microRNA-19b regulates proliferation & patterning in the avian forebrain

Reviewer #1 (Public review):

Summary:

This study provides new insights into the role of miR-19b, an oncogenic microRNA, in the developing chicken pallium. Dynamic expression pattern of miR-19b is associated with its role in regulating cell cycle progression in neural progenitor cells. Furthermore, miR-19b is involved in determining neuronal subtypes by regulating Fezf2 expression during pallial development. These findings suggest an important role for miR-19b in the coordinated spatio-temporal regulation of neural progenitor cell dynamics and its evolutionary conservation across vertebrate species.

Strengths:

The authors identified conserved roles of miR-19 in the regulation of neural progenitor maintenance between mouse and chick, and the latter is mediated by the repression of E2f8 and NeuroD1. Furthermore, the authors found that miR-19b-dependent cell cycle regulation is tightly associated with specification of Fezf1 or Mef2c-positive neurons, in spatio-temporal manners during chicken pallial development. These findings uncovered molecular mechanisms underlying microRNA-mediated neurogenic controls.

Weaknesses:

Although the authors in this study claimed striking similarities of miR-19a/b in neurogenesis between mouse and chick pallium, a previous study by Bian et al. revealed that miR-19a contributes the expansion of radial glial cells by suppressing PTEN expression in the developing mouse neocortex, while miR-19b maintains apical progenitors via inhibiting E2f2 and NeuroD1 in chicken pallium. Thus, it is still unclear whether the orthologous microRNAs regulate common or species-specific target genes.

The spatiotemporal expression patterns of miR-19b and several genes are not convincing. For example, the authors claim that NeuroD1 is initially expressed uniformly in the subventricular zone (SVZ) but disappears in the DVR region by HH29 and becomes detectable by HH35 (Figure 1). However, the in situ hybridization data revealed that NeuroD1 is highly expressed in the SVZ of the DVR at HH29 (Figure 4F). Thus, perhaps due to the problem of immunohistochemistry, the authors have not been able to detect NeuroD1 expression in Figure 1D, and the interpretation of the data may require significant modification.

It seems that miR-19b is also expressed in neurons (Figure 1), suggesting the role of miR19-b must be different in progenitors and differentiated neurons. The data on the gain- and loss-of-function analysis of miR-19b on the expression of Mef2c should be carefully considered, as it is possible that these experiments disturb the neuronal functions of miR19b rather than in the progenitors.

The regions of chicken pallium were not consistent among figures: in Figure 1, they showed caudal parts of the pallium (HH29 and 35), while the data in Figure 4 corresponded to the rostral part of the pallium (Figure 4B).

The neurons expressing Fezf2 and Mef2 in the chicken pallium are not homologous neuronal subtypes to mammalian deep and superficial cortical neurons. The authors must understand that chicken pallial development proceeds in an outside-in manner. Thus, Mef2c-postive neurons in a superficial part are early-born neurons, while FezF2-positive neurons residing in deep areas are later-born neurons. It should be noted that the expression of a single marker gene does not support cell type homology, and the authors' description "the possibility of primitive pallial lamina formation in common ancestors of birds and mammals" is misleading.

Overexpression of CDKN1A or Sponge-19b induced ectopic expression of Fezf2 in the ventricular zone (Figure 3C, E). Do these cells maintain progenitor statement or prematurely differentiate to neurons? In addition, the authors must explain that the induction of Fezf2 is also detected in GFP-negative cells.

Reviewer #2 (Public review):

Summary:

This paper investigates the general concept that avian and mammalian pallium specifications share similar mechanisms. To explore that idea, the authors focus their attention on the role of miR-19b as a key controlling factor in the neuronal proliferation/differentiation balance. To do so, the authors checked the expression and protein level of several genes involved in neuronal differentiation, such as NeuroD1 or E2f8, genes also expressed in mammals after conducting their functional gene manipulation experiments. The work also shows a dysregulation in the number of neurons from lower and upper layers when miR-19b expression is altered.

To test it, the authors conducted a series of functional experiments of gain and loss of function (G&LoF) and enhancer-reporter assays. The enhancer-reporter assays demonstrate a direct relationship between miR-19b and NeuroD1 and E2f8 which is also validated by the G&LoF experiments. It´s also noteworthy to mention that the way miR-19b acts is maintaining the progenitor cells from the ventricular zone in an undifferentiated stage, thus promoting them into a stage of cellular division.

