Repression of MAPK/Erk signaling by Efnb2-Ephb4-Rasa1 is required for lymphatic valve formation

The lymphatic vascular system plays important roles in various physiological and pathological processes, and lack of lymphatic or lymphovenous valves always causes lymph or blood reflux, and can lead to lymphedema. However, the molecular mechanism underlying the valve formation is poorly understood. Here we report that the MAPK/Erk signaling needs to be repressed during the valve-forming lymphatic endothelial cells (LECs) fate determination, which differs from its positive role in the LECs specification. Up-regulation of MAPK/Erk signaling in ephb4b, efnb2a;efnb2b and rasa1a;rasa1b mutants leads to lymphatic valve defects, whereas simultaneous loss of Erk1 and Erk2 causes valve hyperplasia. Moreover, valve defects in ephb4b or rasa1a;rasa1b mutants are mitigated in the presence of MEK inhibitors, indicating a new function of Efnb2-Ephb4-Rasa1 cassette in lymphatic valve progenitor cells specification by repressing MAPK/Erk activity. Therefore, our findings provide a mechanistic understanding of the lymphatic valve formation and potential drug targets for related lymphatic diseases.

lymphatic vessels, pericardial edema was obvious in ephb4b tsu25 mutants starting from 4 dpf, 132 which sometimes expanded to the gut region ( Figure 1I). Injection of 2,000 kDa Dextran- 133 Fluorescein into the pericardial cavity of wild-type larvae in Tg(lyve1b:TopazYFP) background 134 at 3 dpf led to a rapid appearance of the fluorescent dye within the artery blood vessels only after 135 1 hour post injection (hpi), revealing a rapid dye absorption of the lymphatic system and then 136 subsequent transport to the circulation system. However, when this Dextran-Fluorescein was 137 injected into the ephb4b tsu25 mutant larvae in the same way, only 54.5% (n = 18/33) of embryos 138 showed a similar dye flow pattern even at 24 hpi, whereas the other embryos only exhibited a 139 very mild or even no absorption of the dye ( Figure 1J). More importantly, this defective 140 lymphatic absorption in ephb4b tsu25 mutants was positively corelated to the edema phenotype, 141 which was probably caused by dysfunction of the blood-filled lymphatic vessels.  . Based on the valve morphology at 3.2-3.5 dpf in ephb4b tsu25 mutants, we classified the 164 facial LVs into three types: L1, normal LV with two leaflets; L2, small LV; and L3, little or no 165 LV structure. It was found that, when 29.7% (n = 11/37) of sibling embryos were starting to 166 form LV structure (L1 and L2) at 3.2-3.5 dpf, 7.3% (n = 3/41) of ephb4b tsu25 mutants only that the expression of Prox1a, the zebrafish homolog of the mammalian Prox1, together with 184 gata2:EGFP, was much higher in valve-forming LECs in the LV and LVV structure at 3.2 dpf.

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In ephb4b tsu25 mutants, however, the number of valve-forming LECs with high-level Prox1a in 186 the LVs was reduced to some extent ( Figure 2D and E). These results indicate that ephb4b might 187 be involved in the initiation of the lymphatic valve formation.    4A and B). rasa1a tsu38 or rasa1b tsu39 homozygous mutants developed normally to adulthood 236 without visible morphological changes. However, rasa1a tsu38 ;rasa1b tsu39 double mutants 237 exhibited blood filling within lymphatic vessels and severe pericardial edema at 4 dpf, and could 238 not survive over 6 dpf ( Figure 4C). In addition, we obtained another rasa1a tsu40 mutant allele  Simultaneous loss of Erk1 and Erk2 leads to valve hyperplasia 266 The above data indicate that Efnb2-Ephb4-Rasa1 regulates the development of both the LV and 267 LVV formation in the zebrafish, but how they function is still unclear. It is reported that Rasa1   Figure 5A and B). Likely due to compensatory function of Erk1 and Erk2, 278 either erk1 tsu45 or erk2 tsu46 homozygous mutants developed normally to adulthood. We then 279 generated erk1 tsu45 ;erk2 tsu46 zygotic double mutants to observe potential phenotypic changes.

