The genes encoding proteins involved in cilia formation and function are thought to be well conserved, but ciliopathies are associated with a broad range of tissue-specific phenotypes. A new paper in Development investigates differences in ciliary gene expression across different tissues and developmental stages. To hear more about the story, we caught up with first author Kelsey Elliott and her doctoral supervisor Samantha Brugmann, Professor at Cincinnati Children's Hospital Medical Center.

Kelsey Elliott (L) and Samantha Brugmann (R)

Samantha, can you give us your scientific biography and the questions your lab is trying to answer?

SB: I received my B.S. in Cell and Molecular Biology from Tulane University in New Orleans. For my doctoral training, I joined the lab of Dr Sally Moody at George Washington University in Washington DC. There, I earned my Ph.D. in Genetics, focusing on the role of the transcription factor Six1 in cranial placode development. After obtaining my Ph.D., I went to Stanford University for my postdoctoral training. At Stanford, I worked in the labs of Dr Jill Helms and Dr Joanna Wysocka, studying neural crest cell (NCC) development and differentiation in the context of craniofacial development and disease. In 2011, I joined Cincinnati Children's Hospital Medical Center as an Assistant Professor with a dual appointment in the Divisions of Developmental Biology and Plastic Surgery.

The long-term goal of my laboratory is to help children with craniofacial anomalies by understanding the molecular mechanisms of development and disease, and generating tissue amenable for surgical repair. To achieve this goal, my lab specifically focuses on the role of the primary cilium during craniofacial development and the craniofacial anomalies that arise when the cilium does not function properly (ciliopathies).

Kelsey, how did you come to work in Samantha's lab and what drives your research today?

KE: After having several meaningful experiences at the bench during my undergraduate studies, both coincidentally focused on key developmental biology organisms, sea urchin and Drosophila, I knew I loved being in the lab. While both of my undergraduate mentors were great advisors, they were both male, so I was very interested in having my Ph.D. studies guided by a female. Sam is a great mentor, both scientifically and personally, and when I joined the lab, it was an all-female crew, which was great exposure to how females can succeed at all levels in academia. My research today is driven by the same things that drove me as an undergraduate – how do we end up with all our organs and tissue in the right place!?

Before your work, what was known about ciliary gene expression during development?

KE: Several papers had come out over the years describing tissue specificity of individual ciliary genes/proteins, frequently describing the situation as a rarity. While many in the field described the cilia as a ‘ubiquitous organelle’, we knew from the variety of phenotypes and pleiotropy presented in both ciliopathic patients and models used by our lab and others that there had to be more heterogeneity in ciliary composition than the handful of single-gene reports that were out there.

SB: There had also been several screens done to generate a comprehensive ciliome, many of which we used or cited in our paper. However, several of them were performed in vitro or focused on one organ system. On a transcriptional level, Foxj1 and the RFX family of transcription factors had been identified as contributing to the transcriptional control of ciliary biogenesis. Furthermore, a nice review published in Development in 2014 had schematized the structural diversity between different types of cilia (Choksi et al., 2014).

Can you give us the key results of the paper in a paragraph?

KE: We identified unique tissue and temporal-specific heterogeneity in the genes that compose and contribute to functionality of the primary cilia. In addition to characterizing this heterogeneity and generating an extremely user-friendly resource for the community, we show that this ciliary heterogeneity is likely a key driver in organismal and tissue-specific functionalization, suggesting primary cilia play an even larger role in tissue- and cell type-specific differentiation and activity than previously appreciated.

“Ciliary heterogeneity is likely a key driver in organismal and tissue-specific functionalization, suggesting primary cilia play an even larger role in tissue- and cell-type specific differentiation and activity than previously appreciated”

SB: Across organ systems frequently affected in ciliopathies, ∼30% of the ciliome is differentially expressed (DE). Genes within the DE ciliome exhibited a lower level of functional constraint across species, suggesting organism and cell-specific function adaptation. In addition to this spatial heterogeneity, observations of multipotent NCCs differentiating over time also revealed a temporal heterogeneity that correlates with tissue-specific function. To allow users easy access to this data, we generated a customizable and searchable platform (https://research.cchmc.org/Ciliome_Gene_Expression/).

