Transient abnormal myelopoiesis (TAM) is a myeloid proliferation resembling acute megakaryoblastic leukemia (AMKL), mostly affecting perinatal infants with Down syndrome. Although self-limiting in a majority of cases, TAM may evolve as non-self-limiting AMKL after spontaneous remission (DS-AMKL). Pathogenesis of these Down syndrome-related myeloid disorders is poorly understood, except for GATA1 mutations found in most cases. Here we report genomic profiling of 41 TAM, 49 DS-AMKL and 19 non-DS-AMKL samples, including whole-genome and/or whole-exome sequencing of 15 TAM and 14 DS-AMKL samples. TAM appears to be caused by a single GATA1 mutation and constitutive trisomy 21. Subsequent AMKL evolves from a pre-existing TAM clone through the acquisition of additional mutations, with major mutational targets including multiple cohesin components (53%), CTCF (20%), and EZH2, KANSL1 and other epigenetic regulators (45%), as well as common signaling pathways, such as the JAK family kinases, MPL, SH2B3 (LNK) and multiple RAS pathway genes (47%).

Drosophila sine oculis and eyes absent genes synergize in compound-eye formation. The murine homologues of these genes, Six and Eya, respectively, show overlapping expression patterns during development. We hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites. A clear synergistic activation of these promoters was observed in certain combinations of Six and Eya. To investigate the molecular basis for the cooperation, we first examined the intracellular distribution of Six and Eya proteins in transfected COS7 cells. Coexpression of Six2, Six4, or Six5 induced nuclear translocation of Eya1, Eya2, and Eya3, which were otherwise distributed in the cytoplasm. In contrast, coexpression of Six3 did not result in nuclear localization of any Eya proteins. Six and Eya proteins were coimmunoprecipitated from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, were necessary and sufficient for the nuclear translocation of Eya. In contrast, the Eya domain, a conserved domain among Eya proteins, was not sufficient for the translocation. A specific interaction between the Six domain and homeodomain of Six4 and Eya2 was observed by yeast two-hybrid analysis. Our results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes.Six genes are mouse homologues of the Drosophila sine oculis (so) gene, which is essential for compound-eye formation (9, 31). Six members of the Six family of genes have so far been identified in the mouse (17,18,27,28,35). Each Six gene shows a specific expression pattern during development of the mouse embryo. Six1 and Six2 show expression in mesenchymal cells around E8.5 to E10.5 and in muscles and limb tendons in later stages (28). Six3 is expressed in the rostral forebrain in earlier stages and is confined to the prospective eye region (27). Six4 proteins are distributed in the peripheral region of the mantle layer of the developing brain and spinal cord and in various ganglia between E9.5 and E14.5 (25). Six5 mRNA is expressed as early as E7 and is abundantly expressed in neonatal heart and skeletal muscles (24). Human SIX5 resides downstream of a CTG repeat, whose expansion leads to myotonic dystrophy (DM) (7). Since SIX5 is expressed in several tissues affected by DM and the transcription of SIX5 is repressed by the causative a CTG repeat expansion, it has been proposed that SIX5 is involved in som...

A similar gene network was found to control chick myogenesis, in which Six1, Eya2 and Dach2 synergistically regulate the expression of myogenic genes such as myogenin and MyoD (Heanue et Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wildtype otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1-and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.

To determine the dendritic fields, mitral, displaced mitral, middle tufted, and granule cells in the rabbit olfactory bulb were stained by intracellular injection of HRP. The secondary dendrites of mitral cells were distributed mostly in the inner half of the external plexiform layer (EPL). Those of displaced mitral cells extended mainly into the middle and superficial sublayers in the EPL. The secondary dendrites of middle tufted cells were distributed mostly in the superficial portion of the EPL. Mitral cells extended their secondary dendrites in virtually all directions within a plane tangential to the mitral cell layer (MCL) and thus had a disklike projection field with a radius of about 850 microns. Displaced mitral cells had similar dendritic projection fields in the tangential plane but with somewhat distorted shapes. The secondary dendrites of middle tufted cells had a tendency to extend in particular directions. From the projection pattern of the gemmules on the peripheral processes, granule cells were classified into three types. Type I granule cells had gemmules both in the superficial and in the deep sublayers of the EPL. The peripheral processes of Type II granule cells were confined to the deep half of the EPL. The gemmules of Type III granule cells ere distributed in the superficial half of the EPL. The differing dendritic ramification among mitral, displaced mitral, and middle tufted cells suggests the separation of the dendrodendritic synaptic interactions with granule cells in different sublayers in the EPL. It also suggests a functional separation of the sublayers of the EPL.

Homeobox genes constitute a large family of transcription factors that are essential during normal development and are often dysregulated in cancer. However, the molecular mechanisms by which homeobox genes influence cancer remain largely unknown. Here we show that the tissue-restricted cyclin A1 is a transcriptional target of the Six1 homeoprotein. Both genes are expressed in the embryonic but not the terminally differentiated mammary gland, and Six1-knockout mice show a dramatic reduction of cyclin A1 in the embryonic mammary gland. In addition, both genes are reexpressed in breast cancers. Six1 overexpression increases cyclin A1 mRNA levels and activity, cell proliferation, and tumor volume, whereas Six1 down-regulation decreases cyclin A1 mRNA levels and proliferation. Overexpression of Six1 in wild-type mouse embryonic fibroblasts, but not in knockout variants lacking the cyclin A1 gene, induces cell proliferation. Furthermore, inhibition of cyclin A1 in Six1-overexpressing mammary carcinoma cells decreases proliferation. Together these results demonstrate that cyclin A1 is required for the proliferative effect of Six1. We conclude that Six1 overexpression reinstates an embryonic pathway of proliferation in breast cancer by up-regulating cyclin A1.