Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology and disease.Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here we present an optimized method for non-invasive imaging of Drosophila melanogaster at high resolution using micro computed tomography (µ-CT). Our method allows for rapid processing of intact animals at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. We then use the power of µ-CT imaging to model human diseases through the characterization of microcephaly in the fly. Our work demonstrates that µ-CT is a versatile and accessible tool that complements standard imaging techniques, capable of uncovering novel biological mechanisms that have remained undocumented due to limitations of current methods.

Cell division is critical for development, organ growth, and tissue repair. The later stages of cell division include the formation of the microtubule (MT)-rich central spindle in anaphase, which is required to properly define the cell equator, guide the assembly of the acto-myosin contractile ring, and ultimately ensure complete separation and isolation of the two daughter cells via abscission. Much is known about the molecular machinery that forms the central spindle, including proteins needed to generate the antiparallel overlapping interzonal MTs. One critical protein that has garnered great attention is Protein Regulator of Cytokinesis 1 (PRC1), or Fascetto (Feo) in Drosophila, which forms a homodimer to crosslink interzonal MTs, ensuring proper central spindle formation and cytokinesis. Here, we report on a new direct protein interactor and regulator of Feo we named Fascetto Interacting Protein (FIP). Loss of FIP results in a significant reduction in Feo localization, rapid disassembly of interzonal MTs, and several cytokinesis defects. Simultaneous reduction in Feo and FIP results in tumor-like, DNA-filled masses in the brain. In aggregate our data show that FIP functions upstream of, and acts directly on, Feo to ensure fully accurate cell division.

Microcephaly is a developmental failure to achieve proper brain size and neuron number. Mutations in diverse genes are linked to microcephaly, including several with DNA damage repair (DDR) functions; however, it is not well understood how these DDR gene mutations limit brain size. One such gene is TRAIP, which has multiple known functions in DDR. We characterized the Drosophila ortholog Traip, finding that loss of Traip causes a brain-specific defect in the Mushroom Body (MB). Traip mutant (traip-) MBs had reduced size and fewer neurons, but no neurodegeneration, consistent with human primary microcephaly disorders. Reduced neuron numbers in traip- were explained by premature caspase-dependent cell death of MB neuroblasts (MB-NBs). Many traip- MB-NBs had prominent chromosome bridges in anaphase, along with polyploidy, aneuploidy, or micronuclei. We found no evidence for an interphase DNA repair role for Traip in MB-NBs; instead, proper MB development requires Traip function during mitosis, where Traip localizes to centrosomes and mitotic spindles. Our results suggest that proper brain size is ensured by the recently described role for TRAIP in unloading stalled replication forks in mitosis, which suppresses DNA bridges and neural stem cell death to promote proper neuron number. Further, the mitotic nature of traip- MB-NB defects and Traip localization suggest a closer etiological link between DDR microcephaly genes like Traip and the centrosome/spindle-related genes more commonly associated with microcephaly.