Identification of proteins that are developmentally regulated during early cerebral corticogenesis in the rat

Abstract

Between embryonic day 14 (E14) and embryonic day 21 (E21), the rat neopallium develops from a relatively homogeneous band of mitotic precursor cells into a complex laminated structure containing diverse classes of neurons. In order to identify some of the molecular components underlying this process, 2-dimensional PAGE was used to compare proteins expressed before cortical neurons are born (E14) with those expressed during neurogenesis and neuronal migration (E17 and E21). This approach has permitted the identification of 15 proteins that show greater than 3-fold changes in their rate of accumulation between E14 and E21. Six proteins show consistent up-regulation, ranging from 3.2- to 10.7-fold. Five proteins show consistent down-regulation ranging from 9- to 22-fold. Four proteins that appear at E21 are not detectable on fluorograms of E14 cortex, even after long exposures, and thus are up-regulated more than 200-fold from E14 to E21 and may be considered to appear de novo. The molecular weights and isoelectric points of most of these 15 suggest that they are previously unreported, developmentally regulated proteins. Comparisons of gels of cortex to gels of lung and heart suggest that several of these proteins are enriched in brain relative to non-neural tissues. This analysis also indicates that, despite the large morphogenic changes observed during this developmental period, few proteins (less than 3%) among the total spectrum analyzed show large changes in their rates of synthesis.

Cdk5‐mediated phosphorylation of CRMP‐2 enhances its interaction with CaV2.2

“…Initially identified in chick sensory neurons as a protein responsible for growth cone retraction in response to negative guidance signals in the semaphorin 3A (Sema3A) pathway [1], CRMP-2 analogues were subsequently identified in Caenorhabditis elegans (uncoordinated protein-33 (Unc-33)) [2], in rodents ((named turned on after development 64 (TOAD-64)) in rats and Unc-33 like phosphoprotein (Ulip) in mice) [3][4][5], in humans (HUlip) [6,7] and in Drosophila Melanogaster [8]. Together with its established roles in neurite growth and retraction and kinesin-dependent axonal transport, mapping the CRMP-2 interactome has revealed previously unappreciated functions including affecting microtubule dynamics, protein endocytosis and vesicle recycling, as well as synaptic assembly (see reviews by Hensley [9] and Strittmatter [10]).…”

Changes in protein expression during neural development analyzed by two‐dimensional gel electrophoresis

“…In spite of these limitations, the authors were able to identify several area-specific proteins. Most earlier development studies of brain development detected few changes during this early period, or only detected changes in known, abundant proteins such as tubulin [26, 43, 441. Because of this limited success, we introduced several methodological features that we hoped would increase the likelihood of identifying proteins that played a significant role in the development of a complex tissue like the brain [6]. First, we restricted our analysis to one specific brain area, the cerebral cortex.…”

“…To further reduce the complexity of the analysis and focus on the cell fraction most likely to contain proteins involved in processes requiring cell-cell interactions, membrane and membrane-associated proteins were prepared and in our initial analysis soluble proteins were ignored. Approximately 1000 proteins were resolved on 2-D gels and over 400 proteins were quantitatively analyzed using a BioImage Gel scanner and software [6].…”

“…This can be a particularly serious problem for studies of proteins that are expressed differentially in the brain, either between brain areas or between different times in development, where low abundance proteins may play important roles in developmental or cell-type-specific processes. While 2-DE offers the sensitivity to detect rare molecules, which may account for as little as 0.01-0.0005% of total mRNA in a cell [6,141, subsequent purification and characterization of these proteins remains a daunting task. Despite these limitations, 2-DE has led to the purification of neural proteins that in some cases are the products of entirely new classses of genes, as described below.…”

“…2-DE has been used to find cell phenotype specific markers by comparing the spectrum of proteins expressed in different neural cell lines [lo, 11, 251. Developmental studies of a variety of central nervous system (CNS) areas and tissues have compared the patterns of proteins synthesized at different embryonic and adult stages of neocortex or other brain regions [6,7,[26][27][28][29], as well as proteins regulated by specific pharmacological agents [3, 301. Here, we will attempt to highlight a few of the large number of studies using 2-DE in neural development, concentrating on some of the innovative approaches that have used well-defined systems to concentrate an analysis on particular cell classes, protein families, or subcellular compartments to identify proteins of interest. We will then focus in more detail on studies that have been carried out in our lab to identify novel proteins associated with the acquisition of neuronal phenotype in the developing cerebral cortex.…”