Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways

The transcriptional effector SMAD4 is a core component of the TGF-β family signaling pathways. However, its role in vertebrate embryo development remains unresolved. To address this, we deleted Smad4 in zebrafish and investigated the consequences of this on signaling by the TGF-β family morphogens, BMPs and Nodal. We demonstrate that in the absence of Smad4, dorsal/ventral embryo patterning is disrupted due to the loss of BMP signaling. However, unexpectedly, Nodal signaling is maintained, but lacks robustness. This Smad4-independent Nodal signaling is sufficient for mesoderm specification, but not for optimal endoderm specification. Furthermore, using Optical Projection Tomography in combination with 3D embryo morphometry, we have generated a BMP morphospace and demonstrate that Smad4 mutants are morphologically indistinguishable from embryos in which BMP signaling has been genetically/pharmacologically perturbed. Smad4 is thus differentially required for signaling by different TGF-β family ligands, which has implications for diseases where Smad4 is mutated or deleted.

Single-Shot Optical Projection tomography for high-speed volumetric imaging

We present a single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic samples. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio but the sequential acquisition of projection images required for volumetric reconstruction typically requires samples to be immobilised during the acquisition of an OPT data set. We present a proof-of-principle system capable of single-shot imaging of a 1 mm diameter volume, demonstrating camera-limited rates of up to 62.5 volumes/second, which we have applied to 3D imaging of a freely-swimming zebrafish embryo. This is achieved by recording 8 projection views simultaneously on 4 low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under 5,000GBP. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including fluid dynamics.

Optical projection tomography as a quantitative tool for analysis of cell morphology and density in 3D hydrogels

Assessing cell morphology and function, as well as biomaterial performance in cell cultures, is one of the key challenges in cell biology and tissue engineering (TE) research. In TE, there is an urgent need for methods to image actual three-dimensional (3D) cell cultures and access the living cells. This is difficult using established optical microscopy techniques such as wide-field or confocal microscopy. To address the problem, we have developed a new protocol using Optical Projection Tomography (OPT) to extract quantitative and qualitative measurements from hydrogel cell cultures. Using our tools, we demonstrated the method by analyzing cell response in three different hydrogel formulations in 3D with 1.5 mm diameter samples of: gellan gum (GG), gelatin functionalized gellan gum (gelatin-GG), and Geltrex. We investigated cell morphology, density, distribution, and viability in 3D living cells. Our results showed the usability of the method to quantify the cellular responses to biomaterial environment. We observed that an elongated morphology of cells, thus good material response, in gelatin-GG and Geltrex hydrogels compared with basic GG. Our results show that OPT has a sensitivity to assess in real 3D cultures the differences of cellular responses to the properties of biomaterials supporting the cells.

Frame localisation optical projection tomography

We present a tomographic reconstruction algorithm (flOPT), which is applied to Optical Projection Tomography (OPT) images, that is robust to mechanical jitter and systematic angular and spatial drift. OPT relies on precise mechanical rotation and is less mechanically stable than large-scale computer tomography (CT) scanning systems, leading to reconstruction artefacts. The algorithm uses multiple (5+) tracked fiducial beads to recover the sample pose and the image rays are then back-projected at each orientation. The quality of the image reconstruction using the proposed algorithm shows an improvement when compared to the Radon transform. Moreover, when adding a systematic spatial and angular mechanical drift, the reconstruction shows a significant improvement over the Radon transform.