Analysis of Dip2B Expression in Adult Mouse Tissues Using the LacZ Reporter Gene

Disconnected (disco)-interacting protein 2 homolog B (Dip2B) is a member of the Dip2 superfamily and plays an essential role in axonal outgrowth during embryogenesis. In adults, Dip2B is highly expressed in different brain regions, as shown by in situ analysis, and may have a role in axon guidance. However, the expression and biological role of Dip2B in other somatic tissues remain unknown. To better visualize Dip2B expression and to provide insight into the roles of Dip2B during postnatal development, we used a Dip2btm1a(wtsi)komp knock-in mouse model, in which a LacZ-Neo fusion protein is expressed under Dip2b promoter and allowed Dip2B expression to be analyzed by X-gal staining. qPCR analyses showed that Dip2b mRNA was expressed in a variety of somatic tissues, including lung and kidney, in addition to brain. LacZ staining indicated that Dip2B is broadly expressed in neuronal, reproductive, and vascular tissues as well as in the kidneys, heart, liver, and lungs. Moreover, neurons and epithelial cells showed rich staining. The broad and intense patterns of Dip2B expression in adult mice provide evidence of the distribution of Dip2B in multiple locations and, thereby, its implication in numerous physiological roles.

A new mouse model for retinal degeneration due to Fam161a deficiency

FAM161A mutations are the most common cause of inherited retinal degenerations in Israel. We generated a knockout (KO) mouse model, Fam161atm1b/tm1b, lacking the major exon #3 which was replaced by a construct that include LacZ under the expression of the Fam161a promoter. LacZ staining was evident in ganglion cells, inner and outer nuclear layers and inner and outer-segments of photoreceptors in KO mice. No immunofluorescence staining of Fam161a was evident in the KO retina. Visual acuity and electroretinographic (ERG) responses showed a gradual decrease between the ages of 1 and 8 months. Optical coherence tomography (OCT) showed thinning of the whole retina. Hypoautofluorescence and hyperautofluorescence pigments was observed in retinas of older mice. Histological analysis revealed a progressive degeneration of photoreceptors along time and high-resolution transmission electron microscopy (TEM) analysis showed that photoreceptor outer segment disks were disorganized in a perpendicular orientation and outer segment base was wider and shorter than in WT mice. Molecular degenerative markers, such as microglia and CALPAIN-2, appear already in a 1-month old KO retina. These results indicate that a homozygous Fam161a frameshift mutation affects retinal function and causes retinal degeneration. This model will be used for gene therapy treatment in the future.

Conditional targeting in mice reveals that hepatic homogentisate 1,2-dioxygenase activity is essential in reducing circulating homogentisic acid and for effective therapy in the genetic disease alkaptonuria

Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype. We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a −/− mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy. Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA. This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.

Hematopoietic Specific GATA-1-Improved Cre Mouse for Erythroid-Specific Gene Modification.

Animal models of erythropoiesis related genes have been limited by the fact that some of these genes have non-erythroid expression and other functions in addition to erythropoiesis and thus their knock-out may be embryonic lethal. Tissue specific knock-out or knock-in mice models employing GATA-1-Cre and other constructs showed that these promoters are also active in non-hematopoietic tissues, i.e. GATA-1 has activity in early embryonic development and in neuronal tissue. Suzuki et al (Blood, 2002, 100; 2279) isolated the GATA-1 locus hematopoietic regulatory domain (GATA-1-HRD) and demonstrated that the expression of a transgene under its control is limited to the hematopoietic tissue. We generated a transgenic mouse expressing an improved Cre (iCre) under GATA-1-HRD promoter control. This mouse was crossbred with ROSA 26 mouse and the progeny was examined for tissue specificity of iCre expression using beta-galactosidase staining. Brain, spleen, kidney, heart, thymus, liver, lung and ovary were examined for whole organ LacZ staining. All tested organs were negative except kidney and spleen where some positivity was observed. Subsequently, we prepared tissue sections from kidney, spleen and bone marrow and stained with LacZ and anti-beta-galactosidase antibody. Only the bone marrow EpoR expressing cells were positive; the kidney and the spleen cells were negative. Although Suzuki et al previously showed expression of the GATA-1-HRD driven erythropoietin receptor in spleen using RT-PCR, we were not able to find iCre expression in the splenic cells using these approaches. We demonstrate that our transgenic mouse (GATA-1-HRD-iCre) showed a restricted iCre expression in hematopoietic tissue that differs from previous studies of other hematopoiesis specific cre mouse. We conclude that this mouse model should be useful in studies of function of erythroid specific genes.

Tie-2 Activation Is Required for Regeneration of Marrow Vasculature, Supporting Hematopoietic Reconstitution.

