Uncovering new dimensions of brain development: Decoding cortical layer identity with spatial long-read technology

From cells to tissue layers—with precision, not assumptions

Puberty reshapes cortical plasticity, but how splicing and poly(A)-site usage shift across specific layers and cell types—and whether these spatial changes contribute equally to neuronal plasticity—has remained unclear. Cell-type and brain-region specificity in isoform expression, including dramatic developmental isoform changes in brain have been studied, yet it has been difficult to conclusively determine the layer in which an individual cortical neuron resides, especially in the human brain. To answer the question of which cortical layers are most strongly altered by splicing and poly(A)-site choice, a high-resolution spatial technology is required.  

Long reads hold the promise to reveal combination patterns of variable sites but are not sufficient for gene expression-based analyses, while cell-type information is better determined with short-read sequencing due to enhanced read depth and gene expression profiles. Thus, both sequencing modalities and a spatial technology with the appropriate resolution (spot size) and capture efficiency are required in tandem to study alternative splicing and polyadenylation in their full biological context. Yet, with many commonly available spatial technologies, most spots cover multiple cells, thus hindering a cell type-specific view of spatially regulated isoforms.

Advancing beyond current limitations with Spl-ISO-Seq

In a new study, Foord et al. introduce spatial isoform sequencing (Spl-ISO-Seq), a powerful approach that combines long-read sequencing with Slide-SeqV2 (Seeker™ technology), ensuring that RNA molecules are spatially barcoded based on tissue placement. Foord et al. showed that Spl-ISO-Seq:

• Enriches for long, exon-spanning cDNAs and couples them with spatial barcoding. This results in 2–3 times longer read lengths compared to standard preparations, significantly improving isoform recovery 
• Utilizes a spatial technology (Seeker technology) that helps achieve single-cell resolution for true unbiased discovery not limited by predetermined targets. A tightly packed monolayer of beads ensures a continuous view of regions of interest at 10 um resolution
• Enables precise barcode deconvolution and isoform quantification

Together, these innovations provided a much clearer picture of transcript diversity within complex tissues.

Insights into brain development: revealing regulations in the human cortex

To uncover changes in RNA biology, Foord et al. applied this technology to compare human post-mortem prepuberty samples (8–11 years) vs postpuberty (16–19 years) visual cortex samples. The results linked developmental splicing changes during puberty in specific layers and cell types. The researchers found:

• Stronger regulation of splicing and polyadenylation in cortical layers compared to adjacent white matter
• Pronounced developmental changes exhibited in the cortical Layer 4 excitatory neurons
• Greater developmental splicing changes for oligodendrocytes in white matter
• Enrichment of genes associated with postsynaptic function, with many of these events associated with genes previously implicated in autism spectrum disorder
• No association with genes attributed to Alzheimer’s or schizophrenia, which could be due to these conditions being associated with older ages or different cortical regions, thus not the period or anatomical area investigated

Looking ahead

Spatial long-read technology sets a new benchmark for understanding gene regulation in development and disease, supplying a view of splicing changes that are rooted in specific cell types and specific structures within anatomical regions. The advantages of this technology to study any complex tissue are clear; unbiased (not limited by predetermined targets), sensitive to spatial context, and primed to reveal previously hidden biology. 

Researchers can now map how RNA processing shapes tissue function with unprecedented clarity in cancer, neurodegeneration, neuropsychiatric disorders, and many other conditions.

Reference 

Foord, C. et al. A spatial long-read approach at near-single-cell resolution reveals developmental regulation of splicing and polyadenylation sites in distinct cortical layers and cell types. Nat Commun 16, 8093 (2025).

Takara Bio products used in this exciting research:

Seeker Spatial Transcriptomics Kit