Rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3) is a cytoskeletal protein that promotes directional cell migration and angiogenesis in vitro and is implicated in human carcinomas and coronary artery disease. To study the role of Rudhira during development in vivo, we generated the first knockout mouse for rudhira and show that Rudhira is essential for mouse development. Rudhira null embryos die at embryonic day (E) 9.5 accompanied by severe vascular patterning defects in embryonic and extra-embryonic tissues. To identify the molecular processes downstream of rudhira, we analyzed the transcriptome of intact knockout yolk sacs. Genome-wide transcriptome analysis showed that Rudhira functions in angiogenesis and its related processes such as cell adhesion, extracellular matrix organization, peptidase activity and TGFβ signaling. Since Rudhira is also expressed in endothelial cells (ECs), we further generated Tie2Cre-mediated endothelial knockout (CKO) of rudhira. CKO embryos survive to E11.5 and similar to the global knockout, display gross vascular patterning defects, showing that endothelial Rudhira is vital for development. Further, Rudhira knockdown ECs in culture fail to sprout in a spheroid-sprouting assay, strongly supporting its role in vascular patterning. Our study identifies an essential role for Rudhira in blood vessel remodeling and provides a mouse model for cardiovascular development.

Lamins are essential components of the nuclear envelope and have been studied for decades due to their involvement in several devastating human diseases, the laminopathies. Despite intensive research, the molecular basis behind the disease state remains mostly unclear with a number of conflicting results regarding the different cellular functions of nuclear lamins being published. The field of developmental biology is no exception. Across model organisms, the types of lamins present in early mammalian development have been contradictory over the years. Due to the long half-life of the lamin proteins, which is a maternal factor that gets carried over to the zygote after fertilization, investigators are posed with challenges to dive into the functional aspects and significance of lamins in development. Due to these technical limitations, the role of lamins in early mammalian embryos is virtually unexplored. This review aims in converging results that were obtained so far in addition to the complex functions that ceases if lamins are mutated.

23Vascular development involves de novo formation of a capillary plexus, which is then pruned and 24 remodeled by angiogenic events. Cytoskeletal remodeling and directional endothelial migration are 25 essential for developmental and pathological angiogenesis. Smad-dependent TGF signaling controls 26 vascular patterning and is negatively regulated by microtubules. Here we show that a positive regulator 27 of TGF signaling is essential for developmental vascular patterning and microtubule stability. 28 Rudhira/BCAS3 is known to bind microtubules and to play a nodal role in cytoskeletal remodeling and 29 directional endothelial cell (EC) migration in vitro. We demonstrate that the molecular and cellular 30 function of Rudhira is deployed at critical steps in vascular patterning. We generated the first floxed 31 mice for rudhira and find that global or endothelial knockout of rudhira results in mid-gestation lethality 32 due to aberrant embryonic and extra-embryonic vessel patterning and defective cardiac morphogenesis. 33Rudhira null yolk sac ECs show random and retarded migration. Yolk sac transcriptome analysis revealed 34 key mediators of angiogenic processes and TGF receptor signaling were perturbed in rudhira null 35 mutants. Molecular and biochemical analyses showed that rudhira depletion reduced microtubule 36 stability but increased expression of pathway inhibitors leading to high levels of SMAD2/3 ubiquitination 37 and reduced activation. These effects were not rescued by exogenous TGFβ. However, TGF treatment 38 of wild type ECs increased Rudhira expression. Further, exogenous Rudhira, which promotes directional 39 cell migration, caused increased SMAD2/3 nuclear translocation and reduced inhibitor levels. Therefore, 40we propose that Rudhira and TGF signaling are mutually dependent. Rudhira has a dual function in 41 promoting TGF signaling, possibly by sequestering microtubules and simultaneously preventing 42 SMAD2/3 ubiquitination to permit EC migration and vascular patterning. TGF signaling and aberrant 43 human Rudhira (Breast Cancer Amplified Sequence 3, BCAS3) expression are both associated with 44 tumour metastasis. Our study identifies a cytoskeletal, cell type-specific modulator of TGF signaling 45 important in development and cancer. 46 3 47 Author Summary 48Remodeling and fine patterning of the blood vasculature requires controlled and co-ordinated 49 endothelial cell (EC) migration. This is achieved by the tight regulation of complex signaling pathways in 50 developmental and adult vascular patterning. The TGFβ pathway, in particular, is important for this 51 process. Here we show that the cytoskeletal protein Rudhira plays a critical role in promoting TGF 52 signaling in EC. We generated "knockout" mice and show that Rudhira is crucial for EC migration in 53 mouse developmental vascular patterning. In the absence of Rudhira there is increased degradation of 54 TGFβ pathway effectors resulting in reduced signaling. This affects target gene expression and 55 angiogenic processes, espe...