Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore.

By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.

Cdh5/VE-cadherin promotes endothelial cell interface elongation via cortical actin polymerization during angiogenic sprouting.

Organ morphogenesis requires the coordination of cell behaviors. Here, we have analyzed dynamic endothelial cell behaviors underlying sprouting angiogenesis in vivo. Two different mechanisms contribute to sprout outgrowth: tip cells show strong migratory behavior, whereas extension of the stalk is dependent upon cell elongation.

Directed evolution of a far-red fluorescent rhodopsin.

Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging.

Blood flow changes coincide with cellular rearrangements during blood vessel pruning in zebrafish embryos.

After the initial formation of a highly branched vascular plexus, blood vessel pruning generates a hierarchically structured network with improved flow characteristics. We report here on the cellular events that occur during the pruning of a defined blood vessel in the eye of developing zebrafish embryos. Time-lapse imaging reveals that the connection of a new blood vessel sprout with a previously perfused multicellular endothelial tube leads to the formation of a branched, Y-shaped structure.

In vivo analysis reveals a highly stereotypic morphogenetic pathway of vascular anastomosis.

Organ formation and growth requires cells to organize into properly patterned three-dimensional architectures. Network formation within the vertebrate vascular system is driven by fusion events between nascent sprouts or between sprouts and pre-existing blood vessels. Here, we describe the cellular activities that occur during blood vessel anastomosis in the cranial vasculature of the zebrafish embryo.

Distinct cellular mechanisms of blood vessel fusion in the zebrafish embryo.

Although many of the cellular and molecular mechanisms of angiogenesis have been intensely studied [1], little is known about the processes that underlie vascular anastomosis. We have generated transgenic fish lines expressing an EGFP-tagged version of the junctional protein zona occludens 1 (ZO1) to visualize individual cell behaviors that occur during vessel fusion and lumen formation in vivo. These life observations show that endothelial cells (ECs) use two distinct morphogenetic mechanisms, cell membrane invagination and cord hollowing to generate different types of vascular tubes.

The novel transmembrane protein Tmem2 is essential for coordination of myocardial and endocardial morphogenesis.

Coordination between adjacent tissues plays a crucial role during the morphogenesis of developing organs. In the embryonic heart, two tissues - the myocardium and the endocardium - are closely juxtaposed throughout their development. Myocardial and endocardial cells originate in neighboring regions of the lateral mesoderm, migrate medially in a synchronized fashion, collaborate to create concentric layers of the heart tube, and communicate during formation of the atrioventricular canal.

Semaphorin-PlexinD1 signaling limits angiogenic potential via the VEGF decoy receptor sFlt1.

Sprouting angiogenesis expands the embryonic vasculature enabling survival and homeostasis. Yet how the angiogenic capacity to form sprouts is allocated among endothelial cells (ECs) to guarantee the reproducible anatomy of stereotypical vascular beds remains unclear. Here we show that Sema-PlxnD1 signaling, previously implicated in sprout guidance, represses angiogenic potential to ensure the proper abundance and stereotypical distribution of the trunk's segmental arteries (SeAs).

Vascular morphogenesis in the zebrafish embryo.

During embryonic development, the vertebrate vasculature is undergoing vast growth and remodeling. Blood vessels can be formed by a wide spectrum of different morphogenetic mechanisms, such as budding, cord hollowing, cell hollowing, cell wrapping and intussusception. Here, we describe the vascular morphogenesis that occurs in the early zebrafish embryo.

Complex cell rearrangements during intersegmental vessel sprouting and vessel fusion in the zebrafish embryo.

The formation of intersegmental blood vessels (ISVs) in the zebrafish embryo serves as a paradigm to study angiogenesis in vivo. ISV formation is thought to occur in discrete steps. First, endothelial cells of the dorsal aorta migrate out and align along the dorsoventral axis.