Developmentally programmed histone H3 expression regulates cellular plasticity at the parental-to-early embryo transition.

During metazoan development, the marked change in developmental potential from the parental germline to the embryo raises an important question regarding how the next life cycle is reset. As the basic unit of chromatin, histones are essential for regulating chromatin structure and function and, accordingly, transcription. However, the genome-wide dynamics of the canonical, replication-coupled (RC) histones during gametogenesis and embryogenesis remain unknown. In this study, we use CRISPR-Cas9-mediated gene editing in Caenorhabditis elegans to investigate the expression pattern and role of individual RC histone H3 genes and compare them to the histone variant, H3.3. We report a tightly regulated epigenome landscape change from the germline to embryos that are regulated through differential expression of distinct histone gene clusters. Together, this study reveals that a change from a H3.3- to H3-enriched epigenome during embryogenesis restricts developmental plasticity and uncovers distinct roles for individual H3 genes in regulating germline chromatin.

Epigenetic dynamics during germline development: insights from Drosophila and C. elegans.

Gametogenesis produces the only cell type within a metazoan that contributes both genetic and epigenetic information to the offspring. Extensive epigenetic dynamics are required to express or repress gene expression in a precise spatiotemporal manner. On the other hand, early embryos must be extensively reprogrammed as they begin a new life cycle, involving intergenerational epigenetic inheritance. Seminal work in both Drosophila and C. elegans has elucidated the role of various regulators of epigenetic inheritance, including (1) histones, (2) histone-modifying enzymes, and (3) small RNA-dependent epigenetic regulation in the maintenance of germline identity. This review highlights recent discoveries of epigenetic regulation during the stepwise changes of transcription and chromatin structure that takes place during germline stem cell self-renewal, maintenance of germline identity, and intergenerational epigenetic inheritance. Findings from these two species provide precedence and opportunity to extend relevant studies to vertebrates.

Human Marfan and Marfan-like Syndrome associated mutations lead to altered trafficking of the Type II TGFß receptor in Caenorhabditis elegans.

The transforming growth factor-ß (TGFß) family plays an important role in many developmental processes and when mutated often contributes to various diseases. Marfan syndrome is a genetic disease with an occurrence of approximately 1 in 5,000. The disease is caused by mutations in fibrillin, which lead to an increase in TGFß ligand activity, resulting in abnormalities of connective tissues which can be life-threatening. Mutations in other components of TGFß signaling (receptors, Smads, Schnurri) lead to similar diseases with attenuated phenotypes relative to Marfan syndrome. In particular, mutations in TGFß receptors, most of which are clustered at the C-terminal end, result in Marfan-like (MFS-like) syndromes. Even though it was assumed that many of these receptor mutations would reduce or eliminate signaling, in many cases signaling is active. From our previous studies on receptor trafficking in C. elegans, we noticed that many of these receptor mutations that lead to Marfan-like syndromes overlap with mutations that cause mis-trafficking of the receptor, suggesting a link between Marfan-like syndromes and TGFß receptor trafficking. To test this hypothesis, we introduced three of these key MFS and MFS-like mutations into the C. elegans TGFß receptor and asked if receptor trafficking is altered. We find that in every case studied, mutated receptors mislocalize to the apical surface rather than basolateral surface of the polarized intestinal cells. Further, we find that these mutations result in longer animals, a phenotype due to over-stimulation of the nematode TGFß pathway and, importantly, indicating that function of the receptor is not abrogated in these mutants. Our nematode models of Marfan syndrome suggest that MFS and MFS-like mutations in the type II receptor lead to mis-trafficking of the receptor and possibly provides an explanation for the disease, a phenomenon which might also occur in some cancers that possess the same mutations within the type II receptor (e.g. colon cancer).

Mutagenesis and Imaging Studies of BMP Signaling Mechanisms in C. elegans.

C. elegans has played a central role in the elucidation of the TGFß pathway over the last two decades. This is due to the high conservation of the pathway components and the power of genetic and cell biological approaches applied toward understanding how the pathway signals. In Subheading 3, we detail approaches to study the BMP branch of the TGFß pathway in C. elegans.

