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Probing the relationship between BTBD9 and MEIS1 in C. elegans and mouse

Subject: Life Science and Biomedicine

Published online by Cambridge University Press:  31 March 2020

Shangru Lyu Open the ORCID record for Shangru Lyu [Opens in a new window] ,
Atbin Doroodchi ,
Yi Sheng ,
Mark P. DeAndrade ,
Youfeng Yang ,
Yuning Liu Open the ORCID record for Yuning Liu [Opens in a new window] ,
Michael A. Miller ,
Rui Xiao  and
Yuqing Li Open the ORCID record for Yuqing Li [Opens in a new window]
Shangru Lyu
Affiliation:
Norman Fixel Institute for Neurological Diseases, Department of Neurology, College of Medicine, University of Florida, Gainesville, FL, 32610, USA
Atbin Doroodchi
Affiliation:
Department of Cell, Developmental and Integrative Biology, the University of Alabama at Birmingham, Birmingham, AL, 35294, USA
Yi Sheng
Affiliation:
Department of Aging and Geriatric Research, College of Medicine, University of Florida, Gainesville, FL, 32610, USA
Mark P. DeAndrade
Affiliation:
Norman Fixel Institute for Neurological Diseases, Department of Neurology, College of Medicine, University of Florida, Gainesville, FL, 32610, USA
Youfeng Yang
Affiliation:
Department of Cell, Developmental and Integrative Biology, the University of Alabama at Birmingham, Birmingham, AL, 35294, USA
Yuning Liu
Affiliation:
Norman Fixel Institute for Neurological Diseases, Department of Neurology, College of Medicine, University of Florida, Gainesville, FL, 32610, USA
Michael A. Miller
Affiliation:
Department of Cell, Developmental and Integrative Biology, the University of Alabama at Birmingham, Birmingham, AL, 35294, USA
Rui Xiao
Affiliation:
Department of Aging and Geriatric Research, College of Medicine, University of Florida, Gainesville, FL, 32610, USA
Yuqing Li*
Affiliation:
Norman Fixel Institute for Neurological Diseases, Department of Neurology, College of Medicine, University of Florida, Gainesville, FL, 32610, USA
*
*Corresponding author. E-mail: yuqing.li@neurology.ufl.edu

Abstract

Restless legs syndrome (RLS) is a neurological disorder characterized by an urge to move and uncomfortable sensations. Genetic studies have identified polymorphisms in up to 19 risk loci, including MEIS1 and BTBD9. Rodents deficient in either homolog show RLS-like phenotypes. However, whether MEIS1 and BTBD9 interact in vivo is unclear. Here, with C. elegans, we observed that the hyperactive egg-laying behavior caused by loss of BTBD9 homolog was counteracted by knockdown of MEIS1 homolog. This was further investigated in mutant mice with Btbd9, Meis1, or both knocked out. The double knockout mice showed an earlier onset of the motor deficit in a wheel running test but did not have increased sensitivity to heat stimuli as observed in single knock outs. Meis1 protein level was not influenced by Btbd9 deficiency, and Btbd9 transcription was not affected by Meis1 haploinsufficiency. Our results demonstrate that MEIS1 and BTBD9 do not regulate each other.

Keywords

restless legs syndromehpo-9unc-62Btbd9Meis1

Information

Type
Research Article
Information
Experimental Results , Volume 1 , 2020 , e8
Result type: Novel result
Creative Commons
Creative Common License - CCCreative Common License - BY
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copyright
© The Author(s) 2020

Introduction

RLS is characterized by a strong urge to move and uncomfortable sensations in lower limbs, which can be relieved by movements. Genome-wide association studies have implicated up to 19 risk loci for RLS, across which two of the candidate genes are MEIS1 and BTBD9 (Schormair et al., Reference Schormair, Zhao, Bell, Tilch, Salminen, Putz, Dauvilliers, Stefani, Högl, Poewe, Kemlink, Sonka, Bachmann, Paulus, Trenkwalder, Oertel, Hornyak, Teder-Laving, Metspalu and Winkelmann2017).

