The cardiopharyngeal mesoderm contributes to lymphatic vessel development in mouse

  1. Kazuaki Maruyama  Is a corresponding author
  2. Sachiko Miyagawa-Tomita
  3. Yuka Haneda
  4. Mayuko Kida
  5. Fumio Matsuzaki
  6. Kyoko Imanaka-Yoshida
  7. Hiroki Kurihara  Is a corresponding author
  1. Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Japan
  2. Department of Pathology and Matrix Biology, Graduate School of Medicine, Mie University, Japan
  3. Department of Animal Nursing Science, Yamazaki University of Animal Health Technology, Japan
  4. Heart Center, Department of Pediatric Cardiology, Tokyo Women's Medical University, Japan
  5. Laboratory for Cell Asymmetry, RIKEN Center for Biosystems Dynamics Research, Japan

Abstract

Lymphatic vessels are crucial for tissue homeostasis and immune responses in vertebrates. Recent studies have demonstrated that lymphatic endothelial cells (LECs) arise from both venous sprouting (lymphangiogenesis) and de novo production from non-venous origins (lymphvasculogenesis), which is similar to blood vessel formation through angiogenesis and vasculogenesis. However, the contribution of LECs from non-venous origins to lymphatic networks is considered to be relatively small. Here, we identify the Islet1 (Isl1)-expressing cardiopharyngeal mesoderm (CPM) as a non-venous origin of craniofacial and cardiac LECs. Genetic lineage tracing with Isl1Cre/+ and Isl1CreERT2/+ mice suggested that a subset of CPM cells gives rise to LECs. These CPM-derived LECs are distinct from venous-derived LECs in terms of their developmental processes and anatomical locations. Later, they form the craniofacial and cardiac lymphatic vascular networks in collaboration with venous-derived LECs. Collectively, our results demonstrate that there are two major sources of LECs, the cardinal vein and the CPM. As the CPM is evolutionarily conserved, these findings may improve our understanding of the evolution of lymphatic vessel development across species. Most importantly, our findings may provide clues to the pathogenesis of lymphatic malformations, which most often develop in the craniofacial and mediastinal regions.

Editor's evaluation

This paper provides fundamental insight into the developmental source of lymphatic endothelial cells, which has been debated for over a century. This important work characterises the development of the mouse craniofacial lymphatics, and provides compelling evidence for a non-venous source of craniofacial lymphatic endothelial cells. The manuscript is well presented and will be of interest to developmental, vascular and lymphatic vascular biologists.

https://doi.org/10.7554/eLife.81515.sa0

Introduction

The lymphatic vascular system plays diverse roles in the maintenance of tissue fluid balance, immune surveillance, lipid absorption from the gut, and tumor metastasis (Oliver and Alitalo, 2005). Furthermore, recent progress in molecular and cellular characterization has unveiled the processes involved in the development of the lymphatic vasculature and the roles played by the lymphatic vasculature in various pathophysiological conditions (Klaourakis et al., 2021; Maruyama et al., 2021; Maruyama and Imanaka-Yoshida, 2022; Oliver et al., 2020; Oliver and Alitalo, 2005).

The origin of lymphatic endothelial cells (LECs) has been discussed since the 1900s (Huntington and McClure, 1910; Sabin, 1902). Recent studies using genetic lineage tracing confirmed Sabin’s hypothesis that lymphatic vessels originate from the embryonic cardinal vein through lymphangiogenesis (Srinivasan et al., 2007; Yang et al., 2012). In mice, lymphatic vessels arise from the common cardinal vein between embryonic day (E) 9.5 and E10.0 during the partial expression of prospero homeobox transcription protein 1 (Prox1), which is the master regulator of LEC differentiation (Srinivasan et al., 2007; Wigle and Oliver, 1999). After that, vascular endothelial growth factor C induces the sprouting and expansion of vascular endothelial growth factor receptor 3 (VEGFR3)+ LECs from the common cardinal veins to form the first lymphatic plexus (Hägerling et al., 2013; Karkkainen et al., 2004). In contrast, in tadpoles and avian embryos, mesenchymal cells also contribute to lymphatic vessel development (Ny et al., 2005; Schneider et al., 1999; Wilting et al., 2006), supporting Huntington and McClure’s suggestion that lymphatic vessels form via the differentiation of mesenchymal cells into LECs, which later develop into a primary lymph sac and connect to the venous system. Supporting these findings, recent studies involving various genetic lineage-tracing models have revealed that non-venous sources of LECs also contribute to the lymphatic vasculature in the skin, mesentery, and heart through lymphvasculogenesis (Klotz et al., 2015; Lioux et al., 2020; Mahadevan et al., 2014; Martinez-Corral et al., 2015; Maruyama et al., 2019; Pichol-Thievend et al., 2018; Stanczuk et al., 2015). Despite the accumulation of studies on non-venous sources of LECs, it remains uncertain how much they contribute to lymphatic vessels throughout the body. Our previous studies showed that LIM-homeodomain protein Islet1 (Isl1)-expressing second heart field cells contribute to ventral cardiac lymphatic vessels as a non-venous source of LECs (Maruyama et al., 2019). Isl1-expressing second heart field cells have been found to overlap with the progenitor populations that give rise to the pharyngeal muscles in mice and chicks, and they are collectively known as the cardiopharyngeal mesoderm (CPM), which contributes to broad regions of the heart, cranial musculature, and connective tissue (Diogo et al., 2015; Grimaldi et al., 2022; Harel et al., 2009; Heude et al., 2018; Tirosh-Finkel et al., 2006; Tzahor and Evans, 2011). The CPM is composed of the paraxial and splanchnic mesodermal cells surrounding the pharynx. Later, the CPM cells migrate to form the mesodermal cores of the pharyngeal arches. Before their differentiation, CPM cells express both immature skeletal and cardiac muscle markers, including Isl1, myocyte-specific enhancer factor 2C (Mef2c), and T-box transcription factor (Tbx1). In mice, these molecules have been shown to be markers of a subset of CPM cells, which play important roles in cardiovascular and skeletal muscle development (Adachi et al., 2020; Diogo et al., 2015; Grimaldi et al., 2022; Harel et al., 2009; Heude et al., 2018; Lescroart et al., 2010; Nathan et al., 2008; Tzahor and Evans, 2011).

Herein, we identified that the CPM contributes to broader regions of the facial, laryngeal, and cardiac lymphatic vasculature using Isl1Cre/+ and Isl1CreERT2/+ mice, which can be used to study developmental processes involving the CPM. According to our developmental analysis, CPM-derived progenitor cells have the capacity to differentiate into LECs for a limited embryonic period. CPM-derived LECs are spatiotemporally distinct from venous-derived LECs during the early embryonic period. Later, they coordinate to form capillary lymphatics in and around the cranial and cardiac regions. Conditional knockout (KO) of Prox1 in Isl1-expressing cells resulted in decreased numbers of lymphatic vessels in the tongue and altered the proportions of Isl1+ and Isl1- LECs in facial lymphatic vessels. These results suggest that a subpopulation of LECs may share a common mesodermal origin with cardiopharyngeal components. In addition, as the CPM is conserved across vertebrates, they may provide clues regarding the evolution of lymphatic vessel development. From a clinical viewpoint, head and neck regions contributed by the CPM are the most common sites of lymphatic malformations (LMs) (Perkins et al., 2010). Collectively, the present findings provide a fundamental basis for our understanding of lymphatic vessel development and lymphatic system-related diseases.

Results

Isl1+ CPM-derived LECs contribute to cardiac, facial, and laryngeal lymphatic vessel development

To assess the regional contribution of Isl1+ CPM cells to lymphatic vessel development, we crossed Isl1Cre/+ mice, which express Cre recombinase under the control of the Isl1 promoter and in which second heart field derivatives are effectively labeled, with the transgenic reporter line Rosa26tdTomato/+ and analyzed at E16.5, when lymphatic networks are distributed throughout the whole body (Srinivasan et al., 2007). Co-immunostaining of platelet endothelial cell adhesion molecule (PECAM) and VEGFR3, which we confirmed its colocalization with lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) at E14.5 and E16.5 (Figure 1—figure supplement 1), revealed tdTomato+ LECs in and around the larynx, the skin of the lower jaw, the tongue, and the cardiac outflow tracts, at various frequencies, whereas no such cells were found on the dorsal side of the ventricles, which agrees with our previous study (Maruyama et al., 2019; Figure 1A–H). We also confirmed that tdTomato+ cells were present in populations of PECAM+ blood and aortic endothelial cells in these regions (Figure 1A–F).

Figure 1 with 2 supplements see all
Isl1+ lineages contribute to cranial and cardiac lymphatic vessels.

(A–G’) Sagittal sections of Isl1Cre/+;Rosa26tdTomato/+ embryos, in which platelet endothelial cell adhesion molecule (PECAM), tdTomato, and vascular endothelial growth factor receptor 3 (VEGFR3) were labeled at embryonic day (E) 16.5, are shown. (A–F) tdTomato colocalized with PECAM/VEGFR3 (white arrows) and PECAM (yellow arrows) in and around the larynx (B), the skin of the lower jaw (C), the tongue (D), and the cardiac outflow tracts (F) (n=3). (G) tdTomato did not colocalize with VEGFR3 in the LECs on the dorsal side of the ventricles (white arrowhead, n=3). (H) The results of quantitative analysis of the percentage of the area in tdTomato+/VEGFR3+ lymphatic vessels among all VEGFR3+ lymphatic vessels are shown. (I–O’) Sagittal sections of Wnt1-Cre;Rosa26eYFP/+ embryos, in which PECAM, eYFP, and VEGFR3 were labeled at E16.5, are shown. There were no eYFP+/VEGFR3+ lymphatic vessels in or around the larynx, the skin of the lower jaw, the tongue, or the heart (the white arrowheads indicate cardiac lymphatic vessels around the cardiac outflow tracts and the dorsal side of the ventricles; n=3). Each dot represents a value obtained from one sample. L, larynx; T, tongue; Ao, aorta; PA, pulmonary artery; V, ventricle. Scale bars, 100 μm (B–D, F, G, J–L, N, O), 1 mm (A, E, I, M).

Figure 1—source data 1

Quantification of Isl1+ lineages contribution to craniofacial and cardiac lymphatic vessels.

https://cdn.elifesciences.org/articles/81515/elife-81515-fig1-data1-v2.xlsx

To examine whether the Isl-marked LECs originated from the neural crest (Engleka et al., 2012), we used Wnt1-Cre mice, in which neural crest cells were effectively marked, crossed with the Rosa26eYFP/+ reporter line. We detected broad eYFP+ cell contributions to the bone, cartilage, and mesenchymal tissues in the head and neck regions as well as the cardiac outflow tract wall, but no eYFP+ LECs were found in these regions (Figure 1I–O), excluding the possibility of them originating from the neural crest. We then investigated the possible contribution of myogenic CPM populations (Harel et al., 2012; Harel et al., 2009; Heude et al., 2018) to LECs using Myf5CreERT2/+ mice crossed with the Rosa26tdTomato/+ line. After tamoxifen was administered at E8.5, tdTomato+ cells were broadly detected in the skeletal muscle in the head and neck regions at E16.5, indicating effective Cre recombination in CPM-derived musculatures (Figure 1—figure supplement 2A–E). By contrast, no tdTomato+ LECs were detected in the cardiopharyngeal region (Figure 1—figure supplement 2A–E). These results indicate that Isl1+ CPM cells contribute to LECs in the cardiopharyngeal region through progenitors distinct from the Myf5+ myogenic lineage.

