dact1/2 modifies noncanonical Wnt signaling and calpain 8 expression to regulate convergent extension and craniofacial development

Reviewer #1 (Public Review):

Summary:
This study delves into the roles of dact1 and dact2 during zebrafish embryonic axis formation and craniofacial morphogenesis. The researchers seek to unravel the mechanisms by which dact1/2 influences Wnt signaling modulation throughout embryonic development and patterning. They propose distinct spatiotemporal roles for Dact1 and Dact2 proteins in zebrafish embryonic development, highlighting their involvement in modulating noncanonical Wnt signaling during convergent extension events. Their findings demonstrate that dact1 and dact2 exhibit distinct spatiotemporal expression domains during development and that dact1/2 mutation leads to convergent extension defects. Furthermore, the study attempts to establish a link between convergent extension defects resulting from dact1/2 mutation and subsequent craniofacial abnormalities during development. To investigate the connection between dact1 and dact2, compound mutants were employed since single mutants did not exhibit craniofacial phenotypes. Additionally, the research encompasses comprehensive transcriptomics and pathway analyses of differentially expressed genes in dact1/2 mutants. This analysis reveals the overexpression of a calcium-dependent cysteine protease, calpain 8. The study suggests a connection between the upregulation of calpain 8 and the observed craniofacial dysmorphology in dact1/2 mutants, implying a potential link between the altered expression of calpain 8 and the craniofacial abnormalities observed in these mutants.

Strengths:
The study beautifully recapitulates previous findings on the role of dact1/2 in modulating convergent extension during zebrafish embryogenesis.

A combination of multiple approaches, including in vivo time-lapse imaging, has been employed to elucidate the etiology of the rod-like neurocranial phenotype in dact1/2 double mutant.
This study utilizes and discusses several 'traditional' mutant lines and newly created ones, analyzing them through single-cell transcriptomics.

Weaknesses:
1. Enhancing Reproducibility and Robustness:
To enhance the reproducibility and robustness of the findings, it would be valuable for the authors to provide specific numbers of animals used in each experiment.
Explicitly stating the penetrance of the rod-like neurocranial shape in dact1/2-/- animals would provide a clearer understanding of the consistency of this phenotype.

2. Strengthening Single-Cell Data Interpretation:
To further validate the single-cell data and strengthen the interpretation of the gene expression patterns, I recommend the following:
-Provide a more thorough explanation of the rationale for comparing dact1/2 double mutants with gpc4 mutants.
-Employ genotyping techniques after embryo collection to ensure the accuracy of animal selection based on phenotype and address the potential for contamination of wild-type "delayed" animals.
-Supplement the single-cell data with secondary validation using RNA in situ or immunohistochemistry techniques.

3. Directly Investigating Non-Cell-Autonomous Effects:
To directly assess the proposed non-cell-autonomous role of dact1/2, I suggest conducting transplantation experiments to examine the ability of ectodermal/neural crest cells from dact1/2 double mutants to form wild-type-like neurocranium.

4. Further Elucidating Calpain 8's Role:
To strengthen the evidence supporting the critical role of Calpain 8, I recommend conducting overexpression experiments using a sensitized background to enhance the statistical significance of the findings.

Reviewer #2 (Public Review):

Summary:
Non-canonical Wnt signaling plays an important role in morphogenesis, but how different components of the pathway are required to regulate different developmental events remains an open question. This paper focuses on elucidating the overlapping and distinct functions of dact1 and dact2, two Dishevelled-binding scaffold proteins, during zebrafish axis elongation and craniofacial development. By combining genetic studies, detailed phenotypic analysis, lineage tracing, and single-cell RNA-sequencing, the authors aimed to understand (1) the relative function of dact1/2 in promoting axis elongation, (2) their ability to modulate phenotypes caused by mutations in other non-canonical wnt components, and (3) pathways downstream of dact1/2.

Strong qualitative evidence was provided to support dact1/2's role in genetically modulating non-canonical wnt signaling to regulate body axis elongation and the morphology of the anterior neurocranium (ANC). However, there is currently insufficient evidence supporting the author's claim that suppression of calpain 8 by dact1/2 is important for craniofacial development and that "embryonic fields determined during gastrulation affect the CNCC ability to contribute to the craniofacial skeleton".

Strengths:
(1) The generation of dact1/2 germline mutants and the use of genetic approaches to dissect their genetic interactions with wnt11f2 and gpc4 provide unambiguous and consistent results that inform the relative functions of dact1 and dact2, as well as their combined effects.

(2) Because the anterior neurocranium exhibits a spectrum of phenotypes in different genetic mutants, it is a useful system for studying how tissue morphology can be modulated by different components of the same pathway, as demonstrated in this study.

(3) The authors leveraged lineage tracing by photoconversion to dissect how dact1/2 differentially impacts the ability of different cranial neural crest populations to contribute to the anterior neurocranium. This revealed that distinct mechanisms can lead to similar phenotypes in different mutants.

