@article {89660, title = {Differential activation of JAK-STAT signaling reveals functional compartmentalization in blood progenitors.}, journal = {Elife}, volume = {10}, year = {2021}, month = {2021 Feb 17}, abstract = {

Blood cells arise from diverse pools of stem and progenitor cells. Understanding progenitor heterogeneity is a major challenge. The larval lymph gland is a well-studied model to understand blood progenitor maintenance and recapitulates several aspects of vertebrate hematopoiesis. However in-depth analysis has focused on the anterior lobe progenitors (AP), ignoring the posterior progenitors (PP) from the posterior lobes. Using expression mapping, developmental and transcriptome analysis we reveal PP heterogeneity and identify molecular-genetic tools to study this abundant progenitor population. Functional analysis shows that PP resist differentiation upon immune challenge, in a JAK-STAT-dependent manner. Upon wasp parasitism, AP downregulate JAK-STAT signaling and form lamellocytes. In contrast, we show that PP activate STAT92E and remain undifferentiated, promoting survival. knockdown or genetically reducing JAK-STAT signaling permits PP lamellocyte differentiation. We discuss how heterogeneity and compartmentalization allow functional segregation in response to systemic cues and could be widely applicable.

}, issn = {2050-084X}, doi = {10.7554/eLife.61409}, author = {Rodrigues, Diana and Renaud, Yoan and VijayRaghavan, K and Waltzer, Lucas and Inamdar, Maneesha S} } @article {103403, title = {Intact in situ Preparation of the Drosophila melanogaster Lymph Gland for a Comprehensive Analysis of Larval Hematopoiesis }, journal = {Bio Protoc}, volume = {11}, year = {2021}, month = {11/2021}, pages = {e4204}, abstract = {

Blood cells have a limited lifespan and are replenished by a small number of hematopoietic stem and progenitor cells (HSPCs). Adult vertebrate hematopoiesis occurs in the bone marrow, liver, and spleen, rendering a comprehensive analysis of the entire HSPC pool nearly impossible. The Drosophila blood system is well studied and has developmental, molecular, and functional parallels with that of vertebrates. Unlike vertebrates, post-embryonic hematopoiesis in Drosophila is essentially restricted to the larval lymph gland (LG), a multi-lobed organ that flanks the dorsal vessel. Because the anterior-most or primary lobes of the LG are easy to dissect out, their cellular and molecular characteristics have been studied in considerable detail. The 2-3 pairs of posterior lobes are more delicate and fragile and have largely been ignored. However, posterior lobes harbor a significant blood progenitor pool, and several hematopoietic mutants show differences in phenotype between the anterior and posterior lobes. Hence, a comprehensive analysis of the LG is important for a thorough understanding of Drosophila hematopoiesis. Most studies focus on isolating the primary lobes by methods that generally dislodge and damage other lobes. To obtain preparations of the whole LG, including intact posterior lobes, here we provide a detailed protocol for larval fillet dissection. This allows accessing and analyzing complete LG lobes, along with dorsal vessel and pericardial cells. We demonstrate that tissue architecture and integrity is maintained and provide methods for quantitative analysis. This protocol can be used to quickly and effectively isolate complete LGs from first instar larval to pupal stages and can be implemented with ease.

}, doi = {10.21769/BioProtoc.4204 }, author = {Rodrigues, Diana and Vijayraghavan, K. and Waltzer, Lucas and Inamdar, Maneesha S} } @article {55792, title = {Asrij/OCIAD1 suppresses CSN5-mediated p53 degradation and maintains mouse hematopoietic stem cell quiescence.}, journal = {Blood}, year = {2019}, month = {2019 Apr 05}, abstract = {

Inactivation of the tumor suppressor p53 is essential for unrestrained growth of cancers. However, only 11\% of hematological malignancies have mutant p53. Mechanisms that cause wild type p53 dysfunction and promote leukemia are inadequately deciphered. The stem cell protein Asrij/OCIAD1 is misexpressed in several human hematological malignancies and implicated in the p53 pathway and DNA damage response. However, Asrij function in vertebrate hematopoiesis remains unknown. We generated the first asrij null (knockout, KO) mice and show that they are viable and fertile with no gross abnormalities. However, by six months, they exhibit increased peripheral blood cell counts, splenomegaly and an expansion of bone marrow hematopoietic stem cells (HSCs) with higher myeloid output. HSCs lacking Asrij are less quiescent and more proliferative with higher repopulation potential as observed from serial transplantation studies. However, stressing KO mice with sub-lethal gamma irradiation or multiple injections of 5-fluorouracil results in reduced survival and rapid depletion of hematopoietic stem/progenitor cells (HSPCs) by driving them into proliferative exhaustion. Molecular and biochemical analyses revealed increased polyubiquitinated protein levels, Akt/STAT5 activation and CSN5 (COP9 signalosome subunit 5)-mediated p53 ubiquitination and degradation in KO HSPCs. Further, we show that Asrij sequesters CSN5 via its conserved OCIA domain, thereby preventing p53 degradation. In agreement, Nutlin-3 treatment of KO mice restored p53 levels and reduced high HSPC frequencies. Thus, we provide a new mouse model resembling myeloproliferative disease and identify a post-translational regulator of wild type p53 essential for maintaining HSC quiescence that could be a potential target for pharmacological intervention.

}, issn = {1528-0020}, doi = {10.1182/blood.2019000530}, author = {Sinha, Saloni and Dwivedi, Tirath Raj and Yengkhom, Roja and Bheemsetty, Venkata Anudeep and Abe, Takaya and Kiyonari, Hiroshi and VijayRaghavan, K and Inamdar, Maneesha S} } @article {32474, title = {Rudhira/BCAS3 is essential for mouse development and cardiovascular patterning.}, journal = {Sci Rep}, volume = {8}, year = {2018}, month = {2018 Apr 04}, pages = {5632}, abstract = {

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.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-24014-w}, author = {Shetty, Ronak and Joshi, Divyesh and Jain, Mamta and Vasudevan, Madavan and Paul, Jasper Chrysolite and Bhat, Ganesh and Banerjee, Poulomi and Abe, Takaya and Kiyonari, Hiroshi and VijayRaghavan, K and Inamdar, Maneesha S} }