Combined lineage mapping and gene expression profiling of embryonic brain patterning using ultrashort pulse microscopy and image registration

Abstract

During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.

Optical Coherence Tomography for Brain Imaging and Developmental Biology

“…OCM has been combined with two-photon microscopy [217] to provide high-resolution registered images of brain patterning and morphogenesis in live zebrafish embryos. OCT has also been combined with photoacoustic tomography to image tissue optical scattering and absorption profiles simultaneously [218, 219].…”

Midbrain-Hindbrain Boundary Morphogenesis: At the Intersection of Wnt and Fgf Signaling

“…The failure of the constriction to continue morphogenesis in the maintenance phase is due to aberrant cell behaviors in two groups of cells. By imaging a transgenic wnt1 reporter line (Gibbs et al, 2014b ) in the ace(fgf8a) background, we identified one group of cells that fails to maintain wnt1 expression in the posterior midbrain, and to subsequently coordinate the proper morphogenesis of the PML and boundary tegmentum, and another group that fails to suppress wnt1 expression in the dorsal part of r1 to correctly specify the cerebellar plate (Figures 2 , 3 ). This observation, based on identification of individual cells, supports previous reports of an isthmo/cerebellar-to-tectal transformation in molecular identity of the presumptive cerebellum that occurs with genetic reprogramming during the maintenance phase (Jászai et al, 2003 ; Gibbs, 2014 ), though with live imaging we observed that this reprogramming caused by a lack of fgf8a does not preclude the previous initiation of the morphogenesis of the MHB during the activation phase.…”

“…Generated two-photon excited fluorescence is collected in back-reflected geometry by the microscope objective and directed to photon-counting photomultiplier tubes for image rendering. In this configuration, our UPM system is point-scanning wherein images are rendered digitally pixel-by-pixel (Gibbs et al, 2014b ).…”

“…For example, the evolution of the volume and morphology of different brain compartments at various time points and distribution of gene reporters across the brain and their time evolution are imperative relationships that would vary in pathological development including neurodevelopmental diseases. Further, in order to build a “canonical” model of zebrafish brain development, measurements described above have to be made across images of many different embryos, and averaged at each time point, which requires co-registration (Gibbs et al, 2014b ). Building such canonical models is a major goal of structural bioimage informatics.…”

Use of combined polarization-sensitive optical coherence tomography and Mueller matrix imaging for the polarimetric characterization of excised biological tissue

“…OCT has been explored in many applications over the past decade, including ophthalmology, cardiovascular, oncology, and dermatology [28][29][30][31] as well as embryogenesis, angiogenesis, and tissue engineering. [32][33][34][35][36] Polarization-sensitive OCT (PS-OCT), 37,[38][39][40] as a functional extension of OCT, uses the information encoded in the polarization state of the recorded interference fringe intensity to provide additional contrast. In birefringent materials, a phase delay between the two orthogonally polarized wave components is caused by the difference of the refractive indices n o and n e of the ordinary and extraordinary wave Δn ¼ n o − n e , resulting in different phase velocities of both wave components.…”