Cyclin B2-null mice develop normally and are fertile whereas cyclin B1-null mice die in utero

Two B-type cyclins, B1 and B2, have been identified in mammals. Proliferating cells express both cyclins, which bind to and activate p34cdc2. To test whether the two B-type cyclins have distinct roles, we generated lines of transgenic mice, one lacking cyclin B1 and the other lacking cyclin B2. Cyclin B1 proved to be an essential gene; no homozygous B1-null pups were born. In contrast, nullizygous B2 mice developed normally and did not display any obvious abnormalities. Both male and female cyclin B2-null mice were fertile, which was unexpected in view of the high levels and distinct patterns of expression of cyclin B2 during spermatogenesis. We show that the expression of cyclin B1 overlaps the expression of cyclin B2 in the mature testis, but not vice versa. Cyclin B1 can be found both on intracellular membranes and free in the cytoplasm, in contrast to cyclin B2, which is membrane-associated. These observations suggest that cyclin B1 may compensate for the loss of cyclin B2 in the mutant mice, and implies that cyclin B1 is capable of targeting the p34cdc2 kinase to the essential substrates of cyclin B2.

Galanin regulates prolactin release and lactotroph proliferation

The neuropeptide galanin is predominantly expressed by the lactotrophs (the prolactin secreting cell type) in the rodent anterior pituitary and in the median eminence and paraventricular nucleus of the hypothalamus. Prolactin and galanin colocalize in the same secretory granule, the expression of both proteins is extremely sensitive to the estrogen status of the animal. The administration of estradiol-17β induces pituitary hyperplasia followed by adenoma formation and causes a 3,000-fold increase in the galanin mRNA content of the lactotroph. To further study the role of galanin in prolactin release and lactotroph growth we now report the generation of mice carrying a loss-of-function mutation of the endogenous galanin gene. There is no evidence of embryonic lethality and the mutant mice grow normally. The specific endocrine abnormalities identified to date, relate to the expression of prolactin. Pituitary prolactin message levels and protein content of adult female mutant mice are reduced by 30–40% compared with wild-type controls. Mutant females fail to lactate and pups die of starvation/dehydration unless fostered onto wild-type mothers. Prolactin secretion in mutant females is markedly reduced at 7 days postpartum compared with wild-type controls with an associated failure in mammary gland maturation. There is an almost complete abrogation of the proliferative response of the lactotroph to high doses of estrogen, with a failure to up-regulate prolactin release, STAT5 expression or to increase pituitary cell number. These data further support the hypothesis that galanin acts as a paracrine regulator of prolactin expression and as a growth factor to the lactotroph.

Phenotypic alterations in insulin-deficient mutant mice

Two mouse insulin genes, Ins1 and Ins2, were disrupted and lacZ was inserted at the Ins2 locus by gene targeting. Double nullizygous insulin-deficient pups were growth-retarded. They did not show any glycosuria at birth but soon after suckling developed diabetes mellitus with ketoacidosis and liver steatosis and died within 48 h. Interestingly, insulin deficiency did not preclude pancreas organogenesis and the appearance of the various cell types of the endocrine pancreas. The presence of lacZ expressing β cells and glucagon-positive α cells was demonstrated by cytochemistry and immunocytochemistry. Reverse transcription-coupled PCR analysis showed that somatostatin and pancreatic polypeptide mRNAs were present, although at reduced levels, accounting for the presence also of δ and pancreatic polypeptide cells, respectively. Morphometric analysis revealed enlarged islets of Langherans in the pancreas from insulin-deficient pups, suggesting that insulin might function as a negative regulator of islet cell growth. Whether insulin controls the growth of specific islet cell types and the molecular basis for this action remain to be elucidated.

