3 Retinoic Acid-Regulated Target Genes During Development …
79
Future Directions
The combination of NGS with a variety of modern DNA/RNA enrichment technologies has provided a means for interrogating genome-wide transcriptional activity
(RNA-seq), functional protein–genome interactions (ChIP-seq), chromatin accessibility (DNase-seq, FAIRE-seq, ATAC-seq), and more recently, the three-dimensional
organization of chromatin (Hi-C, ChIA-PET). Some of these approaches have been
carried out using ECCs and ESCs.
Today, the challenge is to reconstruct the RA-regulated gene networks during the
development of specialized embryonic tissues. The aim is to perform a meta-analysis,
combining the results from multiple studies (ECCs, ESCs and specialized embryonic
tissues) in order to recapitulate the RA-regulated programs leading to common or
specialized cell fates.
Most genomic profiling studies performed with embryonic tissues have analyzed
heterogeneous cell populations using bulk samples (whole embryos or explants).
Now, the fundamental question is how individual cells differentiate to form tissues
or organs during a developmental continuum with plastic transitions between several
cell types and diffusion of morphogen gradients. One solution is to isolate and analyze
specific subpopulations of cells at distinct functional points in their differentiation
process (Fig. 3.5).
Several NGS protocols have been recently reconfigured to enable their use with
single cells or single nuclei by improving the throughput (number of cells), the
robustness (performance in samples of varying quality) and the accuracy (level of
noise) of the assays (Dirks et al. 2016; Tanay and Regev 2017). After micro dissection
of a specialized tissue, followed by cell or nuclei dissociation and separation into
separate wells, it is possible to collect genome-wide profiles of single cells (sc) or
single nuclei (sn) by sc and snRNA-seq (Bakken et al. 2018), snChIP-seq (CUT and
RUN) (Skene et al. 2018), sc and snATAC-seq (Buenrostro et al. 2015), scDNase-seq
(Jin et al. 2015), sc and snHi-C (Nagano et al. 2013; Padmarasu et al. 2019).
Remarkably, more recent methods encapsulate individual cells in droplets in a
microfluidic device (Briggs et al. 2018, Wagner et al. 2018). Barcodes can be also
incorporated during the reverse transcription reaction converting RNAs to cDNAs
(scRNA-seq) or in transposase (ATAC-seq). Consequently, each droplet carries a
DNA “barcode” that uniquely labels the DNAs derived from a single cell. Once the
reaction is complete, the DNA fragments from many cells can be mixed together
for sequencing. Consequently, transcripts from a particular cell are identified by the
unique barcode. Such a strategy has been recently used for single-nucleus analysis
of accessible chromatin in developing mouse forebrain (Preissl et al. 2018).
In conclusion, novel NGS techniques offer promise for the analysis of the RAregulated programs in developmental tissues. Ultimately, combining single-cell
omics with experimental perturbations of the RA signaling system (knockout models,
morpholinos) should help to predict the RA-regulated programs in rare tissues.
79
Future Directions
The combination of NGS with a variety of modern DNA/RNA enrichment technologies has provided a means for interrogating genome-wide transcriptional activity
(RNA-seq), functional protein–genome interactions (ChIP-seq), chromatin accessibility (DNase-seq, FAIRE-seq, ATAC-seq), and more recently, the three-dimensional
organization of chromatin (Hi-C, ChIA-PET). Some of these approaches have been
carried out using ECCs and ESCs.
Today, the challenge is to reconstruct the RA-regulated gene networks during the
development of specialized embryonic tissues. The aim is to perform a meta-analysis,
combining the results from multiple studies (ECCs, ESCs and specialized embryonic
tissues) in order to recapitulate the RA-regulated programs leading to common or
specialized cell fates.
Most genomic profiling studies performed with embryonic tissues have analyzed
heterogeneous cell populations using bulk samples (whole embryos or explants).
Now, the fundamental question is how individual cells differentiate to form tissues
or organs during a developmental continuum with plastic transitions between several
cell types and diffusion of morphogen gradients. One solution is to isolate and analyze
specific subpopulations of cells at distinct functional points in their differentiation
process (Fig. 3.5).
Several NGS protocols have been recently reconfigured to enable their use with
single cells or single nuclei by improving the throughput (number of cells), the
robustness (performance in samples of varying quality) and the accuracy (level of
noise) of the assays (Dirks et al. 2016; Tanay and Regev 2017). After micro dissection
of a specialized tissue, followed by cell or nuclei dissociation and separation into
separate wells, it is possible to collect genome-wide profiles of single cells (sc) or
single nuclei (sn) by sc and snRNA-seq (Bakken et al. 2018), snChIP-seq (CUT and
RUN) (Skene et al. 2018), sc and snATAC-seq (Buenrostro et al. 2015), scDNase-seq
(Jin et al. 2015), sc and snHi-C (Nagano et al. 2013; Padmarasu et al. 2019).
Remarkably, more recent methods encapsulate individual cells in droplets in a
microfluidic device (Briggs et al. 2018, Wagner et al. 2018). Barcodes can be also
incorporated during the reverse transcription reaction converting RNAs to cDNAs
(scRNA-seq) or in transposase (ATAC-seq). Consequently, each droplet carries a
DNA “barcode” that uniquely labels the DNAs derived from a single cell. Once the
reaction is complete, the DNA fragments from many cells can be mixed together
for sequencing. Consequently, transcripts from a particular cell are identified by the
unique barcode. Such a strategy has been recently used for single-nucleus analysis
of accessible chromatin in developing mouse forebrain (Preissl et al. 2018).
In conclusion, novel NGS techniques offer promise for the analysis of the RAregulated programs in developmental tissues. Ultimately, combining single-cell
omics with experimental perturbations of the RA signaling system (knockout models,
morpholinos) should help to predict the RA-regulated programs in rare tissues.