Genetic analysis of the neuronal and ubiquitous AP-3 adaptor complexes reveals divergent functions in brain.

Neurons express adaptor (AP)-3 complexes assembled with either ubiquitous (beta3A) or neuronal-specific (beta3B) beta3 isoforms. However, it is unknown whether these complexes indeed perform distinct functions in neuronal tissue. Here, we explore this hypothesis by using genetically engineered mouse models lacking either beta3A- or beta3B-containing AP-3 complexes. Somatic and neurological phenotypes were specifically associated with the ubiquitous and neuronal adaptor deficiencies, respectively. At the cellular level, AP-3 isoforms were localized to distinct neuronal domains. beta3B-containing AP-3 complexes were preferentially targeted to neuronal processes. Consistently, beta3B deficiency compromised synaptic zinc stores assessed by Timm's staining and the synaptic vesicle targeting of membrane proteins involved in zinc uptake (ZnT3 and ClC-3). Surprisingly, despite the lack of neurological symptoms, beta3A-deficient mouse brain possessed significantly increased synaptic zinc stores and synaptic vesicle content of ZnT3 and ClC-3. These observations indicate that the functions of beta3A- and beta3B-containing complexes are distinct and divergent. Our results suggest that concerted nonredundant functions of neuronal and ubiquitous AP-3 provide a mechanism to control the levels of selected membrane proteins in synaptic vesicles.

Nrl is required for rod photoreceptor development.

The protein neural retina leucine zipper (Nrl) is a basic motif-leucine zipper transcription factor that is preferentially expressed in rod photoreceptors. It acts synergistically with Crx to regulate rhodopsin transcription. Missense mutations in human NRL have been associated with autosomal dominant retinitis pigmentosa. Here we report that deletion of Nrl in mice results in the complete loss of rod function and super-normal cone function, mediated by S cones. The photoreceptors in the Nrl-/- retina have cone-like nuclear morphology and short, sparse outer segments with abnormal disks. Analysis of retinal gene expression confirms the apparent functional transformation of rods into S cones in the Nrl-/- retina. On the basis of these findings, we postulate that Nrl acts as a 'molecular switch' during rod-cell development by directly modulating rod-specific genes while simultaneously inhibiting the S-cone pathway through the activation of Nr2e3.

Deficiency of reproductive tract alpha(1,2)fucosylated glycans and normal fertility in mice with targeted deletions of the FUT1 or FUT2 alpha(1,2)fucosyltransferase locus.

The fucose alpha(1-->2) galactose beta structure is expressed by uterine epithelial cells in the mouse and has been implicated in blastocyst adhesion events thought to be required for murine implantation. Fucalpha(1-->2)Galbeta moieties and cognate fucosyltransferases are also expressed by epithelial cells of the male reproductive tract and have been implicated in sperm maturation events that may contribute to fertilization. To determine directly if Fucalpha(1-->2)Galbeta moieties are required for fertility, we have generated strains of mice that are deficient in genes encoding FUT1 and FUT2, a pair of GDP-L-fucose:beta(1-->4)-D-galactosyl-R 2-alpha-L-fucosyltransferase enzymes (EC 2.4.1.69) responsible for Fucalpha(1-->2)Galbeta synthesis and expression. FUT1 null mice and FUT2 null mice develop normally and exhibit no gross phenotypic abnormalities. The Fucalpha(1-->2)Galbeta epitope is absent from the uterine epithelia of FUT2 null mice and from the epithelia of the epididymis of FUT1 null mice. Fully normal fertility is observed in FUT1 null intercrosses and in FUT2 null intercrosses. These observations indicate that Fucalpha(1-->2)Galbeta moieties are not essential to blastocyst-uterine epithelial cell interactions required for implantation and are not required for sperm maturation events that permit fertilization and that neither the FUT loci nor their cognate fucosylated glycans are essential to normal development.

Altered podocyte structure in GLEPP1 (Ptpro)-deficient mice associated with hypertension and low glomerular filtration rate.

