Giorgio R. Merlo

Polyethylenimine-based intravenous delivery of transgenes to mouse lung

Generally, cationic vector-based intravenous delivery of DNA is hindered by interactions of positively charged complexes with serum proteins. However, if optimally formulated, cationic vectors can provide reasonable levels of transfection in the lung either by intravenous or intrapulmonary routes. We investigated the in vivo transfection capacity of a cationic polymer: linear, 22 kDa polyethylenimine. PEI/DNA complexes were formulated in 5% glucose and delivered into adult mice through the tail vein. Two marker genes were used, β-galactosidase and luciferase. High levels of luciferase expression (107 RLU/mg protein) were found in the lung when DNA was complexed with PEI at a ratio of 4 nitrogen equivalents per DNA phosphate. Lower levels of transfection were found in the heart, spleen, liver and kidney. Expression was dose- and time-dependent in all tissues examined. In the lung, β-galactosidase staining showed transgene expression in clusters of 10 or more pulmonary cells including the alveolar endothelium, squamous and great alveolar epithelial cells (type I and II pneumocytes) and septal cells. These findings indicate that the complexes pass the capillary barrier in the lung. Although the delivery mechanism requires elucidation, linear PEI has promise as a vector for intravenous transfer of therapeutic genes.

Differential regulation of the zinc finger genes Krox-20 and Krox-24 (Egr-1) suggests antagonistic roles in Schwann cells

Krox‐20 and Krox‐24 (Egr‐1) encode closely related zinc finger transcription factors, which interact with the same DNA target sequences. Krox‐20 is required for myelination in the peripheral nervous system. Using lacZ knock‐in mutant mouse lines as well as immunohistochemical analyses, we have studied the expression of Krox‐20 and Krox‐24 in the Schwann cell lineage during normal development and following nerve lesion in the mouse and in human neuropathies. During embryogenesis, the two genes are expressed in a successive and mutually exclusive manner, Krox‐24 being restricted to Schwann cell precursors and Krox‐20 to mature Schwann cells. At birth, Krox‐24 is reactivated and the two genes are coexpressed. In the adult, Krox‐20 is expressed in myelinating cells, while Krox‐24 is restricted to nonmyelinating cells. Following nerve lesion, Krox‐24 is strongly induced in Schwann cells, reinforcing the link between its expression and the nonmyelinating and/or proliferative state, whereas Krox‐20 is downregulated. These data are consistent with Krox‐20 and Krox‐24 playing antagonistic roles during the development of the Schwann cell lineage. In particular, their balance of expression might participate in the choice between myelinating and nonmyelinating pathways. J. Neurosci. Res. 50:702–712, 1997. © 1997 Wiley‐Liss, Inc.

A regulatory feedback loop involving p63 and IRF6 links the pathogenesis of 2 genetically different human ectodermal dysplasias.

The human congenital syndromes ectrodactyly ectodermal dysplasia-cleft lip/palate syndrome, ankyloblepharon ectodermal dysplasia clefting, and split-hand/foot malformation are all characterized by ectodermal dysplasia, limb malformations, and cleft lip/palate. These phenotypic features are a result of an imbalance between the proliferation and differentiation of precursor cells during development of ectoderm-derived structures. Mutations in the p63 and interferon regulatory factor 6 (IRF6) genes have been found in human patients with these syndromes, consistent with phenotypes. Here, we used human and mouse primary keratinocytes and mouse models to investigate the role of p63 and IRF6 in proliferation and differentiation. We report that the DeltaNp63 isoform of p63 activated transcription of IRF6, and this, in turn, induced proteasome-mediated DeltaNp63 degradation. This feedback regulatory loop allowed keratinocytes to exit the cell cycle, thereby limiting their ability to proliferate. Importantly, mutations in either p63 or IRF6 resulted in disruption of this regulatory loop: p63 mutations causing ectodermal dysplasias were unable to activate IRF6 transcription, and mice with mutated or null p63 showed reduced Irf6 expression in their palate and ectoderm. These results identify what we believe to be a novel mechanism that regulates the proliferation-differentiation balance of keratinocytes essential for palate fusion and skin differentiation and links the pathogenesis of 2 genetically different groups of ectodermal dysplasia syndromes into a common molecular pathway.

