Laminin and Fibronectin contribute to Guided Endothelial Self-Organization during Intersegmental Vessel Formation

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   Review Commons

   Oct 16, 2026

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   We thank both reviewers for their careful evaluations. Their comments have led us to strengthen the experimental analysis, sharpen the conceptual framework, and qualify the mechanistic interpretation.

   REVIEWER 1

   Summary

   In this study, the authors investigate how extracellular matrix (ECM) properties and repulsive guidance cues cooperate to regulate vessel growth and patterning during angiogenesis. To address this question, they develop a mathematical framework that integrates endothelial cell network formation, ECM mechanics, and semaphorin-mediated repulsive signaling. Their simulations predict that reductions in ECM stiffness or density impair …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   We thank both reviewers for their careful evaluations. Their comments have led us to strengthen the experimental analysis, sharpen the conceptual framework, and qualify the mechanistic interpretation.

   REVIEWER 1

   Summary

   In this study, the authors investigate how extracellular matrix (ECM) properties and repulsive guidance cues cooperate to regulate vessel growth and patterning during angiogenesis. To address this question, they develop a mathematical framework that integrates endothelial cell network formation, ECM mechanics, and semaphorin-mediated repulsive signaling. Their simulations predict that reductions in ECM stiffness or density impair vessel extension, while reductions in either ECM density or semaphorin gradient distribution promote vessel fusion, suggesting that ECM properties and semaphorin signaling act together to guide vascular sprouts and maintain vessel patterning.

   The authors then test these predictions experimentally using zebrafish intersegmental vessels (ISVs) as an in vivo model of sprouting angiogenesis. Through morpholino-mediated depletion of laminin 1, laminin 4, fibronectin 1a, and fibronectin 1b, alone and in combination, they examine the consequences of ECM disruption on ISV growth and organization. They report defects in vessel extension and patterning, including increased ectopic vessel fusion, and conclude that laminin- and fibronectin-dependent ECM organization is required for proper ISV migration, growth, and patterning.

   The study combines computational modeling with experimental analysis to address an important question in vascular development and provides a potentially valuable framework for understanding how mechanical and guidance cues are integrated during angiogenic sprouting.

   Major comments

   Comment 1

   1. The evidence does not convincingly demonstrate that laminin/fibronectin directly regulate ISV patterning

   The authors developed a mathematical model incorporating endothelial cell network formation, ECM mechanics, and semaphorin-mediated repulsive signaling. The simulations predict that reduced ECM stiffness or density slows vessel growth, whereas reduction of either ECM density or the semaphorin gradient promotes vessel fusion, suggesting that ECM properties and semaphorin signaling cooperate to guide vessel trajectories.

   The experimental data, however, do not convincingly establish that the observed ISV patterning defects are a direct consequence of laminin/fibronectin loss. As acknowledged by the authors, combined depletion of laminin and fibronectin severely disrupts somite and notochord morphogenesis. Given the well-established role of somites and semaphorin-mediated repulsive signaling in restricting ISV growth and preventing ectopic branching or fusion, it remains unclear whether the vascular phenotypes arise from direct endothelial-ECM defects or indirectly from altered trunk patterning.

   The authors argue that ISV disorganization is still observed in embryos with comparatively preserved somite morphology (e.g., MO L14Fb). However, the assessment of somite preservation is based on relatively low-magnification images, and it is unclear whether molecular patterning within the somites remains intact. In particular, the manuscript does not address whether semaphorin expression or distribution is altered in MO L14Fa, MO L14Fb, or the corresponding rescue conditions. This is an important omission because semaphorin gradients are explicitly incorporated into the mathematical model and are predicted to strongly influence vessel guidance by preventing ectopic vessel fusion.

   Therefore, to support the conclusion that laminin/fibronectin directly regulate ISV patterning, it would be important to determine whether semaphorin expression and/or distribution are preserved following laminin/fibronectin depletion. Without such data, the possibility remains that the vascular mispatterning phenotype is secondary to defects in somite and notochord development.

   Furthermore, previous studies disrupting endothelial-ECM adhesion have generally reported defects in ISV growth and elongation without accompanying vessel mispatterning:

   • Talin1-deficient zebrafish (tln1 uqal−/−) exhibit impaired ISV growth and an increased frequency of disconnected ISVs but do not display ectopic branching or aberrant vessel patterning (Chau et al., 2022; doi:10.1242/dev.200454).

   • A second talin1 mutant (fl02k) similarly shows defects in vascular development without ISV mispatterning (Wu et al., 2015; doi:10.1096/fj.15-273409).

   • Loss of integrin β1b results in impaired ISV elongation but does not disrupt ISV patterning (Iida et al., 2018; doi:10.1111/gtc.1264).

   These studies suggest that impaired endothelial-ECM interactions alone are insufficient to produce the extensive ISV fusion and mispatterning observed here, raising the possibility that secondary defects in trunk tissue organization contribute substantially to the phenotype.

   Authors’ response

   We agree that the original manuscript did not sufficiently distinguish a direct endothelial-ECM effect from indirect effects arising from disruption of the surrounding trunk tissues. We have therefore substantially revised both the experimental analysis and our interpretation.

   First, we added whole-embryo bright-field imaging to document gross morphology across the principal ECM perturbation conditions. These analyses show that MO L14Fa displays pronounced trunk and somite abnormalities, whereas MO L14Fb retains comparatively preserved gross segmental morphology despite strong vascular disorganization.

   Second, because Semaphorin/Plexin signaling is an established regulator of ISV pathfinding and is explicitly represented in our mathematical model, we directly examined sema3aa and sema3ab expression. Whole-mount HCR RNA-FISH showed that both transcripts remained detectable in the trunk. The sema3ab spatial expression pattern, which is implicated in ISV guidance, appeared broadly preserved, whereas sema3aa showed more apparent local differences in the more severely affected MO L14Fa embryos. Complementary RT-qPCR performed on ΔCt values detected no significant differences in overall sema3aa or sema3ab transcript abundance between Control, MO L14Fa, and MO L14Fb. Nevertheless, these transcript-level analyses cannot exclude changes in local Semaphorin protein distribution or activity.

   We therefore interpret these data as arguing against wholesale loss of sema3aa/sema3ab expression as the sole explanation for the vascular phenotype, while explicitly acknowledging that local tissue disorganization and altered presentation of guidance signals may still contribute, particularly in MO L14Fa.

   Third, we added genetic validation experiments in which MO L14 was combined with homozygous fn1a or fn1b mutant backgrounds. Laminin depletion strongly increased aberrant ISV patterning in both fibronectin mutant backgrounds. These experiments provide independent genetic support for a laminin-fibronectin interaction, although we explicitly acknowledge that the laminin component remains morpholino-based.

   Fourth, chimeric fn1a and fn1b mRNA rescue significantly reduced the vascular patterning phenotype in the corresponding combined perturbation backgrounds.

   In response to the reviewer’s central concern, we have also revised the wording throughout the manuscript. We no longer claim that the experiments establish a purely endothelial or cell-autonomous mechanism. The revised Results explicitly state that the combined genetic, rescue, and Semaphorin analyses support an ECM-dependent contribution to ISV patterning “without establishing a purely endothelial or cell-autonomous mechanism.”

