Immunohistochemical and molecular genetic analysis of p 53 in oral squamous cell carcinoma (scc) in Hospital University Science Malaysia: a preliminary study

The role of p53 mutation in oral cancer cases has not been studied in Malaysia. This study was designed to focus on over expression of p53 nuclear protein and gene mutation in oral squamous cell carcinoma (SCC) cases in Hospital University Science Malaysia (HUSM). Twenty randomly selected cases previously diagnosed as oral SCC lesions in HUSM since 2002 were included in this study. Patient’s folders were reviewed to study clinical history and tumor staging. Extracted DNA amplified using PCR. Gene sequencing had done to detect genetic mutation. Protein overexpression detected using the Streptavidin-biotin Peroxidase technique. P53 gene mutations, analyzed within exons 5- 8, were observed in 10 out of 20 cases (50%) of oral SCC. The positive nuclear staining by immunohistochemical was observed in 18 cases (18/20, 90%). These data indicate the highly incidence of p53 protein overexpression as well as specific mutations in oral SCC patients in Malaysia. However this preliminary study had been based on analysis of relatively few cases of oral squamous cell carcinoma, so further study require to including large number of cases from different states in Malaysia.

Lithium stabilizes the polarized lens epithelial phenotype and inhibits proliferation, migration, and epithelial mesenchymal transition.

Posterior capsule opacification (PCO) is a common complication of cataract surgery caused by epithelial mesenchymal transition (EMT) and aberrant lens cell growth. One path to prevention depends on maintaining the quiescent lens epithelial phenotype. Here we report that lithium chloride (LiCl) is a potent stabilizer of the lens epithelial phenotype. In lens epithelial explants (controls), at low cell density, cells readily depolarized, spread out, and proliferated. By contrast, in the presence of LiCl, cells did not spread out or exhibit migratory behaviour. Using concentrations of 1-30 mM LiCl we also showed that cell proliferation is inhibited in a dose-dependent manner. Confocal microscopy and immunohistochemistry for ZO-1 and E-cadherin showed that LiCl treatment maintained tight junctions at the apical margins of cells. Taken together with measurements of cell heights, this showed that the cells in LiCl-treated explants maintained the apical baso-lateral polarity and cobblestone-like packing that is characteristic of lens epithelial cells in vivo. Significantly, the effects of LiCl also extended to blocking the potent EMT/cataract-promoting effects of transforming growth factor beta (TGFbeta) on lens epithelial cells. In TGFbeta-treated explants, cells progressively dissociated from one another, taking on various elongated spindle shapes and strongly expressing alpha-smooth muscle actin (alpha-SMA). These features are characteristic of PCO. In both rat and human capsulorhexis explants, LiCl treatment effectively blocked the accumulation of alpha-SMA and maintained the cells in a polarized, adherent, cobblestone-packed monolayer. These findings highlight the feasibility of applying molecular strategies to stabilize lens epithelial cells and prevent aberrant differentiation and growth that leads to cataract.

Distinct regulators control the expression of the mid-hindbrain organizer signal FGF8.

Local expression of FGF8 at the mid/hindbrain boundary (MHB) governs the development of multiple neurons and support cells. Here we show that the paired-domain protein Pax2 is necessary and sufficient for the induction of FGF8 in part by regulating the expression of Pax5&8. A network of transcription and secreted factors, including En1, Otx2, Gbx2, Grg4 and Wnt1&4, that is established independently of Pax2, further refines the expression domain and level of FGF8 at the MHB through opposing effects on Pax2 activity. Our results indicate that the expression of local organizing factors is controlled by combinatorial interaction between inductive and modulatory factors.

Tissue-specific deletion of Foxa2 in pancreatic beta cells results in hyperinsulinemic hypoglycemia.

We have used conditional gene ablation to uncover a dramatic and unpredicted role for the winged-helix transcription factor Foxa2 (formerly HNF-3 beta) in pancreatic beta-cell differentiation and metabolism. Mice that lack Foxa2 specifically in beta cells (Foxa2(loxP/loxP); Ins.Cre mice) are severely hypoglycemic and show dysregulated insulin secretion in response to both glucose and amino acids. This inappropriate hypersecretion of insulin in the face of profound hypoglycemia mimics pathophysiological and molecular aspects of familial hyperinsulinism. We have identified the two subunits of the beta-cell ATP-sensitive K(+) channel (K(ATP)), the most frequently mutated genes linked to familial hyperinsulinism, as novel Foxa2 targets in islets. The Foxa2(loxP/loxP); Ins.Cre mice will serve as a unique model to investigate the regulation of insulin secretion by the beta cell and suggest the human FOXA2 as a candidate gene for familial hyperinsulinism.