Weaknesses:
(1) While the qualitative data show altered morphologies in each mutant, quantifications of these phenotypes are lacking in several instances, making it difficult to gauge reproducibility and penetrance, as well as to assess the novel ANC forms described in certain mutants.

(2) Germline mutations limit the authors' ability to study a gene's spatiotemporal functional requirement. They therefore cannot concretely attribute nor separate early-stage phenotypes (during gastrulation) to/from late-stage phenotypes (ANC morphological changes).

(3) Given that dact1/2 can regulate both canonical and non-canonical wnt signaling, this study did not specifically test which of these pathways is altered in the dact1/2 mutants, and it is currently unclear whether disrupted canonical wnt signaling contributes to the craniofacial phenotypes, even though these phenotypes are typical non-canonical wnt phenotypes.

(4) The use of single-cell RNA sequencing unveiled genes and processes that are uniquely altered in the dact1/2 mutants, but not in the gpc4 mutants during gastrulation. However, how these changes lead to the manifested ANC phenotype later during craniofacial development remains unclear. The authors showed that calpain 8 is significantly upregulated in the mutant, but the fact that only 1 out of 142 calpain-overexpressing animals phenocopied dact1/2 mutants indicates the complexity of the system.

(5) Craniofacial phenotypes observed in this study are attributed to convergent extension defects but convergent extension cell movement itself was not directly examined, leaving open if changes in other cellular processes, such as cell differentiation, proliferation, or oriented division, could cause distinct phenotypes between different mutants.

Reviewer #3 (Public Review):

Summary:
In this manuscript, the authors explore the roles of dact1 and dact2 during zebrafish gastrulation and craniofacial development. Previous studies used morpholino (MO) knockdowns to show that these scaffolding proteins, which interact with disheveled (Dsh), are expressed during zebrafish gastrulation and suggested that dact1 promotes canonical Wnt/B-catenin signaling, while dact2 promotes non-canonical Wnt/PCP-dependent convergent-extension (Waxman et al 2004). This study goes beyond this work by creating loss-of-function mutant alleles for each gene and unlike the MO studies finds little (dact2) to no (dact1) phenotypic defects in the homozygous mutants. Interestingly, dact1/2 double mutants have a more severe phenotype, which resembles those reported with MOs as well as homozygous wnt11/silberblick (wnt11/slb) mutants that disrupt non-canonical Wnt signaling (Heisenberg et al., 1997; 2000). Further analyses in this paper try to connect gastrulation and craniofacial defects in dact1/2 mutants with wnt11/slb and other wnt-pathway mutants. scRNAseq conducted in mutants identifies calpain 8 as a potential new target of dact1/2 and Wnt signaling.

Strengths:
When considered separately the new mutants are an improvement over the MOs and the paper contains a lot of new data.

Weaknesses:
The hypotheses are very poorly defined and misinterpret key previous findings surrounding the roles of wnt11 and gpc4, which results in a very confusing manuscript. Many of the results are not novel and focus on secondary defects. The most novel result of overexpressing calpain8 in dact1/2 mutants is preliminary and not convincing.

Major Comments:

1. One major problem throughout the paper is that the authors misrepresent the fact that wnt11f2 and gpc4 act in different cell populations at different times. Gastrulation defects in these mutants are not similar: wnt11 is required for anterior mesoderm CE during gastrulation but not during subsequent craniofacial development while gpc4 is required for posterior mesoderm CE and later craniofacial cartilage morphogenesis (LeClair et al., 2009). Overall, the non-overlapping functions of wnt11 and gpc4, both temporally and spatially, suggest that they are not part of the same pathway.

2. There are also serious problems surrounding attempts to relate single-cell data with the other data in the manuscript and many claims that lack validation. For example, in Fig 1 it is entirely unclear how the Daniocell scRNA-seq data have been used to compare dact1/2 with wnt11f2 or gpc4. With no labeling in panel 1E of this figure these comparisons are impossible to follow. Similarly, the comparisons between dact1/2 and gpc4 in scRNA-seq data in Fig. 6 as well as the choices of DEGs in dact1/2 or gpc4 mutants in Fig. 7 seem arbitrary and do not make a convincing case for any specific developmental hypothesis. Are dact1 and gpc4 or dact2 and wnt11 co-expressed in individual cells? Eyeballing similarity is not acceptable.

3. Many of the results in the paper are not novel and either confirm previous findings, particularly Waxman et al (2004), or even contradict them without good evidence. The authors should make sure that dact2 loss-of-function is not compensated for by an increase in dact1 transcription or vice versa. Testing genetic interactions, including investigating the expression of wnt11f2 in dact1/2 mutants, dact1/2 expression in wnt11f2 mutants, or the ability of dact1/2 to rescue wnt11f2 loss of function would give this work a more novel, mechanistic angle.

4. The identification of calpain 8 overexpression in Dact1/2 mutants is interesting, but getting 1/142 phenotypes from mRNA injections does not meet reproducibility standards.