Endocardial TIE1 synergizes with TIE2 to regulate the atrial internal muscular network assembly

  1. Kai Ding
  2. Beibei Xu
  3. Xinhao Yu
  4. Xiwen Jia
  5. Taotao Li
  6. Xin Shen
  7. Junda Li
  8. Xudong Cao
  9. Yahui Liu
  10. Zhen Zhang
  11. Yulong He

  • Curated by eLife

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    eLife Assessment

    This study reports the relative importance of Tie1 and Tie2 signaling for atrial versus ventricular trabeculation. It is an important study and is one of the few works to date that have carefully and simultaneously analyzed these two processes. In line with a previous study in zebrafish, the authors demonstrate key differences between atrial and ventricular trabeculation. While the imaging and quantitative analyses are conducted using solid and validated methodologies throughout the manuscript, future studies using additional experimental approaches will be invaluable to further establish the underlying mechanisms.

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Abstract

Atrial cardiomyopathy is characterized by altered atrial structures, yet its underlying genetic basis remains inadequately explored. TIE1 mutations are reported in lymphedema patients, but whether they exhibit cardiac defects alongside lymphatic abnormalities remains unknown. Here, single-cell RNA sequencing showed high Tie1 and Tek expression in atrial endocardial cells. TIE1 deficiency severely disrupted atrial morphogenesis, with only minor ventricular defects. Bulk RNA-seq showed reduced transcripts for endothelial development and cardiac trabeculation in Tie1 mutants. Separate RNA-seq analysis of the atria and ventricles further confirmed these findings, revealing a more pronounced downregulation of trabecular genes, including Tek, in the atria upon Tie1 loss. Endothelial deletion of Tek impaired trabeculation, particularly in atria. Consistently, Tie1 loss combined with a single null Tek allele disrupted both atrial and ventricular trabeculation. Strikingly, atrial morphogenesis defects appeared within 48 hours of induced endothelial Tie1 loss coupled with Tek heterozygous deletion, implying a critical endocardial role in organizing the atrial muscular network. The synergy of TIE1 and TIE2 in remodeling atrial trabeculae was confirmed postnatally, whereas TIE1 insufficiency alone had no effect. Together, TIE1 is differentially required for atrial versus ventricular development, acting synergistically with TIE2 to regulate endocardial cell-coordinated assembly of atrial internal muscular network.

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  1. Version published to 10.7554/elife.111156.2 on eLife
  2. Version published to 10.7554/elife.111156 on eLife
  3. eLife

    eLife Assessment

    This study reports the relative importance of Tie1 and Tie2 signaling for atrial versus ventricular trabeculation. It is an important study and is one of the few works to date that have carefully and simultaneously analyzed these two processes. In line with a previous study in zebrafish, the authors demonstrate key differences between atrial and ventricular trabeculation. While the imaging and quantitative analyses are conducted using solid and validated methodologies throughout the manuscript, future studies using additional experimental approaches will be invaluable to further establish the underlying mechanisms.

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Ding et al. use genetic mouse models to demonstrate that atrial trabeculation is more dependent on Tie1/Tie2 signaling than ventricular trabeculation. With additional experimentation that would support the current claims, the results may hold significant value, as atrial trabeculation remains an understudied phenomenon in cardiac biology with potential implications for atrial cardiomyopathy and atrial fibrillation.

    Strengths:

    Detailed characterization of atrial versus ventricular trabeculation across different developmental timepoints, and the use of appropriate animal models to address the scientific question at hand.

    Comments on revised version.

    I thank the authors for their detailed comments on the reviewers' comments. There however remain some issues that should be properly addressed to make the manuscript stronger.

    (1) The authors state that Tie1 and Tek levels are noticeably higher in atrial vs ventricular endocardial cells. Can the authors provide the appropriate statistics to give strength to this claim, since the Supplemental Figure 1F does not indicate robust chamber-specific differences? As normal statistics (t-test, Mann-Whitney, ANOVA) cannot be applied to raw RNAseq data, the authors should collect independent samples and perform qRT-PCR analyses on these.

    (2) When Tek is depleted in endothelial cells at E12.5, ventricular defects are also observed at later stages. Presumable, this could mean that earlier depletion of Tek, prior to initiation of ventricular trabeculation, might have resulted in equally severe atrial and ventricular defects. Similarly, they observe that, when Tie1 is deleted early on (tamoxifen administration at E10.5), combined with the heterozygous deletion of Tek, both atrial and ventricular defects are observed. While their postnatal studies do suggest a differential impact of Tie2 on atrial vs ventricular trabeculation (or the maintenance thereof), a more thorough analysis is needed to truly support the authors' claim that Tie1 and Tie2 synergistically impact atrial trabeculation more dramatically than ventricular trabeculation. However, to give weight to this claim, they should perform EC-specific depletion of Tie1 and Tie2 (or at least 3 out of 4 alleles) at different developmental stages, where tamoxifen is administered prior to ventricular or atrial trabeculation.

    (3) Earlier reports indicate that a part of Tie1 null mutants (app. 15%) die in utero. Did the authors also observe this phenomenon in their mutant lines, and did these animals have more severe atrial defects?

    (4) Although the reviewers did not request detailed mechanistic insights into the molecular mechanisms that drive Tie1-mediated atrial trabeculation, several of the reviewers' requests (e.g. YAP/TAZ staining) were unfortunately not performed. This is a shame, as peer review is instrumental to strengthen manuscripts and increase their validity. It would have therefore been nice if the authors had at least made an attempt to address some of outstanding reviewers' questions.

  5. eLife

    Reviewer #2 (Public review):

    Ding et al. examine the role of TIE1 in cardiac chamber morphogenesis using genetic mouse models targeting Tie1, Tek, or both, and analyzing endocardial cell-mediated chamber formation across multiple embryonic developmental and postnatal stages, supported by analysis of published single cell datasets and new bulk RNA seq analyses of murine cardiac tissue. The authors find that Tie1 and Tek expression is higher in atrial than ventricular endocardial cells. Notably, endothelial TIE1 is required for atrial trabeculation at E12.5, but is less critical in ventricular trabeculation. TIE1 also acts synergistically with TIE2 during atrial trabeculation. While Tie1 deficiency alone does not cause defects at E10.5, combined heterozygous deletion of Tek disrupts both atrial and ventricular development at E10.5. This synergy is further supported by analyses at later embryonic stages and in postnatal hearts.

    Strengths:

    The study is well designed, clearly written, and supported by high-quality figures. The performed experiments demonstrate a previously unrecognized role for TIE1 in cardiac development and identify synergistic control of cardiac morphogenesis by TIE1 and TIE2. This synergy is consistent with the previously identified roles of TIE1 and TIE2 in venous development and with TIE1 involvement in angiopoietin-dependent postnatal vascular and lymphatic remodeling. Together, these findings support a role for TIE1 as a contributor to ANGPT1-TIE2 signaling during heart development.

    Weaknesses:

    The manuscript does not include direct mechanistic studies; however, RNA seq analysis of atria and ventricles showed reduced expression of Tek, Dll1, and Notch1 upon Tie1 deficiency in developing hearts. Although no direct mechanistic experiments are performed, potential mechanisms based on previously published work such as TIE1-TIE2 heterodimer formation, effects on endothelial junctions, migration, survival and venous development are discussed in the context of TIE1-TIE2 synergy.

