Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins

The mechanisms by which cells obtain instructions to precisely re-create the missing parts of an organ remain an unresolved question in regenerative biology. Urodele limb regeneration is a powerful model in which to study these mechanisms. Following limb amputation, blastema cells interpret the proximal-most positional identity in the stump to reproduce missing parts faithfully. Classical experiments showed the ability of retinoic acid (RA) to proximalize blastema positional values. Meis homeobox genes are involved in RA-dependent specification of proximal cell identity during limb development. To understand the molecular basis for specifying proximal positional identities during regeneration, we isolated the axolotl Meis homeobox family. Axolotl Meis genes are RA-regulated during both regeneration and embryonic limb development. During limb regeneration, Meis overexpression relocates distal blastema cells to more proximal locations, whereas Meis knockdown inhibits RA proximalization of limb blastemas. Meis genes are thus crucial targets of RA proximalizing activity on blastema cells.     

Cellular heterogeneity during vertebrate skeletal muscle development

Although skeletal muscles appear superficially alike at different anatomical locations, in reality there is considerably more diversity than previously anticipated. Heterogeneity is not only restricted to completely developed fibers, but is clearly apparent during development at the molecular, cellular and anatomical level. Multiple waves of muscle precursors with different features appear before birth and contribute to muscular diversification. Recent cell lineage and gene expression studies have expanded our knowledge on how skeletal muscle is formed and how its heterogeneity is generated. This review will present a comprehensive view of relevant findings in this field.     

OLFML3 expression is decreased during prenatal muscle development and regulated by microRNA-155 in pigs

The Olfactomedin-like 3 (OLFML3) gene has matrix-related function involved in embryonic development. MicroRNA-155 (miR-155), 21- to 23-nucleotides (nt) noncoding RNA, regulated myogenesis by target mRNA. Our LongSAGE analysis suggested that OLFML3 gene was differently expressed during muscle development in pig. In this study, we cloned the porcine OLFML3 gene and detected its tissues distribution in adult Tongcheng pigs and dynamical expression in developmental skeletal muscle (12 prenatal and 10 postnatal stages) from Landrace (lean-type) and Tongcheng (obese-type) pigs. Subsequently, we analyzed the interaction between OLFML3 and miR-155. The OLFML3 was abundantly expressed in liver and pancreas, moderately in lung, small intestine and placenta, and weakly in other tissues and postnatal muscle. There were different dynamical expression patterns between Landrace and Tongcheng pigs during prenatal skeletal muscle development. The OLFML3 was down-regulated (33-50 days post coitus, dpc), subsequently up-regulated (50-70 dpc), and then down-regulated (70-100 dpc) in Landrace pigs, while in Tongcheng pigs, it was down-regulated (33-50 dpc), subsequently up-regulated (50-55 dpc) and then down-regulated (55-100 dpc). There was higher expression in Tongcheng than Landrace in prenatal muscle from 33 to 60 dpc, and opposite situation from 65 to 100 dpc. Dual luciferase assay and real time PCR documented that OLFML3 expression was regulated by miR-155 at mRNA level. Our research indicated that OLFML3 gene may affect prenatal skeletal muscle development and was regulated by miR-155. These finding will help understanding biological function and expression regulation of OLFML3 gene in mammal animals.     

Stress resilience is established during development and is regulated by complement factors

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Identification of collagen fibril fusion during vertebrate tendon morphogenesis. The process relies on unipolar fibrils and is regulated by collagen-proteoglycan interaction

The synthesis of an extracellular matrix containing long (approximately mm in length) collagen fibrils is fundamental to the normal morphogenesis of animal tissues. In this study we have direct evidence that fibroblasts synthesise transient early fibril intermediates (approximately 1 micrometer in length) that interact by tip-to-tip fusion to generate long fibrils seen in older tissues. Examination of early collagen fibrils from tendon showed that two types of early fibrils occur: unipolar fibrils (with carboxyl (C) and amino (N) ends) and bipolar fibrils (with two N-ends). End-to-end fusion requires the C-end of a unipolar fibril. Proteoglycans coated the shafts of the fibrils but not the tips. In the absence of proteoglycans the fibrils aggregated by side-to-side interactions. Therefore, proteoglycans promote tip-to-tip fusion and inhibit side-to-side fusion. This distribution of proteoglycan along the fibril required co-assembly of collagen and proteoglycan prior to fibril assembly. The study showed that collagen fibrillogenesis is a hierarchical process that depends on the unique structure of unipolar fibrils and a novel function of proteoglycans.     

MicroRNA调控猪骨骼肌发育的研究进展

骨骼肌的生长发育是影响猪肉产量和品质的重要因素,其受遗传和营养等众多因素的精细调控。MicroRNA (miRNA)是一种长度约为22 nt的非编码RNA,通过与靶基因的mRNA 3′UTR序列结合,调控其转录后的表达发挥作用。近年来,大量的研究表明miRNA参与机体的生长发育、生殖、疾病等多种生命过程。本文对miRNA在猪骨骼肌发育调控中的作用进行了综述,以期为猪的遗传改良提供参考和借鉴。     

Mitochondrial genome content is regulated during nematode development

Growth and development rely on the mitochondrial respiratory chain (MRC) as the major source of ATP. We measured the mitochondrial DNA (mtDNA) copy number of each of the Caenorhabditis elegans developmental stages. Embryos, L1, L2, and L3 larvae all have approximately 25,000 copies of maternally derived mtDNA. The copy number increases fivefold in L4 larvae and a further sixfold in adult hermaphrodites, but only twofold in adult males. The majority of mtDNA in adult worms is germline associated, and germline-deficient mutants show markedly reduced mtDNA contents. With sperm-deficient or oocyte-deficient mutants, we confirm that mtDNA amplification is primarily associated with oocyte production. When mtDNA replication is inhibited, a quantitative and homogeneous arrest as L3 larvae occurs. Thus, mtDNA amplification is a necessary component of normal development and its regulation may involve an energy-sensing decision or checkpoint that can be invoked when mitochondrial energy generation is impaired.     

