No abstract

The retinal pigment epithelium (RPE) exhibits a diverse range of plasticity across vertebrates and is a potential source of cells for the regeneration of retinal neurons. Embryonic amniotes possess a transitory ability to regenerate neural retina through the reprogramming of RPE cells in an FGF-dependent manner. Chicken RPE can regenerate neural retina at embryonic day 4 (E4), but RPE neural competence is lost by embryonic day 5 (E5). To identify mechanisms that underlie loss of regenerative competence, we performed RNA and ATAC sequencing using E4 and E5 chicken RPE, as well as at both stages following retinectomy and FGF2 treatment. We find that genes associated with neural retina fate remain FGF2-inducible in the non-regenerative E5 RPE. Coinciding with fate restriction, RPE cells stably exit the cell cycle and dampen the expression of cell cycle progression genes normally expressed during regeneration, including E2F1. E5 RPE exhibits progressive activation of gene pathways associated with mature function independently of retinectomy or FGF2 treatment, including retinal metabolism, pigmentation synthesis, and ion transport. Moreover, the E5 RPE fails to efficiently repress OTX2 expression in response to FGF2. Predicted OTX2 binding motifs undergo robust accessibility increases in E5 RPE, many of which coincide with putative regulatory elements for genes known to facilitate RPE differentiation and maturation. Together, these results uncover widespread alterations in gene regulation that culminate in the loss of RPE neural competence and implicate OTX2 as a key determinant in solidifying the RPE fate. These results yield valuable insight to the basis of RPE lineage restriction during early development and will be of importance in understanding the varying capacities for RPE-derived retinal regeneration observed among vertebrates.

Tissue growth is a common characteristic of carcinogenesis and regeneration. Here we show that suprabasal expression of human papillomavirus (HPV)16 E6/E7 oncogenes in Tg(K6b-E6/E7) mice, similar to that observed in HPV-infected human tissue, and estradiol increased cervical epithelium growth and ear-hole closure efficiency. Oncogenes in combination with estradiol had a significant contribution to the proliferation of suprabasal cells of cervical epithelium that correlated with an increased expression of keratin genes. Remarkably, long-term treatments with estradiol resulted in evident cellular and tissue abnormalities indicative of a precancerous phenotype. Regenerating ear epithelium of transgenic mice also showed increased suprabasal cell proliferation and expression of keratin genes. Unexpectedly, we observed higher ear regeneration efficiency in adult than in young female mice, which was further increased by E6/E7 oncogenes. Supporting a role of estradiol in this phenomenon, ovariectomy and treatment with an estrogen receptor inhibitor caused a significant reduction in regenerative capacity. Our data suggest that Tg(K6b-E6/E7) mice are unique to mimic the initial stages of HPV-mediated cervical carcinogenesis, and ear regeneration could facilitate the elucidation of mechanisms involved.

Thyrotropin-releasing hormone (TRH) is expressed in the brain, and a few peripheral tissues. Apart from controlling TSH secretion from the pituitary, it has an anorexic effect. TRH is a short-lived intercellular signaling molecule, hydrolyzed by the TRH-degrading ectoenzyme (TRH-DE), a narrow specificity peptidase which only known biological substrate is TRH. TRH-DE activity is mainly detected in brain, with an uneven distribution among regions; at the base of the third ventricle, Trhde is expressed by β2 tanycytes whose end-feet form synaptoid contacts with TRH terminals in the external layer of the median eminence (ME). Tanycyte TRH-DE activity is sensitive to thyroid hormone feedback and energy balance clues and may contribute to adjust the thyroid axis to changing energy needs. To test whether this enzyme is relevant for energy balance, we used mice generated in the B6/129S background in which Trhde exon 2 was deleted and backcrossed to C57BL/6NJ background for 11 generations. TRH-DE activity was reduced in heterozygote (HT) and eliminated in homozygote (KO) compared to wild type (WT) animals. Male mice (70-75 days old) were switched from a standard diet to a high fat high fructose diet (HFFD) for 9 weeks. On HHFD, KO animals ingested less kcal and gained less body weight (BW) than WT animals. Bio-impedance data indicated a lower fat mass in KO mice, compared to WT or HT mice. Glucose tolerance was higher in KO mice than in WT or HT mice. To clarify the specific relevance of tanycyte TRH-DE, we used adeno-associated virus (AAV) vectors in male rats to either knock down (with TRH-DE*, a dominant negative isoform) or overexpress TRH-DE in ME tanycytes. Serum TSH concentration increased when TRH-DE activity decreased, and the converse occurred when TRH-DE activity increased. Thus, TRH-DE activity from β2-tanycytes controls the concentration of TSH in the circulation, probably because it regulates the turnover of TRH before entry into the hypothalamus-pituitary portal vessels. However, 2- or 3-weeks treatment with AAV-TRH-DE or AAV-TRH-DE* did not affect BW. To further test the role of peripheral TRH-DE activity, a phosphinic analogue of TRH (P-TRH) was administered to adult male HFFD mice during 28 days through osmotic pumps connected to an intraperitoneal catheter. P-TRH treatment reduced TRH-DE activity in serum, but not inside the blood brain barrier, compared with mice treated with vehicle; however, treatment with P-TRH did not change food consumption and BW. In conclusion, ablation of TRH-DE impedes diet-induced obesity in male mice, possibly through enhanced thyroid axis activity and amplification of the anorectic effect of TRH. Supported in part by grants from DGAPA-UNAM (PAPIIT IN206712, IN206416, IN212719), and CONACYT (CB154931, CB254960 and PN562).