SVA Retrotransposons and a Low Copy Repeat in Humans and Great Apes: A Mobile Connection.

Segmental duplications (SDs) constitute a considerable fraction of primate genomes. They contribute to genetic variation and provide raw material for evolution. Groups of SDs are characterized by the presence of shared core duplicons. One of these core duplicons, low copy repeat (lcr)16a, has been shown to be particularly active in the propagation of interspersed SDs in primates. The underlying mechanisms are, however, only partially understood. Alu short interspersed elements (SINEs) are frequently found at breakpoints and have been implicated in the expansion of SDs. Detailed analysis of lcr16a-containing SDs shows that the hominid-specific SVA (SINE-R-VNTR-Alu) retrotransposon is an integral component of the core duplicon in Asian and African great apes. In orang-utan, it provides breakpoints and contributes to both interchromosomal and intrachromosomal lcr16a mobility by inter-element recombination. Furthermore, the data suggest that in hominines (human, chimpanzee, gorilla) SVA recombination-mediated integration of a circular intermediate is the founding event of a lineage-specific lcr16a expansion. One of the hominine lcr16a copies displays large flanking direct repeats, a structural feature shared by other SDs in the human genome. Taken together, the results obtained extend the range of SVAs' contribution to genome evolution from RNA-mediated transduction to DNA-based recombination. In addition, they provide further support for a role of circular intermediates in SD mobilization.

LINE-1 ORF1p does not determine substrate preference for human/orangutan SVA and gibbon LAVA.

BACKGROUND: Non-autonomous VNTR (Variable Number of Tandem Repeats) composite retrotransposons - SVA (SINE-R-VNTR-Alu) and LAVA (L1-Alu-VNTR-Alu) - are specific to hominoid primates. SVA expanded in great apes, LAVA in gibbon. Both SVA and LAVA have been shown to be mobilized by the autonomous LINE-1 (L1)-encoded protein machinery in a cell-based assay in trans. The efficiency of human SVA retrotransposition in vitro has, however, been considerably lower than would be expected based on recent pedigree-based in vivo estimates. The VNTR composite elements across hominoids - gibbon LAVA, orangutan SVA_A descendants and hominine SVA_D descendants - display characteristic structures of the 5' Alu-like domain and the VNTR. Different partner L1 subfamilies are currently active in each of the lineages. The possibility that the lineage-specific types of VNTR composites evolved in response to evolutionary changes in their autonomous partners, particularly in the nucleic acid binding L1 ORF1-encoded protein, has not been addressed. RESULTS: Here I report the identification and functional characterization of a highly active human SVA element using an improved mneo retrotransposition reporter cassette. The modified cassette (mneoM) minimizes splicing between the VNTR of human SVAs and the neomycin phosphotransferase stop codon. SVA deletion analysis provides evidence that key elements determining its mobilization efficiency reside in the VNTR and 5' hexameric repeats. Simultaneous removal of the 5' hexameric repeats and part of the VNTR has an additive negative effect on mobilization rates. Taking advantage of the modified reporter cassette that facilitates robust cross-species comparison of SVA/LAVA retrotransposition, I show that the ORF1-encoded proteins of the L1 subfamilies currently active in gibbon, orangutan and human do not display substrate preference for gibbon LAVA versus orangutan SVA versus human SVA. Finally, I demonstrate that an orangutan-derived ORF1p supports only limited retrotransposition of SVA/LAVA in trans, despite being fully functional in L1 mobilization in cis. CONCLUSIONS: Overall, the analysis confirms SVA as a highly active human retrotransposon and preferred substrate of the L1-encoded protein machinery. Based on the results obtained in human cells coevolution of L1 ORF1p and VNTR composites does not appear very likely. The changes in orangutan L1 ORF1p that markedly reduce its mobilization capacity in trans might explain the different SVA insertion rates in the orangutan and hominine lineages, respectively.

Phylogenomic analysis reveals splicing as a mechanism of parallel evolution of non-canonical SVAs in hominine primates.

