Specificity Protein 1 is essential for the limb trajectory of ephrin-mediated spinal motor axons

Reviewer #1 (Public review):

The manuscript by Liao et al investigates the mechanisms that induce ephrin expression in spinal cord lateral motor column (LMC) neurons to facilitate axon guidance into the dorsal and ventral limb. The authors show that Sp1 and its co-activators p300 and CBP are required to induce ephrin expression to modulate the responsiveness of motor neurons to external ephrin cues. The study is well done and convincingly demonstrates the role of Sp1 in motor neuron axon guidance.

Further discussion and clarification of some results would further improve the study.

(1) The mechanism that the authors propose (Figure 7) and is also supported by their data is that Sp1 induces ephrinA5 in LMCm and ephrinB2 in LMCl to attenuate inappropriate responses to external ephrins in the limb. Therefore, deletion of Sp1 should result in mistargeting of LMCl and LMCm axons, as shown in the mouse data, but no overt changes in the number of axons in the ventral and dorsal limb. From the mouse backfills, it seems that an equal number of LMCm/LMCl project into the wrong side of the limb. However, the chick data show an increase of axons projecting into the ventral limb in the Sp1 knockout. Is this also true in the mouse? The authors state that medial and lateral LMC neurons differ in their reliance on Sp1 function but that is not supported by the mouse backfill data (27% vs 32% motor neurons mistargeted). Also, the model presented in Figure 7 does not explain how Sp1 overexpression leads to axon guidance defects.

(2) The authors do not directly show changes in ephrin expression in motor neurons, either in chick or mouse, after Sp1 knockout, which is the basis of their model. The experiment in Figure 4G seems to be Sp1 overexpression rather than knockdown (as mentioned in the results) and NSC-34 cells may not be relevant to motor neurons in vivo. NSC-34 experiments are also not described in the methods.

(3) There is no information about how the RNA-sequencing experiment was done (which neurons were isolated, how, at what age, how many replicates, etc) so it is hard to interpret the resulting data.

(4) It is unclear why the authors chose to use a Syn1-cre driver rather than a motor neuron restricted cre driver. Since this is a broad neuronal cre driver, the behavioral defects shown in Figure 7 may not be solely due to Sp1 deletion in motor neurons. Are there other relevant neuronal populations that express Sp1 that are targeted by this cre-mediated deletion?

Reviewer #2 (Public review):

Summary:

This study shows that transcription factor Sp1 is required for correct ventral vs. dorsal targeting of limb-innervating LMC motor neurons using mouse and chick as model systems. In a wild-type embryo, lateral LMC axons specifically target dorsal muscles while medial LMC axons target ventral muscles. The authors convincingly show that this specificity is lost when Sp1 is knocked down or knocked out - axons of both lateral and medial LMC motor neurons project to both dorsal and ventral muscles in mutant conditions. The authors then conduct RNA-seq and ChIP experiments to show that Sp1 loss of function disrupts Ephrin-Epha receptor signaling pathway genes. These molecules are known to provide attractive or repulsive cues to guide LMC axons to their targets. The authors show that attraction/repulsion properties of medial and lateral LMC axons to specific Ephrin/Epha molecules are in fact disrupted in Sp1 mutants using ex vivo explant studies. Finally, the authors show that behaviors like coordinated movement and grip strength are also affected in Sp1 mutant mice. This study convincingly shows that Sp1 is important for correct circuit wiring of LMC neurons, and moves the field forward by elucidating a new level of transcriptional regulation required in this process. However, the claims made by the authors that the mode of Sp1-mediated regulation is through cis-attenuation of Epha activity is not well supported. These and additional strengths and weaknesses in approach and in data interpretation are discussed below.

Strengths:

(1) The study convincingly shows that wildtype levels of Sp1 are necessary for LMC axon targeting specificity. The combination of the following approaches is a strength:
a) Both loss of function and gain of function experiments are performed for Sp1 and show complementary effects on the axon targeting phenotype.
b) Retrograde labeling of LMC neurons from dorsal and ventral muscles shows that Sp1 mutants clearly lose the specificity of LMC axon targeting.
c) The authors also use explant experiments to show that both loss of Sp1 and gain of Sp1 show clear changes in attraction and repulsion to specific ephrin and epha receptor molecules.
d) The Sp1 loss and gain of function experiments are well controlled to show that the changes in axon wiring observed are not due to cell death, cell fate switches, or due to unequal numbers of medial and lateral LMC neurons being labeled in the experiments.

(2) It is also convincing that Sp1 requires cofactors p300 and CBP for its function. In the absence of these cofactors, the gain of function phenotypes of Sp1 are subdued.

Weaknesses:

(1) The robustness of RNAseq and ChIP experiments is difficult to judge as methods are not described. For example, it is unclear if RNAseq is performed on purified motor neurons or on whole spinal cords. This is an important consideration as Sp1 is a broadly expressed protein.

(2) The authors state that expression of Ephrin A5 and Ephrin B2 is reduced based on RNAseq data, however, it is not shown that this reduction occurs specifically in LMC neurons.