Overall, the paper argues that the expression of miR-19b in the ventricular zone promotes the cells in a proliferative phase and inhibits the expression of differentiation genes such as E2f8 and NeurD1. The authors claim that a decrease in the progenitor cell pool leads to an increase and decrease in neurons in the lower and upper layers, respectively.

Strengths:

(1) Novelty Contribution
The paper offers strong arguments to prove that the neurodevelopmental basis between mammals and birds is quite the same. Moreover, this work contributes to a better understanding of brain evolution along the animal evolutionary tree and will give us a clearer idea about the roots of how our brain has been developed. This stands in contrast to the conventional framing of mammal brain development as an independent subject unlinked to the "less evolved species". The authors also nicely show a concept that was previously restricted to mammals - the role of microRNAs in development.

(2) Right experimental approach
The authors perform a set of functional experiments correctly adjusted to answer the role of miR-19b in the control of neuronal stem cell proliferation and differentiation. Their histological, functional, and genetic approach gives us a clear idea about the relations between several genes involved in the differentiation of the neurons in the avian pallium. In this idea, they maintain the role of miR-19b as a hub controller, keeping the ventricular zone cells in an undifferentiated stage to perpetuate the cellular pool.

(3) Future directions
The findings open a door to future experiments, particularly to a better comprehension of the role of microRNAs and pallidal genetic connections. Furthermore, this work also proves the use of avians as a model to study cortical development due to the similarities with mammals.

Weaknesses:

While there are questions answered, there are still several that remain unsolved. The experiments analyzed here lead us to speculate that the early differentiation of the progenitor cells from the ventricular zone entails a reduction in the cellular pool, affecting thereafter the number of latter-born neurons (upper layers). The authors should explore that option by testing progenitor cell markers in the ventricular zone, such as Pax6. Even so, it remains possible that miR-19b is also changing the expression pattern of neurons that are going to populate the different layers, instead of their numbers, so the authors cannot rule that out or verify it. Since the paper focuses on the role of miR-19b in patterning, I think the authors should check the relationship and expression between progenitors (Pax6) and intermediate (Tbr2) cells when miR-19b is affected. Since neuronal expression markers change so fast within a few days (HH24-HH35), I don't understand why the authors stop the functional experiments at different time points.

Reviewer #3 (Public review):

Summary:

This is a timely article that focuses on the molecular machinery in charge of the proliferation of pallial neural stem cells in chicks, and aims to compare them to what is known in mammals. miR19b is related to controlling the expression of E2f8 and NeuroD1, and this leads to a proper balance of division/differentiation, required for the generation of the right number of neurons and their subtype proportions. In my opinion, many experiments do reflect an interaction between all these genes and transcription factors, which likely supports the role of miR19b in participating in the proliferation/differentiation balance.

Strengths:

Most of the methodologies employed are suitable for the research question, and present data to support their conclusions.

The authors were creative in their experimental design, in order to assess several aspects of pallial development.

Weaknesses:

However, there are several important issues that I think need to be addressed or clarified in order to provide a clearer main message for the article, as well as to clarify the tools employed. I consider it utterly important to review and reinterpret most of the anatomical concepts presented here. The way the are currently used is confusing and may mislead readers towards an understanding of the bird pallium that is no longer accepted by the community.

Major Concerns:

(1) Inaccurate use of neuroanatomy throughout the entire article. There are several aspects to it, that I will try to explain in the following paragraphs:

a) Figure 1 shows a dynamic and variable expression pattern of miR19b and its relation to NeuroD1. Regardless of the terms used in this figure, it shows that miR19b may be acting differently in various parts of the pallium and developmental stages. However, all the rest of the experiments in the article (except a few cases) abolish these anatomical differences. It is not clear, but it is very important, where in the pallium the experiments are performed. I refer here, at least, to Figures 2C, E, F, H, I; 3D, E; 4C, D, G, I. Regarding time, all experiments were done at HH22, and the article does not show the native expression at this stage. The sacrifice timing is variable, and this variability is not always justified. But more importantly, we don't know where those images were taken, or what part of the pallium is represented in the images. Is it always the same? Do results reflect differences between DVR and Wulst gene expression modifications? The authors should include low magnification images of the regions where experiments were performed. And they should consider the variable expression of all genes when interpreting results.