SVA (SINE-R-VNTR-Alu) elements are non-autonomous non-LTR (Long Terminal Repeat) retrotransposons. They are found in all hominoid primates but did not amplify to appreciable numbers in gibbons. Recently, phylogenetic networks of hominid (orangutan, gorilla, chimpanzee, human) SVA elements based on comparison of overall sequence identity have been reported. Here I present a detailed phylogeny of SVA_D elements in gorilla, chimpanzee and humans based on sorting of co-segregating substitutions. Complementary comparative genomics analysis revealed that the majority (1763 out of 1826-97%) of SVA_D elements in gorilla represent species-specific insertions - indicating very low activity of the subfamily before the gorilla/chimpanzee-human split. The origin of the human-specific subfamily SVA_F could be traced back to a source element in the hominine common ancestor. The major expanding lineage-specific subfamilies were found to differ between chimpanzee and humans. Precursors of the dominant chimpanzee SVA_D subfamily are present in humans; however, they did not expand to appreciable levels. The analysis also uncovered that one of the chimpanzee-specific subfamilies was formed by splicing of the STK40 first exon to the SVA Alu-like region. Many of the 94 subfamily members contain additional 5' transductions - among them exons of 8 different other genes. Striking similarities to the MAST2-containing human SVA_F1 suggest parallel evolution of non-canonical SVAs in chimpanzees and humans.

Mobile Element Evolution Playing Jigsaw - SINEs in Gastropod and Bivalve Mollusks.

SINEs (Short INterspersed Elements) are widely distributed among eukaryotes. Some SINE families are organized in superfamilies characterized by a shared central domain. These central domains are conserved across species, classes, and even phyla. Here we report the identification of two novel such superfamilies in the genomes of gastropod and bivalve mollusks. The central conserved domain of the first superfamily is present in SINEs in Caenogastropoda and Vetigastropoda as well as in all four subclasses of Bivalvia. We designated the domain MESC (Romanian for MElc-snail and SCoica-mussel) because it appears to be restricted to snails and mussels. The second superfamily is restricted to Caenogastropoda. Its central conserved domain-Snail-is related to the Nin-DC domain. Furthermore, we provide evidence that a 40-bp subdomain of the SINE V-domain is conserved in SINEs in mollusks and arthropods. It is predicted to form a stable stem-loop structure that is preserved in the context of the overall SINE RNA secondary structure in invertebrates. Our analysis also recovered short retrotransposons with a Long INterspersed Element (LINE)-derived 5' end. These share the body and/or the tail with transfer RNA (tRNA)-derived SINEs within and across species. Finally, we identified CORE SINEs in gastropods and bivalves-extending the distribution range of this superfamily.

Lineage specific evolution of the VNTR composite retrotransposon central domain and its role in retrotransposition of gibbon LAVA elements.

BACKGROUND: VNTR (Variable Number of Tandem Repeats) composite retrotransposons - SVA (SINE-R-VNTR-Alu), LAVA (LINE-1-Alu-VNTR-Alu), PVA (PTGR2-VNTR-Alu) and FVA (FRAM-VNTR-Alu) - are specific to hominoid primates. Their assembly, the evolution of their 5' and 3' domains, and the functional significance of the shared 5' Alu-like region are well understood. The central VNTR domain, by contrast, has long been assumed to represent a more or less random collection of 30-50 bp GC-rich repeats. It is only recently that it attracted attention in the context of regulation of SVA expression. RESULTS: Here we provide evidence that the organization of the VNTR is non-random, with conserved repeat unit (RU) arrays at both the 5' and 3' ends of the VNTRs of human, chimpanzee and orangutan SVA and gibbon LAVA. The younger SVA subfamilies harbour highly organized internal RU arrays. The composition of these arrays is specific to the human/chimpanzee and orangutan lineages, respectively. Tracing the development of the VNTR through evolution we show for the first time how tandem repeats evolve within the constraints set by a functional, non-autonomous non-LTR retrotransposon in two different families - LAVA and SVA - in different hominoid lineages. Our analysis revealed that a microhomology-driven mechanism mediates expansion/contraction of the VNTR domain at the DNA level. Elements of all four VNTR composite families have been shown to be mobilized by the autonomous LINE1 retrotransposon in trans. In case of SVA, key determinants of mobilization are found in the 5' hexameric repeat/Alu-like region. We now demonstrate that in LAVA, by contrast, the VNTR domain determines mobilization efficiency in the context of domain swaps between active and inactive elements. CONCLUSIONS: The central domain of VNTR composites evolves in a lineage-specific manner which gives rise to distinct structures in gibbon LAVA, orangutan SVA, and human/chimpanzee SVA. The differences observed between the families and lineages are likely to have an influence on the expression and mobilization of the elements.

Variant rs1801157 in the 3'UTR of SDF-1ß does not explain variability of healthy-donor G-CSF responsiveness.