   We have additionally incorporated the studies cited by the reviewer concerning Talin1 and integrin β1. In the revised Discussion, we distinguish perturbation of a single endothelial adhesion component from combined laminin/fibronectin perturbation, which can simultaneously influence ligand availability, ECM organization and mechanics, neighboring tissues, and multiple receptor systems.

   Comment 2

   1. Quantification of ISV growth using "ISV volume" requires further justification

   The authors estimate average ISV volume by dividing the total segmented trunk vascular volume by the number of ISVs. This metric raises several concerns.

   First, the segmented vascular volume appears to include non-ISV structures such as the dorsal longitudinal anastomotic vessel (DLAV). Consequently, the calculated parameter does not exclusively represent ISV volume and may not accurately reflect ISV growth.

   Second, several morpholino conditions (e.g., MO Fn1ab, MO L14, MO lama1/4 fn1a, and MO lama1/4 fn1b) exhibit pronounced developmental delay and shortened trunk length. Under these conditions, reduced vascular volume may simply reflect overall embryonic growth retardation rather than a specific defect in ISV growth. Therefore, it is difficult to distinguish primary vascular phenotypes from secondary consequences of reduced embryo size.

   Finally, ISV growth is normalized to the dorsal-ventral axis length. Because overall embryonic dimensions are reduced in several experimental groups, this normalization may partially mask genuine ISV growth defects.

   The authors should therefore provide additional justification for this metric and discuss its limitations. Direct measurements of ISV length may provide more robust assessments of ISV growth.

   Authors’ response

   We agree with this criticism and have replaced the previous volume-based analysis.

   The revised manuscript no longer uses average segmented vascular volume or normalization to the embryo dorsal-ventral axis as the primary measure of ISV growth. Instead, ISV extension was measured directly in micrometers during time-lapse imaging from approximately 22–28 hpf.

   For each embryo, three ISVs that had only recently initiated dorsal extension from the dorsal aorta at the beginning of the recording were selected and followed over time. ISVs that had already undergone substantial extension were excluded. The three measurements were averaged to obtain an embryo-level value at each time point, and an embryo-level growth rate in µm/h was calculated over the six-hour interval. Thus, the revised analysis directly measures ISV elongation and avoids the concerns associated with inclusion of non-ISV vascular structures, differences in overall embryo dimensions, and dorsal-ventral normalization.

   Minor comments

   Minor comment 1

   1. When describing tip-cell filopodia and lamellipodia dynamics in ISVs, the authors should cite Phng et al., 2013 (doi:10.1242/dev.097352).

   Authors’ response

   Added. Phng et al., 2013, is now cited together with the relevant literature on endothelial tip-cell behavior.

   Minor comment 2

   1. Page 3 contains a "?" in place of a reference citation.

   Authors’ response

   Corrected during revision of the Introduction and references.

   Minor comment 3

   1. Transgenic line nomenclature should be italicized throughout the manuscript.

   Authors’ response

   The nomenclature was reviewed and standardized throughout the manuscript.

   Minor comment 4

   1. mRNA transcript names should be italicized throughout the manuscript.

   Authors’ response

   The nomenclature was reviewed and standardized throughout the manuscript.

   Minor comment 5

   1. Figure 1E is presented at insufficient magnification to evaluate ISV morphology and patterning. Higher-magnification images similar to those shown in Figures 5 and 6 would be helpful.

   Authors’ response

   Figure 1 has been reorganized and now contains higher-magnification representative trunk images that allow individual ISV trajectories and patterning defects to be evaluated more clearly.

   Minor comment 6

   1. Figure 3 legend refers to a fourth simulation condition ("reduction of both fiber stiffness and fiber density"), but this condition is not shown in the figure. This statement should be removed unless the corresponding data are presented.

   Authors’ response

   Corrected. The revised figure and legend describe only the simulation conditions that are presented.

   Minor comment 7

   1. Statistical comparisons should be provided for Figure 3C-F.

   Authors’ response

   Added. The revised Figure 3 legend now reports the statistical tests, multiple-comparison procedures, sample sizes, and adjusted p-values for the relevant comparisons.

   Minor comment 8

   1. In Figure 3B-F, reductions in ECM density and ECM stiffness both decrease vessel length and growth speed. Both perturbations also reduce focal adhesion number, yet only ECM stiffness reduction decreases focal adhesion lifetime. The authors should clarify how these distinct focal adhesion phenotypes converge on a similar vascular outcome.

   Authors’ response

   We have clarified this point in the revised Results and Discussion. Both decreased stiffness and decreased fiber density reduce the total number of focal adhesions and slow vessel extension, but reduced stiffness produces a stronger reduction in focal-adhesion lifetime. We now interpret reduced density primarily as decreasing the availability of attachment sites, while reduced stiffness has a particularly strong effect on focal-adhesion stabilization. Although these perturbations influence focal-adhesion dynamics differently, both ultimately reduce efficient sprout extension.

   Minor comment 9

   1. In Figure 5F, the control group should be presented first (leftmost position) for consistency with the other panels.

   Authors’ response

   The control group is now presented in the leftmost position in the corresponding revised figure panel.

   Minor comment 10

   1. In Figure 5C (lower panels), image orientation should be standardized such that anterior is positioned on the left side.

   Authors’ response

   Image orientation was standardized. Embryo and trunk images are now consistently presented with anterior to the left and dorsal up, and this orientation is specified in the figure legends.

   Minor comment 11

   1. In Figure 6A, fn1a and fn1b mRNA overexpression appear to exert different effects on ISV volume. Specifically, fn1a overexpression increases ISV volume relative to control, whereas fn1b overexpression appears to decrease it. The authors should discuss the basis for these differing effects.

   Authors’ response

   We agree that the original presentation encouraged comparisons between experiments that had been performed independently. The fn1a and fn1b rescue series were carried out as separate experiments in different endothelial reporter backgrounds and are now analyzed and interpreted independently.

   Because these rescue experiments were performed independently and in different endothelial reporter backgrounds, we do not interpret differences between the fn1a and fn1b mRNA-only groups as evidence of distinct biological effects of the two fibronectin paralogs. The revised analysis therefore evaluates each mRNA condition against its corresponding experimental control, rather than making direct comparisons across the two rescue series.

   Minor comment 13

   1. The phenotypic severity of MO L14Fa and MO L14Fb appears inconsistent between Figures 5 and 6. In Fig. 5B, ISV volume of MO L14Fb is bigger than MO L14Fa while in Fig. 6A, ISV volume of MO L14Fb is smaller than MO L14Fa. In Fig. 5C, aberrant ISV number in MO L14Fb is significantly smaller than MO L14Fa while in Fig. 6A, they are comparable. The authors should clarify whether these experiments were performed independently and explain the source of these discrepancies.

   Authors’ response

   We agree that the original presentation encouraged comparisons between experiments that had been performed independently. The fn1a and fn1b rescue series were carried out as separate experiments in different endothelial reporter backgrounds and are now analyzed and interpreted independently.

