Showing papers in "Genes & Development in 1995"

Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse.

Abstract: Bone morphogenetic protein-4 (BMP-4) is a member of the TGF-beta superfamily of polypeptide signaling molecules, closely related to BMP-2 and to Drosophila decapentaplegic (DPP). To elucidate the role of BMP-4 in mouse development the gene has been inactivated by homologous recombination in ES cells. Homozygous mutant Bmp-4tm1blh embryos die between 6.5 and 9.5 days p.c., with a variable phenotype. Most Bmp-4tm1blh embryos do not proceed beyond the egg cylinder stage, do not express the mesodermal marker T(Brachyury), and show little or no mesodermal differentiation. Some homozygous mutants develop to the head fold or beating heart/early somite stage or beyond. However, they are developmentally retarded and have truncated or disorganized posterior structures and a reduction in extraembryonic mesoderm, including blood islands. These results provide direct genetic evidence that BMP-4 is essential for several different processes in early mouse development, beginning with gastrulation and mesoderm formation. Moreover, in the presumed absence of zygotic ligand, it appears that homozygous mutants can be rescued partially by related proteins or by maternal BMP-4.

Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities.

Abstract: Although progesterone has been recognized as essential for the establishment and maintenance of pregnancy, this steroid hormone has been recently implicated to have a functional role in a number of other reproductive events. The physiological effects of progesterone are mediated by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors. In most cases the PR is induced by estrogen, implying that many of the in vivo effects attributed to progesterone could also be the result of concomitantly administered estrogen. Therefore, to clearly define those physiological events that are specifically attributable to progesterone in vivo, we have generated a mouse model carrying a null mutation of the PR gene using embryonic stem cell/gene targeting techniques. Male and female embryos homozygous for the PR mutation developed normally to adulthood. However, the adult female PR mutant displayed significant defects in all reproductive tissues. These included an inability to ovulate, uterine hyperplasia and inflammation, severely limited mammary gland development, and an inability to exhibit sexual behavior. Collectively, these results provide direct support for progesterone's role as a pleiotropic coordinator of diverse reproductive events that together ensure species survival.

Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway.

Abstract: The transcription factor NF-kappa B is sequestered in the cytoplasm by the inhibitor protein I kappa B alpha. Extracellular inducers of NF-kappa B activate signal transduction pathways that result in the phosphorylation and subsequent degradation of I kappa B alpha. At present, the link between phosphorylation of I kappa B alpha and its degradation is not understood. In this report we provide evidence that phosphorylation of serine residues 32 and 36 of I kappa B alpha targets the protein to the ubiquitin-proteasome pathway. I kappa B alpha is ubiquitinated in vivo and in vitro following phosphorylation, and mutations that abolish phosphorylation and degradation of I kappa B alpha in vivo prevent ubiquitination in vitro. Ubiquitinated I kappa B alpha remains associated with NF-kappa B, and the bound I kappa B alpha is degraded by the 26S proteasome. Thus, ubiquitination provides a mechanistic link between phosphorylation and degradation of I kappa B alpha.

A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye.

Abstract: BMP-7/OP-1, a member of the transforming growth factor-beta (TGF-beta) family of secreted growth factors, is expressed during mouse embryogenesis in a pattern suggesting potential roles in a variety of inductive tissue interactions. The present study demonstrates that mice lacking BMP-7 display severe defects confined to the developing kidney and eye. Surprisingly, the early inductive tissue interactions responsible for establishing both organs appear largely unaffected. However, the absence of BMP-7 disrupts the subsequent cellular interactions required for their continued growth and development. Consequently, homozygous mutant animals exhibit renal dysplasia and anophthalmia at birth. Overall, these findings identify BMP-7 as an essential signaling molecule during mammalian kidney and eye development.

Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5.