Knockout (KO) animals of BTBD9 or MEIS1 homologs exhibit RLS-like phenotypes. For instance, homozygous Btbd9 KO mice have motor restlessness, disrupted sleep, and altered sensory perception (DeAndrade et al., Reference DeAndrade, Johnson, Unger, Zhang, L., van Groen, Gamble and Li2012). Loss of BTBD9 homolog in Drosophila melanogaster results in increased motor activity, decreased dopamine levels, and disrupted sleep (Freeman et al., Reference Freeman, Pranski, Miller, Radmard, Bernhard, Jinnah, Betarbet, Rye and Sanyal2012). Heterozygous Meis1 KO mice are hyperactive (Meneely et al., Reference Meneely, Dinkins, Kassai, Lyu, Liu, Lin, Brewer, Li and Clemens2018). C. elegans with decreased MEIS1 homolog show increased expression of ferritin (Catoire et al., Reference Catoire, Dion, Xiong, Amari, Gaudet, Girard, Noreau, Gaspar, Turecki, Montplaisir, Parker and Rouleau2011). Therefore, both BTBD9 and MEIS1 may play a role in the development of RLS, yet whether and how the two genes interact is not known.

Objective

Our goal was to define the relationships between BTBD9 and MEIS1 in the pathogenesis of RLS. Egg retention assay in C. elegans was used to determine if there are genetic interactions between hpo-9, a BTBD9 homolog, and unc-62, a MEIS1 homolog. Furthermore, we created mouse models by knocking out BTBD9 homolog, Btbd9, MEIS1 homolog, Meis1, or both. Their motor-sensory responses were compared by wheel-running and tail-flick tests. Homozygous Meis1 KO was not included because of embryonic lethality (Spieler et al., Reference Spieler, Kaffe, Knauf, Bessa, Tena, Giesert, Schormair, Tilch, Lee, Horsch, Czamara, Karbalai, Toerne, Waldenberger, Gieger, Lichtner, Claussnitzer, Naumann, Müller-Myhsok and Winkelmann2014). The transcription of Btbd9 in Meis1 KO and the level of Meis1 protein in Btbd9 KO animals were studied.

Methods

C. elegans were maintained using standard methods (Catoire et al., Reference Catoire, Dion, Xiong, Amari, Gaudet, Girard, Noreau, Gaspar, Turecki, Montplaisir, Parker and Rouleau2011). The wildtype (WT) used was Bristol N2. The hpo-9 KO, hpo-9(tm3719), was obtained from the National BioResource Project (Japan) and backcrossed four times to the N2 background. RNAi against unc-62 (unc-62 RNAi) and the empty vector (EV) were used according to a standard feeding method with HT115 bacterial strain. Egg retention assay was performed according to published protocols (Chase & Koelle, Reference Chase and Koelle2004) and analyzed by a Students’ t-test (supplementary material).

Heterozygous Btbd9 KO (Lyu et al., Reference Lyu, Xing, DeAndrade, Liu, Perez, Yokoi, Febo, Walters and Li2019) were bred with Meis1 KO animals (Meneely et al., Reference Meneely, Dinkins, Kassai, Lyu, Liu, Lin, Brewer, Li and Clemens2018) to generate double heterozygotes, which were bred with heterozygous Btbd9 KO mice to generate experimental mice. Behavioral tests were conducted as described (Lyu et al., Reference Lyu, Xing, DeAndrade, Liu, Perez, Yokoi, Febo, Walters and Li2019) and analyzed by SAS GENMOD or mixed model ANOVA. Western blot and quantitative RT-PCR were performed and analyzed as described (Yokoi et al., Reference Yokoi, Dang, Liu, Gandre, Kwon, Yuen and Li2015) using striatal tissues (supplementary material).

Results

Worms: Figure 1 shows that unc-62 knockdown led to an increased number of eggs retained in both N2 and hpo-9(tm3719) as described (Kamath et al., Reference Kamath, Fraser, Dong, Poulin, Durbin, Gotta, Kanapin, Le Bot, Moreno, Sohrmann, Welchman, Zipperlen and Ahringer2003). The hpo-9 mutation caused fewer eggs retained in the presence or absence of unc-62 RNAi. Additionally, hpo-9(tm3719) treated with unc-62 RNAi retained a similar number of eggs as N2.

Figure 1.

Egg retention assay. Bars represent the mean ± standard error of the mean (SEM) for 12 animals for each strain. ***, p < 0.001.

