Exploring the link between prenatal nutrition and congenital craniofacial malformations
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Exploring the link between prenatal nutrition and congenital craniofacial malformations

A recent study published in Nature Communications conducted a comprehensive genome-wide analysis, using CAGE-sequencing on the facial mesenchyme of human embryos and comparing the results with genes linked to facial appearance by GWAS, to understand the complex development of craniofacial skeletal structures and improve treatments for congenital craniofacial malformations.

Study: The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling.  Image Credit: SeventyFour/Shutterstock.comStudy: The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling. Image Credit: SeventyFour/Shutterstock.com


Facial recognition is a crucial aspect of most social communications in humans, and congenital craniofacial malformations profoundly impact social interactions. The viscerocranium harbors crucial structures and supports sensory organs. The interplay between genetic, environmental, and epigenetic factors forms the craniofacial skeleton.

Alcohol consumption during pregnancy is a known factor influencing facial morphogenesis. The viscerocranium forms from neural crest cell (NCC) descendants in all Gnathostomataincluding mice, zebrafish, and humans.

Several NCC-derived mesenchymal subpopulations condense and differentiate into osteoblasts and chondrocytes. The shapes of the mesenchymal chondrogenic condensations determine the shapes of craniofacial skeletal elements.

A complex interplay between the facial ectoderm, placodes, NCCs, neuroepithelium, and endoderm orchestrates accurate viscerocranium sculpting; this involves continuous expression changes in thousands of genes.

Moreover, the proper migration and differentiation of NCCs and their interactions with neighboring tissues involve conserved signaling pathways.

The signaling pathways and related morphogens form a system responsible for viscerocranium sculpting. Nevertheless, the capability of the signaling pathways to sense and integrate environmental cues/signals into facial morphogenesis remains unknown.

Nutritional sensing by the mTORC1 pathway is highly evolutionarily conserved. Further, changes in mTORC1 activity can influence the shape of craniofacial structures.

The study and findings

The present study hypothesized and tested that mTORC1 signaling may mediate interactions between environmental cues and craniofacial morphogenesis.

First, human embryonic facial material was sequenced to identify actively transcribed enhancers involved in facial development between gestational weeks 3 and 12.

Enhancers were cross-checked and enriched against those previously identified. The team noted enrichment in phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT)/mTORC1/autophagy pathway components.

Next, the mTORC1 signaling pathway was manipulated during facial development to investigate the mechanisms underlying craniofacial shaping.

To this end, mTORC1 signaling was activated by crossing tuberous sclerosis 1 (Tsc1)-floxed mice with SRY-box transcription factor 10 (Sox10)-CreERT2 strain, wherein a tamoxifen pulse on embryonic day 8.5 (E8.5) induces recombination in NCCs.

Micro-computed tomography images revealed the altered thickness of skeletal elements and minor developmental abnormalities by E17.5.

Previously, the researchers demonstrated that the craniofacial shape in mice is established at mesenchymal condensation. Embryos were stained on E12.5 to illustrate the shape of mesenchymal condensations.

The overall shape remained preserved, but thicker nasal capsule compartments were observed. When Tsc1 was ablated, nasal chondrocyte clones appeared as large bulky clusters with extensive dispersion and misalignment.

These findings indicated that the activation of the mTORC1 pathway modulated chondrogenic condensation and clonal arrangement. Further analyzes suggested that the mTORC1 pathway was involved in craniofacial shaping at a stage before or during chondrogenic condensations.

Next, mTORC1 was inhibited using rapamycin in pregnant dams on E10.5; this led to a slightly elongated snout in embryos on E17.5.

Moreover, reduced thickness of chondrogenic mesenchymal condensations was observed on E12.5. Next, the team investigated whether these effects of mTORC1 were conserved among species.

As such, they selected zebrafish and exposed the larvae to rapamycin at several time points during development. Rapamycin exposure before or during chondrogenic condensation did not affect the overall facial skeleton size, although the cartilaginous structures were narrowed.

Further, when exposed before condensation, a slight curvature of the ethmoid plate was observed, with repositioning of various cartilage elements.

Next, the researchers examined whether alterations in mTORC1 activity through diets with varying protein levels might influence the offspring’s craniofacial shaping. Accordingly, pregnant mice consumed isocaloric diets with 4%, 20%, or 40% protein, beginning E6.5.

The lowest and most pronounced mTORC1 activity was observed in embryos from low- and high-protein diet recipients.

Protein levels in the maternal diet influenced Meckel’s cartilage length and the nasal capsule’s width and length in embryos. The thickness of the nasal capsule cartilage increased with higher protein in the diet.


In sum, the study illustrated the mechanisms of mTORC1-dependent shaping of craniofacial skeletal elements in mice and zebrafish, which mainly occurs along with chondrogenic mesenchymal condensations, with fine-tuning during the intercalation of chondroprogenitors.

Moreover, protein content in maternal diets modulated the mTORC1 activity in mouse embryos. Overall, the results offer insights into the craniofacial shaping and its phenotypic plasticity.

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