   The revised manuscript therefore does not use the relative severity of MO L14Fa versus MO L14Fb across these independent rescue experiments as a biological comparison. Instead, rescue is assessed within each matched experimental series.

   Moreover, the original manuscript had figure 5C with an absolute number of aberrant ISVs whereas figure 6A had it as a percentage of aberrant ISVs. Now we show all aberrant ISVs as a percentage of the total ISVs analyzed.

   Significance assessment — Nature and significance of the advance

   This work represents a primarily conceptual advance in the field of angiogenesis by proposing a mechanism through which ECM mechanical properties and semaphorin-mediated repulsive signaling cooperate to guide vessel growth and patterning. The integration of computational modeling with in vivo experiments is a notable strength and has the potential to provide new insights into how multiple environmental cues are combined to regulate angiogenic behavior.

   The study builds upon a substantial body of work demonstrating that ECM composition and mechanics influence endothelial migration and sprouting, as well as studies showing that semaphorin signaling restricts aberrant vessel branching and fusion during zebrafish vascular development. The novelty of the present work lies in its attempt to unify these mechanisms within a single theoretical and experimental framework. However, the extent to which the experimental data support a direct role for laminin and fibronectin in vessel patterning, as opposed to indirect effects arising from disrupted somite and notochord development, remains an important question that should be addressed before the proposed mechanism can be fully established.

   Significance assessment — Audience

   The findings are likely to be of interest to researchers working in vascular biology, developmental biology, angiogenesis, collective cell migration, extracellular matrix biology, mechanobiology, and computational modeling of morphogenesis.

   Reviewer expertise

   My expertise includes vascular development, sprouting angiogenesis, endothelial cell biology, mechanobiology and zebrafish models of vascular development.

   I am comfortable evaluating the developmental biology, vascular phenotyping, endothelial cell biology, and interpretation of the zebrafish experiments. While I can assess the biological assumptions and interpretation of the computational framework, I do not have specialized expertise in mathematical modeling and therefore cannot fully evaluate the technical implementation, parameterization, or numerical aspects of the model.

   REVIEWER 2

   Summary and general assessment

   This manuscript combines zebrafish genetics, live imaging, rescue experiments, and mathematical modeling to investigate the roles of laminin and fibronectin during intersegmental vessel (ISV) development. The integration of experimental and computational approaches is a strength of the study, and the manuscript presents potentially interesting findings suggesting that extracellular matrix (ECM) components, including laminin and fibronectin, cooperatively contribute to ISV growth and guidance. However, the current experimental evidence lacks sufficient rigor to convincingly support this conclusion and does not provide adequate support for the broader mechanistic and conceptual framework of ECM-guided self-organization proposed by the authors.

   Overall, the observations presented in this manuscript may be of potential interest to the vascular biology field, but the manuscript requires substantial improvements in its presentation, conceptual framework, mechanistic evidence, and experimental rigor to support its main conclusions.

   My major concerns regarding these points are outlined below. In its current form, the manuscript reads more like a dissertation chapter than a primary research article, with the excessively long Introduction and the Results section containing extensive methodological descriptions and discussions that obscure the main experimental findings. More importantly, while the experimental observations suggest a role for laminin and fibronectin during ISV development, the proposed mechanistic and conceptual framework of "ECM-guided self-organization" is neither sufficiently defined nor experimentally supported. Finally, several aspects of the experimental design require additional rigor, including developmental stage normalization, independent validation of morpholino-mediated phenotypes, and more careful interpretation of the observed vascular defects. Addressing these concerns would substantially improve both the clarity and scientific impact of the manuscript.

   Major comments

   Comment 1

   1. The manuscript would benefit from substantial reorganization from its current dissertation-style structure into the format of a primary research article.

   The manuscript appears to retain much of the organization and level of detail expected for a dissertation chapter rather than being revised into the concise format of a primary research article. A major reorganization is needed to substantially improve the scientific presentation of the study. The Introduction is considerably longer than necessary and contains an extensive literature review before introducing the specific biological question. Likewise, the Results frequently include lengthy methodological descriptions, literature comparisons, and biological interpretation, making it difficult to distinguish the main experimental findings from the discussion. Streamlining the Introduction, moving methodological details to the Methods or Supplementary Information where appropriate, reducing repetitive discussion throughout the Results, and presenting the experiments in a more concise and sequential manner would greatly improve the readability of the manuscript and allow readers to more readily appreciate its principal scientific findings.

   Authors’ response

   We agree and have substantially reorganized the manuscript.

   The Introduction has been shortened and rewritten around a more focused progression: the relationship between angiogenic sprouting and endothelial network-forming behavior; the conceptual role of local endothelial interactions; ISV development as the experimental system; known Semaphorin and ECM guidance mechanisms; the specific laminin/fibronectin hypothesis; and the principal experimental strategy.

   Extensive methodological information has been moved from the Results to the Materials and Methods, and literature discussion and interpretation have been reduced within individual Results sections. The Results now follow the experimental sequence more directly.

   We believe this substantially improves the distinction between experimental findings, methodological details, and broader biological interpretation.

   Comment 2

   1. The central concept of "ECM-guided self-organization" is not clearly defined, and its conceptual advance is currently difficult to appreciate.

   One of my primary concerns is the conceptual framework proposed throughout the manuscript. The term "self-organization" is never clearly defined, making it difficult to understand precisely what biological mechanism the authors intend to describe. Although the Introduction discusses endothelial morphogenesis as a process involving both endothelial cell-intrinsic programs and tissue-derived extrinsic regulatory mechanisms, it subsequently introduces the concept of "ECM-guided self-organization" without clearly explaining how this framework differs from existing models of angiogenic sprouting or endothelial morphogenesis.

   This ambiguity is particularly important because endothelial sprouting is generally understood to result from integration of endothelial cell-intrinsic programs together with tissue-derived extrinsic mechanisms, including extracellular matrix components, angiogenic factors, and guidance cues. Indeed, the Introduction itself acknowledges this principle. Consequently, it remains unclear to me how the proposed model of "ECM-guided self-organization" differs conceptually from the current understanding of endothelial morphogenesis or whether it establishes a fundamentally new biological principle.

   More broadly, the conceptual advance of the study remains difficult to appreciate. The importance of extracellular matrix components during endothelial migration, angiogenesis, and vascular morphogenesis is already well established. The present work aims to provide additional evidence that laminin and fibronectin contribute to ISV sprouting and patterning, but it is difficult to identify what new mechanistic or conceptual insight is established beyond the recognized roles of the extracellular matrix. A clearer definition of "self-organization" and a more explicit explanation of how the proposed model advances beyond existing concepts would greatly strengthen the manuscript and help readers better appreciate its conceptual contribution.

   Authors’ response

   We agree that the term 'guided self-organization' was insufficiently defined in the original manuscript.

   The revised Introduction now distinguishes angiogenesis from vasculogenesis while noting that both involve a shared repertoire of dynamic endothelial interactions. We use “self-organization” to refer to the capacity of endothelial collectives to generate organized multicellular arrangements through local interactions between endothelial cells, without implying that angiogenesis and vasculogenesis are identical developmental processes.

