Abnormal lung development and cleft palate in mice lacking TGF-beta 3 indicates defects of epithelial-mesenchymal interaction.

A broad spectrum of biological activities has been proposed for transforming growth factor-beta 3 (TGF-beta 3). To study TGF-beta 3 function in development, TGF-beta 3 null mutant mice were generated by gene-targeting. Within 20 hours of birth, homozygous TGF-beta 3-/- mice die with unique and consistent phenotypic features including delayed pulmonary development and defective palatogenesis. Unlike other null mutants with cleft palate, TGF-beta 3-/- mice lack other concomitant craniofacial abnormalities. This study demonstrates an essential function for TGF-beta 3 in the normal morphogenesis of palate and lung, and directly implicates this cytokine in mechanisms of epithelial-mesenchymal interaction.

Periderm Fate during Palatogenesis: TGF-ß and Periderm Dedifferentiation.

Failure of palatogenesis results in cleft palate, one of the most common congenital disabilities in humans. During the final phases of palatogenesis, the protective function of the peridermal cell layer must be eliminated for the medial edge epithelia to adhere properly, which is a prerequisite for the successful fusion of the secondary palate. However, a deeper understanding of the role and fate of the periderm in palatal adherence and fusion has been hampered due to a lack of appropriate periderm-specific genetic tools to examine this cell type in vivo. Here we used the cytokeratin-6A (Krt-6a) locus to develop both constitutive (Krt6ai-Cre) and inducible (Krt6ai-CreERT2) periderm-specific Cre driver mouse lines. These novel lines allowed us to achieve both the spatial and temporal control needed to dissect the periderm fate on a cellular resolution during palatogenesis. Our studies suggest that, already before the opposing palatal shelves contact each other, at least some palatal periderm cells start to gradually lose their squamous periderm-like phenotype and dedifferentiate into cuboidal cells, reminiscent of the basal epithelial cells seen in the palatal midline seam. Moreover, we show that transforming growth factor-ß (TGF-ß) signaling plays a critical periderm-specific role in palatogenesis. Thirty-three percent of embryos lacking a gene encoding the TGF-ß type I receptor (Tgfbr1) in the periderm display a complete cleft of the secondary palate. Our subsequent experiments demonstrated that Tgfbr1-deficient periderm fails to undergo appropriate dedifferentiation. These studies define the periderm cell fate during palatogenesis and reveal a novel, critical role for TGF-ß signaling in periderm dedifferentiation, which is a prerequisite for appropriate palatal epithelial adhesion and fusion.

A mouse model for the human lysosomal disease aspartylglycosaminuria.

Aspartylglycosaminuria (AGU), the most common disorder of glycoprotein degradation in humans, is caused by mutations in the gene encoding the lysosomal enzyme glycosylasparaginase (Aga). The resulting enzyme deficiency allows aspartylglucosamine (GlcNAc-Asn) and other glycoasparagines to accumulate in tissues and body fluids, from early fetal life onward. The clinical course is characterized by normal early development, slowly progressing to severe mental and motor retardation in early adulthood. The exact pathogenesis of AGU in humans is unknown and neither therapy nor an animal model for this debilitating and ultimately fatal disease exists. Through targeted disruption of the mouse Aga gene in embryonic stem cells, we generated mice that completely lack Aga activity. At the age of 5-10 months a massive accumulation of GlcNAc-Asn was detected along with lysosomal vacuolization, axonal swelling in the gracile nucleus and impaired neuromotor coordination. A significant number of older male mice had massively swollen bladders, which was not caused by obstruction, but most likely related to the impaired function of the nervous system. These findings are consistent with the pathogenesis of AGU and provide further data explaining the impaired neurological function in AGU patients.

TGF-beta3-induced palatogenesis requires matrix metalloproteinases.