Abstract: The murine homeo box gene Nkx2-5 is expressed in precardiac mesoderm and in the myocardium of embryonic and fetal hearts. Targeted interruption of Nkx2-5 resulted in abnormal heart morphogenesis, growth retardation and embryonic lethality at approximately 9-10 days postcoitum (p.c.). Heart tube formation occurred normally in mutant embryos, but looping morphogenesis, a critical determinant of heart form, was not initiated at the linear heart tube stage (8.25-8.5 days p.c.). Commitment to the cardiac muscle lineage, expression of most myofilament genes and myofibrillogenesis were not compromised. However, the myosin light-chain 2V gene (MLC2V) was not expressed in mutant hearts nor in mutant ES cell-derived cardiocytes. MLC2V expression normally occurs only in ventricular cells and is the earliest known molecular marker of ventricular differentiation. The regional expression in mutant hearts of two other ventricular markers, myosin heavy-chain beta and cyclin D2, indicated that not all ventricle-specific gene expression is dependent on Nkx2-5. The data demonstrate that Nkx2-5 is essential for normal heart morphogenesis, myogenesis, and function. Furthermore, this gene is a component of a genetic pathway required for myogenic specialization of the ventricles.

LXR, a nuclear receptor that defines a distinct retinoid response pathway.

Abstract: We have identified a new retinoid response pathway through which 9-cis retinoic acid (9cRA) activates transcription in the presence of LXR alpha, a member of the nuclear receptor superfamily. LXR alpha shows a specific pattern of expression in visceral organs, thereby restricting the response to certain tissues. Retinoid trans-activation occurs selectively on a distinct response element termed an LXRE. Significantly, neither RXR homodimers nor RXR/RAR heterodimers are able to substitute for LXR alpha in mediating this retinoid response. We provide evidence that the retinoid response on the LXRE is the result of a unique interaction between LXR alpha and endogenous RXR, which, unlike in the RXR/RAR heterodimer, makes RXR competent to respond to retinoids. Thus, the interaction with LXR alpha shifts RXR from its role described previously as a silent, DNA-binding partner to an active ligand-binding subunit in mediating retinoid responses through target genes defined by LXREs.

p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage

Abstract: Expression of p21 has been shown to be up-regulated by the p53 tumor suppressor gene in vitro in response to DNA-damaging agents. However, p21 expression can be regulated independently of p53, and here we show that expression of p21 in various tissues during development and in the adult mouse occurs in the absence of p53 function. However, most tissues tested did require p53 for p21 induction following exposure of the whole animal to gamma irradiation. These results show that normal tissue expression of p21 to high levels is not dependent on p53 and confirm that induction of p21 by DNA-damaging agents does require p53. p21 is expressed upon differentiation of p53-deficient murine erythroleukemia (MEL) cells, and the kinetics of induction of p21 in this system suggest that it may be involved in the growth arrest that precedes terminal differentiation. The gene is up-regulated in mouse fibroblasts in response to serum restimulation but the kinetics and levels of induction differ between wild-type and mutant cells. Expression of p21 message following serum restimulation is superinducible by cycloheximide in wild-type but not in p53-deficient cells. The increases in p21 mRNA are reflected in changes in p21 protein levels. p21 expression also appears to be regulated at the post-transcriptional level because moderate increases in mRNA expression, during differentiation of MEL cells and upon serum restimulation of fibroblasts, are followed by large increases in protein levels. Regulation of the mouse p21 promoter by p53 depends on two critical p53-binding sites located 1.95 and 2.85 kb upstream from the transcriptional initiation site. The sequences mediating serum responsiveness of the promoter map to a region containing the proximal p53 site. p53 appears to play a critical role in p21 induction following DNA damage. Moreover, p21 can be regulated independently of p53 in several situations including during normal tissue development, following serum stimulation, and during cellular differentiation.

BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning.

Abstract: Bone morphogenetic proteins (BMPs) are multifunctional growth factors originally identified by their ability to induce ectopic bone formation. To investigate the function of one of the BMPs, BMP-7, we have generated BMP-7-deficient mice using embryonic stem cell technology. BMP-7-deficient mice die shortly after birth because of poor kidney development. Histological analysis of mutant embryos at several stages of development revealed that metanephric mesenchymal cells fail to differentiate, resulting in a virtual absence of glomerulus in newborn kidneys. In situ hybridization analysis showed that the absence of BMP-7 affects the expression of molecular markers of nephrogenesis, such as Pax-2 and Wnt-4 between 12.5 and 14.5 days postcoitum (dpc). This identifies BMP-7 as an inducer of nephrogenesis. In addition, BMP-7-deficient mice have eye defects that appear to originate during lens induction. Finally, BMP-7-deficient mice also have skeletal patterning defects restricted to the rib cage, the skull, and the hindlimbs.

Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta.