  6. eLife

    Reviewer #3 (Public review):

    Summary:

    Ding et al. investigate the roles of TIE1 and TEK (Tie2) in mouse cardiac development, with a particular focus on atrial trabeculation. The authors employ multiple genetic models, including Tie1ICDflox/flox (with Cdh5-CreERT2), a knockout-first allele (EUCOMM, Tie1 tm1a/tm1a), and a Tek deletion model.

    Based on the dataset from Feng et al. 2022 Nat Commun, the authors report increased expression of Tie1 and Tek transcripts in atrial endocardial cells compared to ventricular cells at embryonic day (E) 14.5. Loss of Tie1 leads to early atrial trabeculation defects detectable at E12.5, whereas ventricular defects appear later and are less pronounced at E14.5. Chamber-specific RNA sequencing reveals stronger transcriptional changes in atrial tissue.

    Conditional deletion of Tek results in a similar phenotype, with more pronounced atrial defects. Combined deletion of Tie1 and Tek (Tie1 ΔICD/ΔICD; Tek+/-) leads to earlier and more severe defects in both atrial and ventricular trabeculation and results in embryonic lethality around E12.5, suggesting a synergistic interaction between the two genes.

    Conditional endothelial deletion of Tie1 combined with heterozygous global Tek at later embryonic stages allows analysis at later time points and again shows more severe defects in atrial trabeculation. Postnatal analysis of this model reveals reduced heart-to-body weight ratios and potential mild atrial abnormalities.

    Strength:

    (1) The authors address chamber-specific signaling mechanisms underlying atrial versus ventricular trabeculation, an area of high developmental and clinical relevance.

    (2) The study provides a comprehensive temporal analysis across multiple embryonic stages.

    (3) The use of multiple genetic models strengthens the overall conclusions and allows comparative interpretation.

    (4) While focusing on trabeculation, the authors also include observations on coronary vessel development, increasing the broader relevance of the work. The findings are therefore of interest to the wider cardiovascular research community.

    Weakness:

    (1) Limited mechanistic insight
    While the authors aim to investigate underlying mechanisms, the current study is largely descriptive and based on mRNA expression and genetic interaction analyses (Tie1/Tek co-deletion). Direct mechanistic insights into signaling pathways remain limited. However, the dataset provides a valuable foundation for future mechanistic studies, which should be more clearly acknowledged in the discussion.

  7. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    eLife Assessment:

    This study reports the relative importance of Tie1 and Tie2 signaling for atrial versus ventricular trabeculation. It is an important study and is one of the few works to date that have carefully and simultaneously analyzed these two processes. In line with a previous study in zebrafish, the authors demonstrate key differences between atrial and ventricular trabeculation. While the imaging and quantitative data were conducted with solid and validated methodology throughout the manuscript, the work would benefit from more rigourous approaches where Tie1/2 signaling is disrupted prior to the onset of atrial/ventricular trabeculation, to allow for a more direct comparison.

    We thank the editors for the eLife assessment. We would like to request that the following statement be modified: “…the work would benefit from more rigourous approaches where Tie1/2 signaling is disrupted prior to the onset of atrial/ventricular trabeculation, to allow for a more direct comparison”. We request this change for the following reasons:

    We utilized two distinct genetic mouse models in this study (as summarized in Fig. 7I), comprising conventional knockouts (Tie1tm1a/tm1a, Tie1ΔICD/ΔICD and Tie1ΔICD/ΔICD;Tek+/-) and inducible gene deletion models (TekiECKO, Tie1ICDiECKO, and Tie1ICDiECKO;Tek+/-) [1-3].

    The Tie1tm1a/tm1a line is equivalent to the previously published Tie1-/- mouse line as demonstrated in our prior work and by others [1, 4-6]. For the Tie1ΔICD/ΔICD line, as described in the previous report [2], the expression level of Tie1ΔICD decreased dramatically compared with that of wild-type mice, which may result from the nonsense-mediated mRNA decay. Based on the lymphatic and venous phenotypes [1, 2], we conclude that the Tie1ΔICD/ΔICD line is almost equivalent to Tie1 null mice.

    Therefore, the Tie1 or Tek alleles were inactivated prior to the onset of atrial and ventricular trabeculations, as shown in Fig. 1, Fig. 2, Fig. 3, Fig. 5A-D, and Supplemental Fig. 3. Based on these findings, we propose that TIE1 is differentially required for atria versus ventricle morphogenesis, and acts synergistically with TIE2 during cardiac trabeculation.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Ding et al. use genetic mouse models to demonstrate that atrial trabeculation is more dependent on Tie1/Tie2 signaling than ventricular trabeculation. With additional experimentation that would support the current claims, the results may hold significant value, as atrial trabeculation remains an understudied phenomenon in cardiac biology with potential implications for atrial cardiomyopathy and atrial fibrillation.

    Strengths:

    Detailed characterization of atrial versus ventricular trabeculation across different developmental time-points, and the use of appropriate animal models to address the scientific question at hand.

    Weaknesses:

    The authors have consistently treated mice with tamoxifen after ventricular, but not atrial, trabeculation has already started. As such, the observed cardiac phenotypes - where predominantly atrial trabeculation is affected - might be a mere consequence of the precise time window in which Tie1/2 signaling was impaired, rather than a direct measurement of its relative importance for atrial versus ventricular trabeculation. The conclusions of the paper may thus be significantly strengthened by depleting Tie1/2 signaling prior to the onset of ventricular trabeculation, as is done for atrial trabeculation.

    We thank the reviewer for the comments.

    Regarding the timeline of gene deletion and tamoxifen treatment, we would like to provide the following clarification.

    Fig. 1-3: As described in the Methods and Materials, Tie1tm1a/tm1a is a knockout first mouse model established from EUCOMM embryonic stem cells (EPD0735-3B07) targeting Tie1 gene. Therefore, the Tie1tm1a/tm1a line is equivalent to the previously published Tie1 null mice (Tie1-/-). The Tie1Flox/Flox mouse line (with exon 8 floxed) was generated when the lacZ reporter and neo-cassette were excised using the FLPeR mice.

    Fig. 5A-D: To investigate the synergy of TIE1 and TIE2 in cardiac trabeculation, we utilized the Tek+/- and Tie1ΔICD/+ mouse lines and they were crossbred to generate double mutant mice harboring a homozygous Tie1 mutation and a single null Tek allele (Tie1ΔICD/ΔICD;Tek+/-). Although no obvious defects were observed in atrial or ventricular structures following Tie1 deficiency alone at E10.5, both atrial and ventricular development were disrupted in Tie1ΔICD/ΔICD;Tek+/- mutants at the same stage (Fig. 5A-D).

    Supplemental Fig. 3: To verify the role of TIE1 in atrial development, we employed alternative knockout mouse line targeting the Tie1 intracellular domain by floxing exons 15 and exon 16 (Tie1ICDFlox/Flox). Mutants harboring these null alleles are designated as Tie1ΔICD/ ΔICD. As detailed in the previous publication, the line is also equivalent to the previously published Tie1 null mice (Tie1-/-). The cardiac phenotypes shown in Supplemental Fig. 3 are indeed similar to those of Tie1tm1a/tm1a mutant mice.

    For the inducible knockouts targeting Tie1, Tek and both, the results are shown in Fig. 4, Fig. 5E-H, Fig. 6, and Fig. 7.

