Achaete-scute like 2 (ascl2) is a target of Wnt signalling and is upregulated in intestinal neoplasia.

Achaete-scute like (ASCL)2 is a basic helix-loop-helix transcription factor essential for the maintenance of proliferating trophoblasts during placental development. Using oligonucleotide microarrays we identified ascl2 as a gene significantly upregulated in colorectal adenocarcinomas (n=36 cancers, n=16 normals; 15-fold, P<0.0001). This finding was confirmed by quantitative reverse transcriptase (RT)-PCR on large intestinal cancers (n=29 cancers, n=16 normals; 10-fold, P<0.0001). In situ hybridization for ascl2 demonstrated expression at the base of small and large intestinal crypts (n=304), but in no other normal tissues excepting placenta. By in situ hybridization, 52-71% of colorectal adenomas (n=187), 50-73% of large (n=327) and 33-64% of small intestinal adenocarcinomas (n=124) were positive for ascl2 expression. Upregulation of murine ascl2 was also observed using oligonucleotide microarrays, quantitative RT-PCR and in situ hybridization on apcmin/+ and apc1638N/+ smad4-/+ tumours. Tumour cell lines stably transfected with LEF1(DN) or APC2, or transiently transfected with short-interfering RNA (siRNA) against beta-catenin showed a significant downregulation of ascl2. Colocalization of ascl2 with nuclear beta-catenin was observed in 73 small intestinal adenocarcinomas (P=0.0008) and apcmin/+ tumours. Preliminary in vitro data suggest ascl2 may promote progression through the G2/M cell cycle checkpoint. In summary, ascl2 is a putative regulator of proliferation that is overexpressed in intestinal neoplasia.

Expression of vascular endothelial growth factor, hypoxia inducible factor 1alpha, and carbonic anhydrase IX in human tumours.

AIMS: To measure vascular endothelial growth factor (VEGF-A) mRNA in a large, diverse cohort of tumours and to investigate whether VEGF-A expression is associated with markers of hypoxia, including hypoxia inducible factor 1alpha (HIF-1alpha) and carbonic anhydrase IX (CA9). METHODS: The expression of VEGF-A and CA9 was assessed in 5067 fresh frozen human tissue samples and 238 cell lines by DNA microarray analysis. In addition, tissue microarrays were constructed from 388 malignancies to investigate the expression of VEGF-A and HIF-1alpha by in situ hybridisation and immunohistochemistry, respectively. RESULTS: VEGF-A was significantly upregulated in primary malignancies of the breast, cervix, colon and rectum, oesophagus, head and neck, kidney, ovary, skin, urinary system, and white blood cells by DNA microarray analysis. However, VEGF-A expression only correlated with CA9 expression in renal tissues. In the tissue microarrays, HIF-1alpha positive cores showed a significant increase in VEGF-A expression in lung, ovary, soft tissue, and thyroid malignancies. CONCLUSIONS: The expression of VEGF-A is upregulated in a large proportion of human malignancies, and may be associated with markers of hypoxia. VEGF-A expression can be induced in the absence of hypoxia and hypoxia does not always provoke VEGF-A upregulation in tumours.

Quantitative analysis of colorectal tissue microarrays by immunofluorescence and in situ hybridization.

The accuracy and reliability of in situ studies may be compromised by qualitative interpretations. Quantitation imposes a greater degree of objectivity, is more reproducible, and facilitates the clarity of definitions. The aim of this study was to validate the utility of laser imaging systems for the in situ quantitative analysis of gene expression in tissue microarrays. Immunofluorescence was employed to quantify the expression of the tumour suppressor p53, a marker of proliferation (Ki67), an endothelial cell marker (CD31), and the mismatch repair proteins human Mut L homologue 1 and human Mut S homologue 2 in an arrayed series of colorectal tissues (n = 110). Quantitative data on this panel of antigens were compared objectively with qualitative scoring of immunohistochemical chromogen deposition. In addition, the expression of vascular endothelial growth factor (VEGF)-A, placental growth factor, hepatocyte growth factor, and c-Met mRNA was quantified by phosphor image analysis of in situ hybridization reactions. The quantified data on p53, Ki67, and CD31 expression were significantly associated with the pathologist's score (p < or = 0.001). While hepatocyte growth factor and placental growth factor were not up-regulated, c-Met expression was increased up to 2.5-fold and the median VEGF-A expression was elevated 4-fold (p = 0.003) in this series of colorectal tumours. Laser imaging systems are therefore feasible for high-throughput, quantitative profiling of tissue microarrays.

Detection of novel gene expression in paraffin-embedded tissues by isotopic in situ hybridization in tissue microarrays.

