Control of spinal motor neuron terminal differentiation through sustained Hoxc8 gene activity
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Evaluation Summary:
This manuscript will be of interest to developmental geneticists interested in neuroscience, as it demonstrates how spinal motor neurons maintain their unique identities in adulthood after fate decisions are made in the embryo. The work here suggests that a Hox transcription factor acts as a terminal selector to control motor neuron identity, thus mirroring recent studies in C. elegans, and pointing towards this type of gene regulation as important in building diverse nervous systems.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Spinal motor neurons (MNs) constitute cellular substrates for several movement disorders. Although their early development has received much attention, how spinal MNs become and remain terminally differentiated is poorly understood. Here, we determined the transcriptome of mouse MNs located at the brachial domain of the spinal cord at embryonic and postnatal stages. We identified novel transcription factors (TFs) and terminal differentiation genes (e.g. ion channels, neurotransmitter receptors, adhesion molecules) with continuous expression in MNs. Interestingly, genes encoding homeodomain TFs (e.g. HOX, LIM), previously implicated in early MN development, continue to be expressed postnatally, suggesting later functions. To test this idea, we inactivated Hoxc8 at successive stages of mouse MN development and observed motor deficits. Our in vivo findings suggest that Hoxc8 is not only required to establish, but also maintain expression of several MN terminal differentiation markers. Data from in vitro generated MNs indicate Hoxc8 acts directly and is sufficient to induce expression of terminal differentiation genes. Our findings dovetail recent observations in Caenorhabditis elegans MNs, pointing toward an evolutionarily conserved role for Hox in neuronal terminal differentiation.
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Author Response
Reviewer #1 (Public Review):
The manuscript by Catela is a very interesting study investigating terminal selection factors in spinal motor neurons. While the field has focused largely on the development and specification of motor neurons, much less attention has been garnered on gene expression programs that endow the mature motor neuron with adult stage terminal characteristics. This question has recently been tackled in the nematode roundworm C. elegans, but a terminal selector code in mammals is lacking. The work here showing sustained activity of Hoxc8 acting as a terminal selector is interesting and may point towards this kind of encoding being a general rule throughout nervous systems, from invertebrates to vertebrates. In addition to the strengths of this work
We are glad the reviewer finds our study …
Author Response
Reviewer #1 (Public Review):
The manuscript by Catela is a very interesting study investigating terminal selection factors in spinal motor neurons. While the field has focused largely on the development and specification of motor neurons, much less attention has been garnered on gene expression programs that endow the mature motor neuron with adult stage terminal characteristics. This question has recently been tackled in the nematode roundworm C. elegans, but a terminal selector code in mammals is lacking. The work here showing sustained activity of Hoxc8 acting as a terminal selector is interesting and may point towards this kind of encoding being a general rule throughout nervous systems, from invertebrates to vertebrates. In addition to the strengths of this work
We are glad the reviewer finds our study interesting and comments on its evolutionary implications.
However, this work would benefit from added approaches beyond RNAseq and RNA in situ to strengthen the data. Additional neuroanatomical, physiology, and behavior data would certainly also strengthen this work - especially since one would expect to see more phenotypes in hoxc8 mutants beyond only misexpression of downstream genes. There are a wealth of motor behavior/motor acuity tasks that can be performed in the mouse and adding such experiments would certainly strengthen this paper.
We agree. Our new behavioral data (Fig. 6) nicely complement the molecular analysis of brachial motor neurons in Hoxc8 MNΔearly and Hoxc8 MNΔlate mice.
Reviewer #2 (Public Review):
Different motor neurons located in different parts of the spinal cord are known to perform distinct functions. It is known that these differences are specified during embryonic development by the expression of different transcription factors. But it is unknown how these differences are maintained in the adult spinal cord. In this manuscript, Kratsios and colleagues propose that the Hox transcription factor Hoxc8 acts as a terminal selector for brachial motor neurons in the developing mouse spinal cord. They perform a series of experiments in which Hoxc8 is deleted from embryonic (e12) and early postnatal (p8) motor neurons and show that this transcription factor is required for the establishment and/or maintenance of a set of terminal differentiation genes in this motor neuron population. Notably, a similar and larger body of work from this lab has previously focused on the role of terminal selector genes, including Hox factors, in the worm C elegans nervous system development. The main conclusions of this current study are 1) Hox factors also control mouse neuronal terminal differentiation, suggesting an evolutionarily conserved role; 2) Hox genes such as Hoxc8 act through multiple downstream effectors, including other transcription factors such as Irx family; and 3) single transcription factors such as Hoxc8 can have multiple distinct roles in terminal differentiation based on the timing of their expression/action. This latter point is perhaps the most interesting conclusion of this paper as it helps to uncover why Hox gene expression may be maintained in post-mitotic neurons beyond initial cell specification.