Abstract: G1 progression in mammalian cells requires the activity of the cyclin D-dependent kinases Cdk4 and/or Cdk6 and the cyclin E-dependent kinase Cdk2. Proliferating Mv1Lu mink lung epithelial cells and human keratinocytes contain high levels of the universal Cdk inhibitor p27Kip1 distributed in complexes with Cdk2, Cdk4, and Cdk6. Addition of the antimitogenic cytokine transforming growth factor-beta (TGF-beta) elevates expression of the Cdk4/6-specific inhibitor p15Ink4B and induces the release of p27 from Cdk4 and Cdk6. In Mv1Lu cells, this release of p27 coincides with increased binding of p27 to Cdk2. Recombinant p15 inhibits p27 binding to Cdk4 in vitro, and p15 overexpression induces the transfer of p27 from Cdk4 to Cdk2 in vivo, suggesting that the release of Cdk4-bound p27 in TGF-beta-treated cells is caused by the surge in p15 levels. In keratinocytes, TGF-beta increases not only p15 but also p21Cip1, which binds to Cdk2. These events correlate with Cdk2 inhibition and cell cycle arrest and occur without a loss of G1 Cdk components. The results suggest that TGF-beta induces G1 arrest in these two epithelial cell types by inhibiting various cyclin-Cdk kinases through the cooperative action of an Ink4 Cdk inhibitor and a Cip/Kip Cdk inhibitor. Subsequent to cell cycle arrest, Cdk2 and Cdk4 levels decline as part of a second set of events that may represent a program of cell adaptation to the quiescent state.

Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins.

Abstract: Terminal differentiation of cultured 3T3-L1 fibroblasts to the adipogenic phenotype is potently stimulated by dexamethasone (DEX) and methylisobutylxanthine (MIX). Previous studies have shown that these hormones induce the expression of genes encoding two members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. In the absence of new protein synthesis DEX activates the gene encoding C/EBP~. Likewise, MIX is a direct inducer of C/EBP~ gene expression. Optimal conditions for differentiation entail a 2-day period wherein confluent fibroblasts are exposed to DEX and MIX, followed by removal of the hormones and subsequent culture in the presence of insulin and fetal bovine serum. During the early phase of differentiation, high levels of C/EBP6 and C/EBP~ accumulate. These transcription factors diminish during the terminal phase of differentiation and come to be replaced by a third member of the C/EBP family, C/EBP~. Conclusive evidence has already shown that C/EBPot regulates terminal adipocyte differentiation, turning on the battery of fat-specific genes required for the synthesis, uptake, and storage of long chain fatty acids. Here we provide evidence that C/EBP~ and C/EBP~ play early catalytic roles in the differentiation pathway, relaying the effects of the hormonal stimulants DEX and MIX in a cascade-like fashion, leading to the activation of the gene encoding C/EBP~. Conditions facilitating the precocious expression of either C/EBP6 or C/EBPI3 were observed to accelerate adipogenesis and, in the case of C/EBPI3, relieve dependence on the early hormonal stimulants. Likewise, conditions that prevented the expression of functional C/EBP~ effectively blocked terminal differentiation. Finally, we have discovered that ectopic expression of C/EBPI3 in multipotential NIH-3T3 cells results in their conversion into committed adipoblasts capable, upon hormonal stimulation, of synchronous and uniform differentiation into fat-laden adipocytes.

p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene.

Abstract: Cyclin-dependent kinases (Cdks) are positive regulators of cell proliferation, whereas Cdk inhibitors (CKIs) inhibit proliferation. We describe a new CKI, p57KIP2, which is related to p21CIP1 and p27KIP1. p57KIP2 is a potent, tight-binding inhibitor of several G1 cyclin/Cdk complexes, and its binding is cyclin dependent. Unlike CIP1, KIP2 is not regulated by p53. Overexpression of p57KIP2 arrests cells in G1. p57KIP2 proteins have a complex structure. Mouse p57KIP2 consists of four structurally distinct domains: an amino-terminal Cdk inhibitory domain, a proline-rich domain, an acidic-repeat region, and a carboxy-terminal domain conserved with p27KIP1. Human p57KIP2 appears to have conserved the amino- and carboxy-terminal domains but has replaced the internal regions with sequences containing proline-alanine repeats. In situ hybridization during mouse embryogenesis revealed that KIP2 mRNA displays a striking pattern of expression during development, showing high level expression in skeletal muscle, brain, heart, lungs, and eye. Most of the KIP2-expressing cells are terminally differentiated, suggesting that p57KIP2 is involved in decisions to exit the cell cycle during development and differentiation. Human KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a familial cancer syndrome, marking it as a candidate tumor suppressor. The discovery of a new member of the p21CIP1 inhibitor family with novel structural features and expression patterns suggests a complex role for these proteins in cell cycle control and development.

Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation

Abstract: The role of the glucocorticoid receptor (GR) in glucocorticoid physiology and during development was investigated by generation of GR-deficient mice by gene targeting. GR -/- mice die within a few hours after birth because of respiratory failure. The lungs at birth are severely atelectatic, and development is impaired from day 15.5 p.c. Newborn livers have a reduced capacity to activate genes for key gluconeogenic enzymes. Feedback regulation via the hypothalamic-pituitary-adrenal axis is severely impaired resulting in elevated levels of plasma adrenocorticotrophic hormone (15-fold) and plasma corticosterone (2.5-fold). Accordingly, adrenal glands are enlarged because of hypertrophy of the cortex, resulting in increased expression of key cortical steroid biosynthetic enzymes, such as side-chain cleavage enzyme, steroid 11 beta-hydroxylase, and aldosterone synthase. Adrenal glands lack a central medulla and synthesize no adrenaline. They contain no adrenergic chromaffin cells and only scattered noradrenergic chromaffin cells even when analyzed from the earliest stages of medulla development. These results suggest that the adrenal medulla may be formed from two different cell populations: adrenergic-specific cells that require glucocorticoids for proliferation and/or survival, and a smaller noradrenergic population that differentiates normally in the absence of glucocorticoid signaling.

Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution.

Abstract: Progression through the cell cycle is catalyzed by cyclin-dependent kinases (CDKs) and is negatively controlled by CDK inhibitors (CDIs). We have isolated a new member of the p21CIP1/p27KIP1 CDI family and named it p57KIP2 to denote its apparent molecular mass and higher similarity to p27KIP1. Three distinct p57 cDNAs were cloned that differ at the start of their open reading frames and correspond to messages generated by the use of distinct splice acceptor sites. p57 is distinguished from p21 and p27 by its unique domain structure. Four distinct domains follow the heterogeneous amino-terminal region and include, in order, a p21/p27-related CDK inhibitory domain, a proline-rich (28% proline) domain, an acidic (36% glutamic or aspartic acid) domain, and a carboxy-terminal nuclear targeting domain that contains a putative CDK phosphorylation site and has sequence similarity to p27 but not to p21. Most of the acidic domain consists of a novel, tandemly repeated 4-amino acid motif. p57 is a potent inhibitor of G1- and S-phase CDKs (cyclin E-cdk2, cyclin D2-cdk4, and cyclin A-cdk2) and, to lesser extent, of the mitotic cyclin B-Cdc2. In mammalian cells, p57 localizes to the nucleus, associates with G1 CDK components, and its overexpression causes a complete cell cycle arrest in G1 phase. In contrast to the widespread expression of p21 and p27 in human tissues, p57 is expressed in a tissue-specific manner, as a 1.5-kb species in placenta and at lower levels in various other tissues and a 7-kb mRNA species observed in skeletal muscle and heart. The expression pattern and unique domain structure of p57 suggest that this CDI may play a specialized role in cell cycle control.

The Drosophila Tumor Suppressor Gene Warts Encodes a Homolog of Human Myotonic Dystrophy Kinase and Is Required for the Control of Cell Shape and Proliferation

