In this portfolio, I want to showcase some of the group work we did in class. I have never appreciate group work before this class. Perhaps other class where there is group work, the work division is not even. It could be stressful to work in groups.
In this class I really enjoyed the group work and discussion portion. I enjoyed listening to what other people think and also discussion what I think on the lecture material
Below is one of the very first group assignments we did.
Note how we all contributed relatively equally to the work.
1. Please list the names of all the group members who participated:
Sophie Boerlage, Madeline Iseminger, Rosalie Ho, Giulio Sucar Pregnolato
2. Please provide the complete reference for your assigned article (any format that includes authors, year, title and journal is fine).
Collins, D. H. et al. MicroRNAs Associated with Caste Determination and
Differentiation in a Primitively Eusocial Insect. Sci. Rep. 7, 45674; doi: 10.1038/srep45674 (2017).
3. Is your paper mostly an investigation of a factor that affects the developmental trajectory of honeybees, or of a mechanism responsible for it? Please indicate the specific factor or mechanism investigated.
The paper investigates a factor, miRNA, that affects developmental trajectory. The paper explores miRNAs that are differentially expressed between worker-destined and queen-destined larvae, but doesn’t investigate the mechanisms of action or the genes inhibited by the miRNA. It doesn’t investigate factors outside the larvae, such as the components of Royal Jelly, but it neither does it investigate the mechanisms of the miRNA (the mechanism is assumed to be what is already known about miRNA). The authors look for any potential miRNA that may be differentially expressed, then further investigate the validated miRNA (Bte-miR-6001–5p and Bte-miR-6001-3p) to determine where and when in development they’re expressed, and what genes could potentially be inhibited by them.
4. Before reading the article, what were your hypotheses about the main factor and/or mechanism responsible for queen vs. worker developmental trajectory?
We hypothesized that the main factor in developmental determination of bee caste was nutrition, since we knew about the existence of royal jelly, a highly nutritious feed exclusive to potential queen bees.
5. Analyze each of the data figures presented in the paper. For each one, please answer the following questions:
A. What was the experiment that lead to the results? (What were the authors asking, what did they do, what did they measure, what were the controls).
B. What do the data show?
C. What can we conclude from the data?
Figure 1:
a) sRNA-Seq was used to determine the differential expression of miRNA molecules between early- and late-instar worker- or queen-destined larva. The authors were asking whether there are one or more miRNAs expressed at higher concentrations in either worker or queen larvae, but not both. If high differential expression levels are found, it may imply that the expression of the miRNA is in some way triggered by the queen pheromones (or lack thereof), and may target genes involved in developing into a worker or a queen. They sequenced the miRNA (after preparing it and transcribing it into DNA) and normalized the results to the median number of reads (and aligned the reads to the genome). They then compared the number of reads for each stage of development (early and late) between worker and queen destined larvae to find differential expression. If there is a difference between queen and worker bees it would indicate a potential miRNA involved in developing into a queen or worker, and differential expression between stages would indicate the timing of the effect. The authors do not make a clear definition of “early-instar” and “late-instar” considering that Bombus terrestris have four instar phases of development, so it is unclear whether differentiation between worker and queen bees is expected to begin in the early-instar stage. If it is not, then that acts as a technical and biological control for the miRNA in question between queen and worker bees. The authors would expect to see the same levels (within bounds of error) of miRNA expression between the early-instar worker-destined and early-instar queen-destined larvae. For further controls, they took data from eight different colonies, four of which didn’t have a queen present to ensure that the larvae would develop to be queens. This allowed them to harvest four biological replicates of each condition, although they excluded one library from each replicate after checking the efficiency of the normalization. To control for differences in sequencing they could have used technical replicates and sequenced the miRNA of one larvae twice, then compared the expression level of the same miRNA between replicates. They did not do this, but they did use biological replicates from different colonies. Genetic differences between members of the two colonies may result in differential expression of miRNA. The error bars, which are quite large in cases of highly expressed miRNA, are representative of both biological and technical replicates. They also failed to control for tissue type. Large differential expression in one type of tissue may be flattened out by lower expression in other tissue types after normalization.