The allocation and differentiation of mouse primordial germ cells.

Analysis of the lineage potency of epiblast cells of the early-streak stage mouse embryo reveals that the developmental fate of the cells is determined by their position in the germ layer. Epiblast cells that are fated to become neuroectoderm can give rise to primordial germ cells (PGCs) and other types of somatic cells when they were transplanted to the proximal region of the epiblast. On the contrary, proximal epiblast cells transplanted to the distal region of the embryo do not form PGCs. Therefore, the germ line in the mouse is unlikely to be derived from a predetermined progenitor population, but may be specified as a result of tissue interactions that take place in the proximal epiblast of the mouse gastrula. The initial phase of the establishment of the PGC population requires, in addition to BMP activity emanating from the extraembryonic ectoderm, normal Lim1 and Hnf3beta activity in the germ layers. The entire PGC population is derived from a finite number of progenitor cells and there is no further cellular recruitment to the germ line after gastrulation. The XX PGCs undergo X-inactivation at the onset of migration from the gut endoderm and re-activate the silenced X-chromosome when they enter the urogenital ridge. Germ cells that are localised ectopically in extragonadal sites do not re-activate the X-chromosome, even when nearly all germ cells in the fetal ovary have restored full activity of both X-chromosomes. XXSxr germ cells can re-activate the X-chromosome in the sex-reversed testis, suggesting that the regulation of X-chromosome activity is independent of ovarian morphogenesis.

Role of the anterior visceral endoderm in restricting posterior signals in the mouse embryo.

Recent genetic and embryological experiments have demonstrated that head formation in the mouse embryo is dependent on signals provided by two organising centers during gastrulation, the anterior visceral endoderm (AVE) and the anterior primitive streak (also called the Early Gastrula Organiser, EGO). However the molecular nature of the signals triggering anterior neural formation from the epiblast is not clearly understood. The analysis of mouse mutants has allowed the identification of some of the molecular players involved in the process of head formation. In this review, we describe different mutant embryos in which impairment of visceral endoderm function leads to similar defects in antero-posterior axis specification. These phenotypes are consistent with a role of the AVE in protecting anterior embryonic regions from signals that promote posterior development. We propose that a genetic cascade in the AVE, involving HNF3beta, Lim1, Otx2, Smad2 and ActRIB, leads to the production of secreted TGFbeta antagonists that protect the anterior epiblast region from Nodal signalling.

Defects of the body plan of mutant embryos lacking Lim1, Otx2 or Hnf3beta activity.

The orientation of the anterior-posterior (A-P) axis was examined in gastrula-stage Hnf3beta, Otx2 and Lim1 null mutant embryos that display defective axis development. In situ hybridization analysis of the expression pattern of genes associated with the posterior germ layer tissues and the primitive streak (T, Wnt3 and Fgf8) and anterior endoderm (Cer1 and Sox17) revealed that the A-P axis of mutant embryos remains aligned with the proximo-distal plane of the gastrula. Further analysis revealed that cells which express Chrd activity are either absent in Hnf3beta mutant embryos or localised in heterotopic sites in Lim1 and Otx2 null mutants. Lim1-expressing cells are present in the Hnf3beta mutant embryo albeit in heterotopic sites. In all three mutants, Gsc-expressing cells are missing from the anterior mesendoderm. These findings suggest that although some cells with organizer activity may be present in the mutant embryo, they are not properly localised and fail to contribute to the axial mesoderm of the head. By contrast, in T/T mutant embryos that display normal head fold development, the expression domains of organizer, primitive streak and anterior endoderm genes are regionalised correctly in the gastrula.

Otx2 and HNF3beta genetically interact in anterior patterning.