This is an exciting paper and will be of broad interest to the spinal cord field. Their demonstration of similar logic in mouse as to what they reported earlier for C. elegans demonstrates the evolutionarily conserved mechanism by which Hox genes function in terminal differentiation of spinal motor neurons. Strengths of this study include the detailed transcriptomics analyses at different time points in mouse and functional studies using conditional mouse knockouts.
Overall, the conclusions in this paper are well-supported and of high quality. However, one of the authors' main conclusions is that Hoxc8 expression helps control terminal differentiation of spinal motor neurons. But they identify relatively few potential terminal effector genes (8) that seem to require Hoxc8 at any stage. This is especially evident when examined in the context of the initial RNA seq analysis of wildtype e12 vs p8 motor neurons, in which there are >3000 differentially expressed genes between those time points. Therefore, while Hoxc8 may have some role in brachial motor neuron differentiation, it appears to not be a very significant role, or at least does not seem so based on the analyses presented here. The authors might wish to either clarify this point or try to put their findings in a broader context so the readers can appreciate the importance of Hoxc8 in motor neuron differentiation and the potential involvement of other collaborating factors.
We completely agree and have modified the text and figures (Fig. 4F, new Fig. 5 - figure supplement 1) accordingly to clarify the importance of Hoxc8 in motor neuron terminal differentiation and the involvement of Hoxc8 collaborators.
Of note, we found six other Hox genes to be expressed continuously in brachial MNs - these could act as Hoxc8 collaborators. Based on studies conducted in mice and chick embryos at early stages of MN development, the strongest candidate is Hoxc6 (Catela et al., 2016, PMID: 26904955; Jung et al., 2010, PMID: 20826310). Deletion of either Hoxc6 or Hoxc8 at the MN progenitor stage (with Olig2-Cre) results in similar axon guidance defects in brachial MNs, and these early defects can be molecularly explained by Hoxc6 and Hoxc8 collaborating to control the expression of Ret, an axon guidance molecule (Catela et al., 2016, PMID: 26904955). Interestingly, when we interrogated available ChIP-Seq datasets from mouse ESC-derived motor neurons in which Hoxc6 or Hoxc8 expression is induced with doxycycline (Bulajic et al., 2020, we found Hoxc6 and Hoxc8 bind on the same cis-regulatory regions of the terminal differentiation markers (e.g., Mcam, Pappa, Glra2) we identified with our in vivo genetic studies (new Fig. 5; new Fig. 5 – figure supplement 1).
We have modified the Results and Discussion to clarify this important point, as well as highlight the potential involvement of additional factors outside the Hox family, such Islet-1.
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Evaluation Summary:
This manuscript will be of interest to developmental geneticists interested in neuroscience, as it demonstrates how spinal motor neurons maintain their unique identities in adulthood after fate decisions are made in the embryo. The work here suggests that a Hox transcription factor acts as a terminal selector to control motor neuron identity, thus mirroring recent studies in C. elegans, and pointing towards this type of gene regulation as important in building diverse nervous systems.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Catela is a very interesting study investigating terminal selection factors in spinal motor neurons. While the field has focused largely on the development and specification of motor neurons, much less attention has been garnered on gene expression programs that endow the mature motor neuron with adult stage terminal characteristics. This question has recently been tackled in the nematode roundworm C. elegans, but a terminal selector code in mammals is lacking. The work here showing sustained activity of Hoxc8 acting as a terminal selector is interesting and may point towards this kind of encoding being a general rule throughout nervous systems, from invertebrates to vertebrates.
