MEMO1 drives cranial endochondral ossification and palatogenesis

Abstract

The cranial base is a component of the neurocranium and has a central role in the structural integration of the face, brain and vertebral column. Consequently, alteration in the shape of the human cranial base has been intimately linked with primate evolution and defective development is associated with numerous human facial abnormalities. Here we describe a novel recessive mutant mouse strain that presented with a domed head and fully penetrant cleft secondary palate coupled with defects in the formation of the underlying cranial base. Mapping and non-complementation studies revealed a specific mutation in Memo1 - a gene originally associated with cell migration. Expression analysis of Memo1 identified robust expression in the perichondrium and periosteum of the developing cranial base, but only modest expression in the palatal shelves. Fittingly, although the palatal shelves failed to elevate in Memo1 mutants, expression changes were modest within the shelves themselves. In contrast, the cranial base, which forms via endochondral ossification had major reductions in the expression of genes responsible for bone formation, notably matrix metalloproteinases and markers of the osteoblast lineage, mirrored by an increase in markers of cartilage and extracellular matrix development. Concomitant with these changes, mutant cranial bases showed an increased zone of hypertrophic chondrocytes accompanied by a reduction in both vascular invasion and mineralization. Finally, neural crest cell-specific deletion of Memo1 caused a failure of anterior cranial base ossification indicating a cell autonomous role for MEMO1 in the development of these neural crest cell derived structures. However, palate formation was largely normal in these conditional mutants, suggesting a non-autonomous role for MEMO1 in palatal closure. Overall, these findings assign a new function to MEMO1 in driving endochondral ossification in the cranium, and also link abnormal development of the cranial base with more widespread effects on craniofacial shape relevant to human craniofacial dysmorphology.

AP-2α and AP-2β cooperatively orchestrate homeobox gene expression during branchial arch patterning

“…As previously stated (Van Otterloo et al, 2016), this study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Colorado -Denver.…”

“…For western blot analysis, E12.5 whole heads (Sox2:CRE experiments) or E10.5 craniofacial prominences (Wnt1:CRE experiments) were lysed, on ice, in RIPA buffer containing protease inhibitors (ThermoScientific Protease Inhibitor Cocktail). Briefly, as described previously (Van Otterloo et al, 2016), tissue was first minced with a disposable pestle followed by further disruption using a tissue homogenizer (Pro200 homogenizer, PRO Scientific). Following homogenization, samples were allowed to lyse on ice for ∼30 min.…”

“…Embryos were collected at appropriate time points and processed using Alizarin Red and Alcian Blue stains as previously described (Van Otterloo et al, 2016).…”

“…The anti-neurofilament antibody (Dodd et al, 1988) was used at a 1:100 dilution (IgG clone 2H3, obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA, USA). Hematoxylin and Eosin (H&E) staining was carried out as previously described (Cardiff et al, 2014;Van Otterloo et al, 2016). In situ hybridization was carried out as previously described (Simmons et al, 2014;Van Otterloo et al, 2016).…”

Differential 3’ Processing of Specific Transcripts Expands Regulatory and Protein Diversity Across Neuronal Cell Types

“…Pcp2/L7-Cre, Neurod1-Cre and Math1-Cre mice were obtained from The Jackson Laboratory (catalog numbers: 004146, 028364 and 011104). Memo1 mutant mice were generated using ES-cells from the European Conditional Mouse Mutagenesis (EUCOMM) repository as described previously (Van Otterloo et al, 2016). Mouse studies were allocated by genotype, and were not blinded.…”