POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types.

Pax3 modulates expression of the c-Met receptor during limb muscle development.

Pax3 is a transcription factor whose expression has been used as a marker of myogenic precursor cells arising in the lateral somite destined to migrate to and populate the limb musculature. Accruing evidence indicates that the embryologic origins of axial and appendicular muscles are distinct, and limb muscle abnormalities in both mice and humans harboring Pax3 mutations support this distinction. The mechanisms by which Pax3 affects limb muscle development are unknown. The tyrosine kinase receptor for hepatocyte growth factor/scatter factor encoded by the c-met protooncogene is also expressed in limb muscle progenitors and, like Pax-3, is required in the mouse for limb muscle development. Here, we show that c-met expression is markedly reduced in the lateral dermomyotome of Splotch embryos lacking Pax3. We show that Pax3 can stimulate c-met expression in cultured cells, and we identify a potential Pax3 binding site in the human c-MET promoter that may contribute to direct transcriptional regulation. In addition, we have found that several cell lines derived from patients with rhabdomyosarcomas caused by a t(2;13) chromosomal translocation activating PAX3 express c-MET, whereas those rhabdomyosarcoma cell lines examined without the translocation do not. These results are consistent with a model in which Pax3 modulates c-met expression in the lateral dermomyotome, a function that is required for the appropriate migration of these myogenic precursors to the limb where the ligand for c-met (hepatocyte growth factor/scatter factor) is expressed at high levels.

Complexity of the erythroid transcription factor NF-E2 as revealed by gene targeting of the mouse p18 NF-E2 locus.

High-level globin expression in erythroid precursor cells depends on the integrity of NF-E2 recognition sites, transcription factor AP-1-like protein-binding motifs, located in the upstream regulatory regions of the alpha- and beta-globin loci. The NF-E2 transcription factor, which recognizes these sites, is a heterodimer consisting of (i) p45 NF-E2 (the larger subunit), a hematopoietic-restricted basic leucine zipper protein, and (ii) a widely expressed basic leucine zipper factor, p18 NF-E2, the smaller subunit. p18 NF-E2 protein shares extensive homology with the maf protooncogene family. To determine an in vivo role for p18 NF-E2 protein we disrupted the p18 NF-E2-encoding gene by homologous recombination in murine embryonic stem cells and generated p18 NF-E2-/- mice. These mice are indistinguishable from littermates throughout all phases of development and remain healthy in adulthood. Despite the absence of expressed p18 NF-E2, DNA-binding activity with the properties of the NF-E2 heterodimer is present in fetal liver erythroid cells of p18 NF-E2-/- mice. We speculate that another member of the maf basic leucine zipper family substitutes for the p18 subunit in a complex with p45 NF-E2. Thus, p18 NF-E2 per se appears to be dispensable in vivo.

Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis.

Presomitic and 3- to 12-somite pair cultured mouse embryos were deprived of retinoic acid (RA) by yolk-sac injections of antisense oligodeoxynucleotides for retinol binding protein (RBP). Inhibition of yolk-sac RBP synthesis was verified by immunohistochemistry, and the loss of activity of a lacZ-coupled RA-sensitive promoter demonstrated that embryos rapidly became RA-deficient. This deficiency resulted in malformations of the vitelline vessels, cranial neural tube, and eye, depending upon the stage of embryonic development at the time of antisense injection. Addition of RA to the culture medium at the time of antisense injection restored normal development implicating the role of RBP in embryonic RA synthesis. Furthermore, the induced RA deficiency resulted in early down-regulation of developmentally important genes including TGF-beta1 and Shh.

Immunolocalization of estrogen receptor protein in the mouse blastocyst during normal and delayed implantation.

We previously showed that estrogen receptor (ER) mRNA is present in preimplantation mouse embryos. The apparent synthesis of ER mRNA by the blastocyst at the time of implantation when estrogen is required was of special interest. A demonstration of the presence of ER protein would support the idea that estrogen can act directly on the embryo. The mouse embryo at the blastocyst stage is differentiated into two cell types, the trophectoderm and the inner cell mass. To determine whether ER mRNA is translated into ER protein and its cell-specific distribution, immunocytochemical analyses were performed in mouse blastocysts. ER protein was detected in all cell types of the normal, dormant, or activated blastocyst. To trace the fate of ER in these cell types, immunocytochemistry was performed in implanting blastocysts and early egg cylinder stage embryos developed in culture. Again, ER was detected in all cells of the implanting blastocyst. At the early egg cylinder stage, continued expression of ER was observed in cells derived from the inner cell mass or the trophoblast. In trophoblast giant cells, ER was concentrated in small regions of the nucleus, possibly the nucleoli, which was similar to that observed in dormant and activated blastocysts. The embryonic expression of ER at such early stages in a broad array of cells suggests that ER may have a general role during early development.

