An ATF3-CreERT2 knock-in mouse for axotomy-induced genetic editing: proof of principle.

ABSTRACT

Genome editing techniques have facilitated significant advances in our understanding of fundamental biological processes, and the Cre-Lox system has been instrumental in these achievements. Driving Cre expression specifically in injured neurons has not been previously possible: we sought to address this limitation in mice using a Cre-ERT2 construct driven by a reliable indicator of axotomy, Activating Transcription Factor 3 (ATF3). When crossed with reporter mice, a significant amount of recombination was achieved (without tamoxifen treatment) in peripherally-projecting sensory, sympathetic, and motoneurons after peripheral nerve crush in hemizygotes (65-80% by 16 days) and was absent in uninjured neurons. Importantly, injury-induced recombination did not occur in Schwann cells distal to the injury, and with a knockout-validated antibody we verified an absence of ATF3 expression. Functional recovery following sciatic nerve crush in ATF3-deficient mice (both hemi- and homozygotes) was delayed, indicating previously unreported haploinsufficiency. In a proof-of-principle experiment, we crossed the ATF3-CreERT2 line with a floxed PTEN line and show significantly improved axonal regeneration, as well as more complete recovery of neuromuscular function. We also demonstrate the utility of the ATF3-CreERT2 hemizygous line by characterizing recombination after lateral spinal hemisection (C8/T1), which identified specific populations of ascending spinal cord neurons (including putative spinothalamic and spinocerebellar) and descending supraspinal neurons (rubrospinal, vestibulospinal, reticulospinal and hypothalamic). We anticipate these mice will be valuable in distinguishing axotomized from uninjured neurons of several different classes (e.g. via reporter expression), and in probing the function of any number of genes as they relate to neuronal injury and regeneration.

SIGNIFICANCE STATEMENT Understanding reactions to neurotrauma and overcoming obstacles to neural regeneration should benefit from the ability to genetically label or otherwise edit the genome of injured neurons. We sought to achieve this in mice by driving Cre recombinase expression under the control of Activating Transcription Factor (ATF3), which is robustly induced by axotomy in several populations of peripheral and central neurons. When crossed with reporter mice, recombination occurred only in injured neurons following sciatic nerve injury or spinal hemisection. Peripheral nerve injury-induced neuronal PTEN excision also resulted in improved regeneration and more complete functional recovery. These results demonstrate the feasibility and utility of axotomy-induced recombination and represent a new tool for investigating genetic control of injury responses and regeneration.