Understanding how Notch influences the development and fate of the hemogenic endothelium using genetic mosaics

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 18-10-2019Esta tesis tiene embargado el acceso al texto completo hasta el 18-04-2021Notch is an important signaling pathway in the intraembryonic hematopoietic wave that occurs between E9.5-E11.5. Although most studies so far have claimed that its activity in endothelial cells (ECs) of the dorsal aorta (DA) is necessary for hematopoiesis, more recent evidence has suggested that Notch signaling must be downregulated for endothelialto-hematopoietic transition (EHT) to occur. However, the exact molecular and cellular mechanisms are still not well characterized. In this thesis we have used a wide range of novel genetic tools and imaging approaches to analyze the role of Notch in the EHT process with higher cellular, temporal, and molecular resolution. Our results show that embryos with increased Notch activity in the dorsal endothelium at E10.5 have a decrease in c-Kit+ hematopoietic stem progenitor cells (HSPCs). This is caused by a decrease in hemogenic specification, or CD31+/Runx1+ cells. Additionally, HSPCs with high Notch activation presented proliferation defects as they tended to form unicellular clusters. We also found that embryos with loss of Jag1 in DA ECs had higher Dll4/Notch activity, which may explain why the loss of this Notch ligand also induces a decrease in hematopoiesis. This differential Jagged1/Dll4 Notch activity could be due to the known inhibitory role of Fringes on Jagged1/Notch signaling. Indeed overexpression of Manic Fringe in DA ECs resulted in a decrease in Dll4/Notch signaling and an increased number of HSPCs in the DA. To determine the single cell-autonomous role of Notch during EHT, we induced Notch genetic mosaics in individual DA ECs. Single ECs with Notch loss-of-function (LOF) undergo EHT with more frequency compared to wildtype ECs. Comparative transcriptional profiling revealed that they had similar arterial endothelial identity (Sox17, Cx40) and hematopoietic progenitor (CD41) expression, however, we found that Myc and Mycn expression was significantly deregulated. Analysis of embryos with Myc and Mycn deletion in DA ECs surprisingly revealed that Mycn is a strong regulator of EHT, whereas Myc is dispensable. Mycn mutants presented a drastic loss of HSPCs in the DA whereas Myc mutants only displayed a late hematopoiesis progression phenotype. Analysis of recently published single cell transcriptomic data allowed us to validate several of the experimental findings described above and to propose a model for the regulation of EHT by Notch. During EHT, most DA ECs have high Notch signaling induced by Jagged1 and Dll4 ligands and they are quiescent (KI67-). Among these cells, some upregulate Mfng, which decreases the signaling ability of the surrounding Jagged1 ligands and lowers Notch signaling cell-autonomously. These cells with lower Notch signaling upregulate Mycn, which is necessary to induce EHT. The Mycn induced EHT is associated with an entry into cell cycle which is subsequently maintained by the expression of the homologous gene Myc in the HSPCs.Notch es una vía de señalización importante en la onda hematopoyética intraembrionaria que se produce entre E9.5-E11.5. Aunque se especula que la señalización de Notch baja en las células endoteliales (EC) para que ocurra la transición endotelial-a-hematopoyética (EHT), el mecanismo molecular debajo de Notch en la aorta dorsal (DA) aún no está bien caracterizado. En este tesis, hemos utilizado una amplia gama de herramientas genéticas novedosas.para analizar el papel de Notch en el proceso EHT con mayor resolución celular, temporal, y molecular. Nuestro resultados muestran que en en los embriones con mayor actividad de Notch en el endotelio dorsal tenían defectos en el sistema hematopoyético definitivo en la aorta gónada mesonefros (AGM) en embriones de E10.5. Vimos una pérdida prejudicial de células madres progenitoras hematopoyético c-Kit+ (HSPC) en embriones con alta señalización de Notch. Los embriones con alta señalización de Notch tenían menos especificación hemogénica y un pequeño porcentaje de células endoteliales con alta activación celular autónoma de Notch también eran Runx1+. Además, las HSPCs con alta activación de Notch presentan defectos de proliferación. También hemos visto que en los mutantes Jag1 habia una alta tinción de Dll4/Notch1 y tiene un fenotipo similar a los embriones con alta señalización de Notch. Nosotros creíamos que los resultos implican un papel para Fringe. En efecto, sobreexpresión de Manic Fringe en el aorta bajan los niveles de la señalización de Dll4/Notch y suben los números de HSPCs en el aorta dorsal. Utilizando mosaico genéticos de ratones para investigar el papel de Notch en el endotelio hemogénico (HE), hemos determinado que las ECs con pérdida de función de Notch (LOF) se someten a EHT con más frecuencia en comparación con las ECs de controles. Al asilar estas células, hemos encontrado que, aunque tenían una expresión de endotelio arterial (Sox17, CX40) y de progenitor hematopoyético (CD41+) similar a las ECs de controles, tanto Myc como Mycn se modificaron drásticamente. Utilizamos los mutantes Myc y Mycn para determinar si los dos factores de trascripción desempeñaron un papel en el desarrollo temprano del sistema hematopoyético. Los mutantes de Mycn presentaron una pérdida drástica de HSPCs en la aorta, mientras mutantes de Myc tenían defectos hematopoyéticos en adultos. El análisis de los datos transcriptómicos de las células del aorta, tanto las HSPCs como las endoteliales, nos ayuda en proponer un modelo para la regulación de EHT por Notch. Durante EHT, la mayoría de las células del aorta dorsal tienen alta señalización de Notch inducido por los ligandos Jagged1 y Dll4 y bajo niveles de proliferación (Ki67-). Dentro de estas células, algunos suben la expresión de Manic Fringe y después bajan los niveles de Notch en la célula de una forma autónoma. Estas células luego suben los niveles de Mycn, lo cual es necesaria para inducer EHT. El EHT inducido por Mycn se asocia con una entrada en el ciclo celular que posteriormente se mantiene mediante la expresión del gen homólogo Myc en las HSPCs

