A Krüppel-like factor is required for development and regeneration of germline and yolk cells from somatic stem cells in planarians.

Sexually reproducing animals segregate their germline from their soma. In addition to gamete-producing gonads, planarian and parasitic flatworm reproduction relies on yolk cell-generating accessory reproductive organs (vitellaria) supporting development of yolkless oocytes. Despite the importance of vitellaria for flatworm reproduction (and parasite transmission), little is known about this unique evolutionary innovation. Here, we examine reproductive system development in the planarian Schmidtea mediterranea, in which pluripotent stem cells generate both somatic and germ cell lineages. We show that a homolog of the pluripotency factor Klf4 is expressed in primordial germ cells (PGCs), presumptive germline stem cells (GSCs), and yolk cell progenitors. Knockdown of this klf4-like (klf4l) gene results in animals that fail to specify or maintain germ cells; surprisingly, they also fail to maintain yolk cells. We find that yolk cells display germ cell-like attributes and that vitellaria are structurally analogous to gonads. In addition to identifying a new proliferative cell population in planarians (yolk cell progenitors) and defining its niche, our work provides evidence supporting the hypothesis that flatworm germ cells and yolk cells share a common evolutionary origin.

The good, the bad, and the ugly: From planarians to parasites.

Platyhelminthes can perhaps rightly be described as a phylum of the good, the bad, and the ugly: remarkable free-living worms that colonize land, river, and sea, which are often rife with color and can display extraordinary regenerative ability; parasitic worms like schistosomes that cause devastating disease and suffering; and monstrous tapeworms that are the stuff of nightmares. In this chapter, we will explore how our research expanded beyond free-living planarians to their gruesome parasitic cousins. We start with Schistosoma mansoni, which is not a new model; however, approaching these parasites from a developmental perspective required a reinvention that may hold generalizable lessons to basic biologists interested in pivoting to disease models. We then turn to our (re)establishment of the rat tapeworm Hymenolepis diminuta, a once-favorite model that had been largely forgotten by the molecular biology revolution. Here we tell our stories in three, first-person narratives in order to convey personal views of our experiences. Welcome to the dark side.

Region-specific regulation of stem cell-driven regeneration in tapeworms.

Tapeworms grow at rates rivaling the fastest-growing metazoan tissues. To propagate they shed large parts of their body; to replace these lost tissues they regenerate proglottids (segments) as part of normal homeostasis. Their remarkable growth and regeneration are fueled by adult somatic stem cells that have yet to be characterized molecularly. Using the rat intestinal tapeworm, Hymenolepis diminuta, we find that regenerative potential is regionally limited to the neck, where head-dependent extrinsic signals create a permissive microenvironment for stem cell-driven regeneration. Using transcriptomic analyses and RNA interference, we characterize and functionally validate regulators of tapeworm growth and regeneration. We find no evidence that stem cells are restricted to the regeneration-competent neck. Instead, lethally irradiated tapeworms can be rescued when cells from either regeneration-competent or regeneration-incompetent regions are transplanted into the neck. Together, the head and neck tissues provide extrinsic cues that regulate stem cells, enabling region-specific regeneration in this parasite.

Prospecting for Planarian Pluripotency.

Planarians are renowned for extraordinary regenerative abilities that are driven by stem cells maintained throughout their lives. In this issue of Cell, Zeng et al. report the prospective isolation of planarian pluripotent stem cells. Their work opens new directions for understanding how these remarkable cells are established, maintained, and activated.

A confocal microscopy-based atlas of tissue architecture in the tapeworm Hymenolepis diminuta.

Tapeworms are pervasive and globally distributed parasites that infect millions of humans and livestock every year, and are the causative agents of two of the 17 neglected tropical diseases prioritized by the World Health Organization. Studies of tapeworm biology and pathology are often encumbered by the complex life cycles of disease-relevant tapeworm species that infect hosts such as foxes, dogs, cattle, pigs, and humans. Thus, studies of laboratory models can help overcome the practical, ethical, and cost-related difficulties faced by tapeworm parasitologists. The rat intestinal tapeworm Hymenolepis diminuta is easily reared in the laboratory and has the potential to enable modern molecular-based experiments that will greatly contribute to our understanding of multiple aspects of tapeworm biology, such as growth and reproduction. As part of our efforts to develop molecular tools for experiments on H. diminuta, we have characterized a battery of lectins, antibodies, and common stains that label different tapeworm tissues and organ structures. Using confocal microscopy, we have assembled an "atlas" of H. diminuta organ architecture that will be a useful resource for helminthologists. The methodologies we describe will facilitate characterization of loss-of-function perturbations using H. diminuta. This toolkit will enable a greater understanding of fundamental tapeworm biology that may elucidate new therapeutic targets toward the eradication of these parasites.

Diverse functions of kindlin/fermitin proteins during embryonic development in Xenopus laevis.