Isl1+ CPM cells can differentiate into LECs in a limited developmental period

To determine the timing of the differentiation of CPM into LECs, we employed tamoxifen-inducible Isl1CreERT2/+ mice crossed with either the Rosa26tdTomato/+ or Rosa26eYFP/+ reporter line to conditionally label descendants of Isl1-expressing cells. We intraperitoneally injected tamoxifen into pregnant female mice at several timepoints. We first injected tamoxifen at E6.5 and analyzed the embryos at E16.5 by examining sagittal sections that had been immunostained for PECAM and VEGFR3. We injected tamoxifen into three pregnant female mice; however, due to the toxicity of tamoxifen, we could only obtain two embryos from one mouse. In the embryos, tdTomato+ cells broadly contributed to LECs in and around the larynx, the skin of the lower jaw, the tongue, and the cardiac outflow tracts at various frequencies, but not in the back skin or on the dorsal side of the ventricles (Figure 2—figure supplement 1A–I). We then injected tamoxifen at E8.5 and analyzed the embryos at E16.5. Although the contribution of tdTomato+ cells to LECs was smaller, tdTomato+ cells broadly contributed to LECs, as was found for the tamoxifen-treated embryos analyzed at E6.5 (Figure 2A–H). In contrast, the number of tdTomato+ LECs seen at E16.5 was markedly decreased when tamoxifen was injected at E11.5 (Figure 2I–P). These results indicate that the differentiation of CPM cells into LECs occurs before E11.5.

Figure 2 with 1 supplement see all
The differentiation of Isl1+ cardiopharyngeal mesoderm (CPM) cells into lymphatic endothelial cells (LECs) occurs during the early embryonic period.

(A–G’, I–O’) Sagittal sections of Isl1CreERT2/+;Rosa26tdTomato/+ embryos, in which platelet endothelial cell adhesion molecule (PECAM), tdTomato, and vascular endothelial growth factor receptor 3 (VEGFR3) were labeled at embryonic day (E) 16.5, are shown. Tamoxifen was administered at E8.5 (A–G’) or E11.5 (I–O’). (A–G’) Both tdTomato+ (white arrows) and tdTomato- (white arrowheads) VEGFR3+ lymphatic vessels were observed in and around the larynx (B), the skin of the lower jaw (C), the tongue (D), and the cardiac outflow tracts (F) (n=5). (G) tdTomato did not colocalize with VEGFR3 in the LECs on the dorsal side of the ventricles (white arrowhead, n=5). (I–O’) Almost all of the VEGFR3+ lymphatic vessels in and around the larynx (J), the skin of the lower jaw (K), the tongue (L), the cardiac outflow tracts (N), and the dorsal side of the ventricles (O) were tdTomato- (white arrows, n=3). (H, P) The results of a quantitative analysis of the percentage of the area of tdTomato+/VEGFR3+ lymphatic vessels among all VEGFR3+ lymphatic vessels are shown. Tamoxifen was administered at E8.5 (H) or E11.5 (P). Each dot represents a value obtained from one sample. L, larynx; T, tongue; Ao, aorta; PA, pulmonary artery; V, ventricle. Scale bars, 100 μm (B–D, F, G, J–L, N, O), 1 mm (A, E, I, M).

Figure 2—source data 1

Quantification of Isl1+ lineages contribution to craniofacial and cardiac lymphatic vessels at embryonic day (E) 16.5 with tamoxifen treatment at E8.5.

https://cdn.elifesciences.org/articles/81515/elife-81515-fig2-data1-v2.xlsx
Figure 2—source data 2

Quantification of Isl1+ lineages contribution to craniofacial and cardiac lymphatic vessels at embryonic day (E) 16.5 with tamoxifen treatment at E11.5.

https://cdn.elifesciences.org/articles/81515/elife-81515-fig2-data2-v2.xlsx

We then performed whole-mount immunostaining of hearts from E16.5 Isl1CreERT2/+;Rosa26eYFP/+ embryos for PECAM, VEGFR3, and eYFP to show the spatial contribution of Isl1+ CPM cells to lymphatic vessels. In the embryos from the mice treated with tamoxifen at E8.5, eYFP+ cells were detected in VEGFR3+ lymphatic vessels around the cardiac outflow tracts. In contrast, we could not detect eYFP+ cells in the VEGFR3+ lymphatic vessels around the cardiac outflow tracts when tamoxifen was administered at E11.5, or on the dorsal side of the ventricles when tamoxifen was administered at E8.5 or E11.5 (Figure 3A–H). These results indicate that Isl1+ CPM cells gradually lose their capacity to differentiate into LECs as development progresses.

Figure 3 with 1 supplement see all
Isl1+ cardiopharyngeal mesoderm (CPM) cells contribute to cardiac lymphatic vessel development.

(A–H) Whole-mount confocal images of Isl1CreERT2/+;Rosa26eYFP/+ hearts, in which platelet endothelial cell adhesion molecule (PECAM), eYFP, and vascular endothelial growth factor receptor 3 (VEGFR3) were labeled at embryonic day (E) 16.5, are shown. Tamoxifen was administered at E8.5 (A–D) or E11.5 (E–H). (A–D) Many eYFP+ cells were observed around the cardiac outflow tracts, and they contributed to lymphatic vessels (white arrow) (n=9/9 hearts [100%]) (A–C’). (D) There were no eYFP+ lymphatic vessels on the dorsal side of the ventricles (n=9/9 hearts [100%]). The cardiac nerves were also positive for eYFP after tamoxifen treatment at E8.5 (white arrowhead) (D). (E–H) Fewer eYFP+ cells were observed around the cardiac outflow tracts (E–G’), and there were no eYFP+ lymphatic vessels around the cardiac outflow tracts (F) or on the dorsal side of the ventricles (n=0/8) (H). Ao, aorta; PA, pulmonary artery. Scale bars, 100 μm (A–C, E–G), 500 μm (D, H).

Isl1+ CPM-derived LECs are spatiotemporally distinct from vein-derived LECs

We next analyzed the early stages of the spatiotemporal development of Isl1+ CPM-derived lymphatic vessels using Isl1Cre/+;Rosa26eYFP/+ mice by performing immunostaining for PECAM, Prox1, and eYFP. At E11.5, eYFP+/Prox1+ cells were detected around the eYFP+ mesodermal core region of the first and second pharyngeal arches in Isl1Cre/+;Rosa26eYFP/+ mice (Figure 4A and B). At E12.0, eYFP+/Prox1+ cells were distributed in the pharyngeal arch region and extended toward the lymph sac-forming domain composed of eYFP/Prox1+ cells, which were considered to emerge from the cardinal vein and intersomitic vessels, as reported previously (Yang et al., 2012; Figure 4C and D). At E14.5, eYFP+/Prox1+/PECAM+ LECs formed lymphatic capillaries in the lower jaw and the tongue, which are derived from the first pharyngeal arch (Figure 4E–G).

The spatiotemporal development of Isl1+ cardiopharyngeal mesoderm (CPM)-derived lymphatic endothelial cells (LECs).

(A–G’) Whole-mount and sagittal section images of Isl1Cre/+;Rosa26eYFP/+ embryos, in which platelet endothelial cell adhesion molecule (PECAM), eYFP, and Prox1 were labeled at embryonic day (E) 11.5, E12.0, or E14.5, are shown. (A, B) eYFP+/Prox1+ cells were observed around the cores of the first and second pharyngeal arches at E11.5 (white arrowheads). (C–D’’) eYFP+/Prox1+ LECs migrated from the first and second pharyngeal arches to the lymph sac-forming domain (white dotted region) adjacent to the anterior cardinal vein at E12.0 (white arrows). (E–G’) Some of the eYFP+/Prox1+ LECs expressed PECAM and formed lymphatic capillaries in the lower jaw and tongue at E14.5 (white arrows). (H–N’) Whole-mount and sagittal section images of Isl1CreERT2/+;Rosa26eYFP/+ embryos, in which PECAM, eYFP, and Prox1 were labeled at E12.0, are shown. Tamoxifen was administered at E8.5 (H–L’) or E9.5 (M–N’). (I, I’) eYFP+/Prox1+ LECs (white arrows) migrated from the first and second pharyngeal arches to the lymph sac-forming domain (white dotted region) at E12.0. (J) There were no eYFP+/Prox1+ cells in or around the cardinal vein (n=4). (K–N) eYFP+/Prox1+/PECAM+ LECs were seen in the first and second pharyngeal arches of the embryos at E12.0, when tamoxifen was administered at E8.5 or E9.5 (white arrows), although the number of these cells was decreased in the E9.5 group (M–N’). (O) The results of a quantitative analysis of the percentage of eYFP+/Prox1+ cells among Prox1+ cells in the first and second pharyngeal arches at E12.0 after tamoxifen treatment at E8.5 (the number of eYFP+/Prox1+ cells [10.83 (mean)±1.249 (SEM)]: Prox1+ cells [30.83±4.549]) or E9.5 (the number of eYFP+/Prox1+ cells [2.833±1.108]: Prox1+ cells [35.50±5.847]) are shown. **p=0.0022. All of the data are presented as the mean ± SEM, and statistical analyses were performed using the non-parametric Mann-Whitney U-test. V, ventricle; PA1, first pharyngeal arch; PA2, second pharyngeal arch; CV, cardinal vein; LS, lymph sac-forming domain; L, liver; T, tongue; RA, right atrium; OFT, cardiac outflow tract. Scale bars, 100 μm (A, B, D, F, G, I, J, L, N), 500 μm (C, E, H, K, M); **p<0.01.

Figure 4—source data 1

Quantification of eYFP+/Prox1+ cells among Prox1+ cells in the first and second pharyngeal arches at embryonic day (E) 12.0 with tamoxifen treatment at E8.5 and E9.5.

https://cdn.elifesciences.org/articles/81515/elife-81515-fig4-data1-v2.xlsx

We then analyzed Isl1CreERT2/+;Rosa26eYFP/+ embryos at E12.0. After tamoxifen was administered at E8.5, eYFP+/Prox1+ cells were found in and around the pharyngeal mesodermal condensation, close to the lymph sac-forming region, as was seen in the Isl1Cre/+;Rosa26eYFP/+ embryos (Figure 4H, I). No eYFP+/Prox1+ cells were observed in or around the cardinal veins (Figure 4J). Some of the eYFP+/Prox1+ cells in the pharyngeal arch region expressed PECAM at E12.0, indicating that they had endothelial characteristics (Figure 4K and L). After tamoxifen was administered at E9.5, the number of eYFP+/Prox1+ LECs was significantly decreased compared with that seen in the embryos treated with tamoxifen at E8.5 (Figure 4M–O).

To identify possible Isl1+ LEC progenitors, we investigated the expression patterns of Isl1, Prox1, and vascular endothelial markers (Flk1 and PECAM) by immunostaining sections of E9.0–E11.5 pharyngeal arches and cardinal veins. Consistent with the previous report (Cai et al., 2003), Isl1 was abundantly expressed in the core mesoderm of the first and second pharyngeal arches corresponding to the CPM from E9.0 to E11.5 (Nathan et al., 2008), where Prox1+ cells also aggregated and partially overlapped with Isl1 signals (Figure 3—figure supplement 1A, A’ C, C’ E, E’ G, G’ I, I’). By contrast, Flk1+ or PECAM+ cells were distributed mainly around the CPM and not expressed Isl1 (Figure 3—figure supplement 1A, A’ C, C’ E, E’ G, G’ I, I’). Furthermore, Isl1 was expressed neither in the endothelial layer of the cardinal vein nor in surrounding Prox1+/PECAM+ LECs (Figure 3—figure supplement 1B, B’ D, D’, F, F’, H, H’, J, and J’). Taken together with the result from Myf5CreERT2/+ mice, these results indicate that Isl1+ non-myogenic CPM cells may serve as LEC progenitors independent of venous-derived LECs and the commitment to LEC differentiation occurs before E9.5 in the pharyngeal arch region.