Using CRISPR/Cas9 based genetic screen to understand the role of Flk1 in the specification of endothelial and blood lineages

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 25-05-2020Esta tesis tiene embargado el acceso al texto completo hasta el 25-11-202

A gene trap transposon eliminates haematopoietic expression of zebrafish Gfi1aa, but does not interfere with haematopoiesis

A transposon-mediated gene trap screen identified the zebrafish line qmc551 that expresses a GFP reporter in primitive erythrocytes and also in haemogenic endothelial cells, which give rise to haematopoietic stem and progenitor cells (HSPCs) that seed sites of larval and adult haematopoiesis. The transposon that mediates this GFP expression is located in intron 1 of the gfi1aa gene, one of three zebrafish paralogs that encode transcriptional repressors homologous to mammalian Gfi1 and Gfi1b proteins. In qmc551 transgenics, GFP expression is under the control of the endogenous gfi1aa promoter, recapitulates early gfi1aa expression and allows live observation of gfi1aa promoter activity. While the transposon integration interferes with the expression of gfi1aa mRNA in haematopoietic cells, homozygous qmc551 fish are viable and fertile, and display normal primitive and definitive haematopoiesis. Retained expression of Gfi1b in primitive erythrocytes and upregulation of Gfi1ab at the onset of definitive haematopoiesis in homozygous qmc551 carriers, are sufficient to allow normal haematopoiesis. This finding contradicts previously published morpholino data that suggested an essential role for zebrafish Gfi1aa in primitive erythropoiesi

EGFR is required for Wnt9a-Fzd9b signalling specificity in haematopoietic stem cells.

Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand-receptor (Wnt-Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit β-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a-Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a-Fzd9b-LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt-Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine

Germline competent mesoderm: the substrate for vertebrate germline and somatic stem cells?

In vitro production of tissue-specific stem cells [e.g. haematopoietic stem cells (HSCs)] is a key goal of regenerative medicine. However, recent efforts to produce fully functional tissue-specific stem cells have fallen short. One possible cause of shortcomings may be that model organisms used to characterize basic vertebrate embryology (Xenopus, zebrafish, chick) may employ molecular mechanisms for stem cell specification that are not conserved in humans, a prominent example being the specification of primordial germ cells (PGCs). Germ plasm irreversibly specifies PGCs in many models; however, it is not conserved in humans, which produce PGCs from tissue termed germline-competent mesoderm (GLCM). GLCM is not conserved in organisms containing germ plasm, or even in mice, but understanding its developmental potential could unlock successful production of other stem cell types. GLCM was first discovered in embryos from the axolotl and its conservation has since been demonstrated in pigs, which develop from a flat-disc embryo like humans. Together these findings suggest that GLCM is a conserved basal trait of vertebrate embryos. Moreover, the immortal nature of germ cells suggests that immortality is retained during GLCM specification; here we suggest that the demonstrated pluripotency of GLCM accounts for retention of immortality in somatic stem cell types as well. This article has an associated Future Leaders to Watch interview with the author of the paper

