(A) Cartilage structures of the zebrafish neuro- and viscerocranium at 5 dpf (light and dark blue, respectively), ventral perspective. A number of these structures is used as a homolog to human skeletal features: Meckel’s cartilage for the lower jaw, the ethmoid plate for the hard palate and the palatoquadrate for the middle ear. (B) Intramembranous bones of the 5 dpf zebrafish head, dorsal view.

(A) Wildtype ethmoid plate with associated stacking of chondrocytes. (B) Redrawn examples of ethmoid plate defects observed in zebrafish environmental factor exposure studies (Kuchler et al., 2018; Liu et al., 2020) (Methotrexate 100 μM, Dexamethasone 200 μM, retinoic acid 5 nM and hypoxia). A broad phenotypic range of ethmoid plate defects is observed, indicating that development of the ethmoid plate is affected variously by different compounds. Mild phenotypes include a rough edge to the anterior ethmoid plate, and differential cell morphology (round vs. elongated) and chondrocyte stacking is disordered, drawn from Liu et al. (2020) (scale bar 5 μm). At the other end of the spectrum, phenotypes are observed in which ethmoid plate structures are (partially) missing, indicating effects on migration, differentiation and survival CNCCs. Moreover, some factors affect early as well as late craniofacial development, and various effects can occur though differential exposure times during specific sensitivity windows.

Meckel’s-palatoquadrate (M-PQ) angle is proposed as a reliable high-throughput standard parameter to assess craniofacial outcomes after single or mixed compound exposures. The measurements can be easily obtained by imaging of cartilage stained larvae and proved to be informative on a broad spectrum of craniofacial malformations. The M-PQ angle is especially affected in craniofacial malformations such as microcephaly and micrognathia.

General summary showing the environmental factors smoking, alcohol use, vitamin imbalance, drug use, (xeno) estrogens and pesticides. These factors (represented in the upper part of the figure) differentially affect critical developmental processes such as the formation, survival, delamination, migration, condensation, and differentiation of CNCCs (represented in the lower part of the figure). Evidence of interactions between environmental factors that result in craniofacial malformations has been reported as well, these are indicated by black arrows. Exposure to environmental factors often results in aberrant signaling of essential pathways in craniofacial development including SHH, TGF, FGF, BMP, RA, and WNT. Moreover, gene mutations in these pathways can also interact with environmental factors, complicating the etiology. Known GxE interactions are indicated in this figure with red dotted arrows.