   The revised Introduction further explains that mathematical models based on local endothelial interactions can generate both network-like organization and sprouting depending on initial configuration and environmental constraints.

   Within this framework, we define “guided self-organization” as a situation in which spatially patterned environmental cues channel endothelial network-forming and migratory behavior toward reproducible trajectories.

   We have deliberately moderated the broader conceptual claim. Rather than arguing that angiogenesis and vasculogenesis represent the same biological process, the revised manuscript proposes that they may draw on overlapping endothelial self-organizing mechanisms whose outcomes depend on initial tissue configuration and environmental constraints. The conceptual contribution of our study is therefore not the identification of ECM regulation as a new mechanism in angiogenesis, but the integration of endothelial self-organizing behavior, mechanical cell–ECM interactions, and spatially patterned repulsive guidance within a common framework. This allows us to investigate, in the simulations, how changes in environmental constraints can shift endothelial behavior between directed sprouting and network-like organization. We prefer to retain the phrase “guided self-organization” in the title because the concept is now explicitly defined and constitutes the central framework tested by the combined experimental and modeling work.

   Comment 3

   1. The experimental evidence does not sufficiently support the proposed mechanistic model.

   The current loss-of-function experiments, which primarily rely on morpholino-mediated knockdown, present potentially interesting observations suggesting that laminin and fibronectin contribute to ISV sprouting and vascular patterning. However, the mechanistic conclusions extend considerably beyond what is directly supported by the presented data, resulting in overinterpretation of the experimental findings.

   For example, the manuscript does not determine the cellular source of the relevant ECM components during ISV development, whether endothelial cells directly respond to these matrices through specific receptor-mediated mechanisms, or whether the observed vascular phenotypes arise indirectly through disruption of surrounding tissues or altered presentation of established angiogenic and guidance molecules such as Vegfa or Semaphorins. Consequently, the current data do not distinguish among several possible mechanisms by which laminin and fibronectin may influence vascular morphogenesis.

   Likewise, the manuscript does not investigate how laminin and fibronectin may interact with established endothelial guidance pathways. Developmental mechanisms governing ISV sprouting have been extensively investigated, and similar endothelial migration and misguidance phenotypes have been reported. The present study reports abnormal ISV defects in embryos that also exhibit broader tissue disorganization, making it difficult to determine whether the vascular phenotype represents a primary endothelial guidance defect or is secondary to disruption of tissue architecture. Additional analyses of somite organization and potential interactions with established endothelial guidance pathways would substantially strengthen the mechanistic conclusions.

   Authors’ response

   We agree that the original manuscript extended some mechanistic interpretations beyond what was directly demonstrated.

   The revised manuscript now clearly separates experimentally supported conclusions from mechanistic hypotheses generated by the model.

   Several additional analyses were incorporated:

   1. Genetic fn1a and fn1b mutant backgrounds were combined with laminin depletion to provide orthogonal support for the laminin-fibronectin interaction.

   2. Chimeric fibronectin mRNA rescue was used to test specificity of the fibronectin component.

   3. Whole-embryo morphology was added to evaluate gross developmental and trunk abnormalities.

   4. sema3aa and sema3ab were examined by whole-mount HCR and RT-qPCR.

   5. The contrasting phenotypes of MO L14Fa and MO L14Fb were used to distinguish severe gross trunk disruption from vascular disorganization.

   We have also added a dedicated Discussion of the possible cellular sources of the ECM. We explicitly state that the reporter lines show where laminin and fibronectin proteins accumulate but do not identify the cells responsible for depositing the matrix encountered by extending ISVs.

   Likewise, we do not claim to have identified the relevant endothelial ECM receptors or to have directly demonstrated changes in VEGF, Dll4/Notch, or other candidate guidance pathways. These are now presented as potential mechanisms and limitations rather than established conclusions.

   Comment 4

   1. Several aspects of the experimental design require additional rigor.

   Given the broad developmental functions of laminin and fibronectin, embryonic developmental delay represents an important potential contributing factor when interpreting the observed, impeded growth of ISV sprouting. The manuscript reports these phenotypes at defined time points (e.g., 22-28 hpf and 2 dpf), but it does not clearly describe how developmental stage was determined and normalized prior to quantitative comparisons. This issue is particularly important because ISV sprouting progresses rapidly during this developmental period, and even modest differences in developmental stage could substantially influence measurements of sprout extension, migration, and vascular patterning. Additional staging based on established developmental landmarks (e.g., somite number or comparable morphological criteria) would substantially strengthen the interpretation of the reported vascular phenotypes. Furthermore, because these analyses were performed during early embryogenesis, reporting developmental timing using precise hours post-fertilization (hpf), together with appropriate developmental staging criteria, would be more informative than referring broadly to embryos as "2 dpf."

   Additionally, because the study depends primarily on morpholino-mediated knockdown, potential morpholino off-target effects and/or nonspecific toxicity should be more carefully considered. Although the manuscript describes the utilization of a Cas13d strategy, this approach ultimately does not provide independent validation of the vascular phenotypes. Independent confirmation using complementary genetic approaches (e.g., CRISPR-Cas9-mediated gene disruption) or other orthogonal perturbation methods, such as pharmacological inhibition or analysis of relevant receptor-mediated signaling pathways, would substantially increase confidence in the reported conclusions.

   Together, rigorous developmental stage matching, careful evaluation of neighboring tissue organization, and independent validation using complementary experimental approaches would substantially enhance the scientific rigor of the findings presented in this study.

   Authors’ response

   We agree that developmental staging is particularly important during the rapid period of primary ISV sprouting.

   In the revised analysis, developmental age for quantitative experiments is reported in hours post-fertilization rather than broadly as “2 dpf.” For the 22–28 hpf time-lapse experiments, we additionally selected vessels according to their stage of sprout development: only ISVs that had recently initiated dorsal extension at the beginning of imaging were analyzed, and ISVs that had already undergone substantial elongation were excluded. The same selection criteria were applied to all experimental groups.

   This approach allowed us to compare vessels at a comparable stage of sprout development rather than relying exclusively on anatomical position or embryo size. Although this approach improves comparability at the level of individual sprout development, it does not fully exclude differences in overall embryonic developmental progression.

   We nevertheless acknowledge in the revised Discussion that broader developmental effects of laminin and fibronectin perturbation may still influence sprout dynamics.

   To strengthen independent validation of the morpholino phenotypes, we combined MO L14 with genetic loss of fn1a or fn1b. Both mutant backgrounds showed strongly increased vascular mispatterning following laminin depletion. Together with the independent fibronectin mRNA rescue experiments, these experiments substantially reduce reliance on fibronectin morpholino phenotypes alone.

   We explicitly acknowledge that the laminin component remains morpholino-based and therefore describe this as partial rather than fully genetic validation.

