Non-uniform temporal scaling of developmental processes in the mammalian cortex.

The time that it takes the brain to develop is highly variable across animals. Although staging systems equate major developmental milestones between mammalian species, it remains unclear how distinct processes of cortical development scale within these timeframes. Here, we compare the timing of cortical development in two mammals of similar size but different developmental pace: eutherian mice and marsupial fat-tailed dunnarts. Our results reveal that the temporal relationship between cell birth and laminar specification aligns to equivalent stages between these species, but that migration and axon extension do not scale uniformly according to the developmental stages, and are relatively more advanced in dunnarts. We identify a lack of basal intermediate progenitor cells in dunnarts that likely contributes in part to this timing difference. These findings demonstrate temporal limitations and differential plasticity of cortical developmental processes between similarly sized Therians and provide insight into subtle temporal changes that may have contributed to the early diversification of the mammalian brain.

Combining Multiplex Fluorescence in situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections.

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A "neurochemical signature" to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section. Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei. Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.