Edwin Munro

The segment polarity network is a robust developmental module

All insects possess homologous segments, but segment specification differs radically among insect orders. In Drosophila, maternal morphogens control the patterned activation of gap genes, which encode transcriptional regulators that shape the patterned expression of pair-rule genes. This patterning cascade takes place before cellularization. Pair-rule gene products subsequently ‘imprint’ segment polarity genes with reiterated patterns, thus defining the primordial segments. This mechanism must be greatly modified in insect groups in which many segments emerge only after cellularization. In beetles and parasitic wasps, for instance, pair-rule homologues are expressed in patterns consistent with roles during segmentation, but these patterns emerge within cellular fields. In contrast, although in locusts pair-rule homologues may not control segmentation, some segment polarity genes and their interactions are conserved. Perhaps segmentation is modular, with each module autonomously expressing a characteristic intrinsic behaviour in response to transient stimuli. If so, evolution could rearrange inputs to modules without changing their intrinsic behaviours. Here we suggest, using computer simulations, that the Drosophila segment polarity genes constitute such a module, and that this module is resistant to variations in the kinetic constants that govern its behaviour.

Force Generation, Transmission, and Integration during Cell and Tissue Morphogenesis

Cell shape changes underlie a large set of biological processes ranging from cell division to cell motility. Stereotyped patterns of cell shape changes also determine tissue remodeling events such as extension or invagination. In vitro and cell culture systems have been essential to understanding the fundamental physical principles of subcellular mechanics. These are now complemented by studies in developing organisms that emphasize how cell and tissue morphogenesis emerge from the interplay between force-generating machines, such as actomyosin networks, and adhesive clusters that transmit tensile forces at the cell cortex and stabilize cell-cell and cell-substrate interfaces. Both force production and transmission are self-organizing phenomena whose adaptive features are essential during tissue morphogenesis. A new era is opening that emphasizes the similarities of and allows comparisons between distant dynamic biological phenomena because they rely on core machineries that control universal features of cytomechanics.

Robustness, Flexibility, and the Role of Lateral Inhibition in the Neurogenic Network

BACKGROUND Many gene networks used by developing organisms have been conserved over long periods of evolutionary time. Why is that? We showed previously that a model of the segment polarity network in Drosophila is robust to parameter variation and is likely to act as a semiautonomous patterning module. Is this true of other networks as well? RESULTS We present a model of the core neurogenic network in Drosophila. Our model exhibits at least three related pattern-resolving behaviors that the real neurogenic network accomplishes during embryogenesis in Drosophila. Furthermore, we find that it exhibits these behaviors across a wide range of parameter values, with most of its parameters able to vary more than an order of magnitude while it still successfully forms our test patterns. With a single set of parameters, different initial conditions (prepatterns) can select between different behaviors in the networks repertoire. We introduce two new measures for quantifying network robustness that mimic recombination and allelic divergence and use these to reveal the shape of the domain in the parameter space in which the model functions. We show that lateral inhibition yields robustness to changes in prepatterns and suggest a reconciliation of two divergent sets of experimental results. Finally, we show that, for this model, robustness confers functional flexibility. CONCLUSIONS The neurogenic network is robust to changes in parameter values, which gives it the flexibility to make new patterns. Our model also offers a possible resolution of a debate on the role of lateral inhibition in cell fate specification.

C. elegans PAR-3 and PAR-6 are required for apicobasal asymmetries associated with cell adhesion and gastrulation.

PAR proteins distribute asymmetrically across the anterior-posterior axis of the 1-cell-stage C. elegans embryo, and function to establish subsequent anterior-posterior asymmetries. By the end of the 4-cell stage, anteriorly localized PAR proteins, such as PAR-3 and PAR-6, redistribute to the outer, apical surfaces of cells, whereas posteriorly localized PAR proteins, such as PAR-1 and PAR-2, redistribute to the inner, basolateral surfaces. Because PAR proteins are provided maternally, distinguishing apicobasal from earlier anterior-posterior functions requires a method that selectively prevents PAR activity after the 1-cell stage. In the present study we generated hybrid PAR proteins that are targeted for degradation after the 1-cell stage. Embryos containing the hybrid PAR proteins had normal anterior-posterior polarity, but showed defects in apicobasal asymmetries associated with gastrulation. Ectopic separations appeared between lateral surfaces of cells that are normally tightly adherent, cells that ingress during gastrulation failed to accumulate nonmuscle myosin at their apical surfaces and ingression was slowed. Thus, PAR proteins function in both apicobasal and anterior-posterior asymmetry during the first few cell cycles of embryogenesis.

