The role of the transcription factor Sp1 in embryonic macrophage development

Macrophages are a type of white blood cell which are very important for the health of an organism. These cells have the ability to recognize infectious particles and send out alarm signals that mobilize the bodies? defenses. However, in the embryo they have different functions. Here they function mainly as scavengers and have the ability to ?eat? dead cells and cell debris, and therefore play important roles in shaping the body outline. For example, even in mammals hands are originally webbed and macrophages remove the skin between the fingers. In the project described here we wish to address the question how this cell type is formed in the embryo. All different cells of the organism originate from a single egg; the reason why they are all different is that they all express different sets of genes. Therefore we want to know which genes are responsible for generating embryonic macrophages. To find out which genes are responsible, we will use an embryonic stem (ES) cell line that lacks one specific protein (Sp1) that is responsible for the activity of many genes. We found that normal ES cells can be turned into embryonic macrophages by culturing them in special medium. However, in the absence of Sp1 these cells are not formed. When Sp1 is removed in mice, embryos are unable to develop. The reason for this is unclear, but our work suggests that macrophages may have something to do with it. Sp1 is a so-called transcription factor, which directly binds to the DNA of genes and switches them on. Very little is known about how and where it works. Using novel techniques we can identify all genes within the cell which are bound by Sp1 and we can then test whether they fail to be switched on in its absence. The final outcomes of this work will be (i) a list of genes of which we now know that they are important for forming macrophages, and (ii) a much more detailed understanding of how Sp1 acts in this process. This work is not only of relevance to macrophage biology but also to the understanding of embryogenesis as such.

The different macrophage types found at the different developmental stages of the mammalian organism comprise a highly heterogeneous cell population which can be distinguished by differential mRNA expression, differentiation pathway and functional properties. Each macrophage type is tailored to specific functions within the tissues where they reside. Besides their function in the innate immune system, they also have other roles. These functions are particularly important during embryonic development where phagocytes are involved in tissue remodeling via the removal of cells undergoing programmed cell death.
Using the differentiation of mouse ES cells as a model, we have recently demonstrated that embryonic macrophages are uniquely dependent on the presence of the transcription factor Sp1. Sp1 is a member of the Cys2-His2 type Zn finger class of transcription factors. It is ubiquitously expressed and has been implicated in regulating vast numbers of genes. Germline ablation of Sp1 is lethal in the early mouse embryo, whereby the knockout mutation shows a variable penetrance with embryos dying before E10 with various defects. Sp1-/- ES cells grow normally, however, differentiating Sp1-/- ES cells are unable to differentiate into macrophages. Studies with the Sp1-/- mouse embryos show that they are capable of blood cell development. In addition, preliminary experiments with adult conditional knock-out mice by the group of Sjaak Philipsen in Rotterdam do not show any obvious alterations in macrophage numbers, indicating that the knockout mainly affects embryonic macrophage development. The molecular basis of the in vivo and the in vitro phenotype as well as the target genes which are affected by the absence of Sp1 are completely unknown.
In this proposal and in collaboration with the Philipsen group we therefore plan to address this issue. We will (i) perform ChIP-sequencing to identify Sp1 target genes in embryonic macrophages, (ii) examine the chromatin structure of Sp1 target genes in the presence and absence of Sp1 in ES cells to test whether normally poised genes required for early macrophage differentiation are silenced in Sp1-/- ES cells, and (iii) last but not least, we will investigate whether the related factor Sp3 compensates for the lack of Sp1 on Sp1 target genes at later stages of macrophage differentiation. Our work will lay the molecular groundwork with respect to an elucidation of the role of Sp1 in embryonic development and also for studies investigating whether defects in embryonic macrophages contribute to the multiple developmental defects observed in Sp1 knockout mice.