b) SVZ is not a postmitotic zone (as stated in line 123, and wrongly assigned throughout the text and figures). On the contrary, the SVZ is a secondary proliferative zone, organized in a layer, located in a basal position to the VZ. Both (VZ and SVZ) are germinative zones, containing mostly progenitors. The only postmitotic neurons in VZ and SVZ occupy them transiently when moving to the mantle zone, which is closer to the meninges and is the postmitotic territory. Please refer to the original Boulder committee articles to revise the SVZ definition. The authors, however, misinterpret this concept, and label the whole mantle zone as it this would be the SVZ. Indeed, the term "mantle zone" does not appear in the article. Please, revise and change the whole text and figures, as SVZ statements and photographs are nearly always misinterpreted. Indeed, SVZ is only labelled well in Figure 4F.

The two articles mentioning the expression of NeuroD1 in the SVZ (line 118) are research in Xenopus. Is there a proliferative SVZ in Xenopus?

For the actual existence of the SVZ in the chick pallium, please refer to the recent Rueda-Alaña et al., 2025 article that presents PH3 stainings at different timepoints and pallial areas.

c) What is the Wulst, according to the authors of the article? In many figures, the Wulst includes the medial pallium and hippocampus, whereas sometimes it is used as a synonym of the hyperpallium (which excludes the medial pallium and hippocampus). Please make it clear, as the addition or not of the hippocampus definitely changes some interpretations.

d) The authors compare the entirety of the chick pallium - including the hippocampus (see above), hyperpallium, mesopallium, nidopallium - to only the neocortex of mammals. This view - as shown in Suzuki et al., 2012 - forgets the specificity of pallial areas of the pallium and compares it to cortical cells. This is conceptually wrong, and leads to incorrect interpretations (please refer to Luis Puelles' commentaries on Suzuki et al results); there are incorrect conclusions about the existence of upper-layer-like and deep-layer-like neurons in the pallium of birds. The view is not only wrong according to the misinterpreted anatomical comparisons, but also according to novel scRNAseq data (Rueda-Alaña et al., 2025; Zaremba et al., 2025; Hecker et al., 2025). These articles show that many avian glutamatergic neurons of the pallium have highly diversified, and are not comparable to mammalian cortical cells. The authors should therefore avoid this incorrect use of terminology. There are not such upper-layer-like and deep-layer-like neurons in the pallium of birds.

(2) From introduction to discussion, the article uses misleading terms and outdated concepts of cell type homology and similarity between chick and pallial territories and cells. The authors must avoid this confusing terminology, as non-expert readers will come to evolutionary conclusions which are not supported by the data in this article; indeed, the article does not deal with those concepts.

a) Recent articles published in Science (Rueda-Alaña et al., 2025; Zaremba et al., 2025; Hecker et al., 2025) directly contradict some views presented in this article. These articles should be presented in the introduction as they are utterly important for the subject of this article and their results should be discussed in the light of the new findings of this article. Accordingly, the authors should avoid claiming any homology that is not currently supported. The expression of a single gene is not enough anymore to claim the homology of neuronal populations.

b) Auditory cortex is not an appropriate term, as there is no cortex in the pallium of birds. Cortical areas require the existence of neuronal arrangements in laminae that appear parallel to the ventricular surface. It is not the case of either hyperpallium or auditory DVR. The accepted term, according to the Avian Nomenclature forum, is Field L.

c) Forebrain, a term overused in the article, is very unspecific. It includes vast areas of the brain, from the pretectum and thalamus to the olfactory bulb. However the authors are not researching most of the forebrain here. They should be more specific throughout the text and title.

(3) In the last part of the results, the authors claim miR19b has a role in patterning the avian pallium. What they see is that modifying its expression induces changes in gene expression in certain neurons. Accordingly, the altered neurons would differentiate into other subtypes, not similar to the wild type example. In this sense, miR19b may have a role in cell specification or neuronal differentiation. However, patterning is a different developmental event, which refers to the determination of broad genetic areas and territories. I don't think miR19b has a role in patterning.

(4) Please add a scheme of the molecules described in this article and the suggested interaction between them.

