effect of lithium chloride on the development of zebra fish

Effect of Lithium Chloride on the Development of Zebrafish Embryos

Effect of Lithium Chloride on the Development of Zebrafish Embryos

Done By: Denise Lim, Eugene Ng, Au Shi Yi and Nickolas Teo

Aim of Research ProjectAim of Research Project

To investigate the effects of lithium chloride on the development of Zebrafish Embryos

To use results as justification of the detrimental effects of Lithium Chloride on Human Embryonic Growth

To investigate the effects of lithium chloride on the development of Zebrafish Embryos

To use results as justification of the detrimental effects of Lithium Chloride on Human Embryonic Growth

HypothesisHypothesis

Lithium Chloride has adverse effects on embryonic development and can cause malformations and dysfunctional growth.

Zebrafish embryos exposed to Lithium Chloride during development will experience retarded anterior-posterior development and experience brain damage.

Higher concentrations of LiCl cause increasingly pronounced deformities and increasingly stunted growth. Higher concentrations of teratogen also increase fatality rates of embryos.




Background ResearchBackground Research

1.

Zebrafish as a Model Organism

Small in size, low maintenanceHigh fertility- Zebrafish females are known to produce hundreds of eggs at one timeShort generation time (3-6 months for maturity)External fertilization (easily harvested eggs for study)Completely sequenced genome aids in identification and manipulation of genesTransparent embryos (easily observable organs)

1.

Zebrafish as a Model Organism

Small in size, low maintenanceHigh fertility- Zebrafish females are known to produce hundreds of eggs at one timeShort generation time (3-6 months for maturity)External fertilization (easily harvested eggs for study)Completely sequenced genome aids in identification and manipulation of genesTransparent embryos (easily observable organs)


2. Lithium Chloride as a teratogen

Lithium salts are known teratogens and have proven to have detrimental effects on the central nervous system.

Lithium Chloride is known to inhibit the expression of the HoxGene

Malformations of central nervous system cause dysfunctional nerve and muscle development, causing retardation in movementRetarded Anterior-posterior growth is a result of embryonic exposure to LiCl

First stage of normal cell development and division: Cells go towards anterior.Abnormal development: Cells go towards posterior instead of anterior, causing brain damage as well as underdeveloped nerve , neuron, and muscular systems. ( Brain is the catalyst of the formation of nervous system.)

2. Lithium Chloride as a teratogen

Lithium salts are known teratogens and have proven to have detrimental effects on the central nervous system.

Lithium Chloride is known to inhibit the expression of the HoxGene

Malformations of central nervous system cause dysfunctional nerve and muscle development, causing retardation in movementRetarded Anterior-posterior growth is a result of embryonic exposure to LiCl

First stage of normal cell development and division: Cells go towards anterior.Abnormal development: Cells go towards posterior instead of anterior, causing brain damage as well as underdeveloped nerve , neuron, and muscular systems. ( Brain is the catalyst of the formation of nervous system.)


3. Known mutations caused by Lithium Chloride

-

Bananas exposed to LiCl

turn green and mouldy

and start harboring evil intentions to take over the animal kingdom

-

Sheep exposed to LiCl

turn into Cows-

Humans exposed to LiCl

remain partially human but develop abnormally inflated heads as well as green tails. Tendency to evolve into cowlike

structures after prolonged exposure to sunlight.

-

Cats become dogs, dogs become cats, and rats just go moo.-

Rats develop elephant trunks and begin to spray water out of petri

dishes.

Disclaimer: May be inaccurate…

3. Known mutations caused by Lithium Chloride

-

Bananas exposed to LiCl

turn green and mouldy

and start harboring evil intentions to take over the animal kingdom

-

Sheep exposed to LiCl

turn into Cows-

Humans exposed to LiCl

remain partially human but develop abnormally inflated heads as well as green tails. Tendency to evolve into cowlike

structures after prolonged exposure to sunlight.

-

Cats become dogs, dogs become cats, and rats just go moo.-

Rats develop elephant trunks and begin to spray water out of petri

dishes.

