Blogs

The two main observations that are made in this paper are that there is a low level of thyroid hormone at the time of birth, with a sharp increase on day 6, peaking in day 15 and falling at day 25. This observation is shown in figure 1. The second observation is that in mice with induced hypothyroidism, the rate of mitosis increase compared to controls at day 2 and day 6. This observation is shown in figure 2. In figure 2, the researchers are measuring the cells that are going through DNA synthesis. This, in turn, indicates the cells that are going through mitosis. The difference between P2N and P6N indicates how many cells have been labeled with BrdU, and by extension, how cell division has increased or decreased in control mice brain on day 2 compared to day 6. The difference between P2N and P6N indicates how much cells have been labeled by BrdU and consequently, the amount of cell division has increased or decreased in PTU treated (induced hypothyroidism) mice in the brain at day 2 compared to day 6. P2N/P6N observation relates to the data in figure 1 because the thyroid level of the brain in P2N and P6N  which are normal levels are in figure 1. This indicates that at time of P2N and P6N, the level of thyroid hormone, which is not mentioned specifically, is found in figure 1. From figure 1, the conclusion that can be drawn is that because there was an increase in thyroid hormone from day 2 to day 6, the decrease in mitotic cells in P6N compared P2N indicates that thyroid hormone may be causing a decrease in cell division and DNA synthesis. The hypothesis of this paper hyperthyroidism causes decreased mitotic activity compared to control mice. In the paper, the researchers never explained the biological mechanism of how hyperthyroidism causes a decrease of mitotic activity, only that it does actually cause the decrease rather than just correlate with the decrease.

Weatherbee, S. D., Behringer, R. R., Rasweiler, J. J., & Niswander, L. A. (2006). Interdigital webbing retention in bat wings illustrates genetic changes underlying amniote limb diversification. Proceedings of the National Academy of Sciences, 103(41), 15103–15107. doi: 10.1073/pnas.0604934103

• Discusses the process of interdigital webbing in mammals, including examples of bat forelimb webbing retention and duck hindlimb webbing retention. Inhibition of apoptosis caused by Gre and FGF signaling within interdigital space results in retention of membrane.

Pajni-Underwood, S., Wilson, C. P., Elder, C., Mishina, Y., & Lewandoski, M. (2007). BMP signals control limb bud interdigital programmed cell death by regulating FGF signaling. Development, 134(12), 2359–2368. doi: 10.1242/dev.001677

• Discusses BMPs used as a regulator of programmed cell death within the interdigital membrane. Bmpr1a inactivation in mice after limb bud initiation results in upregulation of Fgf4 and Fgf8. Inactivation of Bmpr1a also results in decreased apoptosis within interdigital membrane, leading to webbed digits.

(Adapted from Developmental Biology essay)

Homo Lontra is a species of hominid located in modern day coastal Europe. Upon increased competition from the Homo Sapien invasion of Eurasia due to their emigration out of Africa, Homo Neanderthalensis was forced to the coast, outcompeted for access to many land animals and foraging areas. There, Homo Lontra evolved from Neanderthals, with visible adaptions for a diet of coastal marine foraging.  This included webbed feet for better aquatic propulsion, a hardened enamel for consuming hard-shelled marine animals, and the re-evolution of dense body hair to protect their bodies from the cold European winters and water. These traits are similar to those of other aquatic mammals-such as otters (Homo Lontra’s namesake)-but a complete molecular and developmental analysis must be performed in order to determine their true origins.

The teeth of Homo Lontra are much tougher than that of Homo Sapiens due to their increased enamel thickness. In mice and humans, the teeth are not continuously grown or renewed after development; therefore, any alteration to tooth structure would have to take place developmentally.¹ Muscle segment homeobox 2 (MSX2) encoded proteins act as repressors; specifically affecting epithelial-mesenchyme interactions as well as ameloblast differentiation, a cell that deposits enamel proteins during tooth development.²Msx2^+/– mutants display stronger expression of amelogenin (proteins related in amelogenesis; enamel development) than that of Msx2^+/+ individuals, with the heterozygote displaying a twofold increase in amelogenin expression.²

1. Li, J., Parada, C., & Chai, Y. (2017). Cellular and molecular mechanisms of tooth root development. Development, 144(3), 374–384. doi: 10.1242/dev.137216
2. Molla, M., Descroix, V., Aïoub, M., Simon, S., Castañeda, B., Hotton, D., … Berdal, A. (2010). Enamel Protein Regulation and Dental and Periodontal Physiopathology in Msx2 Mutant Mice. The American Journal of Pathology, 177(5), 2516–2526. doi: 10.2353/ajpath.2010.091224

Figure 2 shows that the her2 is regulated by the delta notch pathway. The authors came to this conclusion by measuring the expression of her2 in notch deficient embryos. This shows that the her2 is somewhere in the notch pathway because everything else is kept constant. Because neurogenesis occurs in the first 3 days after fertilization, since the drug was added at day 5, her 2 is no longer regulated by Notch signaling at the time that our study is done. However, because the study that we are doing involves not only neurogenesis, but also oligogenesis, and how those two things that are influenced by the thyroid hormones affect stem cells. In the paper, while the paper mentions that her2 is not regulated by notch signaling after oligogenesis, it does not discount that her2 is not affected by the thyroid hormone through some other pathway. Because of this, it is possible that her2 does have an effect on the neural stem cell. However, the only thing that is known is that if her2 does affect neurogenesis and gliogenesis, it will not be through the notch pathway.  I think that her2 is definitely playing a role that can be seen in the class data. The paper mentions that her2 induces glial differentiation and that inhibits the neural differentiation, which was similar to how hypothyroidism affects the neural cells in the papers we have read. However, I am not sure whether her2 directly responds to TH. There is no evidence for me to think that the two are related, especially since her2 is not connected to notch signaling in fish that are past neurogenesis.  I think that her 2 is promoting the formation of glia because the study is involved in glial differentiation, this would imply that her2 is promoting the formation of the glia. 

