Development Of Somatic Tissues Research Articles

Regulation of retrotransposons in <i>Drosophila melanogaster</i> somatic tissues

Regulation of retrotransposon activity in somatic tissues is a complex mechanism that is still not studied in details. It is strongly believed that siRNA interference is main mechanism of retrotransposon activity regulation outside the gonads, but recently was demonstrated that piRNA interference participates in retrotransposon repression during somatic tissue development. In this work, using RT-PCR, we demonstrated that during ontogenesis piRNA interference determinates retrotransposon expression level on imago stage and retrotransposons demonstrate tissue-specific expression. The major factor of retrotransposon tissue-specific expression is presence of transcription factor binding sites in their regulatory regions.

A cascade of bHLH-regulated pathways programs maize anther development.

The spatiotemporal development of somatic tissues of the anther lobe is necessary for successful fertile pollen production. This process is mediated by many transcription factors acting through complex, multi-layered networks. Here, our analysis of functional knockout mutants of interacting basic helix-loop-helix genes Ms23, Ms32, basic helix-loop-helix 122 (bHLH122), and bHLH51 in maize (Zea mays) established that male fertility requires all four genes, expressed sequentially in the tapetum (TP). Not only do they regulate each other, but also they encode proteins that form heterodimers that act collaboratively to guide many cellular processes at specific developmental stages. MS23 is confirmed to be the master factor, as the ms23 mutant showed the earliest developmental defect, cytologically visible in the TP, with the most drastic alterations in premeiotic gene expression observed in ms23 anthers. Notably, the male-sterile ms23, ms32, and bhlh122-1 mutants lack 24-nt phased secondary small interfering RNAs (phasiRNAs) and the precursor transcripts from the corresponding 24-PHAS loci, while the bhlh51-1 mutant has wild-type levels of both precursors and small RNA products. Multiple lines of evidence suggest that 24-nt phasiRNA biogenesis primarily occurs downstream of MS23 and MS32, both of which directly activate Dcl5 and are required for most 24-PHAS transcription, with bHLH122 playing a distinct role in 24-PHAS transcription.

52 Ovarian Follicular Profiling of High- and Low-immunocrit Gilts on Postnatal Day 14

Abstract Colostrum intake by neonatal piglets can be measured using the immunoglobulin immunocrit assay (iCrit). Lactocrine effects occur when maternally derived, milk-borne bioactive factors are transferred to the neonatal circulation with consumption of colostrum during nursing and affect development of somatic tissues, which can have long-term consequences in adulthood. Lactocrine deficiency, indicated by low neonatal iCrit, altered uterine gene expression and reduced fecundity in adult, neonatally lactocrine-deficient gilts. Litter size in pigs is dependent on both ovarian and uterine function. It was hypothesized that lactocrine deficiency affects development of ovarian follicles in gilts. The objective was to determine the number of primordial, primary, and secondary follicles in ovaries of gilts with high (12% ± 0.5; n = 10) or low (1.9% ± 0.4; n = 10) iCrit, determined on postnatal day (PND) 1 after birth. Paired high- and low-iCrit gilts were chosen from the same litters (birth weight; 3.1 ± 0.2 lbs). On PND 14, ovaries were collected and histological sections prepared (3 sections per animal; 30–150 µm apart). Ovarian follicles in each section were staged and the number of follicles in each category were quantified and subjected to ANOVA. Total number of ovarian follicles did not differ with iCrit (P = 0.55; 1,370.6 ± 147.8 follicles per section). The proportion of primordial, primary, and secondary follicles was 89.6 ± 1.15%, 7.7 ± 0.87%, 2.7 ± 0.51%, respectively. The number of primordial (P = 0.55), primary (P = 0.64), and secondary (P = 0.93) follicles did not differ with iCrit. Results indicate that ovarian follicular development of neonatal gilts is not sensitive to immunocrit status. Although lactocrine deficiency did not influence the ovarian follicular profile at PND 14, it remains unknown whether lactocrine programming alters ovarian follicular dynamics in neonatally lactocrine-deficient adults. USDA is an equal opportunity provider and employer.