The genetics responsible for the inter-individually variable G-CSF responsiveness remain elusive. A single nucleotide polymorphism (SNP) in the 3'UTR of CXCL12, rs1801157, was implicated in X4-tropic HiV susceptibility and later, in two small studies, in G-CSR responsiveness in patients and donors. The position of the SNP in the 3'UTR together with in-silico predictions suggested differential binding of micro-RNA941 as an underlying mechanism. In a cohort of 515 healthy stem cell donors we attempted to reproduce the correlation of the CXCL12 3'UTR SNP and mobilization responses and tested the role of miR941 in this context. The SNP was distributed with the expected frequency. Mobilization efficiency for CD34+ cells in WT, heterozygous and homozygous SNP individuals was indistinguishable, even after controlling for gender. miR941 expression in non-hematopoietic bone marrow cells was undetectable and miR941 did not interact with the 3' UTR of CXCL12. Proposed effects of the SNP rs1801157 on G-CSF responsiveness cannot be confirmed in a larger cohort.

Composite non-LTR retrotransposons in hominoid primates.

Composite retrotransposons are widely distributed in the plant and animal kingdoms. Some of the most complex of these are found in hominoid primates. SVA, LAVA, PVA and FVA combine simple repeats, Alu fragments, a VNTR (Variable Number of Tandem Repeats) and variable 3' domains, which are, except for PVA, derived from other retrotransposons. Although a likely precursor of SVA-a "tailed VNTR" named SVA2-had been identified in the Rhesus genome, the exact sequence and mechanism of the assembly of this type of composite retrotransposon had been elusive. The discovery of LAVA, PVA and FVA in gibbons provided the opportunity to delineate the order of assembly of the components of VNTR-containing retrotransposons. Our recent analysis suggests that an extinct "Alu-SVA2" acquired variant 3' ends by splicing. In this commentary I will discuss the mode of assembly of VNTR composites in the context of their capacity to engage in alternative splicing to co-mobilize host RNA sequences and to become exonized. The second part will focus on structural determinants of VNTR composite retrotransposon mobilization in the context of lineage-specific expansion of particular families/subfamilies of these elements.

Hominoid composite non-LTR retrotransposons-variety, assembly, evolution, and structural determinants of mobilization.

SVA (SINE-R-VNTR-Alu) elements constitute the youngest family of composite non-LTR retrotransposons in hominoid primates. The sequence of their assembly, however, remains unclear. Recently, a second family of VNTR-containing composites, LAVA (L1-Alu-VNTR-Alu), has been identified in gibbons. We now report the existence of two additional VNTR composite families, PVA (PTGR2-VNTR-Alu) and FVA (FRAM-VNTR-Alu), in the genome of Nomascus leucogenys. Like LAVA, they share the 5'-Alu-like region and VNTR with SVA, but differ at their 3'-ends. The 3'-end of PVA comprises part of the PTGR2 gene, whereas FVA is characterized by the presence of a partial FRAM element in its 3'-domain. Splicing could be identified as the mechanism of acquisition of the variant 3'-ends in all four families of VNTR composites. SVAs have been shown to be mobilized by the L1 protein machinery in trans. A critical role in this process has been ascribed to their 5'-hexameric repeat/ Alu-like region. The Alu-like region displays specific features in each of the VNTR composite families/subfamilies with characteristic deletions found in the evolutionary younger subfamilies. Using reciprocal exchanges between SVA_E and PVA/FVA elements, we demonstrate that the structure, not the presence of the (CCCTCT)n/ Alu-like region determines mobilization capacity. Combination of LAVA and SVA_E domains does not yield any active elements-suggesting the use of different combinations of host factors for the two major groups of VNTR composites. Finally, we demonstrate that the LAVA 3'-L1ME5 fragment attenuates mobilization capacity.

Gibbon genome and the fast karyotype evolution of small apes.

Gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between Old World monkeys and great apes. Here we present the assembly and analysis of a northern white-cheeked gibbon (Nomascus leucogenys) genome. We describe the propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. We further show that the gibbon genera (Nomascus, Hylobates, Hoolock and Symphalangus) experienced a near-instantaneous radiation ∼5 million years ago, coincident with major geographical changes in southeast Asia that caused cycles of habitat compression and expansion. Finally, we identify signatures of positive selection in genes important for forelimb development (TBX5) and connective tissues (COL1A1) that may have been involved in the adaptation of gibbons to their arboreal habitat.

Inference of transposable element ancestry.