Glomerular epithelial protein 1 (GLEPP1) is a receptor tyrosine phosphatase present on the apical cell surface of the glomerular podocyte. The GLEPP1 gene (PTPRO:) was disrupted at an exon coding for the NH(2)-terminal region by gene targeting in embryonic stem cells. Heterozygote mating produced the expected genotypic ratio of 1:2:1, indicating that the Ptpro(-/-) genotype does not lead to embryonic or neonatal lethality. Kidney and glomerular structure was normal at the gross and light microscopic levels. Scanning and transmission electron microscopy showed that Ptpro(-/-) mice had an amoeboid rather than the typical octopoid structure seen in the wild-type mouse podocyte and that there were blunting and widening of the minor (foot) processes in association with altered distribution of the podocyte intermediate cytoskeletal protein vimentin. Reduced filtration surface area in association with these structural changes was confirmed by finding reduced glomerular nephrin content and reduced glomerular filtration rate in Ptpro(-/-) mice. There was no detectable increase in the urine albumin excretion of Ptpro(-/-) mice. After removal of one or more kidneys, Ptpro(-/-) mice had higher blood pressure than did their wild-type littermates. These data support the conclusion that the GLEPP1 (Ptpro) receptor plays a role in regulating the glomerular pressure/filtration rate relationship through an effect on podocyte structure and function.

Pancreatic function in CCK-deficient mice: adaptation to dietary protein does not require CCK.

A CCK-deficient mouse mutant generated by gene targeting in embryonic stem cells was analyzed to determine the importance of CCK for growth and function of the exocrine pancreas and for pancreatic adaptation to dietary changes. RIAs confirmed the absence of CCK in mutant mice and demonstrated that tissue concentrations of the related peptide gastrin were normal. CCK-deficient mice are viable and fertile and exhibit normal body weight. Pancreas weight and cellular morphology appeared normal, although pancreatic amylase content was elevated in CCK-deficient mice. We found that a high-protein diet increased pancreatic weight, protein, DNA, and chymotrypsinogen content similarly in CCK-deficient and wild-type mice. This result demonstrates that CCK is not required for protein-induced pancreatic hypertrophy and increased proteolytic enzyme content. This is a novel finding, since CCK has been considered the primary mediator of dietary protein-induced changes in the pancreas. Altered somatostatin concentrations in brain and duodenum of CCK-deficient mice suggest that other regulatory pathways are modified to compensate for the CCK deficiency.

Role of Ahch in gonadal development and gametogenesis.

Ahch (also known as Dax1) encodes a transcription factor that has been implicated in sex determination and gonadal differentiation. Mutations in human AHC cause X-linked, adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Duplication of the Xp21 dosage-sensitive sex reversal (DSS) region, which contains the Ahch locus, and transgenic overexpression of Ahch cause male-to-female sex reversal. Using Cre-mediated disruption of Ahch, we have generated a mouse model of AHC-HH that allows the function of Ahch to be examined in both males and females. Although Ahch has been postulated to function as an ovarian determination gene, the loss of Ahch function in females does not affect ovarian development or fertility. Ahch is instead essential for the maintenance of spermatogenesis. Lack of Ahch causes progressive degeneration of the testicular germinal epithelium independent of abnormalities in gonadotropin and testosterone production and results in male sterility. Ahch is thus not an ovarian determining gene, but rather has a critical role in spermatogenesis.

Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene.

The shaker-2 mouse mutation, the homolog of human DFNB3, causes deafness and circling behavior. A bacterial artificial chromosome (BAC) transgene from the shaker-2 critical region corrected the vestibular defects, deafness, and inner ear morphology of shaker-2 mice. An unconventional myosin gene, Myo15, was discovered by DNA sequencing of this BAC. Shaker-2 mice were found to have an amino acid substitution at a highly conserved position within the motor domain of this myosin. Auditory hair cells of shaker-2 mice have very short stereocilia and a long actin-containing protrusion extending from their basal end. This histopathology suggests that Myo15 is necessary for actin organization in the hair cells of the cochlea.

Cell-specific expression of the mouse glycoprotein hormone alpha-subunit gene requires multiple interacting DNA elements in transgenic mice and cultured cells.