Cleft lip and palate syndromes are among the most common congenital malformations in humans. Mammalian palatogenesis is a complex process involving highly regulated interactions between epithelial and mesenchymal cells of the palate to permit correct positioning of the palatal shelves, the remodeling of the extracellular matrix (ECM), and subsequent fusion of the palatal shelves. Here we show that several matrix metalloproteinases (MMPs), including a cell membrane-associated MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were highly expressed by the medial edge epithelium (MEE). MMP-13 was expressed both in MEE and in adjacent mesenchyme, whereas gelatinase A (MMP-2) was expressed by mesenchymal cells neighboring the MEE. Transforming growth factor (TGF)-beta3-deficient mice, which suffer from clefting of the secondary palate, showed complete absence of TIMP-2 in the midline and expressed significantly lower levels of MMP-13 and slightly reduced levels of MMP-2. In concordance with these findings, MMP-13 expression was strongly induced by TGF-beta3 in palatal fibroblasts. Finally, palatal shelves from prefusion wild-type mouse embryos cultured in the presence of a synthetic inhibitor of MMPs or excess of TIMP-2 failed to fuse and MEE cells did not transdifferentiate, phenocopying the defect of the TGF-beta3-deficient mice. Our observations indicate for the first time that the proteolytic degradation of the ECM by MMPs is a necessary step for palatal fusion.

Cellular interactions of CRKL, and SH2-SH3 adaptor protein.

Chronic myelogenous leukemia is characterized by a specific chromosomal translocation, t(9;22), in which the ABL protooncogene and the BCR gene become juxtaposed. The chimeric BCR/ABL gene produces a P210 fusion protein with deregulated tyrosine kinase activity. We have recently isolated a complementary DNA, CRKL, which could code for an adaptor protein consisting of one SH2 and two SH3 domains and lacking any catalytic domain. In the current study, we show that CRKL is highly phosphorylated in the chronic myelogenous leukemia cell line K562 and that it is a substrate for the p210 BCR/ABL and p145 ABL kinases. BCR/ABL and ABL are coimmunoprecipitated with CRKL in vivo, demonstrating that relatively stable complexes are formed. In addition, the nucleotide exchange factor mSOS1 was found to be coimmunoprecipitated with CRKL. These findings establish a putative signal transduction pathway way through which BCR/ABL mediates its oncogenic activity.

Crkl enhances leukemogenesis in BCR/ABL P190 transgenic mice.

The adapter protein Crkl has been implicated in the abnormal signal transduction pathways activated by the Bcr/Abl oncoprotein, which causes Philadelphia-positive leukemias in humans. To investigate the role of Crkl in tumorigenesis, we have generated transgenic mice that express human Crkl from the CRKL promoter. Western blot analysis showed a 4-6-fold overexpression of transgenic Crkl above endogenous crkl in two lines and increased constitutive complex formation between Crkl and C3G, an exchange factor for the small GTPase Rap1. This was associated with a significant increase in integrin-based motility of transgenic macrophages. Overexpression of Crkl was associated with increased incidence of tumor formation, and Rap1 was activated in a metastatic mammary carcinoma. The coexpression of Crkl and Bcr/Abl in mice transgenic for P190 BCR/ABL and CRKL markedly increased the rapidity of development of leukemia/lymphoma, decreasing the average survival by 3.8 months. These results provide direct evidence that Crkl plays a role in tumor development and is important in the leukemogenesis caused by Bcr/Abl.

Bcr/Abl associated leukemogenesis in bcr null mutant mice.

The BCR gene contributes to Philadelphia-positive leukemogenesis via a number of discrete mechanisms, one of which may be through interaction of its normal gene product with the Bcr/Abl oncoprotein. In the current study this hypothesis was tested in vivo by introducing a Bcr/Abl P190 transgene into mice lacking endogenous bcr protein. Our finding, that the P190 BCR/ABL oncogene is still capable of producing leukemia in these mice with indistinguishable latency and clinical pattern as in genetically matched counterparts, rules out any significant or major contribution of the bcr protein as a whole to leukemia development in these mice.

Glycoasparaginase in human urine.

Glycoasparaginase was purified 15,000-fold from human urine. The enzyme is a tetrameric protein of 86 kDa, composed of two heavy chains (25 kDa) and two light chains (18 kDa). Its structure and properties are very similar to those of human leukocyte glycoasparaginase. Glycoasparaginase activity is totally absent from urine of aspartylglycosaminuria patients.

Hemoglobin binding to deglycosylated haptoglobin.

