Specific projects

1. 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.

   Current funding: NIH/NICHD

2. 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.

   Current funding: NIH/NIGMS

3. Development and function of a sensory/adhesive organ that controls larval settlement and metamorphosis

   It has been shown that a set of specialized sensory/adhesive organs termed the papillae at the very anterior of the larval head is indispensable for the attachment of Ciona larvae to a solid substrate, and to trigger the onset of metamorphosis. While adhesive-secreting cells ensure larval attachment, mechanical stimulation of sensory neurons in the papillae is sufficient to trigger metamorphosis, though chemical sensing may also play a role in substrate selection. In collaboration with Ute Rothbächer’s lab at the University of Innsbruck, Austria, we are working to unravel the regulatory networks that pattern these remarkable organs, as well as the molecular mechanisms underlying their sensory and adhesive functions.

4. Evolution of myoblast fusion mechanisms

   Vertebrate skeletal muscles are multinucleated, resulting from the fusion of many mononucleated muscle precursor cells, or myoblasts, during development and regeneration. In collaboration with Pengpeng Bi’s lab at the University of Georgia, we found that tunicates and vertebrates share a unique molecular mechanism for myoblast fusion. In tunicates and vertebrates, myoblast fusion requires the transmembrane protein Myomaker. Remarkably, Ciona Myomaker can rescue myoblast fusion and muscle multinucleation in human Myomaker CRISPR cells. We have further shown that in Ciona, myoblast fusion only occurs in post-metamorphic muscles, not in mononucleated larval muscles, thanks to the selective expression of Myomaker in the former. We have presented evidence that this is due to the cooperative activity of transcription factors EBF and MRF which are co-expressed only in post-metamorphic myoblasts. However, there is still much to uncover about how Myomaker works at the molecular level, including unknown, potentially conserved regulators and cofactors. We are taking advantage of the facultative, not obligate, fusion of post-metamorphic myoblasts in Ciona to identify novel players in this chordate-specific myoblast fusion pathway.

5. Evolutionary loss of locomotor functions in certain tunicate larvae

   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 the central nervous system. While some loss-of-function mutations have been identified in various genes required for the differentiation of these tissues, little is known about the extent of evolutionary “decay” of the regulatory networks that underlie the development of these structures in typical, “swimming” species.

   Based on RNAseq of M. occulta, the closely related swimming species M. oculata, and their interspecific hybrids, we are investigating the evolutionary loss of such gene regulatory networks, in close collaboration with Billie Swalla’s lab at University of Washington and Anna Di Gregorio’s lab at New York University. For instance, we have 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. In another study, we found that the gene encoding the notochord extracellular matrix component Collagen1/2a is still present in the non-swimming species’ genome, but its expression is markedly reduced. CRISPR knockout of the Collagen 1/2a gene in Ciona showed this gene is important for notochord morphogenesis. This suggests that certain genes have not been entirely lost from non-swimming species, but rather certain cis-regulatory changes have affected their expression specifically during larval neurodevelopment, but likely not in the adult phase.