(5) The methods section is way too brief to allow for repeatability of the procedures. This may be due to an editorial policy but if possible, please extend the details of the experimental procedures.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study provides new insights into the role of miR-19b, an oncogenic microRNA, in the developing chicken pallium. Dynamic expression pattern of miR-19b is associated with its role in regulating cell cycle progression in neural progenitor cells. Furthermore, miR-19b is involved in determining neuronal subtypes by regulating Fezf2 expression during pallial development. These findings suggest an important role for miR-19b in the coordinated spatio-temporal regulation of neural progenitor cell dynamics and its evolutionary conservation across vertebrate species.

Strengths:

The authors identified conserved roles of miR-19 in the regulation of neural progenitor maintenance between mouse and chick, and the latter is mediated by the repression of E2f8 and NeuroD1. Furthermore, the authors found that miR-19b-dependent cell cycle regulation is tightly associated with specification of Fezf1 or Mef2c-positive neurons, in spatio-temporal manners during chicken pallial development. These findings uncovered molecular mechanisms underlying microRNA-mediated neurogenic controls.

Weaknesses:

Although the authors in this study claimed striking similarities of miR-19a/b in neurogenesis between mouse and chick pallium, a previous study by Bian et al. revealed that miR-19a contributes the expansion of radial glial cells by suppressing PTEN expression in the developing mouse neocortex, while miR-19b maintains apical progenitors via inhibiting E2f2 and NeuroD1 in chicken pallium. Thus, it is still unclear whether the orthologous microRNAs regulate common or species-specific target genes.

In this study, we have proposed that miR-19b regulates similar phenomena in both species using different targets, such as regulation of proliferation through PTEN in mouse and through E2f8 in the chicken.

The spatiotemporal expression patterns of miR-19b and several genes are not convincing. For example, the authors claim that NeuroD1 is initially expressed uniformly in the subventricular zone (SVZ) but disappears in the DVR region by HH29 and becomes detectable by HH35 (Figure 1). However, the in situ hybridization data revealed that NeuroD1 is highly expressed in the SVZ of the DVR at HH29 (Figure 4F). Thus, perhaps due to the problem of immunohistochemistry, the authors have not been able to detect NeuroD1 expression in Figure 1D, and the interpretation of the data may require significant modification.

While Fig. 1B may suggest that NeuroD1 expression has disappeared from the DVR region by HH29, this is not true in general because we have observed NeuroD1 to be expressed in the DVR at HH29 in images of other sections. In the revised version, we will include improved images for panels of Fig. 1B which accurately show the expression pattern of NeuroD1 and miR19b at stages HH29 and HH35.

It seems that miR-19b is also expressed in neurons (Figure 1), suggesting the role of miR19-b must be different in progenitors and differentiated neurons. The data on the gain- and loss-offunction analysis of miR-19b on the expression of Mef2c should be carefully considered, as it is possible that these experiments disturb the neuronal functions of miR19b rather than in the progenitors.

As pointed out by the reviewer, it is quite possible that upon manipulation of miR19b its neuronal functions are also perturbed in addition to its function in progenitor cells. After introducing gain-of-function construct in progenitor cells, we have observed changes in the morphology of these cells. These data will be included in the revised version.

The regions of chicken pallium were not consistent among figures: in Figure 1, they showed caudal parts of the pallium (HH29 and 35), while the data in Figure 4 corresponded to the rostral part of the pallium (Figure 4B).

We will address this by providing images from a similar region of the pallium showing Fezf2 and Mef2c expression patterns.

The neurons expressing Fezf2 and Mef2 in the chicken pallium are not homologous neuronal subtypes to mammalian deep and superficial cortical neurons. The authors must understand that chicken pallial development proceeds in an outside-in manner. Thus, Mef2c-postive neurons in a superficial part are early-born neurons, while FezF2-positive neurons residing in deep areas are later-born neurons. It should be noted that the expression of a single marker gene does not support cell type homology, and the authors' description "the possibility of primitive pallial lamina formation in common ancestors of birds and mammals" is misleading.

We appreciate this clarification and will modify or remove this statement regarding the “primitive pallial lamina formation” to avoid any confusion and misinterpretation.

Overexpression of CDKN1A or Sponge-19b induced ectopic expression of Fezf2 in the ventricular zone (Figure 3C, E). Do these cells maintain progenitor statement or prematurely differentiate to neurons? In addition, the authors must explain that the induction of Fezf2 is also detected in GFP-negative cells.

We propose to follow up on the fate of these cells by extending the observation period post-overexpression of CDKN1A or Sponge-19b to assess whether they retain progenitor characteristics or differentiate. The presence of Fezf2 in GFP-negative cells could be due to the non-cell-autonomous effects, and we will discuss this possibility in the revised manuscript.