Mice: During the light phase of the wheel running test, neither single KOs exhibited significant difference compared with the WT (Figure 2). However, the double KO showed a robust increase from the WT and a lesser extent of increase from both single KOs. During the dark phase, activity levels were similar among the four groups. Figure 3 shows that the double KO did not have changes in the tail-flick response although both single KOs had reduced latency. Moreover, Meis1 protein levels and Btbd9 mRNA levels were unaffected by Btbd9 knockout and Meis1 deficiency, respectively (Figure 4).

Figure 2.

Wheel running during the light phase (A), and the dark phase (B). The data was not normally distributed and analyzed by SAS GENMOD with a negative binomial distribution. In the scatter plot, each dot is an average value calculated from 4 days’ data for each mouse. Bars represent the median with 95% confidence intervals (CIs). Hourly activity is presented next to the scatter plot. Each dot is an average value calculated from 4 days’ data for each genotype. The activity of the double KO mice shot up right after the light was turned on and right before the light was turned off. In addition, they also showed high levels of activity around the middle of the rest period. The results indicate that the double KO mice may have difficulty in falling asleep and tend to wake up early. WT, n = 7; Btbd9 KO, n = 5; Meis1 KO, n = 4; double KO, n = 6. *, p < 0.05.

Figure 3.

Tail-flick test. The data were normally distributed and analyzed by mixed model ANOVA with repeated measurements. Each dot is an average value calculated from 3 trials for each mouse. Single KO had reduced latency compared with the WT but did not show a significant difference compared with the double KOs. The double KO did not have significant changes compared with the WT. Bars represent the mean ± SEM. WT, n = 7; Btbd9 KO, n = 5; Meis1 KO, n = 4; double KO, n = 6. *, p < 0.05.

Figure 4.

Molecular analysis. (A) Western blot to measure the amount of Meis1, normalized to β-actin, in Btbd9 KO (n = 6) and WT (n = 7) mice. (B) Quantitative RT-PCR to test the level of Btbd9 mRNA, normalized to β-actin, in Meis1 KO (n = 4) and WT (n = 4) mice. Bars represent the mean ± SEM.

Discussion

Wheel-running data from day and night were analyzed separately because RLS symptoms mostly happen at night, which is the day for rodents. With animals at an average age of 3 months, we did not observe increased activity in either single KO as suggested before (DeAndrade et al., Reference DeAndrade, Johnson, Unger, Zhang, L., van Groen, Gamble and Li2012; Meneely et al., Reference Meneely, Dinkins, Kassai, Lyu, Liu, Lin, Brewer, Li and Clemens2018), indicating that the double KO had early-onset deficit while the single KOs were still asymptomatic. It has been shown that Btbd9 expression does not change by Meis1 deficiency (Spieler et al., Reference Spieler, Kaffe, Knauf, Bessa, Tena, Giesert, Schormair, Tilch, Lee, Horsch, Czamara, Karbalai, Toerne, Waldenberger, Gieger, Lichtner, Claussnitzer, Naumann, Müller-Myhsok and Winkelmann2014). This was confirmed by molecular analyses, suggesting that Btbd9 and Meis1 do not regulate each other.

Conclusion

In worms, the augmentation effect of unc-62 knockdown is independent of hpo-9 manipulation and it is also true the other way around. Moreover, hpo-9 knockout and unc-62 knockdown counteract each other. In mice, the wheel running test suggests that there is an additive effect of Meis1 and Btbd9 mutations in the double KO mice. Btbd9 does not influence the Meis1 protein level, and Meis1 cannot alter Btbd9 gene expression. Hence, the two RLS risk genes work independently and have functional interactions in both worms and mice. Protein–protein interaction assays would be ideal to confirm this conclusion in the future.

Acknowledgments

We would like to thank Drs. Neil Copeland and Hesham Sadek for supplying Meis1 loxP mice, Dr. Shohei Mitani for the hpo-9(tm3719) strain, and Fumiaki Yokoi, Lin Zhang, Chad C. Cheetham, Sung Min Han, Jack Vibbert, Pauline Cottee, Jessica Winek, and Hieu Hoang for their technical assistance and stimulating discussions.

