Tbx5a and Tbx5b paralogues act in combination to control separate vectors of migration in the fin field of zebrafish.

The T-box containing family member, TBX5, has been shown to play important functional roles in the pectoral appendages of a variety of vertebrate species. While a single TBX5 gene exists in all tetrapods studied to date, the zebrafish genome retains two paralogues, designated as tbx5a and tbx5b, resulting from a whole genome duplication in the teleost lineage. Zebrafish deficient in tbx5a lack pectoral fin buds, whereas zebrafish deficient in tbx5b exhibit misshapen pectoral fins, showing that both paralogues function in fin development. The mesenchymal cells of the limb/fin bud are derived from the Lateral Plate Mesoderm (LPM). Previous fate mapping work in zebrafish has shown that wildtype (wt) fin field cells are initially located adjacent to somites (s)1-4. The wt fin field cells migrate in opposing diagonal directions placing the limb bud between s2-3 and lateral to the main body. To better characterize tbx5 paralogue functions in zebrafish, time-lapse analyses of the migrations of fin bud precursors under conditions of tbx5a knock-down, tbx5b knock-down and double-knock-down were performed. Our data suggest that zebrafish tbx5a and tbx5b have functionally separated migration direction vectors, that when combined recapitulate the migration of the wt fin field. We and others have shown that loss of Tbx5a function abolishes an fgf24 signaling cue resulting in fin field cells failing to converge in an Antero-Posterior (AP) direction and migrating only in a mediolateral (ML) direction. We show here that loss of Tbx5b function affects initial ML directed movements so that fin field cells fail to migrate laterally but continue to converge along the AP axis. Furthermore, fin field cells in the double Tbx5a/Tbx5b knock-down zebrafish do not engage in directed migrations along either the ML or AP axis. Therefore, these two paralogues may be acting to instruct separate vectors of fin field migration in order to direct proper fin bud formation.

Asymmetric cell convergence-driven zebrafish fin bud initiation and pre-pattern requires Tbx5a control of a mesenchymal Fgf signal.

Tbx5 plays a pivotal role in vertebrate forelimb initiation, and loss-of-function experiments result in deformed or absent forelimbs in all taxa studied to date. Combining single-cell fate mapping and three-dimensional cell tracking in the zebrafish, we describe a Tbx5a-dependent cell convergence pattern that is both asymmetric and topological within the fin-field lateral plate mesoderm during early fin bud initiation. We further demonstrate that a mesodermal Fgf24 convergence cue controlled by Tbx5a underlies this asymmetric convergent motility. Partial reduction in Tbx5a or Fgf24 levels disrupts the normal fin-field cell motility gradient and results in anteriorly biased perturbations of fin-field cell convergence and truncations in the pectoral fin skeleton, resembling aspects of the forelimb skeletal defects that define individuals with Holt-Oram syndrome. This study provides a quantitative reference model for fin-field cell motility during vertebrate fin bud initiation and suggests that a pre-pattern of anteroposterior fate specification is already present in the fin-field before or during migration because perturbations to these early cell movements result in the alteration of specific fates.

Tension-driven multi-scale self-organisation in human iPSC-derived muscle fibers.

Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. More efficient myofiber bundling accelerates the speed of sarcomerogenesis suggesting that tension generated by bundling promotes sarcomerogenesis. We tested this hypothesis by directly probing tension and found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.

The evolution of courtship behaviors through the origination of a new gene in Drosophila.

New genes can originate by the combination of sequences from unrelated genes or their duplicates to form a chimeric structure. These chimeric genes often evolve rapidly, suggesting that they undergo adaptive evolution and may therefore be involved in novel phenotypes. Their functions, however, are rarely known. Here, we describe the phenotypic effects of a chimeric gene, sphinx, that has recently evolved in Drosophila melanogaster. We show that a knockout of this gene leads to increased male-male courtship in D. melanogaster, although it leaves other aspects of mating behavior unchanged. Comparative studies of courtship behavior in other closely related Drosophila species suggest that this mutant phenotype of male-male courtship is the ancestral condition because these related species show much higher levels of male-male courtship than D. melanogaster. D. melanogaster therefore seems to have evolved in its courtship behaviors by the recruitment of a new chimeric gene.

Formation of polarized contractile interfaces by self-organized Toll-8/Cirl GPCR asymmetry.

Interfaces between cells with distinct genetic identities elicit signals to organize local cell behaviors driving tissue morphogenesis. The Drosophila embryonic axis extension requires planar polarized enrichment of myosin-II powering oriented cell intercalations. Myosin-II levels are quantitatively controlled by GPCR signaling, whereas myosin-II polarity requires patterned expression of several Toll receptors. How Toll receptors polarize myosin-II and how this involves GPCRs remain unknown. Here, we report that differential expression of a single Toll receptor, Toll-8, polarizes myosin-II through binding to the adhesion GPCR Cirl/latrophilin. Asymmetric expression of Cirl is sufficient to enrich myosin-II, and Cirl localization is asymmetric at Toll-8 expression boundaries. Exploring the process dynamically, we reveal that Toll-8 and Cirl exhibit mutually dependent planar polarity in response to quantitative differences in Toll-8 expression between neighboring cells. Collectively, we propose that the cell surface protein complex Toll-8/Cirl self-organizes to generate local asymmetric interfaces essential for planar polarization of contractility.

