The absence or dysfunction of primary cilia, which are non-motile protrusions on cells, leads to a group of neurodevelopment disorders called ciliopathies. In a new study, Esther Stoeckli and colleagues identify the role of primary cilium-mediated sonic hedgehog (Shh) signaling in commissural axon guidance in mice and chick embryos. We caught up with first author, Alexandre Dumoulin, and corresponding author, Esther Stoeckli, Professor at the University of Zurich, to find out more about the work.

Alexandre Dumoulin (left) and Esther Stoeckli (right)

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

ES: I have always been fascinated by the idea of understanding learning and memory at the molecular level. This was my motivation to become a neuroscientist. Early on, I realized that we did not know enough about the ‘hardware’, the neural circuits, as a first step towards understanding their function in learning processes. After my PhD, during which I was involved in the discovery and the functional analysis of cell-adhesion molecules in vitro, I wanted to study the molecular basis of neural circuit formation. This was of course only possible with in vivo approaches. In the early 1990s, I considered the possibility of perturbing molecular interactions in chicken embryos the best option for functional in vivo studies. This approach had been spearheaded by Lynn Landmesser. Therefore, I joined her lab as a postdoctoral fellow. Later, in my own lab, we developed in ovo RNAi, an even more powerful approach to perturb gene function with high temporal and spatial precision during neural circuit formation in chicken embryos. The only disadvantage of the chicken embryo as an animal model is the absence of a cortex, and, therefore, the impossibility of directly comparing cell migration and neural circuit formation with the brain of mammals. However, the molecular mechanisms of neural circuit formation are conserved throughout the entire nervous system. Thus, we focus on the spinal cord and the cerebellum for the central nervous system, and on the innervation of the hindlimb for the peripheral nervous system.

A really important development in our quest to understand the molecular basis of neural circuit formation has been the possibility of observing axon guidance with live imaging. Thanks to Alexandre's skills and his perseverance in establishing an ex vivo preparation that allows us to follow commissural axons in the intact spinal cord, we have learned more about the temporal aspects of cell-cell interactions, and the communication between navigating axons and their intermediate targets during axon guidance. For the last couple of years, the learning and memory aspect has become more important again, as my lab at the University of Zurich in Switzerland is part of a research network in the context of the University Research Priority Program ‘Adaptive Brain Circuits in Development and Learning’ (URPP AdaBD) that brings together basic neuroscientists like us with colleagues performing human studies, including clinicians who see patients with developmental delays and intellectual disabilities.

Alexandre, how did you come to work in the lab and what drives your research today?

AD: During my PhD at the Max Delbrück Center Berlin in Germany I uniquely used the mouse model to study axon branching during the development of sensory neurons in vivo. At some point, I began using chick embryos to collect neurons for in vitro assays. I found it highly convenient to have access to abundant embryonic tissue for my experiments, and I became increasingly aware of the potential for in vivo studies with this animal model as well. The chick embryo model appeared to be very versatile, allowing for the investigation of genes of interest in a much shorter time compared to the mouse. When the opportunity to do a postdoc in Esther's lab arose, it was a no-brainer – I wanted to switch to this model to continue exploring axonal development! Now, thanks to in vivo interventions followed by ex vivo cultures, we can visualize developing axons in their intact environment in real time. This approach has become the major driver of my current research. I am passionate about observing living neurons, discovering new features, drawing hypotheses from these observations and testing them with more classical approaches.

What is the background of the field that inspired your work?