Author Contributions

SL, MPD, MAM, RX and YLi conceived and designed the study. SL, AD, YS, MPD, YY and YLiu conducted data gathering. SL and YLiu performed statistical analyses. SL and YLi wrote the article.

Funding Information

This work was supported by a grant from Restless Legs Syndrome Foundation; startup funds from the Departments of Neurology at UAB and UF; and the National Institutes of Health (grant numbers NS37409, NS47466, NS47692, NS54246, NS57098, NS65273, NS72872, NS74423, and NS82244). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publishing Ethics

The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.

Conflicts of Interest

All authors declare none.

Data Availability Statements

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary Materials

To view supplementary material for this article, please visit http://dx.doi.org/10.1017/exp.2020.12.

References

Catoire, H., Dion, P. A., Xiong, L., Amari, M., Gaudet, R., Girard, S. L., Noreau, A., Gaspar, C., Turecki, G., Montplaisir, J. Y., Parker, J. A., & Rouleau, G. A. (2011). Restless legs syndrome-associated MEIS1 risk variant influences iron homeostasis. Annals of Neurology, 70, 170–175.CrossRefGoogle ScholarPubMed
Chase, D. L., & Koelle, M. R. (2004). Genetic analysis of RGS protein function in Caenorhabditis elegans. Methods in Enzymology, 389, 305–320.CrossRefGoogle ScholarPubMed
DeAndrade, M. P., Johnson, R. L., Jr., Unger, E. L., Zhang, , L., van Groen, T., Gamble, K. L., & Li, Y. (2012). Motor restlessness, sleep disturbances, thermal sensory alterations and elevated serum iron levels in Btbd9 mutant mice. Human Molecular Genetics , 21, 3984–3992.CrossRefGoogle ScholarPubMed
Freeman, A., Pranski, E., Miller, R. D., Radmard, S., Bernhard, D., Jinnah, H. A., Betarbet, R., Rye, D. B., & Sanyal, S. (2012). Sleep fragmentation and motor restlessness in a Drosophila model of restless legs syndrome. Current Biology, 22, 1142–1148.CrossRefGoogle Scholar
Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D. P., Zipperlen, P., & Ahringer, J. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature, 421, 231–237.CrossRefGoogle ScholarPubMed
Lyu, S., Xing, H., DeAndrade, M. P., Liu, Y., Perez, P. D., Yokoi, F., Febo, M., Walters, A. S., & Li, Y. (2019). The role of BTBD9 in striatum and restless legs syndrome. eNeuro, 6, 0277-19.2019.CrossRefGoogle ScholarPubMed
Meneely, S., Dinkins, M. L., Kassai, M., Lyu, S., Liu, Y., Lin, C. T., Brewer, K., Li, Y., & Clemens, S. (2018). Differential dopamine D1 and D3 receptor modulation and expression in the spinal cord of two mouse models of restless legs syndrome. Frontiers in Behavioral Neuroscience, 12, 199.CrossRefGoogle ScholarPubMed
Schormair, B., Zhao, C., Bell, S., Tilch, E., Salminen, A. V., Putz, B., Dauvilliers, Y., Stefani, A., Högl, B., Poewe, W., Kemlink, D., Sonka, K., Bachmann, C. G., Paulus, W., Trenkwalder, C., Oertel, W. H., Hornyak, M., Teder-Laving, M., Metspalu, A., & Winkelmann, J. (2017). Identification of novel risk loci for restless legs syndrome in genome-wide association studies in individuals of European ancestry: A meta-analysis. The Lancet Neurology, 16, 898–907.CrossRefGoogle ScholarPubMed
Spieler, D., Kaffe, M., Knauf, F., Bessa, J., Tena, J. J., Giesert, F., Schormair, B., Tilch, E., Lee, H., Horsch, M., Czamara, D., Karbalai, N., von, Toerne, C., Waldenberger, M., Gieger, C., Lichtner, P., Claussnitzer, M., Naumann, R., Müller-Myhsok, B., Winkelmann, J. (2014). Restless legs syndrome-associated intronic common variant in MEIS1 alters enhancer function in the developing telencephalon. Genome Research, 24, 592–603.CrossRefGoogle ScholarPubMed
Yokoi, F., Dang, M. T., Liu, J., Gandre, J. R., Kwon, K., Yuen, R., & Li, Y. (2015). Decreased dopamine receptor 1 activity and impaired motor-skill transfer in Dyt1 DeltaGAG heterozygous knock-in mice. Behavioural Brain Research, 279, 202–210.CrossRefGoogle ScholarPubMed
Figure 0

Figure 1. Egg retention assay. Bars represent the mean ± standard error of the mean (SEM) for 12 animals for each strain. ***, p < 0.001.