Protecting and Diversifying the Germline.

Gametogenesis represents the most dramatic cellular differentiation pathways in both female and male flies. At the genome level, meiosis ensures that diploid germ cells become haploid gametes. At the epigenome level, extensive changes are required to turn on and shut off gene expression in a precise spatiotemporally controlled manner. Research applying conventional molecular genetics and cell biology, in combination with rapidly advancing genomic tools have helped us to investigate (1) how germ cells maintain lineage specificity throughout their adult reproductive lifetime; (2) what molecular mechanisms ensure proper oogenesis and spermatogenesis, as well as protect genome integrity of the germline; (3) how signaling pathways contribute to germline-soma communication; and (4) if such communication is important. In this chapter, we highlight recent discoveries that have improved our understanding of these questions. On the other hand, restarting a new life cycle upon fertilization is a unique challenge faced by gametes, raising questions that involve intergenerational and transgenerational epigenetic inheritance. Therefore, we also discuss new developments that link changes during gametogenesis to early embryonic development-a rapidly growing field that promises to bring more understanding to some fundamental questions regarding metazoan development.

C. elegans SMA-10 regulates BMP receptor trafficking.

Signal transduction of the conserved transforming growth factor-ß (TGFß) family signaling pathway functions through two distinct serine/threonine transmembrane receptors, the type I and type II receptors. Endocytosis orchestrates the assembly of signaling complexes by coordinating the entry of receptors with their downstream signaling mediators. Recently, we showed that the C. elegans type I bone morphogenetic protein (BMP) receptor SMA-6, part of the TGFß family, is recycled through the retromer complex while the type II receptor, DAF-4 is recycled in a retromer-independent, ARF-6 dependent manner. From genetic screens in C. elegans aimed at identifying new modifiers of BMP signaling, we reported on SMA-10, a conserved LRIG (leucine-rich and immunoglobulin-like domains) transmembrane protein. It is a positive regulator of BMP signaling that binds to the SMA-6 receptor. Here we show that the loss of sma-10 leads to aberrant endocytic trafficking of SMA-6, resulting in its accumulation in distinct intracellular endosomes including the early endosome, multivesicular bodies (MVB), and the late endosome with a reduction in signaling strength. Our studies show that trafficking defects caused by the loss of sma-10 are not universal, but affect only a limited set of receptors. Likewise, in Drosophila, we find that the fly homolog of sma-10, lambik (lbk), reduces signaling strength of the BMP pathway, consistent with its function in C. elegans and suggesting evolutionary conservation of function. Loss of sma-10 results in reduced ubiquitination of the type I receptor SMA-6, suggesting a possible mechanism for its regulation of BMP signaling.

Symmetry from Asymmetry or Asymmetry from Symmetry?

The processes of DNA replication and mitosis allow the genetic information of a cell to be copied and transferred reliably to its daughter cells. However, if DNA replication and cell division were always performed in a symmetric manner, the result would be a cluster of tumor cells instead of a multicellular organism. Therefore, gaining a complete understanding of any complex living organism depends on learning how cells become different while faithfully maintaining the same genetic material. It is well recognized that the distinct epigenetic information contained in each cell type defines its unique gene expression program. Nevertheless, how epigenetic information contained in the parental cell is either maintained or changed in the daughter cells remains largely unknown. During the asymmetric cell division (ACD) of Drosophila male germline stem cells, our previous work revealed that preexisting histones are selectively retained in the renewed stem cell daughter, whereas newly synthesized histones are enriched in the differentiating daughter cell. We also found that randomized inheritance of preexisting histones versus newly synthesized histones results in both stem cell loss and progenitor germ cell tumor phenotypes, suggesting that programmed histone inheritance is a key epigenetic player for cells to either remember or reset cell fates. Here, we will discuss these findings in the context of current knowledge on DNA replication, polarized mitotic machinery, and ACD for both animal development and tissue homeostasis. We will also speculate on some potential mechanisms underlying asymmetric histone inheritance, which may be used in other biological events to achieve the asymmetric cell fates.

BMP signaling requires retromer-dependent recycling of the type I receptor.

