Molecular mechanisms of tubulogenesis revealed in the sea star hydro-vascular organ.

A fundamental goal in the organogenesis field is to understand how cells organize into tubular shapes. Toward this aim, we have established the hydro-vascular organ in the sea star Patiria miniata as a model for tubulogenesis. In this animal, bilateral tubes grow out from the tip of the developing gut, and precisely extend to specific sites in the larva. This growth involves cell migration coupled with mitosis in distinct zones. Cell proliferation requires FGF signaling, whereas the three-dimensional orientation of the organ depends on Wnt signaling. Specification and maintenance of tube cell fate requires Delta/Notch signaling. Moreover, we identify target genes of the FGF pathway that contribute to tube morphology, revealing molecular mechanisms for tube outgrowth. Finally, we report that FGF activates the Six1/2 transcription factor, which serves as an evolutionarily ancient regulator of branching morphogenesis. This study uncovers distinct mechanisms of tubulogenesis in vivo and we propose that cellular dynamics in the sea star hydro-vascular organ represents a key comparison for understanding the evolution of vertebrate organs.

Use of Echinoderm Gametes and Early Embryos for Studying Meiosis and Mitosis.

The early embryos of sea urchins and other echinoderms have served as experimental models for the study of cell division since the nineteenth century. Their rapid development, optical clarity, and ease of manipulation continue to offer advantages for studying spindle assembly and cytokinesis. In the absence of transgenic lines, alternative strategies must be employed to visualize microtubules and actin. Here, we describe methods to visualize actin and microtubule using either purified, recombinant proteins, or probes in in vitro-transcribed mRNAs.

A conserved node in the regulation of Vasa between an induced and an inherited program of primordial germ cell specification.

Primordial germ cells (PGCs) are specified by diverse mechanisms in early development. In some animals, PGCs are specified via inheritance of maternal determinants, while in others, in a process thought to represent the ancestral mode, PGC fate is induced by cell interactions. Although the terminal factors expressed in specified germ cells are widely conserved, the mechanisms by which these factors are regulated can be widely diverse. Here we show that a post-translational mechanism of germ cell specification is conserved between two echinoderm species thought to employ divergent germ line segregation strategies. Sea urchins segregate their germ line early by an inherited mechanism. The DEAD-box RNA - helicase Vasa, a conserved germline factor, becomes enriched in the PGCs by degradation in future somatic cells by the E3-ubiquitin-ligase Gustavus (Gustafson et al., 2011). This post-translational activity occurs early in development, substantially prior to gastrulation. Here we test this process in germ cell specification of sea star embryos, which use inductive signaling mechanisms after gastrulation for PGC fate determination. We find that Vasa-GFP protein becomes restricted to the PGCs in the sea star even though the injected mRNA is present throughout the embryo. Gustavus depletion, however, results in uniform accumulation of the protein. These data demonstrate that Gustavus-mediated Vasa turnover in somatic cells is conserved between species with otherwise divergent PGC specification mechanisms. Since Gustavus was originally identified in Drosophila melanogaster to have similar functions in Vasa regulation (Kugler et al., 2010), we conclude that this node of Vasa regulation in PGC formation is ancestral and evolutionarily transposable from the ancestral, induced PGC specification program to an inherited PGC specification mechanism.

Polarized Dishevelled dissolution and reassembly drives embryonic axis specification in sea star oocytes.

The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes.1 However, the mechanisms that generate cellular asymmetries within the oocyte and then transduce that polarity to organismal scale body axes are poorly understood outside of select model organisms. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled (Dvl) is a cytoplasmic Wnt pathway effector required for axis development in diverse species,2-4 but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dvl localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-reassembly mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially regulated Dvl dynamics.

Selective dephosphorylation by PP2A-B55 directs the meiosis I-meiosis II transition in oocytes.

Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in synchronized sea star oocytes from prophase I through the first embryonic cleavage. Although we found that protein levels were broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We found that the phosphatase PP2A-B55 is reactivated at the meiosis I/meiosis II (MI/MII) transition, resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI/MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after MI. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions.