The kindlin/fermitin family includes three proteins involved in regulating integrin ligand-binding activity and adhesion. Loss-of-function mutations in kindlins1 and 3 have been implicated in Kindler Syndrome and Leukocyte Adhesion Deficiency III (LAD-III) respectively, whereas kindlin2 null mice are embryonic lethal. Post translational regulation of cell-cell and cell-ECM adhesion has long been presumed to be important for morphogenesis, however, few specific examples of activation-dependent changes in adhesion molecule function in normal development have been reported. In this study, antisense morpholinos were used to reduce expression of individual kindlins in Xenopus laevis embryos in order to investigate their roles in early development. Kindlin1 knockdown resulted in developmental delays, gross malformations of the gut and eventual lethality by tadpole stages. Kindlin2 morphant embryos displayed late stage defects in vascular maintenance and angiogenic branching consistent with kindlin2 loss of function in the mouse. Antisense morpholinos were also used to deplete maternal kindlin2 protein in oocytes and eggs. Embryos lacking maternal kindlin2 arrested at early cleavage stages due to failures in cytokinesis. Kindlin3 morphant phenotypes included defects in epidermal ciliary beating and partial paralysis at tailbud stages but these embryos recovered eventually as morpholino levels decayed. These results indicate a remarkably diverse range of kindlin functions in vertebrate development.

H2A.Z (Htz1) controls the cell-cycle-dependent establishment of transcriptional silencing at Saccharomyces cerevisiae telomeres.

The establishment of transcriptional silencing in Saccharomyces cerevisiae requires progression through the cell cycle. We have previously found that transit through M-phase is necessary and sufficient to establish silencing at telomeres following induction of the Sir3 silencing factor. In this study we find that halting cell-cycle progression in either G(1) or at the beginning of M-phase limits the ability of Sir3 to associate with a telomere-linked reporter gene and prevents the changes in histone modifications associated with gene repression. Deletion of genes coding for the histone variant H2A.Z (Htz1 in yeast) and histone acetyltransferase Sas2 abolish the cell-cycle progression requirement for the establishment of silencing. Cells blocked in telophase (but not at metaphase) are also able to establish silencing. We show that H2A.Z binds to the promoter of our telomere-linked reporter gene and that this binding diminishes in silenced cells. Finally, we observe a specific displacement of H2A.Z from chromatin in telophase-blocked cells, regardless of the silencing status of the reporter gene. These results suggest that the requirement for M-phase in the establishment of silencing may reflect a cell-cycle regulated relaxation of heterochromatin barriers.

The extracellular matrix in development and morphogenesis: a dynamic view.

The extracellular matrix (ECM) is synthesized and secreted by embryonic cells beginning at the earliest stages of development. Our understanding of ECM composition, structure and function has grown considerably in the last several decades and this knowledge has revealed that the extracellular microenvironment is critically important for cell growth, survival, differentiation and morphogenesis. ECM and the cellular receptors that interact with it mediate both physical linkages with the cytoskeleton and the bidirectional flow of information between the extracellular and intracellular compartments. This review considers the range of cell and tissue functions attributed to ECM molecules and summarizes recent findings specific to key developmental processes. The importance of ECM as a dynamic repository for growth factors is highlighted along with more recent studies implicating the 3-dimensional organization and physical properties of the ECM as it relates to cell signaling and the regulation of morphogenetic cell behaviors. Embryonic cell and tissue generated forces and mechanical signals arising from ECM adhesion represent emerging areas of interest in this field.

The physical state of fibronectin matrix differentially regulates morphogenetic movements in vivo.

This study demonstrates that proper spatiotemporal expression and the physical assembly state of fibronectin (FN) matrix play key roles in the regulation of morphogenetic cell movements in vivo. We examine the progressive assembly and 3D fibrillar organization of FN and its role in regulating cell and tissue movements in Xenopus embryos. Expression of the 70 kD N-terminal fragment of FN blocks FN fibril assembly at gastrulation but not initial FN binding to integrins at the cell surface. We find that fibrillar FN is necessary to maintain cell polarity through oriented cell division and to promote epiboly, possibly through maintenance of tissue-surface tension. In contrast, FN fibrils are dispensable for convergence and extension movements required for axis elongation. Closure of the migratory mesendodermal mantle was accelerated in the absence of a fibrillar matrix. Thus, the macromolecular assembly of FN matrices may constitute a general regulatory mechanism for coordination of distinct morphogenetic movements.

Isolation and characterization of conditional alleles of the yeast SIR2 gene.

Sir2 is a protein deacetylase that mediates transcriptional silencing at the HM loci, telomeres, and rDNA repeats in yeast. To identify functionally significant regions of the Sir2 protein, we have characterized two types of mutations. First, we used random mutagenesis to create temperature-sensitive alleles of the SIR2 gene. Mutations conferring conditional silencing can be isolated throughout the SIR2 gene, causing both enzymatic and protein interaction defects. We used external deletions to identify regions essential for silencing in the non-conserved regions of Sir2. Deletions of the Sir2 N-terminal 89 amino acid residues caused a subtle increase in silencing, while deletions encompassing residues 110-146 caused loss of Sir2 interactions with both Sir4 and Net1. This loss of protein interaction correlates with a loss of Sir2-mediated silencing, and is consistent with a model in which Net1 and Sir4 compete for interaction with Sir2. These results indicate that recognition of the binding partners of Sir2 is a key function of non-conserved sequences.