Abnormal arterial-venous fusions and fate specification in mouse embryos lacking blood flow.

The functions of blood flow in the morphogenesis of mammalian arteries and veins are not well understood. We examined the development of the dorsal aorta (DA) and the cardinal vein (CV) in Ncx1 -/- mutants, which lack blood flow due to a deficiency in a sodium calcium ion exchanger expressed specifically in the heart. The mutant DA and CV were abnormally connected. The endothelium of the Ncx1 -/- mutant DA lacked normal expression of the arterial markers ephrin-B2 and Connexin-40. Notch1 activation, known to promote arterial specification, was decreased in mutant DA endothelial cells (ECs), which ectopically expressed the venous marker Coup-TFII. These findings suggest that flow has essential functions in the DA by promoting arterial and suppressing venous marker expression. In contrast, flow plays a lesser role in the CV, because expression of arterial-venous markers in CV ECs was not as dramatically affected in Ncx1 -/- mutants. We propose a molecular mechanism by which blood flow mediates DA and CV morphogenesis, by regulating arterial-venous specification of DA ECs to ensure proper separation of the developing DA and CV.

Neural crest defects in ephrin-B2 mutant mice are non-autonomous and originate from defects in the vasculature.

Ephrin-B2, a member of the Eph/ephrin family of cell signaling molecules, has been implicated in the guidance of cranial and trunk neural crest cells (NCC) and development of the branchial arches(BA), but detailed examination in mice has been hindered by embryonic lethality of Efnb2 null loss of function due to a requirement in angiogenic remodeling. To elucidate the developmental roles for Efnb2, we generated a conditional rescue knock-in allele that allows rescue of ephrin-B2 specifically in the vascular endothelium (VE), but is otherwise ephrin-B2 deficient. Restoration of ephrin-B2 expression specifically to the VE completely circumvents angiogenic phenotypes, indicating that the requirement of ephrin-B2 in angiogenesis is limited to the VE. Surprisingly, we find that expression of ephrin-B2 specifically in the VE is also sufficient for normal NCC migration and that conversely, embryos in which ephrin-B2 is absent specifically from the VE exhibit NCC migration and survival defects. Disruption of vascular development independent of loss of ephrin-B2 function also leads to defects in NCC and BA development. Together, these data indicate that direct ephrin-B2 signaling to NCCs is not required for NCC guidance, which instead depends on proper organization of the embryonic vasculature.

Large-scale mapping of transposable element insertion sites using digital encoding of sample identity.

Determining the genomic locations of transposable elements is a common experimental goal. When mapping large collections of transposon insertions, individualized amplification and sequencing is both time consuming and costly. We describe an approach in which large numbers of insertion lines can be simultaneously mapped in a single DNA sequencing reaction by using digital error-correcting codes to encode line identity in a unique set of barcoded pools.

Apical-basal polarity proteins are required cell-type specifically to direct photoreceptor morphogenesis.

Insect photoreceptor function is dependent on precise placement of the rhabdomeres, elaborated apical domains specialized for capturing light, within each facet of a compound eye. In Diptera, an asymmetric arrangement of rhabdomeres, combined with a particular pattern of axonal connections, enhances light sensitivity through the principle of neural superposition. To achieve the necessary retinal geometry, different photoreceptors (R cells) have distinct shapes. The Crumbs and Bazooka complexes play critical roles in directing rhabdomere development, but whether they might direct cell-type-specific apical architectures is unknown. We demonstrate that while mutations in Bazooka complex members cause pleiotropic morphogenesis defects in all R cell subtypes, Crumbs (Crb) and Stardust (Sdt) function cell autonomously to direct early stages in rhabdomere assembly in specific subsets of R cells. This requirement is reflected in the cell-type-specific expression of Crb protein and demonstrates that Sdt and Crb can act independently to similar effect. These two genes are also required for zonula adherens (ZA) assembly but display an unusual pattern of cellular redundancy for this function, as each gene is required in only one of two adjoining cells. Our results provide a direct link between fate specification and morphogenetic patterning and suggest a model for ZA assembly.

The cytoskeletal regulator Genghis khan is required for columnar target specificity in the Drosophila visual system.

