Behind the paper and the SciComm story: uncovering the role of CNKSR2 in the chick forebrain
Posted by Niveda Udaykumar, on 17 March 2023
“CNKSR2: Identifying and elucidating the role of a novel downstream effector of RA signalling in chick forebrain morphogenesis“
In the February issue of Development, Niveda and her colleagues report on the identification and function of a previously unreported downstream effector of retinoic acid (RA) signalling in the chick forebrain. Niveda shares some insights into the story behind the paper and the science communication outreach initiated by her department at the Indian Institute of Technology Kanpur.
How did you get started on this project?
The genesis of this project was based on the findings of a study carried out in Prof. Amitabha Bandyopadhyay’s laboratory at the Department of Biological Sciences and Bioengineering (BSBE) at IIT Kanpur, wherein they performed a genome-wide expression screen of Metabolism-Related Genes (MRGs) in the chicken embryo. The result of this experiment was very interesting as the expression of MRGs peaked at the time of differentiation of the various tissues in the embryo. Intrigued by this result, we started to explore the role of Metabolism-Related Genes during the development of the chick brain.
We started the study by first examining the spatiotemporal expression profiling of the MRGs that were reported by the initial genome-wide screen. Our laboratory is interested in understanding the process of midline invagination of the forebrain roof plate, which leads to the formation of the cerebral hemispheres from the single forebrain vesicle. Thus, we were very intrigued when we discovered that one MRG known as Connector Enhancer Kinase Suppressor of Ras 2 (CNKSR2) was expressed very precisely in the middle of the invaginating roof plate of the chick forebrain. We then decided to examine its role in the process of midline invagination.
What was already known about the topic?
Before we started our research, the only information available about the process of separation of the cerebral hemispheres was that certain genes linked to holoprosencephaly, a devastating developmental disorder in humans, may be regulating this process. However, nothing was known about the molecular mechanism involved. In a previous study from our lab, we observed that during the process of midline invagination, the roof plate forms a characteristic W-shaped fold1. Also, it is through this process that the two hemispheres and the medially derived structures such as the hippocampus and choroid plexus are formed. This W-shaped invagination functions as a secondary signalling centre for pathways such as BMP2 and WNT3. In the paper published in 2015, we reported that Retinoic Acid signalling is detected in the middle loop of the W-shaped invagination of the roof plate and its inhibition leads to a flattened forebrain roof plate, a phenotype that resembles the human disorder holoprosencephaly1, where the hemispheres are improperly separated4. In this study, we found that the expression of CNKSR2 exactly coincided with the RA signalling domain in the chick forebrain roof plate, prompting us to investigate the role of this gene in this context.
Can you summarize your findings?
We can summarize our findings as follows: we found the expression of an MRG, CNKSR2, in the middle loop of the invaginating dorsal forebrain roof plate and overlapped with the active RA signalling domain. We manipulated RA signalling in the roof plate and found that the expression of the CNKSR2 transcript changed. This led us to infer that CNKSR2 is a downstream effector of RA signalling in this context.
Further, when we knocked down CNKSR2 from the invaginating roof plate using RNA-interference (RNAi) and obtained roof plate invagination defects which phenocopied loss of RA signalling. We found that the invagination defects upon knockdown of CNKSR2 were related to changes in cell proliferation and patterning of this region. Further, misexpression of mouse CNKSR2 was sufficient to ectopically induce the expression of roof plate midline markers in the lateral forebrain. This led us to conclude that CNKSR2 is necessary and sufficient for roof plate patterning. The final experiment that we performed revealed that CNKSR2 modulates Ras/Raf/MEK signalling to lower levels in the roof plate midline for proper patterning and subsequent chick forebrain morphogenesis.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
We had three eureka moments during our study. The first was when we found that the knockdown of CNKSR2 in the chick embryo forebrain led to invagination defects with a holoprosencephaly-like phenotype. The second, was when the misexpression of the mouse CNKSR2 in the lateral forebrain was sufficient to induce the roof plate marker genes. And the third, and most unexpected result, was when ectopic downregulation of Ras/Raf/MEK in the lateral forebrain was sufficient to induce the expression of the patterning marker, Bmp7. I believe this third result was the final bit of evidence that helped us piece the story together.
Where will this story take the lab?
This story has helped to identify one important molecule-CNKSR2 in the bigger picture of understanding the process of midline invagination in the chick forebrain. Also, this gene may be used as both a proxy for RA signalling in the chick forebrain, as well as a roof plate midline marker. The process of midline invagination is complex with many aspects such as cell adhesion and cytoskeleton rearrangements also likely to be involved. We are currently investigating the possible role of CNKSR2 in each of these functions to understand the forebrain midline invagination process and the resulting separation of the cerebral hemispheres.
Science outreach and its importance
India is a country with enormous linguistic diversity. Our research group comprises members from across the country who are fluent in many languages. As our research is funded by taxpayers, we as a group believe that the general public in the country should be aware of the kind of research taking place in the lab and the resulting publications.
To fulfil this, the Department of Biological Sciences and Bioengineering (BSBE) at the Indian Institute of Technology Kanpur (IITK) decided to start a new initiative wherein the authors of a publication convey their research in their native language and English, all in layman’s terms. The authors of our study are fluent in Bengali, English, Hindi, Nepali and Tamil. Simple animated videos with narration in each of these languages were made and shared across social media for public awareness. We hope to continue this initiative with future publications, and we anticipate that it will be well-received by the viewers. In the end, we aim to inspire young students to actively consider becoming scientists and join us on this exciting journey!
The links for the videos are attached below:
1) Gupta S, Sen J. Retinoic acid signalling regulates development of the chick’s dorsal forebrain midline and the choroid plexus. Development. 2015 Apr 1;142(7):1293-8. doi: 10.1242/dev.122390. Epub 2015 Mar 10. PMID: 25758461.
2) Furuta Y, Piston DW, Hogan BL. Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development. Development. 1997 Jun;124(11):2203-12. doi: 10.1242/dev.124.11.2203. PMID: 9187146.
3) Lee SM, Tole S, Grove E, McMahon AP. A local Wnt-3a signal is required for the development of the mammalian hippocampus. Development. 2000 Feb;127(3):457-67. doi: 10.1242/dev.127.3.457. PMID: 10631167.
4) Roessler E, Muenke M. The molecular genetics of holoprosencephaly. Am J Med Genet C Semin Med Genet. 2010 Feb 15;154C(1):52-61. doi: 10.1002/ajmg.c.30236. PMID: 20104595; PMCID: PMC2815021.