Illustration by Lindsey Leigh © 2017 lindseyleighart.com/
Research overview
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.
Specific projects
Regulation of polarized cell behaviors and morphogenesis in differentiating neurons
We have profiled the transcriptomes of specific neuronal subtypes in the Ciona larva, like the Descending Decussating Neurons (ddNs), proposed homologs of vertebrate Mauthner Cells, or the Bipolar Tail Neurons (BTNs), proposed homologs of vertebrate neural crest-derived Dorsal Root Ganglia neurons. By analyzing their transcriptomes during development, we have identified candidate effectors of their unique morphological characteristics.
We have previously shown that the ddNs uniquely invert their apical/basal polarity relative to neighboring neurons prior to growing an axon towards and across the neural tube midline. We have begun to investigate polarized autocrine deposition of signaling cues such as Netrin1, and the link between this signaling and centrosome stability in establishing these contralateral axon projections.
We are also investigating the role of conserved effectors potentially regulating the highly stereotyped but dynamic polarized cell behaviors in migrating BTNs, which initially migrate and extend an axon anteriorly before inverting their polarity to extend a second axon branch posteriorly. More specifically, we are interested in how these neurons are equipped to respond to extrinsic cues that may be instructive during polarized migration and axon growth, and how surrounding tissues might regulate the bioavailability of these cues. This project is specifically a collaboration between our lab and Christina Cota’s lab at Colby College.
To study candidate effector gene functions in these neurons, we use tissue-specific CRISPR/Cas9 to delete these genes in specific cell lineages and assay resulting protein localization, intracellular polarity, and axon outgrowth defects using new super-resolution time-lapse microscopy techniques in whole embryos.
Funding: NSF, NIH/NICHD
Regulation of quiescence and survival of adult neural stem cells
In the unique biphasic life cycle of tunicates, a swimming larval phase alternates with a sessile adult phase. While the larval central nervous system (CNS) is replaced by an adult CNS during metamorphosis, larval and adult neurons are born from specific compartments of neural precursors that are invariantly specified during embryogenesis. This patterning process closely resembles the compartmentalization of the vertebrate CNS, giving rise to regionalized subsets of neural precursors that differentiate either in the larva or in the adult.
In order for this compartmentalized heterochrony of CNS differentiation to occur, 1) adult neural stem cells need to remain undifferentiated while the larva finds a suitable place to settle, 2) they need to be protected from the wave of cell death that wipes out the larval CNS during metamorphosis, and 3) they need to be released from their quiescence to proliferate and differentiate during post-metamorphic development. We are studying the signaling pathways and gene networks that regulate these processes, in the hopes of identifying chordate-specific mechanisms that can promote cellular quiescence and survival.
Funding: NIH/NIGMS
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, in close collaboration with Billie Swalla’s lab at University of Washington and Anna Di Gregorio’s lab at New York University. 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.
Two Bipolar Tail Neurons (BTNs, in green) migrating away from the the dorsal midline of the tail tip.
Differentiating BTNs showing the acquisition of their typical bipolar morphology. Cell bodies labeled with YFP (green), nuclei with H2B::mCherry (red), and Golgi apparatus with Galnact::CFP (blue).
A pair of Descending Decussating Neurons (ddNs) in the Motor Ganglion.
Non-swimming (Molgula occulta) and swimming (Molgula oculata) larvae.
Molgula occulta non-swimming larva stained with phallodin conjugate (magenta) and anti-acetylated tubulin antibody (green), revealing only 4 ciliated caudal epidermal sensory neurons at the vestigial tail tip (inset).