Regulation of Dlx5 and Dlx6 gene expression by p63 is involved in EEC and SHFM congenital limb defects

The congenital malformation Split Hand-Foot Malformation (SHFM, or ectrodactyly) is characterized by a medial cleft of hands and feet, and missing central fingers. Five genetically distinct forms are known in humans; the most common (type-I) is linked to deletions of DSS1 and the distalless-related homeogenes DLX5 and DLX6. As Dlx5;Dlx6 double-knockout mice show a SHFM-like phenotype, the human orthologs are believed to be the disease genes. SHFM-IV and Ectrodactyly-Ectodermal dysplasia-Cleft lip (EEC) are caused by mutations in p63, an ectoderm-specific p53-related transcription factor. The similarity in the limb phenotype of different forms of SHFM may underlie the existence of a regulatory cascade involving the disease genes. Here, we show that p63 and Dlx proteins colocalize in the nuclei of the apical ectodermal ridge (AER). In homozygous p63- (null) and p63EEC (R279H) mutant limbs, the AER fails to stratify and the expression of four Dlx genes is strongly reduced; interestingly, the p63+/EEC and p63+/- hindlimbs, which develop normally and have a normally stratified AER, show reduced Dlx gene expression. The p63+/EEC mutation combined with an incomplete loss of Dlx5 and Dlx6 alleles leads to severe limb phenotypes, which are not observed in mice with either mutation alone. In vitro, ΔNp63α induces transcription from the Dlx5 and Dlx6 promoters, an activity abolished by EEC and SHFM-IV mutations, but not by Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) mutations. ChIP analysis shows that p63 is directly associated with the Dlx5 and Dlx6 promoters. Thus, our data strongly implicate p63 and the Dlx5-Dlx6 locus in a pathway relevant in the aetio-pathogenesis of SHFM.

Transforming growth factor-α messenger RNA localization in the developing adult rat and human mammary gland by in situ hybridization

Transforming growth factor-alpha (TGF alpha) has been implicated in the autocrine growth control of a number of different rodent and human tumor cells, including breast cancer cells. Although TGF alpha has been detected in a limited number of normal tissues, its distribution and physiological function in the mammary gland are relatively unknown. TGF alpha mRNA expression was detected by in situ hybridization with a labeled TGF alpha antisense RNA probe and quantitated by application of computer-assisted digital image processing in both the ductal and alveolar epithelial cells in the virgin rat and nulliparous and parous human mammary glands. During pregnancy and lactation, the level of TGF alpha mRNA expression in the ductal and alveolar epithelial cells increased two- to threefold, while a heterogeneous yet strong expression of TGF alpha mRNA could also be detected in approximately 10-15% of the surrounding stromal cells in the pregnant mammary gland.

The mitochondrial heme exporter FLVCR1b mediates erythroid differentiation

Feline leukemia virus subgroup C receptor 1 (FLVCR1) is a cell membrane heme exporter that maintains the balance between heme levels and globin synthesis in erythroid precursors. It was previously shown that Flvcr1-null mice died in utero due to a failure of erythropoiesis. Here, we identify Flvcr1b, a mitochondrial Flvcr1 isoform that promotes heme efflux into the cytoplasm. Flvcr1b overexpression promoted heme synthesis and in vitro erythroid differentiation, whereas silencing of Flvcr1b caused mitochondrial heme accumulation and termination of erythroid differentiation. Furthermore, mice lacking the plasma membrane isoform (Flvcr1a) but expressing Flvcr1b had normal erythropoiesis, but exhibited hemorrhages, edema, and skeletal abnormalities. Thus, FLVCR1b regulates erythropoiesis by controlling mitochondrial heme efflux, whereas FLVCR1a expression is required to prevent hemorrhages and edema. The aberrant expression of Flvcr1 isoforms may play a role in the pathogenesis of disorders characterized by an imbalance between heme and globin synthesis.

