SOX9 directly regulates the type-II collagen gene.

Mutations in human SOX9 are associated with campomelic dysplasia (CD), characterised by skeletal malformation and XY sex reversal. During chondrogenesis in the mouse, Sox9 is co-expressed with Col2a1, the gene encoding type-II collagen, the major cartilage matrix protein. Col2a1 is therefore a candidate regulatory target of SOX9. Regulatory sequences required for chondrocyte-specific expression of the type-II collagen gene have been localized to conserved sequences in the first intron in rats, mice and humans. We show here that SOX9 protein binds specifically to sequences in the first intron of human COL2A1. Mutation of these sequences abolishes SOX9 binding and chondrocyte-specific expression of a COL2A1-driven reporter gene (COL2A1-lacZ) in transgenic mice. Furthermore, ectopic expression of Sox9 trans-activates both a COL2A1-driven reporter gene and the endogenous Col2a1 gene in transgenic mice. These results demonstrate that COL2A1 expression is directly regulated by SOX9 protein in vivo and implicate abnormal regulation of COL2A1 during, chondrogenesis as a cause of the skeletal abnormalities associated with campomelic dysplasia.

Hepatocyte-specific activation of NF-kappaB does not aggravate chemical hepatocarcinogenesis in transgenic mice.

The NF-kappaB signalling pathway plays important roles in liver organogenesis and carcinogenesis. Mouse embryos deficient in IKKbeta die in mid-gestation, due to excessive apoptosis of hepatoblasts. Although activation of the NF-kappaB signalling pathway has been demonstrated in human hepatocellular carcinoma, the role of NF-kappaB is controversial. Here, we have generated transgenic mice in which a constitutively active form of IKKbeta was expressed in a hepatocyte-specific manner. Using electrophoretic mobility shift assay, we documented increased NF-kappaB activities and up-regulated levels of NF-kappaB downstream target genes, Bcl-xL and STAT5, in the transgenic mouse livers. These results confirmed that the NF-kappaB pathway was activated in the livers of the transgenic mice. However, there was no significant difference in tumour formation between transgenic and wild-type mice up to an age of 50 weeks. When we treated the transgenic mice with the chemical carcinogen diethylnitrosamine (DEN), we observed no significant differences in the incidence and size of liver tumours formed in these mice with and without DEN treatment at 35 weeks of age, suggesting that the activated NF-kappaB pathway in the livers of the transgenic mice did not enhance hepatocarcinogenesis. Interestingly, some of the transient transgenic embryos (E12.5) had abnormal excessive accumulation of nucleated red blood cells in their developing livers. In summary, NF-kappaB activation in hepatocytes did not significantly affect chemical hepatocarcinogenesis. In addition, the TTR/IKKCA transgenic mice may serve as a useful model for studying the role of NF-kappaB activation in hepatocarcinogenesis as well as inflammatory and metabolic diseases.

Prokineticin-1 modulates proliferation and differentiation of enteric neural crest cells.

Prokineticins (Prok-1 and Prok-2) belong to a newly identified AVIT protein family. They are involved in variety of activities in various tissues, including smooth muscle contraction of the gastrointestinal tract and promoting proliferation of endothelial cells derived from adrenal gland. Importantly, they also act as the survival factors to modulate growth and survival of neurons and hematopoietic stem cells. In this study we demonstrated that Prok-1 (but not Prok-2) protein is expressed in the mucosa and mesenchyme of the mouse embryonic gut during enteric nervous system development. Its receptor, PK-R1 is expressed in the enteric neural crest cells (NCCs). To elucidate the physiological role(s) of Prok-1 in NCCs, we isolated the NCCs from the mouse embryonic gut (E11.5) and cultured them in the form of neurospheres. In an in vitro NCC culture, Prok-1 was able to activate both Akt and MAPK pathways and induce the proliferation and differentiation (but not migration) of NCCs via PK-R1. Knock-down of PK-R1 using siRNA resulted in a complete abolishment of Prok-1 induced proliferation. Taken together, it is the first report demonstrating that Prok-1 acts as a gut mucosa/mesenchyme-derived factor and maintains proliferation and differentiation of enteric NCCs.

The Mll-Een knockin fusion gene enhances proliferation of myeloid progenitors derived from mouse embryonic stem cells and causes myeloid leukaemia in chimeric mice.

