An integrated microfluidic device for multiplexed imaging of spatial gene expression patterns of Drosophila embryos.

To reveal the underlying mechanism of the biological function of multicellular systems, it is important to obtain comprehensive spatial gene expression profiles. Among the emerging single-cell spatial-omics techniques, immunofluorescence (IF)-based iterative multiplexed imaging is a promising approach. However, the conventional method is usually costly, time-consuming, labor-intensive, and has low throughput. Moreover, it has yet to be demonstrated in intact multicellular organisms. Here, we developed an integrated microfluidic system to overcome these challenges for quantitatively measuring multiple protein profiles sequentially in situ in the same Drosophila embryo. We designed an array of hydrodynamic trapping sites to automatically capture over ten Drosophila embryos with orientation selectivity at more than 90% trapping rates. We also optimized the geometry of confinement and the on-chip IF protocol to achieve the same high signal-to-noise ratio as the off-chip traditional IF experiments. Moreover, we developed an efficient de-staining protocol by combining on-chip antibody stripping and fluorophore bleaching. Using the same secondary antibody to sequentially stain different genes, we confirmed that the de-stained genes have no detectable interference with the subsequently stained genes, and the gene expression profiles are preserved after multiple cycles of staining and de-staining processes. This preliminary test shows that our newly developed integrated microfluidic system can be a powerful tool for multiplexed imaging of Drosophila embryos. Our work opens a new avenue to design microfluidic chips for multicellular organisms and single-cell spatial-omics techniques.

Quantifying Temperature Compensation of Bicoid Gradients with a Fast T-Tunable Microfluidic Device.

As a reaction-diffusion system strongly affected by temperature, early fly embryos surprisingly show highly reproducible and accurate developmental patterns during embryogenesis under temperature perturbations. To reveal the underlying temperature compensation mechanism, it is important to overcome the challenge in quantitative imaging on fly embryos under temperature perturbations. Inspired by microfluidics generating temperature steps on fly embryos, here we design a microfluidic device capable of ensuring the normal development of multiple fly embryos as well as achieving real-time temperature control and fast temperature switches for quantitative live imaging with a home-built two-photon microscope. We apply this system to quantify the temperature compensation of the morphogen Bicoid (Bcd) gradient in fly embryos. The length constant of the exponential Bcd gradient reaches the maximum at 25°C within the measured temperatures of 18-29°C and gradually adapts to the corresponding value at new temperatures upon a fast temperature switch. The relaxation time of such an adaptation becomes longer if the temperature is switched in a later developmental stage. This age-dependent temperature compensation could be explained if the traditional synthesis-diffusion-degradation model is extended to incorporate the dynamic change of the parameters controlling the formation of Bcd gradients.

The dynamic transmission of positional information in stau- mutants during Drosophila embryogenesis.

It has been suggested that Staufen (Stau) is key in controlling the variability of the posterior boundary of the Hb anterior domain (xHb). However, the mechanism that underlies this control is elusive. Here, we quantified the dynamic 3D expression of segmentation genes in Drosophila embryos. With improved control of measurement errors, we show that the xHb of stau- mutants reproducibly moves posteriorly by 10% of the embryo length (EL) to the wild type (WT) position in the nuclear cycle (nc) 14, and that its variability over short time windows is comparable to that of the WT. Moreover, for stau- mutants, the upstream Bicoid (Bcd) gradients show equivalent relative intensity noise to that of the WT in nc12-nc14, and the downstream Even-skipped (Eve) and cephalic furrow (CF) show the same positional errors as these factors in WT. Our results indicate that threshold-dependent activation and self-organized filtering are not mutually exclusive and could both be implemented in early Drosophila embryogenesis.

Broadly speaking, all individuals of any animal species share a highly consistent shape and structure. Despite this, the activity of the genes that control these body patterns can vary significantly. There are currently two models that have been proposed for how noisy systems of genes, and the proteins they code, can produce consistent body patterns. The first, suggests the noise is essentially self-compensating so stably produces the same result, while the second invokes localized self-organizing systems that help to refine the structural details. In the early stages of development for the fruit fly, Drosophila melanogaster, one of the proteins that controls body patterns is called Hunchback (often just Hb for short). The Hb proteins are largely found at the front-end of the fly embryo, with a sharp drop near the middle. Normally the position of the drop in Hb varies between flies by around 1% of the total length of the fly embryo. Previous work has linked a gene called staufan (or stau for short) to the distribution of Hb in flies but the mechanism involved is unknown. Yang, Zhu, Kong et al. have now used a technique called light sheet microscopy to accurately measure the location of Hb proteins in fruit fly embryos. Without the stau gene, the average position of the drop in Hb proteins underwent a larger shift towards the rear at a key stage in development. Despite this altered behavior, the extent of variation between flies did not change. Similarly, the variation of other genes that control Hb location and that are controlled by Hb remained unchanged. As such, it seems stau affects Hb positioning but has no impact on variation between individuals. These findings suggest that both models for controlling variation in fly development could still be relevant and may operate together. This study also provides a new method for the more precise measurement of systems like these that may offer insights into the mechanisms involved in early embryonic development.