   Comment 5

   1. Interpretation of the vascular phenotypes and mathematical modeling should be reconsidered.

   The manuscript repeatedly refers to abnormal vascular patterning as "ISV fusion." However, similar vascular phenotypes have been extensively characterized as ISV misguidance or ectopic ISV sprouting following disruptions of several endothelial guidance or inhibitory pathways. Unless the authors provide evidence that the observed phenotype represents a biologically distinct vascular fusion process, the use of established terminology would help avoid confusion in the field.

   Likewise, although the mathematical modeling represents a valuable component of the study, it simplifies many aspects of ISV morphogenesis, including endothelial cell behaviors, tissue architecture, extracellular matrix organization, and multiple signaling pathways that regulate vascular sprouting. Given the limited experimental evidence supporting the proposed mechanistic model, it remains difficult to determine how faithfully the computational model reflects the underlying biological processes occurring during zebrafish vascular development. The computational simulations may generate hypotheses and provide possible explanations for the observed phenotypes. However, the modeling alone cannot establish or validate the proposed in vivo mechanisms. The authors should therefore clearly distinguish experimentally supported conclusions from the hypotheses and predictions generated by computational modeling and avoid drawing mechanistic conclusions that are insufficiently supported by the in vivo experimental data.

   Authors’ response

   We agree with both aspects of this comment.

   The terminology has been revised throughout the manuscript. Morphological abnormalities are now classified as ectopic branching, loss of the normal dorsoventral trajectory, or abnormal lateral/inter-ISV connections. The term “fusion” is reserved for events in which vessel joining is directly demonstrated by time-lapse imaging.

   We added a time-lapse sequence (Supplementary Video 4) showing two initially distinct ISVs extending along abnormal trajectories and subsequently establishing a direct lateral connection. This event is described as direct evidence of an ISV fusion event, whereas similar static phenotypes are not automatically interpreted as fusion.

   We have also revised the presentation of the computational model. The manuscript now explicitly states that ECM stiffness, attachment-site density, and Semaphorin gradients were not directly measured in the morphants. The stiffness- and density-reduction simulations are therefore presented as mechanistic hypotheses rather than literal representations of the knockdown conditions.

   The Discussion further emphasizes that the model is deliberately simplified and should be interpreted as a framework for exploring possible mechanisms rather than as a direct molecular representation of laminin and fibronectin organization in vivo.

   Minor comments

   Minor comment 1

   1. Please include whole-embryo brightfield images from the laminin and fibronectin loss-of-function experiments to demonstrate overall embryonic morphology. These images would allow readers to assess potential gross morphological defects and developmental delays that may contribute to the observed vascular phenotypes.

   Authors’ response

   Added as a new supplementary figure (Supplementary Figure 5). These images document gross morphology and developmental differences among the principal combined ECM perturbation conditions independently of endothelial fluorescence.

   Minor comment 2

   1. The transgenic reporter signals from Tg(lama1:lama1-GFP), Tg(fn1a:fn1a-mNeonGreen),

   and Tg(fn1b:fn1b-mCherry) lines do not identify the cellular sources of these ECM components. To understand the cellular mechanisms underlying ECM-mediated regulation of vascular development, additional histological analyses (e.g., HCR, RNAscope, or characterization using BAC transgenic or knock-in reporter lines) would be valuable. In addition, please include lower-magnification, whole-embryo fluorescent images of these transgenic lines to better understand the overall distribution patterns of these ECM components.

   Authors’ response

   We agree that the reporter lines identify protein localization rather than cellular origin. The revised Discussion now explicitly states this limitation and summarizes available evidence for expression of lama1, lama4, fn1a, and fn1b in surrounding trunk tissues.

   We additionally included lower-magnification, whole-embryo fluorescent images of the ECM reporter lines (Supplementary Figure S6 to provide broader spatial context. These images are intended as qualitative spatial-localization data and are not used for quantitative comparison of reporter abundance across lines.

   Minor comment 3

   1. Better characterize somite organization using an established somite marker to distinguish direct endothelial guidance defects from secondary tissue disorganization.

   Authors’ response

   We agree that a dedicated molecular somite marker would provide additional information. We did not add a separate molecular somite-marker experiment in the present revision.

   Instead, we addressed the underlying concern using several complementary approaches: whole-embryo bright-field imaging, comparison of MO L14Fa and MO L14Fb, HCR analysis of the spatial expression of the established ISV guidance genes sema3aa and sema3ab, quantitative RT-qPCR of the same genes, genetic fibronectin validation, and mRNA rescue.

   These data show that MO L14Fa has more pronounced trunk disruption, whereas MO L14Fb retains comparatively preserved gross segmental morphology despite clear vascular disorganization. We therefore no longer use gross morphology to infer normal molecular somite patterning. The revised text explicitly states that molecular somite patterning was not assessed, and the absence of a dedicated molecular analysis of somite organization is acknowledged as a limitation. Accordingly, our conclusion has been narrowed from a direct endothelial mechanism to the more conservative interpretation that gross trunk disorganization may exacerbate the vascular phenotype, but vascular abnormalities are also observed in embryos with comparatively preserved gross segmental morphology.

   Minor comment 4

   1. Refine the text throughout the manuscript after its reorganization, as it currently contains numerous typos, redundant statements, and awkward or unclear sentence constructions.

   Authors’ response

   The manuscript has been extensively edited and reorganized throughout. Redundant material has been removed, terminology has been standardized, and multiple sentences and figure legends have been rewritten for clarity.

   Minor comment 5

   1. Revise the manuscript title, particularly the phrase "Endothelial Self-Organization", as appropriate following manuscript revision.

   Authors’ response

   We carefully reconsidered the title in light of this comment. We have retained “Endothelial Self-Organization” because the revised Introduction now explicitly defines what we mean by self-organization and distinguishes this concept from the broader developmental distinction between angiogenesis and vasculogenesis.

   The revised manuscript uses this term specifically to describe endothelial collective behaviors arising from local cell interactions whose resulting geometry is constrained by environmental cues. We believe that, with this clarification and the more cautious framing introduced throughout the manuscript, the current title accurately reflects the central question addressed by the combined experimental and mathematical work.

   Significance assessment

   General assessment: The manuscript investigates the roles of laminin and fibronectin during zebrafish intersegmental vessel development using morpholino-mediated gene knockdown, live imaging, and computational modeling. The integration of experimental and computational approaches is a strength of the study, and the work presents potentially interesting observations regarding extracellular matrix regulation of vascular morphogenesis. However, substantial improvements in manuscript organization, experimental rigor, and mechanistic validation are needed before the proposed conclusions can be fully justified.

   Advance: The conceptual and mechanistic advances remain limited in the current form. Developmental mechanisms governing ISV sprouting have been extensively investigated, and essential roles of extracellular matrix components, endothelial guidance pathways, and tissue-derived regulatory cues in vascular morphogenesis are already well established in the vascular biology field. While the present study suggests that laminin and fibronectin cooperatively contribute to ISV development, the current evidence does not clearly establish a mechanism that distinguishes these findings from existing models or demonstrates a clearly new biological principle. Even if the proposed model were further validated, the conceptual advance beyond the current understanding of ISV development would likely remain modest unless clearer mechanistic insights or previously unrecognized regulatory mechanisms are established.

