Xenopus embryonic epidermis as a mucociliary cellular ecosystem to assess the effect of sex hormones in a non-reproductive context

Abstract

BackgroundHow important are sexual hormones beyond their function in reproductive biology has yet to be understood. In this study, we analyzed the effects of sex steroids on the biology of the embryonic amphibian epidermis, which represents an easily amenable model of non-reproductive mucociliary epithelia (MCE). MCE are integrated systems formed by multiciliated (MC), mucus-secreting (MS) and mitochondrion-rich (MR) cell populations that are shaped by their microenvironment. Therefore, MCE could be considered as ecosystems at the cellular scale, found in a wide array of contexts from mussel gills to mammalian oviduct.ResultsWe showed that the natural estrogen (estradiol, E2) and androgen (testosterone, T) as well as the synthetic estrogen (ethinyl-estradiol, EE2), all induced a significant enhancement of MC cell numbers. The effect of E2, T and EE2 extended to the MS and MR cell populations, to varying degrees. They also modified the expression profile of RNA MCE markers, and induced a range of “non-typical” cellular phenotypes, with mixed identities and aberrant morphologies, as revealed by imaging analysis through biomarker confocal detection and scanning electron microscopy. Finally, these hormones also affected tadpole pigmentation, revealing an effect on the entire cellular ecosystem of the Xenopus embryonic skin.ConclusionsThis study reveals the impact in vivo, at the molecular, cellular, tissue and organism levels, of sex steroids on non-reproductive mucociliary epithelium biogenesis, and validates the use of Xenopus as a relevant model system in this field.

CDC20B is required for deuterosome-mediated centriole production in multiciliated cells

“…Whole-mount chromogenic in situ hybridization and whole-mount fluorescent in situ hybridization (FISH) was performed as detailed by Marchal and colleagues 54 , and Castillo-Briceno and Kodjabachian 55 , respectively. For single staining, all RNA probes were labeled with digoxigenin.…”

Lineage commitment of embryonic cells involves MEK1-dependent clearance of pluripotency regulator Ventx2

“…FLDX was detected by incubation with alkaline phosphatase conjugated anti-fluorescein antibody (dilution 1/10,000; Roche). Sections were prepared and immune staining was performed as previously described ( Cibois et al, 2015 ; Castillo-Briceno and Kodjabachian, 2014 ). Primary antibodies were as follows: anti-Myc (9E10; Santa Cruz Biotech, dilution 1/300 RRID: AB_627268 ), anti-GFP (GFP-1020; 2BScientific; dilution 1/1000 RRID: AB_10000240 ), anti-γ-Tubulin (ab16504; Abcam; dilution 1/1000 RRID: AB_443396 ), anti-phospho-MEK1 (9121; Cell Signaling Technology; dilution 1/400 RRID: AB_331648 ).…”

“…Mk-MO injected and uninjected (controls) embryos were grown until gastrula stage 10.5, and then processed for RT-qPCR, as previously described ( Scerbo et al, 2012 ; Castillo-Briceno and Kodjabachian, 2014 ). Ten embryos per biological replicate were used.…”

BMP signalling controls the construction of vertebrate mucociliary epithelia

“…FISH on sections was an adaptation of the whole-mount FISH method. Immunohistochemical staining was performed on whole embryos as described previously (Castillo-Briceno and Kodjabachian, 2014) and adapted for sections.…”

“…Mucociliary epithelia (MCE) are encountered in many bilaterians, where they perform a variety of functions, including animal movement, food particle capture and ingestion, gamete transportation, and protection against pollutants and infectious agents (Castillo-Briceno and Kodjabachian, 2014;Lyons et al, 2006;Rompolas et al, 2013;Silverman et al, 1999;Werner and Mitchell, 2012). One of the most widely studied MCE is the lining of the human upper airways, which ensures the clearance of inhaled noxious particles by means of the mucociliary escalator.…”