b) The data show the normalized read counts (y axis is number of reads per four million reads) over four different conditions (EW, early-instar worker-destined larvae; LW, late-instar worker-destined larvae; EQ, early-instar queen-destined larvae; LQ, late-instar queen-destined larvae). The error bars indicate the variance of the specific miRNA reported between the three biological replicates. Asterixis indicate a log2(OFC) ≥ 1, which means a fold change of expression between phenotypes of greater than two. The expression levels of six miRNAs that each had a difference in expression of more than two-fold are plotted over the four phenotypes. The difference between expression is greatest in the last two plots, for Bte-miR-6001–5p and Bte-miR-6001-3p. For each of these, there is a negligible amount found in EW and EQ. There is very low amount of expression in LW, while there is much more expressed in LQ. While the difference between LE and LQ is great, the absolute amount of LQ is approximately mid-range in expression of the six miRNAs shown (~1500 reads per four million) while the greatest expression levels are found in EW Bte-miR-306 at ~7000 per four million.
c) We can conclude that there are at least six miRNAs that are differentially expressed (by at least two-fold) between worker- and queen destined Bombus terrestris in the early- or late-instar stage of development. We can make individual conclusions for each of the six miRNA, but the most differentially expressed (and therefore most interesting/likely to be a factor) miRNAs are Bte-miR-6001–5p and Bte-miR-6001-3p, which have very similar expression profiles, as they are the 5’ and 3’ arms of the Bte-miR-6001 duplex. We can conclude that this miRNA is not produced in significant quantities in the early-instar phase of development. However, in the late-instar phase they are produced in large quantities in the queen-destined bees, but not the worker-destined bees. This indicates that these miRNA may play a role in inactivating genes involved in developing into a worker (or be involved in more complex interactions, such as inhibiting an inhibitor, etc).
Figure 2:
a) The overall goal of the study was to identify microRNAs that play a role in differentiation of queen and worker bees. After the first experiment with sRNA-Seq, six miRNA sequences appeared to be expressed differently in worker or queen destined larvae. A northern blot experiment was run to confirm the sRNA-Seq results. The northern blot was run on the six miRNAs that appeared to behave differently within queens and workers, as well as four mRNAs with sequence homology to miRNAs associated with caste differences in A. mellifera. They used the probe U6 (which should be expressed equally) to control for sample loading. Each phenotype was analysed in at least two replicates drawn from separate but paired pools.
b) The data show that there is a difference in expression levels of miRNAs between larvae destined to become queens or workers (specifically, Bte-miR-6001-5p and Bte-miR-6001-5p). Bte-miR-6001-5p and Bte-miR-6001-5p are each expressed at a much higher rate in late-instar queens as opposed to late-instar workers, and at a higher rate in late-instar versus early-instar larvae of the same caste. Also, the data show that other miRNAs tested did not seem to express differently in the larvae tested (specifically, Bte-miR-13a, Bte-miR-87a, Bte-miR-100, Bte-miR-306, Bte-miR-9a, Bte-miR-184, Bte-miR-71 and Bte-miR-275).
c) We can conclude that the four miRNAs associated with caste differentiation in A. mellifera are not associated with caste differentiation in B. terrestris. We can also conclude that, of the six miRNAs found to be differentially expressed between B. terrestris caste phenotypes based on sRNA-seq data, only the two arms of the mir-6001 duplex are confirmed to be associated with caste differentiation in B. terrestris.
