Unravelling the wound healing ability and mode of action of pyridine carboxamide oxime using Caenorhabditis elegans as potential prescreen wound model.

AIM: In the current scenario of ethical issues related to animal usage in research, the present study was intended to explore the proficient utility of nematode, Caenorhabditis elegans as wound model in preliminary screening of wound healing therapeutics. MAIN METHODS: In this study, a new wounding protocol and quantitative assessment strategies for various healing parameters [survival, Reactive Oxygen Species (ROS), calcium signals, F-actin dynamics, new collagen synthesis and wound induced anti-microbial peptides] were developed and used for preliminary screening of wound healing actives from natural sources. Wound healing ability of positive lead Tridax procumbens (TP) and its major phytocompounds [Octa decenoic acid (ODA), Pyridine carboxamide oxime, known as Nicotinamide (NA) and Dimethyl Benz[c]acridine (DMB)] were assessed using C. elegans wound model and cell lines scratch wound healing assay. Mode of action of active lead was elucidated using metabolome analysis coupled with MALDI-MS followed by molecular docking. KEY FINDINGS: From the four tested methanolic extracts, TP was chosen as positive lead compared to control, Benzalkonium chloride (BKC) based on survival and new collagen synthesis analyses. Results indicated that the wound healing ability of TP was majorly contributed by NA. Further, it was found that NA acts in chloromethyl nicotinamide derivative form by interacting with the known wound healing biomarker, glycogen synthase kinase 3 (GSK-3) to exert wound healing ability. SIGNIFICANCE: The study evidenced that C. elegans, could be a reliable wound model for high-throughput screening of wound healing actives and to identify their possible mode of action.

Understanding the role of DAF-16 mediated pathway in Caenorhabditis elegans during UV-A mediated photoaging process.

Even though Sun is the major source of energy to all living beings in the universe, continuous and prolonged exposure to sunlight will lead to detrimental effects. Human skin will undergo extrinsic aging, known as photoaging upon prolonged exposure to sunlight which is characterized by wrinkles, dryness, loss of elasticity, and so on. The model nematode Caenorhabditis elegans which is widely used in aging studies, could be used to study photoaging also. Transcription factor DAF-16, which regulates longevity, stress resistance and many other physiological events, mediates the photoaging mechanism in C. elegans. Elevation in extracellular ROS and altered expression of SGK-1 indicates the role of DAF-16 during UV-A exposure. Further, the role of daf-2, the receptor gene and lys-7, an effector gene of DAF-16 were characterized through mutant based studies. The long lived daf-2 mutants upon UV-A exposure showed reduction in lifespan, but the upregulation of daf-16 allowed the other molecular mechanisms like healthspan, antimicrobial and stress resistance to be active. In the case of lys-7 mutants, the lifespan was reduced and all other molecular mechanisms were also downregulated. However, the daf-16 mutants showed no change in lifespan irrespective of UV-A exposure. This signifies the role of DAF-16 during UV-A mediated photoaging in C. elegans. The present study helps in understanding the role of daf-16 in UV-A mediated stress response which will be of considerable importance in the field of pharmacy in designing targets for specific agents against photoaging.

Smad4 is required predominantly in the developmental processes dependent on the BMP branch of the TGF-ß signaling system in the embryonic mouse retina.

PURPOSE: The present study was aimed at defining developmental roles of Smad4, a key mediator of the TGF-ß superfamily signaling system, in the embryonic mouse retina. METHODS: Using a Cre/loxP-mediated conditional gene targeting approach, Smad4 gene function was deleted from the embryonic mouse retina. Mutant phenotypes were morphologically and molecularly examined. RESULTS: Loss of Smad4 in the developing retina led to varying degrees of microphthalmia at birth, presumably because of elevated apoptosis observed transiently at embryonic day 12.5 in the developing retina. This was also associated with an apparent delay in accumulation of retinal ganglion cells. Smad4 conditional mutants also exhibited alterations of retinal spatial patterning along the dorsal-ventral axis, consistent with a known function of BMP signaling in the embryonic retina. However, despite a known role for BMP signaling in retinal cell survival, proliferation, and differentiation, Smad4 mutant retinal progenitor cells were capable of maintaining growth and neurogenesis throughout embryonic development. We also found that the loss of Smad4 led to abnormal targeting of retinal ganglion cell axons to the optic nerve head, a phenotype consistent with reduced BMP signaling in the developing retina. CONCLUSIONS: These results suggest that Smad4 is essential for a subset of, but not all, TGF-ß/BMP-dependent developmental processes in the embryonic retina. In addition, genetic requirements for Smad4 in the embryonic retina are evident predominantly in the developmental events regulated by the BMP branch of the TGF-ß signaling pathway.