The transforming growth factor ß (TGFß) superfamily of signaling pathways, including the bone morphogenetic protein (BMP) subfamily of ligands and receptors, controls a myriad of developmental processes across metazoan biology. Transport of the receptors from the plasma membrane to endosomes has been proposed to promote TGFß signal transduction and shape BMP-signaling gradients throughout development. However, how postendocytic trafficking of BMP receptors contributes to the regulation of signal transduction has remained enigmatic. Here we report that the intracellular domain of Caenorhabditis elegans BMP type I receptor SMA-6 (small-6) binds to the retromer complex, and in retromer mutants, SMA-6 is degraded because of its missorting to lysosomes. Surprisingly, we find that the type II BMP receptor, DAF-4 (dauer formation-defective-4), is retromer-independent and recycles via a distinct pathway mediated by ARF-6 (ADP-ribosylation factor-6). Importantly, we find that loss of retromer blocks BMP signaling in multiple tissues. Taken together, our results indicate a mechanism that separates the type I and type II receptors during receptor recycling, potentially terminating signaling while preserving both receptors for further rounds of activation.

Regulation of genes affecting body size and innate immunity by the DBL-1/BMP-like pathway in Caenorhabditis elegans.

BACKGROUND: Bone morphogenetic proteins (BMPs) are members of the conserved transforming growth factor beta (TGFbeta superfamily, and play many developmental and homeostatic roles. In C. elegans, a BMP-like pathway, the DBL-1 pathway, controls body size and is involved in innate immunity. How these functions are carried out, though, and what most of the downstream targets of this pathway are, remain unknown. RESULTS: We performed a microarray analysis and compared expression profiles of animals lacking the SMA-6 DBL-1 receptor, which decreases pathway signaling, with animals that overexpress DBL-1 ligand, which increases pathway signaling. Consistent with a role for DBL-1 in control of body size, we find positive regulation by DBL-1 of genes involved in physical structure, protein synthesis and degradation, and metabolism. However, cell cycle genes were mostly absent from our results. We also identified genes in a hedgehog-related pathway, which may comprise a secondary signaling pathway downstream of DBL-1 that controls body size. In addition, DBL-1 signaling up-regulates pro-innate immunity genes. We identified a reporter for DBL-1 signaling, which is normally repressed but is up-regulated when DBL-1 signaling is reduced. CONCLUSIONS: Our results indicate that body size in C. elegans is controlled in part by regulation of metabolic processes as well as protein synthesis and degradation. This supports the growing body of evidence that suggests cell size is linked to metabolism. Furthermore, this study discovered a possible role for hedgehog-related pathways in transmitting the BMP-like signal from the hypodermis, where the core DBL-1 pathway components are required, to other tissues in the animal. We also identified the up-regulation of genes involved in innate immunity, clarifying the role of DBL-1 in innate immunity. One of the highly regulated genes is expressed at very low levels in wild-type animals, but is strongly up-regulated in Sma/Mab mutants, making it a useful reporter for DBL-1/BMP-like signaling in C. elegans.

Correlations between local strains and tissue phenotypes in an experimental model of skeletal healing.

Defining how mechanical cues regulate tissue differentiation during skeletal healing can benefit treatment of orthopaedic injuries and may also provide insight into the influence of the mechanical environment on skeletal development. Different global (i.e., organ-level) mechanical loads applied to bone fractures or osteotomies are known to result in different healing outcomes. However, the local stimuli that promote formation of different skeletal tissues have yet to be established. Finite element analyses can estimate local stresses and strains but require many assumptions regarding tissue material properties and boundary conditions. This study used an experimental approach to investigate relationships between the strains experienced by tissues in a mechanically stimulated osteotomy gap and the patterns of tissue differentiation that occur during healing. Strains induced by the applied, global mechanical loads were quantified on the mid-sagittal plane of the callus using digital image correlation. Strain fields were then compared to the distribution of tissue phenotypes, as quantified by histomorphometry, using logistic regression. Significant and consistent associations were found between the strains experienced by a region of the callus and the tissue type present in that region. Specifically, the probability of encountering cartilage increased, and that of encountering woven bone decreased, with increasing octahedral shear strain and, to a lesser extent, maximum principal strain. Volumetric strain was the least consistent predictor of tissue type, although towards the end of the four-week stimulation timecourse, cartilage was associated with increasingly negative volumetric strains. These results indicate that shear strain may be an important regulator of tissue fate during skeletal healing.