Patterning the developing nervous system in the mouse has been proposed to depend on two separate sources of signals, the anterior visceral endoderm (AVE) and the node or organizer. Mutation of the winged-helix gene HNF3beta leads to loss of the node and its derivatives, while mutation of the homeobox gene Otx2 results in loss of head structures, apparently at least partially because of defects in the AVE. To investigate the potential genetic interactions between the two signaling centers, we crossed Otx2+/- and HNF3beta+/- mice and found that very few Otx2+/-;HNF3beta+/- double heterozygous mutants survived to weaning. Normal Mendelian ratios of genotypes were observed during gestation, but more than half the double heterozygotes displayed a severe anterior patterning phenotype that would be incompatible with postnatal survival. The phenotype was characterized by varying degrees of holoprosencephaly, cyclopia with proboscis-like structures, and anterior forebrain truncations. Regional marker analysis revealed that ventral forebrain structures of Otx2+/-;HNF3beta+/- mutant embryos were most severely affected. Shh expression was completely absent in the anterior region of Otx2+/-;HNF3beta+/- embryos, suggesting that Otx2 and HNF3beta genetically interact, directly or indirectly, to regulate Shh expression in the anterior midline. In addition, the forebrain truncations suggest an involvement of both genes in anterior patterning, through their overlapping expression domains in either the AVE and/or the prechordal mesoderm.

Otx2 is required for visceral endoderm movement and for the restriction of posterior signals in the epiblast of the mouse embryo.

Genetic and embryological experiments have demonstrated an essential role for the visceral endoderm in the formation of the forebrain; however, the precise molecular and cellular mechanisms of this requirement are poorly understood. We have performed lineage tracing in combination with molecular marker studies to follow morphogenetic movements and cell fates before and during gastrulation in embryos mutant for the homeobox gene Otx2. Our results show, first, that Otx2 is not required for proliferation of the visceral endoderm, but is essential for anteriorly directed morphogenetic movement. Second, molecules that are normally expressed in the anterior visceral endoderm, such as Lefty1 and Mdkk1, are not expressed in Otx2 mutants. These secreted proteins have been reported to antagonise, respectively, the activities of Nodal and Wnt signals, which have a role in regulating primitive streak formation. The visceral endoderm defects of the Otx2 mutants are associated with abnormal expression of primitive streak markers in the epiblast, suggesting that anterior epiblast cells acquire primitive streak characteristics. Taken together, our data support a model whereby Otx2 functions in the anterior visceral endoderm to influence the ability of the adjacent epiblast cells to differentiate into anterior neurectoderm, indirectly, by preventing them from coming under the influence of posterior signals that regulate primitive streak formation.

Hepatocyte nuclear factor 3beta (Foxa2) is dispensable for maintaining the differentiated state of the adult hepatocyte.

Liver-specific gene expression is controlled by a heterogeneous group of hepatocyte-enriched transcription factors. One of these, the winged helix transcription factor hepatocyte nuclear factor 3beta (HNF3beta or Foxa2) is essential for multiple stages of embryonic development. Recently, HNF3beta has been shown to be an important regulator of other hepatocyte-enriched transcription factors as well as the expression of liver-specific structural genes. We have addressed the role of HNF3beta in maintenance of the hepatocyte phenotype by inactivation of HNF3beta in the liver. Remarkably, adult mice lacking HNF3beta expression specifically in hepatocytes are viable, with histologically normal livers and normal liver function. Moreover, analysis of >8,000 mRNAs by array hybridization revealed that lack of HNF3beta affects the expression of only very few genes. Based on earlier work it appears that HNF3beta plays a critical role in early liver development; however, our studies demonstrate that HNF3beta is not required for maintenance of the adult hepatocyte or for normal liver function. This is the first example of such functional dichotomy for a tissue specification transcription factor.

A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons.

Neurogenin1 (Ngn1), Neurogenin2 (Ngn2), and Mash1 encode bHLH transcription factors with neuronal determination functions. In the telencephalon, the Ngns and Mash1 are expressed at high levels in complementary dorsal and ventral domains, respectively. We found that Ngn function is required to maintain these two separate expression domains, as Mash1 expression is up-regulated in the dorsal telencephalon of Ngn mutant embryos. We have taken advantage of the replacement of the Ngns by Mash1 in dorsal progenitors to address the role of the neural determination genes in neuronal-type specification in the telencephalon. In Ngn2 single and Ngn1; Ngn2 double mutants, a population of early born cortical neurons lose expression of dorsal-specific markers and ectopically express a subset of ventral telencephalic-specific markers. Analysis of Mash1; Ngn2 double mutant embryos and of embryos carrying a Ngn2 to Mash1 replacement mutation demonstrated that ectopic expression of Mash1 is required and sufficient to confer these ventral characteristics to cortical neurons. Our results indicate that in addition to acting as neuronal determinants, Mash1 and Ngns play a role in the specification of dorsal-ventral neuronal identity, directly linking pathways of neurogenesis and regional patterning in the forebrain.