Engrailed homeoprotein secretion is a regulated process

Chicken Engrailed 2 homeoprotein is transported between cells in culture. This intercellular transfer is based on unconventional secretion and internalisation mechanisms: Engrailed 2 has access to vesicles but lacks a signal sequence for secretion and is internalised by a non-endocytic process. We show that phosphorylation of a serine-rich domain within Engrailed 2 by the protein kinase CK2 specifically inhibits Engrailed 2 secretion. The availability of the serine-rich domain to CK2 is highly increased when it is displaced from its normal position to the C terminus of Engrailed 2, leading to a constitutive blockage of Engrailed 2 intercellular transfer. This indicates that intercellular transfer of Engrailed 2 is a highly regulated process.     

Filamentous smooth muscle myosin is regulated by phosphorylation

The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to "fix" dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody- stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1 (Cross, R. A., K. E. Cross, A. Sobieszek. 1986. EMBO [Eur. Mol. Biol. Organ.] J. 5:2637-2641) the rate of product release from dephosphorylated filaments was only 3-12-fold higher, depending on the ionic strength. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin (Sellers, J. R. 1985. J. Biol. Chem. 260:15815-15819) thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.     

USP19 is a ubiquitin-specific protease regulated in rat skeletal muscle during catabolic states

Ubiquitin-dependent proteolysis is activated in skeletal muscle atrophying in response to various catabolic stimuli. Previous studies have demonstrated activation of ubiquitin conjugation. Because ubiquitination can also be regulated by deubiquitinating enzymes, we used degenerate oligonucleotides derived from conserved sequences in the ubiquitin-specific protease (UBP) family of deubiquitinating enzymes in RT-PCR with skeletal muscle RNA to amplify putative deubiquitinating enzymes. We identified USP19, a 150-kDa deubiquitinating enzyme that is widely expressed in various tissues including skeletal muscle. Expression of USP19 mRNA increased by approximately 30-200% in rat skeletal muscle atrophying in response to fasting, streptozotocin-induced diabetes, dexamethasone treatment, and cancer. Increased mRNA levels during fasting returned to normal with refeeding, but 1 day later than the normalization of rates of proteolysis and coincided instead with recovery of muscle mass. Indeed, in all catabolic treatments, USP19 mRNA was inversely correlated with muscle mass and provided an index of muscle mass that may be useful in many pathological conditions, using small human muscle biopsies. The increased expression of this deubiquitinating enzyme under conditions of increased proteolysis suggests that it may play a role in regeneration of free ubiquitin either coincident with or after proteasome-mediated degradation of substrates. USP19 may also be involved in posttranslational processing of polyubiquitin produced de novo in response to induction of the polyubiquitin genes seen under these conditions. Deubiquitinating enzymes thus appear involved in muscle wasting and implicate a widening web of regulation of genes in the ubiquitin system in this process.     

Ndr protein kinase is regulated by phosphorylation on two conserved sequence motifs

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases implicated in the regulation of cell division and cell morphology. This subfamily includes the kinases LATS, Orb6, Cot-1, and Dbf2. We show here that Ndr is potently activated when intact cells are treated with okadaic acid, suggesting that Ndr is normally held in a state of low activity by protein phosphatase 2A. We mapped the regulatory phosphorylation sites of Ndr protein kinase and found that active Ndr is phosphorylated on Ser-281 and Thr-444. Mutation of either site to alanine strongly reduced both basal and okadaic acid-stimulated Ndr activity, while combined mutation abolished Ndr activity completely. Importantly, each of these sites (and also the surrounding sequences) are conserved in the kinase relatives of Ndr, suggesting a general mechanism of activation for kinases of this subfamily. Ser-281 and Thr-444 are also similar to the regulatory phosphorylation sites in several targets of the phosphoinositide-dependent protein kinase PDK1.(1) However, PDK1 does not appear to function as an upstream kinase for Ndr. Thus, Ndr and its close relatives may operate in a novel signaling pathway downstream of an as-yet-unidentified kinase with specificity similar to, but distinct from, PDK1.     

The molecular motor KIFC1 associates with a complex containing nucleoporin NUP62 that is regulated during development and by the small GTPase RAN

KIFC1 is a C-terminal kinesin motor associated with the nuclear membrane and acrosome in round and elongating spermatids. This location in developing spermatids is consistent with possible roles in acrosome elongation and manchette motility or both. Here we describe the association of the KIFC1 motor with a complex containing the nucleoporin NUP62. Formation of this complex is developmentally regulated, being absent before puberty and appearing only after nuclear elongation has begun. In addition, the integrity of this complex is dependent on GTP hydrolysis and the GTP state of the small GTPase RAN. Concomitant with the association of this motor with the NUP62-containing complex is an apparent reorganization of the nuclear pore with loss of NUP62 from larger complexes containing other nucleoporins. The association of KIFC1 with a component of the nuclear membrane is more consistent with a role for this motor in acrosome/manchette transport along the nuclear membrane than for a role for this motor in transport of vesicles along the outer face of the manchette.