The people behind the papers – Matthew Kourakis and William Smith

William Smith (left) and Matthew Kourakis (right)

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

WS: My research interests have changed direction several times over my career. My undergraduate and graduate studies were in mammalian endocrinology. After starting a postdoc studying retinoic acid receptors, my interests drifted to vertebrate embryology, specifically questions surrounding the Spemann organizer. Once starting as an Assistant Professor at UC Santa Barbara, I added a new model system to my research – the tunicate Ciona. After spending most of my career as a PI working on questions of notochord and neural tube development and morphogenesis in Ciona, the focus of my lab changed again, and for about the past 7 years we've focused on questions concerning neural circuits and behavior, again in Ciona. This change in direction was inspired by the publication of the Ciona larval connectome by the Meinertzhagen lab. It was largely through our ongoing mining of the Ciona connectome data that we arrived at our current publication in Development.

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

MK: My graduate and postdoctoral research focused on topics exploring the evolution of development – how changes in development, over time, help to answer the question of how animal body plans have arisen. Not long before I entered the Smith lab, he had begun work on tunicates (specifically, the ascidian Ciona). Ciona research appealed to me as a continuation of my evo-devo interests: Ciona is a member of the sister group to vertebrates and so is an excellent model with which to investigate the evolution of chordates in general and, in particular, to gain insight on the origins of features that characterize the vertebrates. The community of Ciona research has made incredible progress since I joined the lab, but the organism continues to surprise. That, and the collaboration required to make this sort of research successful, are what keep me engaged.

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

WS: While the evolutionary ties between tunicates and vertebrates have been long recognized, these two chordate subphyla have clearly diverged in the hundreds of millions of years since their split. For class Ascidiacea (e.g. Ciona), the sessile adult bodyform is hardly recognizable as belonging to the closest extant relatives of the vertebrates. However, in the tadpole larva of Ciona, a conserved chordate body plan is much more evident. This is particularly true of the Ciona larval central nervous system, which shows strong conservation with vertebrate induction and neurulation. However, the evolutionary relationship of morphological domains in the fully formed Ciona larval CNS to those of vertebrate nervous systems is still not fully resolved. The primary tool for investigating these questions has long been spatiotemporal gene expression analysis. While gene expression analysis has been a useful approach, the connectome adds a new and powerful complementary tool. For example, gene expression data give conflicting results on the presence of a midbrain homolog in Ciona. However, the connectome shows that the putative midbrain homolog of Ciona, called the posterior sensory vesicle, receives and integrates multiple sensory modalities – adding further evidence for a tectum-like domain in the larval CNS. In the current study, we turned our attention to a Ciona larval CNS domain called the caudal nerve cord. Despite the anatomical resemblance of the caudal nerve cord to the vertebrate spinal cord, both the expression of Hox cluster genes, and the apparent absence of motor neurons, argued against spinal cord homology. This was the starting point for the present study.

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

WS: The results of the paper are straightforward. We report that the narrow and elongated caudal domain of the Ciona larval CNS, called the caudal nerve cord, has functional motor neurons throughout its length – 12 in total. This probably should not come as too much of a surprise given the morphological similarity of the caudal nerve cord to a spinal cord, and overall conserved chordate body plan of the Ciona larva. However, given that the prevailing view was that the caudal nerve cord is devoid of motor neurons, and that spinal cord homology was thus in doubt, we felt it was important to demonstrate the identity of these cells by several independent routes. One of the confounding features of the caudal nerve cord motor neurons is that they develop relatively late compared to a previously identified set of motor neurons in the ‘motor ganglion’, the likely hindbrain homolog. To start with, we demonstrated that the 12 putative motor neurons all co-expressed the genes motor neuron and pancreas homeobox 1 (mnx1) and the cholinergic marker vesicular acetylcholine transporter (VAChT). While the expression data was suggestive of motor neuron identity, we followed this up by optogenetically activating the neurons, which resulted in the tail beating. Finally, the Ciona larval connectome project had identified four neurons in the caudal nerve cord that were named ‘midtail neurons’. We were able to definitively identify these as the four rostral-most of the 12 putative caudal nerve cord motor neurons. Serial-section electron micrographs of the midtail neurons showed that they make neuromuscular junctions with tail muscles. Moreover, the connectome ties the midtail neurons to previously studied sensorimotor circuits, supporting their functional significance. In summary, gene expression, functional analysis and synaptic connectivity all support motor neuron identity for the 12 caudal nerve cord neurons – and, by extension, support homology between the caudal nerve cord and the spinal cord.

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

MK: By chance, I found that the motor neuron marker mnx was expressed in cells of the caudal portion of the Ciona larval tail. I had been using it as a label for more anterior cells. Other investigators had seen these tail cells using other markers, but mnx turned on the figurative light bulb that spurred us to look more closely.

Why did you choose to submit this paper to Development?

WS: Deciding on the best journal for our paper was not easy. Our study doesn't fit neatly into a category. It includes elements of evolution, development, behavior and neural circuitry. It also challenges some long-held views on homology relationships between tunicates and vertebrate nervous systems. We decided to take a chance at Development, hoping to get a positive reception.

Matthew, what is next for you after this paper?

MK: I am planning to move to a different region of the Ciona CNS, looking at a unique dorsal assemblage of neurons in the putative hindbrain that is, by connectomics, a signal-processing center. It has some parallels with the vertebrate CNS, notably the cerebellum.

William, where will this story take your lab next?

WS: I feel that the caudal nerve cord story is largely complete. Next? We're focusing on evolutionary relationships between the motor ganglion and the vertebrate hindbrain.