Alfonso Martinez-Arias

Mechanisms of epithelial fusion and repair.

One of the principal functions of any epithelium in the embryonic or adult organism is to act as a self-sealing barrier layer. From the earliest stages of development, embryonic epithelia are required to close naturally occurring holes and to fuse wherever two free edges are brought together, and at the simplest level that is precisely what the epidermis must do to repair itself wherever it is damaged. Parallels can be drawn between the artificially triggered epithelial movements of wound repair and the naturally occurring epithelial movements that shape the embryo during morphogenesis. Recent in vitro and in vivo wound-healing studies and analysis of paradigm morphogenetic movements in genetically tractable embryos, like those of Drosophila and Caenorhabditis elegans, have begun to identify both the signals that initiate these movements and the cytoskeletal machinery that drives motility. We are also gaining insight into the nature of the brakes and stop signals, and the mechanisms by which the confronting epithelial sheets knit together to form a seam.

Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure

Fluctuations in the shape of amnioserosa (AS) cells during Drosophila dorsal closure (DC) provide an ideal system with which to understand contractile epithelia, both in terms of the cellular mechanisms and how tissue behaviour emerges from the activity of individual cells. Using quantitative image analysis we show that apical shape fluctuations are driven by the medial cytoskeleton, with periodic foci of contractile myosin and actin travelling across cell apices. Shape changes were mostly anisotropic and neighbouring cells were often, but transiently, organised into strings with parallel deformations. During the early stages of DC, shape fluctuations with long cycle lengths produced no net tissue contraction. Cycle lengths shortened with the onset of net tissue contraction, followed by a damping of fluctuation amplitude. Eventually, fluctuations became undetectable as AS cells contracted rapidly. These transitions were accompanied by an increase in apical myosin, both at cell-cell junctions and medially, the latter ultimately forming a coherent, but still dynamic, sheet across cells. Mutants with increased myosin activity or actin polymerisation exhibited precocious cell contraction through changes in the subcellular localisation of myosin. thickveins mutant embryos, which exhibited defects in the actin cable at the leading edge, showed similar timings of fluctuation damping to the wild type, suggesting that damping is an autonomous property of the AS. Our results suggest that cell shape fluctuations are a property of cells with low and increasing levels of apical myosin, and that medial and junctional myosin populations combine to contract AS cell apices and drive DC.

The distribution of Ultrabithorax transcripts in Drosophila embryos

We have used in situ hybridization to monitor the distribution of Ultrabithorax (Ubx) transcripts during the early stages of Drosophila embryogenesis. When first detectable, in the late syncytial blastoderm, Ubx transcripts are broadly distributed from 20 to 50% egg length (measured from the posterior pole). By the completion of cellular blastoderm formation, a precisely bounded zone one segment wide is defined by the accumulation of high levels of Ubx transcripts. This zone is probably the primordium for parasegment 6, that is, the posterior compartment of the third thoracic segment (T3) and the anterior compartment of the first abdominal segment (A1). Following gastrulation, seven metameric units of the germ band express Ubx transcripts in most or all cells of both the ectoderm and the mesoderm. This principal domain of Ubx expression is sharply bounded at superficial grooves in the embryo which probably mark the A/P compartment boundaries in T3 and A7, and so consists of parasegments 6‐12. Outside this region, Ubx transcripts can be detected in only a few cells of the ectoderm of parasegments 5 and 13, and in the amnioserosa. This distribution suggests that the major role of Ubx in the early embryo lies in the morphological domain generally ascribed to the control of the bithoraxoid gene. Later in embryogenesis, the pattern of Ubx transcript accumulation is different in each of the major germ layers, suggesting that different controls regulate Ubx in each. In the ectoderm, but not in the mesoderm, Ubx transcripts accumulate differentially in anterior and posterior compartments. Probes prepared from different regions of the Ubx transcription unit show related but different patterns of hybridization. These patterns suggest that the 3.2‐kb Ubx transcript is distributed throughout the wider domain of Ubx expression, but that the 4.7‐kb transcript accumulates preferentially in parasegment 6.

