Genetic evidence: zebrafish hoxba and hoxbb clusters are essential for the anterior-posterior positioning of pectoral fins
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eLife Assessment
This important study advances our understanding of vertebrate forelimb development, specifically the contribution of Hox genes to zebrafish pectoral fin formation. While there are reservations about some of the descriptions and interpretations of the data, the results are mostly convincing. The authors have employed a robust and extensive genetic approach to tackle a key and unresolved question. The findings will be of broad interest to developmental and evolutionary biologists.
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Abstract
Abstract
Vertebrate paired appendages, such as the pectoral fins in fish and the forelimbs in tetrapods, emerge at specific regions along the anterior-posterior axis of the body. Hox genes are considered prime candidates for determining the positioning of these paired appendages during development. Despite extensive phenotypic analyses of numerous single and compound Hox knockout mice, no genetic studies have identified substantial defects in limb positioning, leaving questions unresolved. In a previous study, we generated seven distinct hox cluster-deficient mutants in zebrafish. Here, we provide genetic evidence that zebrafish hoxba;hoxbb cluster-deleted mutants specifically exhibit a complete absence of pectoral fins, accompanied by the absence of tbx5a expression in pectoral fin buds. In these mutants, tbx5a expression in the pectoral fin field of the lateral plate mesoderm fails to be induced at an early stage, suggesting a lack of pectoral fin precursor cells. Furthermore, the competence to respond to retinoic acid is lost in hoxba;hoxbb cluster mutants, indicating that tbx5a expression cannot be induced in the pectoral fin buds. We also identify hoxb4a, hoxb5a, and hoxb5b as pivotal genes underlying this process. Although the frameshift mutations in these hox genes do not recapitulate the absence of pectoral fins, we demonstrate that deletion mutants at these genomic loci show the absence of pectoral fins, albeit with low penetrance. Our results suggest that the positioning of zebrafish pectoral fins is cooperatively determined by hoxb4a, hoxb5a, and hoxb5b within hoxba and hoxbb clusters, which induce tbx5a expression in the restricted pectoral fin field. Our findings also provide insights into the acquisition of paired appendages in vertebrates.
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eLife Assessment
This important study advances our understanding of vertebrate forelimb development, specifically the contribution of Hox genes to zebrafish pectoral fin formation. While there are reservations about some of the descriptions and interpretations of the data, the results are mostly convincing. The authors have employed a robust and extensive genetic approach to tackle a key and unresolved question. The findings will be of broad interest to developmental and evolutionary biologists.
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Reviewer #1 (Public review):
Summary:
The authors have used gene deletion approaches in zebrafish to investigate the function of genes of the hox clusters in pectoral fin "positioning" (but perhaps more accurately pectoral fin "formation").
Strengths:
The authors have employed a robust and extensive genetic approach to tackle an important and unresolved question.
The results are largely presented in a very clear way.
Weaknesses:
The Abstract suggests that no genetic evidence exists in model organisms for a role of Hox genes in limb positioning. There are, however, several examples in mouse and other models (both classical genetic and other) providing evidence for a role of Hox genes in limb position, which is elaborated on in the Introduction.
It would perhaps be more accurate to state that several lines of evidence in a range of model …
Reviewer #1 (Public review):
Summary:
The authors have used gene deletion approaches in zebrafish to investigate the function of genes of the hox clusters in pectoral fin "positioning" (but perhaps more accurately pectoral fin "formation").
Strengths:
The authors have employed a robust and extensive genetic approach to tackle an important and unresolved question.
The results are largely presented in a very clear way.
Weaknesses:
The Abstract suggests that no genetic evidence exists in model organisms for a role of Hox genes in limb positioning. There are, however, several examples in mouse and other models (both classical genetic and other) providing evidence for a role of Hox genes in limb position, which is elaborated on in the Introduction.
It would perhaps be more accurate to state that several lines of evidence in a range of model organisms (including the mouse) support a role for Hox genes in limb positioning. The author's work is not weakened by a more inclusive introduction that cites the current literature more comprehensively.
It would be helpful for the authors to make a clear distinction between "positioning" of the limb/fin and whether a limb/fin "forms" at all, independent of the relative position of this event along the body axis.
Discussion of why the zebrafish is sensitive to Hoxb loss with reference to the fin, but mouse Hoxb mutants do make a limb?
Is this down to exclusive expression of Hoxbs in the zebrafish pectoral fin forming region rather than a specific functional role of the protein? This is important as it has implications for the interpretation of results throughout the paper and could explain some apparently conflicting results.
Why is Hoxba more potent than Hoxbb? Is this because Hoxba has Hox4/5 present, while Hoxbb has only Hoxb5? Hoxba locus has retained many more Hox genes in cluster than hoxbb; therefore, one might expect to see greater redundancy in this locus).