Rearrangement of the mixed lineage leukaemia (MLL) gene with extra eleven nineteen (EEN) was previously identified in an infant with acute myeloid leukaemia. Using homologous recombination, we have created a mouse equivalent of the human MLL-EEN allele and showed that when Mll(Een/+) embryonic stem (ES) cells were induced to differentiate in vitro into haemopoietic cells, there was increased proliferation of myeloid progenitors with self-renewal property. We also generated Mll(Een/+) chimeric mice, which developed leukaemia displaying enlarged livers, spleens, thymuses and lymph nodes owing to infiltration of Mll(Een/+)-expressing leukemic cells. Immunophenotyping of cells from enlarged organs and bone marrow (BM) of the Mll(Een/+) chimeras revealed an accumulation of Mac-1+/Gr-1- immature myeloid cells and a reduction in normal B- and T-cell populations. We observed differential regulation of Hox genes between myeloid cells derived from Mll(Een/+) ES cells and mouse BM leukemic cells which suggested different waves of Hox expression may be activated by MLL fusion proteins for initiation (in ES cells) and maintenance (in leukemic cells) of the disease. We believe studies of MLL fusion proteins in ES cells combined with in vivo animal models offer new approaches to the dissection of molecular events in multistep pathogenesis of leukaemia.

The prenatal expression of secretin receptor.

Secretin is a classical gastrointestinal peptide while its neuroactive functions in the central nervous system have recently been consolidated. In the past, there was little information regarding the expression of secretin receptor in prenatal development. In this article, using mouse embryos and by in situ hybridization, secretin receptor transcripts were detected in several developing brain regions including the cerebellar primordium and choroid plexus. In the developing intestine, secretin receptor is present in the epithelial lining of the villi and the inner circular muscle. Interestingly, the transcripts for secretin receptor were also detected in the epicardium and myocardium of the developing heart as well as the glomerulus and collecting duct in the developing kidney. Taken together, our data suggest a potential pleiotrophic role of secretin during embryonic development.

CDX-1 and CDX-2 are expressed in human colonic mucosa and are down-regulated in patients with Hirschsprung's disease associated enterocolitis.

Caudal type homeobox gene-1 and -2 (CDX-1 and CDX-2), homologues of the Drosophila homeobox gene caudal, encode transcription factors in endoderm derived tissues of the intestine. CDX genes control proliferation and differentiation of intestinal mucosal cells and colon cancer cells. Hirschsprung's Disease (HD) or congenital intestinal aganglionosis, a major developmental anomaly of intestine, which causes functional intestinal obstruction, is frequently associated with enterocolitis. Aetiology of HD-associated enterocolitis (HDEC) remains obscure. Reduction of gut mucosal enteroendocrine cells, and inefficient transfer of the secretory immunoglobulin A across the gut mucosal cell were shown to be associated with enterocolitis in HD patients suggesting that mucosa may directly involve in the pathophysiology of HDEC. This study aims to ascertain whether the CDX-1 and CDX-2 genes, that control the proliferation and differentiation of mucosal cells, play a role in HDEC. Using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridisation, we analysed the expression of CDX-1 and CDX-2 genes in colon specimens of normal controls, necrotising enterocolitis (NEC) infants, and HD patients with and without enterocolitis. We showed for the first time that CDX-1 and CDX-2 genes were expressed in the colonic mucosal epithelium in normal, NEC and in HD infants. However, the expressions of both genes were reduced in patients with HDEC. Our findings suggest that reduced expression of CDX-1 and CDX-2 genes in mucosa may be associated with the development of HDEC.

Secretin, a known gastrointestinal peptide, is widely expressed during mouse embryonic development.

The gastrointestinal functions of the 27-amino acid secretin peptide have been well established. In previous prenatal studies, secretin expression in the rat duodenum was reported after day 17 of gestation while its expression in other organs and its functions in the developing embryos are still unknown. By in situ hybridization and immunohistochemical staining, secretin transcripts and peptides were found to be widely expressed in mouse embryos. Consistent with the idea that secretin is a brain-gut peptide, its expressions are present in several developing brain regions such as cephalic mesenchyme, cerebellar primordium and choroid plexus as well as the epithelial villi lining and inner circular muscle of the developing intestine. Other than these organs, secretin was also detected in the developing heart including the ventricular epicardium and myocardium and certain structures of the developing kidney like ureteric bud, collecting duct and glomerulus. These observations strongly suggest for a functional role of secretin during mouse embryonic development.

The interaction between EEN and Abi-1, two MLL fusion partners, and synaptojanin and dynamin: implications for leukaemogenesis.