Disclaimer: May be inaccurate…

Mutation of Zebrafish after exposure to Lithium Chloride

Mutation of Zebrafish after exposure to Lithium Chloride

LiCl

Materials and ApparatusMaterials and Apparatus

Fish TankZebrafish pair ( it is vital that they be of the opposite gender)SievePetri DishesPartial Pipette9ml of Lithium Chloride SolutionEmbryo Medium ( Distilled water + Methylene Blue –to prevent fungal contamination + DMSO media)NeedlesDissecting MicroscopeMicropipette + disposable tips

Fish TankZebrafish pair ( it is vital that they be of the opposite gender)SievePetri DishesPartial Pipette9ml of Lithium Chloride SolutionEmbryo Medium ( Distilled water + Methylene Blue –to prevent fungal contamination + DMSO media)NeedlesDissecting MicroscopeMicropipette + disposable tips

Methodology :Methodology :

1. Embryo HarvestingPut a pair of male and female Zebrafish in the same tank filled with egg water. Use a separator between female and male fishes to prevent matingRemoval of separator to facilitate mating; collection of fish eggsRemove wire mesh separating fish eggs and sieve to wash out faeces. Empty sieve in petri-dish containing Embryo Medium ( Methylene Blue + Distilled Water)

1. Embryo HarvestingPut a pair of male and female Zebrafish in the same tank filled with egg water. Use a separator between female and male fishes to prevent matingRemoval of separator to facilitate mating; collection of fish eggsRemove wire mesh separating fish eggs and sieve to wash out faeces. Empty sieve in petri-dish containing Embryo Medium ( Methylene Blue + Distilled Water)

Methodology:Methodology:

2. Incubation for 4.7 hours until 30% Epiboly

3. Separation of fish eggs into four separate petri

dishes. All four petri

dishes contained 10ml of Embryo Medium containing Zebrafish eggs.

4. Usage of needles to make small holes in chorion

membrane in order to facilitate penetration of LiCl

to Embryo

5. In order to measure the effects of varying concentrations of LiCl

on Embryonic development, dose response procedures were carried out and three petri-dishes out of four had varying amounts ( 1.5ml, 3ml,4.5ml) of LiCl

added to them. One petri

dish was used as a control reference and LiCl

was

not added.

2. Incubation for 4.7 hours until 30% Epiboly

3. Separation of fish eggs into four separate petri

dishes. All four petri

dishes contained 10ml of Embryo Medium containing Zebrafish eggs.

4. Usage of needles to make small holes in chorion

membrane in order to facilitate penetration of LiCl

to Embryo

5. In order to measure the effects of varying concentrations of LiCl

on Embryonic development, dose response procedures were carried out and three petri-dishes out of four had varying amounts ( 1.5ml, 3ml,4.5ml) of LiCl

added to them. One petri

dish was used as a control reference and LiCl

was

not added.

Methodology:Methodology:

6. 10 minute incubation of Petri Dishes at 280C due to potency of LiCl

7. Usage of partial pipette to remove embryo medium and LiCl

in all petri

dishes excluding control dish. Fresh embryo medium was added to each petri

dish.

8. 24 hours incubation of Petri Dishes at 280C


in all petri


dish.

6. 10 minute incubation of Petri Dishes at 280C due to potency of LiCl


in all petri


dish.

8. 24 hours incubation of Petri Dishes at 280C


in all petri


dish.

MethodologyMethodology

10. Observation and recording of unique embryonic developments in different concentrations of LiCl.

11.Usage of partial pipette to remove embryo medium and LiCl

in all petri

dishes excluding control dish. Fresh embryo medium was added to each Petri dish.

12. Usage of needles to dechorionate

embryos, in order to facilitate clearer observation of embryonic development.

13. Incubation at 280C and noting of embryonic development in

different concentrations of LiCl

10. Observation and recording of unique embryonic developments in different concentrations of LiCl.

11.Usage of partial pipette to remove embryo medium and LiCl

in all petri

dishes excluding control dish. Fresh embryo medium was added to each Petri dish.