However, this work would benefit from added approaches beyond RNAseq and RNA in situ to strengthen the data. …
Reviewer #1 (Public Review):
The manuscript by Catela is a very interesting study investigating terminal selection factors in spinal motor neurons. While the field has focused largely on the development and specification of motor neurons, much less attention has been garnered on gene expression programs that endow the mature motor neuron with adult stage terminal characteristics. This question has recently been tackled in the nematode roundworm C. elegans, but a terminal selector code in mammals is lacking. The work here showing sustained activity of Hoxc8 acting as a terminal selector is interesting and may point towards this kind of encoding being a general rule throughout nervous systems, from invertebrates to vertebrates.
However, this work would benefit from added approaches beyond RNAseq and RNA in situ to strengthen the data. Additional neuroanatomical, physiology, and behavior data would certainly also strengthen this work, especially since one would expect to see more phenotypes in hoxc8 mutants beyond only misexpression of downstream genes. There are a wealth of motor behavior/motor acuity tasks that can be performed in the mouse and adding such experiments would certainly strengthen this paper.
-
Reviewer #2 (Public Review):
Different motor neurons located in different parts of the spinal cord are known to perform distinct functions. It is known that these differences are specified during embryonic development by the expression of different transcription factors. But it is unknown how these differences are maintained in the adult spinal cord. In this manuscript, Kratsios and colleagues propose that the Hox transcription factor Hoxc8 acts as a terminal selector for brachial motor neurons in the developing mouse spinal cord. They perform a series of experiments in which Hoxc8 is deleted from embryonic (e12) and early postnatal (p8) motor neurons and show that this transcription factor is required for the establishment and/or maintenance of a set of terminal differentiation genes in this motor neuron population. Notably, a similar …
Reviewer #2 (Public Review):
Different motor neurons located in different parts of the spinal cord are known to perform distinct functions. It is known that these differences are specified during embryonic development by the expression of different transcription factors. But it is unknown how these differences are maintained in the adult spinal cord. In this manuscript, Kratsios and colleagues propose that the Hox transcription factor Hoxc8 acts as a terminal selector for brachial motor neurons in the developing mouse spinal cord. They perform a series of experiments in which Hoxc8 is deleted from embryonic (e12) and early postnatal (p8) motor neurons and show that this transcription factor is required for the establishment and/or maintenance of a set of terminal differentiation genes in this motor neuron population. Notably, a similar and larger body of work from this lab has previously focused on the role of terminal selector genes, including Hox factors, in the worm C. elegans nervous system development. The main conclusions of this current study are 1) Hox factors also control mouse neuronal terminal differentiation, suggesting an evolutionarily conserved role; 2) Hox genes such as Hoxc8 act through multiple downstream effectors, including other transcription factors such as Irx family; and 3) single transcription factors such as Hoxc8 can have multiple distinct roles in terminal differentiation based on the timing of their expression/action. This latter point is perhaps the most interesting conclusion of this paper as it helps to uncover why Hox gene expression may be maintained in post-mitotic neurons beyond initial cell specification.
This is an exciting paper and will be of broad interest to the spinal cord field. Their demonstration of similar logic in mouse as to what they reported earlier for C. elegans demonstrates the evolutionarily conserved mechanism by which Hox genes function in terminal differentiation of spinal motor neurons. Strengths of this study include the detailed transcriptomics analyses at different time points in mouse and functional studies using conditional mouse knockouts.
Overall, the conclusions in this paper are well-supported and of high quality. However, one of the authors' main conclusions is that Hoxc8 expression helps control terminal differentiation of spinal motor neurons. But they identify relatively few potential terminal effector genes (8) that seem to require Hoxc8 at any stage. This is especially evident when examined in the context of the initial RNA seq analysis of wildtype e12 vs p8 motor neurons, in which there are >3000 differentially expressed genes between those time points. Therefore, while Hoxc8 may have some role in brachial motor neuron differentiation, it appears to not be a very significant role, or at least does not seem so based on the analyses presented here. The authors might wish to either clarify this point or try to put their findings in a broader context so the readers can appreciate the importance of Hoxc8 in motor neuron differentiation and the potential involvement of other collaborating factors.
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Reviewer #3 (Public Review):
The authors of this manuscript analyze, largely using gene expression profiling, the role of Hoxc8 in mouse motor neurons at two different time points, extending previous studies that focus only on early time points. They conclude that some genes continue to be regulated by Hoxc8 at both time points while some are not.
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