Quiescent Cells Actively Replenish CENP-A Nucleosomes to Maintain Centromere Identity and Proliferative Potential.

Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant centromere protein A (CENP-A). Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential.

Distinct transcriptional regulation of Nanos2 in the germ line and soma by the Wnt and delta/notch pathways.

Specification of the primordial germ cells (PGCs) is essential for sexually reproducing animals. Although the mechanisms of PGC specification are diverse between organisms, the RNA binding protein Nanos is consistently required in the germ line in all species tested. How Nanos is selectively expressed in the germ line, however, remains largely elusive. We report that in sea urchin embryos, the early expression of Nanos2 in the PGCs requires the maternal Wnt pathway. During gastrulation, however, Nanos2 expression expands into adjacent somatic mesodermal cells and this secondary Nanos expression instead requires Delta/Notch signaling through the forkhead family member FoxY. Each of these transcriptional regulators were tested by chromatin immunoprecipitation analysis and found to directly interact with a DNA locus upstream of Nanos2. Given the conserved importance of Nanos in germ line specification, and the derived character of the micromeres and small micromeres in the sea urchin, we propose that the ancestral mechanism of Nanos2 expression in echinoderms was by induction in mesodermal cells during gastrulation.

Transient translational quiescence in primordial germ cells.

Stem cells in animals often exhibit a slow cell cycle and/or low transcriptional activity referred to as quiescence. Here, we report that the translational activity in the primordial germ cells (PGCs) of the sea urchin embryo (Strongylocentrotus purpuratus) is quiescent. We measured new protein synthesis with O-propargyl-puromycin and L-homopropargylglycine Click-iT technologies, and determined that these cells synthesize protein at only 6% the level of their adjacent somatic cells. Knockdown of translation of the RNA-binding protein Nanos2 by morpholino antisense oligonucleotides, or knockout of the Nanos2 gene by CRISPR/Cas9 resulted in a significant, but partial, increase (47%) in general translation specifically in the PGCs. We found that the mRNA of the translation factor eEF1A is excluded from the PGCs in a Nanos2-dependent manner, a consequence of a Nanos/Pumilio response element (PRE) in its 3'UTR. In addition to eEF1A, the cytoplasmic pH of the PGCs appears to repress translation and simply increasing the pH also significantly restores translation selectively in the PGCs. We conclude that the PGCs of this sea urchin institute parallel pathways to quiesce translation thoroughly but transiently.

Germ Line Versus Soma in the Transition from Egg to Embryo.

With few exceptions, all animals acquire the ability to produce eggs or sperm at some point in their life cycle. Despite this near-universal requirement for sexual reproduction, there exists an incredible diversity in germ line development. For example, animals exhibit a vast range of differences in the timing at which the germ line, which retains reproductive potential, separates from the soma, or terminally differentiated, nonreproductive cells. This separation may occur during embryonic development, after gastrulation, or even in adults, depending on the organism. The molecular mechanisms of germ line segregation are also highly diverse, and intimately intertwined with the overall transition from a fertilized egg to an embryo. The earliest embryonic stages of many species are largely controlled by maternally supplied factors. Later in development, patterning control shifts to the embryonic genome and, concomitantly with this transition, the maternally supplied factors are broadly degraded. This chapter attempts to integrate these processes--germ line segregation, and how the divergence of germ line and soma may utilize the egg to embryo transitions differently. In some embryos, this difference is subtle or maybe lacking altogether, whereas in other embryos, this difference in utilization may be a key step in the divergence of the two lineages. Here, we will focus our discussion on the echinoderms, and in particular the sea urchins, in which recent studies have provided mechanistic understanding in germ line determination. We propose that the germ line in sea urchins requires an acquisition of maternal factors from the egg and, when compared to other members of the taxon, this appears to be a derived mechanism. The acquisition is early--at the 32-cell stage--and involves active protection of maternal mRNAs, which are instead degraded in somatic cells with the maternal-to-embryonic transition. We collectively refer to this model as the Time Capsule method for germ line determination.