A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.

Previously, a hypomorphic mutation in CD18 was generated by gene targeting, with homozygous mice displaying increased circulating neutrophil counts, defects in the response to chemically induced peritonitis, and delays in transplantation rejection. When this mutation was backcrossed onto the PL/J inbred strain, virtually all homozygous mice developed a chronic inflammatory skin disease with a mean age of onset of 11 weeks after birth. The disease was characterized by erythema, hair loss, and the development of scales and crusts. The histopathology revealed hyperplasia of the epidermis, subcorneal microabscesses, orthohyperkeratosis, parakeratosis, and lymphocyte exocytosis, which are features in common with human psoriasis and other hyperproliferative inflammatory skin disorders. Repetitive cultures failed to demonstrate bacterial or fungal organisms potentially involved in the pathogenesis of this disease, and the dermatitis resolved rapidly after subcutaneous administration of dexamethasone. Homozygous mutant mice on a (PL/J x C57BL/6J)F1 background did not develop the disease and backcross experiments suggest that a small number of genes (perhaps as few as one), in addition to CD18, determine susceptibility to the disorder. This phenotype provides a model for inflammatory skin disorders, may have general relevance to polygenic human inflammatory diseases, and should help to identify genes that interact with the beta2 integrins in inflammatory processes.

Trinucleotide repeats and long homopeptides in genes and proteins associated with nervous system disease and development.

Several human neurological disorders are associated with proteins containing abnormally long runs of glutamine residues. Strikingly, most of these proteins contain two or more additional long runs of amino acids other than glutamine. We screened the current human, mouse, Drosophila, yeast, and Escherichia coli protein sequence data bases and identified all proteins containing multiple long homopeptides. This search found multiple long homopeptides in about 12% of Drosophila proteins but in only about 1.7% of human, mouse, and yeast proteins and none among E. coli proteins. Most of these sequences show other unusual sequence features, including multiple charge clusters and excessive counts of homopeptides of length > or = two amino acid residues. Intriguingly, a large majority of the identified Drosophila proteins are essential developmental proteins and, in particular, most play a role in central nervous system development. Almost half of the human and mouse proteins identified are homeotic homologs. The role of long homopeptides in fine-tuning protein conformation for multiple functional activities is discussed. The relative contributions of strand slippage and of dynamic mutation are also addressed. Several new experiments are proposed.

HLA-G expression during preimplantation human embryo development.

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.

Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles.

Transgenic mice carrying heterologous genes directed by a 670-bp segment of the regulatory sequence from the human transferrin (TF) gene demonstrated high expression in brain. Mice carrying the chimeric 0.67kbTF-CAT gene expressed TF-CAT in neurons and glial cells of the nucleus basalis, the cerebrum, corpus callosum, cerebellum, and hippocampus. In brains from two independent TF-CAT transgenic founder lines, copy number of TF-CAT mRNA exceeded the number of mRNA transcripts encoding either mouse endogenous transferrin or mouse endogenous amyloid precursor protein. In two transgenic founder lines, the chloramphenicol acetyltransferase (CAT) protein synthesized from the TF-CAT mRNA was estimated to be 0.10-0.15% of the total soluble proteins of the brain. High expression observed in brain indicates that the 0.67kbTF promoter is a promising director of brain expression of heterologous genes. Therefore, the promoter has been used to express the three common human apolipoprotein E (apoE) alleles in transgenic mouse brains. The apoE alleles have been implicated in the expression of Alzheimer disease, and the human apoE isoforms are reported to interact with different affinities to the brain beta-amyloid and tau protein in vitro. Results of this study demonstrate high expression and production of human apoE proteins in transgenic mouse brains. The model may be used to characterize the interaction of human apoE isoforms with other brain proteins and provide information helpful in designing therapeutic strategies for Alzheimer disease.

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

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.

Mapping the mouse genome: current status and future prospects.