The glycoprotein hormone alpha-subunit gene is expressed and differentially regulated in pituitary gonadotropes and thyrotropes. Previous gene expression studies suggested that cell specificity may be regulated by distinct DNA elements. We have identified an enhancer region between -4.6 and -3.7 kb that is critical for high level expression in both gonadotrope and thyrotrope cells of transgenic mice. Fusion of the enhancer to -341/+43 mouse alpha-subunit promoter results in appropriate pituitary cell specificity and transgene expression levels that are similar to levels observed with the intact -4.6 kb/+43 construct. Deletion of sequences between -341 and -297 resuited in a loss of high level expression and cell specificity, exhibited by ectopic transgene activation in GH-, ACTH-, and PRL-producing pituitary cells as well as in other peripheral tissues. Consistent with these results, transient cell transfection studies demonstrated that the enhancer stimulated activity of a -341/+43 alpha-promoter in both alphaTSH and alphaT3 cells, but it did not enhance alpha-promoter activity significantly in CV-1 cells. Removal of sequences between -341 and -297 allowed the enhancer to function in heterologous cells. Loss of high level expression and cell specificity may be due to loss of sequences required for binding of the LIM homeoproteins or the alpha-basal element 1. These data demonstrate that the enhancer requires participation by both proximal and distal sequences for high level expression and suggests that sequences from -341 to -297 are critical for restricting expression to the anterior pituitary.

Impaired gastric acid secretion in gastrin-deficient mice.

To further understand the role of the peptide hormone gastrin in the development and function of the stomach, we have generated gastrin-deficient mice by gene targeting in embryonic stem cells. Mutant mice were viable and fertile, without obvious visible abnormalities. However, gastric function was severely affected by the loss of gastrin. Basal gastric acid secretion was abolished and could not be induced by histamine, carbachol, or gastrin. Histological analysis revealed alterations in the two cell types primarily involved in acid secretion, parietal and enterochromaffin-like (ECL) cells. Parietal cells were reduced in number with an accumulation of immature cells lacking H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase). ECL cells were positioned closer to the base of the gastric glands, with markedly lower expression of histidine decarboxylase. Gastrin administration for 6 days reversed the effects of the gastrin deficiency, leading to an increase in the number of mature, H(+)-K(+)-ATPase-positive parietal cells and a partial restoration of acid secretion. The results show that gastrin is critically important for the function of the acid secretory system.

Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V.

Coagulation factor V is a critical cofactor for the activation of prothrombin to thrombin, the penultimate step in the generation of a fibrin blood clot. Genetic deficiency of factor V results in a congenital bleeding disorder (parahaemophilia), whereas inheritance of a mutation rendering factor V resistant to inactivation is an important risk factor for thrombosis. We report here that approximately half of homozygous embryos deficient in factor V (Fv-/-), which have been generated by gene targeting, die at embryonic day (E) 9-10, possibly as a result of an abnormality in the yolk-sac vasculature. The remaining Fv-/- mice progress normally to term, but die from massive haemorrhage within 2 hours of birth. Considered together with the milder phenotypes generally associated with deficiencies of other clotting factors, our findings demonstrate the primary role of the common coagulation pathway and the absolute requirement for functional factor V for prothrombinase activity. They also provide direct evidence for the existence of other critical haemostatic functions for thrombin in addition to fibrin clot formation, and identify a previously unrecognized role for the coagulation system in early mammalian development.

The alpha(1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis.

alpha(1,3)Fucosylated oligosaccharides represent components of leukocyte counterreceptors for E- and P-selectins and of L-selectin ligands expressed by lymph node high endothelial venules (HEV). The identity of the alpha(1,3)fucosyltransferase(s) required for their expression has been uncertain, as has a requirement for alpha(1,3)fucosylation in HEV L-selectin ligand activity. We demonstrate here that mice deficient in alpha(1,3) fucosyltransferase Fuc-TVII exhibit a leukocyte adhesion deficiency characterized by absent leukocyte E- and P-selectin ligand activity and deficient HEV L-selectin ligand activity. Selectin ligand deficiency is distinguished by blood leukocytosis, impaired leukocyte extravasation in inflammation, and faulty lymphocyte homing. These observations demonstrate an essential role for Fuc-TVII in E-, P-, and L-selectin ligand biosynthesis and imply that this locus can control leukocyte trafficking in health and disease.

