Sally Temple, the current director of the Neural Stem Cell Institute, gave an excellent talk later in the morning and into the afternoon. Although my lunch rendezvous prevented my attending the full symposium, I was able to digest her historical overview. Some key points of interest are below. **If anyone was able to attend this full talk, please comment!**
- In 1887, Wilhelm His identified 2 cell types: germinal cells as rounded cells nearest the lumen (neuronal precursors), and columnar spongioblasts (radial glia).
- In 1987, Jack Price and colleagues were the first to monitor cellular development using retroviral lineage tracing with β-galactosidase. The paper in PNAS is beautiful and succinct, and I highly recommend reviewing it for as an introduction to neural lineages. Arnold Kreigstein's lab has also played a pivotal role in describing neural lineages, which Dr. Temple referred to briefly but I believe warrant more obvious credit in the evolution of this field.
- The last piece I caught was in reference to recent software developed by Andy Cohen's lab: a program for tagging and following cell division in vitro via time-lapse microscopy, and easily translating these divisions into lineage trees. To appreciate this fully, consider the giant leaps forward that technological development have allowed in the evolution of scientific industry. The polymerase chain reaction (PCR) is so simple, and used so ubiquitously now that one has to imagine the grueling hours of restriction enzyme assays and semi-quantitative analyses that were necessary before its development in order to realize its gigantic contribution to throughput. Andy Cohen's software is another such landmark in the progress of scientific industry.
- Commissural neurons grow toward the midline and netrin-1 in the floor plate, then are repelled from the floor plate post-crossing. A family of receptor proteins called Robos are key players in this mechanism. Jaffrey's research indicates compartmental expression of Robo3.1 and Robo3.2 receptors in commissural neurons. Robo3.1 suppresses other Robos so that the axon can grow toward the midline, after which point Robo3.2 expression enhances repulsion from the floor plate. If ther eis no Robo3.1, axons don't cross the midline. If there is no Robo3.2, axons cross and then turn backward and grow aberrantly.
- Jaffrey's lab asked if the switch between Robo3.1 and Robo3.2 was temporally dependent, or in the determined in the floor plate. They made open book explants of spinal cord with no floor plate, and observed protein expression at E10. Although Robo3.2 expression was expected by this time point, only Robo3.1 was expressed. When the floor plate was reintroduced, Robo3.2 expression was observed. This indicated a floor plate mechanism as opposed to a temporal switch within the commissural neurons.
- To confirm this suspicion, the spinal cord floor plate was cultured alone, and the resulting conditioned media was applied to the tips of axons in spinal cord explants without a floor plate. With the floor plate media, the commissural axon tips expressed Robo3.2.
- The next obvious question was what mediated this switch. The group predicted a NMD switch based on indicative NMD sequence in the Robo3.2 gene not observe in the Robo3.1 gene. When they expressed the dominant-negative form of NMD proteins, axons crossed the midline but then grew aberrantly. If the NMD mechanism was blocked, more Robo3.2 was produced and there was increased repulsion to both the floor plate and midline. Therefore, an NMD-dependent mechanism of axon guidance in developing commissural neurons was speculated, although the proteins involved in NMD activity in the floor plate is yet to be determined.