The carbohydrate portion of polymeric haptoglobin was gradually removed by exoglycosidases in order to investigate its role in complex formation between haptoglobin and hemoglobin. Total removal of sialic acid diminished the haptoglobin-hemoglobin complex formation 15%. Removal of about 25% of the galactose residues from asialohaptoglobin, i.e., about 40% of the total weight of the carbohydrate moiety, totally inhibited the ability of haptoglobin to form complex with hemoglobin and react with haptoglobin-specific antibodies. Liberation of further galactose residues resulted in slow precipitation of the protein. Removal of a similar part of the carbohydrate moiety from haptoglobin-hemoglobin complex did not liberate hemoglobin from it, and the complex reacted with haptoglobin antibodies. The combined data indicate that the carbohydrate portion is essential for the functionally active form of polymeric haptoglobin to complex with hemoglobin, but it hardly has any direct role in the binding event, and other factors are responsible for the stability of the complex.

Ph-positive leukemia: a transgenic mouse model.

The presence of the BCR/ABL chimeric gene is the hallmark of defined types of human leukemia. To increase our knowledge of the oncogenic processes and to develop a model for this type of leukemia we generated a BCR/ABL (P190) transgenic mouse line. Over 95% of mice of this line die of leukemia or leukemia/lymphoma within 35-200 days of age. Karyotypically visible genetic alterations were absent from the early stages of BCR/ABL generated leukemia. A high frequency of aneuploidy was found in advanced leukemia indicating a primary and pivotal role for BCR/ABL in leukemogenesis. Moreover, the data suggest that BCR/ABL has a destabilizing effect on the regulation of the cell cycle. BCR/ABL expression was also found in tissues other than hematopoietic cells. However, this did not result in the development of solid tumors, strongly suggesting that the oncogenicity of BCR/ABL is limited to the hematopoietic lineage.

Laboratory detection of aspartylglycosaminuria.

New specific methods for laboratory detection of a lysosomal storage disease aspartylglycosaminuria were developed. Aspartylglucosamine, the main metabolite accumulating in the body fluids and tissues in the disease, was measured with high-performance liquid chromatography in urine of aspartylglycosaminuria patients, carriers of the disease and normal controls as well as in amniotic fluid of normal pregnancies and one with the fetus affected by aspartylglycosaminuria. In the diseased patients, the aspartylglucosamine excretion was over 1,000-fold elevated compared to that in the carriers and controls. In the pregnancy with the fetus affected by the disease, the concentration of aspartylglucosamine was only slightly elevated, but lower than that in urine of normal individuals. As a conclusion, the determination of aspartylglucosamine in urine allowed postnatal detection of aspartylglycosaminuria, but in amniotic fluid it was useless in prenatal detection of the disease. The activity of the deficient enzyme in aspartylglycosaminuria, aspartylglycosylaminase, was assayed with a specific gas-chromatographic method. The enzyme activity was shown to lack in plasma, lymphocytes and amniotic fluid of aspartylglycosaminuria patients and the method proved applicable for postnatal and prenatal detection of aspartylglycosaminuria. The enzyme activity in lymphocytes of the carriers fell between those in normal controls and aspartylglycosaminuria patients, and the assay could be used in carrier detection in most of the cases.

Functional redundancy of TGF-beta family type I receptors and receptor-Smads in mediating anti-Mullerian hormone-induced Mullerian duct regression in the mouse.

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.

Analysis of aspartylglucosamine at the picomole level by high-performance liquid chromatography.

A sensitive method for quantitative analysis of aspartylglucosamine as its dabsyl chloride derivative by high-performance liquid chromatography is described. Precolumn-derivatized aspartylglucosamine and internal standard (carboxymethylcysteine) are separated on a reversed-phase column with a mobile phase consisting of phosphate buffer and acetonitrile and monitored by UV-VIS detection at 436 nm. Aspartylglucosamine acts in the assay like a polar amino acid, and it can be separated from interfering substances in urine with a retention time of ca. 13 min. Its detection limit is ca. 0.3 microM in water and 0.5-1.0 microM in urine and other biological samples, which permits its quantitation in normal urine, for example. The within-day coefficient of variation is less than 4.7% and the day-to-day coefficient of variation is less than 8.3%. The present method is applicable to the direct analysis of aspartylglucosamine in body fluids and tissues without any prepurification and, in combination with automated liquid chromatography, allows rapid assay of a large number of samples in the detection of aspartylglycosaminuria. The sensitivity of the assay also allows direct quantitation of aspartylglucosamine in normal urine and leukocytes of aspartylglycosaminuria patients, and may thus be used in metabolic studies of the compound.

Removal of the floxed neo gene from a conditional knockout allele by the adenoviral Cre recombinase in vivo.

