Obox4 regulates the expression of histone family genes and promotes differentiation of mouse embryonic stem cells

Obox genes are preferentially expressed in the ovary, testis and oocyte, and play important roles in many developmental processes. In this study, we report that Obox4 and Obox6 are expressed in mouse embryonic stem cells (mESCs) and that Obox4 regulates histone family gene expression in mESCs. Obox4 protein expressing mESCs formed colonies with a flattened and irregular morphology, and exhibited decreased expression levels of self-renewal related proteins, such as Oct4 and Sox2, as well as reduced alkaline phosphatase activity. The results of microarray analysis and siRNA mediated knockdown experiments suggest that Obox4 is an upstream regulator of the histone gene family.     

Progenitors of skeletal muscle satellite cells express the muscle determination gene, MyoD

Satellite cells are tissue-specific stem cells responsible for skeletal muscle growth and regeneration. Although satellite cells were identified almost 50 years ago, the identity of progenitor populations from which they derive remains controversial. We developed MyoD^iCre knockin mice, and used Cre/lox lineage analysis to determine whether satellite cell progenitors express MyoD, a marker of myogenic commitment. Recombination status of satellite cells was determined by confocal microscopy of isolated muscle fibers and by electron microscopic observation of muscle tissue fixed immediately following isolation, using R26R-EYFP and R26R (β-gal) reporter mice, respectively. We show that essentially all adult satellite cells associated with limb and body wall musculature, as well as the diaphragm and extraocular muscles, originate from MyoD+ progenitors. Neonatal satellite cells were Cre-recombined, but only a small minority exhibited ongoing Cre expression, indicating that most satellite cells had expressed MyoD prenatally. We also show that satellite cell development in MyoD-null mice is not due to functional compensation by MyoD non-expressing lineages. The results suggest that satellite cells are derived from committed myogenic progenitors, irrespective of the anatomical location, embryological origin, or physiological properties of associated musculature.     

Extraordinarily rapid proliferation of cultured muscle satellite cells from migratory birds

Migratory birds experience bouts of muscle growth and depletion as they prepare for, and undertake prolonged flight. Our studies of migratory bird muscle physiology in vitro led to the discovery that sanderling (Calidris alba) muscle satellite cells proliferate more rapidly than other normal cell lines. Here we determined the proliferation rate of muscle satellite cells isolated from five migratory species (sanderling; ruff, Calidris pugnax; western sandpiper, Calidris mauri; yellow-rumped warbler, Setophaga coronata; Swainson''s thrush, Catharus ustulatus) from two families (shorebirds and songbirds) and with different migratory strategies. Ruff and sanderling satellite cells exhibited rapid proliferation, with population doubling times of 9.3 ± 1.3 and 11.4 ± 2 h, whereas the remaining species'' cell doubling times were greater than or equal to 24 h. The results indicate that the rapid proliferation of satellite cells is not associated with total migration distance but may be related to flight bout duration and interact with lifespan.     

Odd-skipped related 2 regulates genes related to proliferation and development

Cell proliferation is a biological process in which chromosomes replicate in one cell and equally divide into two daughter cells. Our previous findings suggested that Odd-skipped related 2 (Osr2) plays an important role in cellular quiescence and proliferation under epigenetic regulation. However, the mechanism used by Osr2 to establish and maintain proliferation is unknown. To examine the functional role of Osr2 in cell proliferation, we analyzed its downstream target genes using microarray analysis following adenovirus-induced overexpression of Osr2 as well as knockdown with Osr2 siRNA, which showed that Osr2 regulates a multitude of genes involved in proliferation and the cell cycle, as well as development. Additional proliferation assays also indicated that Osr2 likely functions to elicit cell proliferation. Together, these results suggest that Osr2 plays important roles in proliferation and development.     

