every 33 babies and are the leading cause of infant death. These problems, present at birth, are caused by genetics, the environment, and other known and unknown causes. Environmental causes of birth defects include chemical and other exposures that occur during pregnancy, including exposure to alcohol and drugs.

Research shows that there is no safe level of alcohol use during pregnancy; even a very small amount can have negative effects on the developing fetus. For this reason, health care providers recommend that women avoid drinking alcohol and eating foods or taking medications that may contain alcohol, such as certain cough syrups, if there is a chance they might be pregnant.

Drug exposure during pregnancy is not limited to illegal drugs, such as cocaine, but includes prescription and over-the-counter drugs and supplements that might disrupt the development of the fetus.The effects of alcohol consumption during pregnancy on the fetus have been known for some time. Such consumption can cause fetal alcohol spectrum disorders (FASDs), which include physical abnormalities and problems with behavior and learning that last throughout a person's life. FAS, which is characterized by more serious growth deficits, neurological defects, and body malformations, including problems with the structure of the head and face, is at the severe end of the FASD spectrum. Previous studies have found some eye abnormalities resulting from prenatal alcohol exposure, but no studies have investigated the effects of heavy alcohol consumption on the eye development of children who do not have FAS.

The children in the study were examined and observed for a period of up to 9 years. None of the children developed FAS even though all had been prenatally exposed to alcohol. The study included a control cohort of children who were not prenatally exposed to alcohol.

Ophthalmologic examination of the children's eyes over the study period of up to 9 years did not show any differences between children who had been exposed to alcohol and those who had not.In the study, the researchers examined 1,355 cases, which included live-born infants, still-born fetuses (fetal death at greater than 20 weeks gestation), and electively terminated fetuses, with birth defects. Control cases included 700 live-born infants with no birth defects or other abnormalities. Women completed computer-based questionnaires during a 4-month periconceptional time, which was defined as from 2 months before conception to 2 months after conception.The results of this study suggested that maternal alcohol intake increases the risk for neural tube, heart, and facial birth defects. Although smoking cigarettes did not contribute to any of the birth defects that were evaluated in the study, smoking during pregnancy is known to increase the risk of miscarriage, stillbirth, preterm birth, low birth weight, Sudden Infant Death Syndrome, lung problems, learning problems, and other short- and long-term health problems.