In‐frame Genetic Disruption of SPECC1L Microtubule‐Interaction Domain Causes Embryonic Tissue Movement and Fusion Defects

Abstract

Movement and fusion events are critical during embryonic development; defects in these events lead to common birth anomalies. For example, failure of neural tube, secondary palate and ventral body wall closure leads to exencephaly, cleft palate, and omphalocele, respectively. Autosomal‐dominant SPECC1L mutations have been identified in patients with syndromic malformations, including hypertelorism, cleft palate and omphalocele. These mutations cluster in the second coiled‐coil (CCD2) and calponin homology (CHD) domains, which interact with microtubule and actin, respectively. To study SPECC1L function in mice, we first generated genomic deletions that resulted in out‐of‐frame truncations. Homozygous mutants for these truncations, including those lacking CCD2 and CHD, died shortly after birth without cleft palate or omphalocele. We then generated a series of targeted in‐frame deletions in CCD2, homozygotes for which also exhibited perinatal lethality but now with ~50% exencephaly, ~50% cleft palate, and ~50% omphalocele. While the omphalocele phenotype segregated equally with the other two phenotypes, exencephaly and cleft palate were never observed in the same embryo, suggesting antagonistic tissue mechanics between neural tube and palate closure. Consistently, we found that the oral cavity was narrower in mutant embryos with exencephaly, allowing for the palatal shelves to close eventually. We have shown previously that SPECC1L deficiency leads to impaired cell speed and directionality. We now show that subcellular SPECC1L‐ΔCCD2 protein distribution was abnormal, accumulating asymmetrically and associating almost exclusively with filamentous actin. Thus, loss of SPECC1L interaction with microtubules – while maintaining actin association – was more detrimental to cell and tissue movement than loss of SPECC1L protein entirely. Taken together, our results showed that in‐frame perturbations of CCD2 in SPECC1L protein specifically affect embryonic closure of several tissues, suggesting that human SPECC1L CCD2 missense mutations are gain‐of‐function. Our data also revealed a novel role for SPECC1L cross‐linking of actin and microtubule cytoskeletons in embryonic tissue movement and fusion events.Support or Funding InformationThis project was supported in part by the National Institutes of Health grants DE026172 (I.S.). Core support and pilot funding was also provided by National Institutes of Health Center of Biomedical Research Excellence (COBRE) grant (P20 GM104936), Kansas IDeA Network for Biomedical Research Excellence (INBRE) grant (P20 GM103418), and Kansas Intellectual and Developmental Disabilities Research Center (IDDRC) grant (P30 HD 002528).