Most common methods for inferring transposable element (TE) evolutionary relationships are based on dividing TEs into subfamilies using shared diagnostic nucleotides. Although originally justified based on the "master gene" model of TE evolution, computational and experimental work indicates that many of the subfamilies generated by these methods contain multiple source elements. This implies that subfamily-based methods give an incomplete picture of TE relationships. Studies on selection, functional exaptation, and predictions of horizontal transfer may all be affected. Here, we develop a Bayesian method for inferring TE ancestry that gives the probability that each sequence was replicative, its frequency of replication, and the probability that each extant TE sequence came from each possible ancestral sequence. Applying our method to 986 members of the newly-discovered LAVA family of TEs, we show that there were far more source elements in the history of LAVA expansion than subfamilies identified using the CoSeg subfamily-classification program. We also identify multiple replicative elements in the AluSc subfamily in humans. Our results strongly indicate that a reassessment of subfamily structures is necessary to obtain accurate estimates of mutation processes, phylogenetic relationships and historical times of activity.

Chitosan-coated triangular silver nanoparticles as a novel class of biocompatible, highly sensitive plasmonic platforms for intracellular SERS sensing and imaging.

There is a need for new strategies for noninvasive imaging of pathological conditions within the human body. The approach of combining the unique physical properties of noble-metal nanoparticles with their chemical specificity and an easy way of conjugation open up new routes toward building bio-nano-objects for biomedical tracking and imaging. This work reports the design and assessment of a novel class of biocompatible, highly sensitive SERS nanotags based on chitosan-coated silver nanotriangles (Chit-AgNTs) labeled with para-aminothiophenol (p-ATP). The triangular nanoparticles are used as Raman scattering enhancers and have proved to yield a reproducible and strong SERS signal. When tested inside lung cancer cells (A549) this class of SERS nanotags presents low in vitro toxicity, without interfering with cell proliferation. Easily internalized by the cells, as demonstrated by imaging using both reflected bright-light optical microscopy and SERS spectroscopy, the particles are proved to be detectable inside cells under a wide window of excitation wavelengths, ranging from visible to near infrared (NIR). Their high sensitivity and NIR availability make this class of SERS nanotags a promising candidate for noninvasive imaging of cancer cells.

The non-autonomous retrotransposon SVA is trans-mobilized by the human LINE-1 protein machinery.

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.

Synergistic antibacterial activity of chitosan-silver nanocomposites on Staphylococcus aureus.

The approach of combining different mechanisms of antibacterial action by designing hybrid nanomaterials provides a new paradigm in the fight against resistant bacteria. Here, we present a new method for the synthesis of silver nanoparticles enveloped in the biopolymer chitosan. The method aims at the production of bionanocomposites with enhanced antibacterial properties. We find that chitosan and silver nanoparticles act synergistically against two strains of Gram-positive Staphylococcus aureus (S. aureus). As a result the bionanocomposites exhibit higher antibacterial activity than any component acting alone. The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of the chitosan-silver nanoparticles synthesized at 0 °C were found to be lower than those reported for other types of silver nanoparticles. Atomic force microscopy (AFM) revealed dramatic changes in morphology of S. aureus cells due to disruption of bacterial cell wall integrity after incubation with chitosan-silver nanoparticles. Finally, we demonstrate that silver nanoparticles can be used not only as antibacterial agents but also as excellent plasmonic substrates to identify bacteria and monitor the induced biochemical changes in the bacterial cell wall via surface enhanced Raman scattering (SERS) spectroscopy.

Expression of estrogen receptor alpha increases leptin-induced STAT3 activity in breast cancer cells.

Adipositas correlates with an enhanced risk of developing malignant diseases such as breast cancer, endometrial tumor or prostate carcinoma, but the molecular basis for this is not well understood. Potential mechanisms include increased bioavailability of adipocytokines (e.g. leptin) and steroid hormones. Here, we investigated cross-talk between ERalpha (estrogen receptor alpha) and leptin-induced activation of signal transducer and activator of transcription 3 (STAT3), a transactivator of important oncogenes. Upon leptin binding to its receptor Ob-RL (obesity receptor), STAT3 tyrosine phosphorylation and transactivation activity were enhanced by simultaneously expressing ERalpha. Downregulation of ERalpha using small interfering RNA abolished leptin-induced STAT3 phosphorylation. Interestingly, leptin-mediated STAT3 activation was unaffected by co-stimulation with the ERalpha ligands estradiol (E2) or estrogen antagonists ICI182,780 and tamoxifen, implying that enhancement of leptin-mediated STAT3 activity is independent of ERalpha ligands. We also detected ERalpha binding to STAT3 and JAK2 (Janus kinase 2), resulting in enhanced JAK2 activity upstream of STAT3 in response to leptin that might lead to an increased ERalpha-dependent cell viability. Altogether, our results indicate that leptin-induced STAT3 activation acts as a key event in ERalpha-dependent development of malignant diseases.

5'-Transducing SVA retrotransposon groups spread efficiently throughout the human genome.

