Mouse models are a critical tool for studying human diseases, particularly developmental disorders¹. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse². Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing³ to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions^4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be ‘decomposable’ through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

Bats are the only mammals capable of self-powered flight, an evolutionary innovation based on the transformation of forelimbs into wings. The bat wing is characterized by an extreme elongation of the second to fifth digits with a wing membrane called the chiropatagium connecting them. Here we investigated the developmental and cellular origin of this structure by comparing bat and mouse limbs using omics tools and single-cell analyses. Despite the substantial morphological differences between the species, we observed an overall conservation of cell populations and gene expression patterns including interdigital apoptosis. Single-cell analyses of micro-dissected embryonic chiropatagium identified a specific fibroblast population, independent of apoptosis-associated interdigital cells, as the origin of this tissue. These distal cells express a conserved gene programme including the transcription factors MEIS2 and TBX3, which are commonly known to specify and pattern the early proximal limb. Transgenic ectopic expression of MEIS2 and TBX3 in mouse distal limb cells resulted in the activation of genes expressed during wing development and phenotypic changes related to wing morphology, such as the fusion of digits. Our results elucidate fundamental molecular mechanisms of bat wing development and illustrate how drastic morphological changes can be achieved through repurposing of existing developmental programmes during evolution.

The placenta is the first embryonic organ, representing the connection between the embryo and the mother, and is therefore necessary for the embryo’s growth and survival. To meet the ever-growing need for nutrient and gas exchange, the maternal spiral arteries undergo extensive remodeling, thus increasing the uteroplacental blood flow by 16-fold. However, the insufficient remodeling of the spiral arteries can lead to severe pregnancy-associated disorders, including but not limited to pre-eclampsia. Insufficient endovascular trophoblast invasion plays a key role in the manifestation of pre-eclampsia; however, the underlying processes are complex and still unknown. Classical histopathology is based on two-dimensional section microscopy, which lacks a volumetric representation of the vascular remodeling process. To further characterize the uteroplacental vascularization, a detailed, non-destructive, and subcellular visualization is beneficial. In this study, we use light sheet microscopy for optical sectioning, thus establishing a method to obtain a three-dimensional visualization of the vascular system in the placenta. By introducing a volumetric visualization method of the placenta, we could establish a powerful tool to deeply investigate the heterogeneity of the spiral arteries during the remodeling process, evaluate the state-of-the-art treatment options, effects on vascularization, and, ultimately, reveal new insights into the underlying pathology of pre-eclampsia.

High-quality three-dimensional (3D) microscopy allows detailed, unrestricted and non-destructive imaging of entire volumetric tissue specimens and can therefore increase the diagnostic accuracy of histopathological tissue analysis. However, commonly used IgG antibodies are oftentimes not applicable to 3D imaging, due to their relatively large size and consequently inadequate tissue penetration and penetration speed. The lack of suitable reagents for 3D histopathology can be overcome by an emerging class of single-domain antibodies, referred to as nanobodies (Nbs), which can facilitate rapid and superior 2D and 3D histological stainings. Here, we report the generation and experimental validation of Nbs directed against the human endothelial cell-selective adhesion molecule (hESAM), which enables spatial visualization of blood vascular networks in whole-mount 3D imaging. After analysis of Nb binding properties and quality, selected Nb clones were validated in 2D and 3D imaging approaches, demonstrating comparable staining qualities to commercially available hESAM antibodies in 2D, as well as rapid and complete staining of entire specimens in 3D. We propose that the presented hESAM-Nbs can serve as novel blood vessel markers in academic research and can potentially improve 3D histopathological diagnostics of entire human tissue specimens, leading to improved treatment and superior patient outcomes.

Pathogenic variants in kinesin KIF11 underlie microcephaly-lymphedema-chorioretinopathy (MLC) syndrome. Although well known for regulating spindle dynamics ensuring successful cell division, the association of KIF11 (encoding EG5) with development of the lymphatic system and how KIF11 pathogenic variants lead to lymphatic dysfunction and lymphedema remain unknown. Using patient-derived lymphoblastoid cells, we demonstrated that patients with MLC carrying pathogenic stop-gain variants in KIF11 have reduced mRNA and protein levels. Lymphoscintigraphy showed reduced tracer absorption, and intestinal lymphangiectasia was detected in one patient, pointing to impairment of lymphatic function caused by KIF11 haploinsufficiency. We revealed that KIF11 is expressed in early human and mouse development with the lymphatic markers VEGFR3, podoplanin, and PROX1. In zebrafish, single-cell RNA-Seq identified kif11 specifically expressed in endothelial precursors. In human lymphatic endothelial cells, EG5 inhibition with ispinesib reduced VEGFC-driven AKT phosphorylation, migration, and spheroid sprouting. KIF11 knockdown reduced PROX1 and VEGFR3 expression, providing for the first time to our knowledge a link between KIF11 and drivers of lymphangiogenesis and lymphatic identity.

Superficial erythematous cutaneous vascular malformations are assumed to be blood vascular in origin, but cutaneous lymphatic malformations can contain blood and appear red. Management may be different and so an accurate diagnosis is important. Cutaneous malformations were investigated through 2D histology and 3D whole-mount histology. Two lesions were clinically considered as port-wine birthmarks and another 3 lesions as erythematous telangiectasias. The aims were (i) to demonstrate that cutaneous erythematous malformations including telangiectasia can represent a lymphatic phenotype, (ii) to determine if lesions represent expanded but otherwise normal or malformed lymphatics, and (iii) to determine if the presence of erythrocytes explained the red color. Microscopy revealed all lesions as lymphatic structures. Port-wine birthmarks proved to be cystic lesions, with nonuniform lymphatic marker expression and a disconnected lymphatic network suggesting a lymphatic malformation. Erythematous telangiectasias represented expanded but nonmalformed lymphatics. Blood within lymphatics appeared to explain the color. Blood-lymphatic shunts could be detected in the erythematous telangiectasia. In conclusion, erythematous cutaneous capillary lesions may be lymphatic in origin but clinically indistinguishable from blood vascular malformations. Biopsy is advised for correct phenotyping and management. Erythrocytes are the likely explanation for color accessing lymphatics through lympho-venous shunts.