[The effect of puerarin on prevention of oxidative damage of cultured lens capsule epithelial cells].

OBJECTIVE: To investigate the peroxynitrite damage to the lens epithelial cells (LEC) and the prevention of this damage by puerarin in vitro. METHODS: This paper was experimental study. Rabbit LEC were isolated and cultured and the third or forth passage LEC were used in this experiment The experiment groups included: (1) CONTROL GROUP: Heat-pathogen free saline (NS) 200 microl was added to the medium; (2) ONOO- group: ONOO- 200 microl was added to obtain the terminal concentration at 0. 5 mmol/L; (3) Puerarin group: 5 microg/ml ONOO- and 10 microg/ml puerarin were added simultaneously. Then, the cells were cultured and collected after 6,12 or 24 hours. The nitrotyrosine (NT), a symbol of the ONOO-, was tested with immunofluorescence technique. The expression of NT protein was examined with Western blot method. The cell morphology was observed with light microscope. Cell apoptosis was examined via DNA ladder, flow cytometry and Fas/FasL immunohistochemical staining. These datas were analyzed by one-way-ANOVA and q test. RESULTS: During the 6 to 24 hours of experiment period, green color could be observed in the cell nucleus and cytoplasm of control group. Staining ranged from yellow to brown-yellow, then to brown color were observed in STZ group. Staining ranged from faint green to yellow green or faint green color were observed in puerarin group. Slight expression of nitrotyrosine (NT) could be seen in the control group. A moderate to strong expression of NT was observed at different stages in the STZ group (A = 77.22 +/- 2.44, 145.00 +/- 3.94, 235. 8 +/- 5.97). At 6 hours, a slight expression of NT could be seen in the control group (A = 72.78 +/- 2.64), this increased at 12 hours (A =89. 94 +/- 3.01) and decreased at 24 hours (A = 74. 44 +/- 3.00). With computer photo-analysis, there were significant differences between the control, STZ and puerarin groups at different period during the experiment (q = 78.12, 82.76, 69.98, P <0. 01). In the control group, cell morphology and gene DNA ladder were normal, minor apoptosis could be observed but no expression of Fas/FasL in the membrane and cytoplasm of the cells. Distinctive cell morphology changes and the typical "ladder bands" as well as the expression of Fas/FasL could be observed in STZ group. All of these aspects were comparatively normal in puerarin group. CONCLUSIONS: The LEC apoptosis induced by ONOO- in vitro could be alleviated by puerarin. Fas/FasL cell signal transduction pathway may affect and strengthen the apoptosis process mediated by ONOO-.

Fat2 and Lar Dance a Pas de Deux during Collective Cell Migration.

What coordinates the internal leading and trailing edges in collectively migrating cells is largely unknown. In this issue of Developmental Cell, Barlan et al. (2017) delineate a Fat2/Lar planar signaling pathway at the basal, motile cell-cell contacts of Drosophila egg chamber follicle cells.

The antagonism of cholecystokinin octapeptide-8 to the peroxynitrite oxidation on a diabetic cataractal rat model.

BACKGROUND: Cataracts is considered be formed because of an abnormal glucose metabolic pathway or oxidative stress. We explored the damaging role of ONOO- and antagonism of cholecystokinin octapeptide-8 (CCK-8) in diabetic cataractal rat lenses. METHODS: A diabetic cataractal animal model was established by peritoneal injection of streptozotocine (STZ). Thirty-six normal SD rats were taken as control group; seventy-two were given STZ (45 mg/kg) and then divided into STZ group and CCK-8 group (peritoneal injection CCK-8). STZ induced diabetic rats were treated with CCK-8 for 60 days. Lenses were examined with slit lamp at 20, 40 and 60 days. Immunofluorescent staining and Western blot analysis were used for determining nitrotyrosine (NT, a marker for ONOO-). PT-PCR and gene array analysis were used for determining the expression of inducible nitric oxide synthetase mRNA (iNOS mRNA) in lens epithelium (LEC). RESULTS: STZ group rats developed lens opacity by 20 days that reached a high level by 60 days after STZ injection. CCK-8 group rats delayed the cataract formation. CCK-8 group rats delayed the cataract formation. There was no distinct expression of NT and iNOS mRNA in control group. In STZ group, there were distinct expression of NT and upregulation of iNOS mRNA; however, CCK-8 group showed weak expression of NT and downregulation of iNOS mRNA. CONCLUSIONS: NT, which may be a new form of oxidative stress, was expressed in diabetic rat LEC although CCK-8 could reverse NT damage in LEC. The results suggested that CCK-8 might be a useful therapeutic agent against diabetic cataract. The antagonizing mechanism of CCK-8 may be related to direct antagonism of ONOO- as well as its inhibition of the expression of iNOS mRNA for production of NO and therefore decrease in the formation of ONOO-.