Anterior pituitary cells defective in the cell-autonomous factor, df, undergo cell lineage specification but not expansion.

The Ames dwarf mouse transmits a recessive mutation (df) resulting in a profound anterior pituitary hypocellularity due to a general lack of thyrotropes, somatotropes and lactotropes. These cell types are also dependent on the pituitary-specific transcription factor, Pit-1. We present evidence that expression of Pit-1 and limited commitment to these cells lineages occurs in df/df pituitaries. Thus, the crucial role of df may be in lineage-specific proliferation, rather than cytodifferentiation. The presence of all three Pit-1-dependent cell types in clonally derived clusters provides compelling evidence that these three lineages share a common, pluripotent precursor cell. Clusters containing different combinations of Pit-1-dependent cell types suggests that the Pit-1+ precursor cells choose from multiple developmental options during ontogeny. Characterization of df/df<-->+/+ chimeric mice demonstrated that df functions by a cell-autonomous mechanism. Therefore, df and Pit-1 are both cell-autonomous factors required for thyrotrope, somatotrope and lactotrope ontogeny, but their relative roles are different.

Vitronectin is not essential for normal mammalian development and fertility.

Vitronectin (VN) is an abundant glycoprotein present in plasma and the extracellular matrix of most tissues. Though the precise function of VN in vivo is unknown, it has been implicated as a participant in diverse biological processes, including cell attachment and spreading, complement activation, and regulation of hemostasis. The major site of synthesis appears to be the liver, though VN is also found in the brain at an early stage of mouse organogenesis, suggesting that it may play an important role in mouse development. Genetic deficiency of VN has not been reported in humans or in other higher organisms. To examine the biologic function of VN within the context of the intact animal, we have established a murine model for VN deficiency through targeted disruption of the murine VN gene. Southern blot analysis of DNA obtained from homozygous null mice demonstrates deletion of all VN coding sequences, and immunological analysis confirms the complete absence of VN protein expression in plasma. However, heterozygous mice carrying one normal and one null VN allele and homozygous null mice completely deficient in VN demonstrate normal development, fertility, and survival. Sera obtained from VN-deficient mice are completely deficient in "serum spreading factor" and plasminogen activator inhibitor 1 binding activities. These observations demonstrate that VN is not essential for cell adhesion and migration during normal mouse development and suggest that its role in these processes may partially overlap with other adhesive matrix components.

Targeted disruption of the pituitary glycoprotein hormone alpha-subunit produces hypogonadal and hypothyroid mice.

Pituitary thyrotropin (TSH) and gonadotropins (LH and FSH) are thought to be critical for thyroid and gonadal development and function. Each of these pituitary hormones is a heterodimer composed of a common alpha-subunit and unique beta-subunit, and heterodimerization is required for function. No mutations in the alpha-subunit or any of the beta-subunit genes have been reported in mice. To assess directly the functional role of TSH, LH, and FSH in thyroid and gonadal development, we created a disruption of the alpha-subunit gene by homologous recombination. The homozygous mutant animals were hypogonadal and exhibited profound hypothyroidism resulting in dwarfism. Thyroid development was arrested in late gestation, but GnRH neuron migration, development of secondary sex organs, and fetal and neonatal gonadal development were normal. This establishes the importance of thyrotropin in ontogeny and reveals that fetal pituitary gonadotropins are not required for sexual differentiation or genital development in male or female fetuses. The pituitary cells that produce TSH beta-subunit exhibited dramatic hypertrophy and hyperplasia as a result of the lack of thyroid function. This proliferation response occurred at the expense of somatotrope and lactotrope cells, consistent with a derivation of these three cell types from a common precursor.

Implementing transgenic and embryonic stem cell technology to study gene expression, cell-cell interactions and gene function.

