Targeting of the ETS factor GABPalpha disrupts neuromuscular junction synaptic function.

The GA-binding protein (GABP) transcription factor has been shown in vitro to regulate the expression of the neuromuscular proteins utrophin, acetylcholine esterase, and acetylcholine receptor subunits delta and epsilon through the N-box promoter motif (5'-CCGGAA-3'), but its in vivo function remains unknown. A single point mutation within the N-box of the gene encoding the acetylcholine receptor epsilon subunit has been identified in several patients suffering from postsynaptic congenital myasthenic syndrome, implicating the GA-binding protein in neuromuscular function and disease. Since conventional gene targeting results in an embryonic-lethal phenotype, we used conditional targeting to investigate the role of GABPalpha in neuromuscular junction and skeletal muscle development. The diaphragm and soleus muscles from mutant mice display alterations in morphology and distribution of acetylcholine receptor clusters at the neuromuscular junction and neurotransmission properties consistent with reduced receptor function. Furthermore, we confirmed decreased expression of the acetylcholine receptor epsilon subunit and increased expression of the gamma subunit in skeletal muscle tissues. Therefore, the GABP transcription factor aids in the structural formation and function of neuromuscular junctions by regulating the expression of postsynaptic genes.

The ETS transcription factor GABPalpha is essential for early embryogenesis.

The ETS transcription factor complex GABP consists of the GABPalpha protein, containing an ETS DNA binding domain, and an unrelated GABPbeta protein, containing a transactivation domain and nuclear localization signal. GABP has been shown in vitro to regulate the expression of nuclear genes involved in mitochondrial respiration and neuromuscular signaling. We investigated the in vivo function of GABP by generating a null mutation in the murine Gabpalpha gene. Embryos homozygous for the null Gabpalpha allele die prior to implantation, consistent with the broad expression of Gabpalpha throughout embryogenesis and in embryonic stem cells. Gabpalpha(+/-) mice demonstrated no detectable phenotype and unaltered protein levels in the panel of tissues examined. This indicates that Gabpalpha protein levels are tightly regulated to protect cells from the effects of loss of Gabp complex function. These results show that Gabpalpha function is essential and is not compensated for by other ETS transcription factors in the mouse, and they are consistent with a specific requirement for Gabp expression for the maintenance of target genes involved in essential mitochondrial cellular functions during early cleavage events of the embryo.

SOX13 exhibits a distinct spatial and temporal expression pattern during chondrogenesis, neurogenesis, and limb development.

SOX13 is a member of the SOX family of transcription factors. SOX proteins play essential roles in development, and some are associated with human genetic diseases. SOX13 maps to a multi-disease locus on chromosome 1q31-32, yet its function is unknown. Here we describe the temporal and spatial expression of SOX13 protein during mouse organogenesis. SOX13 is expressed in the three embryonic cell lineages, suggesting that it may direct various developmental processes. SOX13 is expressed in the developing central nervous system including the neural tube and the developing brain. Expression is also detected in the condensing mesenchyme and cartilage progenitor cells during endochondral bone formation in the limb as well as the somite sclerotome and its derivatives. SOX13 is also detected in the developing kidney, pancreas, and liver as well as in the visceral mesoderm of the extra-embryonic yolk sac and spongiotrophoblast layer of the placenta.

Tissue-specific overexpression of the HSA21 gene GABPalpha: implications for DS.

The ETS transcription factor GABPalpha is encoded by a gene on HSA21 and interacts with an ankyrin repeat-containing beta subunit to form the GABP complex. GABP regulates expression of genes involved in mitochondrial respiration and neuromuscular signalling. When GABPalpha mRNA is overexpressed in human DS fibroblast cell lines, or by tranfection in NIH3T3 cells, no increase in protein level is detected. However, increased Gabpalpha gene dosage in the Ts65Dn segmental trisomy mouse model of DS (DS) results in elevated Gabpalpha protein levels in brain and skeletal muscle only. These findings suggest that GABPalpha protein levels are tightly regulated in a tissue-specific manner, and consequently GABP may play a role in DS pathologies in tissues where GABPalpha protein levels are elevated.

Identification and expression analysis of alternative transcripts of the mouse GA-binding protein (Gabp) subunits alpha and beta1.

The erythroblast transformation specific (ETS) transcription factor GA-binding protein (Gabp) is widely expressed and acts on a diverse range of target genes, including nuclear-encoded mitochondrial proteins and neuromuscular-specific genes. The GABPalpha subunit contains an ETS DNA binding domain and the beta subunit contains a nuclear localization signal (NLS) and transactivation domain. Here, we show coincident expression of Gabpalpha and beta1 throughout mouse embryogenesis, consistent with the gene products functioning in a complex. We have also identified 2 alternatively spliced, tissue-specific exons 1 (5' untranslated regions) of mouse Gabpalpha and 4 alternative 3' polyadenylation signals that, in combination, result in 12 transcripts for Gabpalpha. These alternative transcripts are suggested to have altered stability, subcellular localization and/or translation efficiency. Further, we identified nine differentially expressed splice variants of mouse Gabpbeta1 that encode beta protein forms lacking functional domains, suggesting a dominant negative function. Together, alternative transcripts of Gabpalpha and beta1 provide a mechanism for tissue-specific regulation of Gabp activity.