Ascidian prickle regulates both mediolateral and anterior-posterior cell polarity of notochord cells.

The ascidian notochord follows a morphogenetic program that includes convergent extension (C/E), followed by anterior-posterior (A/P) elongation [1-4]. As described here, developing notochord cells show polarity first in the mediolateral (M/L) axis during C/E, and subsequently in the A/P axis during elongation. Previous embryological studies [3] have shown that contact with neighboring tissues is essential for directing M/L polarity of ascidian notochord cells. During C/E, the planar cell polarity (PCP) gene products prickle (pk) and dishevelled (dsh) show M/L polarization. pk and dsh colocalize at the notochord cell membranes, with the exception of those in contact with neighboring muscle cells. In the mutant aimless (aim), which carries a deletion in pk, notochord morphogenesis is disrupted, and cell polarization is lost. After C/E, there is a dynamic relocalization of PCP proteins in the notochord cells with dsh localized to the lateral edges of the membrane, and pk and strabismus (stbm) at the anterior edges. An A/P polarity is present in the extending notochord cells and is evident by the position of the nuclei, which in normal embryos are invariably found at the posterior edge of each cell. In the aim mutant, all appearances of A/P polarity in the notochord are lost.

A self-organized biomechanical network drives shape changes during tissue morphogenesis

Tissue morphogenesis is orchestrated by cell shape changes. Forces required to power these changes are generated by non-muscle myosin II (MyoII) motor proteins pulling filamentous actin (F-actin). Actomyosin networks undergo cycles of assembly and disassembly (pulses) to cause cell deformations alternating with steps of stabilization to result in irreversible shape changes. Although this ratchet-like behaviour operates in a variety of contexts, the underlying mechanisms remain unclear. Here we investigate the role of MyoII regulation through the conserved Rho1–Rok pathway during Drosophila melanogaster germband extension. This morphogenetic process is powered by cell intercalation, which involves the shrinkage of junctions in the dorsal–ventral axis (vertical junctions) followed by junction extension in the anterior–posterior axis. While polarized flows of medial–apical MyoII pulses deform vertical junctions, MyoII enrichment on these junctions (planar polarity) stabilizes them. We identify two critical properties of MyoII dynamics that underlie stability and pulsatility: exchange kinetics governed by phosphorylation–dephosphorylation cycles of the MyoII regulatory light chain; and advection due to contraction of the motors on F-actin networks. Spatial control over MyoII exchange kinetics establishes two stable regimes of high and low dissociation rates, resulting in MyoII planar polarity. Pulsatility emerges at intermediate dissociation rates, enabling convergent advection of MyoII and its upstream regulators Rho1 GTP, Rok and MyoII phosphatase. Notably, pulsatility is not an outcome of an upstream Rho1 pacemaker. Rather, it is a self-organized system that involves positive and negative biomechanical feedback between MyoII advection and dissociation rates.

Sequential Activation of Apical and Basolateral Contractility Drives Ascidian Endoderm Invagination

BACKGROUND Epithelial invagination is a fundamental morphogenetic behavior that transforms a flat cell sheet into a pit or groove. Previous studies of invagination have focused on the role of actomyosin-dependent apical contraction; other mechanisms remain largely unexplored. RESULTS We combined experimental and computational approaches to identify a two-step mechanism for endoderm invagination during ascidian gastrulation. During Step 1, which immediately precedes invagination, endoderm cells constrict their apices because of Rho/Rho-kinase-dependent apical enrichment of 1P-myosin. Our data suggest that endoderm invagination itself occurs during Step 2, without further apical shrinkage, via a novel mechanism we call collared rounding: Rho/Rho-kinase-independent basolateral enrichment of 1P-myosin drives apico-basal shortening, whereas Rho/Rho-kinase-dependent enrichment of 1P and 2P myosin in circumapical collars is required to prevent apical expansion and for deep invagination. Simulations show that boundary-specific tension values consistent with these distributions of active myosin can explain the cell shape changes observed during invagination both in normal embryos and in embryos treated with pharmacological inhibitors of either Rho-kinase or Myosin II ATPase. Indeed, we find that the balance of strong circumapical and basolateral tension is the only mechanism based on differential cortical tension that can explain ascidian endoderm invagination. Finally, simulations suggest that mesectoderm cells resist endoderm shape changes during both steps, and we confirm this prediction experimentally. CONCLUSIONS Our findings suggest that early ascidian gastrulation is driven by the coordinated apposition of circumapical and lateral endoderm contraction, working against a resisting mesectoderm. We propose that similar mechanisms may operate during other invaginations.

