Retinoic acid fluctuation activates an uneven, direction-dependent network-wide robustness response in early embryogenesis

Abstract

Retinoic acid (RA) is a central developmental signal whose perturbation results in teratogenic outcomes. We performed transient physiological RA signaling disturbances during embryogenesis followed by kinetic RNAseq and high-throughput qPCR analysis of the recovery. Unbiased comparative pattern analysis identified the RA network as a key differentially regulated module aimed at achieving RA signaling robustness. We analyzed this module using a principal curve-based approach determining clutch-wise variability, and organized the results into a robustness efficiency matrix comparing the shifts in RA feedback regulation and hox gene expression. We found that feedback autoregulation was sensitive to the direction of the RA perturbation: RA knock-down exhibited an upper response limit, whereas RA addition did not activate a feedback response below a threshold. These results suggest an asymmetric capacity for robust feedback control of RA signal during early development based on genetic polymorphisms, likely a significant contributor to the manifestation of developmental defects.

Enhanced loss of retinoic acid network genes in Xenopus laevis achieves a tighter signal regulation

“…In a recent extensive data mining effort searching the KEGG and Xenbase databases [44,52] and the literature, we assembled the putative components of the RA metabolic and signaling network during early embryogenesis [1, 6,9]. To determine the composition of the RA network during gastrula in Xenopus laevis embryos we assessed which components are expressed during this developmental stage based on analysis of transcriptomic data sets [27,53,54] and corroborated the gastrula expression by quantitative RT-PCR (qPCR) [55]. This view of the RA metabolic and signaling network exhibits a rather uncommon characteristic that for each enzymatic step, multiple genes encoding enzymes have been described that are capable of performing the same reaction (Fig.…”

“…Analysis of the RA content of Xenopus laevis embryos during early gastrula estimated that they contain about 100-150 nM RA [71][72][73][74][75][76][77]. To perform physiologically-relevant manipulations of RA we increased that level by about 10%-25% using 10 nM and 20 nM treatments, respectively [27]. Embryos were treated from late blastula to early gastrula (st. 10.25) and collected for expression analysis by qPCR.…”

“…The selfregulation of the RA metabolic and signaling network to maintain or restore normal signaling levels is widely accepted, and the transcriptional responses of aldh1a2, cyp26a1, dhrs3, and rdh10 to increased RA levels are the basis of this suggestion [18][19][20][21][22][23][24]70,78]. In a recent study, we performed a detailed analysis of the RA responsiveness and requirement for the RA network genes expressed during early gastrula [27]. While some genes exhibited robust and concentration-dependent responses, others showed no significant changes in response to RA fluctuations.…”

“…Maintenance of normal RA signaling levels is central for the prevention of the teratogenic effects of increased or decreased RA signaling levels [13,21,71,[79][80][81][82]. Thus, homoeolog gene loss might be a "solution" to achieve tighter signaling regulation, i.e., robustness [27]. Several models can be suggested that could drive this gene loss to achieve higher RA signaling robustness.…”

“…These differential responses to RA fluctuation are part of the mechanism to keep this critical signal within an appropriate, non-teratogenic range [27]. One possibility for the preferential loss of homoeologs in genes encoding RA network components is the selective or non-selective reduction of gene copies to achieve tighter regulation of the signal.…”