Figure 3:
a) To better understand the tissue and stage-specificity of the two miRNAs, Bte-miR-6001-5p and Bte-miR-6001-3p, the authors used Northern blots. The RNA samples were extracted from larval and pupal samples. Then, miR-6001-5p and miR-6001-3p probes were utilized to visualize the amount of miRNAs Bte-miR-6001-5p and Bte-miR-6001-3p present in the head, digestive tract and outer cuticle of late-instar queen-destined larvae, and the expression profiles of these miRNAs throughout the bodies of queen-destined larvae and pupae. The U6 control was also performed to act as a loading control as the U6 probe hybridized to a stably expressed nuclear RNA.
b) For all treatments, miR-6001-3p produced a weaker signal than miR-6001-5p which could be an artifact of differential probe efficiency or due to a lower concentration present. In the case of tissue-specific expression profile, both miRNA were found to be expressed highly in the outer cuticle of late-instar queen-destined larvae, while lower levels of expression were detected in the head and digestive tract. In the investigation of stage-specific expression profile, the expression of these miRNAs were found to be higher in late-instar queen-destined larvae and early queen pupae than in late queen pupae.
c) Previous studies have shown that queen differentiation is influenced by the delayed molting of later larval instars, which is contributed to by the elevated expression of genes involved in the biogenesis of larval cuticular. The elevated expression of miR-6001-3p and miR-6001-5p profile in the outer cuticle and during late-instar queen-destined larvae to early queen pupae suggests that miRNA expression of Bte-miR-6001-5p and Bte-miR-6001-3p contributes to the differentiation into queens.
Figure 4:
a) The authors wanted to discover the genomic context in B. terrestris for the two arms of the miR-6001 duplex that were confirmed to be differentially expressed by the Northern blot (fig. 2), Bte-miR-6001-5p and Bte-miR-6001-3p, and to compare this to the genomic context in A. mellifera. To determine the genomic context in B. terrestris, they scanned the B. terrestris genome for the gene bte-mir-6001 using BLAST, and then determined the neighboring genes using GBrowse. This work was repeated with the A. mellifera genome.
b) The data shows that the predicted precursor sequence for the mir-6001 duplex takes up the whole predicted fourth intron of a very high density lipoprotein (Vhdl) gene in both B. terrestris and A. mellifera. It also shows that the predicted nucleotide sequences for mir-6001 are slightly different between the species.
c) We can conclude that mir-6001 is a mirtron, or an intronic miRNA that starts out as pre-miRNA produced via intron splicing. Also, as Vhdl is homologous to a gene for Vitellogenin, which is a type of protein that is induced by Juvenile Hormone and is known to be important for some reproductive processes in certain members of Hymenoptera, it’s possible that Vhdl itself might be associated with caste determination in bees and other eusocial members of Hymenoptera.
6. As a group, select what you think is the most important or impactful data figure presented in your paper. Briefly explain why you think that it is the most important, and how it contributes to furthering our understanding of the regulation of development in female honeybees (and possibly, developmental regulation in general).
Figure 2 is the most important figure because it shows that the miRNAs that are important for caste determination in A. mellifera aren’t necessarily important for caste determination in B. terrestris. It identifies mir-6001 as confirmed to be differentially expressed between late-instar worker- and queen-destined B. terrestris larvae, as well as between early- and late-instar larvae of the same destiny. This figure contributes to our understanding of development in female honeybees by showing that miRNA regulatory mechanisms are not necessarily conserved throughout evolutionary history in bees, and that certain miRNAs likely play a role in caste determination, especially in late-instar larvae.
7. Think about the experience that you just had of working collaboratively on an assignment. As a group, how did you proceed? Was the strategy successful? How did you define “successful”? If you were to work together again, would you do anything differently?
As a group, we read the paper together, discussed the hypothesis and questions briefly. We each took up parts of question 5 and divided the rest of the questions amongst ourselves. We also created a google docs to share our answers and discuss the answers submitted by everyone. The strategy was successful as everyone’s workload was roughly equal. The google docs also facilitated discussion, enabling us to help one another to better understand the figures and overall objectives of the experiments. If we were to work together again we may implement deadlines to draft answers to the questions, which was not needed in this assignment as this is not an elaborate assignment.