The mixed lineage leukaemia gene, MLL (also called HRX, ALL-1) in acute leukaemia is fused to at least 16 identified partner genes that display diverse structural and biochemical properties. Using GST pull down and the yeast two hybrid system, we show that two different MLL fusion partners with SH3 domains, EEN and Abi-1, interact with dynamin and synaptojanin, both of which are involved in endocytosis. Synaptojanin, a member of the inositol phosphatase family that has recently been shown to regulate cell proliferation and survival, is also known to bind to Eps15, the mouse homologue of AF1p, another fusion partner of MLL. Expression studies show that synaptojanin is strongly expressed in bone marrow and immature leukaemic cell lines, very weakly in peripheral blood leukocytes and absent in Raji, a mature B cell line. We found that the SH3 domains of EEN and Abi-1 interact with different proline-rich domains of synaptojanin while the EH domains of Eps15 interact with the NPF motifs of synaptojanin. In vitro competitive binding assays demonstrate that EEN displays stronger binding affinity than Abi-1 and may compete with it for synaptojanin. These findings suggest a potential link between MLL fusion-mediated leukaemogenesis and the inositol-signalling pathway.

Is there a role for the IHH gene in Hirschsprung's disease?

Hirschsprung disease (HSCR) is characterized by the absence of ganglion cells along a variable length of the intestine. HSCR has a complex genetic aetiology with 50% of the patients unexplained by mutations in the major HSCR genes. The Ihh gene is involved in the development of the enteric nervous system (ENS) and Ihh mutant mice present with a phenotype reminiscent of HSCR. The requirement of Ihh signalling for the proper development of the ENS, together with the evidence presented by the Ihh murine model, prompted us to investigate the involvement of the human IHH gene in HSCR. Sequence analysis revealed seven single nucleotide polymorphisms, six of which were new. Allele and haplotype frequencies were compared between cases and controls, and, among the cases, between genders, between different phenotypes, and between patients with different mutation status in the main HSCR genes. Despite the involvement of IHH in the development of the ENS, IHH is not a major gene in HSCR. Nevertheless, as the manifestation of the HSCR phenotype is genetic background dependent, polymorphic loci should be tested simultaneously to characterize gene-gene interaction. The involvement of IHH in other intestinal anomalies should be investigated.

An efficient method of generating transgenic mice by pronuclear microinjection.

Genetic transformation of mice using pronuclear microinjection was demonstrated by a number of groups in rapid succession in the early 1980's. Since that time, studies using transgenic animals have produced major advances in biomedical sciences and molecular genetics. More important, it is possible to study the molecular basis for tissue and stage-specific expression of genes. We recently used this method to generate transgenic mice. DNA fragment (transgene) was injected into the pronucleus of one-cell embryos. We describe this simplified protocol, which is reliable. With the use of buffered medium M2 for the whole process, it is not mandatory to have a CO2 incubator.

Embryonic development of the ganglion plexuses and the concentric layer structure of human gut: a topographical study.

In this study, we performed a detailed topographical study on the development of ganglion plexuses and the smooth muscle layers of human embryonic and fetal gut. Neuron and glia differentiation was investigated with anti-PGP9.5 and anti-S100 antibodies respectively. The differentiation of smooth muscle and interstitial cells of Cajal (ICC) was studied with anti-smooth muscle alpha-actin and anti-C-Kit antibodies respectively. By week 7, rostro-caudal neural crest cell (NCC) colonization of the gut was complete, and NCCs have differentiated into neurons and glia. At the foregut, neurons and glia were aggregated into ganglion plexus in the myenteric region, and the longitudinal and circular muscle layers have started to differentiate; however, neurons and glia were not found in the submucosa. At the hindgut, neurons and glia were dispersed within the mesenchyme. Myenteric plexus, longitudinal and circular muscle layers formed along the entire gut by week 9. Scattered and individual neurons and glia, and small ganglion plexuses were detected in the foregut and midgut submucosa by week 12. Ganglion plexus was not seen in the hindgut submucosa until week 14. Muscularis mucosae was formed at the foregut and midgut by week 12 but was only discernible at the hindgut 2 weeks later. As the gut wall developed, ganglion plexus increased in size with more neurons and glia, and the formation of intra-plexus nerve fascicle. ICCs were localized in the ganglion plexus as early as week 7. ICCs were initially dispersed in the plexus and were preferentially localized at the periphery of the plexus by week 20. The specification of the annular layers of human embryonic and fetal gut follows a strict spatio-temporal pattern in a rostro-caudal and centripetal manner suggesting that interaction between (1) homotypic and/or heterotypic cells; and (2) cells and the extracellular matrix is critical for the embryonic development of the gut mesenchyme and the enteric nervous system.

