The proper development of an animal relies on cells undergoing a variety of coordinated behaviors such as polarization, migration, cell shape changes, and differentiation. Each of these in turn can be broken down into more specific subcellular processes that are performed by specific proteins acting in conserved interaction networks. Genetic deficiencies specifically affecting these cell behaviors are known to underlie certain congenital malformations and neurological disease. We aim to pursue a systems-level understanding of cell behavior during neural development in tunicates, the invertebrates most closely related to humans.
The tunicates are the sister group to the vertebrates, and the development of their nervous system closely mirrors our own. The larval nervous system of the tunicate Ciona has only 177 neurons, one of the smallest nervous systems in the animal kingdom, and its entire "connectome" has been recently mapped. The Ciona genome is also highly compact, with minimal gene duplications. This all around reduction offers an unprecedented opportunity to understand the gene networks responsible for specifying every type of neuron in a chordate nervous system.
We use RNAseq transcriptome profiling to assay global transcriptional dynamics in neural progenitors during Ciona development, and use CRISPR/Cas9 to knock out important transcription factors and their downstream targets to understand how these networks control neuronal specification, morphology, physiology, neurotransmitter identity, and connectivity.
Sensory neuron migration and polarization
We are currently investigating the regulation of stereotyped cell behaviors observed in the Bipolar Tail Neurons (BTNs) of Ciona, a group of only 4 neurons that relay information from touch receptors in the skin to the central nervous system. We have shown that the BTNs are born from progenitors that delaminate from the neural plate borders and migrate along the mesoderm before undergoing a dramatic polarity inversion that gives them their name.
The conserved neural transcription factor Neurogenin is sufficient and necessary for BTN specification. Ectopic BTNs generated by Neurogenin overexpression all engage in the cell behaviors that are typical of the BTNs, providing us a window into the precise developmental regulation of these processes in vivo. We have profiled isolated BTNs by RNAseq and have validated transcriptional targets of Neurogenin in these cells. We are now using CRISPR/Cas9 to delete these genes specifically in the BTN lineage to identify rate-limiting effectors of neuron delamination, migration, axonogenesis, and bipolar morphology
Regulation of Motor Ganglion neuron subtype-specific traits
Within the Ciona larval nervous system, the Motor Ganglion (MG) is a simple neural circuit that drives the swimming behaviors of the larva. Most of the synaptic connections of the MG are formed among a core of 8 bilateral left/right pairs of distinct neuron types, only 2 of which are motor neurons. Stereotyped cell lineage and transcription factor expression information has been documented for most of these neurons, providing a unique opportunity to understand the development of a central pattern generator comprised of conserved neuron subtypes that appear to have closely related homologs in the hindbrain and spinal cord of vertebrates.
We have profiled the transcriptomes of specific MG neuron types, like the Descending Decussating Neuron (ddN), a proposed homolog of vertebrate Mauthner Cells, or the Motor Ganglion Interneuron 2 (MGIN2), which receives inputs from light- and gravity-sensing organs to modulate swimming behavior. By comparing these MG neuron-specific transcriptomes we hope to identify effectors of their unique morphological and physiological characteristics. In particular, we have begun to investigate the potential roles of specific ddN-upregulated genes involved in effecting polarized autocrine extracellular protein deposition and centrosome stability in establishing the contralateral axon projection of the ddNs.
Evolutionary loss of neurodevelopment in non-swimming species
Most tunicates are marine chordates with a biphasic life cycle divided between a swimming larval phase and a sessile juvenile/adult phase. Among the few exceptions to this rule are several species in the genus Molgula that have independently lost the swimming larva and instead develop as tail-less, non-swimming larvae that bypass the typical period of swimming and dispersal, but metamorphose into otherwise normal adults. The larvae of Molgula occulta and other non-swimming species do not fully develop structures that are essential for swimming behavior, including notochord, tail muscles, and otolith, and loss-of-function mutations have been identified in various genes required for the differentiation of these tissues. However, little is known about the extent of development of the larval nervous system in M. occulta.
Based on RNAseq of M. occulta, the closely related swimming species M. oculata, and their interspecific hybrids, we are investigating the specification and patterning of the M. occulta Motor Ganglion. We found that the expression patterns of important regulators of MG neuron subtype specification are conserved in M. occulta, suggesting that the gene networks regulating their expression are largely intact, despite the loss of swimming ability. However, we identified a M. occulta-specific reduction in expression of the important motor neuron terminal selector gene Ebf in the Motor Ganglion. This suggests that certain neurodevelopmental genes have not been entirely lost from non-swimming species, but rather specific cis-regulatory changes have affected their expression specifically during larval neurodevelopment, but likely not in the adult phase.