Homeodomain-containing proteins comprise a superfamily of transcription factors that participate in the regulation of almost all aspects of embryonic development. Here, we describe the mouse embryonic expression pattern of Mohawk, a new member of the TALE superclass of atypical homeobox genes that is most-closely related to the Iroquois class. During mouse development, Mohawk was transcribed in cell lineages derived from the somites. As early as embryonic day 9.0, Mohawk was expressed in an anterior to posterior gradient in the dorsomedial and ventrolateral lips of the dermomyotome of the somites that normally give rise to skeletal muscle. Mohawk transcription in the dorsomedial region required the expression of the transcription factor paraxis. As somites matured, Mohawk transcription was observed in the tendon-specific syndetome and the sclerotome-derived condensing mesenchyme that prefigures the proximal ribs and vertebral bodies. In the limbs, Mohawk was expressed in a pattern consistent with the developing tendons that form along the dorsal and ventral aspect of the phalanges. Finally, Mohawk was detectable in the tips of the ureteric buds in the metanephric kidneys and the testis cords of the male gonad. Together, these observations suggest that Mohawk is an important regulator of vertebrate development. Developmental Dynamics 235:792-801, 2006.

We have demonstrated that Notch genes are expressed in developing mammalian ovarian follicles. Lunatic fringe is an important regulator of Notch signaling. In this study, data are presented that demonstrate that radical fringe and lunatic fringe are expressed in the granulosa cells of developing follicles. Lunatic fringe null female mice were found to be infertile. Histological analysis of the lunatic fringe-deficient ovary demonstrated aberrant folliculogenesis. Furthermore, oocytes from these mutants did not complete meiotic maturation. This is a novel observation because this is the first report describing a meiotic defect that results from mutations in genes that are expressed in the somatic granulosa cells and not the oocytes. This represents a new role for the Notch signaling pathway and lunatic fringe in mammalian folliculogenesis. (Goode et al., 1996a; Goode et al., 1996b;Hicks et al., 2000;Bruckner et al., 2000; Munro and Freeman, 2000;Schwientek, 2002). Brainiac activity is needed in the germ line for proper organization of the follicle (Goode, 1996). Fringe, the homolog of Lfng, is necessary for specification of the polar cells (Grammont and Irvine, 2001). Brainiac has been demonstrated to modify glycosphingolipids by adding GlcNac residues to mannose and galactose moieties on ceramide (Schwientek et al., 2002). In mice, a null mutation of the murine homolog of brainiac demonstrated that this protein is important for very early development, as braniac -/-embryos die prior to implantation (Vollrath et al., 2001). No role for this family of proteins in mammalian folliculogenesis has been described.It has been demonstrated that Lfng is an important regulator of Notch signaling. For example, Lfng null mutants have segmentation defects that are similar to those seen in null mutations of Notch1 and Dll1 (Evrard et al., 1997; Zhang and Gridley, 1997). In somites, where Lfng is the only family member expressed, Notch receptors and ligands are expressed normally in Lfng -/-mutants, but the Notch downstream target gene Hes5 was not detected, indicating a lack of Notch activation in the presence of ligand. However, Hes5 was expressed normally in the neural tube and developing brain of Lfng null mutants, probably due to expression of Mfng and Rfng in these tissues (Evrard et al., 1997). Interestingly, Rfngdeficient mice had no phenotype and Rfng/Lfng double null mutants had only defects associated with a lack of Lfng (Zhang et al., 2000). Folliculogenesis is the process by which oocytes develop in response to hormonal cues. This requires the coordination of the proliferation and differentiation of granulosa cells and the growth and maturation of the oocyte. Primordial follicles consist of a small oocyte surrounded by squamous somatic cells. When recruited to develop, the granulosa cells proliferate and become cuboidal. As these cells continue to proliferate, layers develop around the growing oocyte. Once a follicle has several layers of cells a fluid filled space, the antrum, will begin to form. The antrum sp...