Accumulating evidence from our lab (Nature Med.2004; 10(1): 64–71) and others have shown that myelosuppression not only results in apoptosis of the hematopoietic cells but also in regression of bone marrow (BM) sinusoidal vessels. Remarkably, we have shown that restoration of hematopoiesis is dependent on the regeneration of BM sinusoidal neo-vessels. However, the mechanism whereby vascular reconstitution is regulated is unknown. Because Tie2/Angiopoietins are critical in the remodeling of neo-vessels, we hypothesized that activation of Tie2 plays a role in regeneration of sinusoidal neo-vessels and restoration of hematopoiesis. To this end, we took advantage of transgenic mice where the Tie2 promoter drives the expression of beta-galactosidase (LacZ) in order to follow the expression of Tie2 in hematopoietic progenitor cells after myelosuppression. Under steady-state conditions, only few LacZ/Tie-2+ cells were localized in the endosteal/osteoblastic region, without any expression within the BM sinusoidal neo-vessels. However, after a myelosuppressive dose of 5-Fluorouracil (5-FU) there was a robust expression of Tie2 expression in the regenerating BM sinusoidal neo-vessels. The expression of Tie2 on the regenerating neo-vasculature reached a maximum at day 10 and reverted back to steady-state by day 20 post 5-FU, the time in which the majority of the neo-vessels were functionally assembled. Blocking angiopoietin/Tie2 signaling with the soluble decoy-receptor, Tie2Fc, after 5-FU inhibited platelet recovery as well as vascular reconstitution. There was a paradoxical accumulation of megakaryocytes in the bone-marrow during the prolonged thrombocytopenic phase, which we have previously shown to be due to vascular niche disruption and inability of megakaryocytes to release platelets. To examine the possibility that Tie2+ progenitors can reconstitute BM sinusoids in the recipient mice, lethally irradiated wildtype mice were transplanted with the BM of Tie2-lacZ knock-in mice. After full recovery, the chimeric mice were challenged with a myelosuppressive dose of 5-FU and Tie2+ neo-vessels were detected histologically on day 5, 10, and 14 by LacZ staining. Remarkably, the reconstituted bone marrow showed the presence of Tie2+ vessels, which unambiguously demonstrates the contribution of donor-derived endothelial progenitors to the reconstitution of the regressed vasculature. Together, our data support the hypothesis that Tie2/angiopoietin signaling is essential for functional regeneration of BM sinusoidal neo-vessels and contributes to the reconstitution of hematopoiesis, specifically to thrombopoiesis. In addition, the use of Tie2-LacZ mice provides an invaluable model to quantify the number of regenerating BM neo-vessels. Angiopoietins may be used in clinical setting in conjunction with other lineage specific cytokines to enhance hematopoiesis after myelosuppression.

Genetically tagging endothelial cells in vivo: bone marrow-derived cells do not contribute to tumor endothelium

Tumor growth is dependent in part on “neoangiogenesis.” Functional involvement of bone marrow (BM)-derived cells in this process has been demonstrated. However, it remains controversial as to whether tumor endothelium itself is BM derived. Here we sought to address this issue with an endothelial-specific, inducible transgenic model. We generated Cretransgenic mice (endothelial-SCL-Cre-ERT) using the tamoxifen-inducible Cre-ERT recombinase driven by the 5′ endothelial enhancer of the stem cell leukemia (SCL) locus. These mice were intercrossed with Cre reporter strains in which β-galactosidase (LacZ) or enhanced yellow fluorescent protein (EYFP) are expressed upon Cre-mediated recombination. After tamoxifen administration, endothelial LacZ staining was observed in embryonic and adult tissues. Cre-mediated recombination was also observed in newly generated tumor endothelium. In adult BM cells we could only detect trace amounts of recombination by flow cytometry. Subsequently, BM from endothelial-SCL-Cre-ERT;R26R mice was transplanted into irradiated recipients. When tumors were grown in recipient mice, which received tamoxifen, no tumor LacZ staining was detected. However, when tumors were grown in endothelial-SCL-Cre-ERT;R26R mice 3 weeks after the cessation of tamoxifen treatment, there was widespread endothelial LacZ staining present. Thus, this genetic model strongly suggests that BM cells do not contribute to tumor endothelium and demonstrates the lineage relation between pre-existing endothelium and newly generated tumor endothelial cells. (Blood. 2004;104:1769-1777)

The Drosophila slamdance Gene: A Mutation in an Aminopeptidase Can Cause Seizure, Paralysis and Neuronal Failure

We report here the characterization of slamdance (sda), a Drosophila melanogaster “bang-sensitive” (BS) paralytic mutant. This mutant exhibits hyperactive behavior and paralysis following a mechanical “bang” or electrical shock. Electrophysiological analyses have shown that this mutant is much more prone to seizure episodes than normal flies because it has a drastically lowered seizure threshold. Through genetic mapping, molecular cloning, and RNA interference, we have demonstrated that the sda phenotype can be attributed to a mutation in the Drosophila homolog of the human aminopeptidase N (APN) gene. Furthermore, using mRNA in situ hybridization and LacZ staining, we have found that the sda gene is expressed specifically in the central nervous system at particular developmental stages. Together, these results suggest that the bang sensitivity in sda mutants is caused by a defective APN gene that somehow increases seizure susceptibility. Finally, by using the sda mutation as a sensitized background, we have been able to identify a rich variety of sda enhancers and other independent BS mutations.

LacZ staining in paraffin-embedded tissue sections.

Femora and tibiae of rats carrying leukemia from a LacZ-marked acute promyelocytic leukemia-derived leukemic cell line (LT12NL15) were decalcified using EDTA and routinely embedded in paraffin. Sections were used to develop for the first time an immunostaining method for LacZ, employing catalyzed reporter deposition (CARD) based on the deposition of biotinylated tyramine. This method was used to study homing and adhesion of leukemic cells.