A defining characteristic of neuronal cell type is the growth of axons and dendrites into specific layers and columns of the brain. Although differences in cell surface receptors and adhesion molecules are known to cause differences in synaptic specificity, differences in downstream signaling mechanisms that determine cell type-appropriate targeting patterns are unknown. Using a forward genetic screen in Drosophila, we identify the GTPase effector Genghis khan (Gek) as playing a crucial role in the ability of a subset of photoreceptor (R cell) axons to innervate appropriate target columns. In particular, single-cell mosaic analyses demonstrate that R cell growth cones lacking Gek function grow to the appropriate ganglion, but frequently fail to innervate the correct target column. Further studies reveal that R cell axons lacking the activity of the small GTPase Cdc42 display similar defects, providing evidence that these proteins regulate a common set of processes. Gek is expressed in all R cells, and a detailed structure-function analysis reveals a set of regulatory domains with activities that restrict Gek function to the growth cone. Although Gek does not normally regulate layer-specific targeting, ectopic expression of Gek is sufficient to alter the targeting choices made by another R cell type, the targeting of which is normally Gek independent. Thus, specific regulation of cytoskeletal responses to targeting cues is necessary for cell type-appropriate synaptic specificity.

Complex interactions amongst N-cadherin, DLAR, and Liprin-alpha regulate Drosophila photoreceptor axon targeting.

The formation of stable adhesive contacts between pre- and post-synaptic neurons represents the initial step in synapse assembly. The cell adhesion molecule N-cadherin, the receptor tyrosine phosphatase DLAR, and the scaffolding molecule Liprin-alpha play critical, evolutionarily conserved roles in this process. However, how these proteins signal to the growth cone and are themselves regulated remains poorly understood. Using Drosophila photoreceptors (R cells) as a model, we evaluate genetic and physical interactions among these three proteins. We demonstrate that DLAR function in this context is independent of phosphatase activity but requires interactions mediated by its intracellular domain. Genetic studies reveal both positive and, surprisingly, inhibitory interactions amongst all three genes. These observations are corroborated by biochemical studies demonstrating that DLAR physically associates via its phosphatase domain with N-cadherin in Drosophila embryos. Together, these data demonstrate that N-cadherin, DLAR, and Liprin-alpha function in a complex to regulate adhesive interactions between pre- and post-synaptic cells and provide a novel mechanism for controlling the activity of Liprin-alpha in the developing growth cone.

Germ-line specific variants of components of the mitochondrial outer membrane import machinery in Drosophila.

A search of the Drosophila genome for genes encoding components of the mitochondrial translocase of outer membrane (TOM) complex revealed duplication of genes encoding homologues of Tom20 and Tom40. Tom20 and Tom40 were represented by two differentially expressed homologues in the Drosophila genome. While dtom20 and dtom40 appeared to be expressed ubiquitously, the second variants, called tomboy20 and tomboy40, were expressed only in the male germ-line. Transcripts for tomboy20 and tomboy40 were detected in primary spermatocytes as well as post-meiotic stages. Transcription of tomboy20 and tomboy40 in spermatocytes was not dependent on the transcription factor Cannonball, which is responsible for controlling expression of gene products exclusively required for post-meiotic germ cell differentiation. Epitope-tagging and transient expression of dTom20 and Tomboy40 in mammalian cell culture showed proper targeting to mitochondria.

Differential expression of the Drosophila mitofusin genes fuzzy onions (fzo) and dmfn.

Mitofusins comprise a family of evolutionarily conserved, nuclear encoded mitochondrial guanosine triphoshatases that control mitochondrial fusion and morphology. The fuzzy onions (fzo) and Drosophila mitofusin (dmfn) genes, which encode the only Mitofusin homologs in Drosophila are differentially expressed during development. Dmfn-mRNA was widely expressed during embryogenesis accumulating in the mesoderm and endoderm during gut development, during oogenesis with transcripts maternally deposited into the early embryo and in the male germ line, where dmfn-mRNA was expressed in spermatogonia, spermatocytes and early spermatids. In contrast, expression of the fzo was tightly restricted to the male germ line, with mRNA accumulation in spermatocytes and early spermatids. In addition, expression of dmfn and fzo in the same cell type, primary spermatocytes, was under control of different regulatory mechanisms.