IntroductionSerum response factor (Srf), a nearly ubiquitous transcription factor that is expressed in all hematopoietic cell types, is a founding member of the MCM1-agamous-deficiens-Srf (MADS) domain-containing family of transcription factors, and binds to so called CArG sites (CC/AT-rich/GG, with CCTTATATGG emerging as a major consensus sequence). 1 More than 200 CArG boxes control expression of more than 150 Srf target genes, including genes of the cytoskeleton as well as several immediateearly genes, for example the proto-oncogene c-fos, 2 and also bcl2, 3 whereby Srf participates in apoptosis, cell growth, differentiation, and cell-cycle regulation. Srf is a downstream target of many pathways; for example, the mitogen-activated protein kinase pathway that acts through the ternary complex factors (TCF) as well as Rho signaling, 4 which promotes actin polymerization. Srf plays an important role in the regulation of smooth, skeletal, and cardiac muscle genes 5-8 during development and in adult life including aging. 9,10 Srf can be activated to promote transcription in response to extracellular signals that induce its association with specific cofactors. The 2 families of Srf cofactors are the TCF proteins (Elk1, SAP1, SAP2), 11 and the myocardin family of proteins that includes myocardin, megakaryoblastic leukemia 1 (Mkl1), and Mkl2. 1,12,13 In some cell types, TCF proteins and Mkl1 compete for binding to Srf to activate or inhibit distinct transcription targets. 14,15 In Drosophila, Srf interaction with Mkl (MAL-D) promotes cytoskeletal strength during cellular migration. 16,17 In murine embryonic stem cells, Srf is crucial for actin cytoskeletal organization and focal adhesion assembly. 18 Murine embryos lacking Srf fail to form mesoderm and thus die early in development. 19 Prior work in our laboratory has focused on acute megakaryoblastic leukemia with the t(1;22) translocation, involving fusion between an RNA binding motif protein 15 (RBM15) and Mkl1. Mkl1 is known to act as a cofactor for Srf-mediated gene activation in muscle differentiation. 12,20 We have demonstrated a physiologic role for Mkl1 in megakaryopoiesis. 21 Mkl1 expression increases with normal megakaryocyte (Mk) differentiation, and promotes Mk polyploidization. Mkl1 knockout (KO) mice have normal hematopoietic stem cells and megakaryocyte-erythroid progenitors (Pre-Meg-E). However, there is a dramatic increase in the number of CD41 ϩ megakaryocytes with most of these having low (2N) ploidy. There is a significant decrease in the number of polyploid megakaryocytes and thrombocytopenia. Mkl1 requires Srf to enhance polyploidization during megakaryocytic differentiation. Based on these findings, we have examined the effects of the Mkl1 cofactor Srf in megakaryocyte development.We show, that Srf deletion specifically in the megakaryocytic lineage leads to macrothrombocytopenia, whereas bone marrow (BM) and spleen show significant accumulation of abnormal megakaryocytes. Examination of candidate Srf target genes reveals that several actin cyt...

• RhoA-induced actin polymerization promotes nuclear accumulation of MKL1 and transcriptional activation.• Thrombopoietin activates nuclear accumulation of MKL1 and transcriptional activation in primary megakarocytes.How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for its function as a transcriptional regulator. MKL1 localization is cell-type specific; it is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types and is constitutively nuclear in neuronal cells. In the present study, we report that in megakaryocytes, subcellular localization and regulation of MKL1 is dependent on RhoA activity and actin organization. Induction of megakaryocytic differentiation of human erythroleukemia cells by 12-O-tetradecanoylphorbol-13-acetate and primary megakaryocytes by thrombopoietin promotes MKL1 nuclear localization. This MKL1 localization is blocked by drugs inhibiting RhoA activity or actin polymerization. We also show that nuclear-localized MKL1 activates the transcription of SRF target genes. This report broadens our knowledge of the molecular mechanisms regulating megakaryocyte differentiation. (Blood. 2013;121(7):1094-1101) IntroductionAlthough megakaryoblastic leukemia 1 (MKL1, also known as MRTF-A, MAL, or BSAC) plays a role in normal megakaryocytopoiesis, 1-3 much of what is known about this transcriptional coactivator of serum response factor (SRF) has been defined in fibroblasts and muscle cells. MKL1 promotes musclespecific gene expression, maintains mammary myoepithelial cell differentiation, and contributes to myocardial infarction-induced fibrosis and myofibroblast activation. [4][5][6][7] Other members of the MKL1 family include MKL2 and Myocardin. All 3 genes have been implicated in muscle cell differentiation, but have different patterns of cellular and developmental expression, which likely explains some of the differences in their knockout (KO) phenotypes. Although Mkl2-and Myocardin-KO mice are embryonic lethal with severe cardiac abnormalities, Mkl1-KO mice are viable with a less severe phenotype. Female Mkl1-KO mice have premature mammary gland involution that prevents lactation. 6,8 In addition, Mkl1-KO mice have impaired megakaryocytopoiesis defined by increased numbers of megakaryocytes in the BM, decreased ploidy of BM megakaryocytes, and low peripheral blood platelet counts. 1,3 In fibroblast cell lines, MKL1 activity is regulated posttranslationally by its subcellular localization, which is dependent on the actin cytoskeleton. 9-11 When MKL1 is bound to monomeric (G)-actin via its N-terminal RPEL domains, it is predominantly local...

Lunatic fringe belongs to a family of β1–3 N-acetyltransferases that modulate the affinity of the Notch receptors for their ligands through the elongation of O-fucose moieties on their extracellular domain. A role for Notch signaling in vertebrate fertility has been predicted by the intricate expression of the Notch receptors and their ligands in the oocyte and granulosa cells of the ovary and the spermatozoa and Sertoli cells of the testis. It has been demonstrated that disruption of Notch signaling by inactivation of lunatic fringe led to infertility associated with pleiotropic defects in follicle development and meiotic maturation of oocytes. Lunatic fringe null males were found to be subfertile. Here, we report that gene expression data demonstrate that fringe and Notch signaling genes are expressed in the developing testis and the intratesticular ductal tract, predicting roles for this pathway during embryonic gonadogenesis and spermatogenesis. Spermatogenesis was not impaired in the majority of the lunatic fringe null males; however, spermatozoa were unilaterally absent in the epididymis of many mice. Histological and immunohistochemical analysis of these testes revealed the development of unilateral cystic dilation of the rete testis. Tracer dye experiments confirm a block in the connection between the rete testis and the efferent ducts. Further, the dye studies demonstrated that many lunatic fringe mutant males had partial blocks of the connection between the rete testis and the efferent ducts bilaterally.