Loss of Prox1 in the Isl1+ lineage reveals the importance of Isl1+ CPM-derived LECs for cranial lymphatic vessel development

To investigate the functional importance of Isl1+ CPM-derived LECs for the development of cranial lymphatic vessels (the lymphatics of the lower jaw skin, cheeks, and tongue), we conditionally knocked out Prox1 in CPM populations by crossing Prox1-flox (fl) mice, which expressed eGFP under the control of the Prox1 promoter upon Cre-mediated exon 2 deletion (Iwano et al., 2012), with Isl1Cre/+ mice. When Prox1 is knocked down in the Tek+ lineage, an initial failure in specification of LECs was confirmed at E14.5 with a lack of LECs even at E17.5 (Klotz et al., 2015; Lioux et al., 2020; Maruyama et al., 2019). Therefore, we compared lymphatic vessel phenotypes at E16.5, by which systemic lymphatics formation is normally completed (Srinivasan et al., 2007). To test the recombination efficiency of the Prox1 locus in Isl1+ LECs, we performed whole-mounted and section immunostaining with PECAM, eGFP, and Prox1 in Isl1Cre/+;Prox1fl/+ heterozygous and Isl1Cre/+;Prox1fl/fl homozygous mice at E12.5 and E13.5. At E12.5, eGFP+/Prox1+ cells were observed in the PA1 of Isl1Cre/+;Prox1fl/+ heterozygous mice, whereas the number of Prox1+ cells was decreased and most of eGFP+ cells were negative for Prox1 in Isl1Cre/+;Prox1fl/fl homozygous mice (Figure 5—figure supplement 1A–F), indicating efficient knockdown of Prox1. At E13.5, eGFP+/Prox1+ cells were almost diminished in the tongue, whereas eGFP+/Prox1+ cells were still observed in the lower jaw in Isl1Cre/+;Prox1fl/fl homozygous mice (Figure 5—figure supplement 1G–P). This discrepancy may indicate that the recombination efficiency differs among tissues and that embryos with low recombination efficiency could survive until E13.5. When lymphatic vessel formation in these regions was analyzed in mice that were heterozygous or homozygous for the Prox1fl allele, the number and area of VEGFR3+/PECAM+ lymphatic vessels were significantly lower in the tongues of the Isl1Cre/+;Prox1fl/fl homozygous mice than in those of the Isl1Cre/+;Prox1fl/+ heterozygous mice (Figure 5A–C, F–H, K and L). In contrast, the formation of facial lymphatic vessels in the lower jaw and cheeks was not significantly affected by the deletion of Prox1 in the Isl1+ lineage (Figure 5D–E’’’1–J’’’, O and P). There were no differences in the mean diameter of the tongue or facial lymphatic vessels between the Isl1Cre/+;Prox1fl/fl homozygous mice and Isl1Cre/+;Prox1fl/+ heterozygous mice (Figure 5A–J, M and Q). Next, we examined the contribution of the Isl1+ lineage to regional lymphatic vessel formation by assessing the relative area of eGFP+ cells among VEGFR3+ LECs to determine whether the regional differences in the effects of Isl1+-lineage-specific Prox1 deletion on lymphatic vessel formation were due to differences in compensation by other cell sources. Isl1Cre/+;Prox1fl/+ heterozygous mice showed that the Isl1+ lineage was almost totally responsible for the development of the tongue and facial skin lymphatic vessels (Figure 5A–E’’’, N and R). On the other hand, Isl1Cre/+;Prox1fl/fl homozygous mice showed a lower contribution of the Isl1+ lineage to the facial skin lymphatics (Figure 5F1–J’’’ and R), whereas its contribution to the lymphatic vessels in the tongue was not decreased (Figure 5F–H and N). These results suggested that defects in LEC differentiation and/or maintenance due to Prox1 deletion in the Isl1+ lineage were compensated for by LECs from other cell sources, probably of venous origin, in facial skin, but not in the tongue.

Figure 5 with 1 supplement see all
The inactivation of Prox1 in Isl1+ lineages confirmed the contribution of the cardiopharyngeal mesoderm (CPM) to cranial lymphatic vessel development.

(A–J”’) Coronal sections of Isl1Cre/+;Prox1fl/+ and Isl1Cre/+;Prox1fl/fl mouse embryos, in which platelet endothelial cell adhesion molecule (PECAM), eGFP, and vascular endothelial growth factor receptor 3 (VEGFR3) were labeled at embryonic day (E) 16.5, are shown. (J) The number of eYFP-/VEGFR3+ lymphatic vessels in facial skin was increased in the Isl1Cre/+; Prox1fl/fl homozygous mice (white arrows). (K–R) The results of quantitative analysis of lymphatic vessel formation in the tongue (K–N) and facial skin (O–R) are shown. (**p=0.0043 (K, L, R)). All the data are presented as the mean ± SEM, and statistical analyses were performed using the non-parametric Mann-Whitney U-test. Each dot represents a value obtained from one sample. **p<0.01; T, tongue. Scale bars, 100 μm (B–E, G–J), 1 mm (A, F).

Figure 5—source data 1

Quantification of lymphatic vessels phenotypes in Isl1Cre/+;Prox1fl/+ and Isl1Cre/+;Prox1fl/fl mouse embryos at embryonic day (E) 16.5.

https://cdn.elifesciences.org/articles/81515/elife-81515-fig5-data1-v2.xlsx

Loss of Prox1 in the Tek+ lineage confirms the heterogeneous origins of LECs

To further investigate the regional differences in the contributions of venous and non-venous cell sources to LECs, we crossed Prox1fl mice with endothelial/hematopoietic cell-specific Tek-Cre mice. Immunostaining revealed decreased Prox1 expression in Tek+ LECs in the back skin of Tek-Cre;Prox1fl/fl homozygous mice compared to Tek-Cre;Prox1fl/+ heterozygous mice at E16.5, indicating efficient knockdown of Prox1, whereas Tek LECs were observed similarly (Figure 6—figure supplement 1A, B). We also observed blood-filled lymphatic vessels in the back skin of the Tek-Cre;Prox1fl/fl homozygous mice, indicating the formation of abnormal anastomosis between lymphatic and blood vessels due to Prox1 deficiency, as previously described (Johnson et al., 2008; Figure 6—figure supplement 1A, B). We then immunostained sagittal sections of the resultant embryos for PECAM, green fluorescent protein (GFP), and the LEC marker LYVE1 at E16.5, when lymphatic networks are distributed throughout the whole body (Srinivasan et al., 2007). We identified LECs by their luminal structure and the colocalization of PECAM and LYVE1, the latter of which is also known to be expressed in a subset of macrophages.

We compared lymphatic vessel development in the tongue, the skin of the lower jaw, and back skin between mice that were heterozygous and homozygous for the Prox1fl allele to assess the morphological changes in LYVE1+/PECAM+ lymphatic vessels seen in each tissue. In the tongue, the number of LYVE1+/PECAM+ lymphatic vessels was significantly lower and the mean lymphatic vessel diameter was significantly higher in the Tek-Cre;Prox1fl/fl homozygous mice than in the Tek-Cre;Prox1fl/+ heterozygous mice (Figure 6A and B and Figure 6—figure supplement 2A–C). Almost all of the LYVE1+/PECAM+ lymphatic vessels in the tongue were positive for eGFP in the Tek-Cre;Prox1fl/+ heterozygous mice (Figure 6A and Figure 6—figure supplement 2D), indicating that the majority of LECs derived from Isl1+ CPM cells developed through Tek expression in the tongue. Remarkably, in the tongues of the Tek-Cre;Prox1fl/fl homozygous mice, many of the eGFP+ cells were not incorporated into the LYVE1+/PECAM+ lymphatics, resulting in an increased eGFP+ area to lymphatics area ratio (Figure 6A and B and Figure 6—figure supplement 2D), which was indicative of impaired differentiation of venous cells into LECs or impaired maintenance of LEC identities due to a lack of Prox1 expression in the Tek+ endothelial cells.

Figure 6 with 2 supplements see all
Prox1 knockdown in Tek+ lineages revealed regional differences in lymphatic vessel development.

(A–F) Sagittal sections of Tek-Cre;Prox1fl/+ or Tek-Cre;Prox1fl/fl mouse embryos, in which platelet endothelial cell adhesion molecule (PECAM), eGFP, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), and DAPI were labeled at embryonic day (E) 16.5, are shown. (A, B) In the tongue, the number of LYVE1-/eGFP+/PECAM+ cells in lymphatic vessels was increased in the Tek-Cre;Prox1fl/fl embryos (white arrows). (C, D) In the skin of the lower jaw, the contribution of eGFP+ cells to lymphatic vessels was small in both the Tek-Cre; Prox1fl/+ and Tek-Cre;Prox1fl/fl embryos (white arrows). (E, F) LYVE1-/eGFP+/PECAM+ cells were observed in the back skin of the Tek-Cre;Prox1fl/fl embryos (white arrows). (G) Schematic representation of lineage classification in venous-derived LECs and Isl1+ CPM-derived LECs. The LECs in the tongue are derived from the Isl1+/Tek+ lineage, whereas the LECs in facial skin, including the skin on the lower jaw, are derived from the Isl1+/Tek+ and Isl1+/Tek lineages. Scale bars, 100 μm (A–F).

On the contrary, compared with that seen in the Tek-Cre;Prox1fl/+ heterozygotes lymphatic vessel formation in the lower jaw and back skin was not significantly affected in the Tek-Cre;Prox1fl/fl homozygotes, although the mean vessel diameter in the back skin of the homozygotes was increased (Figure 6C–F and Figure 6—figure supplement 2E–G,I–K). In contrast to the tongue, the eGFP+ area to LYVE1+/PECAM+ lymphatics area ratios in the lower jaw and back skin were relatively low in both the Tek-Cre;Prox1fl/+ heterozygotes and Tek-Cre;Prox1fl/fl homozygotes (Figure 6C–F and Figure 6—figure supplement 2H, L). These results demonstrate that the lymphatic vessels in these regions are composed of Tek+ and Tek LECs, supporting the idea that LECs have heterogeneous origins.

Taken together with the data from the experiments involving Isl1Cre/+ mice, these results suggest that the LECs in the craniofacial region have heterogeneous origins and developmental processes. Specifically, the LECs in the tongue are derived from the Isl1+/Tek+ lineage, whereas the LECs in facial skin, including the skin on the lower jaw, are derived from the Isl1+/Tek+ and Isl1+/Tek lineages, which may compensate for each other when LECs from one lineage are impaired. Similarly, the LECs in back skin are derived from the Tek+ and Tek lineages, which may compensate for each other, whereas the Isl1+ lineage is not involved in the development of these cells.

Discussion

In this study, we demonstrated that Isl1+ CPM cells, which are known to be progenitors of the cranial and cardiac musculature and connective tissue, contribute to the formation of lymphatic vessels in the cardiopharyngeal region, including the tongue, facial skin, larynx, and cardiac outflow tracts. Tamoxifen-inducible genetic lineage tracing further indicated that Isl1+ CPM-derived progenitors only have the potential to differentiate into LECs before E9.5. In accordance with these findings, Isl1+ CPM-derived LECs showed distinct spatiotemporal developmental processes and subsequently coordinated with Isl1 venous-derived LECs arising from the lymph sac-forming domain to form the systemic lymphatic vasculature. In addition, conditional KO of Prox1 in the Isl1+ lineage resulted in lymphatic vessel deficiencies in the tongue. In contrast, in the facial skin, the loss of Isl1+ LECs was compensated for by increased numbers of Isl1 LECs. Accordingly, conditional KO of Prox1 in Tek+ cells produced regional lymphatic phenotypic differences in the tongue, the skin of the lower jaw, and back skin. These results indicate that the LECs in the cardiopharyngeal region are mainly derived from Isl1+ CPM progenitors (Figure 7).

Lymphatic endothelial cells (LECs) are derived from two distinct origins.

Schematic representation of the origins of LECs. LECs are mainly derived from cardinal veins (red dots) and the cardiopharyngeal mesoderm (CPM) (green dots).

