Reduced Retinoic Acid Signaling During Gastrulation Induces Developmental Microcephaly

Abstract

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.

Retinoic Acid is Required for Normal Morphogenetic Movements During Gastrulation

“…The delayed rostral migration of the cells expressing organizer-specific genes at the onset of gastrulation is probably one of the earliest effects of reduced RA signaling, one consequence of which might be altered patterning of the body axis due to delayed or defective inductive signaling, as previously suggested ( Gur et al, 2022 ). This possibility seemed likely because reduced RA signaling gave rise to embryos whose neural plates appeared to be mislocalized to the ventral side.…”

“…We speculate that an underlying result of delaying the involution and rostral movement of LEM/PCM cells is that some of the ectodermal cells are likely to have lost their competence to respond to mesoderm derived signals. Support for this abnormal inductive timing was observed in embryos manipulated for reduced RA signaling that developed microcephaly ( Gur et al, 2022 ). The remarkable versatility of Xenopus explants, including precise dissection of the different mesodermal precursor populations, tissue separation and migration assays, will be instrumental to further characterize the signaling pathways, cell adhesion molecules and substrates that are altered by reduced RA signaling.…”

“…This report and previous studies ( Yelin et al, 2005 ) clearly demonstrate that the elongation of DMZ explants subjected to reduced RA signaling is severely impaired, indicating a reduced capacity of these cells to perform convergence-extension. Interestingly, we previously showed that the gsc -positive PCM cells in ethanol treated embryos eventually reach their cranial position ( Yelin et al, 2007 , 2005 ), but the delay likely affected inductive interactions with the neural ectoderm that should have taken place earlier ( Gur et al, 2022 ). Together, these reports indicate that in addition to its early role in the organizer RA also has an early effect on the morphogenetic movements of the involuting mesoderm.…”

“…Other studies have relied on pharmacological inhibitors of RA biosynthesis to reduce endogenous RA levels and shown similar effects, as discussed below. The similar effects of increased and decreased RA signaling on the progression of gastrulation suggests a complex regulatory role of RA during early embryogenesis ( Gur et al, 2022 ).…”

“…Similar results were observed when embryos were treated with ethanol, another RA biosynthesis inhibitor ( Shabtai et al, 2018 ; Yelin et al, 2007 , 2005 ). Two retinaldehyde dehydrogenases that produce RA, ALDH1A2 , and ALDH1A3, are expressed during early gastrulation ( Chen et al, 2001 ; Blentic et al, 2003 ; Halilagic et al, 2003 ; Lupo et al, 2005 ; Shabtai et al, 2018 ; Gur et al, 2022 ). Mutation of each of these genes in vertebrate embryos results in post-gastrula delays and loss of viability ( Niederreither et al, 1999 ; Begemann et al, 2001 ; Sandell et al, 2007 ).…”