Figure 1

Figure 2. Wheel running during the light phase (A), and the dark phase (B). The data was not normally distributed and analyzed by SAS GENMOD with a negative binomial distribution. In the scatter plot, each dot is an average value calculated from 4 days’ data for each mouse. Bars represent the median with 95% confidence intervals (CIs). Hourly activity is presented next to the scatter plot. Each dot is an average value calculated from 4 days’ data for each genotype. The activity of the double KO mice shot up right after the light was turned on and right before the light was turned off. In addition, they also showed high levels of activity around the middle of the rest period. The results indicate that the double KO mice may have difficulty in falling asleep and tend to wake up early. WT, n = 7; Btbd9 KO, n = 5; Meis1 KO, n = 4; double KO, n = 6. *, p < 0.05.

Figure 2

Figure 3. Tail-flick test. The data were normally distributed and analyzed by mixed model ANOVA with repeated measurements. Each dot is an average value calculated from 3 trials for each mouse. Single KO had reduced latency compared with the WT but did not show a significant difference compared with the double KOs. The double KO did not have significant changes compared with the WT. Bars represent the mean ± SEM. WT, n = 7; Btbd9 KO, n = 5; Meis1 KO, n = 4; double KO, n = 6. *, p < 0.05.

Figure 3

Figure 4. Molecular analysis. (A) Western blot to measure the amount of Meis1, normalized to β-actin, in Btbd9 KO (n = 6) and WT (n = 7) mice. (B) Quantitative RT-PCR to test the level of Btbd9 mRNA, normalized to β-actin, in Meis1 KO (n = 4) and WT (n = 4) mice. Bars represent the mean ± SEM.

Supplementary material: File

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Reviewing editor:  Michael Nevels [Opens in a new window] University of St Andrews, Biomolecular Sciences Building, Fife, United Kingdom of Great Britain and Northern Ireland, KY16 9ST
This article has been accepted because it is deemed to be scientifically sound, has the correct controls, has appropriate methodology and is statistically valid, and met required revisions.

Review 1: Probe the relationship between BTBD9 and MEIS1 in C. elegans and mouse

Published online by Cambridge University Press:  31 March 2020
DOI: https://doi.org/10.1017/exp.2020.12.pr1 [Opens in a new window]
Aaro Salminen [Opens in a new window] Helmholtz Zentrum München, Institute of Neurogenomics, Neuherberg, Germany
Date of review:  05 February 2020
Role: reviewer

Conflict of interest statement

Reviewer declares none

Comments

Comments to the Author: The manuscript details mouse and worm experiments on BTBD9 and MEIS1, two major candidate genes of RLS. As RLS is a polygenic disease, the double knock-out experiment is of great value in determining the relationship as well as a possible additive effect of these genes to the susceptibility to RLS. However, there are concerns relating to the analysis of the data which should be addressed.

In Figures 2 and 3, individual measurements resulting from repeated measurements in the same animal should not be displayed as individual data points, but should be first averaged within animal.

In statistical tests relating to Figs 2-3, was repeated measures ANOVA used? The test used should be stated in the legend. If so, the appropriate way to analyse the data would be to average first within animal and then use for example one-way ANOVA.

Division into light and dark period is used for wheel running activity, as is often done for this kind of data. Rather, the period right before and after the lights-on should be looked at. This would correspond better to human RLS and allow comparison with previous data in MEIS1 knock-out mice.

In the introduction, the sentence describing the human genetics of RLS is somewhat confusing and should be reformulated.

What does “4 repeats for each genotype” mean? Does this mean the last 4 days that were used in the analysis according to Lyu et al. 2019 or did the mice spend four times seven days in the running wheel?

