Neural tube defects (NTDs) are among the most common structural birth defects in humans and result in long- term disability or even death; yet, the underlying genetic causes remain largely unknown. Addressing this gap in knowledge is best achieved in the context of understanding the mechanisms mediating both normal and abnormal development. Experiments conducted nearly 40 years ago indicate that expansion of the cranial mesenchyme, a cell population that resides beneath the neural plate, is required for elevation of the cranial neural folds and neural tube closure. Yet, little is known regarding how expansion of the cranial mesenchyme drives neural fold elevation and how this process can be disrupted to cause NTDs. Moreover, few genes have been implicated in this process. In this proposal we present a novel mouse model of NTDs with a mutation in the Hectd1 gene and describe approaches that will significantly advance our understanding of how cranial mesenchyme expansion can be disrupted contributing to NTDs. Based on our previous and preliminary data we hypothesize that eHSP90 secreted from Hectd1 mutant NC-CM stimulates increase movement of the CM interfering with expansion of the PM-CM and disrupting neural fold elevation (Specific Aims 1 & 2). We further hypothesize that HECTD1 sequence variants associated with human NTDs employ the same pathogenic mechanism (Specific Aim 3). We will test these hypotheses using a combination of innovative tools including: (1) advanced imaging approaches to visualize, at unprecedented resolution expansion of the cranial mesenchyme in real time during neural fold elevation, (2) fluorescently labeled eHSP90 probes to elucidate spatial and temporal patterns of pathogenic eHSP90 production, (3) an ex vivo cranial mesenchyme explant assay amenable to experimental manipulation, (4) a novel allelic series of mouse lines and (5) pharmacological inhibitors to test whether eHSP90 mediates failure of cranial mesenchyme expansion and neural fold elevation in Hectd1 mutant embryos. These innovative tools will be combined with well-established conditional genetic, histological and embryological approaches to delineate, in unprecedented detail, expansion of cranial mesenchyme in normal neural fold elevation and how this process goes awry during failed cranial mesenchyme expansion responsible for NTDs in the Hectd1 mutant embryo. This information will be used to determine the impact of predicted pathogenic sequence variants of HECTD1 identified in human NTD cases and ascertain if variants disrupt HECTD1 function and contribute to NTDs in human patients.
This proposal aims to elucidate the molecular and cellular mechanisms underlying Neural tube defects (NTDs), that are among the most common birth defects in humans. To achieve this, we will utilize a novel mouse model of NTDs that has a failure of cranial mesenchyme expansion and neural fold elevation. Our findings will yield mechanistic insights into the causes of NTDs, elucidate the contribution of an understudied process driving neural tube closure and implicate an important candidate gene for genetic counseling.