Transcription and translation of phloem protein (PP2) during phloem differentiation in Cucurbita maxima.

The synthesis of a major phloem protein, PP2, was investigated by measurement of the mRNA at various stages of phloem development in Cucurbita. Quantitative assays with immuno-electrophoresis showed that the amounts of PP2 in hypocotyls of Cucurbita seedlings increased with the age of seedlings. An increase in mRNA for PP2 during the early stages of seedling growth was also observed by immunoprecipitation of the invitro translation products of hypocotyl polyadenylated RNA. There was close timing in the variations of PP2 synthesised in vivo and in the changes in amounts of translatable PP2-mRNA during the course of seedling growth. A complementary-DNA (cDNA) library to polyadenylated RNA from hypocotyls of 3-d-old Cucurbita seedlings has been constructed. Two cDNA clones, A and B, have been identified by hybrid-release translation to be complementary to the mRNA coding for PP2. The levels of total mRNA for PP2 measured with clone A were found to increase in the first 4 d of seedling growth but decreased to lower levels in older seedlings. Regulatory controls on both transcription and modification of transcripts appeared to occur during the synthesis of PP2.

Hox homeobox genes and regionalisation of the nervous system.

The Hox family of homeobox-containing genes are intimately associated with the processes of axial patterning in vertebrate embryos. This family of transcription factors is widely conserved in evolution and by analogy with their Drosophila counterparts, the HOM-C homeotic genes, may play a role in establishing regional identity in a number of embryonic systems, including the CNS. The patterns of expression of these genes are linked with the generation of rhombomeres and neural crest in the developing hindbrain, and suggest that they provide a molecular system for generating a combinatorial patterning mechanism. Analysis of mouse Hox mutants generated by homologous recombination have clearly demonstrated that the genes have important roles in normal regionalisation of the hindbrain and branchial arches, and this has lead to interest in how their early patterns are established in the nervous system. The Hox genes and their relation to hindbrain segmentation therefore provide a means of examining the cascade of events which regulates pattern formation in early neural development.

Retinoic acid alters hindbrain Hox code and induces transformation of rhombomeres 2/3 into a 4/5 identity.

It has been suggested that Hox genes play an important part in the patterning of limbs, vertebrae and craniofacial structures by providing an ordered molecular system of positional values, termed the Hox code. Little is known about the nature of the signals that govern the establishment and regulation of Hox genes, but retinoic acid can affect the expression of these genes in cell lines and in embryonic tissues. On the basis of experimental and clinical evidence, the hindbrain and branchial region of the head are particularly sensitive to the effects of retinoic acid but the phenotypes are complex and hard to interpret, and how and if they relate to Hox expression has not been clear. Here we follow the changes induced by retinoic acid to hindbrain segmentation and the branchial arches using transgenic mice which contain lacZ reporter genes that reveal the endogenous segment-restricted expression of the Hox-B1 (Hox-2.9), Hox-B2(Hox-2.8) and Krox-20 genes. Our results show that these genes rapidly respond to exposure to retinoic acid at preheadfold stages and undergo a progressive series of changes in segmental expression that are associated with specific phenotypes in hindbrain of first branchial arch. Together the molecular and anatomical alterations indicate that retinoic acid has induced changes in the hindbrain Hox code which result in the homeotic transformation of rhombomeres (r) 2/3 to an r4/5 identity. A main feature of this rhombomeric phenotype is that the trigeminal motor nerve is transformed to a facial identity. Furthermore, in support of this change in rhombomeric identity, neural crest cells derived from r2/3 also express posterior Hox markers suggesting that the retinoic acid-induced transformation extends to multiple components of the first branchial arch.

Regulatory analysis of the mouse Hoxb3 gene: multiple elements work in concert to direct temporal and spatial patterns of expression.

