eLife assessment

Artery muscularization is spatiotemporally regulated in CoW arteries

CoW arteries have spatiotemporal difference in hemodynamics

Blood flow is required for CoW artery muscularization

Blood flow-regulated transcription factor klf2a modulates CoW artery muscularization

In this study, we used confocal live imaging of fluorescence transgenic zebrafish embryos to investigate the spatiotemporal dynamics of VSMC differentiation on CoW, which comprises major arteries supplying blood to the vertebrate brain. Our observations revealed that CoW morphogenesis precedes arterial specification. Mural cell progenitors marked by pdgfrb+ initiate VSMC marker acta2 expression subsequent to their recruitment to CoW arteries. Notably, VSMC differentiation occurs earlier on anterior CoW arteries compared to their posterior counterparts, owing to elevated WSS resulting from higher velocity of RBCs in the incoming blood flow. To investigate the regulatory role of blood flow on spatiotemporal dynamics of VSMC differentiation, we employed in vitro co-culture assay along with genetic manipulation and drug treatment. Our findings indicate that blood flow indeed governs timing and location of VSMC differentiation on CoW arteries. Moreover, we observed that flow-responsive transcription factor klf2a is activated in a gradient manner from anterior to posterior CoW arteries, preceding VSMC differentiation. Knockdown experiments targeting klf2a revealed a delay in VSMC differentiation specifically on anterior CoW arteries. Taken together, these findings highlight endothelial klf2a activation by blood flow as a mechanism that promotes VSMC differentiation on CoW arteries in the vertebrate brain (see Figure 7 for a visual summary).

Previous research shows that in zebrafish trunk, as soon as recruited to dorsal aorta, VSMCs express acta2 and transgelin (tagln), suggesting simultaneous recruitment and differentiation from sclerotome progenitors (Ando et al., 2016; Stratman et al., 2017). On CoW arteries in zebrafish brain, however, VSMC differentiation occurs after pdgfrb+ progenitor recruitment and proceeds from anterior to posterior (Figure 2B–F). The observed spatiotemporal dynamics of CoW VSMC differentiation again highlights organotypic development of blood vessels.

Our data suggest klf2a mediates blood flow regulation of VSMC differentiation on brain arteries, and thus raise the question of how klf2a transduces endothelial signals to mural cell progenitors and VSMCs. Notch signaling appears a plausible downstream effector of klf2a activation, as previous research suggests Notch responds to flow in heart valve development (Fontana et al., 2020), possibly downstream of klf2 (Duchemin et al., 2019). Wnt signaling is another possible downstream effector of klf2a, as previous research suggests endocardial klf2 upregulates Wnt signaling in neighboring mesenchymal cells in heart valve development (Goddard et al., 2017). The roles of Notch and Wnt signaling in organotypic VSMC differentiation on brain arteries remain to be determined.

The expression of klf2a in CoW arterial ECs remained stable from 3 dpf to 4 dpf (Figure 5), which raises an interesting question on whether sustained klf2a expression supports further maturation of VSMCs, as acta2+ VSMCs on CaDI showed distinct morphology at 4 dpf compared with 3 dpf, when they started differentiation from pdgfrb+ mural cell progenitors (Figure 2C, D and Figure 2—figure supplement 1A, B). Previous research found that acta2+ VSMCs on BCA and PCS express pericyte enriched abcc9 (ATP-binding cassette subfamily C member 9) at 4 dpf (Ando et al., 2022), when they started differentiation from pdgfrb+ mural cell progenitors and initiated acta2 expression; abcc9 expression is gradually lost from 5 dpf to 6 dpf (Ando et al., 2022). In addition, acta2+ VSMCs on CaDI, BCA, and PCS still retain expression of pdgfrb as late as 6 dpf (Ando et al., 2021). Thus, it is possible that stable expression of klf2a in CoW arterial ECs supports further VSMC maturation, as indicated by expression of tagln (Colijn et al., 2023). Another aspect of VSMC maturation is its function to confer vascular tone. A previous study found that VSMC covered vessels in zebrafish brain dilate as early as 4 dpf and constrict at 6 dpf (Bahrami and Childs, 2020). Future study may focus on the association between expression of different VSMC markers and VSMC functional maturation.