Somatic mutations and human breast cancer. A Status Report

A systematic study of primay human breast tumor DNA demonstrated that three proto‐oncogenes or regions of the genome c‐myc, int‐2, and c‐erbB2) are frequently amplified and that there is loss of heterozygosity (LOH) on chromosomes 1p(37%), 1q(20%), 3p(30%), 7(41%), 11p(20%), 13q(30%), 17p(49%), 17q(29%), and 18q(34%). Specific subsets of tumors can be defined based on the particular collection of mutations they contain. For instance, LOH on chromosomes 11p, 17p, and 18q frequently occurs in the same tumor. A search for putative tumor suppressor genes within the regions of the genome affected by LOH has been started. In a comprehensive molecular analysis of the p53 gene on chromosome 17p, 46% of the tumors contained a point mutation in the p53 gene. Cancer 1992; 69:1582‐1588.

PI3K Class II α Controls Spatially Restricted Endosomal PtdIns3P and Rab11 Activation to Promote Primary Cilium Function

Summary Multiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. Here, we report that PI3K-C2α is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2α-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2α rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2α regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation.

Claudin-1 is a p63 target gene with a crucial role in epithelial development

The epidermis of the skin is a self-renewing, stratified epithelium that functions as the interface between the human body and the outer environment, and acts as a barrier to water loss. Components of intercellular junctions, such as Claudins, are critical to maintain tissue integrity and water retention. p63 is a transcription factor essential for proliferation of stem cells and for stratification in epithelia, mutated in human hereditary syndromes characterized by ectodermal dysplasia. Both p63 and Claudin-1 null mice die within few hours from birth due to dehydration from severe skin abnormalities. These observations suggested the possibility that these two genes might be linked in one regulatory pathway with p63 possibly regulating Claudin-1 expression. Here we show that silencing of ΔNp63 in primary mouse keratinocytes results in a marked down-regulation of Claudin-1 expression (−80%). ΔNp63α binds in vivo to the Claudin-1 promoter and activates both the endogenous Claudin-1 gene and a reporter vector containing a –1.4 Kb promoter fragment of the Claudin-1 gene. Accordingly, Claudin-1 expression was absent in the skin of E15.5 p63 null mice and natural p63 mutant proteins, specifically those found in Ankyloblepharon–Ectodermal dysplasia–Clefting (AEC) patients, were indeed altered in their capacity to regulate Claudin-1 transcription. This correlates with deficient Claudin-1 expression in the epidermis of an AEC patient carrying the I537T p63 mutation. Notably, AEC patients display skin fragility similar to what observed in the epidermis of Claudin-1 and p63 null mice. These findings reinforce the hypothesis that these two genes might be linked in a common regulatory pathway and that Claudin-1 may is an important p63 target gene involved in the pathogenesis of ectodermal dysplasias.

The Dlx5 homeodomain gene is essential for olfactory development and connectivity in the mouse.

The distalless-related homeogene Dlx5 is expressed in the olfactory placodes and derived tissues and in the anterior-basal forebrain. We investigated the role of Dlx5 in olfactory development. In Dlx5(-/-) mice, the olfactory bulbs (OBs) lack glomeruli, exhibit disorganized cellular layers, and show reduced numbers of TH- and GAD67-positive neurons. The olfactory epithelium in Dlx5(-/-) mice is composed of olfactory receptor neurons (ORNs) that appear identical to wild-type ORNs, but their axons fail to contact the OBs. We transplanted Dlx5(-/-) OBs into a wild-type newborn mouse; wild-type ORN axons enter the mutant OB and form glomeruli, but cannot rescue the lamination defect or the expression of TH and GAD67. Thus, the absence of Dlx5 in the OB does not per se prevent ORN axon ingrowth. In conclusion, Dlx5 plays major roles in the connectivity of ORN axons and in the differentiation of OB interneurons.