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Retinoic acid (RA) is a central signaling molecule regulating multiple developmental decisions during embryogenesis. Excess RA induces head malformations, primarily by expansion of posterior brain structures at the expense of anterior head regions, i.e., hindbrain expansion. Despite this extensively studied RA teratogenic effect, a number of syndromes exhibiting microcephaly, such as DiGeorge, Vitamin A Deficiency, Fetal Alcohol Syndrome, and others, have been attributed to reduced RA signaling. This causative link suggests a requirement for RA signaling during normal head development in all these syndromes. To characterize this novel RA function, we studied the involvement of RA in the early events leading to head formation in Xenopus embryos. This effect was mapped to the earliest RA biosynthesis in the embryo within the gastrula Spemann-Mangold organizer. Head malformations were observed when reduced RA signaling was induced in the endogenous Spemann-Mangold organizer and in the ectopic organizer of twinned embryos. Two embryonic retinaldehyde dehydrogenases, ALDH1A2 (RALDH2) and ALDH1A3 (RALDH3) are initially expressed in the organizer and subsequently mark the trunk and the migrating leading edge mesendoderm, respectively. Gene-specific knockdowns and CRISPR/Cas9 targeting show that RALDH3 is a key enzyme involved in RA production required for head formation. These observations indicate that in addition to the teratogenic effect of excess RA on head development, RA signaling also has a positive and required regulatory role in the early formation of the head during gastrula stages. These results identify a novel RA activity that concurs with its proposed reduction in syndromes exhibiting microcephaly., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gur, Bendelac-Kapon, Shabtai, Pillemer and Fainsod.)

Robustness is a feature of regulatory pathways to ensure signal consistency in light of environmental changes or genetic polymorphisms. The retinoic acid (RA) pathway, is a central developmental and tissue homeostasis regulatory signal, strongly dependent on nutritional sources of retinoids and affected by environmental chemicals. This pathway is characterized by multiple proteins or enzymes capable of performing each step and their integration into a self-regulating network. We studied RA network robustness by transient physiological RA signaling disturbances followed by kinetic transcriptomic analysis of the recovery during embryogenesis. The RA metabolic network was identified as the main regulated module to achieve signaling robustness using an unbiased pattern analysis. We describe the network-wide responses to RA signal manipulation and found the feedback autoregulation to be sensitive to the direction of the RA perturbation: RA knockdown exhibited an upper response limit, whereas RA addition had a minimal feedback-activation threshold. Surprisingly, our robustness response analysis suggests that the RA metabolic network regulation exhibits a multi-objective optimization, known as Pareto optimization, characterized by trade-offs between competing functionalities. We observe that efficient robustness to increasing RA is accompanied by worsening robustness to reduced RA levels and vice versa. This direction-dependent trade-off in the network-wide feedback response, results in an uneven robustness capacity of the RA network during early embryogenesis, likely a significant contributor to the manifestation of developmental defects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Parihar, Bendelac-Kapon, Gur, Abbou, Belorkar, Achanta, Kinberg, Vadigepalli and Fainsod.)