12. Usage of needles to dechorionate

embryos, in order to facilitate clearer observation of embryonic development.

13. Incubation at 280C and noting of embryonic development in different concentrations of LiCl

Day 1 resultsDay 1 results

There were no noticeable changes observed at this point in time.

We had about 20 eggs in each of the four Petri dish, however we killed some eggs in the process of creating a hole in their membrane bypiercing the eggs too hard.

We then placed 1.5ml,3.0ml and 4.0ml of lithium chloride into the first, second and third Petri dish respectively. Leaving the fourth as control with no LiCl added.

Due to the strength of the lithium chloride, we changed the egg water (Methylene Blue and sterile water) after 10 minutes to prevent them from being killed.

There were no noticeable changes observed at this point in time.

We had about 20 eggs in each of the four Petri dish, however we killed some eggs in the process of creating a hole in their membrane bypiercing the eggs too hard.

We then placed 1.5ml,3.0ml and 4.0ml of lithium chloride into the first, second and third Petri dish respectively. Leaving the fourth as control with no LiCl added.

Due to the strength of the lithium chloride, we changed the egg water (Methylene Blue and sterile water) after 10 minutes to prevent them from being killed.

The control


After taking out the eggs from the incubator at 9.10am, we counted and removed the eggs killed by the teratogen.Around 3 to 10 eggs in each petri dish died due to the lithium chloride poisoning.More died after we removed the membrane surrounding the organisms in the four Petri dishes

After taking out the eggs from the incubator at 9.10am, we counted and removed the eggs killed by the teratogen.Around 3 to 10 eggs in each petri dish died due to the lithium chloride poisoning.More died after we removed the membrane surrounding the organisms in the four Petri dishes

0.15 ml solution


This was observed from the organismsIn the Control: the zebra fishes were growing well, and the tails were long, well-developed, and moving about.In the 0.15M of lithium chloride: the zebra fishes were still growing well, apart from the abnormal growth in certain places, there were also a shorter tail and less tail movement .In the 0.30M of lithium chloride: the head of the zebrafisheswere small, they grew a short tail and there was very little movement.In the 0.45M of lithium chloride: the head of the zebrafisheswere extremely small or huge and the tail was non-existent or deformed completely.

This was observed from the organismsIn the Control: the zebra fishes were growing well, and the tails were long, well-developed, and moving about.In the 0.15M of lithium chloride: the zebra fishes were still growing well, apart from the abnormal growth in certain places, there were also a shorter tail and less tail movement .In the 0.30M of lithium chloride: the head of the zebrafisheswere small, they grew a short tail and there was very little movement.In the 0.45M of lithium chloride: the head of the zebrafisheswere extremely small or huge and the tail was non-existent or deformed completely.

0.30ml solution


In the control: the zebrafishes were growing well, the heart was beating normally, there was a long tail and normal circulation. It also moves when stimulatedIn the 0.15ml: the zebrafishes was still growing well, but the head was slightly squashed and out of proportion. The tail was also shorter and moves when stimulated.

In the control: the zebrafishes were growing well, the heart was beating normally, there was a long tail and normal circulation. It also moves when stimulatedIn the 0.15ml: the zebrafishes was still growing well, but the head was slightly squashed and out of proportion. The tail was also shorter and moves when stimulated.

0.45 ml solution


In the 0.30ml: there are weak heartbeats, and poor blood circulation. The tail is also skinner and bent in some zebrafishes.In the 0.45ml: there was very stunted growth , the heart was beating very slowly and little blood circulation was apparent.

In the 0.30ml: there are weak heartbeats, and poor blood circulation. The tail is also skinner and bent in some zebrafishes.In the 0.45ml: there was very stunted growth , the heart was beating very slowly and little blood circulation was apparent.

0.45ml solution

ConclusionConclusion




Therefore, our hypothesis is confirmed.




Therefore, our hypothesis is confirmed.

effect of lithium chloride on the development of zebra fish

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