Gene Family In Development Research Articles

Midkine regulates pleiotrophin organ-specific gene expression: Evidence for transcriptional regulation and functional redundancy within the pleiotrophin/midkine developmental gene family

Midkine (MK) and the highly related cytokine pleiotrophin (PTN) constitute the PTN/MK developmental gene family. The Mk and Ptn genes are essential for normal development of the catecholamine and renin–angiotensin pathways and the synthesis of different collagens. It is not known whether the Ptn and Mk genes regulate each other or whether PTN and MK are functionally redundant in development. We have now compared the levels of expression of Ptn and Mk in genetically deficient Mk −/− and Ptn −/− mice and found highly significant increases in Ptn gene expression in spinal cord, dorsal root ganglia, eye, heart, aorta, bladder, and urethra, but not in brain, bone marrow, testis, and lung of Mk −/− mice compared with wild type mice; a remarkable ∼230-fold increase in Ptn expression levels was found in heart of Mk −/− mice and highly significant but lesser increases were found in six other organs. Differences in levels of Mk gene expression in Ptn −/− mice could not be detected in any of the organs tested. The data demonstrate that MK regulates Ptn gene expression with a high degree of organ specificity, suggesting that Ptn gene expression follows Mk gene expression in development, that the increase in Ptn gene expression is compensatory for the absence of MK in Mk −/− mice, that PTN and MK share a high degree of functional redundancy, and that MK may be very important in the development of heart in mouse.

Midkine, a newly discovered regulator of the renin–angiotensin pathway in mouse aorta: Significance of the pleiotrophin/midkine developmental gene family in angiotensin II signaling

We previously demonstrated that pleiotrophin (PTN the protein, Ptn the gene) highly regulates the levels of expression of the genes encoding the proteins of the renin–angiotensin pathway in mouse aorta. We now demonstrate that the levels of expression of these same genes are significantly regulated in mouse aorta by the PTN family member midkine (MK the protein, Mk the gene); a 3-fold increase in expression of renin, an 82-fold increase in angiotensinogen, a 6-fold decrease in the angiotensin converting enzyme, and a 6.5-fold increase in the angiotensin II type 1 and a 9-fold increase in the angiotensin II type 2 receptor mRNAs were found in Mk−/− mouse aorta in comparison with the wild type (WT, +/+). The results in Mk−/− mice are remarkably similar to those previously reported in Ptn−/− mouse aorta, with the single exception of that the levels of the angiotensinogen gene expression in Ptn−/− mice are equal to those in WT+/+ mouse aorta, and thus, in contrast to Mk gene expression unaffected by levels of Ptn gene expression. The data indicate that MK and PTN share striking but not complete functional redundancy. These data support potentially high levels importance of MK and the MK/PTN developmental gene family in downstream signals initiated by angiotensin II either in development or in the many pathological conditions in which MK expression levels are increased, such as atherosclerosis and many human neoplasms that acquire constitutive endogenous Mk gene expression by mutation during tumor progression and potentially provide a target through the renin–angiotensin pathway to treat advanced malignancies.

Pleiotrophin induces formation of functional neovasculature in vivo

Pleiotrophin (PTN) is a heparin-binding growth/differentiation inducing cytokine that shares 50% amino acid sequence identity and striking domain homology with Midkine (MK), the only other member of the Ptn/ Mk developmental gene family. The Ptn gene is expressed in sites of early vascular development in embryos and in healing wounds and its constitutive expression in many human tumors is associated with an angiogenic phenotype, suggesting that PTN has an important role in angiogenesis during development and in wound repair and advanced malignancies. To directly test whether PTN is angiogenic in vivo, we injected a plasmid to express PTN into ischemic myocardium in rats. Pleiotrophin stimulated statistically significant increases in both normal appearing new capillaries and arterioles each of which had readily detectable levels of the arteriole marker, smooth muscle cell α-actin. Furthermore, the newly formed blood vessels were shown to interconnect with the existent coronary vascular system. The results of these studies demonstrate directly that PTN is an effective angiogenic agent in vivo able to initiate new vessel formation that is both normal in appearance and function. The data suggest that PTN signals the more “complete” new blood vessel formation through its ability to stimulate different functions in different cell types not limited to the endothelial cell.