    Fig. 4: As mice homozygous for Tek mutation (Tek-/-) die before E10.5 [3, 7], we performed studies using the inducible knockout line targeting Tek (TekFlox/-;Cdh5-CreERT2 named as TekiECKO), as shown in Fig. 4.

    Fig. 5-7: To investigate the synergy of TIE1 and TIE2 in the cardiac trabeculation at the later stages of embryogenesis (Fig. 5E-H, Fig. 6) and the postnatal stage (Fig. 7), we used the inducible knockout models targeting Tie1/Tek, including Tie1ICDiECKO (Tie1ICDFlox/-;Cdh5-CreERT2) and Tie1ICDiECKO;Tek+/- (Tie1ICDFlox/-;Cdh5-CreERT2;Tek+/-).

    Reviewer #2 (Public review):

    Summary:

    Ding et al. examine the role of TIE1 in cardiac chamber morphogenesis using genetic mouse models targeting Tie1, Tek, or both, and analyzing endocardial cell-mediated chamber formation across multiple embryonic developmental and postnatal stages, supported by analysis of published single-cell datasets and new bulk RNA seq analyses of murine cardiac tissue. The authors find that Tie1 and Tek expression is higher in atrial than ventricular endocardial cells. Notably, endothelial Tie1 is required for atrial trabeculation at E12.5, but is less critical in ventricular trabeculation. TIE1 also acts synergistically with TIE2 during atrial trabeculation. While Tie1 deficiency alone does not cause defects at E10.5, combined heterozygous deletion of Tek disrupts both atrial and ventricular development at E10.5. This synergy is further supported by analyses at later embryonic stages and in postnatal hearts.

    Strengths:

    The study is well-designed, clearly written, and supported by high-quality figures. The performed experiments demonstrate a previously unrecognized role for Tie1 in cardiac development and identify synergistic control of cardiac morphogenesis by Tie1 and Tie2. This synergy is consistent with the previously identified roles of Tie1 and Tek in venous development and with Tie1 involvement in angiopoietin-dependent postnatal vascular and lymphatic remodeling. Together, these findings support a role for Tie1 as a contributor to Ang1-Tie2 signaling during heart development.

    Weaknesses:

    The manuscript does not include direct mechanistic studies; however, RNA seq analysis of atria and ventricles showed reduced expression of Tek, Dll1, and Notch1 upon Tie1 deficiency in developing hearts. Although previously reported mechanisms, such as TIE1-TIE2 heterodimer formation and effects on endothelial junctions, migration, or survival are discussed, no direct mechanistic experiments are performed. Addressing some of these mechanisms would have clarified the basis of Tie1-Tie2 synergy. As two distinct Tie1 models are used, including one targeting the kinase domain, the authors should state whether phenotypes differed or were similar between models.

    We thank the reviewer for this comment. In this study, we provide genetic evidence that TIE1 is differentially required for atrial versus ventricular trabeculation. Although the precise molecular mechanisms underlying TIE1 function warrant further investigation, we have provided compelling genetic evidence of its synergistic role with TIE2 during this process. Crucially, the two genetic models targeting Tie1 (Tie1tm1a/tm1a, Tie1ΔICD/ΔICD) produced consistent cardiac and vascular phenotypes, as shown in both this study and our previous work [1, 2].

    Regarding endocardial TIE1 in atrial morphogenesis, fully elucidating its downstream mechanism may require the isolation of primary endocardial cells for further study. However, our previous work using HUVECs have demonstrated that TIE1 acts synergistically with its homologue TIE2 to regulate AKT-mediated COUP-TFII protein stability [1, 3]. The essential roles of COUP-TFII in venous and atrial development have been well-documented in the literature [8, 9]. We have expanded our Discussion section to reflect these mechanistic insights.

    Reviewer #3 (Public review):

    Summary:

    Ding et al. investigate the roles of TIE1 and TEK (Tie2) in mouse cardiac development, with a particular focus on atrial trabeculation. The authors employ multiple genetic models, including Tie1ICDflox/flox (with Cdh5-CreERT2), a knockout-first allele (EUCOMM, Tie1 tm1a/tm1a), and a Tek deletion model.

    Based on the dataset from Feng et al. 2022 Nat Commun, the authors report increased expression of Tie1 and Tek transcripts in atrial endocardial cells compared to ventricular cells at embryonic day (E) 14.5. Loss of Tie1 leads to early atrial trabeculation defects detectable at E12.5, whereas ventricular defects appear later and are less pronounced at E14.5. Chamber-specific RNA sequencing reveals stronger transcriptional changes in atrial tissue.

    Conditional deletion of Tek results in a similar phenotype, with more pronounced atrial defects. Combined deletion of Tie1 and Tek (Tie1 ΔICD/ΔICD; Tek+/-) leads to earlier and more severe defects in both atrial and ventricular trabeculation and results in embryonic lethality around E12.5, suggesting a synergistic interaction between the two genes.

    Conditional endothelial deletion of Tie1 combined with heterozygous global Tek at later embryonic stages allows analysis at later time points and again shows more severe defects in atrial trabeculation. Postnatal analysis of this model reveals reduced heart-to-body weight ratios and potential mild atrial abnormalities.

    Strengths:

    (1) The authors address chamber-specific signaling mechanisms underlying atrial versus ventricular trabeculation, an area of high developmental and clinical relevance.

    (2) The study provides a comprehensive temporal analysis across multiple embryonic stages.

    (3) The use of multiple genetic models strengthens the overall conclusions and allows comparative interpretation.

    (4) While focusing on trabeculation, the authors also include observations on coronary vessel development, increasing the broader relevance of the work. The findings are therefore of interest to the wider cardiovascular research community.

    Weaknesses:

    (1) Timing of recombination vs. trabeculation onset

    Ventricular trabeculation begins earlier than atrial trabeculation. Since tamoxifen (in contrast to 4-hydroxytamoxifen) requires metabolic activation, Cre-mediated recombination will occur with a delay. This suggests that atrial trabeculation may be targeted before its onset, whereas ventricular trabeculation may already be underway for 2-3 days at the time of effective gene deletion.

    How do the authors account for this discrepancy in their interpretation?

    Have earlier induction time points been tested to better capture the onset of ventricular trabeculation? This limitation should be explicitly discussed.

    (2) Clarity of genetic models and experimental design

    The study employs several genetic constructs. It would improve clarity if, for each experiment, the specific genetic model and tamoxifen regimen were clearly described before presenting the results.

    We thank the reviewer for the detailed and constructive comments. As detailed above, for studies employing the inducible gene deletion mouse models (models (TekiECKO, Tie1ICDiECKO, and Tie1ICDiECKO;Tek+/-), the genetic models and tamoxifen treatment schemes have been provided in the related figures.

    For the investigation of TIE1 or TIE2 in atria versus ventricle morphogenesis at earlier embryonic stages, we utilized the conventional knockouts targeting Tie1 (Tie1tm1a/tm1a, Tie1ΔICD/ΔICD) or the combined deletion of Tie1 and Tek (Tie1ΔICD/ΔICD;Tek+/-), as detailed above.

    (3) Tie1 tm1a/tm1a phenotype vs. known global knockout

    Previous studies (PMID: 8846781, 7596437) show that complete Tie1 loss leads to severe edema, vascular rupture, and embryonic lethality around E13.5-E14.5.

