Runt Gene Evolution Figure 4. qRT-PCR results of ScRunx1–3 expression. In dogfish the most prominent expression of all three ScRunt genes was in the skin. Also in visceral cartilage ScRunx1–3 were strongly expressed. B: Brain, C-v: Visceral cartilage, D-m: Ductus mesonephric, E: Epididymis, H: Heart, K: Kidney, L: Liver, Mu: Muscle, Oe: Oesophagus, Sc: Spinal column, Sk: Skin, S-a: Anterior stomach, S-m: Middle part of stomach, Sp: Spleen, T: Testis. doi:10.1371/journal.pgen.1000025.g004 the same expression pattern of ScRunx1–3 was found (Figure 5D– 5F). ScRunx1 and ScRunx3 were expressed at a distal position and ScRunx2 was found at a basal position. Figure 5G shows a schematic of the different sites of Runt expression in teeth and placoid scales. In addition, Runt genes were also expressed in the developing skeleton. Runx2 expression was detected in cranial cartilage and skeletal elements of the fin whereas Runx2 and Runx3 expression was found in gill gut cartilage (Figure 6). Expression of Runt during lancelet (B. floridae) development in the notochord, gut and neural tube To be able to reconstruct the Runt expression domains in the chordate stem species and to see if Runt was expressed in ancient skeletal elements such as the notochord, we analyzed Runt gene expression in lancelets, the putative sistergroup of vertebrates. Using whole mount in situ hybridization of early developmental stages (early and late gastrula) a diffuse Runt staining, indicating a maternal Runt expression, was detected, comparable to the description of maternal Runx1, –2b, and –3 expression in zebrafish. [25–27]. Two different probes were used, corresponding to the Runt gene variant starting with exon 1 (transcribed from the distal promoter P1) and the Runt gene variant starting with exon 2 (transcribed from the proximal promoter P2). These two probes showed overlapping staining patterns (Figure 7). The Runt gene variant P1 was expressed at the 8 somite stage (16 h) in the posterior part of the gut, the notochord and the developing neural tube (Figure 7A). At 26 h Runt expression can be predominantly seen in the middle part of the notochord, the midgut and foregut (Figure 7C). An inconsistent staining pattern was also detected at this stage in about 50% of the larvae immediately below the preoral pit (Figure 7C insert). At 33 h the larvae showed persistent expression of the Runt exon 1 variant in the notochord and neural tube, but also in the midgut region (Figure 7E). The Runt gene variant P2 was exclusively expressed in the hindgut at 16 h (Figure 7B). At 26 h the expression domain extended throughout the entire gut and a signal was also found in a confined region of the foregut (Figure 7D). At 33 h Runt expression was found throughout the entire larvae with the most intense signals in the tailbud and in the anterior gut region. (Figure 7F). Runt, SoxE and Hedgehog expression in gill gut region of adult lancelet (B. lanceolatum) Our analysis had shown that Runt genes are expressed in cartilaginous tissue of the hagfish as well as in the notochord of lancelets indicating a possible role in the development of the ancestral skeleton. We next asked the question, if Runt expression can be found in skeletal elements of adult lancelets. Based on the recent observation that adult lancelets express fibrillar collagen in their gill bars [5,28] we hypothesized that the gill bars represent an ancestral form of cartilage regulated by similar pathways of chondrogenesis as in higher vertebrates. We showed previously that in adult lancelets only the Runt exon 2 variant is expressed [9]. As shown in Figure 8A, qRT-PCR demonstrated expression in almost all tissues, a finding that is in accordance with the broad staining pattern of the exon 2 Runt gene variant at 33 h PF (Figure 7F). However, the most intense signals in adult lancelets were found in the gill gut and the gut. Furthermore qRT-PCR showed that the lancelet SoxE gene had its highest expression and Hh its third highest expression in the gill gut region (Figure 8B and 8C). To determine where exactly Runt and Hh genes were expressed in the gill bars we performed in situ hybridization on tissue sections (Figure 8D–8G). We detected Runt and Hh gene expression in the endo- and ectodermal epithelial cells of primary and secondary gill bars (Figure 8D–8J) but not in the mesodermal coelomic cells of the primary gill bars (data not shown). Interestingly Runt and Hh were strongly coexpressed in a cell population between the endodermal epithelium with cilia and the ectodermal gland epithelium directly adjacent to both sites of the acellular matrix (arrows in Figure 8D–8G). The Hh signal was confirmed by immunohistochemistry (data not shown). Direct regulation of lancelet Hedgehog by Runt As both Runt and Hh showed co-expression in the gill gut region, we analyzed whether a functional relationship between both genes, as it is known for the mouse [10], exists in lancelets. Analysis of the B. floridae Hh promoter revealed several putative Runt binding sites (Figure 9A). All of them were capable of binding to B. lanceolatum Runt as shown by electrophoretic mobility shift assays (Figure 9B). To provide further evidence for a Runt dependent regulation of BfHh we cloned different fragments of the BfHh promoter into the pGL3-basic luciferase reporter vector. Both, MmRunx2 and BlRunt were able to activate the different promoter constructs (Figure 9C). Discussion Runt gene evolution in chordates In order to get insight into the molecular mechanisms underlying the evolution of the skeleton we analyzed the evolution of the Runt gene family in various representative species. Runt genes are important regulators of neurogenesis and hematopoiesis [29,30] and they are essential for mammalian skeletogenesis [7,10]. Our analysis revealed that the stem species of chordates harboured most likely only a single Runt gene and as outlined in Figure 2 independent Runt duplications occurred in the clades of sea urchin (SpRunt1, SpRunt2), and bony fish (duplication of Runx2). PLoS Genetics | www.plosgenetics.org 5 March 2008 | Volume 4 | Issue 3 | e1000025