The ZIC gene family in development and disease

The human ZIC gene family is comprised of five members encoding zinc-finger transcription factors, which are the vertebrate homologs of the Drosophila odd-paired gene. Mutations in ZIC genes in humans have recently been implicated in a wide variety of congenital malformations, including Dandy-Walker malformation, holoprosencephaly, neural tube defects, and heterotaxy. Mutant analysis of these genes in mice has underscored the conserved developmental roles of these genes. Further, this analysis has begun to elucidate the molecular and developmental mechanisms underlying these important birth defects.

TIF1δ, a Novel HP1-interacting Member of the Transcriptional Intermediary Factor 1 (TIF1) Family Expressed by Elongating Spermatids

TIF1 (transcriptional intermediary factor 1) proteins are encoded by an expanding family of developmental and physiological control genes that are conserved from flies to man. These proteins are characterized by an N-terminal RING-B box-coiled-coil (RBCC) motif and a C-terminal PHD finger/bromodomain unit, and have been implicated in epigenetic mechanisms of transcriptional repression involving histone modifiers and heterochromatin-binding proteins. We describe here the isolation and functional characterization of a fourth murine TIF1 gene, TIF1delta. The predicted TIF1delta protein displays all the structural hallmarks of a bona fide TIF1 family member and resembles the other TIF1s in that it can exert a deacetylase-dependent silencing effect when tethered to a promoter region. Moreover, like TIF1alpha and TIF1beta, TIF1delta can homodimerize and contains a PXVXL motif necessary and sufficient for HP1 (heterochromatin protein 1) binding. Although TIF1alpha and TIF1beta also bind nuclear receptors and Kruppel-associated boxes specifically and respectively, TIF1delta appears to lack nuclear receptor- and Kruppel-associated box binding activity. Furthermore, TIF1delta is unique among the TIF1 family proteins in that its expression is largely restricted to the testis and confined to haploid elongating spermatids, where it associates preferentially with HP1 isotype gamma (HP1gamma) and forms discrete foci dispersed within the centromeric chromocenter and the surrounding nucleoplasm. Collectively, these data are consistent with specific, nonredundant functions for the TIF1 family members in vivo and suggest a role for TIF1delta in heterochromatin-mediated gene silencing during postmeiotic phases of spermatogenesis.

SOX8 Is Expressed during Testis Differentiation in Mice and Synergizes with SF1 to Activate the Amh Promoter in Vitro

Sox8 is a member of the Sox family of developmental transcription factor genes and is closely related to Sox9, a key gene in the testis determination pathway in mammals. Like Sox9, Sox8 is expressed in the developing mouse testis around the time of sex determination, suggesting that it might play a role in regulating the expression of testis-specific genes. An early step in male sex differentiation is the expression of anti-Müllerian hormone (AMH) in Sertoli cells. Expression of the Amh gene during sex differentiation requires the interaction of several transcription factors, including SF1, SOX9, GATA4, WT1, and DAX1. Here we show that SOX8 may also be involved in regulating the expression of Amh. Expression of Sox8 begins just prior to that of Amh at 12 days post coitum (dpc) in mouse testes and continues beyond 16 dpc in Sertoli cells. In vitro assays showed that SOX8 binds specifically to SOX binding sites within the Amh minimal promoter and, like SOX9, acts synergistically with SF1 through direct protein-protein interaction to enhance Amh expression, albeit at lower levels compared with SOX9. SOX8 and SOX9 appear to have arisen from a common ancestral gene and may have retained some common functions during sexual development. Our data provide the first evidence that SOX8 may partially compensate for the reduced SOX9 activity in campomelic dysplasia and substitute for Sox9 where Sox9 is either not expressed or expressed too late to be involved in sex determination or regulation of Amh expression.