The zinc finger gene Xblimp1 controls anterior endomesodermal cell fate in Spemann's organizer.

The anterior endomesoderm of the early Xenopus gastrula is a part of Spemann's organizer and is important for head induction. Here we describe Xblimp1, which encodes a zinc finger transcriptional repressor expressed in the anterior endomesoderm. Xblimp1 represses trunk mesoderm and induces anterior endomesoderm in a cooperative manner with the pan-endodermal gene Mix.1. Furthermore, Xblimp1 can cooperate with the BMP inhibitor chordin to induce ectopic heads, while a dominant-negative Xblimp1 inhibits head formation. The head inducer cerberus is positively regulated by Xblimp1 and is able to rescue microcephalic embryos caused by dominant-negative Xblimp1. Our results indicate that Xblimp1 is required for anterior endomesodermal cell fate and head induction.

Neural induction and patterning in the mouse in the absence of the node and its derivatives.

The signals which induce vertebrate neural tissue and pattern it along the anterior-posterior (A-P) axis have been proposed to emanate from Spemann's organizer, which in mammals is a structure termed the node. However, mouse embryos mutant for HNF3 beta lack a morphological node and node derivatives yet undergo neural induction. Gene expression domains occur at their normal A-P axial positions along the mutant neural tubes in an apparently normal temporal manner, including the most anterior and posterior markers. This neural patterning occurs in the absence of expression of known organizer genes, including the neural inducers chordin and noggin. Other potential signaling centers in gastrulating mutant embryos appear to express their normal constellation of putative secreted factors, consistent with the possibility that neural-inducing and -patterning signals emanate from elsewhere or at an earlier time. Nevertheless, we find that the node and the anterior primitive streak, from which the node derives, are direct sources of neural-inducing signals, as judged by expression of the early midbrain marker Engrailed, in explant-recombination experiments. Similar experiments showed the neural-inducing activity in HNF3 beta mutants to be diffusely distributed. Our results indicate that the mammalian organizer is capable of neural induction and patterning of the neural plate, but that maintenance of an organizer-like signaling center is not necessary for either process.

Sequential roles for Otx2 in visceral endoderm and neuroectoderm for forebrain and midbrain induction and specification.

The homeobox gene Otx2 is a mouse cognate of the Drosophila orthodenticle gene, which is required for development of the brain, rostral to rhombomere three. We have investigated the mechanisms involved in this neural function and specifically the requirement for Otx2 in the visceral endoderm and the neuroectoderm using chimeric analysis in mice and explant recombination assay. Analyses of chimeric embryos composed of more than 90% of Otx2-/- ES cells identified an essential function for Otx2 in the visceral endoderm for induction of the forebrain and midbrain. The chimeric studies also demonstrated that an anterior neural plate can form without expressing Otx2. However, in the absence of Otx2, expression of important regulatory genes, such as Hesx1/Rpx, Six3, Pax2, Wnt1 and En, fail to be initiated or maintained in the neural plate. Using explant-recombination assay, we could further demonstrate that Otx2 is required in the neuroectodem for expression of En. Altogether, these results demonstrate that Otx2 is first required in the visceral endoderm for the induction, and subsequently in the neuroectoderm for the specification of forebrain and midbrain territories.

Murine cerberus homologue mCer-1: a candidate anterior patterning molecule.

