The directionality of local blood flow in the zebrafish embryonic heart is essential for proper heart valve formation.

The pattern of blood flow has long been thought to play a significant role in vascular morphogenesis, yet the flow-sensing mechanism that is involved at early embryonic stages, when flow forces are low, remains unclear. It has been proposed that endothelial cells use primary cilia to sense flow, but this has never been tested in vivo. Here we show, by noninvasive, high-resolution imaging of live zebrafish embryos, that endothelial cilia progressively deflect at the onset of blood flow and that the deflection angle correlates with calcium levels in endothelial cells. We demonstrate that alterations in shear stress, ciliogenesis, or expression of the calcium channel PKD2 impair the endothelial calcium level and both increase and perturb vascular morphogenesis. Altogether, these results demonstrate that endothelial cilia constitute a highly sensitive structure that permits the detection of low shear forces during vascular morphogenesis.

The embryonic vertebrate heart begins pumping blood long before the development of discernable chambers and valves. At these early stages, the heart tube has been described as a peristaltic pump. Recent advances in confocal laser scanning microscopy and four-dimensional visualization have warranted another look at early cardiac structure and function. We examined the movement of cells in the embryonic zebrafish heart tube and the flow of blood through the heart and obtained results that contradict peristalsis as a pumping mechanism in the embryonic heart. We propose a more likely explanation of early cardiac dynamics in which the pumping action results from suction due to elastic wave propagation in the heart tube.

Over the course of development, the vertebrate heart undergoes a series of complex morphogenetic processes that transforms it from a simple myocardial epithelium to the complex 3D structure required for its function. One of these processes leads to the formation of trabeculae to optimize the internal structure of the ventricle for efficient conduction and contraction. Despite the important role of trabeculae in the development and physiology of the heart, little is known about their mechanism of formation. Using 3D time-lapse imaging of beating zebrafish hearts, we observed that the initiation of cardiac trabeculation can be divided into two processes. Before any myocardial cell bodies have entered the trabecular layer, cardiomyocytes extend protrusions that invade luminally along neighboring cell-cell junctions. These protrusions can interact within the trabecular layer to form new cell-cell contacts. Subsequently, cardiomyocytes constrict their abluminal surface, moving their cell bodies into the trabecular layer while elaborating more protrusions. We also examined the formation of these protrusions in trabeculationdeficient animals, including erbb2 mutants, tnnt2a morphants, which lack cardiac contractions and flow, and myh6 morphants, which lack atrial contraction and exhibit reduced flow. We found that, compared with cardiomyocytes in wild-type hearts, those in erbb2 mutants were less likely to form protrusions, those in tnnt2a morphants formed less stable protrusions, and those in myh6 morphants extended fewer protrusions per cell. Thus, through detailed 4D imaging of beating hearts, we have identified novel cellular behaviors underlying cardiac trabeculation.

Abstract. Being able to acquire, visualize, and analyze 3D time series ͑4D data͒ from living embryos makes it possible to understand complex dynamic movements at early stages of embryonic development. Despite recent technological breakthroughs in 2D dynamic imaging, confocal microscopes remain quite slow at capturing optical sections at successive depths. However, when the studied motion is periodicsuch as for a beating heart-a way to circumvent this problem is to acquire, successively, sets of 2D + time slice sequences at increasing depths over at least one time period and later rearrange them to recover a 3D + time sequence. In other imaging modalities at macroscopic scales, external gating signals, e.g., an electro-cardiogram, have been used to achieve proper synchronization. Since gating signals are either unavailable or cumbersome to acquire in microscopic organisms, we have developed a procedure to reconstruct volumes based solely on the information contained in the image sequences. The central part of the algorithm is a least-squares minimization of an objective criterion that depends on the similarity between the data from neighboring depths. Owing to a wavelet-based multiresolution approach, our method is robust to common confocal microscopy artifacts. We validate the procedure on both simulated data and in vivo measurements from living zebrafish embryos.

Abstract-We propose a construction of new wavelet-like bases that are well suited for the reconstruction and processing of optically generated Fresnel holograms recorded on CCD-arrays. The starting point is a wavelet basis of 2 to which we apply a unitary Fresnel transform. The transformed basis functions are shift-invariant on a level-by-level basis but their multiresolution properties are governed by the special form that the dilation operator takes in the Fresnel domain. We derive a Heisenberg-like uncertainty relation that relates the localization of Fresnelets with that of their associated wavelet basis. According to this criterion, the optimal functions for digital hologram processing turn out to be Gabor functions, bringing together two separate aspects of the holography inventor's work.We give the explicit expression of orthogonal and semi-orthogonal Fresnelet bases corresponding to polynomial spline wavelets. This special choice of Fresnelets is motivated by their near-optimal localization properties and their approximation characteristics. We then present an efficient multiresolution Fresnel transform algorithm, the Fresnelet transform. This algorithm allows for the reconstruction (backpropagation) of complex scalar waves at several user-defined, wavelength-independent resolutions. Furthermore, when reconstructing numerical holograms, the subband decomposition of the Fresnelet transform naturally separates the image to reconstruct from the unwanted zero-order and twin image terms. This greatly facilitates their suppression. We show results of experiments carried out on both synthetic (simulated) data sets as well as on digitally acquired holograms.

We present a new digital two-step reconstruction method for off-axis holograms recorded on a CCD camera. First, we retrieve the complex object wave in the acquisition plane from the hologram's samples. In a second step, if required, we propagate the wave front by using a digital Fresnel transform to achieve proper focus. This algorithm is sufficiently general to be applied to sophisticated optical setups that include a microscope objective. We characterize and evaluate the algorithm by using simulated data sets and demonstrate its applicability to real-world experimental conditions by reconstructing optically acquired holograms.

We report an accurate method for studying the functional dynamics of the beating embryonic zebrafish heart. The fast cardiac contraction rate and the high velocity of blood cells have made it difficult to study cellular and subcellular events relating to heart function in vivo. We have devised a dynamic three-dimensional acquisition, reconstruction, and analysis procedure by combining (1) a newly developed confocal slit-scanning microscope, (2) novel strategies for collecting and synchronizing cyclic image sequences to build volumes with high temporal and spatial resolution over the entire depth of the beating heart, and (3) data analysis and reduction protocols for the systematic extraction of quantitative information to describe phenotype and function. We have used this approach to characterize blood flow and heart efficiency by imaging fluorescent protein-expressing blood and endocardial cells as the heart develops from a tube to a multichambered organ. The methods are sufficiently robust to image tissues within the heart at cellular resolution over a wide range of ages, even when motion patterns are only quasiperiodic. These tools are generalizable to imaging and analyzing other cyclically moving structures at microscopic scales. Developmental Dynamics 235:2940 -2948, 2006.