Notch Signaling Pathway

Notch is a receptor that mediates intercellular signaling through a pathway conserved across the metazoa. It is involved in cell fate assignation and pattern formation during development. The receptor acts as a membrane-tethered transcription factor and is activated by members of the Delta, Serrate, Lag-2 family of Notch ligands, which trigger two successive proteolytic cleavages of the receptor. The second cleavage releases the intracellular domain of Notch, which translocates to the nucleus, where it interacts with the CSL family of transcriptional regulators and forms part of a Notch target gene–activating complex. In the absence of signaling, CSL [CBF1, Su(H), Lag-1] regulators repress Notch target genes through interactions with several transcriptional co-repressors that recruit histone deacetylases and other chromatin-modifying enzymes. After forming, the transcription-activating binary Notch intracellular domain–CSL complex recruits several proteins that facilitate transcription, among them the coactivator MAM and histone acetylases. Transcription of target genes is terminated when the Notch intracellular domain is degraded in a proteasome-dependent manner.

Interplay between gene expression noise and regulatory network architecture

Complex regulatory networks orchestrate most cellular processes in biological systems. Genes in such networks are subject to expression noise, resulting in isogenic cell populations exhibiting cell-to-cell variation in protein levels. Increasing evidence suggests that cells have evolved regulatory strategies to limit, tolerate or amplify expression noise. In this context, fundamental questions arise: how can the architecture of gene regulatory networks generate, make use of or be constrained by expression noise? Here, we discuss the interplay between expression noise and gene regulatory network at different levels of organization, ranging from a single regulatory interaction to entire regulatory networks. We then consider how this interplay impacts a variety of phenomena, such as pathogenicity, disease, adaptation to changing environments, differential cell-fate outcome and incomplete or partial penetrance effects. Finally, we highlight recent technological developments that permit measurements at the single-cell level, and discuss directions for future research.

An activity of Notch regulates JNK signalling and affects dorsal closure in Drosophila

BACKGROUND The Drosophila Notch protein is a receptor that controls cell fate during embryonic development, particularly in lateral inhibition, a process that acts on groups of cells that share a particular developmental potential to restrict the number of cells that will adopt that cell fate. The process of lateral inhibition is implemented by the nuclear protein Suppressor of Hairless (Su(H)) and is triggered by the ligand Delta. Recent results have shown that the interaction between Delta and Notch triggers the cleavage of the intracellular domain of Notch which then translocates to the nucleus and binds to Su(H). RESULTS We find that Notch plays a role in the patterning of the dorsal epidermis of the Drosophila embryo and that this function of Notch is independent of Su(H), requires Notch at the plasma membrane and targets the c-Jun N-terminal kinase (JNK) signalling pathway. Notch mutants show high levels of JNK activity and can rescue the effects of lowered JNK signalling resulting from mutations in the hemipterous and basket genes. Two regions of the intracellular domain of Notch are involved: the Cdc10/ankyrin repeats, which downregulate signalling through the JNK pathway, and a region carboxy-terminal to these repeats, which regulates this negative function. CONCLUSIONS Our results reveal a novel signalling activity of Notch that does not require its cleavage and acts by modulating signalling through the JNK pathway. In the Drosophila embryo, this activity plays an important role in the morphogenetic movements that drive dorsal closure.

Drosophila midgut homeostasis involves neutral competition between symmetrically dividing intestinal stem cells

The Drosophila adult posterior midgut has been identified as a powerful system in which to study mechanisms that control intestinal maintenance, in normal conditions as well as during injury or infection. Early work on this system has established a model of tissue turnover based on the asymmetric division of intestinal stem cells. From the quantitative analysis of clonal fate data, we show that tissue turnover involves the neutral competition of symmetrically dividing stem cells. This competition leads to stem‐cell loss and replacement, resulting in neutral drift dynamics of the clonal population. As well as providing new insight into the mechanisms regulating tissue self‐renewal, these findings establish intriguing parallels with the mammalian system, and confirm Drosophila as a useful model for studying adult intestinal maintenance.