Mice cloned from embryonic stem cells

Cloning allows the asexual reproduction of selected individuals such that the offspring have an essentially identical nuclear genome. Cloning by nuclear transfer thus far has been reported only with freshly isolated cells and cells from primary cultures. We previously reported a method of cloning mice from adult somatic cells after nuclear transfer by microinjection. Here, we apply this method to clone mice from widely available, established embryonic stem (ES) cell lines at late passage. With the ES cell line R1, 29% of reconstructed oocytes developed in vitro to the morula/blastocyst stage, and 8% of these embryos developed to live-born pups when transferred to surrogate mothers. We thus cloned 26 mice from R1 cells. Nuclei from the ES cell line E14 also were shown to direct development to term. We present evidence that the nuclei of ES cells at G1- or G2/M-phases are efficiently able to support full development. Our findings demonstrate that late-passage ES cells can be used to produce viable cloned mice and provide a link between the technologies of ES cells and animal cloning. It thus may be possible to clone from a single cell a large number of individuals over an extended period.

Uncompensated polyuria in a mouse model of Bartter's syndrome

We have used homologous recombination to disrupt the mouse gene coding for the NaK2Cl cotransporter (NKCC2) expressed in kidney epithelial cells of the thick ascending limb and macula densa. This gene is one of several that when mutated causes Bartter's syndrome in humans, a syndrome characterized by severe polyuria and electrolyte imbalance. Homozygous NKCC2−/− pups were born in expected numbers and appeared normal. However, by day 1 they showed signs of extracellular volume depletion (hematocrit 51%; wild type 37%). They subsequently failed to thrive. By day 7, they were small and markedly dehydrated and exhibited renal insufficiency, high plasma potassium, metabolic acidosis, hydronephrosis of varying severity, and high plasma renin concentrations. None survived to weaning. Treatment of −/− pups with indomethacin from day 1 prevented growth retardation and 10% treated for 3 weeks survived, although as adults they exhibited severe polyuria (10 ml/day), extreme hydronephrosis, low plasma potassium, high blood pH, hypercalciuria, and proteinuria. Wild-type mice treated with furosemide, an inhibitor of NaK2Cl cotransporters, have a phenotype similar to the indomethacin-rescued −/− adults except that hydronephrosis was mild. The polyuria, hypercalciuria, and proteinuria of the −/− adults and furosemide-treated wild-type mice were unresponsive to inhibitors of the renin angiotensin system, vasopressin, and further indomethacin. Thus absence of NKCC2 in the mouse causes polyuria that is not compensated elsewhere in the nephron. The NKCC2 mutant animals should be valuable for uncovering new pathophysiologic and therapeutic aspects of genetic disturbances in water and electrolyte recovery by the kidney.

Neonatal lead exposure impairs development of rodent barrel field cortex

Childhood exposure to low-level lead can permanently reduce intelligence, but the neurobiologic mechanism for this effect is unknown. We examined the impact of lead exposure on the development of cortical columns, using the rodent barrel field as a model. In all areas of mammalian neocortex, cortical columns constitute a fundamental structural unit subserving information processing. Barrel field cortex contains columnar processing units with distinct clusters of layer IV neurons that receive sensory input from individual whiskers. In this study, rat pups were exposed to 0, 0.2, 1, 1.5, or 2 g/liter lead acetate in their dam's drinking water from birth through postnatal day 10. This treatment, which coincides with the development of segregated columns in the barrel field, produced blood lead concentrations from 1 to 31 μg/dl. On postnatal day 10, the area of the barrel field and of individual barrels was measured. A dose-related reduction in barrel field area was observed (Pearson correlation = −0.740; P < 0.001); mean barrel field area in the highest exposure group was decreased 12% versus controls. Individual barrels in the physiologically more active caudoventral group were affected preferentially. Total cortical area measured in the same sections was not altered significantly by lead exposure. These data support the hypothesis that lead exposure may impair the development of columnar processing units in immature neocortex. We demonstrate that low levels of blood lead, in the range seen in many impoverished inner-city children, cause structural alterations in a neocortical somatosensory map.