AD: In some ciliopathies, such as Joubert syndrome, it is clear that brain development is impaired. However, little is known about the molecular mechanisms underlying these developmental issues. So far, the use of animal models has been the most powerful way to gain insights into the possible etiology of the disease. Moreover, the cilia research community is incredibly supportive, maintaining close connections with clinicians and patients, and actively fostering basic research efforts to advance knowledge about ciliopathies, as seen at EMBO cilia conferences. We strongly believe that the crosstalk between all stakeholders should be adopted also to study other neurodevelopmental disorders. Our research has revealed over and over again that maintaining just one perspective is not sufficient to understand the contributions of different genes to neurodevelopmental disorders. Neural circuit formation is a four-dimensional process: time is important! Therefore, animal models that allow temporal and spatial control, like the chicken embryo, can contribute important aspects to functional studies of gene mutations in nervous system development.

The cilia research community is incredibly supportive, maintaining close connections with clinicians and patients, and actively fostering basic research efforts to advance knowledge about ciliopathies

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

AD: We gathered evidence supporting the idea that the tiny primary cilium of commissural neurons, which is located hundreds of microns away from the guidance cues sensed by the growth cone at the tip of their axons, is responsible for the change in behavior of the axons at a choice point. On the one hand, our results describe the mechanism by which Shh signaling steers axons by changing its own receptor on commissural axons during midline crossing in a temporally and spatially controlled manner. On the other hand, our results provide an explanation for the observed cognitive problems that are seen in most patients with ciliopathies.

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

AD: Confirming the involvement of the primary cilium in axon guidance at an intermediate target through Arl13b functional analysis and rescue experiments was particularly satisfying, especially as it validated the Ift88 silencing results we obtained years earlier. I'm grateful to a reviewer who suggested that we conduct this experiment – it made a significant difference! Additionally, the experiments demonstrating retrograde Shh transport from the axonal tip to the soma of commissural neurons were especially exciting for me. They highlighted how signals can travel long distances within neurons to produce an output.

Immunostaining of primary cilia (Arl13b, green) and Lhx2-positive dI1 neurons (reddish) on transverse cryosection of an E11.5 mouse embryo trunk, counterstained with Hoechst

Immunostaining of primary cilia (Arl13b, green) and Lhx2-positive dI1 neurons (reddish) on transverse cryosection of an E11.5 mouse embryo trunk, counterstained with Hoechst

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

AD: The primary cilium is very tiny in neurons of the developing spinal cord, and visualizing it in vivo can be challenging. This required a long and tedious process of adjusting protocols to make it possible. The same was true with visualizing secreted Shh. It can take months or even years before a breakthrough idea finally makes everything work. The key is to never give up!

Why did you choose to submit this paper to Development?

ES: Development is a leading non-profit journal led by scientists, where any developmental biologist would aspire to publish. From our own experience, it also has a fair and constructive review process.

Alexandre, what is next for you after this paper?

AD: I plan to focus on further live imaging studies to uncover how guidepost cells deliver guidance cues. There's still much to explore in this area, and I'm excited to share the next phase of our research soon, so stay tuned!

Esther, where will this story take your lab next?

ES: We will continue to combine our approach of temporal and spatial control of gene functions with the analyses of genes linked to neurodevelopmental disorders. We have always used both methods: a basic science-driven approach, where we want to understand axonal behavior during neural circuit formation; but also a more applied approach – from gene mutations identified in patients to an understanding of the neurodevelopmental steps affected by these genes.

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

AD: Spending time with my wife and son is my top priority! I also love sports. I used to practice capoeira a lot, but now I've gotten back into mountain biking. The Zurich area is amazing for that!

ES: I love the mountains. Thus, I try to spend as much time as possible hiking and biking either on the road or on trails together with my husband.

A.D. & E.S.: Department of Molecular Life Sciences and Neuroscience Center Zurich, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

E.S.: University Research Priority Program ‘Adaptive Brain Circuits in Development and Learning’ (URPP AdaBD), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

E-mail: [email protected]

Dumoulin
,
A.
,
Wilson
,
N. H.
,
Tucker
,
K. L.
and
Stoeckli
,
E. T.
(
2024
).
A cell-autonomous role for primary cilium-mediated signaling in long-range commissural axon guidance
.
Development
151
,
dev202788
.