Abstract: Homozygous loss of the warts (wts) gene of Drosophila, caused by mitotic recombination in somatic cells, leads to the formation of cell clones that are fragmented, rounded, and greatly overgrown compared with normal controls. Therefore, the gene is required for the control of the amount and direction of cell proliferation as well as for normal morphogenesis. The absence of wts function also results in apical hypertrophy of imaginal disc epithelial cells. Secretion of cuticle over and between the domed apical surfaces of these cells leads to a honeycomb-like structure and gives the superficial wart-like phenotype of mitotic clones on the adult. One wts allele allows survival of homozygotes to the late larval stage, and these larvae show extensive imaginal disc overgrowth. Because of the excess growth and abnormalities of differentiation that follow homozygous loss, we consider wts to be a tumor suppressor gene. The wts gene is defined by the breakpoints of overlapping deficiencies in the right telomeric region of chromosome 3, region 100A, and by lethal P-element insertions and excisions. It encodes a protein kinase that is most similar to human myotonic dystrophy kinase, the Neurospora cot-1 protein kinase, two cell-cycle regulated kinases of yeast, and several putative kinases from plants. These proteins define a new subfamily of protein kinases that are closely related to but distinct from the cyclic AMP-dependent kinases. Although myotonic dystrophy is defined by a neuromuscular disorder, it is sometimes associated with multiple pilomatrixomas, which are otherwise rare epithelial tumors, and with other tumors including neurofibromas and parathyroid adenomas. Our results raise the possibility that homozygous loss of the myotonic dystrophy kinase may contribute to the development of these tumors.

Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis.

Abstract: Bone morphogenetic proteins (BMPs) are secreted proteins that interact with cell-surface receptors and are believed to play a variety of important roles during vertebrate embryogenesis. Bmpr, also known as ALK-3 and Brk-1, encodes a type I transforming growth factor-beta (TGF-beta) family receptor for BMP-2 and BMP-4. Bmpr is expressed ubiquitously during early mouse embryogenesis and in most adult mouse tissues. To study the function of Bmpr during mammalian development, we generated Bmpr-mutant mice. After embryonic day 9.5 (E9.5), no homozygous mutants were recovered from heterozygote matings. Homozygous mutants with morphological defects were first detected at E7.0 and were smaller than normal. Morphological and molecular examination demonstrated that no mesoderm had formed in the mutant embryos. The growth characteristics of homozygous mutant blastocysts cultured in vitro were indistinguishable from those of controls; however, embryonic ectoderm (epiblast) cell proliferation was reduced in all homozygous mutants at E6.5 before morphological abnormalities had become prominent. Teratomas arising from E7.0 mutant embryos contained derivatives from all three germ layers but were smaller and gave rise to fewer mesodermal cell types, such as muscle and cartilage, than controls. These results suggest that signaling through this type I BMP-2/4 receptor is not necessary for preimplantation or for initial postimplantation development but may be essential for the inductive events that lead to the formation of mesoderm during gastrulation and later for the differentiation of a subset of mesodermal cell types.

Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3.

Abstract: Fibroblast growth factor 4 (FGF-4) has been shown to be a signaling molecule whose expression is essential for postimplantation mouse development and, at later embryonic stages, for limb patterning and growth. The FGF-4 gene is expressed in the blastocyst inner cell mass and later in distinct embryonic tissues but is transcriptionally silent in the adult. In tissue culture FGF-4 expression is restricted to undifferentia ted embryonic stem (ES) cells and embryonal carcinoma (EC) cell lines. Previously, we determined that EC cell-specific transcriptional activation of the FGF-4 gene depends on a synergistic interaction between octamer-binding proteins and an EC-specific factor, Fx, that bind adjacent sites on the FGF-4 enhancer. Through the cloning and characterization of an F9 cell cDNA we now show that the latter activity is Sox2, a member of the Sry-related Sox factors family. Sox2 can form a ternary complex with either the ubiquitous Oct-1 or the embryonic-specific Oct-3 protein on FGF-4 enhancer DNA sequences. However, only the Sox2/Oct-3 complex is able to promote transcriptional activation. These findings identify FGF-4 as the first known embryonic target gene for Oct-3 and for any of the Sox factors, and offer insights into the mechanisms of selective gene activation by Sox and octamer-binding proteins during embryogenesis.

Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements.

Abstract: The crucifer Arabidopsis thaliana has been used widely as a model organism for the study of plant development. We describe here the development of an efficient insertional mutagenesis system in Arabidopsis that permits identification of genes by their patterns of expression during development. Transposable elements of the Ac/Ds system carrying the GUS reporter gene have been designed to act as enhancer traps or gene traps. A novel selection scheme maximizes recovery of unlinked transposition events. In this study 491 plants carrying independent transposon insertions were generated and screened for expression patterns. One-half of the enhancer trap insertions and one-quarter of the gene trap insertions displayed GUS expression in seedlings or flowers, including expression patterns specific to organs, tissues, cell types, or developmental stages. The patterns identify genes that act during organogenesis, pattern formation, or cell differentiation. Transposon insertion lines with specific GUS expression patterns provide valuable markers for studies of Arabidopsis development and identify new cell types or subtypes in plants. The diversity of gene expression patterns generated suggests that the identification and cloning of Arabidopsis genes expressed in any developmental process is feasible using this system.

Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease.

Abstract: Poly(ADP-ribosyl)ation is catalyzed by NAD+: protein(ADP-ribosyl) transferase (ADPRT), a chromatin-associated enzyme which, in the presence of DNA breaks, transfers ADP-ribose from NAD+ to nuclear proteins. This post-translational modification has been implicated in many fundamental processes, like DNA repair, chromatin stability, cell proliferation, and cell death. To elucidate the biological function of ADPRT and poly(ADP-ribosyl)ation in vivo the gene was inactivated in the mouse germ line. Mice homozygous for the ADPRT mutation are healthy and fertile. Analysis of mutant tissues and fibroblasts isolated from mutant fetuses revealed the absence of ADPRT enzymatic activity and poly(ADP-ribose), implying that no poly(ADP-ribosyl)ated proteins are present. Mutant embryonic fibroblasts were able to efficiently repair DNA damaged by UV and alkylating agents. However, proliferation of mutant primary fibroblasts as well as thymocytes following gamma-radiation in vivo was impaired. Moreover, mutant mice are susceptible to the spontaneous development of skin disease as approximately 30% of older mice develop epidermal hyperplasia. The generation of viable ADPRT-/-mice negates an essential role for this enzyme in normal chromatin function, but the impaired proliferation and the onset of skin lesions in older mice suggest a function for ADPRT in response to environmental stress.

Consequences of lack of beta 1 integrin gene expression in mice.

Abstract: beta 1 integrins are cell-surface receptors that mediate cell-cell and cell-matrix interactions. We have generated a null mutation in the gene for the beta 1 integrin subunit in mice and embryonic stem (ES) cells. Heterozygous mice are indistinguishable from normal littermates. Homozygous null embryos develop normally to the blastocyst stage, implant, and invade the uterine basement membrane but die shortly thereafter. Using beta 1 integrin-deficient ES cells we have established chimeric embryos and adult mice. Analysis of the chimeric embryos demonstrated the presence of beta 1 integrin-deficient cells in all germ layers indicating that beta 1-null cells can differentiate and migrate in a context of normal tissue. When evaluated at embryonic day 9.5 (E9.5), embryos with a beta 1-null cell contribution below 25% were developing normally, whereas embryos with a contribution above this threshold were distorted and showed abnormal morphogenesis. In adult chimeric mice beta 1 integrin-deficient cells failed to colonize liver and spleen but were found in all other tissues analyzed at levels from 2%-25%. Immunostaining of chimeric mice showed that in cardiac muscle, there were small, scattered patches of myocytes that were beta 1-null. In contrast, many myotubes showed some beta 1-null contribution as a result of fusion between wild-type and mutant myoblasts to form mixed myotubes. The adult chimeric brain contained beta 1-null cells in all regions analyzed. Also, tissues derived from the neural crest contained beta 1 integrin-deficient cells indicating that migration of neuronal cells as well as neural crest cells can occur in the absence of beta 1 integrins.

Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression.

Abstract: The c-rel proto-oncogene, which is expressed predominantly in hemopoietic cells encodes a subunit of the NF-kappa B-like family of transcription factors. In mice with an inactivated c-rel gene, whereas development of cells from all hemopoietic lineages appeared normal, humoral immunity was impaired and mature B and T cells were found to be unresponsive to most mitogenic stimuli. Phorbol ester and calcium ionophore costimulation, in contrast to certain membrane receptor-mediated signals, overcame the T cell-proliferative defect, demonstrating that T cell proliferation occurs by Rel-dependent and -independent mechanisms. The ability of exogenous interleukin-2 to restore T Cell, but not B cell, proliferation indicates that Rel regulates the expression of different genes in B and T cells that are crucial for cell division and immune function.

Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects.

