Spatio‐temporal regulation of signalling pathways plays a key role in generating diverse responses during the development of multicellular organisms. The role of signal dynamics in transferring signalling information in vivo is incompletely understood. Here, we employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto‐Delta), which replaces both copies of the endogenous wild‐type locus. Using clonal analysis, we show that optogenetic activation blocks Notch activation through cis‐inhibition in signal‐receiving cells. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals differential tissue‐ and cell‐scale regulatory modes. While at the tissue‐level the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation acts through a switch‐like mechanism. Thus, time confers regulatory properties to Notch signalling that exhibit integrative digital behaviours during tissue differentiation.

Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitored Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging platforms with single-molecule Fluorescence Correlation Spectroscopy (FCS), we demonstrate that Fgf8a, produced at the embryonic margin, propagates by free diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a mediated patterning. Manipulation of the Fgf8a input alters the signaling outcome, thereby establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, using diffusion-hindered versions of Fgf8a, we demonstrate that extracellular diffusion of the protein from the source is critical for it to achieve its morphogenic potential.

Spatio-temporal regulation of signalling pathways plays a key role in generating diverse responses during the development of multicellular organisms. The role of signal dynamics in transferring signalling information in vivo is incompletely understood. Here we employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto-Delta), which replaces both copies of the endogenous wild type locus. Using clonal analysis, we show that optogenetic activation blocks Notch activation through cisinhibition in signal-receiving cells. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals differential tissue-and cell-scale regulatory modes. While at the tissuelevel the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation acts through a switch-like mechanism. Thus, time confers regulatory properties to Notch signalling that exhibit integrative digital behaviours during tissue differentiation.We thank all members of the De Renzis laboratory for helpful discussion. We thank C. Tischer for helping with image analysis and the data pipeline developed in Cell Profiler for quantifying Delta clustering kinetics. We thank A. Aulehla, J.Crocker, J. Hartmann, and T. Hiiragi for helpful discussions and critical reading of the manuscript. We thank the advanced light microscopy for their advice and assistance. We thank the Bloomington Drosophila Stock Center and the Drosophila Genomics Resource Center for providing fly stocks and cDNAs. We thank M. Levine for providing fly stocks. Author contributionsThe experiments were conceived and designed by R.V., A.N., P.N. and S.D.R.

Monensin Sensitive 1 (Mon1) is a component of the Mon1:Ccz1 complex that mediates Rab5 to Rab7 conversion in eukaryotic cells by serving as a guanine nucleotide exchange factor for Rab7 during vesicular trafficking. We find that Mon1 activity modulates the complexity of Class IV dendritic arborization (da) neurons during larval development. Loss of Mon1 function leads to an increase in arborization and complexity, while increased expression, leads to reduced arborization. The ability of Mon1 to influence dendritic development is possibly a function of its interactions with Rab family GTPases that are central players in vesicular trafficking. Earlier, these GTPases, specifically Rab1, Rab5, Rab10, and Rab11 have been shown to regulate dendritic arborization. We have conducted genetic epistasis experiments, by modulating the activity of Rab5, Rab7, and Rab11 in da neurons, in Mon1 mutants, and demonstrate that the ability of Mon1 to regulate arborization is possibly due to its effect on the recycling pathway. Dendritic branching is critical for proper connectivity and physiological function of the neuron. An understanding of regulatory elements, such as Mon1, as demonstrated in our study, is essential to understand neuronal function.