Transcript Zebrafish Embryo - Arne Christensen | Anna Maria College

The Effects of Varying Alcohol Concentrations on Chicken
and Zebrafish Embryonic Development
Kyla-Gaye Pinnock, Mareneth Banzon, and Arne Christensen
BIO 452, Developmental Biology, York College CUNY, Queens, NY 11451, USA
In this experiment the effects of ethanol, at concentrations that are
physiologically relevant to human alcohol consumption, were tested
on vertebrate embryonic heart development. Two model systems
that were used are the chick (Gallus gallus) and zebrafish (Danio
rerio) embryos. In the chick, exposure to different concentrations of
ethanol on heart rate were observed, by pipetting ethanol to
unshelled embryos and measuring the heart beats per minute. In
the zebrafish model, we observed how ethanol exposure affects
heart structure. Freshly fertilized transgenic (cardiac-GFP) zebrafish
eggs were placed in Petri dishes containing different concentrations
of ethanol. Results indicated that the ethanol exposure in chicks
reduced the heart rates significantly, while in zebrafish, physical
deformities of the heart and body structure were observed. In both
cases, ethanol had adverse effects on embryonic development.
Chick Embryo:
A chick egg that was previously incubated for approximately 3 days
was cracked open slightly to observe the embryo inside. Increasing
concentrations of ethanol – 0.00%, 0.01% ,0.05%, or 0.09%, were
added to the embryo and the heart rate was subsequently recorded.
Zebrafish Embryo:
Transgenic zebrafish Tg(cmlc2:egfp) embryos with a heart-specific
promoter driving cardiac GFP expression were provided by Dr.
Nathalia Glickman Holtzman from Queens College / CUNY. Viable
eggs were selected from stock eggs, and were placed in egg water
containing increasing concentrations of ethanol; 0.0, 0.5, or 1.0 %.
Approximately 40 eggs were used for each treatment, and
observations were made five days after the introduction of alcohol
into the systems of the zebrafish.
INTRODUCTION
The heart is derived from mesoderm and forms in the neck area,
moving posterior to the thoracic region as the head elongates. At
first, it starts off as a single tube, with the conus arteriosus at the
anterior end followed by the atrium, ventricle and ending with the
cinus venosus at the posterior. In birds and mammals, the heart
takes on a four chambered shape: atrium and ventricle split into left
and right, conus arteriosus splits into aorta and pulmonary veins and
the cinus venosus develops into the sinoatrial node or pacemaker
which regulates heart rate. After 48 hours, the heart of the chick
embryo starts to beat. It is at this time, that we performed our
experiment with ethanol.
Ethanol is a depressant. By binding to nerve receptors, ethanol
blocks neurotransmitters from being sent. In the case of the heart,
neurons stimulating the sinoatrial node control whether the heart
beats faster or slower. Alcohol limits the impulses generated by
neurons causing the heart rate to decrease. Our hypothesis is that
the embryonic heart rate should slow down after ethanol is given to
the embryo and that the effect will increase with the concentration.
Zebrafish make good model organisms due to their rapid
development and the fact that they can be easily maintained in a 2528 ̊C range. While chick embryos can be observed for their heart rate
when unshelled, the progress of zebra fish can be seen in a Petri dish
so we could compare ethanol exposed embryos to normal embryos.
The results are essential in understanding how alcohol affects
embryonic development.
RESULTS: ZEBRAFISH
MATERIALS/METHODS
ABSTRACT
RESULTS: CHICK
Effects of ethanol on heart rate (BPM)
EtOH (%)
0.00
0.00
0.01
0.05
0.09
read 1
76
88
87
100
86
Heart Rate (BPM) Before Treatment
read 2
read 3
read 4
read 5
75
75
74
72
NA
NA
NA
NA
77
71
72
73
100
98
94
94
81
80
82
72
Average
74.4
88.0
76.0
97.2
80.2
EtOH (%)
0.00
0.00
0.01
0.05
0.09
Heart Rate (BPM) Immediately After Treatment
read 1
read 2
read 3
read 4
read 5
78
68
66
62
72
75
70
62
68
64
70
67
75
73
71
80
79
75
72
70
73
82
73
75
74
Average
69.2
69.3
71.2
75.2
75.4
EtOH (%)
0.00
0.00
0.01
0.05
0.09
Heart Rate (BPM) 10 Minutes After Treatment
read 1
read 2
read 3
read 4
read 5
79
76
68
75
74
NA
NA
NA
NA
NA
65
66
65
66
66
79
76
78
75
74
29
31
31
35
35
Average
74.4
NA
65.6
76.4
32.2
Figure 1: Chick embryos were treated with different concentrations of ethanol (EtOH)
– 0.00, 0.01 ,0.05, or 0.09 %. Heart rate was determined before treatment, immediately
after treatment, and 10 minutes after treatment. Heart rate was determined five times
and then averaged (last table column).
Figure 2: Pictures of the zebrafish heart expressing Green Florescent
Protein. A: The left panel shows a regular two chambered zebrafish
heart. B: The right panel shows the deformed heart, and enlarged
pericardial sac, of a zebrafish exposed to ethanol. C: The left panel
shows normal zebrafish heart structure. D: The right panel shows the
deformed heart of a zebrafish exposed to ethanol. The atrium and the
ventricle are somewhat indistinguishable.
DISCUSSION/CONCLUSION
• Alcohol Negatively affects both chick and zebrafish embryos
• High concentrations of ethanol showed reduced heart rate
in chick embryos (Figure 1, 0.09% ethanol 10 minutes after
treatement).
• Ethanol induced developmental defects in zebrafish
including fattened tails, a decrease in responsiveness,
swelling of the pericardial sac, and the elongation of the
heart atrium and ventricle
• Concrete conclusions cannot be drawn from one experiment
but it was observed that alcohol causes developmental
defects in both chick and zebrafish embryos.
REFERENCES
Gilbert, S. F. (2010). Developmental Biology (9th ed.). N.p.:
Sinauer Associates, Inc.
Tyler, M. S. (2010). Developmental Biology: A Guide For
Experimental Study (3rdrd ed.). Saunderland, MA: Sinauer
Associates, Inc.
Dlugos, C.A and Rabin, R. A. (2010). R.A Structural and
Functional Effects of Developmental Exposure to Ethanol on the
Zebrafish Heart. Alcohol Clin Exp Res. 34, 1013-1021.