Epithelial-mesenchymal transition contrast in the amputated tail and limb of the northern house gecko, Hemidactylus flaviviridis.

The northern house gecko Hemidactylus flaviviridis exhibits appendage-specific responses to injuries. The autotomized tail regenerates, whereas the severed limb fails to regrow. Many site-specific cellular processes influence tail regeneration. Herein, we analyzed the epithelial-mesenchymal transition contrast in the lizard's amputated appendages (tail and limb). Morphological observations in the healing frame indicated the formation of regeneration blastema in the tail and scar formation in limb. Histology of the tail showed that epithelial cells closer to mesenchyme appeared less columnar and loosely packed, with little intercellular matrix. Whereas in the limb, the columnar epithelial cells remained tightly packed. Collagen deposition was seen in the limb at the intersection of wound epithelium and mesenchyme, favoring scarring by blocking the epithelial-mesenchymal transition. Markers for epithelial-mesenchymal transition were assessed at transcript and protein levels. The regenerating tail showed upregulation of N-cadherin, vimentin, and PCNA, favoring epithelial-mesenchymal transition, cell migration, and proliferation, respectively. In contrast, the scarring limb showed persistently elevated levels of E-cadherin and EpCAM, indicating retention of epithelial characteristics. An attempt was made to screen the resident epithelial stem cell population in both appendages to check their potential role in the epithelial-mesenchymal transition (EMT), hence the differential wound healing. Upregulation in transcript and protein levels of Nanog and Sox2 was observed in the regenerating tail. Fluorescence-activated cell sorting (FACS) provided supporting evidence that the epithelial stem cell population in tail remained significantly higher than in limb. Thus, this study focuses on the mechanistic role of the epithelial-mesenchymal transition in wound healing, highlighting the molecular details of regeneration and scarring events.

De Novo Transcriptome Sequencing and Analysis of Differential Gene Expression among Various Stages of Tail Regeneration in Hemidactylus flaviviridis.

Across the animal kingdom, lizards are the only amniotes capable of regenerating their lost tail through epimorphosis. Of the many reptiles, the northern house gecko, Hemidactylus flaviviridis, is an excellent model system that is used for understanding the mechanism of epimorphic regeneration. A stage-specific transcriptome profile was generated in the current study following an autotomized tail with the HiSeq2500 platform. The reads obtained from de novo sequencing were filtered and high-quality reads were considered for gene ontology (GO) annotation and pathway analysis. Millions of reads were recorded for each stage upon de novo assembly. Up and down-regulated transcripts were categorized for early blastema (EBL), blastema (BL) and differentiation (DF) stages compared to the normal tail (NT) by differential gene expression analysis. The transcripts from developmentally significant pathways such as FGF, Wnt, Shh and TGF-ß/BMP were present during tail regeneration. Additionally, differential expression of transcripts was recorded from biological processes, namely inflammation, cell proliferation, apoptosis and cell migration. Overall, the study reveals the stage-wise transcriptome analysis in conjunction with cellular processes as well as molecular signaling pathways during lizard tail regeneration. The knowledge obtained from the data can be extrapolated to configure regenerative responses in other amniotes, including humans, upon loss of a complex organ.

Site-specific variation in the activity of COX-2 alters the pattern of wound healing in the tail and limb of northern house gecko by differentially regulating the expression of local inflammatory mediators.

The role of COX-2 induced PGE2 in the site-specific regulation of inflammatory mediators that facilitate disparate wound healing in the tail and limb of a lizard was studied by analysing their levels during various stages of healing. The activity of COX-2 and concentration of PGE2 surged during the early healing phase of tail along with the parallel rise in EP4 receptor. PGE2-EP4 interaction is corelated to early resolution (by 3 dpa) of inflammation by rising the antiinflammatory mediator IL-10. This likely causes reduction in proinflammatory mediators viz., iNOS, TNF-α, IL-6, IL-17 and IL-22. Conversely, in the limb, COX-2 derived PGE2 likely causes rise in inflammation through EP2 receptor-based signalling, as all the proinflammatory mediators stay elevated through the course of healing (till 9 dpa), while expression of IL-10 is reduced. This study brings to light the novel roles of IL-17 and IL-22 in programming wound healing. As IL-17 reduces in tail, IL-22 behaves in reparative way, causing conducive environment for scar-free wound healing. On the contrary, synergic elevation of both IL-17 and Il-22 form a micro-niche suitable for scarred wound healing in limb, thus obliterating its regenerative potential.

Inhibition of Cyclooxygenase-2 Alters Craniofacial Patterning during Early Embryonic Development of Chick.

