He Q, Zhang C, Wang L, Zhang P, Ma D, Lv D, Liu F. Inflammatory signaling regulates hematopoietic stem and progenitor cell emergence in vertebrates. Blood 2015; 125: 1098-1106.

Prepared by: Zahra A. Adahman, Fall 2015.

LAYMAN SUMMARY

The field of hematopoietic stem and progenitor cells (HSPCs) research has grown and expanded over the years. The findings in the field have played a major role in the discovery of treatment to blood-and hematopoietic -related cancers. Scientists are involved in research studies to understand how the earliest hematopoietic stem and progenitor cells are regulated. The emergence of the earliest HSPCs begins early the development of an embryo. The process occurs in the aorta-gonad-mesonephron (AGM) regions. The hemogenic endothelium begins to transform to hematopoietic stem and progenitor cells. Interestingly, the microenvironment or niche shows that hemogenic endothelium (HE) cells of the AGM release inflammatory signals, the same signals released when the immune system is combating an infection.

The main goal of paper was to identify the key signaling pathways in the induction of the inflammatory signals to regulate embryonic HSPCs emergence. The result from their experiment seals the gap between the inflammatory signaling and HSPCs emergence during development. The authors of the research study focused on the evolutionary conserved toll-like receptor 4 (TLR4)-nuclear factor κ-light chain enhancer of activated B core (NF-κB) signaling pathway. This signaling pathway promotes the gene expression of the proteins (cytokines and chemokine), which serve as the inflammatory signals in most eukaryotes.

During development the TLR4-NF-κB pathways is automatically activated to release inflammatory signals for the emergence of HSPCs. If there is an infection, this same TLR4 is involved in cell to cell signaling and communication during inflammation, when activated by an antigen. The researcher engineered the zebrafish embryos model to genetically express less of the TLR4 protein, and other components of the TLR4-NF-κB pathways to analysis the effect of the loss of function of the constituents of the pathway. The data shows that the embryos modified to express less of these proteins had defects in the number and gene expression profile of the HSPCs. Downstream of the conserved TLR4-NF-κB pathways is the highly conserved notch signaling pathway which involves in differentiation of cells during embryogenesis. Further loss of function experiment was done by modifying gene expression of some proteins involved in the notch signaling pathway and the results also showed defects in the number and gene expression profile of HSPCs. Gain of function experiment was performed by genetically increasing the expression of the endogenous inhibitor protein of the NF-κB. The results show a rescue of the effect of loss of function of the components of the conserved TLR4-NF-κB and notch signaling pathways on the number and gene expression profile of HSPCs.

In conclusion, experiments to test the functionality of the HSPCs in embryos genetically modified not to express TLR 4 were conducted by transplanting the HSPCs from the AGM of the embryos into irradiated mice. The results were collected after two months. Only one out of six mice who were recipients of HSPCs from genetically modified embryo survived, while five out of six subjects who received the normal HSPCs survived. In conclusion, the results show that the TLR4-NF-κB and notch signaling pathways are essential and evolutionary conserved to shown to regulate the emergence and development of the earliest HSCs via inflammatory signals. The results of the study can be applied to the expansion of hematopoietic cells transplant for treatment of for bone marrow failure.

SCIENTIFIC SUMMARY

Infection can induce inflammatory signals so that the immune system is equipped to eradicate infections. These same signals seem to be similar during the development of the hematopoietic system. This study linked the gap between inflammatory signals and the process of hematopoietic stem/progenitor cell (HSPCs) development. The authors studied how the process is affected by loss of function studies of the highly conserved toll-like receptor 4 (TLR4)­ nuclear factor κ-light chain enhancer of activated B core (NF-κB) and notch signaling pathways. The effect of knocking down these genes was examined on HSPCs during embryonic development. The results showed evidence to advance the techniques for bone marrow transplantation as well as in vitro expansion of hematopoietic stem cells, which is yet to be accomplished in the field.

The technology of morpholinos was used to manipulate the expression of components of the TLR4-MyD88-NF­κB pathway. TLR4, MyD88, and (inhibitor of NF-κB) IκBaa morphants were engineered using zebrafish embryos. At 36 hours post-fertilization, the authors performed whole mount in-situ hybridization (WISH) to analyze the expression profile of the (runt-related transcription factor 1) runx1 gene in the aorta-gonad-mesonephron (AGM) region of the embryos. The number of cells expressing the runx1 gene was less in the TLR4 and MyD88 morphants than the control. The opposite was the case in the iκBaa morphants. There were more cells expressing runx1 gene in the AGM of the IκBaa morphants; by silencing the endogenous inhibitor of the NF-κB, it upregulates the amount of NF-κB allowing the pathway to progress. Co-injection experiments were done to test the effect of attenuating TLR4, MyD88 and iκBaa at the same time. The results showed that absence of IκBaa rescued the effect of the expression of runx1 in the TLR4 and MyD88 morphants. The authors concluded that NF-κB is an important component in the TLR4 pathway and tampering with the pathway affected the emergence of HSPCs.

Downstream of the TLR4-MyD88-NF-κB pathway is the notch signaling pathway, known to be involved in cell differentiation. Morphants for proteins her15.1, hey1 and hey2 were used to analyze for runx1 expression. There was a reduction in the number of cells having runx1 expression in the morphants as compared to control. The results established that notch signaling is required for the expression of inflammatory signals necessary for the development of HSPCs. Further experiment was done to analyze the functionality of the HSPCs in embryos with malfunctions in the TLR4 receptor expression. HSCs were harvested from the AGM region of tlr4^+/- and tlr4^-/- zebrafish embryos and transplanted into irradiated mice. Five out of six recipients of stem cells from tlr4^+/- embryos survived, while only one out of six recipients of the tlr4^-/- HSCs survived two months post-transplant. This end result illustrates and confirms that the TLR4-MyD88-NF-κB and notch signaling pathway are evolutionarily conserved and required for proper development of HSPCs via inflammatory signals.