- Sep 2016
These results indicate that all three characteristics (epidermal thickening, expression of epidermal placode markers, and expression of dermal Bmp4), that is, the presence of an anatomical placode, are required for proper development of scales in reptiles.
the presence of an anatomical placode, are required for proper development of scales in reptiles. as shown all the molecular markers of anatomical placode presents in the sca; just the same as wild type. but due to the inability of sca to form anatomical placode because of another mutation; they fail to form scales properly.
TAB ONE - Panel A
A picture taken from above of the wild-type lizard and the scaleless mutant.
The white arrows show the large spines protruding from the sides on the wild-type individual.
In contrast the Scaleless mutant doesn't have any scales protruding.
TAB TWO - Panel B
A picture taken from below of the wild-type lizard and the scaleless mutant.
The white arrows show the position of secretary glands surrounded by rows of scales that stick out from the inner thigh of the wild-type.
The absence of arrows show that these glands are not found in the Scaleless mutant.
TAB THREE - Micro X-Ray images METHOD
Micros-rays use electromagnetic radiation which give them a high penetrating imaging system. This allows the inside of objects and living organisms to be imaged. This is done at a microscopic level, allowing very small elements to be imaged, such as in this case, where a lizard skull and ‘hand’ are imaged
Computed tomography takes the combination of multiple x-ray images taken at different angles to then create a cross sectional ( a slice) image, as seen in Panel C.
TAB THREE - Panel C
X-ray images of the skull and 'hands'.
The white frames on the skulls show the position of the pleurodont ( regenerating lizard teeth).The wild-type show normal teeth while the scaleless mutant have smaller and fewer teeth.
The double-headed arrows show the length of the claws. The wild-type can be seen to have shorter claws than the scaleless mutants.
TAB FOUR - Panel D
The image shows a simplified representation of the EDA protein (which is made up of amino acids).
The EDA protein is made up two distinct conserved areas- a collagen region and a TNF (tumour necrosis factor). the ares highlighted in red is the most conserved region.
In the wild-type, all the domains are found to be intact.
In the scaleless mutant, 15 amino acids are missing from the extremely conserved area in the TNF domain, leaving only 3 amino acids of the high conserved area.
The highly conserved area (displayed as a red area in the higher panel) is represented in red letters. This area is compared in different species (mouse, chicken, pogona lizard).
This sequence is found in all the species except in the mutant. Because it is found across many different species, this proves that is highly conserved. this usually indicates that it plays an important function.
TAB FIVE - Splicing Process
DNA is assembled in a code so that genetic information can be stored. Genes which are made up of DNA, act as individual units of information. The DNA of a gene will be ‘read’ and turned into RNA, which act as a messenger. This information will then be ‘read’ to form proteins. Proteins are functional and can perform actions and functions e.g. causing reactions, degrading etc.
However each gene is highly processed in a process called splicing. This process will edit the RNA; intron regions (non functional/ non coding regions) of a gene are removed and exons (regions that are read) are linked together.
Incorrect splicing can lead to a non-functional protein.
TAB 6 - RT PCR
Reverse transcription polymerase chain reaction
TAB 7 - Panel E
The image above shows a representation of the structure the EDA gene at specific area. The area represented is composed of Exons 7 & 8 and the intron between these two exons.
The length of the intron that is removed is indicated (1.2 kb). the donor site (gt) and acceptor site (ag) are the sites where the DNa is cut and these two sites are then joined together.
Primers used in RT-PCR are indicated in blue: F1, F2, R1 and R2.
In this experiment, the splicing outcomes are verified and compared between the wild-type and the scaleless mutant. This is done using RT-PCR with the primers indicated with blue arrows: F1, F2, R1 and R2.
The results indicate that there are large differences between the wild-type and the mutant. This is due to the presence of a transposon in Exon 7, outlines in red in the image on the top.
TAB SEVEN - Panel F
The cells of the wild-type’s back (dorsal) and front (lateral), shown in the two leftside images, were stained and compared to the same stained cells of the mutant, shown on the two right side images.
Three photos were taken of each place of each lizard
On the top row, H and E staining
The wild-type, show protruding skin sections, indicating presence of scales
ON the middle row. Immuno-flourescent staining was used.
This highlighted the proteins: α-keratins (α-k) and β-keratins (β-k) or laminin. Keratin is a protein found in scales whereas laminin is found on the outer layers of skin. The wild-type has thick beta keratin layer which is almost entirely missing in the mutant. The laminins show a weird formation in the mutants, as they are much more bumpy.
ON the last row, Toluidine blue (TB) staining was used on the dorsum (back)and scanning electron microscopy (SEM) was used on skin molts;
TB staining shows a clear scale outline in the wild-type whilst it is completely flat in the mutant. The skin molts clearly have different textures between the wild-type and mutant.
TAB EIGHT - Panel G
The wild-type lizard (P. vitticeps) embryo at different stages of development (24, 28, 38, 44 days after beginning of development). The image in the corner show the overall embryo with a red arrow indicating where the image of the cell stained image was taken. The cell staining images show when the scale starts to form.
TAB NINE - Conclusion Overall, these images show the difference between the wild-type lizard and the mutant lizard. The mutant has no scales (panel A and F), not as many teetch and longer claws (panel B and C). It also has a an inserted transposon in the gene (panel E) leading to a mutated EDA protein (panel D).