Molecular cloning, characterization, mRNA expression changes and nucleocytoplasmic shuttling during kidney embryonic development of SOX9 in Alligator sinensis

SOX9 plays a crucial, extensive and conservative role in the process of somatic tissue development and adult regeneration through the positive self-regulation mediated by SOM across all vertebrates. In this study, we have cloned SOX9 from the kidney of hatchling Alligator sinensis. The full-length of SOX9 cDNA is 3878 bp with an open reading frame encoding 494 amino acids. Amino acid alignment analyses indicated that the SOX9 exhibit highly conserved functional domains. Using the droplet digital PCR, the mRNA abundances of SOX9 during nephrogenesis in A. sinensis showed prominent changes in the embryonic development, suggesting that SOX9 might combines a vital role in the regulation of complex renal development. Interestingly, we detected the nucleocytoplasmic shuttling of SOX9 protein using immunofluorescence, implying that nucleocytoplasmic shuttling is critical to the regulation of SOX9 in the renal embryonic development. Collectively, these data provide an important foundation for further studies on renal developmental biology and molecular biology of non-mammalian SOX9. Furthermore, it provides new insights into the phenomenon of SOX9 nucleocytoplasmic shuttling in Alligator sinensis, which is probably of great significance to the development of kidney metanephros embryo.

Mad1 influences interphase nucleoplasm organization and chromatin regulation in Drosophila.

The Drosophila Mad1 spindle checkpoint protein helps organize several nucleoplasmic components, and flies lacking Mad1 present changes in gene expression reflecting altered chromatin conformation. In interphase, checkpoint protein Mad1 is usually described as localizing to the inner nuclear envelope by binding the nucleoporin Tpr, an interaction believed to contribute to proper mitotic regulation. Whether Mad1 has other nuclear interphase functions is unknown. We found in Drosophila that Mad1 is present in nuclei of both mitotic and postmitotic tissues. Three proteins implicated in various aspects of chromatin organization co-immunoprecipitated with Mad1 from fly embryos: Mtor/Tpr, the SUMO peptidase Ulp1 and Raf2, a subunit of a Polycomb-like complex. In primary spermatocytes, all four proteins colocalized in a previously undescribed chromatin-associated structure called here a MINT (Mad1-containing IntraNuclear Territory). MINT integrity required all four proteins. In mad1 mutant spermatocytes, the other proteins were no longer confined to chromatin domains but instead dispersed throughout the nucleoplasm. mad1 flies also presented phenotypes indicative of excessive chromatin of heterochromatic character during development of somatic tissues. Together these results suggest that Drosophila Mad1, by helping organize its interphase protein partners in the nucleoplasm, contributes to proper chromatin regulation.

Parallel evolution of male germline epigenetic poising and somatic development in animals.

Changes in gene regulation frequently underlie changes in morphology during evolution, and differences in chromatin state have been linked with changes in anatomical structure and gene expression across evolutionary time. Here we assess the relationship between evolution of chromatin state in germ cells and evolution of gene regulatory programs governing somatic development. We examined the poised (H3K4me3/H3K27me3 bivalent) epigenetic state in male germ cells from five mammalian and one avian species. We find that core genes poised in germ cells from multiple amniote species are ancient regulators of morphogenesis that sit at the top of transcriptional hierarchies controlling somatic tissue development, whereas genes that gain poising in germ cells from individual species act downstream of core poised genes during development in a species-specific fashion. We propose that critical regulators of animal development gained an epigenetically privileged state in germ cells, manifested in amniotes by H3K4me3/H3K27me3 poising, early in metazoan evolution.

Dissecting the Roles of Polycomb Repressive Complex 2 Subunits in the Control of Skin Development

Polycomb repressive complex 2 (PRC2) is an essential regulator of cell physiology. Although there have been numerous studies on PRC2 function in somatic tissue development and stem cell control, these have focused on the loss of a single PRC2 subunit. Recent studies, however, have shown that PRC2 subunits may function independently of the PRC2 complex. To investigate the function of PRC2 in the control of skin development, we generated and analyzed three conditional knockout mouse lines, in which the essential PRC2 subunits embryonic ectoderm development (EED), suppressor of zeste 12 homolog (Suz12), and enhancer of zeste homologs 1 and 2 (Ezh1/2) are conditionally ablated in the embryonic epidermal progenitors that give rise to the epidermis, hair follicles, and Merkel cells. Our studies showed that the observed loss-of-function phenotypes are shared between the three knockouts, indicating that in the skin epithelium, EED, Suz12, and Ezh1/2 function largely as subunits of the PRC2 complex. Interestingly, the absence of PRC2 results in dramatically different phenotypes across the different skin lineages: premature acquisition of a functional epidermal barrier, formation of ectopic Merkel cells, and defective postnatal development of hair follicles. The strikingly different roles of PRC2 in the formation of three lineages exemplify the complex outcomes that the lack of PRC2 can have in a somatic stem cell system.

