To ensure normal development and maintenance of homeostasis, the extensive developmental potential of stem cells must be functionally distinguished from the limited developmental potential of transit amplifying cells. Yet the mechanisms that restrict the developmental potential of transit amplifying cells are poorly understood. Here we show that the evolutionarily conserved transcription factor dFezf/Earmuff (Erm) functions cell-autonomously to maintain the restricted developmental potential of the intermediate neural progenitors generated by type II neuroblasts in Drosophila larval brains. Although erm mutant intermediate neural progenitors are correctly specified and show normal apical-basal cortical polarity, they can dedifferentiate back into a neuroblast state, functionally indistinguishable from normal type II neuroblasts. Erm restricts the potential of intermediate neural progenitors by activating Prospero to limit proliferation and by antagonizing Notch signaling to prevent dedifferentiation. We conclude that Erm dependence functionally distinguishes intermediate neural progenitors from neuroblasts in the Drosophila larval brain, balancing neurogenesis with stem cell maintenance.

Mesoderm induction has been studied in many systems and some of the factors involved have been identified. Although the heart is mesodermal in origin, the molecular basis of heart development is essentially unknown. The Drosophila heart is a simple tubular structure similar to the early heart tube in vertebrates. The homeobox gene, tinman, has been shown to be crucial for heart formation in Drosophila. Several genes with considerable sequence similarities to tinman are expressed in cardiac primordial tissue of vertebrates and are likely to be required for heart development of higher organisms as well. In addition to transcriptional control factors, heart development might also depend on inductive signals. Here, we demonstrate that the gene wingless (wg), which is known to specify segmental polarity and neuroblast identity in Drosophila, has a novel role in mesoderm development: wg function is specifically required for heart development. A temperature-sensitive mutation of wg was used to inactivate wg function during precise developmental time periods. Elimination of wg function for a short time period after gastrulation results in the selective loss of heart precursors, without significantly affecting the formation of the body wall or visceral muscles, although some pattern defects are observed. This developmental requirement of wg for cardiac organogenesis is distinct from its function in segmentation and neurogenesis. We conclude that wg signaling is a crucial component of heart formation.

Heart development in both vertebrates and Drosophila is initiated by bilaterally symmetrical primordia that may be of equivalent embryological origin: the anterior lateral plate mesoderm in vertebrates and the dorsal-most mesoderm in arthropods. These mesodermal progenitors then merge into a heart tube at the ventral midline (vertebrates) or the dorsal midline (Drosophila). These observations suggest that there may be similarities between vertebrate and invertebrate heart development. The homeobox gene, tinman, is required for heart as well as visceral mesoderm formation in Drosophila, and at least one of several vertebrate genes with similarities in protein sequence and cardiac expression to tinman is crucial for heart development in vertebrates. Inductive signals are also required for Drosophila heart development: The secreted gene product of wingless (wg) is critical for heart development during a time period distinct from its function in segmentation and neurogenesis. Here, we show that wg is epistatic to hedgehog (hh), another secreted segmentation gene product, in its requirement for heart formation. We also provide evidence show that downstream of wg in the signal transduction cascade, dishevelled (dsh, a pioneer protein) and armadillo (arm, beta-catenin homolog) are mediating the cardiogenic Wg signal. In particular, overexpression of dsh can restore heart formation in the absence of wg function. We discuss the possibility that Wg signaling is part of a combinatorial mechanism to specify the cardiac mesoderm.

The cut locus of Drosophila encodes a diverged homeodomain-containing protein that is required for the development of external sensory (es) organs and other tissues. A homologous gene (Cux-1) that encodes a transcriptional repressor has been identified in the mouse and other mammals. We have identified a second murine homeobox-containing gene (designated Cux-2) that is structurally related to Drosophila cut. The murine Cux-2 homeobox was similar to Drosophila cut and encoded a homeodomain that contained a characteristic histidine residue at position 50. The predicted Cux-2 protein contained 1426 amino acids and included three internal 60-amino acid repeats (Cut repeats) that were previously found in Drosophila Cut and murine Cux-1. Unlike Cux-1, expression of Cux-2 was restricted to neural tissue. In the adult brain, Cux-2 was prominently expressed in neurons in the thalamus and limbic system. In embryos, Cux-2 was expressed in the developing central and peripheral nervous systems, including the telencephalon and peripheral ganglia of the trigeminal and glossopharyngeal nerves. A glutathione S-transferase fusion protein containing the carboxyl-terminal Cut repeat and homeodomain of Cux-2 exhibited sequence-specific binding to oligonucleotides derived from the promoter of the Ncam gene. Using an interspecific backcross panel, Cux-1 and Cux-2 were mapped to distinct loci that were genetically linked on distal chromosome 5. These results demonstrate that a family of homeobox genes related to Drosophila cut is located on chromosome 5 in the mouse. Cux-2 is expressed exclusively in the central and peripheral nervous systems, and the Cux-2 gene product binds to DNA in a sequence-specific manner. Cux-2 may encode a transcription factor that is involved in neural specification in mammals.

Deep vein thrombosis and pulmonary embolism, collectively defined as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Common genetic variants conferring increased varying degrees of VTE risk have been identified by genome-wide association studies (GWAS). Rare mutations in the anticoagulant genes PROC, PROS1 and SERPINC1 result in perinatal lethal thrombosis in homozygotes and markedly increased VTE risk in heterozygotes. However, currently described VTE variants account for an insufficient portion of risk to be routinely utilized for clinical decision making. To identify new rare VTE risk variants, we performed a whole exome study of 393 individuals with unprovoked VTE and 6114 controls. This study identified four genes harboring an excess number of rare damaging variants in patients with VTE; PROS1, STAB2, PROC and SERPINC1. At STAB2, 7.8% of VTE cases and 2.4% of controls had a qualifying rare variant. In cell culture, VTE associated variants of STAB2 had a reduced surface expression compared to reference STAB2. Common variants in STAB2 have been previously associated with plasma von Willebrand factor and coagulation factor VIII levels in GWAS, suggesting that haploinsufficiency of stabilin-2 may increase VTE risk through elevated levels of these procoagulants. In an independent cohort, we found higher von Willebrand factor levels and equivalent propeptide levels in individuals with rare STAB2 variants compared to controls. Taken together, this study demonstrates the utility of gene-based collapsing analyses to identify loci harboring an excess of rare variants with functional connections to a complex thrombotic disease.