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These results support the idea that the inhibition of MAPK/Erk signaling results in lymphatic 298 valve hyperplasia at early stages of lymphatic system development.  304 To confirm the role of Efnb2-Ephb4-Rasa1 signaling in lymphatic valve formation by inhibiting 305 the Ras-Raf-Mek-Erk pathway, we examined phospho-Erk1/2 (p-Erk1/2) levels by 306 immunofluorescence in isolated gata2:EGFP/lyve1b:DsRed2 double-positive putative valve-307 forming LECs at 3.2 dpf from sibling or ephb4b tsu25 mutant larvae in the 308 Tg(gata2:EGFP;lyve1b:DsRed2) transgenic background. Results showed that p-Erk1/2 signal 309 was higher in ephb4b tsu25 mutants than in siblings ( Figure 6A and B). Considering that valve-  Figure 6D). However, elevated p-Erk1/2 levels in 318 the FCLV and aLFL endothelial cells were observed in ephb4b tsu25 , efnb2a tsu41 ;efnb2b tsu42 and 319 rasa1a tsu38 ;rasa1b tsu39 mutant larvae ( Figure 6D). Taken together, these data suggest that Efnb2-  Next, we tried to use a set of MEK inhibitors to rescue the lymphatic valve defects in 326 ephb4b tsu25 mutants. We applied four different MEK inhibitors, including Selumetinib, 327 Cobimetinib, Trametinib, and U0126-EtOH, at different concentrations to ephb4b tsu25 mutants 328 from 2 dpf to 4 dpf and used pericardial edema as the phenotypic readout ( Figure 7A). As a 329 result, we found that all the four MEK inhibitors could partially rescue the edema phenotype of 330 ephb4b tsu25 mutants at proper concentrations ( Figure 7B). In contrast, the mTOR inhibitors  In this study, we discover that inhibition of the Ras-Raf-Mek-Erk cascade by Efnb2-Ephb4-359 Rasa1 signaling is required for the fate specification of valve-forming LECs during the formation 360 of the zebrafish lymphatic system. Lack of efnb2, ephb4b, or rasa1 leads to the increased Erk1/2 361 activation and defective LV or LVV formation, whereas simultaneous loss of Erk1 and Erk2 362 causes hyperplasia of the valve-forming LECs at the expense of some LECs (Figure 8).

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Moreover, the abnormal lymphatic valves due to ephb4b or rasa1 deficiency can be partially  signaling. Here our findings not only illustrate a possible mechanism underlying the lymphatic 399 abnormalities in such diseases, but also establish several zebrafish genetic disease models, such 400 as ephb4b tsu25 , efnb2a tsu41 ;efnb2b tsu42 and rasa1a tsu38 ;rasa1b tsu39 to further understand the 401 pathogenesis of human lymphedema and evaluate potential drugs. More importantly, we found 402 that treatment with MEK inhibitors significantly improves lymphedema phenotype and restores 403 the lymphatic valve formation in ephb4b tsu25 or rasa1a tsu40 ;rasa1b tsu39 mutant larvae, providing a 404 potential treatment strategy for valve-deficient disorders that currently lack specific molecular 405 treatments.

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We noted that LVs and LVVs almost fail to form in efnb2a tsu41 ;efnb2b tsu42 or 408 rasa1a tsu38 ;rasa1b tsu39 double mutants, but partially form in ephb4b tsu25 mutant larvae, which is   Table S2 for primer information.  Table S1 for additional allele information 445 and Table S2 for mutant identification.   Table S2.  Table S2). The images were then viewed and processed by Imaris 487 software 9.1. Single plane or 3D images were generated by Snapshot and then processed by 488 Adobe Photoshop 2020. Movies were generated using Imaris 9.1, imported into Adobe Premiere 489 Pro 2020 for labeling, and exported as .mp4 files. Coulter). The cells were collected in 5% FBS in PBS, and then concentrated by centrifugation.

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The concentrated cell suspension was applied on the glass slide for pERK immunostaining. The 500 slides were observed on a Dragonfly confocal microscope (Andor) and analyzed by Imaris.    The unpaired student's t-test, chi-squared test and Fisher's exact test were used to calculate 517 significance. The sample size (n) and P value for each experimental group are described in

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The amino acid sequence changes after 57 aa. The ephb4a tsu37 mutant protein only has a partial 975 ligand binding domain (LBD). (B) ephb4a tsu37 mutants exhibit abnormal blood vessel formation 976 and blood accumulation in the tail region at 2 dpf. Scale bars, 500 μm. The ratio of embryos with exhibited valve structure is indicated. Siblings were defined as neither 994 rasa1a tsu40 mutant nor rasa1b tsu39 mutants. Scale bars, 50 μm.