A recent study from our lab revealed that the inhibition of cyclooxygenase-2 (COX-2) exclusively reduces the level of PGE2 (Prostaglandin E2) among prostanoids and hampers the normal development of several structures, strikingly the cranial vault, in chick embryos. In order to unearth the mechanism behind the deviant development of cranial features, the expression pattern of various factors that are known to influence cranial neural crest cell (CNCC) migration was checked in chick embryos after inhibiting COX-2 activity using etoricoxib. The compromised level of cell adhesion molecules and their upstream regulators, namely CDH1 (E-cadherin), CDH2 (N-cadherin), MSX1 (Msh homeobox 1), and TGF-ß (Transforming growth factor beta), observed in the etoricoxib-treated embryos indicate that COX-2, through its downstream effector PGE2, regulates the expression of these factors perhaps to aid the migration of CNCCs. The histological features and levels of FoxD3 (Forkhead box D3), as well as PCNA (Proliferating cell nuclear antigen), further consolidate the role of COX-2 in the migration and survival of CNCCs in developing embryos. The results of the current study indicate that COX-2 plays a pivotal role in orchestrating craniofacial structures perhaps by modulating CNCC proliferation and migration during the embryonic development of chicks.

New insights into the obligatory nature of cyclooxygenase-2 and PGE2 during early chick embryogenesis.

Temporal expression patterns and activity of two cyclooxygenase (COX-1 and COX-2) isoforms were analysed during early chick embryogenesis to evaluate their roles in development. COX-2 inhibition with etoricoxib resulted in significant structural anomalies such as anophthalmia (born without one or both eyes), phocomelia (underdeveloped or truncated limbs), and gastroschisis (an opening in the abdominal wall), indicating its significance in embryogenesis. Furthermore, the levels of PGE2, PGD2, PGF2α, and TXB2 were assessed using quantitative LC-MS/MS to identify which effector prostanoid (s) had their synthesis initiated by COX-2. COX-2 inhibition was only shown to reduce the level of PGE2 significantly, and hence it could be inferred that the later could be largely under the regulation of activated COX-2 in chick embryos. The compensatory increase in the activity of COX-1 observed in the etoricoxib-treated group helped to maintain the levels of PGD2, PGF2α, and TXB2. Though the roles of these three prostanoids in embryogenesis need to be further clarified, it appears that their contribution to the observed developmental anomalies is minimal. This study has shown that COX-2 is functionally active during chick embryogenesis, and it plays a central role in the structural configuration of several organs and tissues through its downstream effector molecule PGE2.

Early embryonic exposure to chlorpyrifos-cypermethrin combination induces pattern deficits in the heart of domestic hen.

Exposure to chlorpyrifos-cypermethrin combination during early development resulted in defective looping and ventricular noncompaction of heart in domestic chicken. The study was extended to elucidate the molecular basis of this novel observation. The primary culture of chicken embryonic heart cells showed a concentration-dependent loss of viability when challenged with this combination of technical-grade insecticides. Comet assay, DNA ladder assay, and analyses of appropriate markers at transcript and protein levels, revealed that chlorpyrifos-cypermethrin combination induced cell death by activating apoptosis. Parallelly, the tissues derived from control and experimental group hearts were checked for apoptotic markers, and the result was much similar to that of the in-vitro study. Further analysis showed that chlorpyrifos-cypermethrin combination deranged the expression pattern of the transcriptional regulators of cardiogenesis, namely TBX20, GATA5, HAND2, and MYOCD. This, together with heightened apoptosis, could well be the reason behind the observed structural anomalies in the heart of chlorpyrifos-cypermethrin poisoned embryos.

Chick embryonic cells as a source for generating in vitro model of muscle cell dystrophy.

Chick embryonic cells can be used to develop an easy and economical in vitro model for conducting studies on the disease muscle dystrophy (MD). For this, the limb myoblasts from 11th day chick embryo were isolated and cultured. To this muscle cell culture, anti-dystroglycan antibody (IIH6) was added so as to target the α-dystroglycan and disrupt the connection between the cytoskeletal proteins and the extracellular matrix (which is a characteristic feature of MD). Cells were allowed to differentiate further and the morphometrics and mRNA expression were studied. The IIH6-treated muscle cells displayed changes in morphometry, contractibility, and also atrophy was observed when compared to the control cultures. Concomitant gene expression studies showed an upregulation in TGF-ß expression, while the muscle sculpture genes MYOD1, MYF5, LAMA2 and MYOG were downregulated resembling the MD in vivo. This simple and cost-effective method can be useful in studies to further understand the disease mechanism and also in conducting initial studies on effect of novel pharmacological agents.