Different Ectopic Hoxa2 Expression Levels in Mouse Cranial Neural Crest Cells Result in Distinct Craniofacial Anomalies and Homeotic Phenotypes

Providing appropriate positional identity and patterning information to distinct rostrocaudal subpopulations of cranial neural crest cells (CNCCs) is central to vertebrate craniofacial morphogenesis. <i>Hox</i> genes are not expressed in frontonasal and first pharyngeal arch (PA1) CNCCs, whereas a single <i>Hox</i> gene, <i>Hoxa2</i>, is necessary to provide patterning information to second pharyngeal arch (PA2) CNCCs. In frog, chick and mouse embryos, ectopic expression of <i>Hoxa2</i> in <i>Hox</i>-negative CNCCs induced hypoplastic phenotypes of CNCC derivatives of variable severity, associated or not with homeotic transformation of a subset of PA1 structures into a PA2-like identity. Whether these different morphological outcomes are directly related to distinct <i>Hoxa2</i> overexpression levels is unknown. To address this issue, we selectively induced <i>Hoxa2</i> overexpression in mouse CNCCs, using a panel of mouse lines expressing different <i>Hoxa2</i> ectopic expression levels, including a newly generated <i>Hoxa2</i> knocked-in mouse line. While ectopic <i>Hoxa2</i> expression at only 60% of its physiological levels was sufficient for pinna duplication, ectopic <i>Hoxa2</i> expression at 100% of its normal level was required for complete homeotic repatterning of a subset of PA1 skeletal elements into a duplicated set of PA2-like elements. On the other hand, ectopic <i>Hoxa2</i> overexpression at non-physiological levels (200% of normal levels) led to an almost complete loss of craniofacial skeletal structures. Moreover, ectopic <i>Hoxa5</i> overexpression in CNCCs, while also resulting in severe craniofacial defects, did not induce homeotic changes of PA1-derived CNCCs, indicating <i>Hoxa2</i> specificity in repatterning a subset of <i>Hox</i>-negative CNCCs. These results reconcile some discrepancies in previously published experiments and indicate that distinct subpopulations of CNCCs are differentially sensitive to ectopic levels of <i>Hox</i> expression.