The mouse is the best model system for the study of mammalian genetics and physiology. Because of the feasibility and importance of studying genetic crosses, the mouse genetic map has received tremendous attention in recent years. It currently contains over 14,000 genetically mapped markers, including 700 mutant loci, 3500 genes, and 6500 simple sequence length polymorphisms (SSLPs). The mutant loci and genes allow insights and correlations concerning physiology and development. The SSLPs provide highly polymorphic anchor points that allow inheritance to be traced in any cross and provide a scaffold for assembling physical maps. Adequate physical mapping resources--notably large-insert yeast artificial chromosome (YAC) libraries--are available to support positional cloning projects based on the genetic map, but a comprehensive physical map is still a few years away. Large-scale sequencing efforts have not yet begun in mouse, but comparative sequence analysis between mouse and human is likely to provide tremendous information about gene structure and regulation.

Selective neuronal toxicity of cocaine in embryonic mouse brain cocultures.

Cocaine exposure in utero causes severe alterations in the development of the central nervous system. To study the basis of these teratogenic effects in vitro, we have used cocultures of neurons and glial cells from mouse embryonic brain. Cocaine selectively affected embryonic neuronal cells, causing first a dramatic reduction of both number and length of neurites and then extensive neuronal death. Scanning electron microscopy demonstrated a shift from a multipolar neuronal pattern towards bi- and unipolarity prior to the rounding up and eventual disappearance of the neurons. Selective toxicity of cocaine on neurons was paralleled by a concomitant decrease of the culture content in microtubule-associated protein 2 (MAP2), a neuronal marker measured by solid-phase immunoassay. These effects on neurons were reversible when cocaine was removed from the culture medium. In contrast, cocaine did not affect astroglial cells and their glial fibrillary acidic protein (GFAP) content. Thus, in embryonic neuronal-glial cell cocultures, cocaine induces major neurite perturbations followed by neuronal death without affecting the survival of glial cells. Provided similar neuronal alterations are produced in the developing human brain, they could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following in utero exposure to cocaine.

Mouse model for Usher syndrome: linkage mapping suggests homology to Usher type I reported at human chromosome 11p15.

Usher syndrome is a group of diseases with autosomal recessive inheritance, congenital hearing loss, and the development of retinitis pigmentosa, a progressive retinal degeneration characterized by night blindness and visual field loss over several decades. The causes of Usher syndrome are unknown and no animal models have been available for study. Four human gene sites have been reported, suggesting at least four separate forms of Usher syndrome. We report a mouse model of type I Usher syndrome, rd5, whose linkage on mouse chromosome 7 to Hbb and tub has homology to human Usher I reported on human chromosome 11p15. The electroretinogram in homozygous rd5/rd5 mouse is never normal with reduced amplitudes that extinguish by 6 months. Auditory-evoked response testing demonstrates increased hearing thresholds more than control at 3 weeks of about 30 decibels (dB) that worsen to about 45 dB by 6 months.

Changes in expression of putative antigens encoded by pigment genes in mouse melanomas at different stages of malignant progression.

Cutaneous melanomas of Tyr-SV40E transgenic mice (mice whose transgene consists of the tyrosinase promoter fused to the coding regions of simian virus 40 early genes) strikingly resemble human melanomas in their development and progression. Unlike human melanomas, the mouse tumors all arise in genetically identical individuals, thereby better enabling expression of specific genes to be characterized in relation to advancing malignancy. The products of pigment genes are of particular interest because peptides derived from these proteins have been reported to function as autoantigens with immunotherapeutic potential in some melanoma patients. However, the diminished pigmentation characteristic of many advanced melanomas raises the possibility that some of the relevant products may no longer be expressed in the most malignant cells. We have therefore investigated the contributions of several pigment genes in melanotic vs. relatively amelanotic components of primary and metastatic mouse melanomas. The analyses reveal marked differences within and among tumors in levels of mRNAs and proteins encoded by the wild-type alleles at the albino, brown, slaty, and silver loci. Tyrosinase (the protein encoded by the albino locus) was most often either absent or undetectable as melanization declined. The protein encoded by the slaty locus (tyrosinase-related protein 2) was the only one of those tested that was clearly present in all the tumor samples. These results suggest that sole reliance on targeting tyrosinase-based antigens might selectively favor survival of more malignant cells, whereas targeting the ensemble of the antigens tested might contribute toward a more inclusive and effective antimelanoma strategy.