Correlating altered gene expression patterns with particular disease states is a critical step in understanding disease processes and developing treatment strategies. Many thousands of novel gene sequences have recently been annotated in public and private databases and are now available for analysis. Tissue-specific expression patterns of these sequences can be evaluated physically on DNA arrays and other high throughput assays, or virtually by bioinformatics mining of expressed sequence tag (EST) databases. As a secondary screening tool, in situ hybridisation (ISH) not only confirms tissue specificity, but also reveals what is often valuable information about cell-type expression patterns of nov16l sequences. Due to their availability and long-term stability at room temperature, formalin-fixed paraffin-embedded clinical specimens provide an invaluable resource for evaluating expression patterns of novel human genes. We describe a high-throughput approach for identifying and quantifying the expression of novel genes in paraffin-embedded human tissues using isotopic in situ hybridisation and tissue microarrays (TMA).

Overexpression of the retinoic acid-responsive gene Stra6 in human cancers and its synergistic induction by Wnt-1 and retinoic acid.

Genetic defects in the Wnt-1 signaling pathway contribute to human tumor progression and are especially prevalent in colorectal cancer. We screened mouse C57MG cells to isolate mRNAs induced by Wnt-1 and identified Stra6, an mRNA known to be up-regulated by retinoic acid. Up-regulation of Stra6 mRNA was also observed in hyperplastic mammary tissue and mammary gland tumors from transgenic mice expressing Wnt-1 and in human tumors that frequently harbor defects in Wnt-1 signaling. Stimulation of C57MG cells with retinoic acid plus Wnt-1 resulted in expression of Stra6 transcript to levels greatly exceeding that observed with either stimulus alone. This synergy could be explained in part by the up-regulation of retinoic acid receptor-gamma that was observed in response to Wnt-1 signaling. Accordingly, treatment of human colorectal cancer cell lines with retinoic acid resulted in the up-regulation of Stra6 mRNA and accumulation of Stra6 protein at the cell membrane. The data support a model in which Wnt-1 signaling synergizes with retinoids to activate retinoic acid receptor-gamma-responsive genes in human cancers.

FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family.

Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation.

Distinct expression patterns of notch family receptors and ligands during development of the mammalian inner ear.

The cochlea and vestibular structures of the inner ear labyrinth develop from the otic capsule via step-wise regional and cell fate specification. Each inner ear structure contains a sensory epithelium, composed of hair cells, the mechanosensory transducers, and supporting cells. We examined the spatio-temporal expression of genes in the Notch signaling pathway, Notch receptors (Notch1-4) and two ligands, Jagged1 and Delta1, in the developing mammalian inner ear. Our results show that Notch1 and Jagged1 are first expressed in the otic vesicle, likely involved in differentiation of the VIIIth nerve ganglion neurons, and subsequently within the inner ear sensory epithelia, temporally coincident with initial hair cell differentiation. Notch1 expression is specific to hair cells and Jagged1 to supporting cells. Their expression persists into adult. Notch2, Notch3, Notch4, and Delta1 are excluded from the inner ear epithelia. These data support the hypothesis that Notch signaling is involved in hair cell differentiation during inner ear morphogenesis.

Restriction of late cerebral cortical progenitors to an upper-layer fate.

Early in development, neural progenitors in cerebral cortex normally produce neurons of several layers during successive cell divisions. The laminar fate of their daughters depends on environmental cues encountered just before mitosis. At the close of neurogenesis, however, cortical progenitors normally produce neurons destined only for the upper layers. To assess the developmental potential of these cells, upper-layer progenitors were transplanted into the cerebral cortex of younger hosts, in which deep-layer neurons were being generated. These studies reveal that late cortical progenitors are not competent to generate deep-layer neurons and are instead restricted to producing the upper layers.

Presence of calbindin D28K and GAD67 mRNAs in both orthotopic and ectopic Purkinje cells of staggerer mice suggests that staggerer acts after the onset of cytodifferentiation.

We used in situ hybridization to study the expression of GAD67 and calbindin D28K mRNAs in developing mouse cerebellar Purkinje cells. Both genes are expressed prenatally; calbindin D28K mRNAs can be detected in Purkinje cells of embryonic day (E) 15 mice, whereas GAD67 mRNAs first appear slightly later, in E16 mice. The stunted Purkinje cells of staggerer (sg/sg) mutant mice maintain calbindin D28K and GAD67 expression. Our data suggest that the sg/sg mutation does not interfere with the transcriptional activation of these two genes, and might therefore act after the induction of specific gene expression in developing Purkinje cells.

Otx1 and Otx2 define layers and regions in developing cerebral cortex and cerebellum.