Polymorphism patterns in two tightly linked developmental genes, Idgf1 and Idgf3, of Drosophila melanogaster.

A new developmental gene family, recently identified in D. melanogaster, has been called imaginal disc growth factors (IDGF) because the proteins promote growth of cell lineages derived from imaginal discs. These are the first genes reported that encode polypeptide factors with mitotic activity in invertebrates. Characteristics such as similar arrangement of introns and exons, small size, and different cytological localization make this family an excellent candidate for evolutionary studies. We focus on the loci Idgf1 and Idgf3, two genes that possess the most distinctive features. We examine the pattern of intra- and interspecific nucleotide variation in the sequences from 20 isogenic lines of D. melanogaster and sequences from D. simulans and D. yakuba. While MK, HKA, and Tajima's tests of neutrality fail to reject a neutral model of molecular evolution, Fu and Li's test with outgroup and McDonald's test suggest that balancing selection is modulating the evolution of the Idgf1 locus. The rate of recombination between the two loci is high enough to uncouple any linkage disequilibrium arising between Idgf1 and Idgf3, despite their close physical proximity.

Wnt-7a is upregulated by norethisterone in human endometrial epithelial cells: a possible mechanism by which progestogens reduce the risk of estrogen-induced endometrial neoplasia

Progestogens are added to oestrogen in hormone replacement therapy regimens to reduce the risk of endometrial cancer. We have performed in vitro studies analysing gene expression of isolated normal endometrial epithelia cells (NEE) treated with estradiol and the progestogen norethisterone acetate (NETA). We report here for the first time upregulation of the Wnt-7a gene by NETA in estrogen treated NEE. Wnt genes are a large family of developmental genes associated with cellular responses such as oncogenesis. We therefore suggest that upregulation of Wnt-7a may be associated with the antineoplastic effects of progestogens on the endometrium.

Expression of the developmental and oncogenic PAX2 gene in human prostate cancer.

In the human prostate cancer cell lines LNCaP, DU145 and PC3, 27 primary prostate cancers, 10 benign prostatic hyperplasia specimens and 5 normal prostates we investigated the expression pattern of PAX2, a member of the PAX family of developmental control genes. PAX2 is expressed at high levels in developing undifferentiated cells of the urogenital system and is repressed upon terminal differentiation with no expression in normal adult cells. It is also been shown to be a proto-oncogene in mice and is expressed in human renal cell carcinoma. PAX2 expression was assessed at the RNA level by reverse transcriptase-polymerase chain reaction and Southern blot analysis using specific sets of nucleotides. The expression pattern of PAX2 was reconfirmed at the protein level by immunofluorescence in the cell lines, and by Western blot analysis in primary human prostate cancers and benign prostatic tissue. Using reverse transcription-polymerase chain reaction combined with Southern hybridization PAX2 expression was detected in 52% of primary cancers and all 3 cell lines. PAX2 expression in these samples was confirmed at a protein level using immunoblotting and immunofluorescence. PAX2 messenger RNA was not detected in any benign or normal prostatic samples. Immunoblotting of protein from benign prostatic hyperplasia samples confirmed the lack of expression of PAX2 protein. The expression of PAX2 in prostate cancer compared to nonmalignant prostates is statistically significant (Fisher's exact test p = 0.0004). These results suggest a possible role for PAX2 in prostate cancer. Although previous studies have suggested a role for PAX2 for supporting proliferation in undifferentiated cells, no correlation of PAX2 expression with Gleason score was found in prostate cancer.

