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

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.

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.

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.

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 (Tie1^tm1a/tm1a, Tie1^ΔICD/ΔICD and Tie1^ΔICD/ΔICD;Tek^+/-) and inducible gene deletion models (Tek^iECKO, Tie1ICD^iECKO, and Tie1ICD^iECKO;Tek^+/-) [1-3]. The Tie1^tm1a/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, Tie1^tm1a/tm1a is a knockout first mouse model established from EUCOMM embryonic stem cells (EPD0735-3B07) targeting Tie1 gene. Therefore, the Tie1^tm1a/tm1a line is equivalent to the previously published Tie1 null mice (Tie1^-/-). The Tie1^Flox/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 Tie1^ΔICD/+ mouse lines and they were crossbred to generate double mutant mice harboring a homozygous Tie1 mutation and a single null Tek allele (Tie1^ΔICD/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 Tie1^ΔICD/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 (Tie1ICD^Flox/Flox). Mutants harboring these null alleles are designated as Tie1^{ΔICD/ 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 Tie1^tm1a/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 (Tek^Flox/-;Cdh5-Cre^ERT2 named as Tek^iECKO), 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 Tie1ICD^iECKO (Tie1ICD^Flox/-;Cdh5-Cre^ERT2) and Tie1ICD^iECKO;Tek^+/- (Tie1ICD^Flox/-;Cdh5-Cre^ERT2;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 (Tie1^tm1a/tm1a, Tie1^ΔICD/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 (Tie1^tm1a/tm1a, Tie1^ΔICD/ΔICD and Tie1^ΔICD/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.

Tie1^tm1a/tm1a is equivalent to the full knockout (Tie1^-/-). As demonstrated in our prior work, the Tie1^ΔICD/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.