RESUMO
Confocal micro X-ray fluorescence (CMXRF) spectroscopy is a non-destructive, depth-resolved, and element-specific technique that is used to analyze the elemental composition of a sample. For this, a focused beam of mono- or polychromatic X-rays is applied to excite the atoms in the sample, causing them to emit fluorescence radiation which is detected with focusing capillary optics. The confocal design of the instrument allows for depth-resolved analysis, in most cases with a resolution in the lower micrometer dimension after collecting X-rays from a predefined volume within the sample. The element-specific nature of the technique allows information to be obtained about the presence and concentration of specific elements in this volume. This makes CMXRF spectroscopy a valuable tool for a wide range of applications, especially when samples with an inhomogeneous distribution of elements and a relatively light matrix have to be analyzed, which are typical examples in materials science, geology, and biology. The technique is also commonly used in the art and archaeology fields to analyze the elemental composition of historical artifacts and works of art, helping to provide valuable insights into their provenance, composition, and making. Recent technical developments to increase sensitivity and efforts to improve quantification in three-dimensional samples will encourage wider use of this method across a multitude of fields of application in the near future. Confocal micro X-ray fluorescence (CMXRF) is based on the confocal overlap of two polycapillary lens foci, creating a depth-sensitive and non-destructive probing volume. Three-dimensional resolved element distribution images can be obtained by measuring the fluorescence intensity as function of the three-dimensional position.
RESUMO
Congenital dyserythropoietic anemia type I (CDA I) is an autosomal recessive disease characterized by moderate to severe macrocytic anemia and pathognomonic morphologic abnormalities of the erythroid precursors, including spongy heterochromatin. The disease is mainly caused by mutations in CDAN1 (encoding for Codanin-1). No patients with homozygous null type mutations have been described, and mouse null mutants die during early embryogenesis prior to the initiation of erythropoiesis. The cellular functions of Codanin-1 and the erythroid specificity of the phenotype remain elusive. To investigate the role of Codanin-1 in erythropoiesis, we crossed mice carrying the Cdan1 floxed allele (Cdan fl/fl ) with mice expressing Cre-recombinase under regulation of the erythropoietin receptor promoter (ErGFPcre). The resulting CdanΔEry transgenic embryos died at mid-gestation (E12.5-E13.5) from severe anemia, with very low numbers of circulating erythroblast. Transmission electron microscopy studies of primitive erythroblasts (E9.5) revealed the pathognomonic spongy heterochromatin. The morphology of CdanΔEry primitive erythroblasts demonstrated progressive development of dyserythropoiesis. Annexin V staining showed increases in both early and late-apoptotic erythroblasts compared to controls. Flow cytometry studies using the erythroid-specific cell-surface markers CD71 and Ter119 demonstrated that CdanΔEry erythroid progenitors do not undergo the semi-synchronous maturation characteristic of primitive erythroblasts. Gene expression studies aimed to evaluate the effect of Cdan1 depletion on erythropoiesis revealed a delay of ζ to α globin switch compared to controls. We also found increased expression of Gata2, Pu.1, and Runx1, which are known to inhibit terminal erythroid differentiation. Consistent with this data, our zebrafish model showed increased gata2 expression upon cdan1 knockdown. In summary, we demonstrated for the first time that Cdan1 is required for primitive erythropoiesis, while providing two experimental models for studying the role of Codanin-1 in erythropoiesis and in the pathogenesis of CDA type I.