Processing bodies and germ granules are distinct RNA granules that interact in C. elegans embryos

In somatic cells, untranslated mRNAs accumulate in cytoplasmic foci called processing bodies or P-bodies. P-bodies contain complexes that inhibit translation and stimulate mRNA deadenylation, decapping, and decay. Recently, certain P-body proteins have been found in germ granules, RNA granules specific to germ cells. We have investigated a possible connection between P-bodies and germ granules in Caenorhabditis elegans. We identify PATR-1, the C. elegans homolog of the yeast decapping activator Pat1p, as a unique marker for P-bodies in C. elegans embryos. We find that P-bodies are inherited maternally as core granules that mature differently in somatic and germline blastomeres. In somatic blastomeres, P-bodies recruit the decapping activators LSM-1 and LSM-3. This recruitment requires the LET-711/Not1 subunit of the CCR4-NOT deadenylase and correlates spatially and temporally with the onset of maternal mRNA degradation. In germline blastomeres, P-bodies are maintained as core granules lacking LSM-1 and LSM-3. P-bodies interact with germ granules, but maintain distinct dynamics and components. The maternal mRNA nos-2 is maintained in germ granules, but not in P-bodies. We conclude that P-bodies are distinct from germ granules, and represent a second class of RNA granules that behaves differently in somatic and germline cells.

Cellular Symmetry Breaking during Caenorhabditis elegans Development

The nematode worm Caenorhabditis elegans has produced a wellspring of insights into mechanisms that govern cellular symmetry breaking during animal development. Here we focus on two highly conserved systems that underlie many of the key symmetry-breaking events that occur during embryonic and larval development in the worm. One involves the interplay between Par proteins, Rho GTPases, and the actomyosin cytoskeleton and mediates asymmetric cell divisions that establish the germline. The other uses elements of the Wnt signaling pathway and a highly reiterative mechanism that distinguishes anterior from posterior daughter cell fates. Much of what we know about these systems comes from intensive study of a few key events-Par/Rho/actomyosin-mediated polarization of the zygote in response to a sperm-derived cue and the Wnt-mediated induction of endoderm at the four-cell stage. However, a growing body of work is revealing how C. elegans exploits elements/variants of these systems to accomplish a diversity of symmetry-breaking tasks throughout embryonic and larval development.

Quantitative Variation in Autocrine Signaling and Pathway Crosstalk in the Caenorhabditis Vulval Network

BACKGROUND Biological networks experience quantitative change in response to environmental and evolutionary variation. Computational modeling allows exploration of network parameter space corresponding to such variations. The intercellular signaling network underlying Caenorhabditis vulval development specifies three fates in a row of six precursor cells, yielding a quasi-invariant 3°3°2°1°2°3° cell fate pattern. Two seemingly conflicting verbal models of vulval precursor cell fate specification have been proposed: sequential induction by the EGF-MAP kinase and Notch pathways, or morphogen-based induction by the former. RESULTS To study the mechanistic and evolutionary system properties of this network, we combine experimental studies with computational modeling, using a model that keeps the network architecture constant but varies parameters. We first show that the Delta autocrine loop can play an essential role in 2° fate specification. With this autocrine loop, the same network topology can be quantitatively tuned to use in the six-cell-row morphogen-based or sequential patterning mechanisms, which may act singly, cooperatively, or redundantly. Moreover, different quantitative tunings of this same network can explain vulval patterning observed experimentally in C. elegans, C. briggsae, C. remanei, and C. brenneri. We experimentally validate model predictions, such as interspecific differences in isolated vulval precursor cell behavior and in spatial regulation of Notch activity. CONCLUSIONS Our study illustrates how quantitative variation in the same network comprises developmental patterning modes that were previously considered qualitatively distinct and also accounts for evolution among closely related species.