Daniel W. Stuckey

BMP signalling inhibits premature neural differentiation in the mouse embryo

The specification of a subset of epiblast cells to acquire a neural fate constitutes the first step in the generation of the nervous system. Little is known about the signals required for neural induction in the mouse. We have analysed the role of BMP signalling in this process. We demonstrate that prior to gastrulation, Bmp2/4 signalling via Bmpr1a maintains epiblast pluripotency and prevents precocious neural differentiation of this tissue, at least in part by maintaining Nodal signalling. We find that during gastrulation, BMPs of the 60A subgroup cooperate with Bmp2/4 to maintain pluripotency. The inhibition of neural fate by BMPs is independent of FGF signalling, as inhibition of FGF signalling between 5.5 and 7.5 days post-coitum does not block neural differentiation in the mouse embryo. Together, our results demonstrate that inhibition of BMP signalling has a central role during neural induction in mammals and suggest that FGFs do not act as neural inducers in the post-implantation mouse embryo.

FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.

In vivo fluorescence lifetime tomography of a FRET probe expressed in mouse

Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct.

Coordination of cell proliferation and anterior-posterior axis establishment in the mouse embryo

During development, the growth of the embryo must be coupled to its patterning to ensure correct and timely morphogenesis. In the mouse embryo, migration of the anterior visceral endoderm (AVE) to the prospective anterior establishes the anterior-posterior (A-P) axis. By analysing the distribution of cells in S phase, M phase and G2 from the time just prior to the migration of the AVE until 18 hours after its movement, we show that there is no evidence for differential proliferation along the A-P axis of the mouse embryo. Rather, we have identified that as AVE movements are being initiated, the epiblast proliferates at a much higher rate than the visceral endoderm. We show that these high levels of proliferation in the epiblast are dependent on Nodal signalling and are required for A-P establishment, as blocking cell division in the epiblast inhibits AVE migration. Interestingly, inhibition of migration by blocking proliferation can be rescued by Dkk1. This suggests that the high levels of epiblast proliferation function to move the prospective AVE away from signals that are inhibitory to its migration. The finding that initiation of AVE movements requires a certain level of proliferation in the epiblast provides a mechanism whereby A-P axis development is coordinated with embryonic growth.

Fluorescence lifetime readouts of Troponin-C-based calcium FRET sensors: a quantitative comparison of CFP and mTFP1 as donor fluorophores.

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.

Correct patterning of the primitive streak requires the anterior visceral endoderm.

Anterior-posterior axis specification in the mouse requires signalling from a specialised extra-embryonic tissue called the anterior visceral endoderm (AVE). AVE precursors are induced at the distal tip of the embryo and move to the prospective anterior. Embryological and genetic analysis has demonstrated that the AVE is required for anterior patterning and for correctly positioning the site of primitive streak formation by inhibiting Nodal activity. We have carried out a genetic ablation of the Hex-expressing cells of the AVE (Hex-AVE) by knocking the Diphtheria toxin subunit A into the Hex locus in an inducible manner. Using this model we have identified that, in addition to its requirement in the anterior of the embryo, the Hex-AVE sub-population has a novel role between 5.5 and 6.5dpc in patterning the primitive streak. Embryos lacking the Hex-AVE display delayed initiation of primitive streak formation and miss-patterning of the anterior primitive streak. We demonstrate that in the absence of the Hex-AVE the restriction of Bmp2 expression to the proximal visceral endoderm is also defective and expression of Wnt3 and Nodal is not correctly restricted to the posterior epiblast. These results, coupled with the observation that reducing Nodal signalling in Hex-AVE ablated embryos increases the frequency of phenotypes observed, suggests that these primitive streak patterning defects are due to defective Nodal signalling. Together, our experiments demonstrate that the AVE is not only required for anterior patterning, but also that specific sub-populations of this tissue are required to pattern the posterior of the embryo.

