Both oriented cell divisions and cell rearrangements are critical for proper embryogenesis and organogenesis. However, little is known about how these two cellular events are integrated. Here we examine the linkage between these processes in chick limb cartilage. By combining retroviral-based multicolor clonal analysis with live imaging, the results show that single chondrocyte precursors can generate both single-column and multi-column clones through oriented division followed by cell rearrangements. Focusing on single column formation, we show that this stereotypical tissue architecture is established by a pivot-like process between sister cells. After mediolateral cell division, N-cadherin is enriched in the post-cleavage furrow; then one cell pivots around the other, resulting in stacking into a column. Perturbation analyses demonstrate that planar cell polarity signaling enables cells to pivot in the direction of limb elongation via this N-cadherin-mediated coupling. Our work provides new insights into the mechanisms generating appropriate tissue architecture of limb skeleton.

Fluorescence lifetime imaging microscopy (FLIM) with phasor analysis provides easy visualization and analysis of fluorophores’ lifetimes which is valuable for multiple applications including metabolic imaging, STED imaging, FRET imaging and functional imaging. However, FLIM imaging typically suffers from low photon budgets, leading to unfavorable signal to noise ratios which in many cases prevent extraction of information from the data. Traditionally, median filters are applied in phasor analysis to tackle this problem. This unfortunately degrades high spatial frequency FLIM information in the phasor analysis. These high spatial frequency components are typically edges of features and puncta, which applies to membranes, mitochondria, granules and small organelles in a biological sample. To tackle this problem, we propose a filtering strategy with complex wavelet filtering and Anscombe transform for FLIM phasor analysis. This filtering strategy preserves fine structures and reports accurate lifetimes in photon starved FLIM imaging. Moreover, this filter outperforms median filters and makes FLIM imaging with lower laser power and faster imaging possible.

Significance: An integrative approach is presented for studying cell biology in vivo, assessing protein dynamics and cell behavior, and offering in situ analyses of cytokinesis, daughter-cell polarity, and stereotyped tissue morphogenesis. Tagging endogenous Discs Large 1 (Dlg1) in cartilage using intrabody technology permits in situ 3D time-lapse imaging and reveals Dlg1 enrichment at the midbody during cytokinesis. Functional significance is tested by disrupting Dlg1 multimerization and its midbody localization by using an ablating intrabody, DLGE3. Building on prior work on Dlg1 in epithelia, our work reveals that Dlg1 propagates cell polarity in proliferative mesenchymal tissues and suggests that multiple mechanisms act in concert at distinct phases of the cell cycle to transmit and maintain cell polarity.

Fluorescence lifetime imaging microscopy (FLIM) evaluates the metabolic state of tissue based on reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) can image the fundus of the eyes, but cannot detect NAD(P)H. We used multiphoton FLIM to study the metabolic state of the retina in fixed eyes of wild-type mice C57BL6/J. We sectioned the eye using a polyacrylamide gel-embedding technique and estimated the percentage of bound NAD(P)H. We found that oxidative phosphorylation was the predominant metabolic state, particularly in the inner retina, when a fixed retina was used. We also demonstrated the feasibility of FAD imaging of the retina. In addition, we demonstrated that autofluorescence and various FLIM channels, such as hemoglobin, melanin and collagen, can be used to evaluate the structure of the retina and other parts of the eye without any special staining.