Luis M. Escudero

Live imaging of Drosophila imaginal disc development

Live imaging has revolutionized the analysis of developmental biology over the last few years. The ability to track in real time the dynamic processes that occur at tissue and cellular levels gives a much clearer view of development, and allows greater temporal resolution, than is possible with fixed tissue. Drosophila imaginal discs are a particularly important model of many aspects of development, but their small size and location inside the larva and pupa has prevented live imaging techniques from extensively being used in their study. Here, we introduce the use of viscous culture medium to enable high resolution imaging of imaginal disc development. As a proof of principle, we have analyzed the transformation that occurs during metamorphosis of the wing imaginal disc into the mature wing and report several previously unobserved stages of this model of organogenesis. These imaging methods are especially useful to study the complex and dynamic changes that occur during morphogenesis, but we show that they can also be used to analyze other developmental and cellular events. Moreover, our viscous medium creates a platform for future adaptation of other tissue culture conditions to allow imaging of a wide range of developmental events and systems.

ATM specifically mediates repair of double-strand breaks with blocked DNA ends

Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders.

Mechanism of G1 arrest in the Drosophila eye imaginal disc

BackgroundMost differentiating cells are arrested in G1-phase of the cell cycle and this proliferative quiescence appears important to allow differentiation programmes to be executed. An example occurs in the Drosophila eye imaginal disc, where all cells are synchronized and arrested in G1 phase prior to making a fate choice either to initiate the first round of photoreceptor differentiation or to re-enter one terminal mitosis.ResultsWe have analysed the mechanism of this temporally regulated G1-phase in order to develop an integrated model of this proliferative regulation. We find that an overlapping set of cell cycle inhibitors combine to form an efficient barrier to cell cycle progression. This barrier depends on both the primary secreted signals that drive retinal development, Dpp and Hh. Each of these has distinct, as well as partially overlapping functions, in ensuring that Cyclin E and dE2F1 are kept in check. Additionally, inhibition of Cyclin A by Roughex is essential, and this regulation is independent of Dpp and Hh.ConclusionOne implication of these results is to further support the idea that Cyclin A has important functions in S-phase entry as well as in mitosis. The unexpectedly complex network of regulation may reflect the importance of cells being uniformly ready to respond to the inductive signals that coordinate retinal differentiation.

Charlatan, a Zn-finger transcription factor, establishes a novel level of regulation of the proneural achaete/scute genes of Drosophila

The proneural genes achaete (ac) and scute (sc) are necessary for the formation of the external sensory organs (SOs) of Drosophila. ac and sc are expressed in proneural clusters and impart their cells with neural potential. For this potential to be realized, and the SO precursor cell (SOP) to arise within a cluster, sufficient proneural protein must accumulate in the cluster. Here we describe a novel gene, charlatan (chn), which encodes a zinc finger transcription factor that facilitates this accumulation by forming a stimulatory loop with ac/sc. We find that loss of function of chn decreases the accumulation of Sc in proneural clusters and partially removes notum macrochaetae, while overexpression of chn enhances ac/sc expression and the formation of extra SOs. Moreover, chn is activated by ac/sc in proneural clusters. Chn apparently stimulates ac/sc by physically interacting with the proneural cluster-specific enhancers and increasing enhancer efficiency, thus acting as a stimulator of ac/sc expression in proneural clusters. chn is also required for the proper development of the embryonic peripheral nervous system, as its absence leads to loss of neurons and causes aberrant development of chordotonal organs.

Fundamental physical cellular constraints drive self‐organization of tissues

Morphogenesis is driven by small cell shape changes that modulate tissue organization. Apical surfaces of proliferating epithelial sheets have been particularly well studied. Currently, it is accepted that a stereotyped distribution of cellular polygons is conserved in proliferating tissues among metazoans. In this work, we challenge these previous findings showing that diverse natural packed tissues have very different polygon distributions. We use Voronoi tessellations as a mathematical framework that predicts this diversity. We demonstrate that Voronoi tessellations and the very different tissues analysed share an overriding restriction: the frequency of polygon types correlates with the distribution of cell areas. By altering the balance of tensions and pressures within the packed tissues using disease, genetic or computer model perturbations, we show that as long as packed cells present a balance of forces within tissue, they will be under a physical constraint that limits its organization. Our discoveries establish a new framework to understand tissue architecture in development and disease.

Age-mediated transcriptomic changes in adult mouse substantia nigra.