    How does the Tie1 tm1a/tm1a allele differ, given that animals appear to survive longer? Is this allele hypomorphic rather than a full knockout?

    This point requires clarification.

    Tie1tm1a/tm1a is equivalent to the full Tie1 knockout (Tie1-/-). As demonstrated in our prior work, the Tie1ΔICD/ΔICD model produced lymphatic and blood vascular phenotypes similar to those of Tie1-/- mutants [1, 2, 5, 6].

    (4) Limited mechanistic insight

    While the authors aim to investigate underlying mechanisms, the current study is largely descriptive and based on mRNA expression and genetic interaction analyses (Tie1/Tek co-deletion). Direct mechanistic insights into signaling pathways remain limited. However, the dataset provides a valuable foundation for future mechanistic studies, which should be more clearly acknowledged in the discussion.

    We thank the reviewer for the comment. We have incorporated this discussion into the revised manuscript

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    The conclusions of the paper may be significantly strengthened by addressing several of the following comments:

    Major critiques:

    The authors state that Angpt1 is more prominently expressed in atrial versus ventricular myocardium and that this might explain the larger impact of Tie1 deletion on atrial versus ventricular trabeculation. However, other ligands for Tie1/Tie2 have been described, such as Svep1, Angpt2, and Lect2. It would be great if the authors could show the relative expression of these ligands in atrial versus ventricular cardiomyocytes over time.

    We appreciate the reviewer's valuable suggestion. To address this, we analyzed the scRNA-seq dataset to compare the expression profiles of key ligands (Angpt1, Angpt2, Svep1, and Lect2) between atrial and ventricular cardiac cells during embryonic development. These new data have been integrated into Supplemental Fig. 1F. Our analysis reveals that Angpt1 is differentially enriched in atrial cells relative to ventricular cells. However, no distinct inter-chamber differences were detected for Svep1 or Angpt2. Notably, Lect2 transcripts were virtually absent in both chambers; this is fully congruent with our bulk RNA-seq data, which showed no detectable Lect2 expression in whole-heart lysates.

    Likewise, the authors indicate that ANGPT1-TIE2 signaling is stronger in atria than ventricles at E14.5. Could this reflect a temporal rather than spatial difference? How does ANGPT1-TIE2 signaling compare between atria and ventricles during different steps of the trabeculation process?

    We thank the reviewer for this insightful question regarding spatial versus temporal dynamics. To address whether the observed differences reflect a temporal shift or a sustained spatial pattern, we evaluated Angpt1 expression across a developmental timeline spanning the major stages of trabeculation, from initiation through remodeling (E10.5 to E18.5).

    As shown in the newly added Supplemental Fig. 1F, Angpt1 is consistently and differentially enriched in atrial cells relative to ventricular cells throughout all examined embryonic stages. This sustained divergence confirms that elevated ANGPT1-TIE2 signaling in the atria is a distinct spatial feature rather than a transient temporal shift. This finding aligns with our ligand-receptor interactome analysis between cardiomyocytes and endocardial cells (Supplemental Fig. 1E). Furthermore, a specialized role for ANGPT1-mediated signaling in atrial morphogenesis has been independently demonstrated by Kim et al. (Cell Rep, 2018), supporting our model that this pathway is uniquely critical for the assembly of the atrial muscular network [10].

    Ding and colleagues demonstrate that loss of endothelial/endocardial Tie1 results in absent atrial trabeculae, while ventricular trabeculae are only moderately impacted. As ventricular trabeculation is initiated much earlier than atrial trabeculation, this discrepancy might, however, be secondary to the precise time point within trabecular network formation at which Tie1 was deleted. The make sure that Tie1 does not prevent ventricular trabeculation to an equal extent as atrial trabeculation, the authors should conduct additional experiments in which Tie1 is deleted before the onset of ventricular trabeculation (i.e., E8.5). Consistent with this, Tie1 knockout mice display substantial ventricular trabeculation defects (Figure 5). This comment extends to the analysis of the Tie2 knockout mice, where knocking out Tie2 starting from E12.5 does not affect ventricular trabeculation at E14.5 but impairs ventricular trabeculation when hearts are analyzed later (at E17.5). Additionally, Figure 5 clearly demonstrates that ventricular trabeculation is impacted in the combined absence of Tie1 and Tie2. Overall, a more detailed analysis of the relative importance of Tie1/Tie2 signaling for atrial versus ventricular trabeculation with more appropriate tamoxifen regimens needs to be performed to support the claims made by the authors.

    While one can deduce from the Methods section that tamoxifen was administered at E10-5-E11.5 for the results depicted in Figure 1, no information is provided regarding the tamoxifen regimens for the following figures, obscuring proper interpretation of the results. The authors should clearly state the precise time window in which they depleted endothelial Tie1 for each Figure in both text and legends, as this essential information is missing in Figures 2 and 3.

    We thank the reviewer for pointing this out. To rigorously investigate the differential requirement of TIE1, as well as its synergy with TIE2, during the distinct stages of atria versus ventricle trabeculation, we utilized both conventional (constitutive) and inducible gene knockout models. For the inducible genetic models, the exact timelines of gene deletion and tamoxifen treatments are detailed within their respective figure panels and legends. Below is a brief overview of the constitutive null models used in this study.

    Fig. 1-3 (constitutive null models): As described in the Materials and Methods, the Tie1tm1a/tm1a line is a “knockout-first” mouse model generated from EUCOMM embryonic stem cells (EPD0735-3B07) targeting Tie1 gene. Consequently, the Tie1tm1a/tm1a line is functionally equivalent to previously published Tie1 null mice (Tie1-/-). The Tie1Flox/Flox mouse line (harboring a floxed exon 8) was generated by excising the lacZ reporter and neo-cassette using FLPeR delete mice. As described above, the Tie1ΔICD/ΔICD line is also equivalent to Tie1 null mice.

    Fig. 5A-D: To investigate the genetic synergy between TIE1 and TIE2 during the initiation phase of cardiac trabeculation, we crossbred Tek+/- and Tie1ΔICD/+ lines to generate compound mutants harboring a homozygous Tie1 mutation paired with a single null Tek allele (Tie1ΔICD/ΔICD;Tek+/-). While Tie1 deficiency alone yielded no obvious defects in atrial or ventricular structures at E10.5, compound Tie1ΔICD/ΔICD;Tek+/- mutants exhibited severe disruption in both atrial and ventricular development at the same developmental stage (Fig. 5A-D).

    In the last paragraph, Ding et al. propose a model in which Tie1/Tie2 signaling drives atrial versus ventricular endothelial/endocardial specification. To substantiate this claim, the authors should identify several genes that differentiate between these two and demonstrate that their asymmetric expression pattern is perturbed in Tie1/2 mutant embryos. Alternatively, single-cell RNA-seq in control and Tie1/2 mutant embryos could provide a more holistic view of endothelial specification processes and their impact on myocardial shape and function.

    We thank the reviewer for these constructive suggestions. We agree that a comprehensive single-cell RNA-seq analysis of Tie1/Tek mutant embryos would provide a holistic view of endocardial specification. While performing this extensive sequencing is beyond the scope of our current study, we believe our present combination of chamber-specific bulk RNA-seq and functional genetic models provides a strong foundation for these mechanisms. Further studies are certainly warranted to provide deeper single-cell insights in this direction, and we have added this point to our revised Discussion section.