Mechanical stimulation alters tissue differentiation and molecular expression during bone healing.

Further understanding of how mechanical cues modulate skeletal tissue differentiation can identify potential means of enhancing repair following injury or disease. Prior studies examined the effects of mechanical loading on osteogenesis, chondrogenesis, and fibrogenesis in an effort to enhance bony union. However, exploring how mechanical stimuli can divert the bone healing process towards formation of other mesenchymal tissues, as an endpoint, may elucidate new avenues for repair and regeneration of tissues such as cartilage and fibrous tissue. This study investigated the use of mechanical stimulation to promote cartilage rather than bone formation within an osteotomy. Our overall goal was to define skeletal tissue distribution and molecular expression patterns induced by the stimulation. Retired breeder Sprague-Dawley rats (n = 85) underwent production of a mid-diaphyseal, transverse femoral osteotomy followed by external fixation. Beginning on postoperative day 10 and continuing for 1, 2, or 4 weeks, a cyclic bending motion (+35 degrees/-25 degrees at 1 Hz) was applied in the sagittal plane for 15 min/day for 5 consecutive days/week. Control animals experienced continuous rigid fixation. Histological and molecular analyses indicated that stimulation substantially altered normal bone healing. Stimulated specimens exhibited an increase in cartilage volume over time, while control specimens demonstrated bony bridging. Stimulation induced upregulation of cartilage-related genes (COL2A1 and COL10A1) and downregulation of bone morphogenetic proteins (BMPs) -4, -6 and -7. However, BMP-3 was upregulated with stimulation. These findings illustrate that mechanical cues can selectively modulate osteogenesis and chondrogenesis in vivo, and suggest a potential basis for treatment regimens for injured or diseased cartilaginous tissues.

Oncogenic activities of metabotropic glutamate receptor 1 (Grm1) in melanocyte transformation.

Previously, we reported a transgenic mouse line, TG-3, that develops spontaneous melanoma with 100% penetrance. We demonstrated that ectopic expression of Grm1 in melanocytes was sufficient to induce melanoma in vivo. In this present study, the transforming properties of Grm1 in two cultured immortalized melanocytes were investigated. We showed that, in contrast to parental melanocytes, these Grm1-clones have lost their requirement of TPA supplement for proliferation and have acquired the ability to form colonies in semi-solid medium. Xenografts of these cells formed robust tumors in both immunodeficient nude and syngeneic mice with a short latency (3-5 days). The malignancy of these cells was demonstrated by angiogenesis and invasion to the muscle and the intestine. The requirement of Grm1 expression for the maintenance of transformation was demonstrated by an inducible siRNA system. Induction of expression of siRNA for Grm1 reduced the number of proliferating/viable cells in vitro and suppressed in vivo xenografted tumor growth in comparison with control. Taken together, these results showed that expression of exogeneously introduced Grm1 is sufficient to induce full transformation of immortalized melanocytes.

Mechanotransduction and fracture repair.

Fracture-healing is regulated in part by mechanical factors. Study of the processes by which the mechanical environment of a fracture modulates healing can yield new strategies for the treatment of bone injuries. This article focuses on several key unanswered questions in the study of mechanotransduction and fracture repair. These questions concern identifying the mechanical stimuli that promote bone-healing, defining the mechanisms that are involved in this process, and examining the potential for cross-talk between investigations of mechanotransduction in bone-healing and in healing of other mesenchymally derived tissues. Several approaches to obtain accurate estimates of the mechanical stimuli present within a fracture callus are proposed, and our current understanding of the mechanotransduction processes involved in bone-healing is reviewed. Further study of mechanotransduction mechanisms is needed in order to identify those that are most critical and active during the various phases of fracture repair. A better understanding of the effect of mechanical factors on bone-healing will also benefit the study of healing, regeneration, and engineering of other skeletal tissues.