Clark CM*, Leifer AM*, Ji N, Florman JT, Mizes K, Samuel ADT, Alkema MJ. Synaptic chain model for an escape response motor sequence. Submitted;
Venkatachalam V*, Ji N*, Wang X, Mitchell J, Klein M, Tabone C, Greenwood JSF, Chisholm A, Srinivasan J, Alkema MJ, Zhen M, Samuel ADT. Pan-neuronal Imaging in Roaming Animals [Internet]. PNAS 2016;113(8):e1082-1088. Publisher's VersionAbstract

We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ∼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal’s posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.

Cell intrinsic modulation of Wnt signaling controls neuroblast migration in C. elegans
Mentink RA, Middelkoop TC, Rella L, Ji N, Chung YT, Betist MC, van Oudenaarden A, Korswagen HR. Cell intrinsic modulation of Wnt signaling controls neuroblast migration in C. elegans [Internet]. Developmental Cell 2014;31(2):188-201. Publisher's VersionAbstract

Members of the Wnt family of secreted signaling proteins are key regulators of cell migration and axon guidance. In the nematode C. elegans, the migration of the QR neuroblast descendants requires multiple Wnt ligands and receptors. We found that the migration of the QR descendants is divided into three sequential phases that are each mediated by a distinct Wnt signaling mechanism. Importantly, the transition from the first to the second phase, which is the main determinant of the final position of the QR descendants along the anteroposterior body axis, is mediated through a cell-autonomous process in which the time-dependent expression of a Wnt receptor turns on the canonical Wnt/β-catenin signaling response that is required to terminate long-range anterior migration. Our results show that, in addition to direct guidance of cell migration by Wnt morphogenic gradients, cell migration can also be controlled indirectly through cell-intrinsic modulation of Wnt signaling responses.

Tan RZ, Ji N, Mentink RA, Korswagen HR, van Oudenaarden A. Deconvolving the roles of Wnt ligands and receptors in sensing and amplification [Internet]. Molecular Systems Biology 2013;9(631) Publisher's VersionAbstract

Establishment of cell polarity is crucial for many biological processes including cell migration and asymmetric cell division. The establishment of cell polarity consists of two sequential processes: an external gradient is first sensed and then the resulting signal is amplified and maintained by intracellular signaling networks usually using positive feedback regulation. Generally, these two processes are intertwined and it is challenging to determine which proteins contribute to the sensing or amplification process, particularly in multicellular organisms. Here, we integrated phenomenological modeling with quantitative single‐cell measurements to separate the sensing and amplification components of Wnt ligands and receptors during establishment of polarity of the Caenorhabditis elegans P cells. By systematically exploring how P‐cell polarity is altered in Wnt ligand and receptor mutants, we inferred that ligands predominantly affect the sensing process, whereas receptors are needed for both sensing and amplification. This integrated approach is generally applicable to other systems and will facilitate decoupling of the different layers of signal sensing and amplification.

Feedback control of gene expression variability in the Caenorhabditis elegans Wnt pathway
Ji N*, Middelkoop TC*, Mentink RA, Betist MC, Tonegawa S, Mooijman D, Korswagen HR, van Oudenaarden A. Feedback control of gene expression variability in the Caenorhabditis elegans Wnt pathway [Internet]. Cell 2013;155(4):869-880. Publisher's VersionAbstract

Variability in gene expression contributes to phenotypic heterogeneity even in isogenic populations. Here, we used the stereotyped, Wnt signaling-dependent development of the Caenorhabditis elegans Q neuroblast to probe endogenous mechanisms that control gene expression variability. We found that the key Hox gene that orients Q neuroblast migration exhibits increased gene expression variability in mutants in which Wnt pathway activity has been perturbed. Distinct features of the gene expression distributions prompted us on a systematic search for regulatory interactions, revealing a network of interlocked positive and negative feedback loops. Interestingly, positive feedback appeared to cooperate with negative feedback to reduce variability while keeping the Hox gene expression at elevated levels. A minimal model correctly predicts the increased gene expression variability across mutants. Our results highlight the influence of gene network architecture on expression variability and implicate feedback regulation as an effective mechanism to ensure developmental robustness.

Single molecule fluorescent in situ hybridization (smFISH) of C. elegans Worms and Embryos
Ji N, van Oudenaarden A. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans Worms and Embryos [Internet]. WormBook 2012; Publisher's Version
Middelkoop TC, Williams L, Yang P-T, Luchtenberg J, Betist MC, Ji N, van Oudenaarden A, Kenyon C, Korswagen HR. The thrombospondin repeat containing protein MIG-21 controls a left-right asymmetric Wnt signaling response in migrating C. elegans neuroblasts [Internet]. Developmental Biology 2012;361:338-348. Publisher's VersionAbstract

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. Although several Wnt signal transduction mechanisms have been described in detail, it is still largely unknown how cells are specified to adopt such different Wnt signaling responses. Here, we have used the stereotypic migration of the C. elegans Q neuroblasts as a model to study how two initially equivalent cells are instructed to activate either β-catenin dependent or independent Wnt signaling pathways to control the migration of their descendants along the anteroposterior axis. We find that the specification of this difference in Wnt signaling response is dependent on the thrombospondin repeat containing protein MIG-21, which acts together with the netrin receptor UNC-40/DCC to control an initial left–right asymmetric polarization of the Q neuroblasts. Furthermore, we show that the direction of this polarization determines the threshold for Wnt/β-catenin signaling, with posterior polarization sensitizing for activation of this pathway. We conclude that MIG-21 and UNC-40 control the asymmetry in Wnt signaling response by restricting posterior polarization to one of the two Q neuroblasts.

Scott BB, Gardner T, Ji N, Fee MS, Lois C. Wandering neuronal migration in the postnatal vertebrate forebrain [Internet]. Journal of Neuorscience 2012;32:1436-1446. Publisher's VersionAbstract

Most non-mammalian vertebrate species add new neurons to existing brain circuits throughout life, a process thought to be essential for tissue maintenance, repair, and learning. How these new neurons migrate through the mature brain and which cues trigger their integration within a functioning circuit is not known. To address these questions, we used two-photon microscopy to image the addition of genetically labeled newly generated neurons into the brain of juvenile zebra finches. Time-lapse in vivo imaging revealed that the majority of migratory new neurons exhibited a multipolar morphology and moved in a nonlinear manner for hundreds of micrometers. Young neurons did not use radial glia or blood vessels as a migratory scaffold; instead, cells extended several motile processes in different directions and moved by somal translocation along an existing process. Neurons were observed migrating for ∼2 weeks after labeling injection. New neurons were observed to integrate in close proximity to the soma of mature neurons, a behavior that may explain the emergence of clusters of neuronal cell bodies in the adult songbird brain. These results provide direct, in vivo evidence for a wandering form of neuronal migration involved in the addition of new neurons in the postnatal brain.