Abstract: Mammalian hairy and Enhancer of split homolog-1 (HES-1) encodes a helix-loop-helix (HLH) factor that is thought to act as a negative regulator of neurogenesis. To directly investigate the functions of HES-1 in mammalian embryogenesis, we performed a targeted disruption of the HES-1 locus. Mice homozygous for the mutation exhibited severe neurulation defects and died during gestation or just after birth. In the developing brain of HES-1-null embryos, expression of the neural differentiation factor Mash-1 and other neural HLH factors was up-regulated and postmitotic neurons appeared prematurely. These results suggest that HES-1 normally controls the proper timing of neurogenesis and regulates neural tube morphogenesis.

The head involution defective gene of Drosophila melanogaster functions in programmed cell death.

Abstract: Deletions of chromosomal region, 75C1,2 block virtually all programmed cell death (PCD) in the Drosophila embryo. We have identified a gene previously in this interval, reaper (rpr), which encodes an important regulator of PCD. Here we report the isolation of a second gene in this region, head involution defective (hid), which plays a similar role in PCD. hid mutant embryos have decreased levels of cell death and contain extra cells in the head. We have cloned the hid gene and find that its expression is sufficient to induce PCD in cell death defective mutants. The hid gene appears to encode a novel 410-amino-acid protein, and its mRNA is expressed in regions of the embryo where cell death occurs. Ectopic expression of hid in the Drosophila retina results in eye ablation. This phenotype can be suppressed completely by expression of the anti-apoptotic p35 protein from baculovirus, indicating that p35 may act genetically downstream from hid.

Mice lacking cyclin D1 are small and show defects in eye and mammary gland development.

Abstract: Using homologous recombination, mice lacking cyclin D1 were generated by replacing most of the first exon of the Cyl-1 gene with sequences encoding neomycin resistance. Cyl-1(-1-) mice were viable and fertile but consistently smaller than their heterozygous or wild-type littermates. The nullizygous animals also showed two distinctive abnormalities: a severe retinopathy caused by impaired development of all layers of the retina and, in the mammary gland during pregnancy, a marked reduction in acinar development accompanied by a failure to lactate. Approximately 50% of animals also had a malformation of the jaw that manifested itself as a misalignment of the incisor teeth. Mouse embryo fibroblasts isolated from 14 day nullizygous, heterozygous, or wild-type embryos and grown under standard conditions showed similar cell-cycle and growth characteristics. Thus although cyclin D1 kinase activity may facilitate G1 progression, it is not essential for the development of most tissues and organs, and only a few specialized cell lineages are demonstrably sensitive to its absence.

Mouse Otx2 functions in the formation and patterning of rostral head.

Abstract: The anterior part of the vertebrate head expresses a group of homeo box genes in segmentally restricted patterns during embryogenesis. Among these, Otx2 expression covers the entire fore- and midbrains and takes place earliest. To examine its role in development of the rostral head, a mutation was introduced into this locus. The homozygous mutants did not develop structures anterior to rhombomere 3, indicating an essential role of Otx2 in the formation of the rostral head. In contrast, heterozygous mutants displayed craniofacial malformations designated as otocephaly; affected structures appeared to correspond to the most posterior and most anterior domains of Otx expression where Otxl is not expressed. The homo- and heterozygous mutant phenotypes suggest Otx2 functions as a gap-like gene in the rostral head where Hox code is not present. The evolutionary significance of Otx2 mutant phenotypes was discussed for the innovation of the neurocranium and the jaw.

The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability.

Abstract: Genomic silencing is a fundamental mechanism of transcriptional regulation, yet little is known about conserved mechanisms of silencing. We report here the discovery of four Saccharomyces cerevisiae homologs of the SIR2 silencing gene (HSTs), as well as conservation of this gene family from bacteria to mammals. At least three HST genes can function in silencing; HST1 overexpression restores transcriptional silencing to a sir2 mutant and hst3 hst4 double mutants are defective in telomeric silencing. In addition, HST3 and HST4 together contribute to proper cell cycle progression, radiation resistance, and genomic stability, establishing new connections between silencing and these fundamental cellular processes.

Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo.