Ets2 is expressed during morphogenesis of the somite and limb in the mouse embryo.

Ets2 is a member of the ETS family of transcription factors. In order to address the developmental function of Ets2, we have examined its expression pattern in E8.5 to E13.5 embryos using RNA whole-mount in situ hybridization. In the paraxial mesoderm, Ets2 is expressed uniformly in the presomitic mesoderm and then restricted to the cells in the rostral portion of the segmenting and the next two recently formed somites. In the developing limb, Ets2 expression in the mesenchyme reflects the progressive formation of the hand or foot plate and the digital skeleton. In addition, Ets2 is expressed in the otic vesicle and its derivatives, the dorsal (posterior) root ganglia, the neuroepithelium in the dorsal part of the caudal neural tube and the inter-segmental vasculature.

Making better transgenic models: conditional, temporal, and spatial approaches.

Over the last decade transgenic mouse models have become a common experimental tool for unraveling gene function. During this time there has been a growing expectation that transgenes resemble the in vivo state as much as possible. To this end, a preference away from heterologous promoters has emerged, and transgene constructs often utilize the endogenous promoter and gene sequences in BAC, PAC and YAC form without the addition of selectable markers, or at least their subsequent removal. There has been a trend toward controlled integration by homologous recombination, either at a characterized chromosomal localization or in some cases within the allele of interest. Markers such as green fluorescent protein (GFP), beta-galactosidase (LacZ), and alkaline phosphatase (AP) continue to be useful to trace transgenic cells, or transgene expression. The development of technologies such as RNA interference (RNAi), are introducing new ways of using transgenic models. Future developments in RNAi technology may revolutionize tissue specific inactivation of gene function, without the requirement of generating conditionally targeted mice and tissue specific recombinase mice. Transgenic models are biological tools that aid discovery. Overall, the main consideration in the generation of transgenic models is that they are bona fide biological models that best impart the disease model or biological function of the gene that they represent. The main consideration is to make the best model for the biological question at heart and this review aims to simplify that task somewhat. Here we take a historical perspective on the development of transgenic models, with many of the important considerations to be made in design and development along the way.

Cysteine-rich secretory protein 2 binds to mitogen-activated protein kinase kinase kinase 11 in mouse sperm.

Cysteine-rich secretory protein (CRISP) 2 (previously TPX1) is a testis-enriched member of the CRISP family, and has been localized to both the sperm acrosome and tail. Like all members of the mammalian CRISP family, its expression pattern is strongly suggestive of a role in male fertility, but functional support for this hypothesis remains limited. In order to determine the biochemical pathways within which CRISP2 is a component, the putative mature form of CRISP2 was used as bait in a yeast two-hybrid screen of a mouse testis expression library. One of the most frequently identified interacting partners was mitogen-activated protein kinase kinase kinase 11 (MAP3K11). Sequencing and deletion experiments showed that the carboxyl-most 20 amino acids of MAP3K11 interacted with the CRISP domain of CRISP2. This interaction was confirmed using pull-down experiments and the cellular context was supported by the localization of CRISP2 and MAP3K11 to the acrosome of the developing spermatids and epididymal spermatozoa. Interestingly, mouse epididymal sperm contained an approximately 60-kDa variant of MAP3K11, which may have been a result of proteolytic cleavage of the longer 93-kDa form seen in many tissues. These data raise the possibility that CRISP2 is a MAP3K11-modifying protein or, alternatively, that MAP3K11 acts to phosphorylate CRISP2 during acrosome development.

Inactivation of the transcription factor Elf3 in mice results in dysmorphogenesis and altered differentiation of intestinal epithelium.

BACKGROUND & AIMS: The mammalian small intestine is lined by a highly specialized epithelium that functions in the digestion and absorption of nutrients. The molecular mechanisms that direct intestinal epithelial cell morphogenesis and terminal differentiation are poorly understood. We have previously identified Elf3 (E74-like factor-3) as a member of the ETS transcription factor family strongly expressed in small intestinal epithelium. The aim of this study is to investigate the biological roles of Elf3 in vivo. METHODS: Mice with a null mutation of Elf3 were generated through targeted gene disruption. Characterization of intestinal development was performed by histologic and immunohistochemical techniques. RESULTS: Targeted disruption of Elf3 resulted in fetal lethality of about 30% at around embryonic day 11.5. Seventy percent of the Elf3-deficent progeny were born and displayed severe alterations of tissue architecture in the small intestine, manifested by poor villus formation and abnormal morphogenesis and terminal differentiation of absorptive enterocytes and mucus-secreting goblet cells. Crypt cell proliferation, however, appeared intact in Elf3-deficient mice.Elf3-deficient enterocytes express markedly reduced levels of the transforming growth factor beta type II receptor (TGF-beta RII), an inducer of intestinal epithelial differentiation. CONCLUSIONS: Elf3 is an important regulator of morphogenesis and terminal differentiation of epithelial cell lineages in the small intestine.