Conditional and tissue specific gene targeting using the Cre-loxP recombination system in combination with established ES cell techniques has become a standard for in vivo loss of function studies. In a typical flox and delete gene targeting strategy, the loxP-neo-loxP cassette is inserted into an intron and an additional loxP site is located in one of the homology arms so that loxP sites surround a functionally essential part of the gene. The neo cassette in usually removed by transient expression of the Cre recombinase in ES cells to avoid selection gene interference and genetic ambiquity. However, this causes a significant increase in manipulation of ES cells and often compromises ES cell pluripotency. Here we describe a method in which the floxed neo gene is removed from a knockout allele by infecting 16-cell-stage morulae by the recombinant Cre adenovirus. This virus provides only transient Cre expression and does not integrate into the mouse genome. Produced mosaic mice transmitted the desired allele without the neo cassette with high frequency to their offspring. This method is rapid and easy and does not require any special equipment. Moreover, because superovulated mice can be used as donors, this method does not necessitate a large number of mice.

Assay of aspartylglycosylaminase by high-performance liquid chromatography.

An aspartylglycosylaminase assay based on high-performance liquid chromatographic analysis of the substrate aspartylglucosamine and product aspartate is described. Aspartylglucosamine and aspartate are derivatized with phenylisothiocyanate and resolved by reverse-phase chromatography. The detection limit for the compounds is 2 pmol. The method can be used for analysis of aspartylglycosylaminase activity in crude cell extracts and tissue samples.

Amniotic fluid glycoasparagines in fetal aspartylglycosaminuria.

Midterm amniotic fluid samples from one pregnancy with the fetus affected by aspartylglycosaminuria and from 11 normal pregnancies were analysed for glycoasparagines accumulating in urine in aspartylglycosaminuria. The aspartylglucosamine concentration in the affected pregnancy was about five times higher than in the controls, but the absolute value was very low being only about one-thousandth of that in urine in aspartylglycosaminuria and one-tenth of that in urine samples from normal adults. In total monosaccharide analysis, only galactose content in the affected amniotic fluid was slightly elevated compared to controls, indicating that higher glycoasparagines typical of urine in aspartylglycosaminuria were not accumulated in significant amounts. The data demonstrate that the analysis of glycoasparagines in amniotic fluid is not likely to permit reliable prenatal diagnosis of aspartylglycosaminuria.

A fluorometric assay for glycosylasparaginase activity and detection of aspartylglycosaminuria.

Recent experimental work on the mechanism of action of glycosylasparaginase suggests that the enzyme specifically reacts toward the L-asparagine or L-aspartic acid moiety of its substrates. Based on this, a new sensitive assay for glycosylasparaginase activity has been developed using L-aspartic acid beta-(7-amido-4-methylcoumarin) as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. At pH 7.5, Km = 93 microM, and as little as 1 ng of glycosylasparaginase could be detected with the assay. Hydrolysis of the substrate was inhibited by diazo-oxonorvaline, a specific inhibitor of glycosylasparaginase. In biological samples, the fluorometric assay is 40-100 times more sensitive than other published methods for glycosylasparaginase. This new assay enables a rapid enzymatic diagnosis of aspartylglycosaminuria--a genetic deficiency of glycosylasparaginase activity--with leukocyte and fibroblast samples.

Differential expression of TGF-beta isoforms during postlactational mammary gland involution.

After cessation of lactation, the mammary gland undergoes involution, which is characterized by a massive epithelial cell death and proteolytic degradation of the extracellular matrix. Whereas the expression patterns and also the function of TGF-beta isoforms during mammary gland branching morphogenesis and lactation are well understood, their expression during postlactational involution and therefore a possible role in this process is poorly known. In this study we show that TGF-beta3 expression is dramatically induced (>fivefold) during mouse mammary gland involution when compared to that of virgin mouse, reaching a maximal expression level at day 4 after weaning. In contrast, other TGF-beta isoforms do not display significant increase in expression during involution (TGF-beta1, 1.3-fold and TGF-beta2, <1.5-fold) when compared to that of virgin or lactating mice. During mammary gland involution, TGF-beta3 is expressed in the epithelial layer and particularly in myoepithelial cells. A comparison of the kinetics of TGF-beta3 expression to that of programmed cell death and degradation of the basement membrane suggests that TGF-beta3 functions in the remodeling events of the extracellular matrix during the second stage of involution.