Presentation

Overall score 3.2 out of 5
Is the article written in clear and proper English? (30%)
4 out of 5
Is the data presented in the most useful manner? (40%)
2 out of 5
Does the paper cite relevant and related articles appropriately? (30%)
4 out of 5

Context

Overall score 4.2 out of 5
Does the title suitably represent the article? (25%)
4 out of 5
Does the abstract correctly embody the content of the article? (25%)
5 out of 5
Does the introduction give appropriate context? (25%)
4 out of 5
Is the objective of the experiment clearly defined? (25%)
4 out of 5

Analysis

Overall score 3.8 out of 5
Does the discussion adequately interpret the results presented? (40%)
4 out of 5
Is the conclusion consistent with the results and discussion? (40%)
4 out of 5
Are the limitations of the experiment as well as the contributions of the experiment clearly outlined? (20%)
3 out of 5

Review 2: Probe the relationship between BTBD9 and MEIS1 in C. elegans and mouse

Published online by Cambridge University Press:  31 March 2020
DOI: https://doi.org/10.1017/exp.2020.12.pr2 [Opens in a new window]
Guy A. Rouleau [Opens in a new window] McGill University, Neurology and Neurosurgery, Montreal, Canada, H3A 0G4
Date of review:  05 February 2020
Role: reviewer

Conflict of interest statement

Reviewer declares none.

Comments

Comments to the Author: In this paper Lyu et al. investigated the impact of two RLS candidate genes in the sensory-motor characteristics of C. elegans and mice. They also probed the potential interactions of these two genes based on the sensory-motor effects of their double KOs in the animals. This is an interesting paper that can provide useful information on the roles of MEIS1 and BTBD9 as two significant genes in RLS genetics.

The paper is well written, and the experiments are well conducted. The Western blot analysis results are not included in the supplemental data, and only graphs with quantitative measurement of the protein levels are provided.

Minor revisions:

  1. 1. Lines 39-41 "The double knockout mice showed an earlier onset of the motor deficit in the wheel running test but did not have increased sensitivity to the heat stimuli as observed in single KOs." Based on publications by Salminen et al. 2017, Meneely et al. 2018 and Spieler et al. 2014, the sensitivity to heat stimuli was not observed in mice with Meis1 deficiency. The authors could provide references supporting the observation of increased sensitivity to heat stimuli in BOTH KOs.

  2. 2. Could the authors elaborate on why Meis1 was only measured at protein level (WB), but Btbd9 at mRNA level (q-RT-PCR)? Would have been ideal if both genes were measured at both levels. The reason and limitations should be discussed in the paper.

  3. 3. Lines 84-85 "With or without unc-62 RNAi, hpo-9(tm3719) retained fewer eggs than N2." According to Figure 1, “N2” should be “N2(unc-62)”.

  4. 4. To make it easier for the readers, in the results section, it should be more clearly stated that which paragraph refers to which animal.

  5. 5. Line 87 "fo" is a typo?

  6. 6. Lines 90-91. The two sentences should be separated more clearly. The second sentence seems to continue the previous sentence about dark phase activity.

  7. 7. Line 122. "the two RLS risk genes work independently and have functional interactions in both worms and mice." The authors are suggesting that there are functional interactions between the two proteins. A protein-protein interaction assay would be ideal to confirm this interaction. If not feasible, the limitations of this conclusion should be discussed in the manuscript.

  8. 8. Please provide all the original Western blot figures in the supplemental data.

Presentation

Overall score 4.6 out of 5
Is the article written in clear and proper English? (30%)
5 out of 5
Is the data presented in the most useful manner? (40%)
4 out of 5
Does the paper cite relevant and related articles appropriately? (30%)
5 out of 5

Context

Overall score 4.8 out of 5
Does the title suitably represent the article? (25%)
4 out of 5
Does the abstract correctly embody the content of the article? (25%)
5 out of 5
Does the introduction give appropriate context? (25%)
5 out of 5
Is the objective of the experiment clearly defined? (25%)
5 out of 5

Analysis

Overall score 4 out of 5
Does the discussion adequately interpret the results presented? (40%)
4 out of 5
Is the conclusion consistent with the results and discussion? (40%)
4 out of 5
Are the limitations of the experiment as well as the contributions of the experiment clearly outlined? (20%)
4 out of 5