Abstract: Hox genes were first recognized for their role in embryonic development and may also play important lineage-specific functions in a variety of somatic tissues including the hematopoietic system. We have recently shown that certain members of the Hox A and B clusters, such as HOXB3 and HOXB4, are preferentially expressed in subpopulations of human bone marrow that are highly enriched for the most primitive hematopoietic cell types. To assess the role these genes may play in regulating the proliferation and/or differentiation of such cells, we engineered the overexpression of HOXB4 in murine bone marrow cells by retroviral gene transfer and analyzed subsequent effects on the behavior of various hematopoietic stem and progenitor cell populations both in vitro and in vivo. Serial transplantation studies revealed a greatly enhanced ability of HOXB4.transduced bone marrow cells to regenerate the most primitive hematopoietic stem cell compartment resulting in 50-fold higher numbers of transplantable totipotent hematopoietic stem cells in primary and secondary recipients, compared with serially passaged neo-infected control cells. This heightened expansion in vivo of HOXB4.transduced hematopoietic stem cells was not accompanied by identifiable anomalies in the peripheral blood of these mice. Enhanced proliferation in vitro of day-12 CFU-S and clonogenic progenitors was also documented. These results indicate HOXB4 to be an important regulator of very early but not late hematopoietic cell proliferation and suggest a new approach to the controlled amplification of genetically modified hematopoietic stem cell populations.

Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethality.

Abstract: Integrin receptors for extracellular matrix receptors are important effectors of cell adhesion, differentiation, and migration in cultured cells and are believed to be critical effectors of these processes during development. To determine when beta 1 integrins become critical during embryonic development, we generated mutant mice with a targeted disruption of the beta 1 integrin subunit gene. Heterozygous mutant mice were normal. Homozygous loss of beta 1 integrin expression was lethal during early postimplantation development. Homozygous embryos lacking beta 1 integrins formed normal-looking blastocysts and initiated implantation at E4.5. However, the E4.5 beta 1-null embryos in situ had collapsed blastocoeles, and whereas the trophoblast penetrated the uterine epithelium, extensive invasion of the decidua was not observed. Laminin-positive endoderm cells were detected in the inner cell mass area, but endoderm morphogenesis and migration were defective. By E5.5 beta 1-null embryos had degenerated extensively. In vitro analysis showed that trophoblast function in beta 1-null peri-implantation embryos was largely normal, including expression of tissue-specific markers, and outgrowth on fibronectin- and vitronectin-coated, although not on laminin-coated substrates. In contrast, the inner cell mass region of beta 1-null blastocyst outgrowths, and inner cell masses isolated from beta 1-null blastocysts, showed highly retarded growth and defective extraembryonic endoderm morphogenesis and migration. These data suggest that beta 1 integrins are required for normal morphogenesis of the inner cell mass and are essential mediators of growth and survival of cells of the inner cell mass. Failure of continued trophoblast development in beta 1-null embryos after inner cell mass failure could be attributable to either an intrinsic requirement for beta 1 integrins for later stages of trophoblast development, or to the lack of trophic signals from the beta 1-null inner cell mass.

Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor.

Abstract: The insulin gene is one of the best paradigms of tissue-specific gene expression. It is developmentally regulated and is expressed exclusively in the pancreatic beta-cell. This restricted expression is directed by a tissue-specific enhancer, within the promoter, which contains an E-box sequence. The insulin E-box binds an islet-specific protein complex, termed 3a1. E-boxes bind proteins belonging to the basic helix-loop-helix (bHLH) family of transcription factors. The bHLH proteins function as potent transcriptional activators of tissue-specific genes by forming heterodimers between ubiquitous and cell-restricted family members. In addition, the cell-restricted bHLH members play an important role in specifying cell fate. To isolate the tissue-specific bHLH factor controlling insulin gene expression and study its role in islet cell differentiation, a modified yeast two-hybrid system was utilized to clone a novel bHLH factor, BETA2 (beta-cell E-box trans-activator 2), from a hamster insulin tumor (HIT) cell cDNA library. Northern analysis demonstrates that high-level expression of the BETA2 gene is restricted to pancreatic alpha- and beta-cell lines. As expected of tissue-specific bHLH members, BETA2 binds to the insulin E-box sequence with high affinity as a heterodimer with the ubiquitous bHLH factor E47. More importantly, antibody supershift experiments clearly show that BETA2 is a component of the native insulin E-box-binding complex. Transient transfection assays demonstrate that the BETA2/E47 heterodimer synergistically interacts with a neighboring beta-cell-specific complex to activate an insulin enhancer. In contrast, other bHLH factors such as MyoD and E47, which can bind to the insulin E-box with high affinity, fail to do so. Thus, a unique, cooperative interaction is the basis by which the insulin E-box enhancer discriminates between various bHLH factors to achieve tissue-specific activation of the insulin gene.