No abstract

We propose that developmental evolution is primarily governed by selection and/or selection-independent constraints, not stochastic processes such as drift in unconstrained phenotypic space.

The mechanisms regulating the extension of small unicellular tubes remain poorly defined. Here we identify several steps in Caenorhabditis elegans excretory canal growth, and propose a model for lumen extension. Our results suggest that the basal and apical excretory membranes grow sequentially: the former extends first like an axon growth cone; the latter extends next as a result of an osmoregulatory activity triggering peri-apical vesicles (a membrane reservoir) to fuse with the lumen. An apical cytoskeletal web including intermediate filaments and actin crosslinking proteins ensures straight regular lumen growth. Expression of several genes encoding proteins mediating excretory lumen extension, such as the osmoregulatory STE20-like kinase GCK-3 and the intermediate filament IFB-1, is regulated by ceh-26 (here referred to as pros-1), which we found essential for excretory canal formation. Interestingly, PROS-1 is homologous to vertebrate Prox1, a transcription factor controlling lymphatic vessel growth. Our findings have potential evolutionary implications for the origin of fluid-collecting organs, and provide a reference for lymphangiogenesis.

Despite the identification of essential processes in which cell fusion plays spectacular roles such as in fertilization and development of muscle, bone, and placenta, there are no identified proteins that directly mediate developmental cell fusion reactions. C. elegans has recently become among the best-characterized models to use for studying developmental cell fusion. The eff-1 (epithelial fusion failure) gene encodes novel type I membrane proteins required for epithelial cell fusion. Analysis of eff-1 mutants showed that cell fusion normally restricts routes for cell migration and establishes body and organ shape and size [ 5, 8, 9, 11]. Here, we explored cell fusion by using time-lapse confocal and electron microscopy of different organs. We found that ectopic expression of eff-1 is sufficient to fuse epithelial cells that do not normally fuse. This ectopic fusion results in cytoplasmic content mixing and disappearance of apical junctions, starting less than 50 min after the start of eff-1 transcription. We found that eff-1 is necessary to initiate and expand multiple microfusion events between pharyngeal muscle cells. Surprisingly, eff-1 is not required to fuse the gonadal anchor cell to uterine cells. Thus, eff-1 is sufficient and essential for most but not all cell fusion events during C. elegans development.

The small ubiquitin-like modifier (SUMO) modification alters the subcellular distribution and function of its substrates. Here we show the major role of SUMO during the development of the Caenorhabditis elegans reproductive system. smo-1 deletion mutants develop into sterile adults with abnormal somatic gonad, germ line, and vulva. SMO-1ϻGFP reporter is highly expressed in the somatic reproductive system. smo-1 animals lack a vulval-uterine connection as a result of impaired ventral uterine -cell differentiation and anchor cell fusion. Mutations in the LIN-11 LIM domain transcription factor lead to a uterine phenotype that resembles the smo-1 phenotype. LIN-11 is sumoylated, and its sumoylation is required for its activity during uterine morphogenesis. Expression of a SUMO-modified LIN-11 in the smo-1 background partially rescued -cell differentiation and retained LIN-11 in nuclear bodies. Thus, our results identify the reproductive system as the major SUMO target during postembryonic development and highlight LIN-11 as a physiological substrate whose sumoylation is associated with the formation of a functional vulval-uterine connection.[Keywords: SUMO; somatic gonad; Supplemental material is available at http://www.genesdev.org. (Schwarz et al. 1998). SUMO conjugation has been shown to affect subcellular localization of the modified substrate, thereby affecting its activity and stability (Matunis et al. 1996;Mahajan et al. 1997; Muller at al. 1998). Several transcription factors are modified by sumoylation. Whereas SUMO modification negatively regulates the androgen receptor, SP3, c-Jun, and p53 (Gostissa et al. 1999; Muller et al. 2000;Poukka et al. 2000;Schmidt and Muller 2002), sumoylation of the glucocorticoid receptor increases its transcriptional activities (LeDrean et al. 2002). Sumoylation also affects transcriptional activities indirectly. For example, SUMO conjugation to class II histone deacetylase impairs its transcription-repressing function (Kirsch et al. 2002). Alternatively, sumoylation has also been shown to affect nuclear and subnuclear (nucleolar or PML nuclear body) localization of regulatory proteins primarily implicated in transcriptional control (Sternsdorf et al. 1997;Pichler et al. 2002).The SUMO conjugation system is essential for viability in Saccharomyces cerevisiae (Melchoir 2000). Phenotypes observed upon aberrant sumoylation in S. cerevisiae include impaired septin ring formation, chromosomal segregation, and progression of the cell cycle through G 2 -M (Johnson and Blobel 1999). Studies in Arabidopsis suggest that the SUMO conjugation system has a role in protection against stress and/or repair of stressrelated damage (Kurepa et al. 2002). In Drosophila melanogaster, the loss-of-function mutation of semushi, the UBC9 (SUMO-conjugating enzyme) ortholog, prevents nuclear import of the transcription factor Bicoid (Bcd) and results in impaired embryogenesis (Epps and Tanda 1998).

To sense the outside world, some neurons protrude across epithelia, the cellular barriers that line every surface of our bodies. To study the morphogenesis of such neurons, we examined the C. elegans amphid, in which dendrites protrude through a glial channel at the nose. During development, amphid dendrites extend by attaching to the nose via DYF-7, a type of protein typically found in epithelial apical ECM. Here, we show that amphid neurons and glia exhibit epithelial properties, including tight junctions and apical-basal polarity, and develop in a manner resembling other epithelia. We find that DYF-7 is a fibril-forming apical ECM component that promotes formation of the tube-shaped glial channel, reminiscent of roles for apical ECM in other narrow epithelial tubes. We also identify a requirement for FRM-2, a homolog of EPBL15/moe/Yurt that promotes epithelial integrity in other systems. Finally, we show that other environmentally exposed neurons share a requirement for DYF-7. Together, our results suggest that these neurons and glia can be viewed as part of an epithelium continuous with the skin, and are shaped by mechanisms shared with other epithelia.

Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest).Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron-microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high-pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 μm distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of noncorrelative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron-microscopic protocols, making them more accessible to a wider range of researchers.Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time-demanding procedure of specimen preparation for any kind of TEM.

The interplay between signalling pathways and metabolism is crucial for tissue growth. Yet, it remains poorly understood. Here, we studied the consequences of modulating iron metabolism on the growth of Drosophila imaginal discs. We find that reducing the levels of the ferritin heavy chain in the larval wing discs leads to drastic growth defects, whereas light chain depletion causes only minor defects. Mutant cell clones for the heavy chain lack the ability to compete against Minute mutant cells. Reactive oxygen species (ROS) accumulate in wing discs with reduced heavy chain levels, causing severe mitochondrial defects and ferroptosis. Preventing ROS accumulation alleviates some of the growth defects. We propose that the increased expression of ferritin in hippo mutant cells may protect against ROS accumulation.