The present study further indicates that the LECs in the tongue are derived from Tek-expressing cells among the Isl1+ lineage. Although it is unclear whether Isl1+-derived cells at the Tek-expressing stage represent a venous endothelial identity, this result means that Tek+ LECs are not equivalent to cardinal vein-derived LECs (Figure 6G). Furthermore, Tek-Cre;Prox1fl/fl homozygotes exhibited greater numbers of eGFP+/LYVE1/PECAM+ cells than Tek-Cre;Prox1fl/+ heterozygotes (Figures 5A–C,F–H6A). As a previous study found that conditional KO of Prox1 could reprogram LECs to become blood endothelial cells (BECs) (Johnson et al., 2008), the number of BECs (eGFP+/LYVE1/PECAM+ cells) may be increased in the tongues of Tek-Cre;Prox1fl/fl homozygous mice due to impaired maintenance of the LEC identity of Isl1+ CPM-derived cells that would have normally expressed Prox1. In contrast, the Isl1+ CPM-derived LECs in facial skin were not labeled by Tek-Cre, indicating that there is heterogeneity within Isl1+ CPM-derived LECs. Although further studies are needed to examine the phenotypic differences between the LECs in the tongue and facial skin, it is possible that differentiation processes may be affected by the extracellular environment, for example, through interactions with the surrounding cells and extracellular matrix.

A recent study has suggested that Pax3+ paraxial mesoderm-derived cells contribute to the cardinal vein and therefore venous-derived LECs originate from the Pax3+ lineage (Stone and Stainier, 2019). The same group has further argued that the Pax3+ lineage gives rise to lymphatic vessels on the trunk side through lymphvasculogenesis (Lupu et al., 2022). Therefore, the Isl1+ and Pax3+ lineages may complement each other to form systemic lymphatic vessels. In experiments involving Myf5Cre/+ mice, it was also suggested that the Myf5+ lineage contributes to LECs in embryonic lymph sacs (Stone and Stainier, 2019); however, we could not identify Myf5+ LECs in Myf5CreERT2/+ mice after tamoxifen treatment at E8.5 (Figure 1—figure supplement 2). This discrepancy may have been caused by differences in the mouse lines, the timing of Cre recombination, the genetic background, or the breeding environment.

Several studies have confirmed that Isl1+ LECs contribute to the ventral side of the heart (Lioux et al., 2020; Maruyama et al., 2019). Milgrom-Hoffman et al. identified Flk1+/Isl1+ endothelial populations in the second pharyngeal arch in mouse embryos from E7.5 to E9.5 (Milgrom-Hoffman et al., 2011). These cell populations may serve as progenitors for LECs and BECs in the cardiopharyngeal region through the sequential expression of Flk1, Tek, and/or Prox1 in response to different cues, leading to diverse fate determination, as revealed by recent single-cell RNA-sequence analyses (Nomaru et al., 2021; Wang et al., 2019).

Heart development and pharyngeal muscle development are known to be tightly linked, suggesting that these tissues share common evolutionary origins (Diogo et al., 2015; Tzahor and Evans, 2011). As the CPM is conserved in various species, CPM-derived LECs may also be evolutionarily conserved. In agreement with this, several studies involving zebrafish have indicated that facial and cardiac LECs originate from distinct cell sources from venous-derived LECs (Eng et al., 2019; Gancz et al., 2019).

LMs are congenital lesions, in which enlarged and/or irregular lymphatic connections do not function properly. The causes of LMs are unknown, but LMs commonly occur in the head and neck regions (Perkins et al., 2010). Accumulating evidence has shown that mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene are found more frequently in LECs than in fibroblasts in LM patients (Blesinger et al., 2018). Given the fact that CPM-derived LECs were distributed around the head and neck and were found in the lymph sac-forming domain in the cervical region in the present study (Figures 14), LMs may be caused by PIK3CA mutations in CPM-derived LECs. In addition to LMs, several blood vessel malformations frequently occur in the neck and facial regions. Thus, some types of blood vessel malformations may also be caused by mutations in CPM progenitors.

In summary, the present study supports the idea that Isl1+ CPM cells are progenitors of LECs, which broadly contribute to cranial, neck, and cardiac lymphatic vessels in concert with venous-derived LECs (Figure 7). These findings are expected to shed new light on the cellular origins of lymphatic vessels and the molecular mechanisms of lymphatic vessel development, which may increase our understanding of the evolution of lymphatic vessels and the pathogenesis of lymphatic system-related diseases.

Materials and methods

Mouse strains

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The following mouse strains were used: Isl1Cre/+ (Cai et al., 2003) (Cat# JAX:024242, RRID:IMSR_JAX:024242), Isl1CreERT2/+ (Isl1MerCreMer/+ is described as Isl1CreERT2/+) (Laugwitz et al., 2005) (Cat# JAX:029566, RRID:IMSR_JAX:029566), Wnt1-Cre (Jiang et al., 2000) (Cat# JAX:007807, RRID:IMSR_JAX:007807), Myf5CreERT2/+ (Biressi et al., 2013) (Cat# JAX:023342, RRID:IMSR_JAX: 023342), Rosa26eYFP/+ (Srinivas et al., 2001) (Cat# JAX:006148, RRID:IMSR_JAX:006148), Rosa26tdTomato/+ (Madisen et al., 2010) (Cat# JAX:007914, RRID:IMSR_JAX:007914), Tek-Cre (Kisanuki et al., 2001) (Cat# JAX:008863, RRID: IMSR_JAX:008863), and Prox1fl/+(Iwano et al., 2012). All mice were maintained on a mixed genetic background (C57BL/6J × Crl:CD1(ICR)), and both sexes were used (the mice were randomly selected). The genotypes of the mice were determined via the polymerase chain reaction using tail-tip or amnion DNA and the primers listed in Supplementary file 1. The mice were housed in an environmentally controlled room at 23 ± 2°C, with a relative humidity level of 50–60%, under a 12 hr light:12 hr dark cycle. Embryonic stages were determined by timed mating, with the day of the appearance of a vaginal plug being designated E0.5. All animal experiments were approved by the University of Tokyo (ethical approval number: H17-250) and Mie University (ethical approval number: 728) animal care and use committee, and were performed in accordance with institutional guidelines.

Immunohistochemistry, histology, confocal imaging, and quantification

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For the histological analyses, hearts and embryos were collected, fixed in 4% paraformaldehyde for 1 hr at 4°C, and stored in phosphate-buffered saline or embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan). Sixteen-μm-thick frozen sections were stained with hematoxylin (Merck) and eosin (Kanto Chemical, Tokyo, Japan). Immunostaining of 16-μm-thick frozen sections was performed using primary antibodies against CD31 (553370, BD Pharmingen, RRID: AB_394816, 1:100), Flk1 (555307, BD Pharmingen, RRID:AB_395720, 1:100), Isl1 (AF1837, R&D systems, RRID:AB_2126324, 1:250), Prox1 (11-002, AngioBio, RRID: AB_10013720, 1:200; AF2727, R&D Systems, RRID: AB_2170716, 1:200), LYVE1 (11-034, AngioBio, 1:200; AF2125, R&D Systems, RPID: AB_2297188, 1:150), VEGFR3 (AF743, R&D Systems, RRID: AB_355563, 1:150), and GFP (GFP-RB-AF2020, FRL, RRID:AB_2491093, 1:500). Alexa Fluor-conjugated secondary antibodies (Abcam, RRID:AB_2636877, RRID:AB_2636997, RRID:AB_2752244, 1:400) were subsequently applied. The same protocol was followed for whole-mounted hearts and embryos, with the primary and secondary antibody incubation periods extended to two nights. Immunofluorescence imaging was conducted using a Nikon C2 confocal microscope or Keyence BZ-700. All images were processed using the ImageJ and Nikon NIS Elements software.

Tamoxifen injection

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For the lineage tracing using the Isl1CreERT2/+ or Myf5CreERT2/+ lines, tamoxifen (20 mg/mL; Sigma) was dissolved in corn oil. Pregnant mice were injected intraperitoneally with 125 mg/kg body weight of tamoxifen at the indicated timepoints.

Statistical analysis

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Data are presented as the mean ± standard error of the mean (SEM). Mann-Whitney U-tests were used for comparisons between two groups. p-Values of <0.05 were considered statistically significant. Data were analyzed using GraphPad Prism version 9 (GraphPad Software).

Quantification of the section and whole-mount images

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For the quantification of section immunostaining at E16.5 embryos, the average of two 16-μm-thick sections taken every 50 μm and ×10 power field of views (0.42 mm2/field) for each anatomical part (the larynx, the skin of the lower jaw, the tongue, and the cardiac outflow tracts) (Figure 1H, Figure 2H and P, and Figure 2—figure supplement 1I) or one middle section containing the same anatomical part (Figure 6—figure supplement 2) were subjected to the analyses. In the facial skin, lymphatic vessels in superficial layers of dermis were subjected to the analyses. The middle sagittal sections, including the aorta, larynx, and tongue, which were selected as hallmarks of midline, was chosen from created sections. The coronal sections, including both eyes, tongue, and olfactory lobes with left and right symmetrical features, were selected and subjected to the analyses (Figure 5K and R). For E12.5 embryos (Figure 4O), two 16-μm-thick sagittal sections taken every 50 μm, including the first and second pharyngeal arches and outflow tracts, were subjected to analyses. The area and the number of cells were measured manually using ImageJ software. For the whole-mount immunostaining of embryos and the heart, the whole samples were scanned every 20 μm and confirmed eYFP contribution to LECs (Figure 3) and cardinal veins (Figure 4J, and Figure 3—figure supplement 1B, D, F, H, J).

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figure 1—source data 1, Figure 2—figure supplement 1—source data 1, Figure 2—source data 1 and 2, Figure 4—source data 1, Figure 5—figure supplement 1—source data 1 and 2, Figure 5—source data 1, and Figure 6—figure supplement 2—source data 1.

References

Decision letter

  1. Oliver A Stone
    Reviewing Editor; University of Oxford, United Kingdom
  2. Didier YR Stainier
    Senior Editor; Max Planck Institute for Heart and Lung Research, Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

[Editors' note: this paper was reviewed by Review Commons.]

Thank you for submitting your article "The cardiopharyngeal mesoderm contributes to lymphatic vessel development" for consideration by eLife. Your article has been reviewed by 3 peer reviewers at Review Commons, and the evaluation at eLife has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor.

Based on the previous reviews and the revisions, the manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

– The data presented in Figure 5 should be interpreted carefully as residual VEGFR3+ GFP+ cells likely indicate incomplete recombination of the Prox1 locus (probably just one allele) by the Isl1-Cre line, as a lack of PROX1 expression during differentiation should lead to an absence of LECs. Thus, it's likely that recombination of Prox1 is less efficient in the progenitors of LECs found in the lower jaw and cheeks than in the tongue. The expression of PROX1 protein should be assessed in these samples to understand the level of knockout achieved.

– Similarly, GFP expression in LYVE1 positive lymphatic vessels in Tie2-Cre;Prox1 fl/fl animals again suggests incomplete recombination of Prox1. The identity of the GFP+ cells that are not incorporated into lymphatic vessels should be clarified by imaging at higher magnification as currently, the data are not clear. The analyses in Figure 6 should be repeated with a more specific marker of lymphatic endothelial cells than LYVE1. Either PROX1 or VEGFR3. The PECAM1 staining in the sections presented in Figure 6A-B should be improved.