This review highlights the use of transgenic mice and gene targeting in the study of reproduction, pituitary gene expression, and cell lineage. Since 1980 numerous applications of transgenic animal technology have been reported. Altered phenotypes resulting from transgene expression demonstrated that introduced genes can exert profound effects on animal physiology. Transgenic mice have been important for the study of hormonal and developmental control of gene expression because gene expression in whole animals often requires more DNA sequence information than is necessary for expression in cell cultures. This point is illustrated by studies of pituitary glycoprotein hormone alpha- and beta-subunit gene expression (Kendall et al., Mol Endocrinol 1994; in press [1]. Transgenic mice have also been invaluable for producing animal models of cancer and other diseases and testing the efficacy of gene therapy. In addition, cell-cell interactions and cell lineage relationships have been explored by cell-specific expression of toxin genes in transgenic mice. Recent studies suggest that attenuated and inducible toxins hold promise for future transgene ablation experiments. Since 1987, embryonic stem (ES) cell technology has been used to create numerous mouse strains with targeted gene alterations, contributing enormously to our understanding of the functional importance of individual genes. For example, the unexpected development of gonadal tumors in mice with a targeted disruption of the inhibin gene revealed a potential role for inhibin as a tumor suppressor (Matzuk et al., Nature 1992:360: 313-319 [2]. The transgenic and ES cell technologies will undoubtedly continue to expand our understanding and challenge our paradigms in reproductive biology.

The proximal promoter of the bovine luteinizing hormone beta-subunit gene confers gonadotrope-specific expression and regulation by gonadotropin-releasing hormone, testosterone, and 17 beta-estradiol in transgenic mice.

Transient transfection studies have proven useful in unraveling the molecular mechanisms underlying gonadotrope-specific expression and hormonal regulation of the gene encoding the alpha-subunit of the glycoprotein hormones. In contrast, similar studies performed with the LH beta gene have been less informative. When assayed by transient transfection into alpha T3-1 cells, activity of a 776-basepair bovine LH beta promoter-chloramphenicol acetyltransferase fusion gene (bLH beta CAT) was no greater than that of a promoterless control. To determine whether limited activity in vitro reflected the absence of critical regulatory elements, we examined activity of bovine LH beta fusion genes after stable integration in transgenic mice. In contrast to transient transfection studies, the LH beta promoter targeted high levels of CAT expression specifically to the pituitary. In addition, a bLH beta TK fusion gene was active only in gonadotropes. The bLH beta CAT transgene was also evaluated for responsiveness to gonadal steroids and GnRH. Testosterone and 17 beta-estradiol were capable of suppressing activity 70-80% in castrated males, despite the absence of high affinity binding sites for androgen or estrogen receptors. This suggests that feedback inhibition of LH beta CAT transgene expression by gonadal steroids may occur through an indirect mechanism, possibly at the level of the hypothalamus. To address whether the bLH beta CAT transgene could be regulated by GnRH, we treated ovariectomized females with antide, a GnRH antagonist. Antide suppressed transgene activity by 60%. Thus, the proximal promoter of the bovine LH beta subunit gene directs appropriate patterns of cell-specific expression and retains responsiveness to gonadal steroids and GnRH. In light of the robust activity of the LH beta promoter in transgenic mice, we suggest that this animal model can be exploited further to dissect the complex mechanisms that underlie gonadotrope-specific expression and hormonal regulation of the LH beta gene.

Targeted ablation of pituitary gonadotropes in transgenic mice.

