Profile of a population-based diabetic macular oedema study: the Liverpool Eye and Diabetes Study (Sydney).

PURPOSE: The population prevalence of diabetic macular oedema (DME) is unclear. Previous estimates have depended on photographic grading of clinically significant macular oedema, which is subjective and has resulted in widely varying estimates. With the advent of optical coherence tomography (OCT), the presence and severity of DME can now be assessed objectively and accurately. METHODS: The Liverpool Eye and Diabetes Study (LEADS) is a cross-sectional population-based study of patients with type 1 and type 2 diabetes in a multi-ethnic region of Sydney, Australia, to determine the population prevalence of OCT-defined DME, how this varies by ethnicity and association with systemic factors. This report describes the rationale, methodology and study aims. RESULTS: To date 646 patients out of an expected sample size of 2000 have been recruited. Baseline data are presented for patients with type 1 (n=75, 11.8%) and type 2 (n=562, 88.2%) diabetes recruited to date. Patients with type 1 diabetes were younger (39.5vs60.7 years), with longer duration of diabetes (18.1vs11.7 years), slightly worse glycaemic control (HbA1c 9.0vs8.3), and less likely to have hypertension (30.7vs71.4%), hypercholesterolaemia (33.3vs74.6%) and obesity (31.1vs51.5%, respectively, all p<0.05). CONCLUSIONS: The LEADS will provide objective estimates of the population prevalence of DME, how this varies with ethnicity and associations with systemic disease.

Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation.

During mouse gastrulation, endoderm cells of the dorsal foregut are recruited ahead of the ventral foregut and move to the anterior region of the embryo via different routes. Precursors of the anterior-most part of the foregut and those of the mid- and hind-gut are allocated to the endoderm of the mid-streak-stage embryo, whereas the precursors of the rest of the foregut are recruited at later stages of gastrulation. Loss of Mixl1 function results in reduced recruitment of the definitive endoderm, and causes cells in the endoderm to remain stationary during gastrulation. The observation that the endoderm cells are inherently unable to move despite the expansion of the mesoderm in the Mixl1-null mutant suggests that the movement of the endoderm and the mesoderm is driven independently of one another.

Regionalization of cell fates and cell movement in the endoderm of the mouse gastrula and the impact of loss of Lhx1(Lim1) function.

Investigation of the developmental fates of cells in the endodermal layer of the early bud stage mouse embryo revealed a regionalized pattern of distribution of the progenitor cells of the yolk sac endoderm and the embryonic gut. By tracing the site of origin of cells that are allocated to specific regions of the embryonic gut, it was found that by late gastrulation, the respective endodermal progenitors are already spatially organized in anticipation of the prospective mediolateral and anterior-posterior destinations. The fate-mapping data further showed that the endoderm in the embryonic compartment of the early bud stage gastrula still contains cells that will colonize the anterior and lateral parts of the extraembryonic yolk sac. In the Lhx1(Lim1)-null mutant embryo, the progenitors of the embryonic gut are confined to the posterior part of the endoderm. In particular, the prospective anterior endoderm was sequestered to a much smaller distal domain, suggesting that there may be fewer progenitor cells for the anterior gut that is poorly formed in the mutant embryo. The deficiency of gut endoderm is not caused by any restriction in endodermal potency of the mutant epiblast cells but more likely the inadequate allocation of the definitive endoderm. The inefficient movement of the anterior endoderm, and the abnormal differentiation highlighted by the lack of Sox17 and Foxa2 expression, may underpin the malformation of the head of Lhx1 mutant embryos.

Cited1 is required in trophoblasts for placental development and for embryo growth and survival.

Cited1 is a transcriptional cofactor that interacts with Smad4, estrogen receptors alpha and beta, TFAP2, and CBP/p300. It is expressed in a restricted manner in the embryo as well as in extraembryonic tissues during embryonic development. In this study we report the engineering of a loss-of-function Cited1 mutation in the mouse. Cited1 null mutants show growth restriction at 18.5 days postcoitum, and most of them die shortly after birth. Half the heterozygous females, i.e., those that carry a paternally inherited wild-type Cited1 allele, are similarly affected. Cited1 is normally expressed in trophectoderm-derived cells of the placenta; however, in these heterozygous females, Cited1 is not expressed in these cells. This occurs because Cited1 is located on the X chromosome, and thus the wild-type Cited1 allele is not expressed because the paternal X chromosome is preferentially inactivated. Loss of Cited1 resulted in abnormal placental development. In mutants, the spongiotrophoblast layer is irregular in shape and enlarged while the labyrinthine layer is reduced in size. In addition, the blood spaces within the labyrinthine layer are disrupted; the maternal sinusoids are considerably larger in mutants, leading to a reduction in the surface area available for nutrient exchange. We conclude that Cited1 is required in trophoblasts for normal placental development and subsequently for embryo viability.

Conserved requirement of Lim1 function for cell movements during gastrulation.

To investigate Lim1 function during gastrulation, we used transcript depletion through DEED antisense oligonucleotides in Xenopus and cell transplantation in mice. Xenopus embryos depleted of Lim1 lack anterior head structures and fail to form a proper axis as a result of a failure of gastrulation movements, even though mesodermal cell identities are specified. Similar disruption of cell movements in the mesoderm is also observed in Lim1(-/-) mice. Paraxial protocadherin (PAPC) expression is lost in the nascent mesoderm of Lim1(-/-) mouse embryos and in the organizer of Lim1-depleted Xenopus embryos; the latter can be rescued to a considerable extent by supplying PAPC exogenously. We conclude that a primary function of Lim1 in the early embryo is to enable proper cell movements during gastrulation.

Introduction of cell markers into germ layer tissues of the mouse gastrula by whole embryo electroporation.

We have optimized the technique of electroporation for introducing genetic markers into cells of the gastrulating mouse embryo to follow cell fates, tissue movement, and lineage differentiation. Using a plate-needle electrode combination and specific route of plasmid delivery, labeling could be targeted to discrete regions of the epiblast or the endoderm of the late gastrula. Among the various types of fluorescent and chromogenic reporter constructs tested, those driven by CMV promoter are efficient and strong expression can be detected as soon as 2-3 h after electroporation. The efficacy of marking cell lineages by CRE-mediated activation of reporters proved to be inefficient for tracking cell lineages due to an obligatory 8-9-h lag from the electroporation of constructs to the expression of reporter. This significant time lag also raises concern of the temporal precision at which tissue- or stage-specific knock-out or activation of genetic activity may be achieved by the Cre-loxP mechanism.