Another question is how flow activates klf2a in brain arteries. In a hypoxia-induced pulmonary hypertension model, Klf2 is activated by G-protein-coupled receptor-mediated Apelin signaling (Chandra et al., 2011). In heart valve development, endocardial klf2a is upregulated by membrane-bound mechanosensitive channels trpp2 and trpv4 (Heckel et al., 2015). The pathway through which flow activates klf2a in brain arterial ECs remains unknown. The CoW VSMC phenotype of klf2a morphants is similar to, but do not fully recapitulate, the phenotypes of tnnt2a morphants or nifedipine-treated embryos (Figure 4E–K and Figure 6). A proximal explanation is compensation by paralogous klf2b in zebrafish. Further characterization of CoW VSMC development in klf2a and klf2b genetic mutants (Rasouli et al., 2018; Steed et al., 2016) may help determine whether klf2b compensates klf2a in CoW VSMC differentiation.

In addition to WSS, transmural pressure and associated mechanical stretch is another mechanical input of vascular wall that may contribute to VSMC differentiation. VSMCs autonomously sense and adapt to mechanical stretch (Haga et al., 2007). Cyclic stretching activates arterial VSMC production of certain components of extracellular matrix (ECM) (Leung et al., 1976). In turn, specific interactions between ECM and integrins enable VSMCs to sense mechanical stretch (Goldschmidt et al., 2001; Wilson et al., 1995). Connection to ECM is essential for VSMC force generation (Milewicz et al., 2017). VSMCs express stretch activated Trpp2, which enables myogenic response to autoregulate resting arterial diameter (Sharif-Naeini et al., 2009). Interestingly, ex vivo stretching promotes expression of contractile proteins, such as Acta2 and Tagln in VSMCs on murine portal veins (Albinsson et al., 2004; Turczyńska et al., 2013). In addition, excessive mechanical stretch promotes VSMC dedifferentiation and inflammation (Cao et al., 2017; Wang et al., 2018). Further investigation is needed to determine the potential role of increasing transmural pressure and associated mechanical stretch during development in VSMC differentiation.

There are a few limitations to our current study. Genetic manipulation and drug treatment that reduce heart rate would also reduce nutrients carried by blood flow, and the effects of nutrient and flow reduction could not be uncoupled in live zebrafish embryos. In addition, these methods are only capable of qualitative reduction of flow but not specific dampening of pulsations. In vitro three-dimensional (3D) vascular culture models, which combine ECs and mural cells (Mirabella et al., 2017; Vila Cuenca et al., 2021), could be further optimized to simulate complex geometry of brain arteries. Combining these models with microfluidics, which allows precise calibration of nutrient composition in culture media, flow velocity, and pulse, would enable more thorough analysis of endothelial mechanotransduction and its contribution to VSMC differentiation (Abello et al., 2022; Gray and Stroka, 2017; Griffith et al., 2020).

We used nifedipine to temporally reduce heart rate and blood flow. Nifedipine is a blocker of L-type voltage-dependent calcium channels (VDCCs) (Quevedo et al., 1998). A previous study shows that ML218, a T-type VDCC-selective inhibitor, tends to increase VSMC differentiation (Ando et al., 2022). In addition to the difference in drug targets, we also noted that in the previous study, the increase in VSMC differentiation only occur on anterior metencephalic central arteries (AMCtAs) that are more than 40 μm away from the BCA; these AMCtAs are much smaller than CoW arteries and have different geometry (Ando et al., 2022). Although the most obvious effect of nifedipine in zebrafish embryos is heart rate and blood flow reduction (Gierten et al., 2020), it is possible that nifedipine affects VSMC through VDCCs. Further investigation is needed to uncouple the roles of different VDCCs in VSMC differentiation and their association with hemodynamics.

We used Tg(flt4:yfp, kdrl:ras-mcherry)^hu4881/s896 to show CoW arterial specification from primitive venous ECs (Chi et al., 2008; Hogan et al., 2009). kdrl is not the best arterial marker as its expression is only enriched but not restricted to arteries. While the use of the two fluorescence transgenic lines helped us establish the temporal sequence of CoW morphogenesis, arterial specification and VSMC differentiation, analysis of additional arterial and venous markers is needed to fully characterize arterial specification in vertebrate brain vascular development.

In summary, our work identifies flow-induced endothelial klf2a activation as a mechanism that regulates spatiotemporal dynamics of VSMC differentiation on CoW arteries in vertebrate brain. Our data may help advance approaches to regenerate dedifferentiated VSMCs in cerebrovascular disease. It would be important to further investigate mechanisms upstream and downstream of klf2a activation in brain arterial ECs and additional mechanotransduction pathways that may contribute to VSMC differentiation on brain arteries.