– It is likely that Myf5 is expressed transiently and at low levels in any mesodermal progenitor that gives rise to the endothelium. The Myf5-CreERT2 mouse line used in this study was constructed using an IRES-CreERT2 cassette in the 3'UTR of the Myf5 gene, meaning that expression of CreERT2 from this locus is likely significantly lower than in the Myf5-Cre line previously used to investigate LEC origins (Stone and Stainier, Dev Cell, 2019). Thus, the conclusions that can be drawn from the experiment presented in Supplementary figure 2 are limited. We recommend deleting the second sentence of the discussion 'We also showed that Myf5+ myogenic lineages, which were previously suggested to be possible sources of LECs35, did not contribute to lymphatic vasculature formation in Myf5-CreERT2 mice subjected to tamoxifen treatment at E8.5' and leaving the discussion of these analyses presented on lines 288-292, which more accurately place these data in the context of published work.

– Reviewer #3 at Review Commons suggested using a Pax3-Cre to assess the contribution of this lineage to facial lymphatics. These analyses have been published and so it would be sufficient to reference Stone and Stainier, Dev Cell, 2019.

– The following statement "The same group has further argued that the Pax3+ lineage gives rise to lymphatic vessels on the trunk side through lymphangiogenesis (Lupu et al., 2022)" should read "The same group has further argued that the Pax3+ lineage gives rise to lymphatic vessels on the trunk side through lymphvasculogenesis (Lupu et al., 2022)"

https://doi.org/10.7554/eLife.81515.sa1

Author response

1. General Statements

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Please see combined review below in the next section,

Reviewer #1 (Significance (Required)):

This is a descriptive manuscript providing a few new insights into a well-recognized and biologically important phenomenon – the lymphatic endothelial cells have heterogeneous origins in different organs. Overall, the idea of Islet1 lineage contributes to regional lymphatic vessel formation during a particular developmental stage is exciting and proven with detailed and careful lineage tracing. The first observation that Islet1 lineage gives rise to cardiac lymphatic vessels was published by the same group in Dev Bio in 2019 so the novelty here is dampened, although the pharyngeal lymphatics and the exact time of these non-venous origin lymphatic vessels arise were not previously characterized – so the current manuscript does provide new important insights. Both the data quality and manuscript layout need improvements, especially when it comes to defining where Islet1 is expressed at all the stages and statistics.

The following suggestions will deepen the scope of the manuscript:

First of all, we would like to express our appreciation to the reviewer for all the constructive comments. We carefully read the reviewer’s comments and discussed it. We agree with the reviewer that our manuscript needs improvements with changes in layout several additional experiments. We have also included several description and new immunostaining data (e.g., Isl1,VEGFR3 and LYVE1 co-staining), to confirm our new findings and highlight the importance of the current manuscript beyond our previous one in Dev Biol in 2019. We also have included detailed quantification methods, single-channel images with improved data resolution, and improved clarity of the manuscript.

2. Description of the planned revisions

Point 4.

The effect of lineage-specific Prox1 knockout is very descriptive, without any discussion of the potential biological function of such cellular origin heterogeneity. This part may be worth a few follow-up experiments in later embryonic stages or even in postnatal stages. The authors demonstrated that loss of Prox1 in Islet1 lineage decreases the number of lymphatic vessels and leads to lymphangiectasia, but whether this phenotype can be later compensated or shows any clinical impact was not proven. Therefore, the statement made in line 206 is questionable, and whether Islet1 lineage-derived lymphatic endothelial cells are dispensable/indispensable remains unclear.

We agree with the reviewer in that additional follow up experiments using later embryonic or postnatal stages will give an insight into the potential biological function of cellular origin heterogeneity. We are generating lineage-specific Prox1 knockout mice by treating Isl1MerCreMer; Prox1fl/fl mice with tamoxifen at E8.5 to analyze phenotypes in the facial lymphatic vessels.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Point 1 and 2

1.Whether Isl1 lineage is independent of venous-derived endothelial cells.

2.This point is very important: the manuscript does not actually show Isl1 expression through the stages they are inducing. I would want to be sure that lymphatic endothelial cells at this stage don't express Isl1. Another way to get at this is to maybe use other second-heart fields or even broader mesoderm drivers that are known to be never expressed in endothelial cells to confirm the findings.

In our previous work (Maruyama et al., Dev Biol 452:134–143, 2019, Figure 2C), we demonstrated that Isl1+ lineages using Isl1-Cre mice did not contribute to endothelial cells in the cardinal vein and its branches (intersomitic vessels: ISVs), which had been thought to the primary and biggest sources of lymphatic endothelial cells (LECs). In this paper, we confirmed this finding using Isl1MerCreMer mice with tamoxifen treatment at E8.5 (Figure 4J). We have scanned whole embryos and detected no eYFP+ cells in the cardinal vein or ISVs (the detailed quantification methods have been added in Methods section). Consistently, another group (Lioux et al., Dev Cell 52:350-363, 2020) re-evaluated this point using Isl1-Cre mice that the Isl1+ lineage contribute to endothelial cells of the cardinal vein only by less than 2%, which neither explains the abundant contribution of the Isl1+ lineage to coronary lymphatics (>50%) nor its restriction to the ventral heart. Based on these reports, we supposed that the Isl1+ lineage was independent of LECs derived from the cardinal vein and ISVs.

In the revised manuscript, we added new data showing thorough expression patterns of Isl1, Prox1, Flk1, and PECAM in the E9.0 to E11.5 pharyngeal arches and cardinal veins by immunostaining and presented them as Supplemental Figure 4. In these sections, we detected Isl1 and Prox1 expression with partial overlapping within the pharyngeal mesodermal core, whereas Isl1 was co-expressed with Flk1, or PECAM neither in vessel-like structures around the mesodermal core nor in the cardinal vein and their surrounding Prox1+/PECAM+ LECs (Supplemental Figure 4H’ and J’) confirming the independency. These findings have been described in the manuscript as follows:

“To identify possible Isl1+ LEC progenitors, we investigated the expression patterns of Isl1, Prox1, and vascular endothelial markers (Flk1 and PECAM) by immunostaining sections of E9.0 to E11.5 pharyngeal arches and cardinal veins. Consistent with the previous report (Cai et al., 2003), Isl1 was abundantly expressed in the core mesoderm of the first and second pharyngeal arches corresponding to the CPM from E9.0 to E11.5 (Nathan et al., 2008), where Prox1+ cells also aggregated and partially overlapped with Isl1 signals (Supplemental Figure 4A, A’ C, C’ E, E’ G, G’ I, I’). By contrast, Flk1+ or PECAM+ cells were distributed mainly around the CPM and not expressed Isl1 (Supplemental Figure 4A, A’ C, C’ E, E’ G, G’ I, I’). Furthermore, Isl1 was expressed neither in the endothelial layer of the cardinal vein nor in surrounding Prox1+/PECAM+ LECs (Supplemental Figure 4B, B’ D, D’, F, F’, H, H’, J, and J’). Taken together with the result from Myf5-CreERT2 mice, these results indicate that Isl1+ non-myogenic CPM cells may serve as LEC progenitors independent of venous-derived LECs and the commitment to LEC differentiation occurs before E9.5 in the pharyngeal arch region.” (Page 6-7, lines 187-200)

Point 3.

The author stated that Islet1 lineage gives rise to lymphatic endothelial cells via the Tie2 mechanism but did not elaborate on this part. What is the potential relationship between Islet1 and Tie2? Or Tie2 just serves as a pan-endothelial lineage marker here?

To clearly demonstrate the relationship between Isl1+ and Tie2+ lineages in facial lymphatics, we added schematic representation in Figure 6G, which showed the differential Tie2 expression in lymphatic vessels in the tongue and facial skin.

Related to point 1 and 2, it has been thought that almost all LECs are formed from cardinal vein-derived Tie2+ endothelial cells. However, we identified the presence of Isl1+/Tie2+ LECs in the tongue, which are apparently not originated from the cardinal vein. In previous reports using Tie2-GFP mice or in situ hybridization of Tie2, Tie2 was not detected in the developing LECs at E9.5, 11.5, 13.5, and E15.5 (Motoike et al., 2000; Srinivasan et al., 2007). In adult mice, Tie2 expression in lymphatics was only observed in restricted regions (Morisada et al., 2005; Tammela et al., 2005). Taken together with our present data that the differentiation fate of Isl1+ CPM-derived LECs was determined between E6.5 and E9.5 (Figure 2-4, Supplemental Figure 3), Tie2 is supposed to be transiently expressed during LEC differentiation in the tongue from early Isl1+ CPM cells, although it remains difficult to identify the Tie2-expressing stage during non-venous LEC differentiation.

It will be an important future subject to identify the stage and implication of transient Tie2 expression in the lineage and, in this paper, we want to just note that the Tie2+ lineage does not always mean the derivation from cardinal vein endothelial cells.

This point has already been included in the manuscript as follows:

“The present study further indicates that the LECs in the tongue are derived from Tie2-expressing cells among the Isl1+ lineage. Although it is unclear whether Isl1+-derived cells at the Tie2expressing stage represent a venous endothelial identity, this result means that Tie2+ LECs are not equivalent to cardinal vein-derived LECs.” (Page 10, 298-301)

Minor Point 1.

The layout of the manuscript needs to be reorganized:

Details in statistical methods and quantification logic were completely missing from the manuscript. For example, definitions of "a sample" (how many sections are taken from one biological sample and how many fields take from one section, etc.), "number of vessels per field", "diameters", and of what parameters the numbers were normalized to, etc. need to be described in the Materials and methods section. For instance, it is not clear how "tomato+ lymphatic vessels per field/Vegfr3+ lymphatic vessels" was defined. First, what proportion of tomato+ cells need to colocalize with Vegfr3 expression cells in a specific vessel to make this vessel being determined as a "tomato+ lymphatic vessel"? Most data provided here are section immunostaining where "multiple vessels" are very likely coming from different cross-sections of one same vessel in the same field. Second, Vegfr3 can stain venous endothelial cells in earlier stages so the specificity of this marker can be controversial. These are some important technical aspects to include in the revised version. Figures needing more description in quantification methods include but are not limited to Figure 1H, 2H, 2P, 5K-R.

We have revised the statistical methods from the ratio of the count of the number to ratio of the area of lymphatic vessels in Figure 1H, 2H, P, and Supplemental Figure 3I to represent more precisely the contribution of Tomato+ cells to lymphatic vessels. We also added more detailed description of the quantification methods in ‘Materials and methods’ section, as follows:

“Quantification of the section and whole mount images

For the quantification of section immunostaining at E16.5 embryos, the average of two 16-μmthick sections taken every 50 μm and 10x power field of views (0.42 mm2/field) for each anatomical part (the larynx, the skin of the lower jaw, the tongue, and the cardiac outflow tracts) were subjected to the analyses. In the facial skin, lymphatic vessels in superficial layers of dermis were subjected to the analyses. The middle sagittal sections, including the aorta, larynx, and tongue, which were selected as hall marks of midline, was chosen from created sections. The coronal sections, including both eyes, tongue, and olfactory lobes with left and right symmetrical features, was selected. For E12.5 embryos (Figure 4O), two 16-μm-thick sagittal sections taken every 50 μm, including the 1st and 2nd pharyngeal arches and outflow tracts, were subjected to analyses. The area and the number of cells were measured manually using ImageJ software. For the whole mount immunostaining of embryos and the heart, the whole samples were scanned every 20 μm and confirmed eYFP contribution to LECs (Figure 3) and cardinal veins (Figure 4J, and Supplemental Figure 4B, D, F, H, J).” (Page 13, lines 402-416)

We also have tested expression patterns of VEGFR3 with Prox1 or LYVE1 as Supplemental Figure 1. At E14.5, VEGFR3 was widely co-expressed with Prox1 in the tongue, facial skin, and around the pulmonary artery (Supplemental Figure 1A-C’). At E16.5, VEGFR3 was co-expressed with LYVE1 in the tongue, facial skin, and around the pulmonary artery. Thus, we thought that VEGFR3 could be used as a marker of LECs in these cardiopharyngeal region.