Deletion of either Hoxa or Hoxd in the background of the Hoxba mutant does have some effect. Is this a reflection of protein function or expression dynamics of Hoxa/Hoxd genes?
Can we really be confident that there is a "transformation of pectoral fin progenitor cells into cardiac cells"?
The failure to repress Nkx2.5 in the posterior (pelvic fin) domain is clear, but have these cells actually acquired cardiac identity? They would be expected to express Tbx5a (or b) as cardiac precursors, but this domain does not broaden. There is no apparent expansion of the heart (field)/domain or progenitors beyond the 16 somite stage. The claimed "migration" of heart precursors in the mutant is not clear. The heart/cardiac domain that does form in the mutant is not clearly expanded in the mutant. The domain of cmlc2 looks abnormal in the mutant, but I am not convinced it is "enlarged" as claimed by the authors. The authors have not convincingly shown that "the cells that should form the pectoral fin instead differentiate into cardiac cells."
The only clear conclusion is the loss of pectoral fin-forming cells rather than these fin-forming cells being "transformed" into a new identity. It would be interesting to know what has happened to the cells of the pectoral fin-forming region in these double mutants.
It is not clear what the authors mean by a "converse" relationship between forelimb/pectoral fin and heart formation. The embryological relationship between these two populations is distinct in amniotes.
The authors show convincing data that RA cannot induce Tbx5a in the absence of Hob clusters, but I am not convinced by the interpretation of this result. The results shown would still be consistent with RA acting directly upstream of tbx5a, but merely that RA acts in concert with hox genes to activate tbx5a. In the absence of one or the other, Tbx5a would not be expressed. It is not necessary that RA and hoxbs act exclusively in a linear manner (i.e., RA regulates hoxb that in turn regulates tbx5a).
The authors have carried out a functional test for the function of hoxb6 and hoxb8 in the hemizygous hoxb mutant background. What is lacking is any expression analysis to demonstrate whether Hoxb6b or Hoxb8b are even expressed in the appropriate pectoral fin territory to be able to contribute to pectoral fin development, either in this assay or in normal pectoral fin development.
(The term "compensate" used in this section is confusing/misleading.)
The authors' confounding results described in Figures 6-7 are consistent with the challenges faced in other model organisms in trying to explore the function of genes in the hox cluster and the known redundancy that exists across paralogous groups and across individual clusters.
Given the experimental challenges in deciphering the actual functions of individual or groups of hox genes, a discussion of the normal expression pattern of individual and groups of hox genes (and how this may change in different mutant backgrounds) could be helpful to make conclusions about likely normal function of these genes and compensation/redundancy in different mutant scenarios.
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Reviewer #2 (Public review):
Summary:
The authors of this manuscript performed a fascinating set of zebrafish mutant analyses on hox cluster deletion and pinpointed the cause of the pectoral fin loss in one combinatorial hox cluster mutant of Hoxba and Hoxbb.
Strengths:
The study is based on a variety of existing experimental tools that enabled the authors' past construction of hox cluster mutants, and is well-designed. The manuscript is well written to report the authors' findings on the mechanism that positions the pectoral fin.
Weaknesses:
The study does not focus on the other hox clusters other than ba and bb, and is confined to the use of zebrafish, as well as the comparison with existing reports from mouse experiments.
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Author response:
We appreciate the reviewers' positive feedback on our paper. We especially thank them for their evaluation of the genetic analysis, which required a significant amount of timef time. We acknowledge that several aspects of our interpretation and description of the results need correction, as noted by both reviewers. Additionally, we recognize the importance of providing a more comprehensive overview of previous findings, including those conducted in mice, in the manuscript. In the revised version, we will thoroughly address the reviewers' concerns.
Both reviewers emphasized the need for further validation to ascertain whether the specific requirement of Hox genes in the Hoxba and Hoxbb clusters for pectoral fin bud formation is due to their expression patterns or the functional roles of Hox proteins. This consideration …
Author response:
We appreciate the reviewers' positive feedback on our paper. We especially thank them for their evaluation of the genetic analysis, which required a significant amount of timef time. We acknowledge that several aspects of our interpretation and description of the results need correction, as noted by both reviewers. Additionally, we recognize the importance of providing a more comprehensive overview of previous findings, including those conducted in mice, in the manuscript. In the revised version, we will thoroughly address the reviewers' concerns.
Both reviewers emphasized the need for further validation to ascertain whether the specific requirement of Hox genes in the Hoxba and Hoxbb clusters for pectoral fin bud formation is due to their expression patterns or the functional roles of Hox proteins. This consideration has been on our agenda for some time; however, our submitted paper does not sufficiently address this aspect. In the revised manuscript, we will conduct a comprehensive analysis of the expression patterns of Hox genes in zebrafish to draw informed conclusions on this matter.
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