Reviewer #2 (Public review):

Summary:

This paper investigates the general concept that avian and mammalian pallium specifications share similar mechanisms. To explore that idea, the authors focus their attention on the role of miR-19b as a key controlling factor in the neuronal proliferation/differentiation balance. To do so, the authors checked the expression and protein level of several genes involved in neuronal differentiation, such as NeuroD1 or E2f8, genes also expressed in mammals after conducting their functional gene manipulation experiments. The work also shows a dysregulation in the number of neurons from lower and upper layers when miR-19b expression is altered.

To test it, the authors conducted a series of functional experiments of gain and loss of function (G&LoF) and enhancer-reporter assays. The enhancer-reporter assays demonstrate a direct relationship between miR-19b and NeuroD1 and E2f8 which is also validated by the G&LoF experiments. It´s also noteworthy to mention that the way miR-19b acts is maintaining the progenitor cells from the ventricular zone in an undifferentiated stage, thus promoting them into a stage of cellular division.

Overall, the paper argues that the expression of miR-19b in the ventricular zone promotes the cells in a proliferative phase and inhibits the expression of differentiation genes such as E2f8 and NeurD1. The authors claim that a decrease in the progenitor cell pool leads to an increase and decrease in neurons in the lower and upper layers, respectively.

Strengths:

(1) Novelty Contribution

The paper offers strong arguments to prove that the neurodevelopmental basis between mammals and birds is quite the same. Moreover, this work contributes to a better understanding of brain evolution along the animal evolutionary tree and will give us a clearer idea about the roots of how our brain has been developed. This stands in contrast to the conventional framing of mammal brain development as an independent subject unlinked to the "less evolved species". The authors also nicely show a concept that was previously restricted to mammals - the role of microRNAs in development.

(2) Right experimental approach

The authors perform a set of functional experiments correctly adjusted to answer the role of miR-19b in the control of neuronal stem cell proliferation and differentiation. Their histological, functional, and genetic approach gives us a clear idea about the relations between several genes involved in the differentiation of the neurons in the avian pallium. In this idea, they maintain the role of miR-19b as a hub controller, keeping the ventricular zone cells in an undifferentiated stage to perpetuate the cellular pool.

(3) Future directions

The findings open a door to future experiments, particularly to a better comprehension of the role of microRNAs and pallidal genetic connections. Furthermore, this work also proves the use of avians as a model to study cortical development due to the similarities with mammals.

Weaknesses:

While there are questions answered, there are still several that remain unsolved. The experiments analyzed here lead us to speculate that the early differentiation of the progenitor cells from the ventricular zone entails a reduction in the cellular pool, affecting thereafter the number of latter-born neurons (upper layers). The authors should explore that option by testing progenitor cell markers in the ventricular zone, such as Pax6. Even so, it remains possible that miR-19b is also changing the expression pattern of neurons that are going to populate the different layers, instead of their numbers, so the authors cannot rule that out or verify it. Since the paper focuses on the role of miR-19b in patterning, I think the authors should check the relationship and expression between progenitors (Pax6) and intermediate (Tbr2) cells when miR-19b is affected. Since neuronal expression markers change so fast within a few days (HH24HH35), I don't understand why the authors stop the functional experiments at different time points.

To address this, we will examine the expression of Pax6 and Tbr2 following both gain-of-function and loss-of-function manipulations of miR-19b. We agree with the reviewer that miR-19b may influence not only the number of neurons but also the expression pattern of neuronal markers. Due to the limitations of our experimental design, we acknowledge that this possibility cannot be ruled out.

Regarding time points chosen for the functional experiments: We selected different stages based on the expression dynamics of specific markers. To detect possible ectopic induction, we analyzed developmental stages where the expression of a given marker is normally absent. Conversely, to detect loss of expression we examined stages in which the marker is typically expressed robustly. This approach allowed us to better interpret the functional consequences of miR-19b manipulation within relevant developmental windows.

Reviewer #3 (Public review):

Summary:

This is a timely article that focuses on the molecular machinery in charge of the proliferation of pallial neural stem cells in chicks, and aims to compare them to what is known in mammals. miR19b is related to controlling the expression of E2f8 and NeuroD1, and this leads to a proper balance of division/differentiation, required for the generation of the right number of neurons and their subtype proportions. In my opinion, many experiments do reflect an interaction between all these genes and transcription factors, which likely supports the role of miR19b in participating in the proliferation/differentiation balance.

