These small, high density cells were additionally distinguishable from the surrounding and larger epithelial cells based expression of pcp, a pupal cuticle gene; only the larger cells expressed pcp. Mitotic activity did not initiate until about 20 24 hrs a.p. and then only at a low level, increasing significantly around 40 48 hrs a.p. and continuing to around 72 80 hrs a.p.
[See Additional file 1 for Supplementary Figure] The success of sequencing based expression profiling is a result of the high sensitivity of transcriptome reads in detecting and counting these abundant genes.
This is even more the case when we limit the extension to analytically based expressions with a minimum of empirical information.
Here we have used a codon-optimised Cas9 nuclease and dual ribozyme-based expression of a single guide RNA (sgRNA) to induce mutations.
Plant-based expression of a number of these candidates has already been achieved, including HIV-1 gp120 envelope glycoprotein [ 8], p24 core protein [ 9] and the regulatory Tat protein [ 10].
While these studies laid the groundwork for generating utilization pathways with titratable responses, they called for extensive genetic manipulations (i.e., plasmid-based expression of a transporter, disruption of multiple genetic loci) that may be difficult to achieve in nonmodel organisms and may not reflect ideal configurations for titratable control.
In another study, Piezo1's N-terminal "propeller" domain was proposed to constitute an intrinsic mechanosensitive module based on expression of a chimera between a pore-forming domain of the mechanically insensitive ASIC1 channel and Piezo1 (Zhao et al., 2016).
Alternatively, a subset of cell bodies can be visualized based on expression of a fluorescent protein marker [47].
Strategies for enrichment have included manual dissection of beating areas [10], [11], Percoll® density gradient sedimentation [7], [12], and fluorescence activated cell sorting (FACS) of cells based on expression of a fluorescent reporter protein from cardiomyocyte gene promoters [11], [13].
They fall apart in three clusters, based on expression of a series of neuronal differentiation genes in clusters C and D and expression of retinal differentiation genes in clusters D and E (see below and Figure 2).
A method for identifying neurons based on expression of a genetically-encoded reporter would provide a powerful and more general approach for probing the role of different neuronal populations in brain circuits.