The expression pattern of the mouse Hoxb3 gene is exceptionally complex and dynamic compared with that of other members of the Hoxb cluster. There are multiple types of transcripts for Hoxb3 gene, and the anterior boundaries of its expression vary at different stages of development. Two enhancers flanking Hoxb3 on the 3' and 5' sides regulate Hoxb2 and Hoxb4, respectively, and these control regions define the two ends of a 28-kb interval in and around the Hoxb3 locus. To assay the regulatory potential of DNA fragments in this interval we have used transgenic analysis with a lacZ reporter gene to locate cis-elements for directing the dynamic patterns of Hoxb3 expression. Our detailed analysis has identified four new and widely spaced cis-acting regulatory regions that can together account for major aspects of the Hoxb3 expression pattern. Elements Ib, IIIa, and IVb control gene expression in neural and mesodermal tissues; element Va controls mesoderm-specific gene expression. The most anterior neural expression domain of Hoxb3 is controlled by an r5 enhancer (element IVa); element IIIa directs reporter expression in the anterior spinal cord and hindbrain up to r6, and the region A enhancer (in element I) mediates posterior neural expression. Hence, the regulation of segmental expression of Hoxb3 in the hindbrain is different from that of Hoxa3, as two separate enhancer elements contribute to expression in r5 and r6. The mesoderm-specific element (Va) directs reporter expression to prevertebra C1 at 12.5 dpc, which is the anterior limit of paraxial mesoderm expression for Hoxb3. When tested in combinations, these cis-elements appear to work as modules in an additive manner to recapitulate the major endogenous expression patterns of Hoxb3 during embryogenesis. Together our study shows that multiple control elements direct reporter gene expression in diverse tissue-, temporal-, and spatially restricted subset of the endogenous Hoxb3 expression domains and work in concert to control the neural and mesodermal patterns of expression.

SOX10 is abnormally expressed in aganglionic bowel of Hirschsprung's disease infants.

BACKGROUND: The primary pathology of Hirschsprung's disease (HD) is a congenital absence of ganglion cells in the caudal most gut. The spastic aganglionic bowel is often innervated by a network of hypertrophied nerve fibres. Recently, mutations of SOX10 have been identified in patients with HD but only in those with Waardenburg-Shah syndrome. AIMS: To understand the molecular basis for the pathogenesis of HD we intended to determine the specific cell lineages in the enteric nervous system which normally express SOX10 but are affected in disease conditions. METHODS: We studied colon biopsies from 10 non-syndromic HD patients, aged three months to four years, and 10 age matched patients without HD as normal controls. The absence of mutation in the SOX10 gene of HD patients was confirmed by DNA sequencing. Expression and cellular distribution of SOX10 in bowel segments of normal and HD infants were examined by reverse transcription-polymerase chain reaction and in situ hybridisation. RESULTS: We found that in normal infants and normoganglionic bowel segments of HD patients, SOX10 was expressed in both neurones and glia of the enteric plexuses and in the nerves among the musculature in normal colon. In the aganglionic bowel segments of patients, SOX10 expression was consistently lower and was found to be associated with the hypertrophic nerve trunks in the muscle and extrinsic nerves in the serosa. CONCLUSION: We conclude that SOX10 is normally required postnatally in the functional maintenance of the entire enteric nervous system, including neurones and glia. In non-syndromic HD patients who do not have the SOX10 mutation, the SOX10 gene expressed in the sacral region may be involved in the pathogenesis of the abnormal nerve trunks through interaction with other factors.

Analysis of the murine Hox-2.7 gene: conserved alternative transcripts with differential distributions in the nervous system and the potential for shared regulatory regions.

In this study we have investigated the organization and regulation of the mouse Hox-2.7 gene. There are several alternative transcripts some of which are conserved between mouse and humans. By Northern and in situ analysis we are able to identify at least three types of transcripts which are different in size and splicing pattern and have distinctly different boundaries of expression in the nervous system. One subset of the endogenous transcripts has a boundary of expression that corresponds to the adjacent Hox-2.8 gene instead of Hox-2.7. In another type of transcript there is an alternative reading frame which predicts a protein that has homology to an enzyme ATPase and suggests that a non-homeobox containing gene may be located in the Hox-2 cluster. A Hox-2.7-lacZ transgene is expressed in a similar pattern to the endogenous gene in that spatially-restricted domains of expression are seen in the branchial arches, neural tube, paraxial mesoderm (somites), cranial ganglia, neural crest and gut. However, the anterior boundaries of transgene expression only correspond to the subset of Hox-2.7 transcripts which map to the Hox-2.8 boundary. The proximity of a Hox-2.7 promoter to regions which regulate the adjacent Hox-2.6 gene and the expression of transgenic and endogenous transcripts in a Hox-2.8 pattern, suggest that regulatory elements may be shared by neighbouring genes to establish the complete expression pattern.