Puerarin decreases lens epithelium cell apoptosis induced partly by peroxynitrite in diabetic rats.

The present study was designed to observe if puerarin decreases lens epithelium cell (LEC) apoptosis induced partly by peroxynitrite (ONOO(-)). One hundred and eight rats were randomly divided into control group (n=36), streptozotocin (STZ) group (n=36) and STZ + puerarin group (n=36). The rats in the control group intraperitoneally (i.p.) received 0.5 ml of saline. The rats in STZ group and STZ + puerarin group received intraperitoneal injection of STZ (45 mg/kg). Three days later, the rats in STZ + puerarin group were given puerarin (140 mg/kg per day, i.p.). On days 20, 40 and 60 of the experiment, morphologic changes of lenses were observed with slit lamp. Then the animals were sacrificed for further analysis. The amount and percentage of apoptotic LECs were determined by flow cytometry. Nitrotyrosine (NT, the foot print of ONOO(-)) was examined by immunohistochemistry. Apoptosis-related genes (iNOS, etc.) were analyzed by gene array. The results showed that in the control group, all the lenses were clear. In STZ group, gradually severe opacity of the lens was observed on days 20, 40 and 60. But in STZ + puerarin group, mild opacity of the lens was observed on day 20 and more severe on day 40, but markedly decreased on day 60. In the control group, mild apoptosis of LECs was observed. In STZ group, time-dependent increase in apoptosis of LECs was observed. In STZ + puerarin group, mild apoptosis of LECs was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of NT in the lens in the control group, but an increased expression of NT in STZ group. In STZ + puerarin group, mild expression of NT was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of iNOS in the lens in the control group, but continuous up-regulation of iNOS expression in STZ group. In STZ + puerarin group, mild expression of iNOS was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. Except the changes of iNOS related to NO production, the other apoptosis-related genes, including BCL-2 and SOD were down-regulated, while NF-kappaB and TNFR1-FADD-caspase signal transduction way were up-regulated in STZ group. The results were opposite in STZ + puerarin group and the control group. These findings show that NT is expressed in diabetic rat lens, which proves that LEC apoptosis in diabetic lens is partly induced by ONOO(-) which may be a new oxidative damage way to form cataract. Puerarin partly decreases LEC apoptosis induced by ONOO(-) and is a potential medicine for therapy of diabetic cataract. The mechanism of puerarin dealing with diabetic cataract may be related to its direct inhibition of LEC apoptosis and antagonism of ONOO(-) in diabetic rats.

Evo-Devo: Universal Toll Pass for the Extension Highway?

The distinction between long-germ and short-germ insects is a classic one in evo-devo, yet a common genetic mechanism may underlie germband extension in all insects, even all arthropods.

Mechanochemical Interplay Drives Polarization in Cellular and Developmental Systems.

Polarity plays important roles in biological processes, such as motility, differentiation, growth, and pattern formation. One major goal in the field of biological polarity is to understand logics used by signaling networks underlying diverse polarization processes: symmetry breaking, amplification, inhibition, and coordination. In this essay, we explore various polarization processes on cell, tissue, and whole-organism scales, aiming to elucidate how diverse mechanical signals, in addition to chemical cues, can be integrated to a common framework of polarization.

[Peroxynitrite-induced formation of diabetic cataract and its prevention by puerarin in rat].

OBJECTIVE: To investigate the effect of peroxynitrite on the formation of diabetic cataract and its reversal by puerarin. in rat. METHODS: Normal Sprague-Dawley (SD) rats were equally divided into control group, streptozotocin group (STZ) and treatment group. STZ and treatment group were treated with STZ to establish animal model of diabetic rat cataract and in treatment group puerarin was given by peritoneal injection. General condition and lens shape of rats were examined at 20th, 40th, 60th day respectively. Lens epithelial cells (LEC) were observed with optical microscope. percentage of apoptotic cells, and fluorescent intensity of positive cells for nitrotyrosine (NT) which is the symbol of peroxynitrite were tested by flow cytometry. The expression of NT in the lens was analyzed by Western blot analysis. RESULTS: On 20th, 40th, 60th day of the experiment, clear, mild and severe lens opacity were found in control group, treatment group, and STZ group respectively and the lens opacity was gradually increased in all of rats included in the study. Under optical microscope, no changes were presented in control group, but mild changes were showed in treatment group and remarkable changes were found in STZ group. On all the specified days, distinguishable differences were found in the amount and percentage of apoptotic cells compared with that of control group in treatment group and STZ groups as well as between treatment and STZ groups (P < 0.05). There was significant increase in the fluorescent intensity and amount of NT in treatment and STZ groups compared with that of control group (P < 0.05), and the similar results were revealed between treatment group and STZ. The amount of expression of NT protein at different time points in different groups as described above was also significant difference (P < 0.001). CONCLUSIONS: Peroxynitrite plays an important role in the formation of diabetic rat cataract and is a participant in the pathogenesis of oxidative stress induced cataract. Puerarin is an effective antioxidative agent in the treatment of early diabetic rat cataract.