We have optimised experimentally the parameters for imaging chick embryos with µMRI over a wide range of developmental stages from early organogenesis. This complements the recent µMRI atlas of quail embryos created by Ruffins et al. (2007)
and extends it by including data from both younger and older embryos. Other novel features of our study include visualisation of live embryos and the detailed analysis of the musculoskeletal system of the chick wing and leg.
The majority of the chick embryo images shown here are spin echo pulse sequence images because comparison of spin echo and gradient echo images of whole fixed embryos showed that the gradient echo image exhibited the least anatomical information (see ). Selection of different TR and TEs allowed specific regions of the embryo to be highlighted in the spin echo images. Thus for example, in , in an image of the chick abdomen acquired using a 3D spin echo sequence with TR/TE = 200/6.3 ms, the internal cavity of the heart and gizzard is grey whereas with TR/TE = 2000/35 ms (), the same regions are white. This change in image contrast is probably due to the T2 relaxation time of the protons in the cavity being longer than those in the surrounding tissue. The T2 of the water protons is highly dependent on local molecular motion and in tissues where this is restricted (for example in regions of small, tightly packed cells) T2 will be shorter. As TE is increased, image intensity falls off at a rate approximately inversely proportional to the T2. Image intensities in short TE and long TR images approximate to relative proton densities (the amount of water present). In long TE images, the relative intensities are weighted by the T2 of the water protons, thus, as might be expected, the behaviour of the MRI image suggests that associated water moves more freely in the cavities than in the surrounding tissues and therefore the relative grey levels are exchanged. gives an indication of the optimum parameters we used to image different tissues.
Parameters used to highlight different tissue types
The best anatomical detail in fixed chick embryo images was obtained using the technique previously described for mouse embryos (Schneider et al. 2003
). Six-day-old chick embryos were soaked in fixative containing the Gd contrast agent and then mounted in agar containing Gd contrast agent and left for at least 3 days before imaging. This allowed the Gd contrast agent to perfuse the sample and selectively collect in the organs, producing good anatomical image contrast (). Although µMRI cannot achieve the image resolution of optical microscopy, our use of a spin echo imaging protocol has allowed the visualisation of most aspects of the gross anatomy albeit not all of the detail seen in histological sections in the chick atlas (Bellairs & Osmond, 2005
). Our preliminary attempts to image 3-day chick embryos were not so successful, although it seems likely that contrast agents will help in visualising early stages in chick development.
MRI is a non-invasive, non-destructive technique and therefore there is the prospect of being able to use µMRI to follow development of living embryos over time. Here we have described parameters for producing detailed anatomical images of first unfixed embryos and then living quail embryos following injection of contrast agents into the egg. So far, the earliest in ovo embryos we have examined have been around 7 days. We would like to use MRI to monitor effects of experimental manipulations on limb development in real time. However, operations, such as implantation of a retinoic acid bead described here, are typically being carried out in 3-day-old embryos, and the challenge will be to improve the imaging of much younger embryos. In this study, the aim was to reveal anatomy and to visualise the live embryo; no observations were made as to any detrimental effects on development which might have been caused by the contrast agent and this, too, must be examined during future studies.
We have shown the potential of MRI for imaging both normal and manipulated chick limb anatomy. The complex tissue anatomy of the chick limb was revealed by using several different MRI acquisition protocols. Separation of the different muscle groups was found to be best visualised using gradient echo protocols whereas spin echo protocols with specific TR
conditions highlighted different connective tissues. Thus, a long TE
was also found to highlight mineralised bone in the chick limb (see ), whereas a shorter TE
highlighted cartilage. Intuitively one might expect the mineralised bone to have a very short T2
(and this is often the case, see Thompson & Chudek, 2007
) but here the T2
of the embryonic bone must be very long to give such a bright image with TE
= 40 ms. One explanation would be that the bone at this stage is still very porous and that the water contained in these pores has a high degree of mobility.
We have demonstrated that, by using a 3D data analysis programme to merge images of the same limb acquired under different MRI conditions, the visualisation of the complex anatomy of different tissues – bone, cartilage and muscle – can be optimised. The anatomy of the duplicated chick wing showed some interesting features. For example, the detailed skeletal anatomy of the epiphyses of the forearm elements showed that the proximal epiphysis of the anterior element resembles that of the radius, whereas the distal epiphysis resembles that of an ulna. It has been well established that retinoic acid can affect not only the pattern of the digits but also the pattern of the forearm (Robson et al. 1994
). Examination of the muscle pattern in the same wing also provided evidence for a duplicated forearm pattern, in that posterior muscles developed ventrally on both sides. Interestingly, in the dorsal part of the wing, the muscle pattern looked more normal. In this context, previous analysis of the effects of experimental manipulations of the chick wing also highlighted dorso-ventral differences in the response to patterning signals and that skeletal and muscle patterns are not always strictly congruent with each other (Akita, 1996
). The ability to visualise detailed anatomy of bone, cartilage and muscle in the same manipulated chick wing using µMRI will permit more extensive investigation of larger numbers of such manipulated limbs and will give new information about the extent to which patterning of these different tissues is co-ordinated during development.
Previous work using µMRI has shown that it is also possible to visualise the vasculature of chick embryos by injecting Gd contrast agent (Smith, 2000
). It also seems likely that manganese based contrast agents could be utilised to visualise nerves (Louie, 2005
). Therefore, it should be possible to work towards producing very high quality anatomical detail of all the tissues of the limb. This methodology can also be applied to other regions of the chick embryo and thus maximise the usefulness of avian embryos in the experimental analysis of development.