# Morphological segmentation with tiling light sheet microscopy to quantitatively analyze the three-dimensional structures of spinal motoneurons

## TL;DR

This study uses advanced imaging to analyze the 3D structure of spinal motoneurons during development, revealing how their morphology changes over time.

## Contribution

The study introduces a high-throughput method using tiling light sheet microscopy to quantitatively analyze spinal motoneuron diversity in 3D.

## Key findings

- Upper limb motoneurons split into two subpopulations by postnatal day 14 based on soma size.
- Dendrite numbers in lumbar motoneurons initially increase with dendritic order but then decline.
- 3D volumetric reconstruction enabled detailed tracing and measurement of dendritic structures.

## Abstract

Spinal motoneurons control muscle fibers contraction and drive all motor behaviors in vertebrates. Although spinal motoneurons share the fundamental role of innervating muscle fibers, they exhibit remarkable diversity that reflects their specific identities. Defining the morphological changes during postnatal development is critical for elucidating this diversity. However, our understanding of the three-dimensional (3D) morphology of spinal motoneurons at these stages remains limited, largely due to the lack of high-throughput imaging tools. Using tiling light sheet microscopy combined with tissue clearing methods, we imaged motoneurons of the lateral and median motor column in the cervical and lumbar cord during postnatal development. By analyzing their soma size, we found that motoneurons innervating the upper limbs differentiate into two subpopulations with distinct soma size by postnatal day 14 (P14), while differentiation of motoneurons innervating the lower limbs is delayed. Furthermore, coupling adenovirus labeling with 3D volumetric reconstruction, we traced and measured the number and lengths of dendrites of flexor and extensor motoneurons in the lumbar cord, finding that the number of dendrites initially increases and subsequently declines as dendritic order rises. Together, these findings provide a quantitative analysis of the 3D morphological changes underlying spinal motoneuron diversity.

The online version contains supplementary material available at 10.1186/s13619-025-00231-3.