Mural cells (MCs) are essential for blood vessel stability and function; however, the mechanisms that regulate MC development remain incompletely understood, in particular those involved in MC specification. Here, we investigated the first steps of MC formation in zebrafish using transgenic reporters. Using pdgfrb and abcc9 reporters, we show that the onset of expression of abcc9, a pericyte marker in adult mice and zebrafish, occurs almost coincidentally with an increment in pdgfrb expression in peri-arterial mesenchymal cells, suggesting that these transcriptional changes mark the specification of MC lineage cells from naïve pdgfrb low mesenchymal cells. The emergence of peri-arterial pdgfrb high MCs required Notch signaling. We found that pdgfrb-positive cells express notch2 in addition to notch3, and although depletion of notch2 or notch3 failed to block MC emergence, embryos depleted of both notch2 and notch3 lost mesoderm-as well as neural crest-derived pdgfrb high MCs. Using reporters that read out Notch signaling and Notch2 receptor cleavage, we show that Notch activation in the mesenchyme precedes specification into pdgfrb high MCs. Taken together, these results show that Notch signaling is necessary for peri-arterial MC specification.

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The migration of lymphatic endothelial cells (LECs) is key for the development of the complex and vast lymphatic vascular network that pervades most tissues in an organism. In zebrafish, arterial intersegmental vessels together with chemokines have been shown to promote lymphatic cell migration from the horizontal myoseptum (HM). We observed that emergence of mural cells around the intersegmental arteries coincides with lymphatic departure from HM which raised the possibility that arterial mural cells promote LEC migration. Our live imaging and cell ablation experiments revealed that LECs migrate slower and fail to establish the lymphatic vascular network in the absence of arterial mural cells. We determined that mural cells are a source for the C-X-C motif chemokine 12 (Cxcl12a and Cxcl12b), Vascular endothelial growth factor C (Vegfc) and Collagen and calcium-binding EGF domain-containing protein 1 (Ccbe1). We showed that chemokine and growth factor signalling function cooperatively to induce robust LEC migration. Specifically, Vegfc-Vegfr3 signalling, but not chemokines, induces extracellular signal-regulated kinase (ERK) activation in LECs, and has an additional pro-survival role in LECs during the migration. Together, the identification of mural cells as a source for signals that guide LEC migration and survival will be important in the future design for rebuilding lymphatic vessels in disease contexts.

In the last decade, China has sharply tightened the monitoring values for wastewater treatment plants (WWTPs). In some regions with sensitive discharge water bodies, the values (24 h composite sample) must be 1.5 mg/L for NH4-N and 10 mg/L for total nitrogen since 2021. Even with the previously less strict monitoring values, around 50% of the wastewater treatment plants in China were permanently unable to comply with the nitrogen monitoring values. Due to the rapid changes on-site to meet the threshold values and the strong relation to energy-intensive aeration strategies to sufficiently remove nitrogen, WWTPs do not always work energy-efficiently. A Chinese WWTP (450,000 Population equivalents or PE) with upstream denitrification, a tertiary treatment stage for phosphorus removal and disinfection, and aerobic sludge stabilisation was modelled in order to test various concepts for operation optimisation to lower energy consumption while meeting and undercutting effluent requirements. Following a comprehensive analysis of operating data, the WWTP was modelled and calibrated. Based on the calibrated model, various approaches for optimising nitrogen elimination were tested, including operational and automation strategies for aeration control. After several tests, a combination of strategies (i.e., partial by-pass of primary clarifiers, NH4-N based control, increase in the denitrification capacity, intermittent denitrification) reduced the air demand by up to 24% and at the same time significantly improved compliance with the monitoring values (up to 80% less norm non-compliances). By incorporating the impact of the strategies on related processes, like the bypass of primary settling tanks, energy consumption could be reduced by almost 25%. Many of the elaborated strategies can be transferred to WWTPs with similar boundary conditions and strict effluent values worldwide.

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