LH, FSH, and TSH are heterodimeric glycoprotein hormones composed of a common alpha-subunit and unique beta-subunits. The alpha-subunit is produced in two distinct specialized cell types of the pituitary gland: gonadotropes, which synthesize LH and FSH, and thyrotropes, which synthesize TSH. We have demonstrated that 313 base pairs of the bovine-alpha subunit promoter direct expression of diphtheria toxin A chain specifically to the gonadotropes in transgenic mice. Animals carrying this transgene generally exhibit reproductive failure and lack of gonadal differentiation, consistent with gonadotrope ablation. Lack of gonadotrope activity was verified by RIA and immunohistochemical staining for LH. The phenotype of these transgenic mice is nearly identical to mice homozygous for the spontaneous mutation, hpg, which is due to a deletion in the gene encoding GnRH. Thyrotrope function was judged normal based on overall growth of the animals, appearance of their thyroids, T4 levels measured by RIA, and immunohistochemical staining for TSH. The ablation of gonadotropes but not thyrotropes suggests that separate cis-acting elements are necessary for expression of the alpha-subunit gene in these two cell types. Pituitary content of ACTH and GH was apparently normal, while PRL synthesis and storage were reduced. Thus, in a pituitary almost completely devoid of gonadotropes, most other pituitary functions were normal. This suggests that most pituitary cells are able to differentiate independently of terminal gonadotrope differentiation and can function in the absence of paracrine signaling provided by gonadotropes.

The Pit-1 transcription factor gene is a candidate for the murine Snell dwarf mutation.

Two nonallelic mouse mutations with severe dwarf phenotypes are characterized by a lack of growth hormone, prolactin, and thyroid stimulating hormone. The cells that normally synthesize these pituitary hormones express a common transcription factor called GHF-1 or Pit-1. Using an intersubspecific backcross, we have demonstrated tight linkage of the Pit-1 and Snell dwarf (dw) genes on mouse chromosome 16. No recombination was observed between Pit-1 and dw in 110 individuals examined. Southern blot analysis of genomic DNA reveals that the Pit-1 gene is rearranged in C3H/HeJ-dwJ/dw mice but not in coisogenic +/+ animals, providing molecular evidence that a lesion in the Pit-1 gene results in the Snell dwarf phenotype. Demonstration of low levels of Pit-1 expression in Ames dwarf (df) mice implies that both Pit-1 and df expression may be required for pituitary differentiation.

Transgenic mouse model of the mild dominant form of osteogenesis imperfecta.

Osteogenesis imperfecta type I is a mild, dominantly inherited, connective tissue disorder characterized by bone fragility. Mutations in type I collagen account for all known cases. In Mov-13 mice, integration of a murine retrovirus within the first intron of the alpha 1(I) collagen gene results in a null allele blocked at the level of transcription. This study demonstrates that mutant mice heterozygous for the null allele are a model of osteogenesis imperfecta type I. A defect in type I collagen production is associated with dominant-acting morphological and functional defects in mineralized and nonmineralized connective tissue and with progressive hearing loss. The model provides an opportunity to investigate the effect of a reduced amount of type I collagen on the structure and integrity of extracellular matrix. It also may represent a system in which therapeutic strategies to strengthen connective tissue can be developed.

Full-length DQ beta cDNA sequences of HLA-DR2/DQw1 subtypes: genetic interactions between two DQ beta loci generate human class II HLA diversity.

Two-dimensional gel electrophoresis of DQ molecules from three different Dw subtypes (Dw2, Dw12, and Dw21/FJO) of the HLA-DR2/DQw1 haplotype reveals that one alpha beta heterodimer of DQ molecule is expressed by each subtype and the DQ beta chain is electrophoretically variable among the three DR2/DQw1 subtypes. We have constructed cDNA libraries from the same homozygous typing cells used in the two-dimensional polyacrylamide gel electrophoresis analyses (HTC VYT for Dw2, HTC DHO for Dw12, and HTC FJO for Dw21/FJO) and isolated DQ beta cDNA clones with full-length coding sequences for each subtype. The deduced amino acid sequences show that the DQ beta chains of these three DR2/DQw1 subtypes are highly polymorphic and confirm their electrophoretic heterogeneity: for a mature protein of 229 amino acids, they differ with each other by 10-17 amino acids in the first domain and by 3-7 residues in the C-terminal sequence. Comparison among the available DQ beta sequences representing the four major DQ specificities (DQw1, DQw2, DQw3, and DQw4) in the DQ subregion as defined by serologic method suggests that (1) DR2,Dw2,DQw1 and DR3,DQw2 haplotypes probably interact with each other to generate the DQw3 and DQw4 beta alleles and (2) an evolutionary scheme may be proposed to relate the various beta alleles of the four major DQ specificities.