Strengths:

Most of the methodologies employed are suitable for the research question, and present data to support their conclusions.

The authors were creative in their experimental design, in order to assess several aspects of pallial development.

Weaknesses:

However, there are several important issues that I think need to be addressed or clarified in order to provide a clearer main message for the article, as well as to clarify the tools employed. I consider it utterly important to review and reinterpret most of the anatomical concepts presented here. The way the are currently used is confusing and may mislead readers towards an understanding of the bird pallium that is no longer accepted by the community.

Major Concerns:

(1) Inaccurate use of neuroanatomy throughout the entire article. There are several aspects to it, that I will try to explain in the following paragraphs:

Figure 1 shows a dynamic and variable expression pattern of miR19b and its relation to NeuroD1. Regardless of the terms used in this figure, it shows that miR19b may be acting differently in various parts of the pallium and developmental stages. However, all the rest of the experiments in the article (except a few cases) abolish these anatomical differences. It is not clear, but it is very important, where in the pallium the experiments are performed. I refer here, at least, to Figures 2C, E, F, H, I; 3D, E; 4C, D, G, I. Regarding time, all experiments were done at HH22, and the article does not show the native expression at this stage. The sacrifice timing is variable, and this variability is not always justified. But more importantly, we don't know where those images were taken, or what part of the pallium is represented in the images. Is it always the same? Do results reflect differences between DVR and Wulst gene expression modifications? The authors should include low magnification images of the regions where experiments were performed. And they should consider the variable expression of all genes when interpreting results.

We agree that precise anatomical context is essential. In the revised version, we propose to:

a) Include schematics of the regions of interest where experimental manipulations were performed.

b) Provide low-magnification panoramic images where appropriate, for anatomical reference.

c) Show the expression patterns of relevant marker genes to better justify stages and region selection.

d) Provide the expression pattern of markers in panoramic view to show differential expression in the DVR and Wulst region and interpret our results accordingly.

b) SVZ is not a postmitotic zone (as stated in line 123, and wrongly assigned throughout the text and figures). On the contrary, the SVZ is a secondary proliferative zone, organized in a layer, located in a basal position to the VZ. Both (VZ and SVZ) are germinative zones, containing mostly progenitors. The only postmitotic neurons in VZ and SVZ occupy them transiently when moving to the mantle zone, which is closer to the meninges and is the postmitotic territory. Please refer to the original Boulder committee articles to revise the SVZ definition. The authors, however, misinterpret this concept, and label the whole mantle zone as it this would be the SVZ. Indeed, the term "mantle zone" does not appear in the article. Please, revise and change the whole text and figures, as SVZ statements and photographs are nearly always misinterpreted. Indeed, SVZ is only labelled well in Figure 4F.

The two articles mentioning the expression of NeuroD1 in the SVZ (line 118) are research in Xenopus. Is there a proliferative SVZ in Xenopus?

For the actual existence of the SVZ in the chick pallium, please refer to the recent Rueda-Alaña et al., 2025 article that presents PH3 stainings at different timepoints and pallial areas.

We appreciate the correction suggested by the reviewer. In the revised manuscript: a) SVZ will be labeled correctly in all figures and descriptions b) The mantle zone terminology will be incorporated appropriately c) The two Xenopus-based references in line 118 will be removed as they are not directly relevant and d) We will refer to the Rueda-Alaña et al., (2025) to guide accurate anatomical labeling and interpretation of proliferative zones.

We also acknowledge that while some proliferative cells exist in the SVZ of the chicken, they are relatively few and do not express typical basal progenitor markers such as Tbr2 (Nomura et al., 2016, Development). We will ensure that this nuance is clearly reflected in the text.

What is the Wulst, according to the authors of the article? In many figures, the Wulst includes the medial pallium and hippocampus, whereas sometimes it is used as a synonym of the hyperpallium (which excludes the medial pallium and hippocampus). Please make it clear, as the addition or not of the hippocampus definitely changes some interpretations.