Parent–progeny recognition as a function of MHC odortype identity

The several linked polymorphic genes of the MHC, which has been proposed as a prime determinant of sensed genetic individuality within species, is known to operate in mice by olfactory recognition in aspects of reproductive behavior that concern mate selection, thereby favoring outbreeding and heterozygosity, and also concern the maintenance of pregnancy. A single base-change can alter an individual MHC odortype, and the potential range of combinatorial MHC-determined odortypes is clearly vast. Following our findings that newborn mice already express their MHC odortype (which is detectable at 9 days of gestational age), we sought to determine whether MHC is involved in behavioral aspects of early development, such as rearing. In the studies presented herein, we report the ability and proclivity of mothers to recognize and preferentially retrieve syngeneic (genetically identical) pups from other pups differing only for MHC. Reciprocally, we report the ability of pups to recognize their familial environment, regardless of whether they had been nursed by their biological mothers or by foster mothers. Early learning experiences of the MHC environment are apparently a key element in survival, assuring maternal protection and promoting outbreeding.

Experimental infection model for Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus)

The relationship between hantaviruses and their reservoir hosts is not well understood. We successfully passaged a mouse-adapted strain of Sin Nombre virus from deer mice (Peromyscus maniculatus) by i.m. inoculation of 4- to 6-wk-old deer mouse pups. After inoculation with 5 ID50, antibodies to the nucleocapsid (N) antigen first became detectable at 14 d whereas neutralizing antibodies were detectable by 7 d. Viral N antigen first began to appear in heart, lung, liver, spleen, and/or kidney by 7 d, whereas viral RNA was present in those tissues as well as in thymus, salivary gland, intestine, white fat, and brown fat. By 14 d nearly all tissues examined displayed both viral RNA and N antigen. We noted no consistent histopathologic changes associated with infection, even when RNA load was high. Viral RNA titers peaked on 21 d in most tissues, then began to decline by 28 d. Infection persisted for at least 90 d. The RNA titers were highest in heart, lung, and brown fat. Deer mice can be experimentally infected with Sin Nombre virus, which now allows provocative examination of the virus-host relationship. The prominent involvement of heart, lung, and brown fat suggests that these sites may be important tissues for early virus replication or for maintenance of the virus in nature.

Requirement for the lpA1 lysophosphatidic acid receptor gene in normal suckling behavior

Although extracellular application of lysophosphatidic acid (LPA) has been extensively documented to produce a variety of cellular responses through a family of specific G protein-coupled receptors, the in vivo organismal role of LPA signaling remains largely unknown. The first identified LPA receptor gene, lpA1/vzg-1/edg-2, was previously shown to have remarkably enriched embryonic expression in the cerebral cortex and dorsal olfactory bulb and postnatal expression in myelinating glia including Schwann cells. Here, we show that targeted deletion of lpA1 results in approximately 50% neonatal lethality, impaired suckling in neonatal pups, and loss of LPA responsivity in embryonic cerebral cortical neuroblasts with survivors showing reduced size, craniofacial dysmorphism, and increased apoptosis in sciatic nerve Schwann cells. The suckling defect was responsible for the death among lpA1(−/−) neonates and the stunted growth of survivors. Impaired suckling behavior was attributable to defective olfaction, which is likely related to developmental abnormalities in olfactory bulb and/or cerebral cortex. Our results provide evidence that endogenous lysophospholipid signaling requires an lp receptor gene and indicate that LPA signaling through the LPA1 receptor is required for normal development of an inborn, neonatal behavior.

Nerve growth factor control of neuronal expression of angiogenetic and vasoactive factors

In postnatal tissues, angiogenesis occurs in nontumoral conditions on appropriate stimuli. In the nervous tissue, hypoxia, neural graft, increased neural function, and synaptic activity are associated with neoangiogenesis. We have investigated the occurrence of neoangiogenesis in the superior cervical ganglia (scg) of newborn rats treated for 8–21 days with 6-hydroxy-dopamine (6-OHDA), nerve growth factor (NGF), or 6-OHDA + NGF. The two latter treatments induced a significant increase in scg size. However, the increase after combined treatment far exceeded that of NGF alone. Similarly, histological and histochemical analysis revealed neuronal hypertrophy and endothelial cell hyperplasia associated with stromal hypertrophy (as described by laminin immunostaining) and increased vascular bed (as revealed by platelet/endothelial cell adhesion molecule-1 immunostaining) in 6-OHDA + NGF-treated pups. NGF, either alone or associated with 6-OHDA, also induced a significant up-regulation of NADPH diaphorase, neuronal nitric oxide synthase, and vascular endothelial growth factor expression in scg neurons. The present investigation suggests that the increase of scg size induced by NGF and 6-OHDA + NGF is associated with neoangiogenesis, and that the induction of vasoactive and angiogenic factors in neurons represents a further and previously undisclosed effect of NGF.

Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility

Little is known about stem cell biology or the specialized environments or niches believed to control stem cell renewal and differentiation in self-renewing tissues of the body. Functional assays for stem cells are available only for hematopoiesis and spermatogenesis, and the microenvironment, or niche, for hematopoiesis is relatively inaccessible, making it difficult to analyze donor stem cell colonization events in recipients. In contrast, the recently developed spermatogonial stem cell assay system allows quantitation of individual colonization events, facilitating studies of stem cells and their associated microenvironment. By using this assay system, we found a 39-fold increase in male germ-line stem cells during development from birth to adult in the mouse. However, colony size or area of spermatogenesis generated by neonate and adult stem cells, 2–3 months after transplantation into adult tubules, was similar (∼0.5 mm2). In contrast, the microenvironment in the immature pup testis was 9.4 times better than adult testis in allowing colonization events, and the area colonized per donor stem cell, whether from adult or pup, was about 4.0 times larger in recipient pups than adults. These factors facilitated the restoration of fertility by donor stem cells transplanted to infertile pups. Thus, our results demonstrate that stem cells and their niches undergo dramatic changes in the postnatal testis, and the microenvironment of the pup testis provides a more hospitable environment for transplantation of male germ-line stem cells.

Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F1 genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F1 ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F1 ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F1 ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F1 ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F1 ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.

The metallochaperone Atox1 plays a critical role in perinatal copper homeostasis

Copper plays a fundamental role in the biochemistry of all aerobic organisms. The delivery of this metal to specific intracellular targets is mediated by metallochaperones. To elucidate the role of the metallochaperone Atox1, we analyzed mice with a disruption of the Atox1 locus. Atox1−/− mice failed to thrive immediately after birth, with 45% of pups dying before weaning. Surviving animals exhibited growth failure, skin laxity, hypopigmentation, and seizures because of perinatal copper deficiency. Maternal Atox1 deficiency markedly increased the severity of Atox1−/− phenotype, resulting in increased perinatal mortality as well as severe growth retardation and congenital malformations among surviving Atox1−/− progeny. Furthermore, Atox1-deficient cells accumulated high levels of intracellular copper, and metabolic studies indicated that this defect was because of impaired cellular copper efflux. Taken together, these data reveal a direct role for Atox1 in trafficking of intracellular copper to the secretory pathway of mammalian cells and demonstrate that this metallochaperone plays a critical role in perinatal copper homeostasis.

Targeted disruption of the Rad51 gene leads to lethality in embryonic mice.

The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.

Fatal embryonic bleeding events in mice lacking tissue factor, the cell-associated initiator of blood coagulation.

Tissue factor (TF) is the cellular receptor for coagulation factor VI/VIIa and is the membrane-bound glycoprotein that is generally viewed as the primary physiological initiator of blood coagulation. To define in greater detail the physiological role of TF in development and hemostasis, the TF gene was disrupted in mice. Mice heterozygous for the inactivated TF allele expressed approximately half the TF activity of wild-type mice but were phenotypically normal. However, homozygous TF-/- pups were never born in crosses between heterozygous mice. Analysis of mid-gestation embryos showed that TF-/- embryos die in utero between days 8.5 and 10.5. TF-/- embryos were morphologically distinct from their TF+/+ and TF+/- littermates after day 9.5 in that they were pale, edematous, and growth retarded. Histological studies showed that early organogenesis was normal. The initial failure in TF-/- embryos appeared to be hemorrhaging, leading to the leakage of embryonic red cells from both extraembryonic and embryonic vessels. These studies indicate that TF plays an indispensable role in establishing and/or maintaining vascular integrity in the developing embryo at a time when embryonic and extraembryonic vasculatures are fusing and blood circulation begins.