In vitro primary satellite cell growth and differentiation within litters of pigs

Postnatal muscle growth is dependent on satellite cell (SC) proliferation, differentiation and fusion to increase the DNA content of existing muscle fibres and thereby the capacity to synthesize protein. The purpose of the present study was to examine the ability of isolated SCs from low, medium and high weaning weight litter mates of pigs to proliferate and differentiate, and to affect protein synthesis and degradation after fusion into myotubes. At 6 weeks of age, SCs from the lowest weight (LW), medium weight (MW) and highest weight (HW) female pigs within eight litters were isolated. Thereby, eight cultures of SCs were established for each of the three weight groups within litter, representing three groups of SCs from pigs exhibiting differences in postnatal muscle growth performance. Proliferation was estimated as the number of viable cells at different time points after seeding. SC differentiation was evaluated by measuring the activity of the muscle-specific enzyme, creatine phosphokinase, and protein synthesis and degradation were measured by incorporation and release of 3H-tyrosine, respectively. A tendency towards a difference in proliferation between SC cultures was found (P = 0.09). This was evident as the number of viable cells at day 3 was lower in cultures from LW pigs than from HW (P < 0.05) and MW (P < 0.01) pigs. Differentiation was significantly different between cultures (P < 0.05). There was a significant difference between LW and MW cultures at 72 h (P < 0.05), and a tendency towards a difference between LW and HW cultures at 45 h (P = 0.07). Protein synthesis per μg protein or per μg DNA did not differ among SC cultures from LW, MW and HW pigs. Neither did protein degradation rate differ significantly among SC cultures from LW, MW and HW pigs. Overall, the results show that SCs from LW pigs seem to proliferate and differentiate at a slower rate than SCs from MW and HW pigs. The results found in this study show no difference in the ability of SCs to affect protein synthesis or degradation between SCs from litter mates exhibiting different growth rates in vivo.     

Identification of candidate genes in Arabidopsis and Populus cell wall biosynthesis using text-mining, co-expression network analysis and comparative genomics

Populus is an important bioenergy crop for bioethanol production. A greater understanding of cell wall biosynthesis processes is critical in reducing biomass recalcitrance, a major hindrance in efficient generation of biofuels from lignocellulosic biomass. Here, we report the identification of candidate cell wall biosynthesis genes through the development and application of a novel bioinformatics pipeline. As a first step, via text-mining of PubMed publications, we obtained 121 Arabidopsis genes that had the experimental evidence supporting their involvement in cell wall biosynthesis or remodeling. The 121 genes were then used as bait genes to query an Arabidopsis co-expression database, and additional genes were identified as neighbors of the bait genes in the network, increasing the number of genes to 548. The 548 Arabidopsis genes were then used to re-query the Arabidopsis co-expression database and re-construct a network that captured additional network neighbors, expanding to a total of 694 genes. The 694 Arabidopsis genes were computationally divided into 22 clusters. Queries of the Populus genome using the Arabidopsis genes revealed 817 Populus orthologs. Functional analysis of gene ontology and tissue-specific gene expression indicated that these Arabidopsis and Populus genes are high likelihood candidates for functional characterization in relation to cell wall biosynthesis.     

Enrichment and terminal differentiation of striated muscle progenitors in vitro

Enrichment and terminal differentiation of mammalian striated muscle cells is severely hampered by fibroblast overgrowth, de-differentiation and/or lack of functional differentiation. Herein we report a new, reproducible and simple method to enrich and terminally differentiate muscle stem cells and progenitors from mice and humans. We show that a single gamma irradiation of muscle cells induces their massive differentiation into structurally and functionally intact myotubes and cardiomyocytes and that these cells can be kept in culture for many weeks. Similar results are also obtained when treating skeletal muscle-derived stem cells and progenitors with Mitomycin C.     