Were you surprised by the proportion of the ciliome that is differentially expressed across embryonic tissues?

KE: I think as a naïve graduate student I first viewed the results and thought ‘I can't believe it isn't more!’. But after many productive lab meetings, we really came to appreciate that while the core components of the primary cilia are absolutely critical for the function of this extremely important organelle, subtle, but impressively significant, changes can really impact how the primary cilia sees and interprets signals from its niche and developmental environment.

SB: At first, I was pleasantly surprised, but after thinking about it for some time, it made a lot of sense that a significant proportion of the ciliome would be differentially expressed to help convey tissue-specific functions.

O9-1 derived osteoblasts co-immunostained for acetylated-tubulin (red, marking the cilium) and Pcm1 (green, a gene in the osteogenic ciliome).

O9-1 derived osteoblasts co-immunostained for acetylated-tubulin (red, marking the cilium) and Pcm1 (green, a gene in the osteogenic ciliome).

What can this ciliary heterogeneity tell us about ciliopathies and other diseases?

KE: I think the philosophy of Sam's approach to truly translational, bench-to-bedside science really shines through with the ciliary heterogeneity discovered in this study. So many individuals with ciliopathies present with phenotypes associated with many signalling pathways that seem impossible to target directly due to very challenging side-effects. This comprehensive understanding of unique tissue- and cell type-specific ciliary structure and gene expression gives us a window of opportunity to treat the root cause of these individuals' phenotypes.

SB: I think this can be an approach to start to think about treatment. Certain ciliary variants will severely impact one tissue versus the other. If we know the molecular environment of the affected tissue and the role of the cilium there, a more-targeted approach is possible.

When doing the research, did you have any particular result or eureka moment that has stuck with you?

KE: Although -omics and -seq based approaches can be very powerful, and provide a lot of data, I think I was truly convinced of the immense heterogeneity by the in situ and western blot results. While these days seem to be consumed by Excel spreadsheets and Venn diagrams, there will never be a substitute to seeing results with your own hands and eyes!

And what about the flipside: any moments of frustration or despair?

KE: Oh my gosh, what is science without moments of frustration and despair! Of course. There were moments when we just felt so bogged down with data, and it wasn't clear what the ‘story’ was. Times when hypotheses needed to be revised, and the initial vision for the project needed to be expanded. Luckily, everyone involved was very invested in the final product, and we had a great team to back us up!

Kelsey, what is next for you after this paper?

KE: Due to Sam's great mentorship, I secured a postdoctoral fellowship in Dr Gage Crump's lab, where I've luckily been able to continue my passion for developmental biology!

Samantha, where will this story take your lab next?

SB: The overarching vision for our research program is to identify how primary cilia function during normal facial development, then to manipulate and employ the cilia to direct NCC differentiation into tissues amenable for surgical repair of craniofacial anomalies. The findings from this manuscript have identified several candidates that we are in the process of testing towards this end.

Finally, let's move outside the lab – what do you like to do in your spare time?

SB: I like being outside and active. Walking, hiking, traveling, playing tennis and gardening are all things I enjoy doing when I'm not in the lab.

KE: Is food a hobby? Ha! I love to cook/bake and explore new restaurants. I have a corgi, who sometimes feels like the only reason I ever leave the lab, and together we enjoy hiking any trail we can find!

K.E. & S.B.: Division of Developmental Biology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA and University of Cincinnati, College of Medicine, Department of Pediatrics, Cincinnati, OH 45229, USA.

S.B.: Division of Plastic Surgery, Department of Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.

E-mail: [email protected]

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