Quantification of gene expression patterns to reveal the origins of abnormal morphogenesis

Author's response

Neus Martinez-Abadias shared

Questions to the authors:

Since the technique is very powerful, I wonder the amount of time the authors dedicated to process the data, and how easily could it be applied in other labs.

As we have developed this technique from scratch, we worked on this project for 2-3 years. Now that all the technical issues have been sorted out, we could reprocess all the data in just 2-3 months. We have optimized the experimentation, imaging, segmentation and landmark processing of the samples. All the details of the “recipe” are now described in the paper. Our next goal is to fully automatize the whole pipeline.

 

The technique is indeed powerful and adaptable to other organs and model systems. It would be great to see other labs applying our approach and discovering new processes and mechanisms that the eyes just can’t see! Whole mount in situ hybridization (WISH) is a technique that is already in use in most molecular labs around the world. Many groups may already have in their “drawers” data on gene expression patterns ready to be quantified. Geometric morphometrics (GM) is also a discipline within everyone’s reach, with a long history, plenty of available resources, user-friendly free software and a big community of users (www.morphometrics.org). It was a pity that GM had not reached the developmental biology community before. Hopefully, our work will eventually bridge these two fields. OPT scanning is perhaps the least accessible technology, but OPT scanners are gradually getting to more and more labs and at EMBL we are always open to collaborate and share our expertise!

 

Which do the authors think are the limitations of the technique?

First, our method is limited to genes that can be labeled by WISH. Then, the limits are those of GM and the type of shape changes occurring during development. GM can only deal with gene expression domains that can be reliably represented as a unique continuously changing shape. However, some gene expression domains have inherently ill-defined shapes, lack homologous landmarks, and/or dramatically change their shape even over short periods of time, with emergence or loss of regions. In those cases, landmark free approaches could offer an alternative.

 

How subtle can the changes be to define a given phenotype?

Very subtle. Indeed, since the shape changes we are searching for are so subtle, we learnt in the process that in order to minimize variations due to experimentation, it is critical to process all the litters in the same experiment and strictly apply the same protocols. Even so, our first results were discouraging. We did not find any differences between the limbs and the gene expression patterns in wildtype and Apert syndrome mice when we first analyzed the pool of samples. Only after analyzing separately forelimbs and hindlimbs from different litters, the “magic” started. It was when we managed to properly control the different sources of variation that we were able to clearly separate between wildtype and mutant mice and detect when the differences first appeared. The resolution of the analysis was so high that we could even detect that altered gene expression occurs first and precedes the limb defects, which emerged only a few hours later.

 

Have they tried to do the analysis with more than one gene?

We have done the analysis with other genes, such as Hoxa11 and Hoxa13 in 2D (Martínez-Abadías et al 2016, https://academic.oup.com/sysbio/article/65/2/194/2427013), and Hand2 in 3D. All the analyses successfully revealed insights into normal and disease-altered limb development. We are also applying the approach to other organs, such as the heart, the face and the brain. The quantitative approach is time and resource-consuming, as it requires large samples of embryos over a short time window, but the effort is totally worth. Otherwise, it would be impossible to detect such critical changes about the origin of diseases and further understand how development translates genetic into phenotypic variation, which is a key process of Developmental Biology.