Laminar Organization of the Anterior Olfactory Nucleus -The Interplay Between Neurogenesis Timing and Neuroblast Migration

Eduardo Martin-Lopez Bowen Brennan Marion Lefèvre Natalie J Spence Kimberly Han Charles A Greer ^*

• Department of Neurosurgery, School of Medicine, Yale University, New Haven, United States

The anterior olfactory nucleus (AON) is a laminar structure embedded within the olfactory peduncle which serves as the conduit for connectivity between the olfactory bulb (OB) and the central processing centers of the brain. The largest portion of the AON is a ring of neurons and fibers that surround the core of the peduncle, the pars principalis (AONpP). The AONpP is further subdivided into an outer plexiform layer, or layer 1 (L1), that contains axons and dendrites, and an inner cell zone, or layer 2 (L2), formed by densely packed pyramidal cells. Relative to other regions of the olfactory system, the development of the AON remains poorly understood. We performed injections of thymidine analogs in pregnant mice from E10 to E18 to determine the timeline of AON neurogenesis. Our analyses established that the earliest neurons targeted to the AON laminae arose at E10 with neurogenesis peaking at E13. In L1, we found a caudal-to-rostral neurogenic gradient not detected in L2. Quantification across the cardinal axes showed no gradients in L2 and a medial-to-lateral gradient for L1. Using immunohistochemistry, we found that AON neurons express the most common cortical markers Tbr1, Ctip2, NeuroD1, Sox5 and Cux1+2 at adult stages without laminar distinction. Tbr1 and NeuroD1 first appeared embryonically at E12, while Ctip2 and Sox5 were present at E13, following a dorsal-ventral pattern. Cux1+2 was not detected embryonically. To better understand migration and differentiation of the AON neurons, we labeled AON precursors using in utero electroporations with the piggyBac transposon into the rostral lateral ganglionic eminence, the embryonic source of AON neurons. Our data revealed that AON neuroblasts use a scaffold of radial glia to migrate to their final destinations in both L1 and L2 through a caudal-to-rostral migratory gradient. For the first time, our data show a comprehensive timeline for the AON neurogenesis and neuroblast migration in mouse. These data are crucial to understanding the embryonic formation and relationship of relay stations along the olfactory pathway.