EXPRESSION OF THE DEVELOPMENTAL AND ONCOGENIC PAX2 GENE IN HUMAN PROSTATE CANCER

Purpose: In the human prostate cancer cell lines LNCaP, DU145 and PC3, 27 primary prostate cancers, 10 benign prostatic hyperplasia specimens and 5 normal prostates we investigated the expression pattern of PAX2, a member of the PAX family of developmental control genes. PAX2 is expressed at high levels in developing undifferentiated cells of the urogenital system and is repressed upon terminal differentiation with no expression in normal adult cells. It is also been shown to be a proto-oncogene in mice and is expressed in human renal cell carcinoma.Materials and Methods: PAX2 expression was assessed at the RNA level by reverse transcriptase-polymerase chain reaction and Southern blot analysis using specific sets of nucleotides. The expression pattern of PAX2 was reconfirmed at the protein level by immunofluorescence in the cell lines, and by Western blot analysis in primary human prostate cancers and benign prostatic tissue.Results: Using reverse transcription-polymerase chain reaction combined with Sout...

Primary renal hypoplasia in humans and mice with PAX2 mutations: evidence of increased apoptosis in fetal kidneys of Pax2(1Neu) +/- mutant mice.

PAX2 mutations cause renal-coloboma syndrome (RCS), a rare multi-system developmental abnormality involving optic nerve colobomas and renal abnormalities. End-stage renal failure is common in RCS, but the mechanism by which PAX2 mutations lead to renal failure is unknown. PAX2 is a member of a family of developmental genes containing a highly conserved 'paired box' DNA-binding domain, and encodes a transcription factor expressed primarily during fetal development in the central nervous system, eye, ear and urogenital tract. Presently, the role of PAX2 during kidney development is poorly understood. To gain insight into the cause of renal abnormalities in patients with PAX2 mutations, kidney anomalies were analyzed in patients with RCS, including a large Brazilian kindred in whom a new PAX2 mutation was identified. In a total of 29 patients, renal hypoplasia was the most common congenital renal abnormality. To determine the direct effects of PAX2 mutations on kidney development fetal kidneys of mice carrying a Pax2 (1Neu)mutation were examined. At E15, heterozygous mutant kidneys were approximately 60% of the size of wild-type littermates, and the number of nephrons was strikingly reduced. Heterozygous 1Neu mice showed increased apoptotic cell death during fetal kidney development, but the increased apoptosis was not associated with random stochastic inactivation of Pax2 expression in mutant kidneys; Pax2 was shown to be biallelically expressed during kidney development. These findings support the notion that heterozygous mutations of PAX2 are associated with increased apoptosis and reduced branching of the ureteric bud, due to reduced PAX2 dosage during a critical window in kidney development.

The expression of PAX5 in human transitional cell carcinoma of the bladder: relationship with de-differentiation.

To investigate the expression of PAX genes, a family of developmental control genes (which encode nine nuclear transcription factors essential for embryogenesis and are proto-oncogenes in mice) in human transitional cell carcinoma (TCC) of the bladder. PAX gene expression was assessed in three established bladder cancer cell lines and 29 primary tumours using the reverse transcriptase-polymerase chain reaction and Southern analysis. All three established TCC cell lines and 79% of primary TCCs expressed PAX5 mRNA. There was a significantly higher proportion of PAX5 expression in malignant than in benign urothelium (P=0.02, Fisher's exact test); nine of 12 pTa tumours (mucosa-confined), seven of eight pT1 (invading lamina propria) and eight of nine pT2 (invading muscle) expressed PAX5. A higher proportion of tumours with increasing de-differentiation expressed PAX5, which correlates well with the expression pattern of PAX5 in development. In well-differentiated tumours (grade 1), half expressed PAX5, compared with 84% of moderately to poorly differentiated tumours (grades 2/3). The odds ratio for PAX5 expression in malignancy suggests that it increases the risk of malignancy four-fold. These data support a role for the PAX family in oncogenesis, by identifying another human neoplasm in which they are inappropriately expressed. PAX5 expression in undifferentiated TCC cells may contribute to pathogenesis by supporting cellular proliferation in the de-differentiated state. Furthermore, the high incidence of PAX5 expression suggests its potential use as a diagnostic tool and therapeutic target in TCC.

The bicoid-related Pitx gene family in development.