   Audience: The study will be primarily of interest to researchers in vascular biology, angiogenesis, extracellular matrix biology, and zebrafish developmental genetics. In its current form, the mechanistic and conceptual advances appear limited, making broader interest outside these specialized research communities less likely.

   Field of expertise

   Field of expertise: Developmental vascular biology, zebrafish genetics, live imaging.

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   Oct 16, 2026

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   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   This manuscript combines zebrafish genetics, live imaging, rescue experiments, and mathematical modeling to investigate the roles of laminin and fibronectin during intersegmental vessel (ISV) development. The integration of experimental and computational approaches is a strength of the study, and the manuscript presents potentially interesting findings suggesting that extracellular matrix (ECM) components, including laminin and fibronectin, cooperatively contribute to ISV growth and guidance. However, the current experimental evidence lacks sufficient rigor to convincingly support this conclusion and does not provide …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   This manuscript combines zebrafish genetics, live imaging, rescue experiments, and mathematical modeling to investigate the roles of laminin and fibronectin during intersegmental vessel (ISV) development. The integration of experimental and computational approaches is a strength of the study, and the manuscript presents potentially interesting findings suggesting that extracellular matrix (ECM) components, including laminin and fibronectin, cooperatively contribute to ISV growth and guidance. However, the current experimental evidence lacks sufficient rigor to convincingly support this conclusion and does not provide adequate support for the broader mechanistic and conceptual framework of ECM-guided self-organization proposed by the authors.

   Overall, the observations presented in this manuscript may be of potential interest to the vascular biology field, but the manuscript requires substantial improvements in its presentation, conceptual framework, mechanistic evidence, and experimental rigor to support its main conclusions.

   My major concerns regarding these points are outlined below. In its current form, the manuscript reads more like a dissertation chapter than a primary research article, with the excessively long Introduction and the Results section containing extensive methodological descriptions and discussions that obscure the main experimental findings. More importantly, while the experimental observations suggest a role for laminin and fibronectin during ISV development, the proposed mechanistic and conceptual framework of "ECM-guided self-organization" is neither sufficiently defined nor experimentally supported. Finally, several aspects of the experimental design require additional rigor, including developmental stage normalization, independent validation of morpholino-mediated phenotypes, and more careful interpretation of the observed vascular defects. Addressing these concerns would substantially improve both the clarity and scientific impact of the manuscript.

   Major Comments

   1. The manuscript would benefit from substantial reorganization from its current dissertation-style structure into the format of a primary research article.

   The manuscript appears to retain much of the organization and level of detail expected for a dissertation chapter rather than being revised into the concise format of a primary research article. A major reorganization is needed to substantially improve the scientific presentation of the study. The Introduction is considerably longer than necessary and contains an extensive literature review before introducing the specific biological question. Likewise, the Results frequently include lengthy methodological descriptions, literature comparisons, and biological interpretation, making it difficult to distinguish the main experimental findings from the discussion. Streamlining the Introduction, moving methodological details to the Methods or Supplementary Information where appropriate, reducing repetitive discussion throughout the Results, and presenting the experiments in a more concise and sequential manner would greatly improve the readability of the manuscript and allow readers to more readily appreciate its principal scientific findings.

   1. The central concept of "ECM-guided self-organization" is not clearly defined, and its conceptual advance is currently difficult to appreciate.

   One of my primary concerns is the conceptual framework proposed throughout the manuscript. The term "self-organization" is never clearly defined, making it difficult to understand precisely what biological mechanism the authors intend to describe. Although the Introduction discusses endothelial morphogenesis as a process involving both endothelial cell-intrinsic programs and tissue-derived extrinsic regulatory mechanisms, it subsequently introduces the concept of "ECM-guided self-organization" without clearly explaining how this framework differs from existing models of angiogenic sprouting or endothelial morphogenesis.

   This ambiguity is particularly important because endothelial sprouting is generally understood to result from integration of endothelial cell-intrinsic programs together with tissue-derived extrinsic mechanisms, including extracellular matrix components, angiogenic factors, and guidance cues. Indeed, the Introduction itself acknowledges this principle. Consequently, it remains unclear to me how the proposed model of "ECM-guided self-organization" differs conceptually from the current understanding of endothelial morphogenesis or whether it establishes a fundamentally new biological principle.

   More broadly, the conceptual advance of the study remains difficult to appreciate. The importance of extracellular matrix components during endothelial migration, angiogenesis, and vascular morphogenesis is already well established. The present work aims to provide additional evidence that laminin and fibronectin contribute to ISV sprouting and patterning, but it is difficult to identify what new mechanistic or conceptual insight is established beyond the recognized roles of the extracellular matrix. A clearer definition of "self-organization" and a more explicit explanation of how the proposed model advances beyond existing concepts would greatly strengthen the manuscript and help readers better appreciate its conceptual contribution.

   1. The experimental evidence does not sufficiently support the proposed mechanistic model.

   The current loss-of-function experiments, which primarily rely on morpholino-mediated knockdown, present potentially interesting observations suggesting that laminin and fibronectin contribute to ISV sprouting and vascular patterning. However, the mechanistic conclusions extend considerably beyond what is directly supported by the presented data, resulting in overinterpretation of the experimental findings.

   For example, the manuscript does not determine the cellular source of the relevant ECM components during ISV development, whether endothelial cells directly respond to these matrices through specific receptor-mediated mechanisms, or whether the observed vascular phenotypes arise indirectly through disruption of surrounding tissues or altered presentation of established angiogenic and guidance molecules such as Vegfa or Semaphorins. Consequently, the current data do not distinguish among several possible mechanisms by which laminin and fibronectin may influence vascular morphogenesis.

   Likewise, the manuscript does not investigate how laminin and fibronectin may interact with established endothelial guidance pathways. Developmental mechanisms governing ISV sprouting have been extensively investigated, and similar endothelial migration and misguidance phenotypes have been reported. The present study reports abnormal ISV defects in embryos that also exhibit broader tissue disorganization, making it difficult to determine whether the vascular phenotype represents a primary endothelial guidance defect or is secondary to disruption of tissue architecture. Additional analyses of somite organization and potential interactions with established endothelial guidance pathways would substantially strengthen the mechanistic conclusions.

   1. Several aspects of the experimental design require additional rigor.

   Given the broad developmental functions of laminin and fibronectin, embryonic developmental delay represents an important potential contributing factor when interpreting the observed, impeded growth of ISV sprouting. The manuscript reports these phenotypes at defined time points (e.g., 22-28 hpf and 2 dpf), but it does not clearly describe how developmental stage was determined and normalized prior to quantitative comparisons. This issue is particularly important because ISV sprouting progresses rapidly during this developmental period, and even modest differences in developmental stage could substantially influence measurements of sprout extension, migration, and vascular patterning. Additional staging based on established developmental landmarks (e.g., somite number or comparable morphological criteria) would substantially strengthen the interpretation of the reported vascular phenotypes. Furthermore, because these analyses were performed during early embryogenesis, reporting developmental timing using precise hours post-fertilization (hpf), together with appropriate developmental staging criteria, would be more informative than referring broadly to embryos as "2 dpf."