Within the cerebral and cerebellar cortices, neurons are organized in layers that segregate neurons with distinctive morphologies and axonal connections, and areas or regions that correspond to distinct functional domains. To explore the molecular underpinnings of pattern formation in layered regions of the CNS, we have characterized the patterns of expression of two homeodomain genes, Otx1 and Otx2, by in situ hybridization during embryonic and postnatal development in the rat. Otx1 and Otx2 are vertebrate homologs of the Drosophila gap gene orthodenticle, and are expressed during the development of the murine CNS (Simeone et al., 1992). Here we report that Otx1 mRNA is expressed in a subpopulation of neurons within cortical layers 5 and 6 during postnatal and adult life. This gene is also expressed by the precursors of deep-layer neurons within the developing cerebral ventricular zone, but is apparently downregulated by the progenitors of upper-layer neurons; Otx1 is never expressed by the neurons of layers 1-4. The spatial and temporal patterns suggest that Otx1 may play a role in the specification or differentiation of neurons in the deep layers of the cerebral cortex. Within the cerebellum, mRNAs for Otx1 and Otx2 are found within the external granular layer (EGL), but in three spatially distinct domains. During postnatal development, Otx1 is expressed within anterior cerebellar lobules; Otx2 mRNA is localized posteriorly, and a region of overlap in mid-cerebellum defines a third domain in which both genes are expressed. The boundaries of Otx1 and Otx2 expression correspond to the major functional boundaries of the cerebellum, and define the vestibulocerebellum, spinocerebellum, and pontocerebellum, respectively. Spatially restricted patterns of hybridization are observed early in postnatal life, at times that correspond roughly to the invasion of spinal and pontine afferents into the cerebellum (Arsénio-Nunes and Sotelo, 1985; Mason, 1987). These results raise the possibility that Otx1 and Otx2 play a role in cerebellar regionalization during early development.

Differential expression of synaptic vesicle protein 2 (SV2) isoforms.

The synaptic vesicle proteins SV2A and SV2B (SV2 = synaptic vesicle protein 2) are two highly related proteins belonging to a family of transporters. As a first step toward identifying the function of the SV2 proteins, we examined the expression of SV2A and SV2B in the rat brain by in situ hybridization, immunohistochemistry, and immunoprecipitation with isoform-specific antibodies. These analyses revealed that one isoform, SV2A, is expressed ubiquitously throughout the brain at varying levels. The other isoform, SV2B, has a more limited distribution with varying degrees of coexpression with SV2A. Immunoprecipitation of brain synaptic vesicles with isoform-specific antibodies followed by Western analyses suggests that both isoforms can be present on the same synaptic vesicle. The expression of the SV2 proteins did not correlate either with neurotransmitter phenotype or with the expression of other synaptic vesicle protein isoforms. SV2B expression was observed to change during development; it is more widely expressed in the immature brain and is found in cells that have yet to establish synaptic contacts. The ubiquitous and overlapping expression of the SV2s suggests that they perform a function common to all synaptic vesicles. Variable and changing coexpression of the SV2 isoforms may indicate that SV2 function is regulated by the isoform composition of synaptic vesicles. The observation that the synaptic vesicle proteins, all occurring in multiple isoforms, are differentially expressed with respect to each other indicates that up to 90 different vesicle types are possible.

Cellular distribution of calbindin D28K mRNAs in the adult mouse brain.

We have determined the cellular distribution of calbindin D28K mRNAs throughout the mouse brain by in situ hybridization. While these studies identified neuronal populations similar to those previously identified in rat brain by immunohistochemistry, some discrepancies exist. These may derive from species differences or from the immunological cross-reactivity of calbindin D28K antiserum with other proteins. We note an intriguing association between the distribution of neurons containing calbindin D28K mRNA and those reported by others to contain the inositol 1,4,5-triphosphate (InsP3) receptor.

Regulation of the POU domain gene SCIP during cerebral cortical development.

The mammalian cerebral cortex is patterned into layers of neurons that share characteristic morphologies, physiological properties, and axonal connections. Neurons in the various layers are thought to acquire their lamina-specific identities shortly before the time of their final mitosis in the cortical ventricular zone. In order to investigate the molecular basis of laminar patterning in the CNS, we have performed in situ hybridization studies of the POU homeodomain gene SCIP (also known as Tst-1 or Oct-6), which is expressed in proliferating Schwann cells in the PNS and O2A progenitor cells in the developing CNS. In the CNS of adult rats, SCIP is expressed at high levels in the cerebral cortex, specifically in layer 5 pyramidal neurons that form subcortical axonal connections. SCIP is both temporally and spatially regulated during cortical development. Its initial expression in the intermediate zone and cortical plate is correlated with the early migration and differentiation of layer 5 neurons. SCIP hybridization was not, however, observed within the ventricular zone during the period of neurogenesis. SCIP is also expressed at high levels in the neurons of cortical layer 2/3, during their migration and differentiation within the cortical plate. This expression in the upper layers is apparently downregulated during postnatal periods, with the adult pattern apparent by postnatal day 30 (P30). POU domain genes are thought to play a role in cell lineage and cell fate decisions in several systems; thus, SCIP may serve a function in generating discrete laminar phenotypes in the developing cerebral cortex. In addition, since SCIP is a putative repressor of myelin gene expression, our results suggest that SCIP plays a role in regulating transcription in differentiated CNS neurons as well as in proliferating glial precursors.