This point has been included in the manuscript as follows:

“Co-immunostaining of platelet endothelial cell adhesion molecule (PECAM) and vascular endothelial growth factor receptor 3 (VEGFR3), which we confirmed its co-localization with lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) at E14.5 and E16.5 (Supplemental Figure 1), revealed tdTomato+ LECs in and around the larynx, the skin of the lower jaw, the tongue, and the cardiac outflow tracts, at various frequencies, whereas no such cells were found on the dorsal side of the ventricles, which agrees with our previous study (Maruyama et al., 2019).” (Page 4, lines 112-119)

Minor point 2.

Data resolution needs to be improved. The magnification of the figures in Figure 1-4 is not sufficient to demonstrate the marker colocalization as described in the texts. Single-channel images (such as the ones shown in Figure 5-6 but in higher magnifications) are also necessary to show the coexpression of markers.

There was a limit on the data capacity when submitting the manuscript. We were therefore obliged to reduce the quality of images and file size. We have revised the figures to add several higher magnification and single-channel images with improved data resolution throughout Figure 1-4.

Minor point 3.

The experimental design is not well-elaborated in the context. For example, the scientific logic of choosing a particular time point/stage for lineage-knockout induction or sample collection needs to be justified. Also, it seems that the authors are using fl/+ as control littermates in most of the experiments. Any specific reason favors using fl/+ heterozygous instead of fl/fl littermates without cre exposure, which is the more commonly used control sample in this kind of comparison, should be addressed.

Knockdown of Prox1 in the Tie2+ lineage has shown to cause an initial failure in specification of LECs at E14.5 with no appearance of lymphatics even at E17.5(Klotz et al., 2015; Lioux et al., 2020; Maruyama et al., 2019), indicating that the effect on lymphatic vessels would not be compensated even at E16.5. In addition, the systemic lymphatic network formation is almost completed at E16.5(Srinivasan et al., 2007), and the lineage trace was also evaluated at this stage. Thus, it was reasonable to compare the phenotype at E16.5.

This point has been addressed in the text as follows:

“When Prox1 is knocked down in the Tie2+ lineage, an initial failure in specification of LECs was confirmed at E14.5 with a lack of LECs even at E17.5(Klotz et al., 2015; Lioux et al., 2020; Maruyama et al., 2019). Therefore, we compared lymphatic vessel phenotypes at E16.5, by which systemic lymphatics formation is normally completed (Srinivasan et al., 2007).” (Page 7, lines 208212)

In Prox1-flox(Prox1fl/+) mice, recombinant cells were labeled with EGFP(Iwano et al., 2012), as already described in the manuscript (Page 7, lines206-208). Therefore, the recombined cells can be visualized by EGFP expression in both heterozygous (fl/+) and homozygous (fl/fl) mice, which enables phenotype analysis referring to the recombined (knocked-out in fl/fl) cells. Importantly, these mice showed no specific phenotypes (Klotz et al., 2015; Maruyama et al., 2019). It is therefore reasonable to use heterozygous mice as controls to compare the phenotype appropriately. Although fl/fl littermates without cre exposure could usually serve as controls, they do not express EGFP in the Prox1 lineage, detracting from their utility (Klotz et al., 2015; Maruyama et al., 2019).

Minor point 4.

Some of the phrases are not clear in the text- either because of the writing style or because the corresponding figures failed to support the statements. These include but are not limited to lines 104-106, 122, 206, 226, and 228-233.

104-106: we crossed Isl1-Cre mice, which express Cre recombinase under the control of the Isl1 promoter and in which second heart field-derivatives are effectively labeled, with the transgenic reporter line R26R-tdTomato at E16.5.

We have re-phrased this sentence as follows:

“We crossed Isl1-Cre mice, which express Cre recombinase under the control of the Isl1 promoter and in which second heart field-derivatives are effectively labeled, with the transgenic reporter line R26R-tdTomato and analyzed at E16.5, when lymphatic networks are distributed throughout the whole body.” (Pages 4, lines 109-112)

122: After tamoxifen was administered at E8.5, tdTomato+ cells were broadly detected in the muscle in the head and neck regions at E16.5, indicating effective Cre-mediated recombination of the target gene.

We have re-phrased this sentence as follows:

“After tamoxifen was administered at E8.5, tdTomato+ cells were broadly detected in the skeletal muscle in the head and neck regions at E16.5, indicating effective Cre recombination in CPMderived musculatures.” (Page 4, lines 130-132)

We have also included red arrowheads, indicating CPM-derived musculatures in Supplemental Figure 2.

206: These results suggested that defects in LEC differentiation and/or maintenance due to Prox1 deletion in the Isl1+ lineage were compensated for by other cell sources, probably of venous origin, in facial skin, but not in the tongue, resulting in impaired lymphatic vessel formation in the tongue.

We have re-phrased this sentence as follows:

“These results suggested that defects in LEC differentiation and/or maintenance due to Prox1 deletion in the Isl1+ lineage were compensated for by LECs from other cell sources, probably of venous origin, in facial skin, but not in the tongue.” (Page 7-8, lines 231-233)

226: Almost all of the LYVE1+/PECAM+ lymphatic vessels in the tongue were positive for eGFP in the Tie2-Cre;Prox1fl/+ heterozygous mice (Figure 6A and Supplemental Figure 3D), indicating that the majority of LECs derived from Isl1+ CPM cells developed through Tie2 expression in the tongue.

We have added new cartoon in Figure 6G to more clearly show the relation of Tie2 expression in Isl1+ lineages. Previous reports have used Tie2-Cre mice to show the vein-derived LECs (Klotz et al., 2015; Srinivasan et al., 2007), because most of cardinal vein endothelium were composed of Tie2+ lineages. In our present study, in the tongue, most of the LECs were derived from Isl1+/Tie2+ lineages (Figure 1D, H, Figure 2D, Figure 4G, Figure 5B, N, Figure 6A and Supplemental Figure 3F, I). These data suggested that there was a group of Tie2+ lineages even though they are derived from non-venous Isl1+ lineages.

Minor point 5.

Reference needed for Myf5-Cre as a driver for Myogenic CPM in the Results section.

We have included several reference, as shown below:

(Harel et al., 2012, 2009; Heude et al., 2018)

Harel et al., Dev cell, 2009

Minor point 6.

In discussion the reference to Pitx2-driven mesenteric lymphatic heterogeneity (Mahadevan et al. 2014) is missing yet Islet1 has been shown downstream of Pitx2 (Davis et al. 2008). The authors should discuss their findings of gut lymphatic heterogeneity in this context, considering that mediastinum is mesentery-derived.

Isl1+ CPM-derived LECs have been distributed to the anterior mediastinum and their relationship to mesenteric lymphatic vessels, which continuous with the thoracic duct in the posterior mediastinum, is currently unclear. However, since this paper is valuable for understanding the heterogeneity of the origins of LECs, we have included the indicated paper (Mahadevan et al., 2014) in ‘Introduction’ section to show gut lymphatic heterogeneity. (Page 3, line 67)

4. Description of analyses that authors prefer not to carry out

Related to point 4.

Regarding the use of other second-heart field drivers as the reviewer recommended,

Lioux et al., have already shown the contribution of Mef2c-AHF+, which marked CPM-derived cells including second heart field, cranial musculatures and connective tissues (Adachi et al., 2020), to ventral cardiac lymphatics. We are also trying to introduce Mef2c-AHF-Cre mice, but it is unfortunately delayed due to the pandemic of COVID-19.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The manuscript entitled “The cardiopharyngeal mesoderm contributes to lymphatic vessel development” identified a novel non-venous origin of craniofacial and cardiac LECs using genetic lineage tracing. Their results also revealed the spatiotemporal difference between CPM- and venous-derived LECs. Overall, the paper is well-organized and has certain implications for understanding lymphatic development. However, some issues still need to be improved:

Reviewer #2 (Significance (Required)):

This study enriched the contribution of CPMs to broader regions of the facial, cardiac and laryngeal lymphatic network and revealed the spatiotemporally difference between CPM- and venous-derived LECs, which provided some basic reference for understanding lymphatic vessel development.

First of all, we would like to express our appreciation to the reviewer for all the constructive comments. We carefully read the reviewer’s comments and discussed it. We agree with the reviewer’s comments to make the text easier to understand and emphasize what we really want to say.

2. Description of the revisions that have already been incorporated in the transferred manuscript

Point

1. Clearly, the introduction needs to be more concise and focused on the main questions you propose to answer and why these questions are important.

We have revised introduction section to be more concise and focus on the developmental process of lymphatic vessels and its relation to CPM. (Page 2-4, lines 41-103)

2. In the Discussion section, you should focus on how the questions have been answered and what they mean. And it would be rash to infer the role of LECs in lymphatic malformation. It would be helpful to validate the changes of CPM-derived LECs in LM patient samples.

We have revised the Discussion section to be more concise. To demonstrate our findings more clearly, we have also revised and added some cartoons in Figure 6G and Figure 7.

3. For the statistical analysis, all the quantitative data should be tested for statistical significance. There are several bar charts lacking P values.

We have included P values in the Figure legends.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Short summary of the findings and key conclusions:

The work from Murayama and colleagues traces the ontogenetic origin of the endothelial cells of the lymphatic vessels in the head and neck region. Using the Cre-lox-based mouse genetics approach, they conclude that the lymphatic endothelial cells (LECs) in this region have mixed origin, with contributions from both the cardiopharyngeal mesoderm (CPM) as well as from cardinal vein. The lineage tracing study is buttressed by assaying LEC formation following selective deletion of the key LEC regulator Prox1 in CPM lineage.

Reviewer #3 (Significance (Required)):

The nature and significance of the advance for the field & the work in the context of the existing literature:

Groups working in the domain of cardiopharyngeal mesoderm (CPM) have focussed on skeletal muscle and heart development. This pool is also known to give rise to skeletal tissues as well as blood vessel endothelium. A recent work Nomaru et al. (Morrow group, Nat Commun 2021) has identified a multi-lineage primed population in the cardiopharyngeal field. In this context, the work from Maruyama and colleagues highlights the versatility of CPM by providing evidence for the emergence of LEC from this multipotent pool. This complex developmental potential of CPM has implications to understand the evolutionary origin of CPM itself.

The connective tissues in the head/neck have mixed origins (Heude et al., 2018 and Grimaldi et al. 2022 from Tajbakhsh group)- from CPM as well as neural crest. This work shows mixed origin for LECs. These works begin to put together the pieces of the puzzle of vertebrate head evolution. Jacob proposed evolution is tinkering. This appears to be true both at the molecular level as well as the cellular level. Head tissues appear to have been put together by exploiting varied sources.

The study is of broad interest to developmental biologists.

Reviewer: A developmental biologist with an interest in understanding the axial patterning of mesoderm early during mammalian development. Not an expert in lymphatic vasculature development.

First of all, we would like to express our appreciation to the reviewer for all the constructive comments. We carefully read the reviewer’s comments and discussed it.

2. Description of the revisions that have already been incorporated in the transferred manuscript

In addition, the article should be revised to include the number of sections and the number of cells counted per embryo in the Figure legend in each case. This will help assess how robust and reliable are the measurements.

