An unexpected role for the molecule netrin1 in organizing the developing spinal cord has been uncovered by scientists at the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA.
The researchers found that netrin1, best known for playing the role of a guidance cue that orients growing nerve fibers, also acts as a brake on BMP signaling in certain parts of the spinal cord. This boundary-setting function is important because this signaling activity must be tightly sequestered to the dorsal region for sensory neurons to develop properly.
Their results, published in Cell Reports, recast how complex spinal circuits are put together during embryonic development, and could provide clues for future therapeutic approaches for spinal cord repair.
“Our work shows that netrin1, long known as a potent neural circuit architect, has an unexpected function in organizing the spinal cord during early development,” Butler, a member of the UCLA Broad Stem Cell Research Center, said. As for the dorsal spinal cord, which handles inputs like touch and pain, relative development is marked by partition and organization. That is, specific neurons must be formed in carefully defined regions for these sensory processes to function. BMP signaling orchestrates this activity only within the dorsal range of the spinal cord.
Producing correct BMP signals is important because if the signals aren’t well contained, they will spread to other parts of the spine, disrupting the formation of other neuron types. Butler and her team found that netrin1 was the critical boundary keeper.
“Signaling molecules like BMP and netrin1 are very important in a regional manner for neural network formation and function,” said Sandy Alvarez, graduate student in Butler’s lab and first author of the study.” Axon transport is also regulated by netrin1, without which we would likely see a disorganized neural network that could impede how, or if, axons reach their targets.”
Netrin1 sets boundaries on BMP signaling to ensure sensory neurons develop away from motor and interneurons in the ventral region and ensure proper relay of sensory input and motor output throughout the body. Butler and her colleagues overturned a long-standing paradigm about axon growth during embryonic development in 2017.
Over decades, scientists had believed that axons—thin ‘wires’ that connect cells of the nervous system across long stretches of space—were guided by cues like netrin1 either by being repelled or attracted from a great distance. However, Butler’s research revealed that netrin1 behaves more like a sticky adhesive surface, directing axon growth along directly followed pathways rather than as a far cue.
Butler’s team couldn’t ignore it: they were prompted to press further. They introduced a traceable form of netrin1 to the developing spinal cord of chicken and mouse embryos, and mouse embryonic stem cells, and watched how the embryos responded to this in gain of function experiments.
At first, Alvarez thought there must have been something wrong, that her experiments had failed. However, when the results were repeated a few times over, she made this surprising connection.
“A lot of work indicated that BMPs are critical for patterning the dorsal spinal cord during embryonic development, but there was very little scientific literature on netrin1 and BMP signaling working together.” “We found that what we were seeing was repression of BMP activity by netrin1 in our animal models.”
In several different animal models, the team was able to use a combination of genetic approaches to show that reducing and increasing netrin1 levels specifically affected how nerve cells pattern in the dorsal area of the spinal cord. “It was so startling that it catapulted netrin1 into being the most powerful architect of the neuronal circuits I have ever worked with,” Butler said. “We will next work to learn how we can deploy netrin1 to regenerate circuitry in patients with damaged nerves or injured spinal cords.”
Netrin1 has a critical role in spinal cord development, influencing neuronal specification and distribution. Misexpression of netrin1 in chicken embryos caused a dose-dependent reduction in the size of Sox2+ NPCs (∼25%) and p27+ neurons (∼33%) without altering spinal cord architecture (p>0.65, p>0.60). At the highest levels, ∼75% of dI1 and ∼50% of dI2/dI3 neurons were lost (p<0.05), with increasing caspase+ cells indicating cell death.
Conversely, netrin1-deficient mice showed a significant expansion of dorsal progenitor domains: dP1 and dP2 areas increased ∼2-fold (p<0.0001) and ∼60% (p<0.0001), respectively. Despite this, there was no change in Ptf1a+ or Olig2+ domains (p>0.85, p>0.25). Extended netrin1 treatment in mESC models reduced dI1 marker expression (p<0.07) while suppressing BMP-dependent dorsalization. These findings highlight netrin1’s dose-dependent effects on spinal cord patterning, limiting dorsal progenitor expansion while affecting neuronal differentiation.
Given our results, Alvarez said there’s a need to reevaluate how netrin1 and BMP act in other systems. “This might be helping us better understand some of those cell-type cancers or developmental issues where BMP and netrin1 are being signaled together.”
Reference: Alvarez S, Gupta S, Mercado-Ayon Y, et al. Netrin1 patterns the dorsal spinal cord through modulation of Bmp signaling. Cell Reports. Published online November 2024:114954.
doi: https://doi.org/10.1016/j.celrep.2024.114954
