The central nervous system can generate various behaviours, including motor responses, which we can observe through video recordings. Recent advancements in genetics, automated behavioural acquisition at scale, and machine learning enable us to link behaviours to their underlying neural mechanisms causally. Moreover, in some animals, such as the Drosophila larva, this mapping is possible at unprecedented scales of millions of animals and single neurons, allowing us to identify the neural circuits generating particular behaviours.These high-throughput screening efforts are invaluable, linking the activation or suppression of specific neurons to behavioural patterns in millions of animals. This provides a rich dataset to explore how diverse nervous system responses can be to the same stimuli. However, challenges remain in identifying subtle behaviours from these large datasets, including immediate and delayed responses to neural activation or suppression, and understanding these behaviours on a large scale. We introduce several statistically robust methods for analyzing behavioural data in response to these challenges: 1) A generative physical model that regularizes the inference of larval shapes across the entire dataset. 2) An unsupervised kernel-based method for statistical testing in learned behavioural spaces aimed at detecting subtle deviations in behaviour. 3) A generative model for larval behavioural sequences, providing a benchmark for identifying complex behavioural changes. 4) A comprehensive analysis technique using suffix trees to categorize genetic lines into clusters based on common action sequences. We showcase these methodologies through a behavioural screen focused on responses to an air puff, analyzing data from 280,716 larvae across 568 genetic lines.Author Summary There is a significant gap in understanding between the architecture of neural circuits and the mechanisms of action selection and behaviour generation.Drosophila larvae have emerged as an ideal platform for simultaneously probing behaviour and the underlying neuronal computation [1]. Modern genetic tools allow efficient activation or silencing of individual and small groups of neurons. Combining these techniques with standardized stimuli over thousands of individuals makes it possible to relate neurons to behaviour causally. However, extracting these relationships from massive and noisy recordings requires the development of new statistically robust approaches. We introduce a suite of statistical methods that utilize individual behavioural data and the overarching structure of the behavioural screen to deduce subtle behavioural changes from raw data. Given our study’s extensive number of larvae, addressing and preempting potential challenges in body shape recognition is critical for enhancing behaviour detection. To this end, we have adopted a physics-informed inference model. Our first group of techniques enables robust statistical analysis within a learned continuous behaviour latent space, facilitating the detection of subtle behavioural shifts relative to reference genetic lines. A second array of methods probes for subtle variations in action sequences by comparing them to a bespoke generative model. Together, these strategies have enabled us to construct representations of behavioural patterns specific to a lineage and identify a roster of ”hit” neurons with the potential to influence behaviour subtly.

Understanding how animals coordinate movements to achieve goals is a fundamental pursuit in neuroscience. Here we explore how neurons that reside in posterior lower-order regions of a locomotor system and project to anterior higher-order regions influence steering and navigation. We characterized the anatomy and functional role of a population of ascending interneurons in the ventral nerve cord of Drosophila larvae. Through electron microscopy reconstructions and light microscopy, we determined that the cholinergic 19f cells receive input primarily from premotor interneurons and synapse upon a diverse array of postsynaptic targets within the anterior segments including other 19f cells. Calcium imaging of 19f activity in isolated CNS preparations in relation to motor neurons revealed that 19f neurons are recruited into most larval motor programmes. 19f activity lags behind motor neuron activity and as a population, the cells encode spatio-temporal patterns of locomotor activity in the larval CNS. Optogenetic manipulations of 19f cell activity in isolated CNS preparations revealed that they coordinate the activity of central pattern generators underlying exploratory headsweeps and forward locomotion in a context and location specific manner. In behaving animals, activating 19f cells suppressed exploratory headsweeps and slowed forward locomotion, while inhibition of 19f activity potentiated headsweeps, slowing forward movement. Inhibiting activity in 19f cells ultimately affected the ability of larvae to remain in the vicinity of an odor source during an olfactory navigation task. Overall, our findings provide insights into how ascending interneurons monitor motor activity and shape interactions amongst rhythm generators underlying complex navigational tasks.

Stem cells rapidly proliferate after injury to repair damaged tissue, and chronic injury predisposes to cancer. However, injury-activated mitogens, the mechanisms that keep them inactive until injury, and their role in cancer are not understood. Here we identify Igf2 as the injury-activated mitogen for neuroendocrine stem cells, a facultative airway stem cell and origin of small cell lung cancer. Igf2 is constitutively produced by the stem cells but sequestered in inactive form by co-expressed Igf binding proteins. Injury releases Igf2 and induces proliferation by activating its receptors and repressing Rb tumor suppressor, which normally enforces stem cell quiescence. Persistent pathway activation initiates oncogenesis. Thus, in addition to its classical hormonal roles in physiology, growth, and aging, Igf operates locally with Igf binding proteins and Rb to control injury-induced stem cell activation and cancer. This pathway may also control related stem cells and cancers of the body and brain.

Building a sizable, complex brain requires both cellular expansion and diversification. One mechanism to achieve these goals is production of multiple transiently amplifying intermediate neural progenitors (INPs) from a single neural stem cell. Like mammalian neural stem cells, Drosophila type II neuroblasts utilize INPs to produce neurons and glia. Within a given lineage, the consecutively born INPs produce morphologically distinct progeny, presumably due to differential inheritance of temporal factors. To uncover the underlying temporal fating mechanisms, we profiled type II neuroblasts' transcriptome across time. Our results reveal opposing temporal gradients of Imp and Syp RNA-binding proteins (descending and ascending, respectively). Maintaining high Imp throughout serial INP production expands the number of neurons and glia with early temporal fate at the expense of cells with late fate. Conversely, precocious upregulation of Syp reduces the number of cells with early fate. Furthermore, we reveal that the transcription factor Seven-up initiates progression of the Imp/Syp gradients. Interestingly, neuroblasts that maintain initial Imp/Syp levels can still yield progeny with a small range of early fates. We therefore propose that the Seven-up-initiated Imp/Syp gradients create coarse temporal windows within type II neuroblasts to pattern INPs, which subsequently undergo fine-tuned subtemporal patterning.

Adult stem cells are responsible for life-long tissue maintenance. They reside in and interact with specialized tissue microenvironments (niches). Using murine hair follicle as a model, we show that when junctional perturbations in the niche disrupt barrier function, adjacent stem cells dramatically change their transcriptome independent of bacterial invasion and become capable of directly signaling to and recruiting immune cells. Additionally, these stem cells elevate cell cycle transcripts which reduce their quiescence threshold, enabling them to selectively proliferate within this microenvironment of immune distress cues. However, rather than mobilizing to fuel new tissue regeneration, these ectopically proliferative stem cells remain within their niche to contain the breach. Together, our findings expose a potential communication relay system that operates from the niche to the stem cells to the immune system and back. The repurposing of proliferation by these stem cells patch the breached barrier, stoke the immune response and restore niche integrity.