Neuronal migration prevents spatial competition in retinal morphogenesis.

The concomitant occurrence of tissue growth and organization is a hallmark of organismal development1-3. This often means that proliferating and differentiating cells are found at the same time in a continuously changing tissue environment. How cells adapt to architectural changes to prevent spatial interference remains unclear. Here, to understand how cell movements that are key for growth and organization are orchestrated, we study the emergence of photoreceptor neurons that occur during the peak of retinal growth, using zebrafish, human tissue and human organoids. Quantitative imaging reveals that successful retinal morphogenesis depends on the active bidirectional translocation of photoreceptors, leading to a transient transfer of the entire cell population away from the apical proliferative zone. This pattern of migration is driven by cytoskeletal machineries that differ depending on the direction: microtubules are exclusively required for basal translocation, whereas actomyosin is involved in apical movement. Blocking the basal translocation of photoreceptors induces apical congestion, which hampers the apical divisions of progenitor cells and leads to secondary defects in lamination. Thus, photoreceptor migration is crucial to prevent competition for space, and to allow concurrent tissue growth and lamination. This shows that neuronal migration, in addition to its canonical role in cell positioning4, can be involved in coordinating morphogenesis.

Deterministic and probabilistic fate decisions co-exist in a single retinal lineage.

Correct nervous system development depends on the timely differentiation of progenitor cells into neurons. While the output of progenitor differentiation is well investigated at the population and clonal level, how stereotypic or variable fate decisions are during development is still more elusive. To fill this gap, we here follow the fate outcome of single neurogenic progenitors in the zebrafish retina over time using live imaging. We find that neurogenic progenitor divisions produce two daughter cells, one of deterministic and one of probabilistic fate. Interference with the deterministic branch of the lineage affects lineage progression. In contrast, interference with fate probabilities of the probabilistic branch results in a broader range of fate possibilities than in wild-type and involves the production of any neuronal cell type even at non-canonical developmental stages. Combining the interference data with stochastic modelling of fate probabilities revealed that a simple gene regulatory network is able to predict the observed fate decision probabilities during wild-type development. These findings unveil unexpected lineage flexibility that could ensure robust development of the retina and other tissues.

Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway.

During brain development, progenitor cells need to balanceproliferation and differentiation in order to generate different neurons in the correct numbers and proportions. Currently, the patterns of multipotent progenitor divisions that lead to neurogenic entry and the factors that regulate them are not fully understood. We here use the zebrafish retina to address this gap, exploiting its suitability for quantitative live-imaging. We show that early neurogenic progenitors arise from asymmetric divisions. Notch regulates this asymmetry, as when inhibited, symmetric divisions producing two neurogenic progenitors occur. Surprisingly however, Notch does not act through an apicobasal activity gradient as previously suggested, but through asymmetric inheritance of Sara-positive endosomes. Further, the resulting neurogenic progenitors show cell biological features different from multipotent progenitors, raising the possibility that an intermediate progenitor state exists in the retina. Our study thus reveals new insights into the regulation of proliferative and differentiative events during central nervous system development.

Stochasticity and determinism in cell fate decisions.

During development, cells need to make decisions about their fate in order to ensure that the correct numbers and types of cells are established at the correct time and place in the embryo. Such cell fate decisions are often classified as deterministic or stochastic. However, although these terms are clearly defined in a mathematical sense, they are sometimes used ambiguously in biological contexts. Here, we provide some suggestions on how to clarify the definitions and usage of the terms stochastic and deterministic in biological experiments. We discuss the frameworks within which such clear definitions make sense and highlight when certain ambiguity prevails. As an example, we examine how these terms are used in studies of neuronal cell fate decisions and point out areas in which definitions and interpretations have changed and matured over time. We hope that this Review will provide some clarification and inspire discussion on the use of terminology in relation to fate decisions.

In vitro modeling of dendritic atrophy in Rett syndrome: determinants for phenotypic drug screening in neurodevelopmental disorders.

Dendritic atrophy, defined as the reduction in complexity of the neuronal arborization, is a hallmark of several neurodevelopmental disorders, including Rett Syndrome (RTT). RTT, affecting 1:10,000 girls worldwide, is mainly caused by mutations in the MECP2 gene and has no cure. We describe here an in vitro model of dendritic atrophy in Mecp2-/y mouse hippocampal primary cultures, suitable for phenotypic drug-screening. Using High-Content Imaging techniques, we systematically investigated the impact of culturing determinants on several parameters such as neuronal survival, total dendritic length, dendritic endpoints, soma size, cell clusterization, spontaneous activity. Determinants included cell-seeding density, glass or polystyrene substrates, coating with poly-Ornithine with/without Matrigel and miniaturization from 24 to 96-half surface multiwell plates. We show that in all plate-sizes at densities below 320 cells/mm2, morphological parameters remained constant while spontaneous network activity decreased according to the cell-density. Mecp2-/y neurons cultured at 160 cells/mm2 density in 96 multiwell plates, displayed significant dendritic atrophy and showed a marked increase in dendritic length following treatment with Brain-derived neurotrophic factor (BDNF) or Mirtazapine. In conclusion, we have established a phenotypic assay suitable for fast screening of hundreds of compounds, which may be extended to other neurodevelopmental diseases with dendritic atrophy.