The bicoid-related Pitx gene family in development.

Expression of Wnt genes in early wound healing.

The Wnt family of developmental genes has previously been shown to be involved in proliferation, differentiation, and cell to cell signaling during embryogenesis. In addition, several Wnt genes have been shown to be expressed during carcinogenesis. We have investigated these genes during the wound-healing process. Wnt-4 gene expression is found in mouse wounds from 2 hours to 30 hours postwounding. The expression of Wnt-4 is also stimulated by direct trauma to murine fibroblasts in culture, and the expression is greatly enhanced by the addition of a short plasmin digest of fibrin. Therefore the regulation of Wnt-4, appears to be complex, with expression being stimulated both by direct trauma and by the influence of clotting and fibrinolysis products. We propose that the expression of Wnt-4 in the early wound, in response to the provisional fibrin matrix, regulates cell movement and proliferation in the creation of new tissue by mechanisms related to those of embryogenesis.

The Genomic Organization and the Full Coding Region of the Human PAX7 Gene

The PAX7 gene encodes a transcription factor that is a member of the PAX family of developmental control genes. In addition to playing a role in embryogenesis, PAX genes appear to be of importance in a number of diverse human diseases and cancers. The PAX7 gene maps to human chromosomal region 1p36 and therefore is a potential candidate for human disorders linked to this region. In particular, a rearrangement of the PAX7 gene by chromosomal translocation is frequently found in alveolar rhabdomyosarcoma tumors. Here, we cloned a cDNA containing the full coding region of the human PAX7 gene and determined its genomic organization. The gene encodes a predicted protein of 520 amino acids that is 47 amino acids longer at the carboxy end than the highly related PAX3 protein. The coding region of the gene is interrupted by seven introns, the positions and lengths of which are similar to those of the corresponding introns of the PAX3 gene. Sequence data for exon/intron boundaries of PAX7 exons 1, 5, 6, 7, and 8 were determined and, together with previously published data for exons 2, 3, and 4, provide the complete sequence information for mutation analysis of the human PAX7 gene.

CDNA Cloning of a Humandishevelled DVL-3Gene, Mapping to 3q27, and Expression in Human Breast and Colon Carcinomas

dishevelled(Dsh) is a member of the segment polarity gene family inDrosophilawhich plays an important role in the early developmental patterning processes. A human homologue ofDsh(DVL-1) has recently been described. Here, we report the cloning of a second human homologue ofDsh(calledDVL-3) by cDNA library screening. The humanDVL-3gene encodes a predicted 716 amino acid protein which exhibits 98% amino acid identity with mouseDvl-3and 49% withDrosophila Dsh. DVL-3was mapped to 3q27. The expression ofDVL-3mRNA was detected in 30 human cell lines and 2 primary cell cultures.DVL-3mRNA was detected in normal human breast tissues (n = 4) and tumours (n = 25). Statistically, there was no difference inDVL-3mRNA level between normal breast tissues and tumours. In human colorectal samples,DVL-3was expressed equally in matched normal tissues, polyps and tumours. The data indicates thatDVL-3is widely expressed in human cells and supports the notion of a new developmental gene family fordishevelledwhich may have a widespread role in signal transduction.

Pax genes and organogenesis.

Pax genes are a family of developmental control genes that encode nuclear transcription factors. They are characterized by the presence of the paired domain, a conserved amino acid motif with DNA-binding activity. Originally, paired-box-containing genes were detected in Drosophila melanogaster, where they exert multiple functions during embryogenesis. In vertebrates, Pax genes are also involved in embryogenesis. Mutations in four out of nine characterized Pax genes have been associated with either congenital human diseases such as Waardenburg syndrome (PAX3), Aniridia (PAX6), Peter's anomaly (PAX6), renal coloboma syndrome (PAX2) or spontaneous mouse mutants (undulated (Pax1), Splotch (Pax3), Small eye (Pax6), Pax2(1)Neu), which all show defects in development. Recently, analysis of spontaneous and transgenic mouse mutants has revealed that vertebrate pax genes are key regulators during organogenesis of kidney, eye, ear, nose, limb muscles, vertebral column and brain. Like their Drosophila counterparts, vertebrate Pax genes are involved in pattern formation during embryogenesis, possibly by determining the time and place of organ initiation or morphogenesis. For most tissues, however, the nature of the primary developmental action of Pax transcription factors remains to be elucidated. One predominant theme is signal transduction during tissue interactions, which may lead to a position-specific regulation of cell proliferation.