Biaxial mechanical characterization of bat wing skin.

The highly flexible and stretchable wing skin of bats, together with the skeletal structure and musculature, enables large changes in wing shape during flight. Such compliance distinguishes bat wings from those of all other flying animals. Although several studies have investigated the aerodynamics and kinematics of bats, few have examined the complex histology and mechanical response of the wing skin. This work presents the first biaxial characterization of the local deformation, mechanical properties, and fiber kinematics of bat wing skin. Analysis of these data has provided insight into the relationships among the structural morphology, mechanical properties, and functionality of wing skin. Large spatial variations in tissue deformation and non-negligible fiber strains in the cross-fiber direction for both chordwise and spanwise fibers indicate fibers should be modeled as two-dimensional elements. The macroscopic constitutive behavior was anisotropic and nonlinear, with very low spanwise and chordwise stiffness (hundreds of kilopascals) in the toe region of the stress-strain curve. The structural arrangement of the fibers and matrix facilitates a low energy mechanism for wing deployment and extension, and we fabricate examples of skins capturing this mechanism. We propose a comprehensive deformation map for the entire loading regime. The results of this work underscore the importance of biaxial field approaches for soft heterogeneous tissue, and provide a foundation for development of bio-inspired skins to probe the effects of the wing skin properties on aerodynamic performance.

Deadenylase depletion protects inherited mRNAs in primordial germ cells.

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.

Membrane muscle function in the compliant wings of bats.

Unlike flapping birds and insects, bats possess membrane wings that are more similar to many gliding mammals. The vast majority of the wing is composed of a thin compliant skin membrane stretched between the limbs, hand, and body. Membrane wings are of particular interest because they may offer many advantages to micro air vehicles. One critical feature of membrane wings is that they camber passively in response to aerodynamic load, potentially allowing for simplified wing control. However, for maximum membrane wing performance, tuning of the membrane structure to aerodynamic conditions is necessary. Bats possess an array of muscles, the plagiopatagiales proprii, embedded within the wing membrane that could serve to tune membrane stiffness, or may have alternative functions. We recorded the electromyogram from the plagiopatagiales proprii muscles of Artibeus jamaicensis, the Jamaican fruit bat, in flight at two different speeds and found that these muscles were active during downstroke. For both low- and high-speed flight, muscle activity increased between late upstroke and early downstroke and decreased at late downstroke. Thus, the array of plagiopatagiales may provide a mechanism for bats to increase wing stiffness and thereby reduce passive membrane deformation. These muscles also activate in synchrony, presumably as a means to maximize force generation, because each muscle is small and, by estimation, weak. Small differences in activation timing were observed when comparing low- and high-speed flight, which may indicate that bats modulate membrane stiffness differently depending on flight speed.

Isolating specific embryonic cells of the sea urchin by FACS.

Isolating cells based on specific gene expression enables a focused biochemical and molecular analysis. While cultured cells and hematopoietic cells, for example, are routinely isolated by fluorescence activated cell sorting (FACS), early embryonic cells are a relatively untapped source for FACS applications often because the embryos of many animals are quite limiting. Furthermore, many applications require genetic model organisms in which cells can be labeled by fluorescent transgenes, or antibodies against cell surface antigens. Here we define conditions in the sea urchin embryo for isolation of embryonic cells based on expression of specific proteins. We use the sea urchin embryo for which a nearly unlimited supply of embryonic cells is available and demonstrate the conditions for separation of the embryo into single cells, fixation of the cells for antibody penetration into the cells, and conditions for FACS of a rare cell type in the embryo. This protocol may be adapted for analysis of mRNA, chromatin, protein, or carbohydrates and depends only on the probe availability for the cell of interest. We anticipate that this protocol will be broadly applicable to embryos of other species.

The biology of the germ line in echinoderms.