Bone morphogenetic proteins, eye patterning, and retinocollicular map formation in the mouse.

Patterning events during early eye formation determine retinal cell fate and can dictate the behavior of retinal ganglion cell (RGC) axons as they navigate toward central brain targets. The temporally and spatially regulated expression of bone morphogenetic proteins (BMPs) and their receptors in the retina are thought to play a key role in this process, initiating gene expression cascades that distinguish different regions of the retina, particularly along the dorsoventral axis. Here, we examine the role of BMP and a potential downstream effector, EphB, in retinotopic map formation in the lateral geniculate nucleus (LGN) and superior colliculus (SC). RGC axon behaviors during retinotopic map formation in wild-type mice are compared with those in several strains of mice with engineered defects of BMP and EphB signaling. Normal RGC axon sorting produces axon order in the optic tract that reflects the dorsoventral position of the parent RGCs in the eye. A dramatic consequence of disrupting BMP signaling is a missorting of RGC axons as they exit the optic chiasm. This sorting is not dependent on EphB. When BMP signaling in the developing eye is genetically modified, RGC order in the optic tract and targeting in the LGN and SC are correspondingly disrupted. These experiments show that BMP signaling regulates dorsoventral RGC cell fate, RGC axon behavior in the ascending optic tract, and retinotopic map formation in the LGN and SC through mechanisms that are in part distinct from EphB signaling in the LGN and SC.

Tissue-specific RNAi reveals that WT1 expression in nurse cells controls germ cell survival and spermatogenesis.

Using a novel tissue-specific RNA interference (RNAi) approach that mimics the principle by which naturally occurring microRNAs (miRNA) are made, we demonstrate that the Wilms' tumor 1 (WT1) transcription factor has an essential role in spermatogenesis. Mice depleted of WT1 in Sertoli nurse cells suffered from increased germ cell apoptosis, loss of adherens junctions, disregulation of adherence junction-associated genes, and impaired fertility. These effects were recapitulated in transgenic mice expressing a dominant-negative form of WT1 in Sertoli cells, demonstrating the validity of our RNAi approach. Our results indicate that the tumor suppressor WT1 promotes Sertoli cell-germ cell signaling events driving spermatogenesis.

Threshold-specific requirements for Bmp4 in mandibular development.

Mandibular development is regulated by an interplay between a specified branchial arch ectoderm and a plastic mesenchyme. Moreover, signaling from the pharyngeal endoderm has been shown to be important for mandibular morphogenesis. To gain insight into the mechanisms regulating mandibular pattern, it is important to investigate the function of the epithelial-derived signals. Bmp4 is expressed in both distal, mandibular arch ectoderm and pharyngeal endoderm. Here, we show that deletion of Bmp4 in the mandibular ectoderm and to a lesser extent in the pharyngeal endoderm, resulted in severe defects in mandibular development. Furthermore, our data uncovered different Bmp4 thresholds for expression of the Bmp-dependent Msx1 and Msx2 genes in mandibular mesenchyme. We also found that ectodermal Fgf8 expression was both activated and repressed by Bmp4 in a dosage-dependent fashion indicating a novel Bmp4 function in threshold-specific regulation of Fgf8 transcription. Lastly, we provide evidence that Prx homeobox genes repress expression of an Msx2 transgene, previously shown to be Bmp4-responsive, revealing a mechanism for differential regulation of Msx1 and Msx2 by Bmp signaling.

Distinct developmental programs require different levels of Bmp signaling during mouse retinal development.

The Bmp family of secreted signaling molecules is implicated in multiple aspects of embryonic development. However, the cell-type-specific requirements for this signaling pathway are often obscure in the context of complex embryonic tissue interactions. To define the cell-autonomous requirements for Bmp signaling, we have used a Cre-loxP strategy to delete Bmp receptor function specifically within the developing mouse retina. Disruption of a Bmp type I receptor gene, Bmpr1a, leads to no detectable eye abnormality. Further reduction of Bmp receptor activity by removing one functional copy of another Bmp type I receptor gene, Bmpr1b, in the retina-specific Bmpr1a mutant background, results in abnormal retinal dorsoventral patterning. Double mutants completely lacking both of these genes exhibit severe eye defects characterized by reduced growth of embryonic retina and failure of retinal neurogenesis. These studies provide direct genetic evidence that Bmpr1a and Bmpr1b play redundant roles during retinal development, and that different threshold levels of Bmp signaling regulate distinct developmental programs such as patterning, growth and differentiation of the retina.