We propose to modify the text and figures to accurately represent the correct location of the Wulst in the chick pallium.

d) The authors compare the entirety of the chick pallium - including the hippocampus (see above), hyperpallium, mesopallium, nidopallium - to only the neocortex of mammals. This view - as shown in Suzuki et al., 2012 - forgets the specificity of pallial areas of the pallium and compares it to cortical cells. This is conceptually wrong, and leads to incorrect interpretations (please refer to Luis Puelles' commentaries on Suzuki et al results); there are incorrect conclusions about the existence of upper-layer-like and deep-layer-like neurons in the pallium of birds. The view is not only wrong according to the misinterpreted anatomical comparisons, but also according to novel scRNAseq data (Rueda-Alaña et al., 2025; Zaremba et al., 2025; Hecker et al., 2025). These articles show that many avian glutamatergic neurons of the pallium have highly diversified, and are not comparable to mammalian cortical cells. The authors should therefore avoid this incorrect use of terminology. There are not such upper-layer-like and deeplayer-like neurons in the pallium of birds.

We acknowledge this conceptual oversight. In the manuscript: a) We will avoid direct comparisons between the entire chick pallium and the mammalian neocortex b) Terms like “upper-layer-like” and deep-layer-like” neurons will be removed or modified d) We will cite and integrate recent findings from Rueda-Alaña et al. (2025), Zaremba et al. (2025), and Hecker et al. (2025), which provide updated insights from scRNAseq analyses into the complexity of avian pallial neurons. Cell types will be described based on marker gene expression only, without unsupported evolutionary or homology claims.

(2) From introduction to discussion, the article uses misleading terms and outdated concepts of cell type homology and similarity between chick and pallial territories and cells. The authors must avoid this confusing terminology, as non-expert readers will come to evolutionary conclusions which are not supported by the data in this article; indeed, the article does not deal with those concepts.

We agree with the reviewer. In the revised version, we will remove the misleading terms and outdated concepts and avoid speculative evolutionary conclusions.

a) Recent articles published in Science (Rueda-Alaña et al., 2025; Zaremba et al., 2025; Hecker et al., 2025) directly contradict some views presented in this article. These articles should be presented in the introduction as they are utterly important for the subject of this article and their results should be discussed in the light of the new findings of this article. Accordingly, the authors should avoid claiming any homology that is not currently supported. The expression of a single gene is not enough anymore to claim the homology of neuronal populations.

In the revised version, these above-mentioned articles (Rueda-Alaña et al., 2025; Zaremba et al., 2025; Hecker et al., 2025) will be included in the introduction and discussion. Our interpretations will be updated to reflect these new insights into neuronal diversity and regionalization in the chick pallium.

Auditory cortex is not an appropriate term, as there is no cortex in the pallium of birds. Cortical areas require the existence of neuronal arrangements in laminae that appear parallel to the ventricular surface. It is not the case of either hyperpallium or auditory DVR. The accepted term, according to the Avian Nomenclature forum, is Field L.

We will replace all instances of “auditory cortex” with “Field L”, as per the accepted terminology in the Avian Nomenclature Forum.

c) Forebrain, a term overused in the article, is very unspecific. It includes vast areas of the brain, from the pretectum and thalamus to the olfactory bulb. However, the authors are not researching most of the forebrain here. They should be more specific throughout the text and title.

In the revised version, we will replace “forebrain” with “Pallium” throughout the manuscript to more accurately reflect the regions studied.

(3) In the last part of the results, the authors claim miR19b has a role in patterning the avian pallium. What they see is that modifying its expression induces changes in gene expression in certain neurons. Accordingly, the altered neurons would differentiate into other subtypes, not similar to the wild type example. In this sense, miR19b may have a role in cell specification or neuronal differentiation. However, patterning is a different developmental event, which refers to the determination of broad genetic areas and territories. I don't think miR19b has a role in patterning.

We agree with the reviewers that an alteration in one marker for a particular cell type may not indicate a change in patterning. However, including the effect of miR-19b gain- and loss-of-function on Pax6 and Tbr2, may strengthen the idea that it affects patterning as suggested by reviewer #2.

(4) Please add a scheme of the molecules described in this article and the suggested interaction between them.

In the revised version, we propose to include a diagram to visually summarize the proposed interactions between miR-19b, E2f8, NeuroD1, and other key regulators.

(5) The methods section is way too brief to allow for repeatability of the procedures. This may be due to an editorial policy but if possible, please extend the details of the experimental procedures.

We will expand the Methods section to provide more detailed protocols and justifications for experimental design, in alignment with journal policy.