## 2 Applications of HREM
### HREM in Embryo Research
#### 2.1.1 Mouse Embryos  
Moderate breeding costs, short generation times and the existence of sophisticated molecular tools to manipulate the mouse genome facilitate efficient engineering of mutant mouse lines by random or targeted disruption of specific genes. Due to the conservation of basic developmental mechanisms and gene function, such lines are then used for studying normal mammalian gene function and the genetic components of diseases. Identifying morphological defects of mouse embryos of genetically engineered mouse lines is an important step to characterise the function of the disrupted gene during embryo development and to define its role in the genesis of hereditary diseases and malformations. HREM was developed to permit the production of 3D volume data of whole E14.5 mouse embryos in a resolution and quality to fit for a holistic qualitative and quantitative analysis of embryo morphology, organ, blood vessel and nerve topology and tissue architecture as shown in Figure 2. In the scope of DMDD [37], which was linked with the International Mouse Phenotyping Consortium (IMPC), HREM was employed to build a virtual resource of fully annotated phenotype data of over 200 normal and over 500 mutant mouse embryos harvested at E14.5 stemming from 87 individual single knock-out or knock-down lines producing lethal or subvital homozygous offspring. Figure 2: 3D visualisation of embryo material. (**A**–**E**). Embryos harvested at embryonic day 14.5 in the scope of the Deciphering the Mechanisms of Developmental Disorders (DMDD) project. Opaque and semitransparent volume models from ventral view (**A**–**D**). Note the intrinsic contrast of the blood-filled liver vasculature inferior vena cava (ivc) and the atria (at) in (**B**) and (**C**). In (**D**), the opaque volume model is coronally sectioned and the stapedal artery (sa) penetrating the developing stages (st) is zoomed in on in the inlay to the top. The bottom inlay provides the aortic valve as detailed as it appears in the coronal 2D resection used as a sectioning plane. (**E**) shows a sagittally sectioned semitransparent volume model from lateral, integrating surface models of the urogenital tract. Choroid plexus (cp) and commissural fibres (cf) are detailed in the zoom-in displayed as an inlay to the top right. The bottom inlay shows a cranial and oblique view of the surface models of the ovaries (violet), ureter, urinary bladder and urethra (yellow), Müller duct (light blue) and Wolff duct (blue). (**F**–**H**). Chick embryo at developmental stage 18 according to [PERSON] and Hamilton. Opaque volume model from the ventrolateral in (**F**) and (**G**) with a zoom-in on the virtually sectioned heart tube (ht) in (**G**). (**H**) shows the virtual 2D resection of the cutting plane in (**G**). ve, ventricles; li, liver; hb, hindbrain; co, cochlea; sv, semilunar valves; rlu, right lung; llu, left lung; te, telencephalon; di, diencephalon; sc, spinal chord; ns, nasal septum; to, tongue; la, larynx; uv, umbilical vein; I, intestine; pa, pharyngeal arches; le, lens; ub, bud of upper limb; lb, bud of lower limb; ec, endocardial cushion. Scale bars: 500 \(\mu\)m.  
In the scope of this program, a standardised and ergonomic protocol for the comprehensive scoring of the morphologic phenotype of E14.5 embryos [38], a novel system for defining developmental substages of E14.5 mouse embryos [39], and reference data for the correct interpretation of phenotype abnormalities and for distinguishing variations from abnormalities [40] were created. Approximately 200 novel mouse phenotype (MP) terms were added to the MP ontology list as a result of HREM allowing for the detection of details that could not be detected with alternative imaging techniques. Numerous publications in highly ranked journals were produced based on HREM data generated in DMDD [38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48].