In vitro characterization of proliferation and differentiation of pig satellite cells

Skeletal muscle contains various muscle fiber types exhibiting different contractile properties based on the myosin heavy chain (MyHC) isoform profile. Muscle fiber type composition is highly variable and influences growth performance and meat quality, but underlying mechanisms regulating fiber type composition remain poorly understood. The aim of the present work was to develop a model based on muscle satellite cell culture to further investigate the regulation of adult MyHC isoforms expression in pig skeletal muscle. Satellite cells were harvested from the mostly fast-twitch glycolytic longissimus (LM) and predominantly slow-twitch oxidative rhomboideus (RM) muscles of 6-week-old piglets. Satellite cells were allowed to proliferate up to 80% confluence, reached after 7 day of proliferation (D7), and then induced to differentiate. Kinetics of proliferation and differentiation were similar between muscles and more than 95% of the cells were myogenic (desmin positive) at D7 with a fusion index reaching 65±9% after 4 day of differentiation. One-dimensional SDS polyacrylamide gel electrophoresis revealed that satellite cells from both muscles only expressed the embryonic and fetal MyHC isoforms in culture, without any of the adult MyHC isoforms that were expressed in vivo. Interestingly, triiodothyronine (T3) induced de novo expression of adult fast and α-cardiac MyHC in vitro making our culture system a valuable tool to study de novo expression of adult MyHC isoforms and its regulation by intrinsic and/or extrinsic factors.     

hunchback and Ikaros-like zinc finger genes control reproductive system development in Caenorhabditis elegans

Here we provide evidence for a C2H2 zinc finger gene family with similarity to Ikaros and hunchback. The founding member of this family is Caenorhabditis elegans ehn-3, which has important and poorly understood functions in somatic gonad development. We examined the expression and function of four additional hunchback/Ikaros-like (HIL) genes in C. elegans reproductive system development. Two genes, ehn-3 and R08E3.4, are expressed in somatic gonadal precursors (SGPs) and have overlapping functions in their development. In ehn-3; R08E3.4 double mutants, we find defects in the generation of distal tip cells, anchor cells, and spermatheca; three of the five tissues derived from the SGPs. We provide in vivo evidence that C. elegans HIL proteins have functionally distinct zinc finger domains, with specificity residing in the N-terminal set of four zinc fingers and a likely protein-protein interaction domain provided by the C-terminal pair of zinc fingers. In addition, we find that a chimeric human Ikaros protein containing the N-terminal zinc fingers of EHN-3 functions in C. elegans. Together, these results lend support to the idea that the C. elegans HIL genes and Ikaros have similar functional domains. We propose that hunchback, Ikaros, and the HIL genes arose from a common ancestor that was present prior to the divergence of protostomes and deuterostomes.     

Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs’ ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2‐Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4‐Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC‐pre‐treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF‐pre‐treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.     

The Sine oculis/Six class family of homeobox genes in jellyfish with and without eyes: development and eye regeneration

The development of visual organs is regulated in Bilateria by a network of genes where members of the Six and Pax gene families play a central role. To investigate the molecular aspects of eye evolution, we analyzed the structure and expression patterns of cognate members of the Six family genes in jellyfish (Cnidaria, Hydrozoa), representatives of a basal, non-bilaterian phylum where complex lens eyes with spherical lens, an epidermal cornea, and a retina appear for the first time in evolution. In the jellyfish Cladonema radiatum, a species with well-developed lens eyes in the tentacle bulbs, Six1/2-Cr and Six3/6-Cr, are expressed in the eye cup. Six4/5-Cr is mainly expressed in the manubrium, the feeding, and sex organ. All three Six genes are expressed in different subsets of epidermal nerve cells, possibly of the RFamide type which are part of a net connecting the different eyes with each other and the effector organs. Furthermore, expression is found in other tissues, notably in the striated muscle. During eye regeneration, expression of Six1/2-Cr and Six3/6-Cr is upregulated, but not of Six4/5-Cr. In Podocoryne carnea, a jellyfish without eyes, Six1/2-Pc and Six3/6-Pc are also expressed in the tentacle bulbs, Six1/2-Pc additionally in the manubrium and striated muscle, and Six3/6-Pc in the mechanosensory nematocytes of the tentacle. The conserved gene structure and expression patterns of all Cladonema Six genes suggest broad conservation of upstream regulatory mechanisms in eye development.     

On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.