Calcium binding protein (calbindin-D28k) and glutamate decarboxylase gene expression after kindling induced seizures.

In order to determine whether calcium binding protein (calbindin-D28k or CaBP) and glutamate decarboxylase (GAD) may be involved in the process underlying the generation of seizure activity, changes in CaBP protein and mRNA and in GAD mRNA were examined in the kindling model of epilepsy. Following amygdaloid (AK) and commissure (CK) kindling significant decreases in the concentration of CaBP of 20% and 30%, respectively, were specifically observed in the hippocampal formation. However, using a cDNA specific to mammalian CaBP, Northern analysis of poly(A+) RNA and slot blot analysis of total RNA revealed no changes in the levels of CaBP mRNA in hippocampus, subcortical area (including amygdala, substantia nigra and striatum) or cerebellum of rats sacrificed 30 min, 1 h, 6 h or 24 h after the last kindled seizure. Similarly when these blots were reprobed with a cDNA specific to mammalian GAD, no changes in GAD gene expression were observed. However, fos gene expression was markedly enhanced at 1 h after seizure. We also tested whether changes in CaBP or GAD mRNA could be detected at any of the various stages of the kindling process. Slot blot analysis of cortex, subcortical structures and hippocampus revealed no changes in CaBP or GAD mRNA during the course of commissure kindling. In situ hybridization studies with GAD and CaBP 35S-labeled antisense probes also indicated no obvious changes upon visual analysis of autoradiographs. However, when silver grains were counted, significant changes in GAD mRNA in individual cells in hippocampus and substantia nigra were noted after kindling induced epilepsy. Our results indicate that, unlike fos gene expression, prominent alterations in GAD and CaBP mRNA in gross brain regions (as measured by slot blot and Northern blot analyses) are not observed in the kindling process. However, our in situ hybridization studies suggest that changes in GAD mRNA in individual cells may be involved in the process underlying kindling induced seizure activity.

Expression of mRNAs that encode D2 dopamine receptor subtypes: Anatomical, developmental, and pharmacological studies.

We used oligonucleotide probes to isolate cDNAs that encode two subtypes of D2 dopamine receptors ("D2(long)" and "D2(short)"). In situ hybridization histochemistry with cRNA probes derived from these cDNAs revealed relatively high concentrations of D2 receptor mRNA in brain regions associated with the nigrostriatal and mesolimbic dopamine systems. D2 receptor mRNA was also present in several other regions including the oculomotor nucleus and areas of the pons and medulla. Hybridizations with a probe specifically recognizing D2(long) mRNA indicated that the fraction of total D2 receptor mRNA that encodes D2(long) receptors is higher in the nigrostriatal and mesolimbic dopamine systems than in the dorsal tegmental nucleus (of Gudden). Northern blot analysis revealed that developmental changes in D2 receptor mRNA concentrations in whole brain roughly parallel previously documented changes in D2 binding site concentrations. In contrast, Northern blot analysis indicated that D2 mRNA concentrations are unaffected by subchronic administration of the D2 receptor antagonist, haloperidol.

Regional distribution of messenger RNAs in postmortem human brain.

The ability to isolate intact RNAs from postmortem human brain permits analysis of gene expression and may help uncover the nature of the molecular lesions in neurological diseases. Starting with poly(A) RNA from postmortem brain of neurologically normal patients, we have constructed two complementary DNA libraries in the plasmid vector pBR322. Each of these libraries contains 2-3 X 10(4) recombinants. One library represents RNA species from the cerebellar cortex, the other from the neostriatum. Using differential colony hybridization, we identified more than 100 relatively abundant RNA species that appeared to be expressed in brain but not in liver. We then used 16 of these clones to analyze brain and liver RNAs by RNA blot hybridization. Thirteen of the 16 clones hybridized to RNAs of both liver and brain. One clone hybridized only to brain RNA, while seven hybridized to RNA species that were present at higher concentrations in brain than in liver. Eleven of the 16 clones hybridized to more than one species of RNA. None of the RNA species examined by RNA blot hybridization was limited to a single brain region, though seven of the cDNA clones hybridized to RNAs that were present at different concentrations in different regions. We have also examined the regional distribution of the RNA encoding glutamic acid decarboxylase, which catalyzes the production of gamma-aminobutyric acid (GABA). GAD RNA showed differential expression among brain regions and was not detectable in liver or kidney. Our data support a model of gene regulation that is based on cell identity, rather than regional specificity.