Forster resonance energy transfer imaging in vivo with approximated radiative transfer equation

We describe a new light transport model, which was applied to three-dimensional lifetime imaging of Förster resonance energy transfer in mice in vivo. The model is an approximation to the radiative transfer equation and combines light diffusion and ray optics. This approximation is well adopted to wide-field time-gated intensity-based data acquisition. Reconstructed image data are presented and compared with results obtained by using the telegraph equation approximation. The new approach provides improved recovery of absorption and scattering parameters while returning similar values for the fluorescence parameters.

Tomographic imaging of flourescence resonance energy transfer in highly light scattering media

Three-dimensional localization of protein conformation changes in turbid media using Förster Resonance Energy Transfer (FRET) was investigated by tomographic fluorescence lifetime imaging (FLIM). FRET occurs when a donor fluorophore, initially in its electronic excited state, transfers energy to an acceptor fluorophore in close proximity through non-radiative dipole-dipole coupling. An acceptor effectively behaves as a quencher of the donors fluorescence. The quenching process is accompanied by a reduction in the quantum yield and lifetime of the donor fluorophore. Therefore, FRET can be localized by imaging changes in the quantum yield and the fluorescence lifetime of the donor fluorophore. Extending FRET to diffuse optical tomography has potentially important applications such as in vivo studies in small animal. We show that FRET can be localized by reconstructing the quantum yield and lifetime distribution from time-resolved non-invasive boundary measurements of fluorescence and transmitted excitation radiation. Image reconstruction was obtained by an inverse scattering algorithm. Thus we report, to the best of our knowledge, the first tomographic FLIM-FRET imaging in turbid media. The approach is demonstrated by imaging a highly scattering cylindrical phantom concealing two thin wells containing cytosol preparations of HEK293 cells expressing TN-L15, a cytosolic genetically-encoded calcium FRET sensor. A 10mM calcium chloride solution was added to one of the wells to induce a protein conformation change upon binding to TN-L15, resulting in FRET and a corresponding decrease in the donor fluorescence lifetime. The resulting fluorescence lifetime distribution, the quantum efficiency, absorption and scattering coefficients were reconstructed.

tomoFLIM - fluorescence lifetime projection tomography

Optical Projection Tomography (OPT) is a wide-field technique for measuring the threedimensional distribution of absorbing/fluorescing species in non-scattering (optically cleared) samples up to ~1cm in size, and as such is the optical analogue of X-ray computed tomography. We have extended the intensity-based OPT technique to measure the three-dimensional fluorescence lifetime distribution (tomoFLIM) in transparent samples. Due to its inherent ratiometric nature, fluorescence lifetime measurements are robust against intensity-based artifacts as well as producing a quantitative measure of the fluorescence signal, making it particularly suited to Förster Resonance Energy Transfer (FRET) measurements. We implement tomoFLIM via OPT by acquiring a series of wide-field time-gated images at different relative time delays with respect to a train of excitation pulses for a range of projection angles. For each time delay, the three-dimensional time-gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime is subsequently determined for each reconstructed horizontal plane by iterative fitting of an appropriate decay model. We present a tomographic reconstruction of a fluorescence lifetime resolved FRET calcium contruct, TN-L15 cytosol suspension, in a silicone phantom. This genetically encoded sensor, TN-L15, comprises the calcium-binding domain of Troponin C, flanked by the fluorophores cyan fluorescent protein and citrine. In the presence of calcium ions TN-L15 changes conformation bringing the two fluorophores into close proximity, resulting in FRET. We also present autofluorescence and fluorescently labelled tomoFLIM reconstructions of chick embryos, including a genetically encoded fluorophore TagRFP-T. The fluorophore was electroporated in ovo into the neural tube of the embryos, which were subsequently dissected two days post-electroporation, fixed in ethanol and optically cleared for OPT/tomoFLIM acquisition. The reconstructed 3-D fluorescence lifetime image provides contrast between the genetically labelled TagRFP-T and the emitted autofluorescence.