Substantia nigra pars compacta (SNpc) is highly sensitive to normal aging and selectively degenerates in Parkinsons disease (PD). Until now, molecular mechanisms behind SNpc aging have not been fully investigated using high throughput techniques. Here, we show early signs of aging in SNpc, which are more evident than in ventral tegmental area (VTA), a region adjacent to SNpc but less affected in PD. Aging-associated early changes in transcriptome were investigated comparing late middle-aged (18 months old) to young (2 months old) mice in both SNpc and VTA. A meta-analysis of published microarray studies allowed us to generate a common “transcriptional signature” of the aged (≥ 24 months old) mouse brain. SNpc of late-middle aged mice shared characteristics with the transcriptional signature, suggesting an accelerated aging in SNpc. Age-dependent changes in gene expression specific to SNpc were also observed, which were related to neuronal functions and inflammation. Future studies could greatly help determine the contribution of these changes to SNpc aging. These data help understand the processes underlying SNpc aging and their potential contribution to age-related disorders like PD.

Echinoid synergizes with the Notch signaling pathway in Drosophila mesothorax bristle patterning

echinoid (ed) encodes an immunoglobulin domain-containing cell adhesion molecule that negatively regulates the Egfr signaling pathway during Drosophila photoreceptor development. We show a novel function of Ed, i.e. the restriction of the number of notum bristles that arise from a proneural cluster. Thus, loss-of-function conditions for ed give rise to the development of extra macrochaetae near the extant ones and increase the density of microchaetae. Analysis of ed mosaics indicates that extra sensory organ precursors (SOPs) arise from proneural clusters of achaete-scute expression in a cell-autonomous way. ed embryos also exhibit a neurogenic phenotype. These phenotypes suggest a functional relation between ed and the Notch (N) pathway. Indeed, loss-of-function of ed reduces the expression of the N pathway effector E(spl)m8 in proneural clusters. Moreover, combinations of moderate loss-of-function conditions for ed and for different components of the N pathway show clear synergistic interactions manifested as strong neurogenic bristle phenotypes. We conclude that Ed is not essential for, but it facilitates, N signaling. It is known that the N and Egfr pathways act antagonistically in bristle development. Consistently, we find that Ed also antagonizes the bristle-promoting activity of the Egfr pathway, either by the enhancement of N signalling or, similar to the eye, by a more direct action on the Egfr pathway.

Epithelial organisation revealed by a network of cellular contacts

The emergence of differences in the arrangement of cells is the first step towards the establishment of many organs. Understanding this process is limited by the lack of systematic characterization of epithelial organisation. Here we apply network theory at the scale of individual cells to uncover patterns in cell-to-cell contacts that govern epithelial organisation. We provide an objective characterization of epithelia using network representation where cells are nodes and cell contacts are links. The features of individual cells, together with attributes of the cellular network produce a defining signature that distinguishes epithelia from different organs, species, developmental stages and genetic conditions. The approach permits characterization, quantification and classification of normal and perturbed epithelia and establishes a framework for understanding molecular mechanisms that underpin the architecture of complex tissues.

Dual role of myosin II during Drosophila imaginal disc metamorphosis

The motor protein non-muscle myosin II is a major driver of the movements that sculpt three dimensional organs from two dimensional epithelia. The machinery of morphogenesis is well established but the logic of its control remains unclear in complex organs. Here we use live imaging and ex vivo culture to report a dual role of myosin II in regulating the development of the Drosophila wing. First, myosin II drives the contraction of a ring of cells that surround the squamous peripodial epithelium, providing the force to fold the whole disc through about 90°. Second, myosin II is needed to allow the squamous cells to expand and then retract at the end of eversion. The combination of genetics and live imaging allows us to describe and understand the tissue dynamics, and the logic of force generation needed to transform a relatively simple imaginal disc into a more complex and three-dimensional adult wing.

Cooperation and competition in the dynamics of tissue architecture during homeostasis and tumorigenesis

The construction of a network of cell-to-cell contacts makes it possible to characterize the patterns and spatial organization of tissues. Such networks are highly dynamic, depending on the changes of the tissue architecture caused by cell division, death and migration. Local competitive and cooperative cell-to-cell interactions influence the choices cells make. We review the literature on quantitative data of epithelial tissue topology and present a dynamical network model that can be used to explore the evolutionary dynamics of a two dimensional tissue architecture with arbitrary cell-to-cell interactions. In particular, we show that various forms of experimentally observed types of interactions can be modelled using game theory. We discuss a model of cooperative and non-cooperative cell-to-cell communication that can capture the interplay between cellular competition and tissue dynamics. We conclude with an outlook on the possible uses of this approach in modelling tumorigenesis and tissue homeostasis.