(3) The authors show Sp1 ChIP peaks at Ephrin B2 promoter, but nothing is mentioned about peaks at Eprin A5 or other types of signaling molecules like Sema7a, which are also differentially expressed in Sp1 mutants. There is also no mention of the correlation between changes in gene expression seen in RNAseq data and the binding profile of Sp1 seen in ChIP data, which could help establish the robustness of these datasets.

(4) The authors conclude that Sp1 functions by activating Ephrin A5 in medial LMC and Ephrin B2 in lateral LMC. The argument, as I understand it, is that this activation leads to cis attenuation of their respective Epha receptors and therefore targeting the correct muscle. Though none of the data presented go against this hypothesis, this hypothesis is also not fully supported. Specifically:
a) It would be important to know that modulation of Sp1 expression leads to changes in EphrinA5 and B2 in LMC lateral/medial neurons.
b) It would also be important to show that none of the other changes caused by Sp1 are responsible for axon mistargeting by performing rescue experiments with Ephrin A5 and Ephrin B2.
c) To make the most convincing case, experiments showing increased or decreased cis-binding of Ephrin molecules with Epha receptors would be necessary. This study would still be compelling without this last experiment, but the language in the abstract would need to be modulated.

(5) All behavior experiments are done in a pan-neuronal knockout of Sp1. As Sp1 is broadly expressed in neurons, a statement describing whether and why the authors think the phenotypes arise from Sp1's function in LMC motor neurons would be helpful. Experimentally, rescue experiments in which Sp1 is restored in LMC neurons or motor neurons would also make this claim more convincing.

Reviewer #3 (Public review):

Summary:

This is a compelling study on the role of Sp1 in motor axon trajectory selection, demonstrating that Sp1 is both necessary and sufficient for correct axon guidance in the limb. Sp1 regulates ephrin ligand expression to fine-tune Eph/ephrin signaling in the lateral motor column (LMC) neurons.

Strengths:

The study integrates multiple approaches. These include in ovo electroporation in chick embryos, conditional knockout mouse models, transcriptomic analyses, and functional assays such as stripe assays and behavioral testing-to provide robust evidence for Sp1's role in axon guidance mechanisms. The manuscript is well-written and scientifically rigorous, and the findings are of broad interest to the developmental neuroscience community.

Weaknesses:

Some aspects of the manuscript could be improved to enhance clarity, ensure logical flow, and strengthen the impact of the findings.

Author response:

Reviewer 1:

(1) Clarification of axon mistargeting patterns and model interpretation

We will clarify the apparent discrepancy between chick and mouse axon mistargeting data. Specifically, we will expand the explanation in the main text and Figure 7 legend and/or revise the model in Figure 7 to better reflect observed phenotypes and clarify how Sp1 overexpression contributes to mistargeting.

(2) Evidence for Sp1-dependent ephrin expression

We agree that demonstrating ephrin expression changes in motor neurons is essential. We will: • Conduct in situ hybridization and/or immunostaining for ephrins in control and Sp1 mutant spinal cords from both chick and mouse embryos.

Clarify and expand the methodological details of the NSC-34 cell experiments shown in Figure 4G.

(3) RNA-seq experiment details

We will revise the Methods section to provide additional experimental details.

(4) Use of Syn1-cre

We acknowledge concerns about the broad expression of Syn1-cre. To address this:

We will clarify our rationale for using Syn1-cre and describe its expression pattern in the spinal cord.

We are evaluating the feasibility of additional experiments using a motor neuron-specific Cre driver to confirm cell-type specificity.

We will include a new paragraph in the Discussion addressing potential contributions from other neuronal populations.

Reviewer 2:

(1) & (2) Clarification and localization of RNA-seq data

We will expand the Methods section to provide greater detail on the RNA-seq approach. In addition, we will validate ephrin downregulation in LMC neurons using in situ hybridization and/or immunostaining.

(3) Integration of ChIP and RNA-seq data We will:

Report additional ChIP peaks for ephrinA5 and other differentially expressed genes such as Sema7a.

Add a summary figure that integrates ChIP and RNA-seq results to strengthen the link between Sp1 binding and transcriptional regulation.

(4) Clarification of the cis-attenuation model

We recognize that our data do not yet directly demonstrate Sp1’s role in cis-attenuation. To address this:

We will revise the abstract and main text to frame Sp1's role in cis-attenuation as a hypothesis. • We are exploring the feasibility of ephrinA5 and B2 rescue experiments in Sp1-deficient embryos to test specificity.

(5) Behavioral phenotypes and cell-type specificity

We will clarify that behavioral phenotypes may result from combined effects across neuron populations due to Syn1-cre expression. To address this:

We are planning rescue experiments with Sp1 expression in chick embryos to test for rescue of axon misrouting.

We will include a new paragraph in the Discussion to highlight this limitation and discuss alternative interpretations.

Reviewer 3:

We appreciate your positive evaluation and support for the rigor of our study.

In response to your suggestions:

We are revising the manuscript to improve clarity and flow, particularly the transitions between datasets.

We will update Figure 7 and the associated text to more clearly convey the working model and avoid overinterpretation.

We thank all reviewers for their constructive feedback and are committed to addressing each point thoroughly. All revisions will be clearly marked in the resubmitted manuscript.