    A recent publication (Albu et al., Nature Communications) found no evidence for Nrg/Erbb and Notch signaling in atrial trabeculation, but reported that YAP signaling was implicated in the latter. This raises important questions: Did the authors observe Yap-mediated signaling in their RNAseq datasets? Could Yap/Taz be investigated in control and Tie1/2 mutant mice? Is there a link between Tie1/2 and Yap/Taz signaling in other systems, such as venous versus arterial endothelial specification?

    We thank the reviewer for this insightful comment. Based on our bulk RNA-seq data, we did not observe an obvious change in Yap1 and Wwtr1 (Taz) transcript expression in the hearts of Tie1 null mice compared to their littermate controls. However, this transcriptomic data does not exclude the possibility of post-translational regulation occurring at the protein level, such as alterations in protein phosphorylation, nuclear translocation, or the activation of downstream YAP/TAZ target genes. We agree that investigating potential alterations in YAP signaling following the induced endothelial deletion of Tek or compound Tie1/2 represents an important direction for future study.

    In contrast to the study by Albu and colleagues, the manuscript presented here observes Notch1 downregulation in the atria but not ventricles (Figures 3F, G). What could be the reason for these discrepancies? Does loss of Tie1 and/or Tie2 impair Nrg1, VEGF, or is Notch signaling more impactful in atria versus ventricles at the protein levels?

    We thank the reviewer for the insightful comment. Using a Zebrofish model, Albu et al., (Nat Comm 2024) [11] reported no evidence for Nrg/ErbB or Notch signaling in atrial muscle network formation. Based on our bulk RNA-seq analysis of heart tissues from Tie1 deficient mice compared to littermate controls, we demonstrated a decrease in Erg1 and Notch1 transcripts in Tie1 mutants. In contrast, we detected an increase in Vegfa transcript levels, which may result from compensatory changes in cardiac angiogenesis induced by hypoxic stress upon TIE1 deficiency. The biological significance of these transcriptomic changes requires further investigation. We have modified the text to include this discussion in the revised manuscript.

    In Figures 5 and 6, it is not clear which genotype was used as a control. Are these controls Tek+/-, Tie1ICDECKO, or WT mice? If there were any differences between these genotypes, these should be reported with the appropriate statistical tests.

    We thank the reviewer for pointing this out. We have added the information in the revised manuscript. Related to Fig. 5 and Fig. 6, the genotype for the control mice used are described in the Methods and Materials section. The genotypes of the Tie1/Tek knockout and control mice are as follows: Tie1ICDFlox/-;Cdh5-CreERT2 named as Tie1ICDiECKO, littermates Tie1ICDFlox/+ or Tie1ICDFlox/+;Cdh5-CreERT2 mice named as Control. TekFlox/-;Cdh5-CreERT2 named as TekiECKO, littermates TekFlox/+ or TekFlox/+;Cdh5-CreERT2 mice named as Control. Tie1ICDFlox/-;Cdh5-CreERT2;Tek+/- named as Tie1ICDiECKO;Tek+/-, littermates Tie1ICDFlox/+;Cdh5-CreERT2;Tek+/-, Tie1ICDFlox/+;Tek+/-, Tie1ICDFlox/+;Cdh5-CreERT2;Tek+/+ or Tie1ICDFlox/+;Tek+/+ named as Control.

    Minor points:

    The resolution of the retinal stainings should be improved.

    We thank the reviewer for pointing this out. We performed the analysis of retinal vascular phenotype in this study, which is for the verification of gene deletion efficiency.

    Sarcomeric defects have been observed in tie1-/- zebrafish. Can the authors check if such sarcomeric defects are also present in their mouse models and if they are more pronounced in atrial versus ventricular cardiomyocytes?

    We thank the reviewer for the interesting comment. We have not studied whether there is any sarcomeric defect in Tie1 null mice, which requires further investigation.

    If Tie1/2 signaling is more critical for atrial than ventricular development and function, is there any evidence for an association between atrial cardiomyopathy and/or atrial fibrillation in humans carrying pathogenic Tie1/Tie2 mutations?

    We thank the reviewer for this insightful comment. Whether human patients carrying pathogenic Tie1/Tie2 mutations have atrial cardiomyopathy and/or atrial fibrillation warrants further investigation.

    Bulk RNAseq reveals that Tie1tm1a/tm1a mice have reduced Nrg1 mRNA levels in the heart (Figure 2I), but this is not reflected in the atrial or ventricular transcriptomics (Figure 3). Could the authors explain this discrepancy in more detail?

    We thank the reviewer for pointing this out. As described above, there was a decrease of Nrg1 transcript level in hearts of Tie1 mutant compared to that of the littermate controls. The biological significance of this change requires further investigation. We have included this discussion in the revised manuscript.

    Reviewer #3 (Recommendations for the authors):

    Unfortunately, line numbers are not provided, which makes it difficult to refer to specific passages.

    We thank the reviewer for pointing this out. We have added line numbers to the revised manuscript t.

    While it is appropriate to mention in the Discussion that Tie1 variants have been associated with lymphatic defects, this information should be removed from the abstract, as the manuscript does not address lymphatic biology, and this statement distracts from the main focus of the study.

    We thank the reviewer for the comment. In the abstract background, we stated: “…TIE1 variants or loss of function mutations were reported in a subset of lymphedema patients, and it is unknown whether the patients have also cardiac defects in addition to the lymphatic abnormality.” By framing the introduction this way, we aimed to establish the central question of our study: whether Tie1 variants or loss-of-function mutations lead to cardiac abnormalities, which could ultimately contribute to or exacerbate systemic lymphedema alongside primary lymphatic defects.

    Figure 5F should include single-channel images for Tie1 staining to allow proper assessment of signal specificity and localization.

    We thank the reviewer for this comment. The co-staining of TIE1 and PECAM1 is intended primarily to demonstrate gene deletion efficiency. As shown In Fig. 5F, TIE1 is efficiently deleted in Tie1ICDiECKO;Tek+/- mice compared to control mice. Furthermore, as previously reported by Cao et al. [1] and Shen et al. [2], the mutant mice (Tie1ICDiECKO;Tek+/-) exhibit disrupted venous development alongside defective lymphatic formation.

    Parts of the manuscript use non-scientific language, for example, "doubly knockout mice" or "atria appear to have some wavy edges." These should be revised for a more formal scientific tone.

    We thank the reviewer to point this out. We have modified the text in the revised manuscript.

    Typo error in Figure 1D: "ventricle"

    We thank the reviewer to point this out. We have made the correction with Fig. 1D.

    Reference

    (1) Cao, X., et al., Endothelial TIE1 Restricts Angiogenic Sprouting to Coordinate Vein Assembly in Synergy With Its Homologue TIE2. Arterioscler Thromb Vasc Biol, 2023. 43(8): p. e323-e338.

    (2) Shen, B., et al., Genetic dissection of tie pathway in mouse lymphatic maturation and valve development. Arterioscler Thromb Vasc Biol, 2014. 34(6): p. 1221-30.

    (3) Chu, M. et al., Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII. Elife, 2016. 5:e21032.

    (4) Rodewald, H.R. and T.N. Sato, Tie1, a receptor tyrosine kinase essential for vascular endothelial cell integrity, is not critical for the development of hematopoietic cells. Oncogene, 1996. 12(2): p. 397-404.