   Additionally, because the study depends primarily on morpholino-mediated knockdown, potential morpholino off-target effects and/or nonspecific toxicity should be more carefully considered. Although the manuscript describes the utilization of a Cas13d strategy, this approach ultimately does not provide independent validation of the vascular phenotypes. Independent confirmation using complementary genetic approaches (e.g., CRISPR-Cas9-mediated gene disruption) or other orthogonal perturbation methods, such as pharmacological inhibition or analysis of relevant receptor-mediated signaling pathways, would substantially increase confidence in the reported conclusions.

   Together, rigorous developmental stage matching, careful evaluation of neighboring tissue organization, and independent validation using complementary experimental approaches would substantially enhance the scientific rigor of the findings presented in this study.

   1. Interpretation of the vascular phenotypes and mathematical modeling should be reconsidered.

   The manuscript repeatedly refers to abnormal vascular patterning as "ISV fusion." However, similar vascular phenotypes have been extensively characterized as ISV misguidance or ectopic ISV sprouting following disruptions of several endothelial guidance or inhibitory pathways. Unless the authors provide evidence that the observed phenotype represents a biologically distinct vascular fusion process, the use of established terminology would help avoid confusion in the field.

   Likewise, although the mathematical modeling represents a valuable component of the study, it simplifies many aspects of ISV morphogenesis, including endothelial cell behaviors, tissue architecture, extracellular matrix organization, and multiple signaling pathways that regulate vascular sprouting. Given the limited experimental evidence supporting the proposed mechanistic model, it remains difficult to determine how faithfully the computational model reflects the underlying biological processes occurring during zebrafish vascular development. The computational simulations may generate hypotheses and provide possible explanations for the observed phenotypes. However, the modeling alone cannot establish or validate the proposed in vivo mechanisms. The authors should therefore clearly distinguish experimentally supported conclusions from the hypotheses and predictions generated by computational modeling and avoid drawing mechanistic conclusions that are insufficiently supported by the in vivo experimental data.

   Minor Comments

   1. Please include whole-embryo brightfield images from the laminin and fibronectin loss-of-function experiments to demonstrate overall embryonic morphology. These images would allow readers to assess potential gross morphological defects and developmental delays that may contribute to the observed vascular phenotypes.
   2. The transgenic reporter signals from Tg(lama1:lama1-GFP), Tg(fn1a:fn1a-mNeonGreen), and Tg(fn1b:fn1b-mCherry) lines do not identify the cellular sources of these ECM components. To understand the cellular mechanisms underlying ECM-mediated regulation of vascular development, additional histological analyses (e.g., HCR, RNAscope, or characterization using BAC transgenic or knock-in reporter lines) would be valuable. In addition, please include lower-magnification, whole-embryo fluorescent images of these transgenic lines to better understand the overall distribution patterns of these ECM components.
   3. Better characterize somite organization using an established somite marker to distinguish direct endothelial guidance defects from secondary tissue disorganization.
   4. Refine the text throughout the manuscript after its reorganization, as it currently contains numerous typos, redundant statements, and awkward or unclear sentence constructions.
   5. Revise the manuscript title, particularly the phrase "Endothelial Self-Organization", as appropriate following manuscript revision.

   Significance

   General assessment: The manuscript investigates the roles of laminin and fibronectin during zebrafish intersegmental vessel development using morpholino-mediated gene knockdown, live imaging, and computational modeling. The integration of experimental and computational approaches is a strength of the study, and the work presents potentially interesting observations regarding extracellular matrix regulation of vascular morphogenesis. However, substantial improvements in manuscript organization, experimental rigor, and mechanistic validation are needed before the proposed conclusions can be fully justified.

   Advance: The conceptual and mechanistic advances remain limited in the current form. Developmental mechanisms governing ISV sprouting have been extensively investigated, and essential roles of extracellular matrix components, endothelial guidance pathways, and tissue-derived regulatory cues in vascular morphogenesis are already well established in the vascular biology field. While the present study suggests that laminin and fibronectin cooperatively contribute to ISV development, the current evidence does not clearly establish a mechanism that distinguishes these findings from existing models or demonstrates a clearly new biological principle. Even if the proposed model were further validated, the conceptual advance beyond the current understanding of ISV development would likely remain modest unless clearer mechanistic insights or previously unrecognized regulatory mechanisms are established.

   Audience: The study will be primarily of interest to researchers in vascular biology, angiogenesis, extracellular matrix biology, and zebrafish developmental genetics. In its current form, the mechanistic and conceptual advances appear limited, making broader interest outside these specialized research communities less likely.

   Field of expertise: Developmental vascular biology, zebrafish genetics, live imaging.

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3. 

   Review Commons

   Oct 16, 2026

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary

   In this study, the authors investigate how extracellular matrix (ECM) properties and repulsive guidance cues cooperate to regulate vessel growth and patterning during angiogenesis. To address this question, they develop a mathematical framework that integrates endothelial cell network formation, ECM mechanics, and semaphorin-mediated repulsive signaling. Their simulations predict that reductions in ECM stiffness or density impair vessel extension, while reductions in either ECM density or semaphorin gradient distribution promote vessel fusion, suggesting that ECM properties and semaphorin signaling act together to guide …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary

   In this study, the authors investigate how extracellular matrix (ECM) properties and repulsive guidance cues cooperate to regulate vessel growth and patterning during angiogenesis. To address this question, they develop a mathematical framework that integrates endothelial cell network formation, ECM mechanics, and semaphorin-mediated repulsive signaling. Their simulations predict that reductions in ECM stiffness or density impair vessel extension, while reductions in either ECM density or semaphorin gradient distribution promote vessel fusion, suggesting that ECM properties and semaphorin signaling act together to guide vascular sprouts and maintain vessel patterning. The authors then test these predictions experimentally using zebrafish intersegmental vessels (ISVs) as an in vivo model of sprouting angiogenesis. Through morpholino-mediated depletion of laminin 1, laminin 4, fibronectin 1a, and fibronectin 1b, alone and in combination, they examine the consequences of ECM disruption on ISV growth and organization. They report defects in vessel extension and patterning, including increased ectopic vessel fusion, and conclude that laminin- and fibronectin-dependent ECM organization is required for proper ISV migration, growth, and patterning. The study combines computational modeling with experimental analysis to address an important question in vascular development and provides a potentially valuable framework for understanding how mechanical and guidance cues are integrated during angiogenic sprouting.