Activity of telomerase and telomeric length in Apis mellifera.

Telomerase is an enzyme that adds repeats of DNA sequences to the ends of chromosomes, thereby preventing their shortening. Telomerase activity is associated with proliferative status of cells, organismal development, and aging. We report an analysis of telomerase activity and telomere length in the honeybee, Apis mellifera. Telomerase activity was found to be regulated in a development and caste-specific manner. During the development of somatic tissues of larval drones and workers, telomerase activity declined to 10% of its level in embryos and remained low during pupal and adult stages but was upregulated in testes of late pupae, where it reached 70% of the embryo level. Upregulation of telomerase activity was observed in the ovaries of late pupal queens, reaching 160% of the level in embryos. Compared to workers and drones, queens displayed higher levels of telomerase activity. In the third larval instar of queens, telomerase activity reached the embryo level, and an enormous increase was observed in adult brains of queens, showing a 70-fold increase compared to a brain of an adult worker. Southern hybridization of terminal TTAGG fragments revealed a high variability of telomeric length between different individuals, although the same pattern of hybridization signals was observed in different tissues of each individual.

A far-upstream (−70 kb) enhancer mediates Sox9 auto-regulation in somatic tissues during development and adult regeneration

SOX9 encodes a transcription factor that presides over the specification and differentiation of numerous progenitor and differentiated cell types, and although SOX9 haploinsufficiency and overexpression cause severe diseases in humans, including campomelic dysplasia, sex reversal and cancer, the mechanisms underlying SOX9 transcription remain largely unsolved. We identify here an evolutionarily conserved enhancer located 70-kb upstream of mouse Sox9 and call it SOM because it specifically activates a Sox9 promoter reporter in most Sox9-expressing somatic tissues in transgenic mice. Moreover, SOM-null fetuses and pups reduce Sox9 expression by 18–37% in the pancreas, lung, kidney, salivary gland, gut and liver. Weanlings exhibit half-size pancreatic islets and underproduce insulin and glucagon, and adults slowly recover from acute pancreatitis due to a 2-fold impairment in Sox9 upregulation. Molecular and genetic experiments reveal that Sox9 protein dimers bind to multiple recognition sites in the SOM sequence and are thereby both necessary and sufficient for enhancer activity. These findings thus uncover that Sox9 directly enhances its functions in somatic tissue development and adult regeneration through SOM-mediated positive auto-regulation. They provide thereby novel insights on molecular mechanisms controlling developmental and disease processes and suggest new strategies to improve disease treatments.

Dynamics of 5-Hydroxymethylcytosine and Chromatin Marks in Mammalian Neurogenesis

DNA methylation in mammals is highly dynamic during germ cell and preimplantation development but is relatively static during the development of somatic tissues. 5-hydroxymethylcytosine (5hmC), created by oxidation of 5-methylcytosine (5mC) by Tet proteins and most abundant in the brain, is thought to be an intermediary toward 5mC demethylation. We investigated patterns of 5mC and 5hmC during neurogenesis in the embryonic mouse brain. 5hmC levels increase during neuronal differentiation. In neuronal cells, 5hmC is not enriched at enhancers but associates preferentially with gene bodies of activated neuronal function-related genes. Within these genes, gain of 5hmC is often accompanied by loss of H3K27me3. Enrichment of 5hmC is not associated with substantial DNA demethylation, suggesting that 5hmC is a stable epigenetic mark. Functional perturbation of the H3K27 methyltransferase Ezh2 or of Tet2 and Tet3 leads to defects in neuronal differentiation, suggesting that formation of 5hmC and loss of H3K27me3 cooperate to promote brain development.

