Neurovascular development in the embryonic zebrafish hindbrain

Abstract

The brain is made of billions of highly metabolically active neurons whose activities provide the seat for cognitive, affective, sensory and motor functions. The cerebral vasculature meets the brain's unusually high demand for oxygen and glucose by providing it with the largest blood supply of any organ. Accordingly, disorders of the cerebral vasculature, such as congenital vascular malformations, stroke and tumors, compromise neuronal function and survival and often have crippling or fatal consequences. Yet, the assembly of the cerebral vasculature is a process that remains poorly understood. Here we exploit the physical and optical accessibility of the zebrafish embryo to characterize cerebral vascular development within the embryonic hindbrain. We find that this process is primarily driven by endothelial cell migration and follows a two-step sequence. First, perineural vessels with stereotypical anatomies are formed along the ventro-lateral surface of the neuroectoderm. Second, angiogenic sprouts derived from a subset of perineural vessels migrate into the hindbrain to form the intraneural vasculature. We find that these angiogenic sprouts reproducibly penetrate into the hindbrain via the rhombomere centers, where differentiated neurons reside, and that specific rhombomeres are invariably vascularized first. While the anatomy of intraneural vessels is variable from animal to animal, some aspects of the connectivity of perineural and intraneural vessels occur reproducibly within particular hindbrain locales. Using a chemical inhibitor of VEGF signaling we determine stage-specific requirements for this pathway in the formation of the hindbrain vasculature. Finally, we show that a subset of hindbrain vessels is aligned and/or in very close proximity to stereotypical neuron clusters and axon tracts. Using endothelium-deficient cloche mutants we show that the endothelium is dispensable for the organization and maintenance of these stereotypical neuron clusters and axon tracts in the early hindbrain. However, the cerebellum's upper rhombic lip and the optic tectum are abnormal in clo. Overall, this study provides a detailed, multi-stage characterization of early zebrafish hindbrain neurovascular development with cellular resolution up to the third day of age. This work thus serves as a useful reference for the neurovascular characterization of mutants, morphants and drug-treated embryos.

In parallelinterconnectivity of the dorsal longitudinal anastomotic vessels requires both VEGF signaling and circulatory flow

“…The first is the formation of a midline vessel from two lateral precursors. This resembles the way in which the midline basilar artery of the hindbrain forms from the two laterally positioned primordial hindbrain channels in zebrafish (Bussmann et al, 2011;Corti et al, 2011;Fujita et al, 2011;Ulrich et al, 2011). The second is 'vessel fusion', a unique process in which vessels coalesce by behaving like highly viscous liquid drops.…”

Reck enables cerebrovascular development by promoting canonical Wnt signaling

“…Most BA-forming sprout remnants are lost by 48 hpf; those that persist become avcs ( Fig. 4D,E; see Bussmann et al, 2011;Corti et al, 2011;Fujita et al, 2011;Fukuhara et al, 2014;Isogai et al, 2001;Ulrich et al, 2011).…”

“…To elucidate the endothelial cellular bases of the intracerebral vascularization deficit of reck mutants (reck − ) we exploited the advantages of the hindbrain (Hb) vasculature as a model for cerebrovascular development, as we reported previously (Ulrich et al, 2011). The WT Hb harbors both extracerebral ( perineural) and intracerebral vessels.…”

“…Unlike drug-based inhibition of circulatory flow, genetic abrogation of circulation increases the number of avcs, even in reck y72 mutants, and has little impact on the abundance of CtAs (Table S2 and Fig. S5; Bussmann et al, 2011;Corti et al, 2011;Fujita et al, 2011;Fukuhara et al, 2014;Isogai et al, 2001;Ulrich et al, 2011). Thus, reck limits the number of avcs, even without flow, strongly suggesting that the overabundance of avcs in reck y72 mutants is unrelated to circulatory pressure gains.…”

A Dicer-miR-107 Interaction Regulates Biogenesis of Specific miRNAs Crucial for Neurogenesis

“…Neurospheres formed mainly from the proliferation of GFAP + radial glia cells that served as neural progenitors able to self-renew and differentiate after withdrawal of growth factors for three consecutive culture passages ( Figure S3E, arrowhead; data not shown). After 4 days in vitro in the absence of growth factors (Div4d), zebrafish neurospheres differentiated into all neural cell types: astrocytelike cells (GFAP + ) (Nielsen and Jørgensen, 2003), oligodendrocytes (Sox-10 + ) (Takada and Appel, 2010), and mature neurons (a-tubulin + ) (Ulrich et al, 2011) and expressed gene markers of several postmitotic neural cells (Figures S3F, arrowheads, and S3G). qRT-PCR analysis indicated that, during the course of neurosphere differentiation, dicer mRNA levels progressively declined, while miR-107 expression increased ( Figures 5A and 5B).…”