    (5) D'Amico, G. et al., Loss of endothelial Tie1 receptor impairs lymphatic vessel development-brief report. Arterioscler Thromb Vasc Biol, 2010. 30(2): p. 207-9.

    (6) Qu, X., et al., Abnormal embryonic lymphatic vessel development in Tie1 hypomorphic mice. Development, 2010. 137(8): p. 1285-95.

    (7) Dumont, D.J., et al., Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev, 1994. 8(16): p. 1897-909.

    (8) You, L.R., et al., Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity. Nature, 2005. 435(7038): p. 98-104.

    (9) Wu, S.P., et al., Atrial identity is determined by a COUP-TFII regulatory network. Dev Cell, 2013. 25(4): p. 417-26.

    (10) Kim, K.H., et al., Myocardial Angiopoietin-1 Controls Atrial Chamber Morphogenesis by Spatiotemporal Degradation of Cardiac Jelly. Cell Rep, 2018. 23(8): p. 2455-2466.

    (11) Albu, M., et al., Distinct mechanisms regulate ventricular and atrial chamber wall formation. Nat Commun, 2024. 15(1): p. 8159.

  8. Version published to 10.64898/2026.02.26.708190v3 on bioRxiv
  9. Version published to 10.64898/2026.02.26.708190v2 on bioRxiv
  10. eLife

    eLife Assessment

    This study reports the relative importance of Tie1 and Tie2 signaling for atrial versus ventricular trabeculation. It is an important study and is one of the few works to date that have carefully and simultaneously analyzed these two processes. In line with a previous study in zebrafish, the authors demonstrate key differences between atrial and ventricular trabeculation. While the imaging and quantitative data were conducted with solid and validated methodology throughout the manuscript, the work would benefit from more rigourous approaches where Tie1/2 signaling is disrupted prior to the onset of atrial/ventricular trabeculation, to allow for a more direct comparison.

  11. eLife

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Ding et al. use genetic mouse models to demonstrate that atrial trabeculation is more dependent on Tie1/Tie2 signaling than ventricular trabeculation. With additional experimentation that would support the current claims, the results may hold significant value, as atrial trabeculation remains an understudied phenomenon in cardiac biology with potential implications for atrial cardiomyopathy and atrial fibrillation.

    Strengths:

    Detailed characterization of atrial versus ventricular trabeculation across different developmental timepoints, and the use of appropriate animal models to address the scientific question at hand.

    Weaknesses:

    The authors have consistently treated mice with tamoxifen after ventricular, but not atrial, trabeculation has already started. As such, the observed cardiac phenotypes - where predominantly atrial trabeculation is affected - might be a mere consequence of the precise time window in which Tie1/2 signaling was impaired, rather than a direct measurement of its relative importance for atrial versus ventricular trabeculation. The conclusions of the paper may thus be significantly strengthened by depleting Tie1/2 signaling prior to the onset of ventricular trabeculation, as is done for atrial trabeculation.

  12. eLife

    Reviewer #2 (Public review):

    Summary:

    Ding et al. examine the role of TIE1 in cardiac chamber morphogenesis using genetic mouse models targeting Tie1, Tek, or both, and analyzing endocardial cell-mediated chamber formation across multiple embryonic developmental and postnatal stages, supported by analysis of published single-cell datasets and new bulk RNA seq analyses of murine cardiac tissue. The authors find that Tie1 and Tek expression is higher in atrial than ventricular endocardial cells. Notably, endothelial Tie1 is required for atrial trabeculation at E12.5, but is less critical in ventricular trabeculation. TIE1 also acts synergistically with TIE2 during atrial trabeculation. While Tie1 deficiency alone does not cause defects at E10.5, combined heterozygous deletion of Tek disrupts both atrial and ventricular development at E10.5. This synergy is further supported by analyses at later embryonic stages and in postnatal hearts.

    Strengths:

    The study is well-designed, clearly written, and supported by high-quality figures. The performed experiments demonstrate a previously unrecognized role for Tie1 in cardiac development and identify synergistic control of cardiac morphogenesis by Tie1 and Tie2. This synergy is consistent with the previously identified roles of Tie1 and Tek in venous development and with Tie1 involvement in angiopoietin-dependent postnatal vascular and lymphatic remodeling. Together, these findings support a role for Tie1 as a contributor to Ang1-Tie2 signaling during heart development.

    Weaknesses:

    The manuscript does not include direct mechanistic studies; however, RNA seq analysis of atria and ventricles showed reduced expression of Tek, Dll1, and Notch1 upon Tie1 deficiency in developing hearts. Although previously reported mechanisms, such as TIE1-TIE2 heterodimer formation and effects on endothelial junctions, migration, or survival are discussed, no direct mechanistic experiments are performed. Addressing some of these mechanisms would have clarified the basis of Tie1-Tie2 synergy. As two distinct Tie1 models are used, including one targeting the kinase domain, the authors should state whether phenotypes differed or were similar between models.

  13. eLife

    Reviewer #3 (Public review):

    Summary:

    Ding et al. investigate the roles of TIE1 and TEK (Tie2) in mouse cardiac development, with a particular focus on atrial trabeculation. The authors employ multiple genetic models, including Tie1ICDflox/flox (with Cdh5-CreERT2), a knockout-first allele (EUCOMM, Tie1 tm1a/tm1a), and a Tek deletion model.

    Based on the dataset from Feng et al. 2022 Nat Commun, the authors report increased expression of Tie1 and Tek transcripts in atrial endocardial cells compared to ventricular cells at embryonic day (E) 14.5. Loss of Tie1 leads to early atrial trabeculation defects detectable at E12.5, whereas ventricular defects appear later and are less pronounced at E14.5. Chamber-specific RNA sequencing reveals stronger transcriptional changes in atrial tissue.

    Conditional deletion of Tek results in a similar phenotype, with more pronounced atrial defects. Combined deletion of Tie1 and Tek (Tie1 ΔICD/ΔICD; Tek+/-) leads to earlier and more severe defects in both atrial and ventricular trabeculation and results in embryonic lethality around E12.5, suggesting a synergistic interaction between the two genes.

    Conditional endothelial deletion of Tie1 combined with heterozygous global Tek at later embryonic stages allows analysis at later time points and again shows more severe defects in atrial trabeculation. Postnatal analysis of this model reveals reduced heart-to-body weight ratios and potential mild atrial abnormalities.

    Strengths:

    (1) The authors address chamber-specific signaling mechanisms underlying atrial versus ventricular trabeculation, an area of high developmental and clinical relevance.

    (2) The study provides a comprehensive temporal analysis across multiple embryonic stages.

    (3) The use of multiple genetic models strengthens the overall conclusions and allows comparative interpretation.

    (4) While focusing on trabeculation, the authors also include observations on coronary vessel development, increasing the broader relevance of the work. The findings are therefore of interest to the wider cardiovascular research community.

    Weaknesses:

    (1) Timing of recombination vs. trabeculation onset

    Ventricular trabeculation begins earlier than atrial trabeculation. Since tamoxifen (in contrast to 4-hydroxytamoxifen) requires metabolic activation, Cre-mediated recombination will occur with a delay. This suggests that atrial trabeculation may be targeted before its onset, whereas ventricular trabeculation may already be underway for 2-3 days at the time of effective gene deletion.