The nucleoporin Seh1 forms a complex with Mio and serves an essential tissue-specific function in Drosophila oogenesis

The nuclear pore complex (NPC) mediates the transport of macromolecules between the nucleus and cytoplasm. Recent evidence indicates that structural nucleoporins, the building blocks of the NPC, have a variety of unanticipated cellular functions. Here, we report an unexpected tissue-specific requirement for the structural nucleoporin Seh1 during Drosophila oogenesis. Seh1 is a component of the Nup107-160 complex, the major structural subcomplex of the NPC. We demonstrate that Seh1 associates with the product of the missing oocyte (mio) gene. In Drosophila, mio regulates nuclear architecture and meiotic progression in early ovarian cysts. Like mio, seh1 has a crucial germline function during oogenesis. In both mio and seh1 mutant ovaries, a fraction of oocytes fail to maintain the meiotic cycle and develop as pseudo-nurse cells. Moreover, the accumulation of Mio protein is greatly diminished in the seh1 mutant background. Surprisingly, our characterization of a seh1 null allele indicates that, although required in the female germline, seh1 is dispensable for the development of somatic tissues. Our work represents the first examination of seh1 function within the context of a multicellular organism. In summary, our studies demonstrate that Mio is a novel interacting partner of the conserved nucleoporin Seh1 and add to the growing body of evidence that structural nucleoporins can have novel tissue-specific roles.

Segregation of mtDNA throughout human embryofetal development: m.3243A>G as a model system

Mitochondrial DNA (mtDNA) mutations cause a wide range of serious diseases with high transmission risk and maternal inheritance. Tissue heterogeneity of the heteroplasmy rate (“mutant load”) accounts for the wide phenotypic spectrum observed in carriers. Owing to the absence of therapy, couples at risk to transmit such disorders commonly ask for prenatal (PND) or preimplantation diagnosis (PGD). The lack of data regarding heteroplasmy distribution throughout intrauterine development, however, hampers the implementation of such procedures. We tracked the segregation of the m.3243A > G mutation (MT-TL1 gene) responsible for the MELAS syndrome in the developing embryo/fetus, using tissues and cells from eight carrier females, their 38 embryos and 12 fetuses. Mutant mtDNA segregation was found to be governed by random genetic drift, during oogenesis and somatic tissue development. The size of the bottleneck operating for m.3243A > G during oogenesis was shown to be individual-dependent. Comparison with data we achieved for the m.8993T > G mutation (MT-ATP6 gene), responsible for the NARP/Leigh syndrome, indicates that these mutations differentially influence mtDNA segregation during oogenesis, while their impact is similar in developing somatic tissues. These data have major consequences for PND and PGD procedures in mtDNA inherited disorders. Hum Mutat 32:116–125, 2011. © 2010 Wiley-Liss, Inc.

Comparative functional genomics of mammalian DNA methyltransferases

DNA methylation involves biochemical modification of DNA by addition of methyl groups onto CpG dinucleotides, and this epigenetic mechanism regulates gene expression in disease and development. Mammalian DNA methyltransferases, DNMT (DNMT1, DNMT3A and DNMT3B), together with the accessory protein DNMT3L establish specific DNA methylation patterns in the genome during gametogenesis, embryogenesis and somatic tissue development. The present study addresses the structural and functional conservation of the DNMT in humans, mice and cattle and the patterns of mRNA abundance of the different enzymes during embryogenesis to improve understanding of epigenetic regulation in early development. The findings showed a high degree of structural and functional conservation among the human, mouse, and bovine DNMT. The results also showed similar patterns of transcript abundance for all of the proteins at different stages of early embryo development. Remarkably, all of the DNMT with an important role in DNA methylation (DNMT1, DNMT3A, DNMT3B, and DNMT3L) show a greater degree of structural similarity between human and bovine than that between human and mouse. These results have important implications for the selection of an appropriate model for study of DNA methylation during early development in humans.

The Layered Structure of Human Mitochondrial DNA Nucleoids

Mitochondrial DNA (mtDNA) occurs in cells in nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde cross-linking was performed to determine which nucleoid proteins are in close contact with the mtDNA. A set of core nucleoid proteins is found in both native and cross-linked nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native nucleoids, including ATAD3, were not observed to cross-link to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA nucleoids in which replication and transcription occur in the central core, whereas translation and complex assembly may occur in the peripheral region.