DNA Methylation and Developmental Genes in Lymphomagenesis—More Questions Than Answers?

There is now considerable evidence suggesting that alterations in the DNA methylating machinery play an important role in tumorigenesis and tumour progression. For example, focal hypermethylation and generalised genomic demethylation are features of many different types of neoplasms. It is thought that tumorigenesis and tumour progression may be caused by hypermethylation induced mutational events and silencing of genes which control cellular proliferation and/or demethylation induced reactivation of genes which may only be required during embryological development. Consequently, we have begun to investigate the role of DNA methylation and developmental genes in malignant lymphoproliferative diseases.Previously, in all cases of non-Hodgkins lymphoma and leukemia studied, we have shown that the myogenic developmental gene Myf-3 is abnormally hypermethylated. In this review we discuss the possible significance of these findings since in vitro studies suggest that Myf-3 may play an important role in control of the cell cycle and therefore lymphomagenesis. In vitro and in vivo evidence suggests that PAX genes may also have oncogenic potential. The PAX family of developmental genes are involved in cellular differentiation, proliferation and cell migration. Expression of PAX3 in particular is associated with cellular mobility. Our previous studies have indicated that alternate regional expression of PAX genes may be controlled by DNA methylation. Therefore, we have proposed that abnormal methylation profiles of PAX3 may be associated with neoplastic transformation and/or metastatic potential. Results thus far reveal that the paired box of PAX3 is abnormally hypermethylated and the homeobox abnormally hypomethylated in lymphomas and leukemias. These new findings are consistent with our postulate and support the idea that inappropriate methylation induced activation or inactivation of developmental genes such as Myf-3 and PAX3 play an important role in lymphomagenesis and disease progression and that inspection of the methylation status of other developmental genes is warranted.

Gen(om)e duplications in the evolution of early vertebrates

Phylogenetic analyses and sequence surveys of developmental regulator gene families indicate that two large-scale gene duplications, most likely genome duplications, occurred in ancestors of vertebrates. Relaxed constraints allowed duplicated and thus redundant genes to diverge in a two stage mechanism. Neutral changes dominated at first but then positively selected regulatory changes evolved the novel and increasingly complex vertebrate developmental program.

The mouse Wnt-10B gene isolated from helper T cells is widely expressed and a possible oncogene in BR6 mouse mammary tumorigenesis

From libraries made from activated mouse T lymphocytes, we have isolated cDNAs encoding Wnt-10B, a new member of the Wnt family of developmental control genes. This protein appears to be the mammalian orthologue of Wnt-10B, first identified in several non-mammalian vertebrates and recently in mouse. The mRNA expression pattern of mouse Wnt-10B indicates that it is induced following activation of helper T cells, but is also expressed in a variety of other tissues and cells of fetal or adult origin. 93 bp at the 5′ end of the cDNA clone are identical to sequences previously reported as 3′ flanking genomic DNA adjacent to a mouse mammary tumor virus (MMTV) provirus in the MMTV-induced BR6 mammary tumor, W26. Sequence analysis of tumor-derived genomic DNA confirms that the entire Wnt-10B gene is immediately adjacent to the provirus, suggesting that MMTV integration drives transcription of Wnt-10B, possibly contributing to the oncogenic process. Consistent with this idea is the detection of hybrid MMTV-Wnt-IOB transcripts in BR6 tumor cells. T cells which produce abundant Wnt-10B mRNA were also found to produce protein.