The efficiency of the mutated RHO allele cleavage is 70.7% whereas the efficiency of the wt RHO allele is 3.8%. In the wt there was 8% insertion and deletion using this method but in the mut, there was 93% insertion-deletion in this method. In the wt, the rho localized on the plasma membrane while the P23H mutant protein remained trapped in the cytoplasm. The two insertion-deletion mutations the antibodies were not seen in both of those mutations. In this experiment, after one month there was a higher thickness of OS was higher in the CAS9-VQR and sgRNA mut compared to cas9-vqr treatment only. There was no change in the ONL thickness did not differ depending on the treatment. At three months, there was a slight difference in ONL thickness, with cas9-VQR and sgRNAmut having thicker ONL layer and cas9-vqr having the least thickness. The effect on the thickness of OS layer remained the same. The CRISPR-VOR the uninjected eye presented better response compared to the treated eye, indicating that there is a detrimental effect that the procedure has on the function of the retina.The injection method allowed for the gene delivery. All subtypes of the retinal cell types were targeted. However, the rod photoreceptor had the highest transduction and the glial cells had the lowest transduction. GFP transduction was robust and diffusion the ONL. The RHO mut gene presented a significant level of cleavage GFP high cells exhibited a higher accumulation of indels High effect in unedited P23H allele. Every delivery system that was compared had the same amount of frameshift mutation in the allele. If the mutation resulted in the stop codon, it was more likely to disrupt the expression. The GFP plus cells had a 3.2% efficiency while te GFP high mutation had a 48% efficiency. 

The goal of the research is to validate a CRISPR/CAS9 inactivation treatment for the P23H mutant for Renintitis pigments and put the treatment to test in vivo using the AAV delivery system Experiment 1 that was shown in the study tested whether RHOgene allele-specific targeting could be achieved in vivo. To do this, the researchers attempted to deliver the CRISPER CAS9 VQR system components in the retina of mice by invivo electroporation. To do this, the researchers devised a vector that pCAG vector was electroporated with GFP reporter in the RHO Het eyes.  The invivo efficiency was assessed by harvesting the eyes and purified by FACS sorting and molecular analysis was done.  The mutation frequency of the wt rho gene was also analyzed to test the effect of treatment on Wt cells. From experiment 1, it has been noted that the P23H mutation had high rates of frameshift mutation which leads to the inactivation of RHO allele. The mutant allele is not effective in treating the disease. To asses, this problem The RHO gene mutations that occurred the most often were overexpressed in mice P19 cell line, and colocalization of the Rho n terminus antibody and Rho c terminus antibody.  because the experiment 1 and two resulted in an efficient delivery of CRISPR/CAS9 elements in the retina, with targeting rate of the mutant gene, the researchers attempted to asses the photoreceptor degeneration in mice that was treated through an histological analysis to see what the result of the treatment in the degeneration of the photoreceptor cell. The histological study was done at 1 month.  The same histological study was done again at 3months. To test the injection method of the treatment, the intravitreal injection of AAV2 variant was given, and the histological studies of the retina were performed for the expression of transgenes in the tissue as opposed to the retina specifically. The AAV-PHP mediated targeting of mutation of the whole retina was used with CRISPER/CAS9 was used and the two different approaches are both used, and the efficiency of treatment was assessed for the genetic change. In this experiment, the possibility of enriching for the editing events using GFP transduction using FACS sorting. Using the data that is observed in experiment 6, the researches subjected the High GFP and low GFP cells to NGS analysis to refine the indel analysis. To do this, a PCR of the RHO allele was done to asses their incidence and the editing frequency in retinal samples. This experiment aimed to confirm the selectivity of the approach for the P24H RHO allele.

Cell splitting is the process in which a cell line is moved to a different flask in order to keep the cell line alive. Cell splitting must be done before the cell population gets too large. If the cell population gets too big, the cells will overcrowd the flask they are in and die as a consequence. In the bioimaging lab, a course I am currently taking. In this class, my lab partner and I are working with a line of LLC-Pk1 epithelial cells., and currently have three flasks of cells for our project. When the time comes for splitting, we follow a specific protocol. First, we prepare the new flask that the cells are going into with medium and correct labeling. We must then remove the medium that is already in the flasks. After that we wash the cells with about 1 mL of warm PBS (phosphate-buffered saline) and after rocking the flask and making sure the PBS has washed thoroughly, the PBS is removed and cells that have died have been removed from the flask. The next step is to add about 0.5 mL of trypsin, and then incubate the flask for about 3 minutes at 37 degrees Celsius. This process removes cells from the bottom of the flask so that they can now be transferred to the next flask. The trypsin reaction is stopped b adding about 1 mL of cold medium to the flask and triturating thoroughly Finally, we can now put 5 or so drops from the old flask into the new flask, where the cells now have more room to grow until they need to be split again.