Chromatin decondensation and nuclear softening accompany Nanog downregulation in embryonic stem cells

The interplay between epigenetic modification and chromatin compaction is implicated in the regulation of gene expression, and it comprises one of the most fascinating frontiers in cell biology. Although a complete picture is still lacking, it is generally accepted that the differentiation of embryonic stem (ES) cells is accompanied by a selective condensation into heterochromatin with concomitant gene silencing, leaving access only to lineage-specific genes in the euchromatin. ES cells have been reported to have less condensed chromatin, as they are capable of differentiating into any cell type. However, pluripotency itself-even prior to differentiation-is a split state comprising a naïve state and a state in which ES cells prime for differentiation. Here, we show that naïve ES cells decondense their chromatin in the course of downregulating the pluripotency marker Nanog before they initiate lineage commitment. We used fluorescence recovery after photobleaching, and histone modification analysis paired with a novel, to our knowledge, optical stretching method, to show that ES cells in the naïve state have a significantly stiffer nucleus that is coupled to a globally more condensed chromatin state. We link this biophysical phenotype to coinciding epigenetic differences, including histone methylation, and show a strong correlation of chromatin condensation and nuclear stiffness with the expression of Nanog. Besides having implications for transcriptional regulation and embryonic cell sorting and suggesting a putative mechanosensing mechanism, the physical differences point to a system-level regulatory role of chromatin in maintaining pluripotency in embryonic development.

The Antennapedia gene is required and expressed in parasegments 4 and 5 of the Drosophila embryo.

Transcripts homologous to the 5′ exon of the Antp gene accumulate in parasegments 4 and 5 of the Drosophila embryo, where analysis of the cuticular phenotype of Antennapedia‐ (Antp‐) embryos shows that there is a genetic requirement for the Antp gene. The pattern of Antp expression is different in different germ layers; the mesoderm expresses Antp only in parasegment 5; in the developing nervous system and the epidermis the pattern of expression is complex and suggests that interactions between different selector genes take place during wild‐type development. Both genetic requirements and gene expression indicate that Antennapedia is the main morphological determinant in parasegment 4; in parasegment 5 it probably acts in concert with other selector genes.

Dissecting ensemble networks in ES cell populations reveals micro-heterogeneity underlying pluripotency

Analysis of transcription at the level of single cells in prokaryotes and eukaryotes has revealed the existence of heterogeneities in the expression of individual genes within genetically homogeneous populations. This variation is an emerging hallmark of populations of Embryonic Stem (ES) cells and has been ascribed to the stochasticity associated with the biochemical events that mediate gene expression. It has been suggested that these heterogeneities play a role in the maintenance of pluripotency. However, for the most part, studies have focused on individual genes in large cell populations. Here we use an existing dataset on the expression of eight genes involved in pluripotency in eighty-three ES cells to create Gene Regulatory Networks (GRNs) at the single cell level. We observe widespread heterogeneities in the expression of the eight genes, but analysis of correlations within individual cells reveals three distinct classes centered on the expression of Nanog, a marker of pluripotency, and Fgf5, a gene associated with differentiation: high levels of Nanog and low levels of Fgf5, low levels of Nanog and high levels of Fgf5, and low levels of both. Each of these classes is associated with a collection of active sub-networks, with differing degrees of connectivity between their elements, which define a cellular state: self-renewal, primed for differentiation or transition between the two. Though every cell should be governed by the same network, the active sub-networks may emerge due to considerations such as variation in (i) the expression level of active transcription factors (e.g. through post-translational modification or ligand/co-factor availability) or (ii) access to the target gene locus (e.g. via changes in chromatin status or epigenetic modifications). We conclude that heterogeneities in gene expression should not be interpreted as representing different states of a single unique network, but as a reflection of the activity of different sub-networks in sub-populations of cells.