Introduction
Previous chapters have investigated the apparent function and m ovem ents o f macrophages in wild-type embryos as they remodel their limbs and kidneys. One good way to address the function o f macrophages in the embryo is to examine em bryos genetically deficient in this cell type, and the P U .l knockout m ouse offers such a m odel. This chapter describes a study o f footplate developm ent in the P U .l null embryo.
The transcription factor P U .l is a member o f the ets family o f transcription factors (review ed in Janknecht and Nordheim , 1993; M oreau-Gachelin, 1994). The prototypic ets gene, e ts - \, was first identified about 15 years ago as an oncogene carried by the avian erythroblastosis virus E26 (Leprince et a l., 1983; Nunn et a l., 1983). The ETS domain has a highly conserved region o f 85 amino acids which binds D N A in a manner similar to the winged helix-turn-helix family o f proteins (Kodandapani et a l., 1996) allow ing it to act as a transcriptional activator (K lem sz et a l., 1990). PU. 1 is expressed exclusively by cells o f the haematopoietic lineage - it has specifically been show n to be expressed by macrophages, B lym phocytes, mast cells, neutrophils and early erythroblasts, but not by T -cells (Galson e t al., 1993; K lem sz e ta l ., 1990).
T w o recently reported P U .l knockout mice give rather conflicting evidence as to w hich o f these cell types are absolutely dependent on P U .l (Scott et a l , 1994; M cKercher et a l., 1996). The first o f these papers describes an embryonic lethal phenotype, with foetuses dying at E l 6.5 apparently o f anaemia (Scott et a l , 1994). The only haematopoietic cell types which are reported to be normal in this PU. 1 deficient m ouse are megakaryocytes and platelets. Erythrocyte precursors develop as normal but they seem unable to mature, and there are no lymphoid or m yeloid progenitors. All organs studied apparently develop normally, as do other tissues including bone (Scott e t a l.,
1994).
In the m ost recent paper, a different region o f the gene was disrupted and knockout mice were born in the expected Mendelian ratio. If kept in normal conditions, these m ice die within 48 hours o f birth o f septicemia, although they can be kept alive for about tw o w eeks on a strict antibiotic regime (McKercher et a l., 1996). Erythrocytes, m egakaryocytes, platelets and mast cells are all apparently normal (McKercher et a l.,
1996). T-lymphocyte development appears to be delayed by eight to ten days in P U .l null m ice, with mature cells not appearing until three days after birth (McKercher et a l.,
1996). Neutrophil developm ent is also retarded by ten or more days, so that neutrophil like cells are not seen until three or four days after birth. O f m ost relevance to this study is that these m ice appear to be almost com pletely m issing cells o f the macrophage lineage. The only F 4/80 positive cells found were in the liver, and they were abnormally large in size and o f uncharacteristic shape. N o macrophages were seen in any other tissues (M cKercher et al., 1996).
These P U .l null mice are also m issing multinucleated osteoclasts and are thus osteopetrotic (Tondravi et al., 1997). Osteoclasts are o f m yeloid origin. In the w ild-type situation marrow macrophages gradually increase their expression o f PU. 1 as they adopt the osteoclast phenotype. Osteoclast differentiation, like that o f macrophages, is affected at the initial m yeloid stage (Tondravi e ta l., 1997). The osteopetrotic effects o f P U .l null
m ice can be rescued by bone marrow transplantation (Tondravi et a l., 1997), and indeed transplants o f this sort w ill, if delivered in the first week o f life, alm ost com pletely rescue the m ouse, such that it lives for close to the normal life span o f a wild-type m ouse (personal comm unication, Scott McKercher, La Jolla, California).
Figure 6.1 summarises the cells involved w hich are affected by the lack o f P U .l, and suggests positions where P U .l might act in the haematopoietic lineage decision pathway.
In all system s examined to date, cells dying by apoptosis are rapidly cleared by adjacent phagocytic cells. In C .elegans it appears that this clearance task is performed by non specialist neighbours (Ellis et a l., 1991). In D rosophila non-specific blood cells, called hem ocytes, which have macrophage characteristics, engulf the apoptotic cells throughout organogenesis (Tepass e ta l., 1994). In vertebrate embryo m odels, various studies have shown that macrophages perform a major role in the clearance o f apoptotic cells found in the nervous system (Cuadros et a l., 1992), the interdigital zones o f the remodelling footplate (H opkinson-W oolley et a l., 1994) and the kidney (Chapter Three; Camp and Martin, 1996).
The PU. 1 embryos offer the ideal opportunity to test the situation when macrophages are not available to perform their clearance functions. A remarkable instance o f cell redundancy is found. There are absolutely no macrophage-like cells in the P U .l knockout footplate at stages when interdigits are regressing, and yet this regression proceeds at precisely the same rate as in wild-types or heterozygotes. Closer examination at the transmission electron m icroscopy (TEM) level show s embryonic mesenchymal cells fulfilling the role o f phagocytes in these zones o f cell death.