   Major Comments

   1. The evidence does not convincingly demonstrate that laminin/fibronectin directly regulate ISV patterning The authors developed a mathematical model incorporating endothelial cell network formation, ECM mechanics, and semaphorin-mediated repulsive signaling. The simulations predict that reduced ECM stiffness or density slows vessel growth, whereas reduction of either ECM density or the semaphorin gradient promotes vessel fusion, suggesting that ECM properties and semaphorin signaling cooperate to guide vessel trajectories. The experimental data, however, do not convincingly establish that the observed ISV patterning defects are a direct consequence of laminin/fibronectin loss. As acknowledged by the authors, combined depletion of laminin and fibronectin severely disrupts somite and notochord morphogenesis. Given the well-established role of somites and semaphorin-mediated repulsive signaling in restricting ISV growth and preventing ectopic branching or fusion, it remains unclear whether the vascular phenotypes arise from direct endothelial-ECM defects or indirectly from altered trunk patterning. The authors argue that ISV disorganization is still observed in embryos with comparatively preserved somite morphology (e.g., MO L14Fb). However, the assessment of somite preservation is based on relatively low-magnification images, and it is unclear whether molecular patterning within the somites remains intact. In particular, the manuscript does not address whether semaphorin expression or distribution is altered in MO L14Fa, MO L14Fb, or the corresponding rescue conditions. This is an important omission because semaphorin gradients are explicitly incorporated into the mathematical model and are predicted to strongly influence vessel guidance by preventing ectopic vessel fusion. Therefore, to support the conclusion that laminin/fibronectin directly regulate ISV patterning, it would be important to determine whether semaphorin expression and/or distribution are preserved following laminin/fibronectin depletion. Without such data, the possibility remains that the vascular mispatterning phenotype is secondary to defects in somite and notochord development. Furthermore, previous studies disrupting endothelial-ECM adhesion have generally reported defects in ISV growth and elongation without accompanying vessel mispatterning:
      • Talin1-deficient zebrafish (tln1 uqal−/−) exhibit impaired ISV growth and an increased frequency of disconnected ISVs but do not display ectopic branching or aberrant vessel patterning (Chau et al., 2022; doi:10.1242/dev.200454).
      • A second talin1 mutant (fl02k) similarly shows defects in vascular development without ISV mispatterning (Wu et al., 2015; doi:10.1096/fj.15-273409).
      • Loss of integrin β1b results in impaired ISV elongation but does not disrupt ISV patterning (Iida et al., 2018; doi:10.1111/gtc.1264). These studies suggest that impaired endothelial-ECM interactions alone are insufficient to produce the extensive ISV fusion and mispatterning observed here, raising the possibility that secondary defects in trunk tissue organization contribute substantially to the phenotype.
   2. Quantification of ISV growth using "ISV volume" requires further justification The authors estimate average ISV volume by dividing the total segmented trunk vascular volume by the number of ISVs. This metric raises several concerns. First, the segmented vascular volume appears to include non-ISV structures such as the dorsal longitudinal anastomotic vessel (DLAV). Consequently, the calculated parameter does not exclusively represent ISV volume and may not accurately reflect ISV growth. Second, several morpholino conditions (e.g., MO Fn1ab, MO L14, MO lama1/4 fn1a, and MO lama1/4 fn1b) exhibit pronounced developmental delay and shortened trunk length. Under these conditions, reduced vascular volume may simply reflect overall embryonic growth retardation rather than a specific defect in ISV growth. Therefore, it is difficult to distinguish primary vascular phenotypes from secondary consequences of reduced embryo size. Finally, ISV growth is normalized to the dorsal-ventral axis length. Because overall embryonic dimensions are reduced in several experimental groups, this normalization may partially mask genuine ISV growth defects. The authors should therefore provide additional justification for this metric and discuss its limitations. Direct measurements of ISV length may provide more robust assessments of ISV growth.

   Minor Comments

   1. When describing tip-cell filopodia and lamellipodia dynamics in ISVs, the authors should cite Phng et al., 2013 (doi:10.1242/dev.097352).
   2. Page 3 contains a "?" in place of a reference citation.
   3. Transgenic line nomenclature should be italicized throughout the manuscript.
   4. mRNA transcript names should be italicized throughout the manuscript.
   5. Figure 1E is presented at insufficient magnification to evaluate ISV morphology and patterning. Higher-magnification images similar to those shown in Figures 5 and 6 would be helpful.
   6. Figure 3 legend refers to a fourth simulation condition ("reduction of both fiber stiffness and fiber density"), but this condition is not shown in the figure. This statement should be removed unless the corresponding data are presented.
   7. Statistical comparisons should be provided for Figure 3C-F.
   8. In Figure 3B-F, reductions in ECM density and ECM stiffness both decrease vessel length and growth speed. Both perturbations also reduce focal adhesion number, yet only ECM stiffness reduction decreases focal adhesion lifetime. The authors should clarify how these distinct focal adhesion phenotypes converge on a similar vascular outcome.
   9. In Figure 5F, the control group should be presented first (leftmost position) for consistency with the other panels.
   10. In Figure 5C (lower panels), image orientation should be standardized such that anterior is positioned on the left side.
   11. In Figure 6A, fn1a and fn1b mRNA overexpression appear to exert different effects on ISV volume. Specifically, fn1a overexpression increases ISV volume relative to control, whereas fn1b overexpression appears to decrease it. The authors should discuss the basis for these differing effects.
   12. The phenotypic severity of MO L14Fa and MO L14Fb appears inconsistent between Figures 5 and 6. In Fig. 5B, ISV volume of MO L14Fb is bigger than MO L14Fa while in Fig. 6A, ISV volume of MO L14Fb is smaller than MO L14Fa. In Fig. 5C, aberrant ISV number in MO L14Fb is significantly smaller than MO L14Fa while in Fig. 6A, they are comparable. The authors should clarify whether these experiments were performed independently and explain the source of these discrepancies.

   Significance

   Nature and Significance of the Advance

   This work represents a primarily conceptual advance in the field of angiogenesis by proposing a mechanism through which ECM mechanical properties and semaphorin-mediated repulsive signaling cooperate to guide vessel growth and patterning. The integration of computational modeling with in vivo experiments is a notable strength and has the potential to provide new insights into how multiple environmental cues are combined to regulate angiogenic behavior. The study builds upon a substantial body of work demonstrating that ECM composition and mechanics influence endothelial migration and sprouting, as well as studies showing that semaphorin signaling restricts aberrant vessel branching and fusion during zebrafish vascular development. The novelty of the present work lies in its attempt to unify these mechanisms within a single theoretical and experimental framework. However, the extent to which the experimental data support a direct role for laminin and fibronectin in vessel patterning, as opposed to indirect effects arising from disrupted somite and notochord development, remains an important question that should be addressed before the proposed mechanism can be fully established.

   The findings are likely to be of interest to researchers working in vascular biology, developmental biology, angiogenesis, collective cell migration, extracellular matrix biology, mechanobiology, and computational modeling of morphogenesis.

   Reviewer Expertise

   My expertise includes vascular development, sprouting angiogenesis, endothelial cell biology, mechanobiology and zebrafish models of vascular development.

   I am comfortable evaluating the developmental biology, vascular phenotyping, endothelial cell biology, and interpretation of the zebrafish experiments. While I can assess the biological assumptions and interpretation of the computational framework, I do not have specialized expertise in mathematical modeling and therefore cannot fully evaluate the technical implementation, parameterization, or numerical aspects of the model.

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4. 

   Version published to 10.64898/2026.03.13.711615 on bioRxiv

   Mar 16, 2026