The formation of the germ line in an embryo marks a fresh round of reproductive potential. The developmental stage and location within the embryo where the primordial germ cells (PGCs) form, however, differs markedly among species. In many animals, the germ line is formed by an inherited mechanism, in which molecules made and selectively partitioned within the oocyte drive the early development of cells that acquire this material to a germ-line fate. In contrast, the germ line of other animals is fated by an inductive mechanism that involves signaling between cells that directs this specialized fate. In this review, we explore the mechanisms of germ-line determination in echinoderms, an early-branching sister group to the chordates. One member of the phylum, sea urchins, appears to use an inherited mechanism of germ-line formation, whereas their relatives, the sea stars, appear to use an inductive mechanism. We first integrate the experimental results currently available for germ-line determination in the sea urchin, for which considerable new information is available, and then broaden the investigation to the lesser-known mechanisms in sea stars and other echinoderms. Even with this limited insight, it appears that sea stars, and perhaps the majority of the echinoderm taxon, rely on inductive mechanisms for germ-line fate determination. This enables a strongly contrasted picture for germ-line determination in this phylum, but one for which transitions between different modes of germ-line determination might now be experimentally addressed.

Notch and Nodal control forkhead factor expression in the specification of multipotent progenitors in sea urchin.

Indirect development, in which embryogenesis gives rise to a larval form, requires that some cells retain developmental potency until they contribute to the different tissues in the adult, including the germ line, in a later, post-embryonic phase. In sea urchins, the coelomic pouches are the major contributor to the adult, but how coelomic pouch cells (CPCs) are specified during embryogenesis is unknown. Here we identify the key signaling inputs into the CPC specification network and show that the forkhead factor foxY is the first transcription factor specifically expressed in CPC progenitors. Through dissection of its cis-regulatory apparatus we determine that the foxY expression pattern is the result of two signaling inputs: first, Delta/Notch signaling activates foxY in CPC progenitors; second, Nodal signaling restricts its expression to the left side, where the adult rudiment will form, through direct repression by the Nodal target pitx2. A third signal, Hedgehog, is required for coelomic pouch morphogenesis and institution of laterality, but does not directly affect foxY transcription. Knockdown of foxY results in a failure to form coelomic pouches and disrupts the expression of virtually all transcription factors known to be expressed in this cell type. Our experiments place foxY at the top of the regulatory hierarchy underlying the specification of a cell type that maintains developmental potency.

A conserved germline multipotency program.

The germline of multicellular animals is segregated from somatic tissues, which is an essential developmental process for the next generation. Although certain ecdysozoans and chordates segregate their germline during embryogenesis, animals from other taxa segregate their germline after embryogenesis from multipotent progenitor cells. An overlapping set of genes, including vasa, nanos and piwi, operate in both multipotent precursors and in the germline. As we propose here, this conservation implies the existence of an underlying germline multipotency program in these cell types that has a previously underappreciated and conserved function in maintaining multipotency.

Bereavement; Silence and Culture within a Peer-Led HIV/AIDS-Prevention Strategy for Vulnerable Children in South Africa

In addressing the psychosocial effects of the HIV and AIDS pandemic among vulnerable children; the issue of bereavement appears inadequately addressed. Amid the global discourse on children orphaned and made vulnerable by HIV and AIDS; this paper explores how cultural contexts and social environments in South Africa shape children's experience of grief. The argument draws on a number of qualitative studies and uses empirical evidence from an evaluation of a peer-led HIV/AIDS-prevention strategy aimed at providing psychosocial support for 10- to 13-year-old South African children living in resource-poor communities. The paper reveals a central paradox regarding how the intervention's objective of talking about death and eliciting memories of deceased loved ones with young children is confounded by cultural practices located in notions of silence and the need to protect children. The paper acknowledges the `culture of silence' surrounding death in some African contexts; but concludes that peer-led strategies have the potential to naturally circumvent these cultural taboos; simultaneously creating a much-needed space for young children to cry and talk among themselves; even if remaining silent at home in the presence of adults