UNDERSTANDING EARLY HEMATOPOIETIC DEVELOPMENT IN THE MOUSE EMBRYO

Ph.DDOCTOR OF PHILOSOPH

Differential Roles of PRDM16 Isoforms in Normal and Malignant Hematopoiesis

PRDM16 is a transcriptional co-regulator that is highly expressed in HSCs and required for their maintenance. It is also involved in translocations in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and T-cell acute lymphoblastic leukemia. Prdm16 is expressed as both full-length (f Prdm16) and short-length (s-Prdm16) isoforms, the latter lacking an N-terminal PR domain homologous to SET methyltransferase domains. The roles of both isoforms in normal and malignant hematopoiesis are unclear. In chromosomal rearrangements involving PRDM16, the PR domain is deleted. Furthermore, overexpression of s-Prdm16, but not f-Prdm16, can cause leukemia in a p53-/- background predisposed to malignancy. Based on this, s-Prdm16 has been proposed as an oncogene whereas f-Prdm16 has been suggested to possess tumor suppressor activity. The aim of this thesis was to more clearly elucidate the role of each Prdm16 isoform in normal and malignant hematopoiesis. We first showed that Prdm16 is essential for adult HSC maintenance using a conditional deletion mouse model specific for hematopoietic cells, as previous findings using an embryonic-lethal global Prdm16-/- mouse demonstrated this only in fetal liver. We then found, using a specific f-Prdm16-/- mouse model, that full-length Prdm16 is essential for HSC maintenance and induces multiple genes involved in GTPase signaling and represses inflammation. Based on a comparison of Prdm16-/- HSCs lacking both isoforms, and f-Prdm16-/- HSCs which express s-Prdm16, we were able to infer some hematopoietic properties of s-Prdm16 – namely that this isoform induces inflammatory gene expression and supports development of a Lineage-Sca1+cKit- lymphoid progenitor distinct from CLPs which predominantly differentiates into marginal zone B cells. s-Prdm16 expression alone, however, was not sufficient to maintain HSCs. We used a mouse model of human MLL-AF9 leukemia and found that leukemia derived from Prdm16-deficient HSCs had extended latency, although expression of Prdm16 decreases during MLL-AF9 transformation and is undetectable in ex vivo leukemic cells. Forced expression of f-Prdm16 in these cells further extended leukemic latency, while forced expression of s-Prdm16 shortened latency. Gene expression profiling using RNAseq indicated that forced expression of f-Prdm16 resulted in altered respiratory metabolism of MLL-AF9 cells, whereas expression of s-Prdm16 induced a strong inflammatory gene signature, comparable to that seen in HSCs expressing only s-Prdm16. Several inflammatory cytokines and chemokines induced by s-Prdm16 are associated with MDS and with a worse prognosis in human AML. Furthermore, leukemia expressing s-Prdm16 had an elevated number of cells with abnormal nuclei, characteristic of dysplasia. Finally, we performed an analysis of PRDM16 in human AML from the publically-available Cancer Genome Atlas dataset, containing clinical and gene expression data for 179 cases of AML. PRDM16 expression negatively correlated with overall survival, both in the entire dataset and in the NPM1 mutated and MLL¬-rearranged subsets, and s-PRDM16 exhibited a stronger correlation than f-PRDM16. HOX gene expression correlated with PRDM16 expression, suggesting that HOX genes may positively regulate PRDM16 expression in AML. In NPM1-mutant and MLL-rearranged subsets of AML, we also found that high PRDM16 expression correlated with an inflammatory gene signature, thus corroborating our findings in mouse MLL-AF9. Our findings demonstrate distinct roles for Prdm16 isoforms in both normal hematopoiesis and AML, and identify s-Prdm16 as one of the drivers of prognostically-adverse inflammatory gene expression in leukemia

Pluripotent Stem Cell Biology

Pluripotent stem cells have the potential to revolutionize treatment options for a range of diseases and conditions. This book presents recent advances in our understanding of the biological mechanisms of stem cell self-renewal, reprograming and regeneration. Also covered are novel methodological advances in the culture, purification and use of stem cells, as well as the ethical and moral dilemmas of embryo donation and adoption. These advances will shape the utilization of stem cells for future basic and applied applications