We have revised the statistical methods from the ratio of the count of the number to the area in Figure 1H, 2H, P, and Supplemental Figure 3I to demonstrate more precisely the contribution of Tomato+ cells in lymphatic vessels. We also added more detailed description of the quantification methods in ‘Materials and methods’ section, as follows:

Quantification of the section and whole mount images

“For the quantification of section immunostaining at E16.5 embryos, the average of two 16-μmthick sections taken every 50 μm and 10x power field of views (0.42 mm2) for each anatomical part (the larynx, the skin of the lower jaw, the tongue, and the cardiac outflow tracts) were subjected to the analyses. In the facial skin, lymphatic vessels in superficial layers of dermis were subjected to the analyses. The middle sagittal sections, including the aorta, larynx, and tongue, which were hall marks of midline, was selected from created sections. The coronal sections, including both eyes, tongue, and olfactory lobes with left and right symmetrical features, was selected. For E12.5 embryos (Figure 4O), two 16-μm-thick sagittal sections taken every 50 μm, including the 1st and 2nd pharyngeal arches and outflow tracts, were subjected to analyses. The area and the number of Prox1+ cells were measured manually using ImageJ software. For the whole mount immunostaining of embryos and the heart, the whole samples were scanned every 20 μm and confirmed eYFP contribution to LECs (Figure 3) and cardinal veins (Figure 4J, and Supplemental Figure 4B, D, F, H, J).” (Pages 13, lines 402-416)

We have also included the number of eYFP+/Prox1+ cells among Prox1+ cells in the first and second pharyngeal in the Figure 4O legends as follows;

“(the number of eYFP+/Prox1+ cells (10.83 (mean) ± 1.249 (SEM)): Prox1+ cells (30.83 ± 4.549)) or E9.5 (the number of eYFP+/Prox1+ cells (2.833 ± 1.108): Prox1+ cells (35.50 ± 5.847)).” (Page 23, lines 684-686)

Minor comments 1.

Several groups have contributed to the CPM literature. The citation of seminal works from Tzahor and Kelly groups is good, however, work from other groups has not been cited. For example, reports such as Heude et al. and Grimaldi et al. from Tajbakhsh group are very relevant to this work.

According to reviewer’s suggestion, we have included following references in the introduction section for the explanation of CPM derivatives. (P3, line 77)

(Heude et al., 2018)

Minor comments 2.

It would help the reader if the authors explain the reasons for selecting specific regions, such as the tongue, and the skin of the lower jaw, for the study.

This is because many lymphatic vessels are distributed in these cardiopharyngeal area and these area is well known as anatomical parts where lymphatic malformation most often occurs. This has been mentioned in the manuscript as follows:

From a clinical viewpoint, head and neck regions contributed by the CPM are the most common sites of lymphatic malformations (LMs) (Page 3-4, lines 99-100)

3. Description of analyses that authors prefer not to carry out

Point 1.

The key conclusions: LECs in the head and neck region derive from CPM. LECs in this region have mixed developmental origins. Both these conclusions are convincingly supported by the study. However, the work would greatly be strengthened by Pax3-Cre lineage tracing. This would complement the Isl1-Cre lineage tracing. As the authors observe, the LEC descendants of Isl1+ cells also appear to go through Tie2+ state. Therefore, Tie2-Cre study has not helped to delineate the LECs of CPM and cardinal vein origins. In this context, tracing with Pax3-Cre is likely to give a very clear picture of LEC origins.

We agree with the reviewer in that the data using Pax3-Cre mice will strengthen our manuscripts. Unfortunately, we could not find out researchers who had this line in our society in Japan. For using this line, we need to get cryo-recovered mice from Jaxon laboratory. It will take at least several months. Therefore it is not realistic for us to use Pax3-Cre mice in this work because of time limitation. Instead, we addressed this issue by rewriting the discussion on the possible complementation with the Pax3-Cre lineage by citing (Lupu et al., 2022; Stone and Stainier, 2019).

This point has been addressed in the text as follows:

“A recent study has suggested that Pax3+ paraxial mesoderm-derived cells contribute to the cardinal vein and therefore venous-derived LECs originate from the Pax3+ lineage (Stone and Stainier, 2019). The same group has further argued that the Pax3+ lineage gives rise to lymphatic vessels on the trunk side through lymphangiogenesis(Lupu et al., 2022). Therefore, the Isl1+ and Pax3+ lineages may complement each other to form systemic lymphatic vessels.” (Page 10, lines 314-319)

Minor comments 3.

The authors should consider presenting the wholemount images, such as those in Figures 3A and 3E for Figures 5 and 6. This would help assess the lymphatic vessel development in a holistic manner.

Although we tried to do the whole mount images of facial and tongue lymphatics, we could not succeed. Antibodies did not penetrate well on the tongue and, as for lymphatics of facial skin, their complicated morphology prevented clear visualization. Whole-mount imaging of the entire head was difficult for the same reason. In our experience, the antibody was useful for immunostaining of the early-stage embryos (up to E11.5) and the surface area of the heart, where lymphatic vessels were distributed on the epicardium. Even in the whole-mount heart, we have not succeeded in clear and estimable imaging of the vascular structure in the myocardium. Instead, we improved the quality of images and statistical comparisons in the revised manuscript, which we believe makes it more convincing.

References.

Adachi N, Bilio M, Baldini A, Kelly RG. 2020. Cardiopharyngeal mesoderm origins of musculoskeletal and connective tissues in the mammalian pharynx. Development

147:dev185256. doi:10.1242/dev.185256

Cai C-L, Liang X, Shi Y, Chu P-H, Pfaff SL, Chen J, Evans S. 2003. Isl1 Identifies a Cardiac

Progenitor Population that Proliferates Prior to Differentiation and Contributes a Majority of

Cells to the Heart. Dev Cell 5:877–889. doi:10.1016/s1534-5807(03)00363-0

Grimaldi A, Comai G, Mella S, Tajbakhsh S. 2022. Identification of bipotent progenitors that give rise to myogenic and connective tissues in mouse. ELife 11:e70235. doi:10.7554/eLife.70235

Harel I, Maezawa Y, Avraham R, Rinon A, Ma H-Y, Cross JW, Leviatan N, Hegesh J, Roy A, Jacob-Hirsch J, Rechavi G, Carvajal J, Tole S, Kioussi C, Quaggin S, Tzahor E. 2012.

Pharyngeal mesoderm regulatory network controls cardiac and head muscle morphogenesis. Proc National Acad Sci 109:18839–18844. doi:10.1073/pnas.1208690109

Harel I, Nathan E, Tirosh-Finkel L, Zigdon H, Guimarães-Camboa N, Evans SM, Tzahor E. 2009. Distinct Origins and Genetic Programs of Head Muscle Satellite Cells. Dev Cell

16:822–832. doi:10.1016/j.devcel.2009.05.007

Heude E, Tesarova M, Sefton EM, Jullian E, Adachi N, Grimaldi A, Zikmund T, Kaiser J, Kardon G, Kelly RG, Tajbakhsh S. 2018. Unique morphogenetic signatures define mammalian neck muscles and associated connective tissues. ELife 7:e40179. doi:10.7554/eLife.40179

Klotz L, Norman S, Vieira JM, Masters M, Rohling M, Dubé KN, Bollini S, Matsuzaki F, Carr CA, Riley PR. 2015. Cardiac lymphatics are heterogeneous in origin and respond to injury. Nature 522:62–67. doi:10.1038/nature14483

Lioux G, Liu X, Temiño S, Oxendine M, Ayala E, Ortega S, Kelly RG, Oliver G, Torres M.

2020. A Second Heart Field-Derived Vasculogenic Niche Contributes to Cardiac Lymphatics. Dev Cell 52:350–363. doi:10.1016/j.devcel.2019.12.006

Lupu I-E, Kirschnick N, Weischer S, Martinez-Corral I, Forrow A, Lahmann I, Riley PR, Zobel T, Makinen T, Kiefer F, Stone OA. 2022. Direct specification of lymphatic endothelium from non-venous angioblasts. Biorxiv 2022.05.11.491403. doi:10.1101/2022.05.11.491403 Mahadevan A, Welsh IC, Sivakumar A, Gludish DW, Shilvock AR, Noden DM, Huss D, Lansford R, Kurpios NA. 2014. The Left-Right Pitx2 Pathway Drives Organ-Specific Arterial and Lymphatic Development in the Intestine. Dev Cell 31:690–706. doi:10.1016/j.devcel.2014.11.002

Maruyama K, Miyagawa-Tomita S, Mizukami K, Matsuzaki F, Kurihara H. 2019. Isl1expressing non-venous cell lineage contributes to cardiac lymphatic vessel development. Dev Biol 452:134–143. doi:10.1016/j.ydbio.2019.05.002

Morisada T, Oike Y, Yamada Y, Urano T, Akao M, Kubota Y, Maekawa H, Kimura Y, Ohmura M, Miyamoto T, Nozawa S, Koh GY, Alitalo K, Suda T. 2005. Angiopoietin-1 promotes

LYVE-1-positive lymphatic vessel formation. Blood 105:4649–4656. doi:10.1182/blood2004-08-3382

Motoike T, Loughna S, Perens E, Roman BL, Liao W, Chau TC, Richardson CD, Kawate T, Kuno J, Weinstein BM, Stainier DYR, Sato TN. 2000. Universal GFP reporter for the study of vascular development. Genesis 28:75–81. doi:10.1002/1526-968x(200010)28:2<75::aidgene50>3.0.co;2-s

Nathan E, Monovich A, Tirosh-Finkel L, Harrelson Z, Rousso T, Rinon A, Harel I, Evans SM, Tzahor E. 2008. The contribution of Islet1-expressing splanchnic mesoderm cells to distinct branchiomeric muscles reveals significant heterogeneity in head muscle development. Development 135:647–57. doi:10.1242/dev.007989

Srinivasan RS, Dillard ME, Lagutin OV, Lin F-J, Tsai S, Tsai M-J, Samokhvalov IM, Oliver G. 2007. Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature. Gene Dev 21:2422–2432. doi:10.1101/gad.1588407

Stone OA, Stainier DYR. 2019. Paraxial Mesoderm Is the Major Source of Lymphatic

Endothelium. Dev Cell 50:247-255.e3. doi:10.1016/j.devcel.2019.04.034

Tammela T, Saaristo A, Lohela M, Morisada T, Tornberg J, Norrmén C, Oike Y, Pajusola K, Thurston G, Suda T, Yla-Herttuala S, Alitalo K. 2005. Angiopoietin-1 promotes lymphatic sprouting and hyperplasia. Blood 105:4642–4648. doi:10.1182/blood-2004-08-3327

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Based on the previous reviews and the revisions, the manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

– The data presented in Figure 5 should be interpreted carefully as residual VEGFR3+ GFP+ cells likely indicate incomplete recombination of the Prox1 locus (probably just one allele) by the Isl1-Cre line, as a lack of PROX1 expression during differentiation should lead to an absence of LECs. Thus, it's likely that recombination of Prox1 is less efficient in the progenitors of LECs found in the lower jaw and cheeks than in the tongue. The expression of PROX1 protein should be assessed in these samples to understand the level of knockout achieved.

To address recombination efficiency of the Prox1 locus and Prox1 expression levels in Isl1+ LECs, we have performed whole-mount and section immunostaining with PECAM, eGFP, and Prox1. In whole-mount immunostaining at E12.5, eGFP+/Prox1+ cells were observed in the PA1 of Isl1Cre/+;Prox1fl/+ heterozygous mice, whereas the number of Prox1+ cells were decreased in Isl1Cre/+;Prox1fl/fl homozygous mice (Figure 5 —figure supplement 1A-F). (The numbers of Prox1+ cells in the first pharyngeal arch were 71.5 (mean) ± 3.5 (SEM) and 7.0 ± 1.0 in Isl1Cre/+;Prox1fl/+ (n=2) and Isl1Cre/+;Prox1fl/fl (n=2) embryos, respectively). Thus, the recombination of the Prox1 locus occurred efficiently. At E13.5, eGFP+/Prox1+ cells were almost disappeared in the tongue, whereas eGFP+/Prox1+ cells were still observed in the lower jaw in Isl1Cre/+;Prox1fl/fl homozygous mice (Figure 5 —figure supplement 1G-P). Notably, the number of homozygous mice was reduced in comparison to that of heterozygous mice at E13.5 (11 heterozygous vs 3 homozygous), indicating that the homozygous genotype was partially lethal and embryos with incomplete recombination might survive.

