Cell survival controlled by lens‐derived Sema3A–Nrp1 is vital on caffeine‐suppressed corneal innervation during chick organogenesis

Abstract

In this study, we investigated the effect of caffeine overexposure on corneal innervation in the early chicken embryo. Caffeine administration restricted corneal innervation by affecting trigeminal nerve development. Immunohistochemistry for phospho‐Histone3 (pHIS3) and C‐caspase3 revealed that cell survival was repressed by caffeine administration. Whole‐mount in situ hybridization against semaphorin 3A (Sema3A) and neuropilin‐1 (Nrp1) showed that both caffeine and 2,2′‐azobis(2‐methylpropionamidine) dihydrochloride (AAPH, a free radical generator) administration upregulates the expression of both Sema3A and Nrp1. Next, we demonstrated that lens ablation in the developing chicken embryos significantly affected NF‐labeled periocular nerve fascicles and innervation to the central eye region. Subsequently, we used a neuroblastoma cell line to investigate in vitro whether or not Sema3A–Nrp1 signaling exerts a key role on the caffeine‐suppressed neuron survival. Knocking‐down Sema3A through transfection with Sema3A‐siRNA dramatically decreased the responsiveness of cells to caffeine administration, as well as cell apoptosis. We suggest that Sema3A–Nrp1 signaling regulates Trp53 and Cdkn1a through Slit2–Robo1 and Ephb2. Taken together, we speculate here that caffeine‐enhanced reactive oxygen species upregulates Sema3A–Nrp1 expression in the lens and periocular tissues, resulting in corneal cell apoptosis, accompanied by its chemorepellent role on the invasion of the developing cornea by trigeminal sensory fibers.

Retinoic Acid Signaling Plays a Crucial Role in Excessive Caffeine Intake-Disturbed Apoptosis and Differentiation of Myogenic Progenitors

“…In rats, maternal coffee consumption could cause the higher accumulation of caffeine in fetal brain than in serum (Wilkinson and Pollard, 1993). Since 100-600 µM of caffeine could affect neurogenesis in the mouse model (Narod et al, 1991;Marret et al, 1997), we applied 15 µmol/egg of caffeine (each egg contains about 50 ml of albumen and yolk) or EC culture mixed with 300 µM of caffeine based on our previous study (Ma et al, 2012(Ma et al, , 2014(Ma et al, , 2016Wang et al, 2019). We do not deny that the concentration of caffeine is hardly representative of the conditions of high caffeine exposure during pregnancy.…”

“…Moreover, the antibiotics penicillin/streptomycin were added to the EC culture (Chapman et al, 2001). For the elder stage embryos, HH10 (about 1.5-day incubation) pre-incubated chicken embryos were exposed to 5, 10, 15, 30 µmol/egg caffeine, or same the amount of avian saline through injection into windowed eggs in vivo (Ma et al, 2012(Ma et al, , 2014(Ma et al, , 2016Wang et al, 2019). The surviving embryos were harvested for further analyses.…”

“…Using chicken embryos, our previous studies have revealed the effects of caffeine exposure on the development of the fetal nervous system, eye, and angiogenesis (Ma et al, 2012(Ma et al, , 2014(Ma et al, , 2016Wang et al, 2019). Since there were no reports about caffeine influence on the process of myogenesis and somitogenesis, we set out to explore the effects and underlying biological mechanisms of caffeine on the critical time period of myogenesis during the early embryo development using the chicken embryo as a model system.…”