SVA elements represent the youngest family of hominid non-LTR retrotransposons, which alter the human genome continuously. They stand out due to their organization as composite repetitive elements. To draw conclusions on the assembly process that led to the current organization of SVA elements and on their transcriptional regulation, we initiated our study by assessing differences in structures of the 116 SVA elements located on human chromosome 19. We classified SVA elements into seven structural variants, including novel variants like 3'-truncated elements and elements with 5'-flanking sequence transductions. We established a genome-wide inventory of 5'-transduced SVA elements encompassing approximately 8% of all human SVA elements. The diversity of 5' transduction events found indicates transcriptional control of their SVA source elements by a multitude of external cellular promoters in germ cells in the course of their evolution and suggests that SVA elements might be capable of acquiring 5' promoter sequences. Our data indicate that SVA-mediated 5' transduction events involve alternative RNA splicing at cryptic splice sites. We analyzed one remarkably successful human-specific SVA 5' transduction group in detail because it includes at least 32% of all SVA subfamily F members. An ancient retrotransposition event brought an SVA insertion under transcriptional control of the MAST2 gene promoter, giving rise to the primal source element of this group. Members of this group are currently transcribed. Here we show that SVA-mediated 5' transduction events lead to structural diversity of SVA elements and represent a novel source of genomic rearrangements contributing to genomic diversity.

Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells.

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.

SCL interacts with VEGF to suppress apoptosis at the onset of hematopoiesis.

During development, hematopoiesis initiates in the yolk sac through a process that depends on VEGF/Flk1 signaling and on the function of the SCL/Tal1 transcription factor. Here we show that VEGF modifies the developmental potential of primitive erythroid progenitors and prolongs their life span. Furthermore, the survival of yolk sac erythrocytes in vivo depends on the dose of VEGF. Interestingly, in Vegf(lo/lo) embryos carrying a hypomorph allele, Flk1-positive cells reach the yolk sac at E8.5, but are severely compromised in their ability to generate primitive erythroid precursors. These observations indicate that during embryonic development, different thresholds of VEGF are required for the migration and clonal expansion of hematopoietic precursors. The near absence of primitive erythroid precursors in Vegf(lo/lo) embryos correlates with low levels of Scl in the yolk sac. Strikingly, gain-of-function of SCL partially complements the hematopoietic defect caused by the hypomorph Vegf(lo) allele, and re-establishes the survival of erythroid cells and the expression of erythroid genes (Gata1 and betaH1). This indicates that SCL functions downstream of VEGF to ensure an expansion of the hematopoietic compartment.

Leptin receptor isoform 219.1: an example of protein evolution by LINE-1-mediated human-specific retrotransposition of a coding SVA element.

Phylogenetically new insertions of repetitive sequences may contribute to genome evolution by altering the function of preexisting proteins. One example is the SVA sequence, which forms the C-terminal coding exon of the human leptin receptor isoform 219.1. Here, we report that the SVA insertion into the LEPR locus has occurred after divergence of humans and chimpanzees. The SVA element was inserted into a Hal-1/LINE element present in all monkeys and apes tested. Structural features point toward an integration event that was mediated by the L1 protein machinery acting in trans. Thus, our findings add evidence to the hypothesis that retrotransposition events are a driving force in genomic evolution and that the presence or absence of specific retroelements are one distinguishing feature that separates humans from chimpanzees.

Cortical and retinal defects caused by dosage-dependent reductions in VEGF-A paracrine signaling.

To determine the function of VEGF-A in nervous system development, we have utilized the Nestin promoter-driven Cre recombinase transgene, in conjunction with a conditional and hypomorphic VEGF-A allele, to lower VEGF-A activity in neural progenitor cells. Mice with intermediate levels of VEGF-A activity showed decreased blood vessel branching and density in the cortex and retina, resulting in a thinner retina and aberrant structural organization of the cortex. Severe reductions in VEGF-A led to decreases in vascularity and subsequent hypoxia, resulting in the specific degeneration of the cerebral cortex and neonatal lethality. Decreased neuronal proliferation and hypoxia was evident at E11.5, leading to increased neuronal apoptosis in the cortex by E15.5. In order to address whether the observed changes in the structural organization of the nervous system were due to a direct and autocrine role of VEGF-A on the neural population, we conditionally inactivated the main VEGF-A receptor, Flk1, specifically in neuronal lineages, by using the Nestin Cre transgene. The normality of these mice ruled out the possibility that VEGF-A/Flk1 signaling has a significant autocrine role in CNS development. VEGF-A dosage is therefore a critical parameter regulating the density of the vascular plexus in the developing CNS that is in turn a key determinant in the development and architectural organization of the nervous system.