Xenopus cerberus (Xcer) is a cytokine expressed in anterior mesendoderm overlapping and surrounding Spemann's gastrula organiser. When misexpressed in blastomeres, Xcer can induce ectopic heads with well-defined brain, cement gland, olfactory placodes, cyclopic eye, and occasionally liver and heart. We report here the identification of mCer-1, a murine gene related to cerberus. Both mCer-1 and Xcer appear to belong to the cystine knot superfamily, which includes TGF beta s and BMPs. In Xenopus animal cap assays, mCer-1 and Xcer induced cement glands and markers of anterior neural tissue and endoderm, characteristic of BMP inhibition. Furthermore, both antagonised the ventrolateral mesoderm-inducing activity of coexpressed BMP4. In mouse embryos, mCer-1 was expressed at early gastrulation in a stripe of primitive endoderm along the future anterior side of the egg cylinder, a region essential for anterior patterning. A second phase of expression was detected in anterior embryonic mesendoderm, and by late-streak stages most of the anterior half of the embryo was positive, except for the node and cardiac progenitors. Expression was later seen in the cranial portion of the two most-recently formed somites and in two stripes within presomitic mesoderm. In embryos lacking Otx2, a homeogene with a demonstrated role in anterior patterning, mCer-1 was still expressed in an anterior zone, although often abnormally. The data suggest that mCer-1 shares structural, functional, and expression characteristics with Xcer and may participate in patterning the anterior of the embryo and nascent somite region, in part, through a BMP-inhibitory mechanism.

Goosecoid and HNF-3beta genetically interact to regulate neural tube patterning during mouse embryogenesis.

The homeobox gene goosecoid (gsc) and the winged-helix gene Hepatic Nuclear Factor-3beta (HNF-3beta) are co-expressed in all three germ layers in the anterior primitive streak and at the rostral end of mouse embryos during gastrulation. In this paper, we have tested the possibility of functional synergism or redundancy between these two genes during embryogenesis by generating double-mutant mice for gsc and HNF-3beta. Double-mutant embryos of genotype gsc(-/-);HNF-3beta(+/-) show a new phenotype as early as embryonic days 8.75. Loss of Sonic hedgehog (Shh) and HNF-3beta expression was observed in the notochord and ventral neural tube of these embryos. These results indicate that gsc and HNF-3beta interact to regulate Shh expression and consequently dorsal-ventral patterning in the neural tube. In the forebrain of the mutant embryos, severe growth defects and absence of optic vesicles could involve loss of expression of fibroblast growth factor-8, in addition to Shh. Our results also suggest that interaction between gsc and HNF-3beta regulates other signalling molecules required for proper development of the foregut, branchial arches and heart.

Mammalian hairy and Enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis.

Mammalian hairy and Enhancer of split homolog 1 (HES1), a basic helix-loop-helix factor gene, is expressed in retinal progenitor cells, and its expression decreases as differentiation proceeds. Retinal progenitor cells infected with HES1-transducing retrovirus did not differentiate into mature retinal cells, suggesting that persistent expression of HES1 blocks retinal development. In contrast, in the retina of HES1-null mutant mice, differentiation was accelerated, and rod and horizontal cells appeared prematurely and formed abnormal rosette-like structures. Lens and cornea development was also severely disturbed. Furthermore, in the mutant retina, bipolar cells extensively died, and finally disappeared. These studies provide evidence that HES1 regulates differentiation of retinal neurons and is essential for eye morphogenesis.

A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain.

Mouse Otx2 is a bicoid-class homeobox gene, related to the Drosophila orthodenticle (otd) gene. Expression of this gene is initially widespread in the epiblast at embryonic day 5.5 but becomes progressively restricted to the anterior end of the embryo at the headfold stage. In flies, loss of function mutations in otd result in deletion of pre-antennal and antennal segments; which leads to the absence of head structures derived from these segments. To study the function of Otx2 in mice, we have generated a homeobox deletion mutation in this gene. Mice homozygous for this mutation show severe defects in gastrulation and in formation of axial mesoderm and loss of anterior neural tissues. These results demonstrate that Otx2 is required for proper development of the epiblast and patterning of the anterior brain in mice, and supports the idea of evolutionary conservation of the function of Otd/Otx genes in head development in flies and mice.

Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects.

Mammalian hairy and Enhancer of split homolog-1 (HES-1) encodes a helix-loop-helix (HLH) factor that is thought to act as a negative regulator of neurogenesis. To directly investigate the functions of HES-1 in mammalian embryogenesis, we performed a targeted disruption of the HES-1 locus. Mice homozygous for the mutation exhibited severe neurulation defects and died during gestation or just after birth. In the developing brain of HES-1-null embryos, expression of the neural differentiation factor Mash-1 and other neural HLH factors was up-regulated and postmitotic neurons appeared prematurely. These results suggest that HES-1 normally controls the proper timing of neurogenesis and regulates neural tube morphogenesis.