Laura Paleari

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.

Msx1 and Dlx5 act independently in development of craniofacial skeleton, but converge on the regulation of Bmp signaling in palate formation.

Msx and Dlx homeoproteins control the morphogenesis and organization of craniofacial skeletal structures, specifically those derived from the pharyngeal arches. In vitro Msx and Dlx proteins have opposing transcriptional properties and form heterodimeric complexes via their homeodomain with reciprocal functional repression. In this report we examine the skeletal phenotype of Msx1; Dlx5 double knock-out (DKO) mice in relationship with their expression territories during craniofacial development. Co-expression of Dlx5 and Msx1 is only observed in embryonic tissues in which these genes have independent functions, and thus direct protein interactions are unlikely to control morphogenesis of the cranium. The DKO craniofacial phenotypes indicate a complex interplay between these genes, acting independently (mandible and middle ear), synergistically (deposition of bone tissue) or converging on the same morphogenetic process (palate growth and closure). In the latter case, the absence of Dlx5 rescues in part the Msx1-dependent defects in palate growth and elevation. At the basis of this effect, our data implicate the Bmp (Bmp7, Bmp4)/Bmp antagonist (Follistatin) signal: in the Dlx5(-/-) palate changes in the expression level of Bmp7 and Follistatin counteract the reduced Bmp4 expression. These results highlight the importance of precise spatial and temporal regulation of the Bmp/Bmp antagonist system during palate closure.

Optimisation of Polyethylenimine-Based Gene Delivery to Mouse Brain

Polyethylenimine (PEI) is proving to be an efficient and versatile vector for gene delivery in vivo. However, a limiting factor is the relatively short duration of gene expression in some sites. Given the particularly high levels of expression seen in the short term we postulated that loss of expression could result from overloading the nucleus with foreign DNA. To address this problem we first followed DNA delivery and localisation with digoxin-labelled plasmid DNA complexed with 22 kD linear PEI and used these complexes for intraventricular injection into brains of anaesthetised newborn mice. At 24 h post-injection, labelled DNA was found exclusively in the nuclear and perinuclear regions. We next carried out a dose response curve using decreasing amounts of DNA, either in a constant volume (2 microl) or at a constant concentration (500 ng/microl). In both conditions, transgene expression yield was maximum at 100 ng DNA per injection. Using this optimal amount of DNA increased yield of transgene expression significantly at 24 h and one week post-injection as compared to 1 microg DNA. A final point addressed was whether co-expressing an anti-apoptotic gene could enhance gene expression in the longer term. Co-expressing bcl-X(L) with luciferase or LacZ significantly increased expression of both these genes at one week post-injection.

Defective neuronogenesis in the absence of Dlx5.

Dlx genes play an important role in the control of the development of the central nervous system (CNS). Single or compound inactivation of Dlx1, Dlx2, or Dlx5 in the mouse causes defects of neuronal migration and differentiation. Dlx5, in particular, is essential for the correct development of the olfactory system. Targeted inactivation of Dlx1 and Dlx2 in the mouse results in abnormal neuronal differentiation in the embryonic subcortical forebrain and is associated to the loss of Dlx5 and Dlx6 expression. So far, however, it has been impossible to investigate the role of Dlx genes on late neurogenesis, as their inactivation leads to perinatal death. We have now generated cultures of neural stem cells (NSCs) derived from embryonic and newborn Dlx5-null mice, and we have compared their capacity to differentiate in vitro to that of equivalent cells derived from normal littermates. We show here that in the absence of Dlx5, NSCs derived from newborn animals have a severely reduced capacity to generate neurons. This is not the case for cells derived from E12.5 embryos. Forced expression of Dlx5 in cultures of newborn mutant NSCs fully restores their neuronogenic potential. Our data suggest that Dlx5 is essential for secondary (postnatal) neuronogenesis.

DLX genes as targets of ALL-1: DLX 2,3,4 down-regulation in t(4;11) acute lymphoblastic leukemias

Dlx genes constitute a gene family thought to be essential in morphogenesis and development. We show here that in vertebrate cells, Dlx genes appear to be part of a regulatory cascade initiated by acute lymphoblastic leukemia (ALL)‐1, a master regulator gene whose disruption is implicated in several human acute leukemias. The expression of Dlx2, Dlx3, Dlx5, Dlx6, and Dlx7 was absent in All‐1 −/− mouse embryonic stem cells and reduced in All‐1 +/− cells. In leukemic patients affected by the t(4;11)(q21;q23) chromosomal abnormality, the expression of DLX2, DLX3, and DLX4 was virtually abrogated. Our data indicate that Dlx genes are downstream targets of ALL‐1 and could be considered as important tools for the study of the early leukemic cell phenotype.

APS8, a Polymeric Alkylpyridinium Salt Blocks α7 nAChR and Induces Apoptosis in Non-Small Cell Lung Carcinoma

Naturally occurring 3-alkylpyridinium polymers (poly-APS) from the marine sponge Reniera sarai, consisting of monomers containing polar pyridinium and nonpolar alkyl chain moieties, have been demonstrated to exert a wide range of biological activities, including a selective cytotoxicity against non-small cell lung cancer (NSCLC) cells. APS8, an analog of poly-APS with defined alkyl chain length and molecular size, non-competitively inhibits α7 nicotinic acetylcholine receptors (nAChRs) at nanomolar concentrations that are too low to be acetylcholinesterase (AChE) inhibitory or generally cytotoxic. In the present study we show that APS8 inhibits NSCLC tumor cell growth and activates apoptotic pathways. APS8 was not toxic for normal lung fibroblasts. Furthermore, in NSCLC cells, APS8 reduced the adverse anti-apoptotic, proliferative effects of nicotine. Our results suggest that APS8 or similar compounds might be considered as lead compounds to develop antitumor therapeutic agents for at least certain types of lung cancer.

Kaposi's Sarcoma and HIV-Tat: Challenges to Antiangiogenesis Research

Kaposis sarcoma (KS) is characterized by an abnormal growth of blood vessels. KS was found mainly in older men of Mediterranean or African origin (classic KS) or in patients after organ transplantation (iatrogenic KS). However, in the early 1980s, an aggressive epidemic form, linked to AIDS, was noticed and was one of the first clues to the existence of HIV-1 pandemy. The link between KS occurrence and HIV has raised multiple hypotheses. The drastic reduction of KS after the introduction of HAART, suggests HIV as a powerful co-factor for KS progression. We and others have contributed to the elucidation of KS cell nature and the possible involvement of extracellular HIV Tat. Tat is proangiogenic and is a true promoter of KS lesions acting as a VEGFR2 ligand both on KS and endothelial cells, in addition Tat is able to bind and activate chemokine receptors on monocytes and granulocytes causing a pro-inflammatory status. Evaluation of the effects of extracellular Tat on KS cells by microarray analysis after 24 h of incubation shows an interesting clustering of gene products involved in signal transduction, especially GTP-ase, Kinase and cAMP activity, confirming that Tat acts extracellularly by ways that are probably unrelated to its nuclear activity. KS occurrence is reduced by HAART but still present and in Africa is one of the most frequent oncologic disease. To find suitable drugs with low toxic impact on KS patients, we have tested several drugs and gene therapy approaches in in vivo models. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005