Molecules that function in the steps of fertilization

Fertilization, the union of mature male and female gametes, requires a precise series of cell–extracellular matrix and cell–cell interactions. It is therefore not surprising that many of the molecules with key roles in fertilization have signaling or adhesive functions. In many cases, the molecular mechanisms of germ cell interactions have important parallels in somatic tissue development and function. Fig. 1 shows a generalized scheme for sperm–egg interactions divided into four sequential steps. After sperm have located the egg, they bind to the egg coat. This initial interaction is similar to the interactions that somatic cells have with their surrounding extracellular matrix. Sperm binding to the egg coat induces the exocytosis of the acrosomal vesicle (acrosome reaction). Sperm then penetrate the egg coat with the aid of enzymes formerly contained in the acrosomal vesicle as well as mechanical force generated by sperm motion. Once under the egg coat, sperm bind and fuse with the egg plasma membrane. This union activates the quiescent egg to commit its stored resources to the developmental program of a new individual. It should be noted that the events depicted in Fig. 1 will vary considerably in detail or may be completely absent in the fertilization of different species. In this review, we examine each of the steps of fertilization with an emphasis on the underlying molecular mechanisms. This is not meant to be an exhaustive review of fertilization model systems and implicated proteins. Rather, we survey the most well studied molecules of fertilization. For more extensive reviews or details on specific species see Yanagimachi [1], Vacquier [2] and Wassarman [3,4]. Most of what is currently know about fertilization comes from studies with marine invertebrates and mammals because of the ease of collecting gametes or the relevance to human fertility. However, important advances in our understanding of fertilization have also come from diverse systems such as green alga, flies, worms and frogs [5–8]. In fact, genetic approaches to the study of sperm–egg interactions in systems such as Drosophila and C. elegans promise to rapidly add to the list of molecules that mediate fertilization [6,7].

Differential expression of nuclear lamin proteins during chicken development.

By immunocytochemistry, quantitative immunoblotting, and two-dimensional gel electrophoresis, we have analyzed the distribution of nuclear lamin proteins during chicken embryonic development. Whereas no qualitative differences in the patterns of expression of lamins A, B1, and B2 were observed during gametogenesis in either the female or the male germ line, profound changes in the composition of the nuclear lamina occurred during the development of somatic tissues. Most unexpectedly, early chicken embryos were found to contain little if any lamin A, although they contained substantial amounts of lamins B1 and B2. During embryonic development, lamin A became increasingly prominent, whereas the amounts of lamin B1 decreased in many tissues. Interestingly, the extent and the developmental timing of these changes displayed pronounced tissue-specific variations. Lamin B2 was expressed in fairly constant amounts in all cell types investigated (except for pachytene-stage germ cells). These results have implications for the purported functional specializations of individual lamin proteins. In addition, they suggest that alterations in the composition of the nuclear lamina may be important for the establishment of cell- or tissue-specific differences in nuclear architecture.

The autonomous function of germ plasm in a somatic region of the Drosophila egg.

Abstract Posterior polar plasm from Binsc/B 6 Y cleavage embryos of Drosophila melanogaster was transferred to the mid-ventral region of mwh e 4 embryos of the same age. Between 15 min and 5 h after cytoplasmic injections, embryos were analyzed at the electron microscopic level for the formation of cells with implanted cytoplasm. Characteristic subcellular organelles, the polar granules, which are exclusively located at the posterior pole of the egg, served as a morphological marker to identify the transferred cytoplasm. The appearance of round nuclear bodies, which are only present in nuclei of cells containing posterior cytoplasm, the so-called pole cells, was used as a further criterion for successful transfers. Polar granules and nuclear bodies could be found in eight and ten cells in two of the seven embryos and analyzed at the blastoderm stage. Experimentally induced pole cells were inserted among pole cells of y sn 3 mal blastoderm hosts in order to determine if they could function normally and give rise to offspring. About 3% of the flies, which were derived from these embryos and mated to mwh e 4 partners, had y sn 3 mal germ cells as well as mwh e 4 ones. In addition, germ-line mosaicism in these flies could be “visualized” by testing histochemically for aldehyde oxidase activity. Mid-ventral blastoderm cells when transferred from non-injected mwh e 4 embryos to the posterior pole of y sn 3 mal blastoderm embryos did not differentiate into germ cells but instead participated in the development of somatic tissues of the hosts. To find out if a pole cell experimentally induced at the mid-ventral region of the egg can be incorporated into the germ line, normal mwh e 4 pole cells were implanted into this area of y sn 3 mal blastoderm hosts. About 5% of the fertile flies were found to have a mosaic germ line. Our results show (1) that cytoplasm of the posterior region of the Drosophila egg contains germ-cell determinants which function autonomously after transplantation to a somatic, i.e. mid-ventral region of the egg; (2) that pole cells either formed in or transferred to this ectopic region can still reach the gonads and contribute to the germ line.