This point is described as following:

“To test the recombination efficiency of the Prox1 locus in Isl1+ LECs, we performed whole-mounted and section immunostaining with PECAM, eGFP, and Prox1 in Isl1Cre/+;Prox1fl/+ heterozygous and Isl1Cre/+;Prox1fl/fl homozygous mice at E12.5 and E13.5. At E12.5, eGFP+/Prox1+ cells were observed in the PA1 of Isl1Cre/+;Prox1fl/+ heterozygous mice, whereas the number of Prox1+ cells was decreased and most of eGFP+ cells were negative for Prox1 in Isl1Cre/+;Prox1fl/fl homozygous mice (Figure 5 —figure supplement 1A-F), indicating efficient knockdown of Prox1. At E13.5, eGFP+/Prox1+ cells was almost diminished in the tongue, whereas eGFP+/Prox1+ cells were still observed in the lower jaw in Isl1Cre/+;Prox1fl/fl homozygous mice (Figure 5 —figure supplement 1G-P). This discrepancy may indicate that the recombination efficiency differs among tissues and that embryos with low recombination efficiency could survive until E13.5. (Page 7, lines211-222)”.

– Similarly, GFP expression in LYVE1 positive lymphatic vessels in Tie2-Cre;Prox1 fl/fl animals again suggests incomplete recombination of Prox1. The identity of the GFP+ cells that are not incorporated into lymphatic vessels should be clarified by imaging at higher magnification as currently, the data are not clear. The analyses in Figure 6 should be repeated with a more specific marker of lymphatic endothelial cells than LYVE1. Either PROX1 or VEGFR3. The PECAM1 staining in the sections presented in Figure 6A-B should be improved.

Regarding the point above, we have performed section immunostaining of Tek-Cre;Prox1fl/fl homozygous (n=3) and Tek-Cre;Prox1fl/+ heterozygous (n=3) embryos for PECAM, eGFP, and Prox1 at E16.5 in the back skin. The Prox1 expression in TeK+ LECs was diminished in Tek-Cre;Prox1fl/fl homozygous embryos compared to Tek-Cre;Prox1fl/+ heterozygous embryos, whereas Tek LECs were observed (Figure 6 —figure supplement 1A-B). Interestingly, blood-filled lymphatic vessels were frequently observed in Tek-Cre;Prox1fl/fl homozygous embryos. Considering a previous report that loss of Prox1 resulted in the formation of anastomosis between blood and lymphatic vessels (Johnson et al., 2008), our observation may indicate the formation of anastomosis between blood and lymphatic vessels. They also indicated that loss of Prox1 in LECs altered marker expression patterns (VE-cadherin and Lyve1 expression were down-regulated in CAGGCre-ERT2;Prox1fl/fl mice (Figure 5C and D)). Thus, our observations that loss of LYVE1 expression in the TeK+ LECs in Tek-Cre;Prox1fl/fl homozygous embryos were consistent with their report (Johnson et al., 2008).

This point was described as follows:

“Immunostaining revealed decreased Prox1 expression in TeK+ LECs in the back skin of Tek-Cre;Prox1fl/fl homozygous mice compared to Tek-Cre;Prox1fl/+ heterozygous mice at E16.5, indicating efficient knockdown of Prox1, whereas Tek LECs were observed similarly (Figure 6 —figure supplement 1A and B). We also observed blood-filled lymphatic vessels in the back skin of the Tek-Cre;Prox1fl/fl homozygous mice, indicating the formation of abnormal anastomosis between lymphatic and blood vessels due to Prox1 deficiency, as previously described (Johnson et al., 2008) (Figure 6 —figure supplement 1A and B).” (Page 8, lines248-255)

We also have improved PECAM immunostaining of Figure 6A and B. We also magnified Figure 6 to show more clearly the correlation of Prox1 knockdown and the decreased LYVE1 expression.

– It is likely that Myf5 is expressed transiently and at low levels in any mesodermal progenitor that gives rise to the endothelium. The Myf5-CreERT2 mouse line used in this study was constructed using an IRES-CreERT2 cassette in the 3'UTR of the Myf5 gene, meaning that expression of CreERT2 from this locus is likely significantly lower than in the Myf5-Cre line previously used to investigate LEC origins (Stone and Stainier, Dev Cell, 2019). Thus, the conclusions that can be drawn from the experiment presented in Supplementary figure 2 are limited. We recommend deleting the second sentence of the discussion 'We also showed that Myf5+ myogenic lineages, which were previously suggested to be possible sources of LECs35, did not contribute to lymphatic vasculature formation in Myf5-CreERT2 mice subjected to tamoxifen treatment at E8.5' and leaving the discussion of these analyses presented on lines 288-292, which more accurately place these data in the context of published work.

We have deleted indicated sentence.

– Reviewer #3 at Review Commons suggested using a Pax3-Cre to assess the contribution of this lineage to facial lymphatics. These analyses have been published and so it would be sufficient to reference Stone and Stainier, Dev Cell, 2019.

We have already included this article as follows:

“A recent study has suggested that Pax3+ paraxial mesoderm-derived cells contribute to the cardinal vein and therefore venous-derived LECs originate from the Pax3+ lineage (Stone and Stainier, 2019).” (Pages 10-11, lines 328-330)

– The following statement "The same group has further argued that the Pax3+ lineage gives rise to lymphatic vessels on the trunk side through lymphangiogenesis (Lupu et al., 2022)" should read "The same group has further argued that the Pax3+ lineage gives rise to lymphatic vessels on the trunk side through lymphvasculogenesis (Lupu et al., 2022)"

We have corrected lymphangiogenesis as lymphvasculogenesis.

References:

Johnson NC, Dillard ME, Baluk P, McDonald DM, Harvey NL, Frase SL, Oliver G. 2008. Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity. Gene Dev 22:3282–3291. doi:10.1101/gad.1727208

Maruyama K, Miyagawa-Tomita S, Mizukami K, Matsuzaki F, Kurihara H. 2019. Isl1-expressing non-venous cell lineage contributes to cardiac lymphatic vessel development. Dev Biol 452:134–143. doi:10.1016/j.ydbio.2019.05.002

https://doi.org/10.7554/eLife.81515.sa2

Article and author information

Author details

  1. Kazuaki Maruyama

    1. Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Japan
    2. Department of Pathology and Matrix Biology, Graduate School of Medicine, Mie University, Mie, Japan
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    k.maruyama0608@gmail.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3935-328X
  2. Sachiko Miyagawa-Tomita

    1. Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Japan
    2. Department of Animal Nursing Science, Yamazaki University of Animal Health Technology, Tokyo, Japan
    3. Heart Center, Department of Pediatric Cardiology, Tokyo Women's Medical University, Tokyo, Japan
    Contribution
    Supervision
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6646-8368
  3. Yuka Haneda

    Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Japan
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  4. Mayuko Kida

    Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Japan
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  5. Fumio Matsuzaki

    Laboratory for Cell Asymmetry, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
    Contribution
    Resources
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7902-4520
  6. Kyoko Imanaka-Yoshida

    Department of Pathology and Matrix Biology, Graduate School of Medicine, Mie University, Mie, Japan
    Contribution
    Supervision
    Competing interests
    No competing interests declared
  7. Hiroki Kurihara

    Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Japan
    Contribution
    Supervision, Validation, Visualization, Writing - review and editing
    For correspondence
    kuri-tky@umin.net
    Competing interests
    No competing interests declared

Funding

Core Research for Evolutional Science and Technology (JPMJCR13W2)

  • Hiroki Kurihara

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank all of the laboratory members for their helpful discussion and encouragement. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (19H01048 to HK and 20K17072 to KM); the Japan Foundation for Applied Enzymology (VBIC to KM); the Miyata Foundation Bounty for Pediatric Cardiovascular Research (KM); the SENSHIN Medical Research Foundation (KM); Takeda Science Foundation (KM); the Platform for Dynamic Approaches to Living Systems of the Ministry of Education, Culture, Sports, Science, and Technology, Japan; and the Core Research for Evolutional Science and Technology (CREST) program of the Japan Science and Technology Agency (JST), Japan (JPMJCR13W2 to HK).

Ethics

All animal experiments were approved by the University of Tokyo (ethical approval number: H17-250) and Mie University (ethical approval number: 728) animal care and use committee, and were performed in accordance with institutional guidelines.

Senior Editor

  1. Didier YR Stainier, Max Planck Institute for Heart and Lung Research, Germany

Reviewing Editor

  1. Oliver A Stone, University of Oxford, United Kingdom

Publication history

  1. Preprint posted: April 1, 2022 (view preprint)
  2. Received: July 4, 2022
  3. Accepted: October 3, 2022
  4. Accepted Manuscript published: October 5, 2022 (version 1)
  5. Version of Record published: October 13, 2022 (version 2)

Copyright

© 2022, Maruyama et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Kazuaki Maruyama
  2. Sachiko Miyagawa-Tomita
  3. Yuka Haneda
  4. Mayuko Kida
  5. Fumio Matsuzaki
  6. Kyoko Imanaka-Yoshida
  7. Hiroki Kurihara
(2022)
The cardiopharyngeal mesoderm contributes to lymphatic vessel development in mouse
eLife 11:e81515.
https://doi.org/10.7554/eLife.81515
  1. Further reading

Further reading

    1. Developmental Biology
    Yanling Xin, Qinghai He ... Shuyi Chen
    Research Article

    N 6-methyladenosine (m6A) is the most prevalent mRNA internal modification and has been shown to regulate the development, physiology, and pathology of various tissues. However, the functions of the m6A epitranscriptome in the visual system remain unclear. In this study, using a retina-specific conditional knockout mouse model, we show that retinas deficient in Mettl3, the core component of the m6A methyltransferase complex, exhibit structural and functional abnormalities beginning at the end of retinogenesis. Immunohistological and single-cell RNA sequencing (scRNA-seq) analyses of retinogenesis processes reveal that retinal progenitor cells (RPCs) and Müller glial cells are the two cell types primarily affected by Mettl3 deficiency. Integrative analyses of scRNA-seq and MeRIP-seq data suggest that m6A fine-tunes the transcriptomic transition from RPCs to Müller cells by promoting the degradation of RPC transcripts, the disruption of which leads to abnormalities in late retinogenesis and likely compromises the glial functions of Müller cells. Overexpression of m6A-regulated RPC transcripts in late RPCs partially recapitulates the Mettl3-deficient retinal phenotype. Collectively, our study reveals an epitranscriptomic mechanism governing progenitor-to-glial cell transition during late retinogenesis, which is essential for the homeostasis of the mature retina. The mechanism revealed in this study might also apply to other nervous systems.

    1. Developmental Biology
    2. Genetics and Genomics
    Xiaodong Li, Patrick J Gordon ... Edward M Levine
    Research Article

    An important question in organogenesis is how tissue-specific transcription factors interact with signaling pathways. In some cases, transcription factors define the context for how signaling pathways elicit tissue- or cell-specific responses, and in others, they influence signaling through transcriptional regulation of signaling components or accessory factors. We previously showed that during optic vesicle patterning, the Lim-homeodomain transcription factor Lhx2 has a contextual role by linking the Sonic Hedgehog (Shh) pathway to downstream targets without regulating the pathway itself. Here, we show that during early retinal neurogenesis in mice, Lhx2 is a multilevel regulator of Shh signaling. Specifically, Lhx2 acts cell autonomously to control the expression of pathway genes required for efficient activation and maintenance of signaling in retinal progenitor cells. The Shh co-receptors Cdon and Gas1 are candidate direct targets of Lhx2 that mediate pathway activation, whereas Lhx2 directly or indirectly promotes the expression of other pathway components important for activation and sustained signaling. We also provide genetic evidence suggesting that Lhx2 has a contextual role by linking the Shh pathway to downstream targets. Through these interactions, Lhx2 establishes the competence for Shh signaling in retinal progenitors and the context for the pathway to promote early retinal neurogenesis. The temporally distinct interactions between Lhx2 and the Shh pathway in retinal development illustrate how transcription factors and signaling pathways adapt to meet stage-dependent requirements of tissue formation.