HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal-to-noise fluorescence.

The expansion of fluorescence bioimaging toward more complex systems and geometries requires analytical tools capable of spanning widely varying timescales and length scales, cleanly separating multiple fluorescent labels and distinguishing these labels from background autofluorescence. Here we meet these challenging objectives for multispectral fluorescence microscopy, combining hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). HyU is efficient and robust, capable of quantitative signal separation even at low illumination levels. In dynamic imaging of developing zebrafish embryos and in mouse tissue, HyU was able to cleanly and efficiently unmix multiple fluorescent labels, even in demanding volumetric timelapse imaging settings. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels. This enables coincident studies of tagged components, cellular behaviors and cellular metabolism within the same specimen, providing more accurate insights into the orchestrated complexity of biological systems.

High-contrast, synchronous volumetric imaging with selective volume illumination microscopy.

Light-field fluorescence microscopy uniquely provides fast, synchronous volumetric imaging by capturing an extended volume in one snapshot, but often suffers from low contrast due to the background signal generated by its wide-field illumination strategy. We implemented light-field-based selective volume illumination microscopy (SVIM), where illumination is confined to only the volume of interest, removing the background generated from the extraneous sample volume, and dramatically enhancing the image contrast. We demonstrate the capabilities of SVIM by capturing cellular-resolution 3D movies of flowing bacteria in seawater as they colonize their squid symbiotic partner, as well as of the beating heart and brain-wide neural activity in larval zebrafish. These applications demonstrate the breadth of imaging applications that we envision SVIM will enable, in capturing tissue-scale 3D dynamic biological systems at single-cell resolution, fast volumetric rates, and high contrast to reveal the underlying biology.

Pre-processing visualization of hyperspectral fluorescent data with Spectrally Encoded Enhanced Representations.

Hyperspectral fluorescence imaging is gaining popularity for it enables multiplexing of spatio-temporal dynamics across scales for molecules, cells and tissues with multiple fluorescent labels. This is made possible by adding the dimension of wavelength to the dataset. The resulting datasets are high in information density and often require lengthy analyses to separate the overlapping fluorescent spectra. Understanding and visualizing these large multi-dimensional datasets during acquisition and pre-processing can be challenging. Here we present Spectrally Encoded Enhanced Representations (SEER), an approach for improved and computationally efficient simultaneous color visualization of multiple spectral components of hyperspectral fluorescence images. Exploiting the mathematical properties of the phasor method, we transform the wavelength space into information-rich color maps for RGB display visualization. We present multiple biological fluorescent samples and highlight SEER's enhancement of specific and subtle spectral differences, providing a fast, intuitive and mathematical way to interpret hyperspectral images during collection, pre-processing and analysis.

Znf385C mediates a novel p53-dependent transcriptional switch to control timing of facial bone formation.

Jaw formation involves an intricate series of molecular events, whereby a chondrogenic scaffold precedes osteogenesis. The mechanisms coupling timing of cartilage maturation to onset of bone differentiation are poorly understood, particularly for neural crest-derived bones of the head. Here we present a novel zebrafish gene/protein-trap Citrine-fusion line that reveals transient expression of the zinc-finger protein Znf385C in maturing chondrocytes of the jaw. Functional analysis shows that loss of Znf385C disrupts a distinct peak of p21(cip1/waf1) expression in the chondrocytes, as well as causes premature ossification of the zebrafish jaw. We find that Znf385C is expressed as two splice variants which act differentially to activate p21(cip1/waf1) and/or interact with p53 in subcellular compartments. Taken together, the results suggest that Znf385C acts as a developmental switch for p53 function that modulates cell cycle arrest of chondrocytes and regulates timing of jaw cartilage maturation and ossification.

Evolutionarily conserved role for SoxC genes in neural crest specification and neuronal differentiation.

Members of the Sox family of transcription factors play a variety of critical developmental roles in both vertebrates and invertebrates. Whereas SoxBs and SoxEs are involved in neural and neural crest development, respectively, far less is known about members of the SoxC subfamily. To address this from an evolutionary perspective, we compare expression and function of SoxC genes in neural crest cells and their derivatives in lamprey (Petromyzon marinus), a basal vertebrate, to frog (Xenopus laevis). Analysis of transcript distribution reveals conservation of lamprey and X. laevis SoxC expression in premigratory neural crest, branchial arches, and cranial ganglia. Moreover, morpholino-mediated loss-of-function of selected SoxC family members demonstrates essential roles in aspects of neural crest development in both organisms. The results suggest important and conserved functions of SoxC genes during vertebrate evolution and a particularly critical, previously unrecognized role in early neural crest specification.

Zebrafish stem/progenitor factor msi2b exhibits two phases of activity mediated by different splice variants.

The Musashi (Msi) family of RNA-binding proteins is important in stem and differentiating cells in many species. Here, we present a zebrafish gene/protein trap line gt(msi2b-citrine)(ct) (57) (a) that expresses a Citrine fusion protein with endogenous Msi2b. Our results reveal two phases of Msi2b expression: ubiquitous expression in progenitor cells in the early embryo and later, tissue-specific expression in differentiating cells in the olfactory organ, pineal gland, and subpopulations of neurons in the central nervous system (CNS). Interestingly, this division between early and late phases is paralleled by differential expression of msi2b alternative splicing products. Whereas the full-length and long variant v3 Msi2b predominate at early stages, the later expression of variants in differentiating tissues appears to be tissue specific. Using the gt(msi2b-citrine)(ct) (57) (a), we characterized tissue-specific expression of Msi2b with cellular resolution in subsets of differentiating cells in the olfactory organ, pineal gland, CNS, and ventral neural tube. By performing transcription activator-like effectors nuclease-mediated biallelic genome editing or morpholino knockdown of Msi2b in zebrafish, our results show that early inactivation of Msi2b results in severe embryonic defects including hypertrophy of the ventricles and shortening of the body, consistent with an important role in cell proliferation and survival. Moreover, specific inactivation of Msi2b full-length indicates that this species is essential for the early role of Msi2b. This line provides a valuable tool both for live imaging of the endogenous Msi2b at subcellular resolution and manipulation of Msi2b-expressing cells.

Laminin ß1a controls distinct steps during the establishment of digestive organ laterality.

Visceral organs, including the liver and pancreas, adopt asymmetric positions to ensure proper function. Yet the molecular and cellular mechanisms controlling organ laterality are not well understood. We identified a mutation affecting zebrafish laminin ß1a (lamb1a) that disrupts left-right asymmetry of the liver and pancreas. In these mutants, the liver spans the midline and the ventral pancreatic bud remains split into bilateral structures. We show that lamb1a regulates asymmetric left-right gene expression in the lateral plate mesoderm (LPM). In particular, lamb1a functions in Kupffer's vesicle (KV), a ciliated organ analogous to the mouse node, to control the length and function of the KV cilia. Later during gut-looping stages, dynamic expression of Lamb1a is required for the bilayered organization and asymmetric migration of the LPM. Loss of Lamb1a function also results in aberrant protrusion of LPM cells into the gut. Collectively, our results provide cellular and molecular mechanisms by which extracellular matrix proteins regulate left-right organ morphogenesis.