    How do the authors account for this discrepancy in their interpretation?

    Have earlier induction time points been tested to better capture the onset of ventricular trabeculation? This limitation should be explicitly discussed.

    (2) Clarity of genetic models and experimental design

    The study employs several genetic constructs. It would improve clarity if, for each experiment, the specific genetic model and tamoxifen regimen were clearly described before presenting the results.

    (3) Tie1 tm1a/tm1a phenotype vs. known global knockout

    Previous studies (PMID: 8846781, 7596437) show that complete Tie1 loss leads to severe edema, vascular rupture, and embryonic lethality around E13.5-E14.5.

    How does the Tie1 tm1a/tm1a allele differ, given that animals appear to survive longer? Is this allele hypomorphic rather than a full knockout?

    This point requires clarification.

    (4) Limited mechanistic insight

    While the authors aim to investigate underlying mechanisms, the current study is largely descriptive and based on mRNA expression and genetic interaction analyses (Tie1/Tek co-deletion). Direct mechanistic insights into signaling pathways remain limited. However, the dataset provides a valuable foundation for future mechanistic studies, which should be more clearly acknowledged in the discussion.

  14. eLife

    Author response:

    eLife Assessment

    This study reports the relative importance of Tie1 and Tie2 signaling for atrial versus ventricular trabeculation. It is an important study and is one of the few works to date that have carefully and simultaneously analyzed these two processes. In line with a previous study in zebrafish, the authors demonstrate key differences between atrial and ventricular trabeculation. While the imaging and quantitative data were conducted with solid and validated methodology throughout the manuscript, the work would benefit from more rigourous approaches where Tie1/2 signaling is disrupted prior to the onset of atrial/ventricular trabeculation, to allow for a more direct comparison.

    We thank the editors for the eLife assessment. We would like to request that the following statement be modified: “…the work would benefit from more rigourous approaches where Tie1/2 signaling is disrupted prior to the onset of atrial/ventricular trabeculation, to allow for a more direct comparison”. We request this change for the following reasons:

    We utilized two distinct genetic mouse models in this study (as summarized in Fig. 7I), comprising conventional knockouts (Tie1tm1a/tm1a, Tie1ΔICD/ΔICD and Tie1ΔICD/ΔICD;Tek+/-) and inducible gene deletion models (TekiECKO, Tie1ICDiECKO, and Tie1ICDiECKO;Tek+/-) [1-3]. The Tie1tm1a/tm1a line is equivalent to the previously published Tie1-/-</sup mouse line, as demonstrated in our prior work and by others [1, 2, 4-6]. Therefore, the Tie1 or Tek alleles were inactivated prior to the onset of atrial and ventricular trabeculations, as shown in Fig. 1, Fig. 2, Fig. 3, Fig. 5A-D, and Supplemental Fig. 3. Based on these findings, we propose that TIE1 is differentially required for atria versus ventricle morphogenesis, and acts synergistically with TIE2 during cardiac trabeculation.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Ding et al. use genetic mouse models to demonstrate that atrial trabeculation is more dependent on Tie1/Tie2 signaling than ventricular trabeculation. With additional experimentation that would support the current claims, the results may hold significant value, as atrial trabeculation remains an understudied phenomenon in cardiac biology with potential implications for atrial cardiomyopathy and atrial fibrillation.

    Strengths:

    Detailed characterization of atrial versus ventricular trabeculation across different developmental timepoints, and the use of appropriate animal models to address the scientific question at hand.

    Weaknesses:

    The authors have consistently treated mice with tamoxifen after ventricular, but not atrial, trabeculation has already started. As such, the observed cardiac phenotypes - where predominantly atrial trabeculation is affected - might be a mere consequence of the precise time window in which Tie1/2 signaling was impaired, rather than a direct measurement of its relative importance for atrial versus ventricular trabeculation. The conclusions of the paper may thus be significantly strengthened by depleting Tie1/2 signaling prior to the onset of ventricular trabeculation, as is done for atrial trabeculation.

    We thank the reviewer for the comments.

    Regarding the timeline of gene deletion and tamoxifen treatment, we would like to provide the following clarification.

    Fig. 1-3: As described in the Methods and Materials, Tie1tm1a/tm1a is a knockout first mouse model established from EUCOMM embryonic stem cells (EPD0735-3B07) targeting Tie1 gene. Therefore, the Tie1tm1a/tm1a line is equivalent to the previously published Tie1 null mice (Tie1-/-). The Tie1Flox/Flox mouse line (with exon 8 floxed) was generated when the lacZ reporter and neo-cassette were excised using the FLPeR mice.

    Fig. 5A-D: To investigate the synergy of TIE1 and TIE2 in cardiac trabeculation, we utilized the Tek+/- and Tie1ΔICD/+ mouse lines and they were crossbred to generate double mutant mice harboring a homozygous Tie1 mutation and a single null Tek allele (Tie1ΔICD/ΔICD;Tek+/-). Although no obvious defects were observed in atrial or ventricular structures following Tie1 deficiency alone at E10.5, both atria and ventricle development were disrupted in Tie1ΔICD/ΔICD;Tek+/- mutants at the same stage (Fig. 5A-D).

    Supplemental Fig. 3: To verify the role of TIE1 in atrial development, we employed alternative knockout mouse line targeting the Tie1 intracellular domain by floxing exons 15 and exon 16 (Tie1ICDFlox/Flox). Mutants harboring these null alleles are designated as Tie1ΔICD/ ΔICD. As detailed in the previous publication [2], the line is also equivalent to the previously published Tie1 null mice (Tie1-/-). The cardiac phenotypes shown in Supplemental Fig. 3 are indeed similar to those of Tie1tm1a/tm1a mutant mice.

    For the inducible knockouts targeting Tie1, Tek and both, the results are shown in Fig. 4, Fig. 5E-H, Fig. 6, Fig. 7.

    Fig. 4: As mice homozygous for Tek mutation (Tek-/-) die before E10.5 [3, 7], we performed studies using the inducible knockout line targeting Tek (TekFlox/-;Cdh5-CreERT2 named as TekiECKO), as shown in Fig. 4.

    Fig. 5-7: To investigate the synergy of TIE1 and TIE2 in the cardiac trabeculation at the later stages of embryogenesis (Fig. 5E-H, Fig. 6) and the postnatal stage (Fig. 7), we used the inducible knockout models targeting Tie1/Tek, including Tie1ICDiECKO (Tie1ICDFlox/-;Cdh5-CreERT2) and Tie1ICDiECKO;Tek+/- (Tie1ICDFlox/-;Cdh5-CreERT2;Tek+/-).

    Reviewer #2 (Public review):

    Summary:

    Ding et al. examine the role of TIE1 in cardiac chamber morphogenesis using genetic mouse models targeting Tie1, Tek, or both, and analyzing endocardial cell-mediated chamber formation across multiple embryonic developmental and postnatal stages, supported by analysis of published single-cell datasets and new bulk RNA seq analyses of murine cardiac tissue. The authors find that Tie1 and Tek expression is higher in atrial than ventricular endocardial cells. Notably, endothelial Tie1 is required for atrial trabeculation at E12.5, but is less critical in ventricular trabeculation. TIE1 also acts synergistically with TIE2 during atrial trabeculation. While Tie1 deficiency alone does not cause defects at E10.5, combined heterozygous deletion of Tek disrupts both atrial and ventricular development at E10.5. This synergy is further supported by analyses at later embryonic stages and in postnatal hearts.

