A DTC morphometrics package for quantification of complex and variable cellular morphology using ImageJ.

Quantification of complex cellular morphology is important for understanding developmental control of cell shape as well as the developmental ramifications of dysregulated cell shape. However, processing and scoring 3D confocal micrographs can be time consuming and prone to errors such as sample-data matching for large datasets, reproducibility between users, and errors introduced by variable image quality. These problems are further compounded where cell shapes vary from sample to sample and intensity dynamic ranges extend over orders of magnitude. Here we present a package of ImageJ macros we developed for analysis of the C. elegans hermaphrodite distal tip cell (DTC) to (a) optimize images for analysis and (b) assist in quantifying various features of the cell by two independent methods, one user-guided and the other unbiased. Together these tools provide functionality for visualization and multiple parameters of quantification which can be easily customized within free open-source ImageJ.

Innexin function dictates the spatial relationship between distal somatic cells in the Caenorhabditis elegans gonad without impacting the germline stem cell pool.

Gap-junctional signaling mediates myriad cellular interactions in metazoans. Yet, how gap junctions control the positioning of cells in organs is not well understood. Innexins compose gap junctions in invertebrates and affect organ architecture. Here, we investigate the roles of gap-junctions in controlling distal somatic gonad architecture and its relationship to underlying germline stem cells in Caenorhabditis elegans. We show that a reduction of soma-germline gap-junctional activity causes displacement of distal sheath cells (Sh1) towards the distal end of the gonad. We confirm, by live imaging, transmission electron microscopy, and antibody staining, that bare regions-lacking somatic gonadal cell coverage of germ cells-are present between the distal tip cell (DTC) and Sh1, and we show that an innexin fusion protein used in a prior study encodes an antimorphic gap junction subunit that mispositions Sh1. We determine that, contrary to the model put forth in the prior study based on this fusion protein, Sh1 mispositioning does not markedly alter the position of the borders of the stem cell pool nor of the progenitor cell pool. Together, these results demonstrate that gap junctions can control the position of Sh1, but that Sh1 position is neither relevant for GLP-1/Notch signaling nor for the exit of germ cells from the stem cell pool.

Germline Stem and Progenitor Cell Aging in C. elegans.

Like many animals and humans, reproduction in the nematode C. elegans declines with age. This decline is the cumulative result of age-related changes in several steps of germline function, many of which are highly accessible for experimental investigation in this short-lived model organism. Here we review recent work showing that a very early and major contributing step to reproductive decline is the depletion of the germline stem and progenitor cell pool. Since many cellular and molecular aspects of stem cell biology and aging are conserved across animals, understanding mechanisms of age-related decline of germline stem and progenitor cells in C. elegans has broad implications for aging stem cells, germline stem cells, and reproductive aging.

Rewiring of an ancestral Tbx1/10-Ebf-Mrf network for pharyngeal muscle specification in distinct embryonic lineages.

Skeletal muscles arise from diverse embryonic origins in vertebrates, yet converge on extensively shared regulatory programs that require muscle regulatory factor (MRF)-family genes. Myogenesis in the tail of the simple chordate Ciona exhibits a similar reliance on its single MRF-family gene, and diverse mechanisms activate Ci-Mrf Here, we show that myogenesis in the atrial siphon muscles (ASMs) and oral siphon muscles (OSMs), which control the exhalant and inhalant siphons, respectively, also requires Mrf We characterize the ontogeny of OSM progenitors and compare the molecular basis of Mrf activation in OSM versus ASM. In both muscle types, Ebf and Tbx1/10 are expressed and function upstream of Mrf However, we demonstrate that regulatory relationships between Tbx1/10, Ebf and Mrf differ between the OSM and ASM lineages. We propose that Tbx1, Ebf and Mrf homologs form an ancient conserved regulatory state for pharyngeal muscle specification, whereas their regulatory relationships might be more evolutionarily variable.

Loss of deeply conserved C-class floral homeotic gene function and C- and E-class protein interaction in a double-flowered ranunculid mutant.

In the model plant Arabidopsis thaliana, a core eudicot, the floral homeotic C-class gene AGAMOUS (AG) has a dual role specifying reproductive organ identity and floral meristem determinacy. We conduct a functional analysis of the putative AG ortholog ThtAG1 from the ranunculid Thalictrum thalictroides, a representative of the sister lineage to all other eudicots. Down-regulation of ThtAG1 by virus-induced gene silencing resulted in homeotic conversion of stamens and carpels into sepaloid organs and loss of flower determinacy. Moreover, flowers exhibiting strong silencing of ThtAG1 phenocopied the double-flower ornamental cultivar T. thalictroides 'Double White.' Molecular analysis of 'Double White' ThtAG1 alleles revealed the insertion of a retrotransposon causing either nonsense-mediated decay of transcripts or alternative splicing that results in mutant proteins with K-domain deletions. Biochemical analysis demonstrated that the mutation abolishes protein-protein interactions with the putative E-class protein ThtSEP3. C- and E-class protein heterodimerization is predicted by the floral quartet model, but evidence for the functional importance of this interaction is scarce outside the core eudicots. Our findings therefore corroborate the importance and conservation of the interactions between C- and E-class proteins. This study provides a functional description of a full C-class mutant in a noncore ("basal") eudicot, an ornamental double flower, affecting both organ identity and meristem determinacy. Using complementary forward and reverse genetic approaches, this study demonstrates deep conservation of the dual C-class gene function and of the interactions between C- and E-class proteins predicted by the floral quartet model.

Development and evolution of the ascidian cardiogenic mesoderm.

The heart and other blood pumping organs are close to being universally essential in the animal kingdom. These organs present a large anatomical, morphological, and cellular diversity, which is thought to have arisen by building developmental modules on a conserved core of ancestral heart regulatory units. In this context, studies using the ascidian model system Ciona intestinalis offer a distinctive set of theoretical and experimental advantages, which we herein discuss in details. Development of the heart and related muscles in Ciona has been analyzed with a cellular to subcellular resolution unprecedented in Chordate model systems. Unique derived developmental characters of the cardiogenic mesoderm appear to be shared between Ciona and vertebrates. Notably, accumulating evidence point to an early Chordate origin of the cardiopharyngeal population of mesoderm cells that may have provided the foundation for the emergence of the second heart field in higher vertebrates.

Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum.

Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.