Laterality defects are influenced by timing of treatments and animal model

Abstract

The timing of when the embryonic left-right (LR) axis is first established and the mechanisms driving this process are subjects of strong debate. While groups have focused on the role of cilia in establishing the LR axis during gastrula and neurula stages, many animals appear to orient the LR axis prior to the appearance of, or without the benefit of, motile cilia. Because of the large amount of data available in the published literature and the similarities in the type of data collected across laboratories, I have examined relationships between the studies that do and do not implicate cilia, the choice of animal model, the kinds of LR patterning defects observed, and the penetrance of LR phenotypes. I found that treatments affecting cilia structure and motility had a higher penetrance for both altered gene expression and improper organ placement compared to treatments that affect processes in early cleavage stage embryos. I also found differences in penetrance that could be attributed to the animal models used; the mouse is highly prone to LR randomization. Additionally, the data were examined to address whether gene expression can be used to predict randomized organ placement. Using regression analysis, gene expression was found to be predictive of organ placement in frogs, but much less so in the other animals examined. Together, these results challenge previous ideas about the conservation of LR mechanisms, with the mouse model being significantly different from fish, frogs, and chick in almost every aspect examined. Additionally, this analysis indicates that there may be missing pieces in the molecular pathways that dictate how genetic information becomes organ positional information in vertebrates; these gaps will be important for future studies to identify, as LR asymmetry is not only a fundamentally fascinating aspect of development but also of considerable biomedical importance.

Figure 1. Distribution of studies in CILIA and EARLY treatments by animal model

A) Distribution of all EARLY and CILIA studies, regardless of endpoint examined. B) Distribution of studies that examined organ situs. C) Distribution of studies that examined Nodal localization. For all panels, the number of treatments examined for each animal model is listed on the graphs. The results clearly indicate that a 2-way ANOVA approach is not an appropriate statistical analysis for this dataset because of minimal overlap of animal models between the EARLY and CILIA groups.

Figure 2. Penetrance of organ situs phenotypes by timing and animal model

Two different measures of organ situs were calculated for comparisons, % heterotaxia + situs inversus and total laterality problems. A) CILIA treatments produced significantly higher values for both measures of organ situs compared to EARLY treatments. p-values on graph are results of independent samples T-tests using weighted data. B) There were significant differences in % heterotaxia + situs inversus and total laterality problems by animal model, with mice having significantly more penetrant defects compared to the other three animal models. The difference between % heterotaxia + situs inversus and total laterality problems for mice suggests that isomerisms are a uniquely important class of laterality defects in this animal. p-values on graph are ANOVA values using weighted data; different letters indicate significant differences between groups (Dunnett T3 posthoc test, p<0.01). C) When the analysis was limited to mutants only, mice still had more penetrant defects for both measures of organ situs compared to fish, the only other animal with significant numbers of mutants available for analysis. p-values on graph are results of independent sample T-tests using weighted data.

Figure 3. Developmental defects have little influence on penetrance of randomized organ situs

A) % heterotaxia + situs inversus in all studies compared to this same measure after treatments that cause malformations and developmental defects were removed from analysis. B) Total laterality problems, again comparing all studies before and after treatments that cause other defects were removed. For both of these measures, the removal of studies that cause developmental defects has very little impact on either EARLY or CILIA treatments, and significant differences between these groups remain. (In both panels, the data shown for “all studies” is the same as reported in Fig. 2A.) p-values on graph are results of independent samples T-tests using weighted data.

Figure 4. Asymmetric gene expression is influenced by how it is calculated, timing, and animal

A) When only right and bilateral gene expression were included in calculations, there were significant differences in incorrect gene expression between CILIA and EARLY treatments two of the three genes examined. B) When right, bilateral and absent gene expression were included in calculations, there were significant differences in all non-left gene expression between CILIA and EARLY treatments for all three genes examined. C) Incorrect gene expression was influenced by animal model for all three genes examined, although the differences in pitx2 did not reach statistical significance. p-values are ANOVA statistics of weighted data; different letters indicate significant differences between groups (Dunnett T3 posthoc test, p<0.01). D) All non-left gene expression was significantly affected by animal model, with mice being the most different. p-values are ANOVA statistics; different letters indicate significant differences between groups (Dunnett T3 posthoc test, p<0.01). For lefty, there were no samples collected in chick. For pitx2, there was only 1 sample for chick, so this group was excluded from analysis.

Figure 5. Gene expression can be used to predict organ situs, but is dependent on several factors

Regression analysis indicates that gene expression data can be used to predict organ situs, but only if the correct analysis is chosen based on timing and animal model. For all panels, total laterality problems are graphed along the X axis. For all graphs in the left column, the Y axis represents all non-left nodal expression (the sum of right, bilateral and absent gene expression). For all graphs in the middle column, the Y axis represents incorrect nodal expression (the sum of right and bilateral expression). For all graphs in the right column, the Y axis represents the predicted organ reversal rate derived from reported localization of Nodal. Yellow panels indicate the best fit regressions for each pathway (CILIA and EARLY) and each animal model. Panels A–C show regressions of all data using the three possible comparisons. Di–iii) The best regression for CILIA treatments is achieved by comparing all non-left nodal expression and total laterality problems. Ei–iii) The best regression for EARLY treatments is achieved by comparing predicted organ reversal rate and total laterality problems, although this relationship did not reach statistical significance. Fi–iii) Data from Xenopus fit two models equally well: regressions comparing all non-left nodal expression and total laterality problems and predicted organ reversal rate and total laterality problems. Gi–iii). Data from fish fit best to the regression between all non-left nodal expression and total laterality problems, but this relationship was not statistically significant. Hi–iii) Unexpectedly, data collected in mouse do not fit any of the regressions well, suggesting poor associations between asymmetric gene expression and organ laterality in this animal model. The best fit was observed between all non-left nodal expression and total laterality problems.