    Strengths:

    The study is well-designed, clearly written, and supported by high-quality figures. The performed experiments demonstrate a previously unrecognized role for Tie1 in cardiac development and identify synergistic control of cardiac morphogenesis by Tie1 and Tie2. This synergy is consistent with the previously identified roles of Tie1 and Tek in venous development and with Tie1 involvement in angiopoietin-dependent postnatal vascular and lymphatic remodeling. Together, these findings support a role for Tie1 as a contributor to Ang1-Tie2 signaling during heart development.

    Weaknesses:

    The manuscript does not include direct mechanistic studies; however, RNA seq analysis of atria and ventricles showed reduced expression of Tek, Dll1, and Notch1 upon Tie1 deficiency in developing hearts. Although previously reported mechanisms, such as TIE1-TIE2 heterodimer formation and effects on endothelial junctions, migration, or survival are discussed, no direct mechanistic experiments are performed. Addressing some of these mechanisms would have clarified the basis of Tie1-Tie2 synergy. As two distinct Tie1 models are used, including one targeting the kinase domain, the authors should state whether phenotypes differed or were similar between models.

    We thank the reviewer for the comments. In this study, we have provided genetic evidence that TIE1 is differentially required for atrial versus ventricular trabeculation. Although the precise molecular mechanisms underlying TIE1 function require further investigation, we have provided compelling genetic evidence of its synergistic role with TIE2 during this process. The two genetic models targeting Tie1 (Tie1tm1a/tm1a, Tie1ΔICD/ΔICD) produced consistent cardiac and vascular phenotypes as shown in this study and our previous work [1, 2].

    Reviewer #3 (Public review):

    Summary:

    Ding et al. investigate the roles of TIE1 and TEK (Tie2) in mouse cardiac development, with a particular focus on atrial trabeculation. The authors employ multiple genetic models, including Tie1ICDflox/flox (with Cdh5-CreERT2), a knockout-first allele (EUCOMM, Tie1 tm1a/tm1a), and a Tek deletion model.

    Based on the dataset from Feng et al. 2022 Nat Commun, the authors report increased expression of Tie1 and Tek transcripts in atrial endocardial cells compared to ventricular cells at embryonic day (E) 14.5. Loss of Tie1 leads to early atrial trabeculation defects detectable at E12.5, whereas ventricular defects appear later and are less pronounced at E14.5. Chamber-specific RNA sequencing reveals stronger transcriptional changes in atrial tissue.

    Conditional deletion of Tek results in a similar phenotype, with more pronounced atrial defects. Combined deletion of Tie1 and Tek (Tie1 ΔICD/ΔICD; Tek+/-) leads to earlier and more severe defects in both atrial and ventricular trabeculation and results in embryonic lethality around E12.5, suggesting a synergistic interaction between the two genes.

    Conditional endothelial deletion of Tie1 combined with heterozygous global Tek at later embryonic stages allows analysis at later time points and again shows more severe defects in atrial trabeculation. Postnatal analysis of this model reveals reduced heart-to-body weight ratios and potential mild atrial abnormalities.

    Strengths:

    (1) The authors address chamber-specific signaling mechanisms underlying atrial versus ventricular trabeculation, an area of high developmental and clinical relevance.

    (2) The study provides a comprehensive temporal analysis across multiple embryonic stages.

    (3) The use of multiple genetic models strengthens the overall conclusions and allows comparative interpretation.

    (4) While focusing on trabeculation, the authors also include observations on coronary vessel development, increasing the broader relevance of the work. The findings are therefore of interest to the wider cardiovascular research community.

    Weaknesses:

    (1) Timing of recombination vs. trabeculation onset

    Ventricular trabeculation begins earlier than atrial trabeculation. Since tamoxifen (in contrast to 4-hydroxytamoxifen) requires metabolic activation, Cre-mediated recombination will occur with a delay. This suggests that atrial trabeculation may be targeted before its onset, whereas ventricular trabeculation may already be underway for 2-3 days at the time of effective gene deletion.

    How do the authors account for this discrepancy in their interpretation?

    Have earlier induction time points been tested to better capture the onset of ventricular trabeculation? This limitation should be explicitly discussed.

    (2) Clarity of genetic models and experimental design

    The study employs several genetic constructs. It would improve clarity if, for each experiment, the specific genetic model and tamoxifen regimen were clearly described before presenting the results.

    We thank the reviewer for the detailed and constructive comments. For studies employing the inducible gene deletion mouse models, the genetic models and tamoxifen treatment schemes have been provided in the related figures. For the rest of studies, we used the conventional knockouts targeting Tie1 and Tek (Tie1tm1a/tm1a, Tie1ΔICD/ΔICD and Tie1ΔICD/ΔICD;Tek+/-), as detailed above.

    (3) Tie1 tm1a/tm1a phenotype vs. known global knockout

    Previous studies (PMID: 8846781, 7596437) show that complete Tie1 loss leads to severe edema, vascular rupture, and embryonic lethality around E13.5-E14.5.

    How does the Tie1 tm1a/tm1a allele differ, given that animals appear to survive longer? Is this allele hypomorphic rather than a full knockout?

    This point requires clarification.

    Tie1tm1a/tm1a is equivalent to the full knockout (Tie1-/-). As demonstrated in our prior work, the Tie1ΔICD/ΔICD model produced lymphatic and blood vascular phenotypes similar to those of Tie1-/- mutants [1, 2, 5, 6].

    (4) Limited mechanistic insight

    While the authors aim to investigate underlying mechanisms, the current study is largely descriptive and based on mRNA expression and genetic interaction analyses (Tie1/Tek co-deletion). Direct mechanistic insights into signaling pathways remain limited. However, the dataset provides a valuable foundation for future mechanistic studies, which should be more clearly acknowledged in the discussion.

    We thank the reviewer for the comments. The manuscript will be revised accordingly, and a detailed response will be provided in our final submission.

    Reference

    (1) Cao, X., et al., Endothelial TIE1 Restricts Angiogenic Sprouting to Coordinate Vein Assembly in Synergy With Its Homologue TIE2. Arterioscler Thromb Vasc Biol, 2023. 43(8): p. e323-e338.

    (2) Shen, B., et al., Genetic dissection of tie pathway in mouse lymphatic maturation and valve development. Arterioscler Thromb Vasc Biol, 2014. 34(6): p. 1221-30.

    (3) Chu, M., et al., Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII. Elife, 2016. 5:e21032.

    (4) Rodewald, H.R. and T.N. Sato, Tie1, a receptor tyrosine kinase essential for vascular endothelial cell integrity, is not critical for the development of hematopoietic cells. Oncogene, 1996. 12(2): p. 397-404.

    (5) D'Amico, G., et al., Loss of endothelial Tie1 receptor impairs lymphatic vessel development-brief report. Arterioscler Thromb Vasc Biol, 2010. 30(2): p. 207-9.

    (6) Qu, X., et al., Abnormal embryonic lymphatic vessel development in Tie1 hypomorphic mice. Development, 2010. 137(8): p. 1285-95.

    (7) Dumont, D.J., et al., Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev, 1994. 8(16): p. 1897-909.

  15. Version published to 10.7554/elife.111156.1 on eLife
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