Bcl11b orchestrates subcerebral projection neuron axon development via cell-autonomous, non-cell-autonomous, and subcellular mechanisms

Reviewer #1 (Public review):

Summary:

This study seeks to investigate the role of the transcription factor Bcl11b/Ctip2 in regulating subcerebral projection neuron (SCPN) axon development through both cell-autonomous and non-cell-autonomous mechanisms. The authors demonstrate that Bcl11b is required within SCPNs for axonal outgrowth and proper entry into the internal capsule, while its expression in medium spiny neurons (MSNs) influences SCPN axon fasciculation and pathfinding in a non-cell-autonomous manner. Notably, through transcriptomic analysis, immunocytochemistry, and in vivo growth cone purification, the study identifies Cdh13 as a downstream mediator of Bcl11b function, localizing along axons and at growth cone surfaces to regulate SCPN axonal outgrowth.

Strengths:

To me the most interesting aspect of this study is how common transcriptional programs across neuronal cell types cooperate to facilitate axon pathfinding, this is a very interesting concept.

Overall, it could be of interest to the brain development field.

Weaknesses:

My main concern is that, as presented in the figures, many phenotypes are too subtle to be convincing and would require quantitative analyses to corroborate the claims of the study.

I also think that the growth cones transcription data needs additional validation to be incorporated into the manuscript. In fact, I am not even sure that it really brings anything to the story.

I also think that the CRISPR in utero electroporation experiments lack appropriate controls.

Reviewer #2 (Public review):

Summary:

Itoh et al. investigate the role of the zinc finger transcription factor Bcl11b/Citp2 on sub cerebral projection neurons (SCPN) development. They dissect Bcl11b cell-autonomous and non-cell-autonomous functions on subcerebral projection neurons. In addition, they identify Cdh13 as a downstream target of Bcl11b in the process of SCPN axon outgrowth.

Strengths:

Itoh et al. take advantage of a mouse CRE/Lox genetic system as a powerful tool to distinguish Bcl11b cell-autonomous function on cortical layer V subcerebral projection neurons and its non-cell-autonomous function mediated by the striatal medium spiny neurons (MSN).

Besides the description of the cellular and anatomical defects of the corticofugal projection neurons' outgrowth and fasciculation, they perform a transcriptomic analysis of SCPN somata to identify Bcl11b target genes. As a result, they find that Cdh13, a membrane-anchored cadherin , is downstream of Bcl11b and mediates its cell-autonomous role on axon outgrowth. To validate the role of Cdh13 as a mediator of Bcl11b on SCPN development, they set up a new technique to identify and quantify superficial antigens on growth cone membranes.

Weaknesses:

While the authors shed light on the role of Bcl11b on SCPN development, they lack to contextualize their findings on the previously described interplay between Bcl11a and b.
In addition, this work is another example of the common practice of picking from a list of differentially expressed genes the most likely ones. This approach, while useful, does not allow the identification of new and unknown players.

Author response:

We appreciate that the reviewers recognize the conceptual novelty of our work and find our work interesting.

Reviewer #1:

We thank Reviewer #1 for making us aware that the image presentation of some of what we see as very clear phenotypes in our work might not have been optimal in the reviewed pdf file, presumably due to the relatively low resolution and lack of appropriately magnified images in the merged pdf file. This issue– if not caught and corrected now– might have caused future readers to similarly not appreciate these clear phenotypes. We will carefully revise the figures and ensure maintenance of appropriate pdf resolution in the merged file so that image presentation is optimal and our findings are appropriately represented.

We appreciate that Reviewer #1 carefully and critically assessed the growth cone transcriptomic data. We agree that future additional validation is warranted, and this will be clearly stated in our revised paper. Because we judge that these data – even in their current form – will be of potential interest to other investigators sooner rather than later, we respectfully offer and request that we should share them in this paper as our attempt so far to identify elements of the relevant growth cone biology, rather than waiting for years before completing additional validation.

Even upon repeated reflection, we judge and respectfully submit that our CRISPR in utero electroporation experiments are, indeed, conducted with appropriate controls. We thought through the potential controls deeply prior to completing these complex experiments. We will describe our reasoning in detail in our point-by-point response.

Reviewer #2:

We thank Reviewer #2 for encouraging us to elaborate on the direction and cross- repressive interplay between Bcl11a and Bcl11b, which we previously identified (Woodworth*, Greig* et al., Cell Rep, 2016). We omitted deep discussion because we had already published this result, cited that work, and did not want to seem overly self- referential, as well as for reasons of length. Though we know and have reported that Bcl11a and Bcl11b are cross-repressive in SCPN development, we currently do not know whether increased Bcl11a expression in Bcl11b-null SCPN contributes to reduced Cdh13 expression. Also, we do not know if there is a similar Bcl11a-Bcl11b cross repression in striatal medium spiny neurons. This will be clarified in our revised paper.

We agree fully with the reviewer that “the common practice of picking from a list of differentially expressed genes the most likely ones” has been useful for and has substantially contributed to the elucidation of molecular mechanisms in many systems, including in CNS development. Indeed, the current paper identifies Cdh13 as a newly recognized functional molecule in SCPN axon development by in part using this approach. Cdh13 belongs to a well-known gene family, and its expression by SCPN was already reported by us (Arlotta*, Molyneauz* et al., Neuron, 2005). Despite these two facts, we newly identify its function in SCPN development, which has never been investigated or reported. We appreciate the reviewer encouraging us to elaborate on this here.

Recent technical advancement allows functional screening of a larger list of genes in vivo (Jin et al., Science, 2020; Ramani et al., bioRxiv, 2024; Zheng et al., Cell, 2024). That said, it is still a challenge to specifically access SCPN in vivo and apply such a high-throughput screening assay for axon development. We agree and predict that future work of this type might likely lead to identification of other new and unknown molecular regulators. We respectfully submit that our work reported here will provide useful foundation for many such future studies.