An embryonic stem cell-based system for rapid analysis of transcriptional enhancers.

With the growing use of genome-wide screens for cis-regulatory elements, there is a pressing need for platforms that enable fast and cost-effective experimental validation of identified hits in relevant developmental and tissue contexts. Here, we describe a murine embryonic stem cell (ESC)-based system that facilitates rapid analysis of putative transcriptional enhancers. Candidate enhancers are targeted with high efficiency to a defined genomic locus via recombinase-mediated cassette exchange. Targeted ESCs are subsequently differentiated in vitro into desired cell types, where enhancer activity is monitored by reporter gene expression. As a proof of principle, we analyzed a previously characterized, Sonic hedgehog (Shh)-dependent, V3 interneuron progenitor (pV3)-specific enhancer for the Nkx2.2 gene, and observed highly specific enhancer activity. Given the broad potential of ESCs to generate a spectrum of cell types, this system can serve as an effective platform for the characterization of gene regulatory networks controlling cell fate specification and cell function.

TWEAK induces liver progenitor cell proliferation.

Progenitor ("oval") cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors.

Transcriptional regulation of pituitary POMC is conserved at the vertebrate extremes despite great promoter sequence divergence.

The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5'-flanking sequences of the teleost fish Tetraodon nigroviridis POMCalpha gene to produce transgenic mice. Tetraodon POMCalpha promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5'-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMCalpha promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence.

Maleylacetoacetate isomerase (MAAI/GSTZ)-deficient mice reveal a glutathione-dependent nonenzymatic bypass in tyrosine catabolism.

In mammals, the catabolic pathway of phenylalanine and tyrosine is found in liver (hepatocytes) and kidney (proximal tubular cells). There are well-described human diseases associated with deficiencies of all enzymes in this pathway except for maleylacetoacetate isomerase (MAAI), which converts maleylacetoacetate (MAA) to fumarylacetoacetate (FAA). MAAI is also known as glutathione transferase zeta (GSTZ1). Here, we describe the phenotype of mice with a targeted deletion of the MAAI (GSTZ1) gene. MAAI-deficient mice accumulated FAA and succinylacetone in urine but appeared otherwise healthy. This observation suggested that either accumulating MAA is not toxic or an alternate pathway for MAA metabolism exists. A complete redundancy of MAAI could be ruled out because substrate overload of the tyrosine catabolic pathway (administration of homogentisic acid, phenylalanine, or tyrosine) resulted in renal and hepatic damage. However, evidence for a partial bypass of MAAI activity was also found. Mice doubly mutant for MAAI and fumarylacetoacetate hydrolase (FAH) died rapidly on a normal diet, indicating that MAA could be isomerized to FAA in the absence of MAAI. Double mutants showed predominant renal injury, indicating that this organ is the primary target for the accumulated compound(s) resulting from MAAI deficiency. A glutathione-mediated isomerization of MAA to FAA independent of MAAI enzyme was demonstrated in vitro. This nonenzymatic bypass is likely responsible for the lack of a phenotype in nonstressed MAAI mutant mice.

Cell-specific translational profiling in acute kidney injury.

Acute kidney injury (AKI) promotes an abrupt loss of kidney function that results in substantial morbidity and mortality. Considerable effort has gone toward identification of diagnostic biomarkers and analysis of AKI-associated molecular events; however, most studies have adopted organ-wide approaches and have not elucidated the interplay among different cell types involved in AKI pathophysiology. To better characterize AKI-associated molecular and cellular events, we developed a mouse line that enables the identification of translational profiles in specific cell types. This strategy relies on CRE recombinase-dependent activation of an EGFP-tagged L10a ribosomal protein subunit, which allows translating ribosome affinity purification (TRAP) of mRNA populations in CRE-expressing cells. Combining this mouse line with cell type-specific CRE-driver lines, we identified distinct cellular responses in an ischemia reperfusion injury (IRI) model of AKI. Twenty-four hours following IRI, distinct translational signatures were identified in the nephron, kidney interstitial cell populations, vascular endothelium, and macrophages/monocytes. Furthermore, TRAP captured known IRI-associated markers, validating this approach. Biological function annotation, canonical pathway analysis, and in situ analysis of identified response genes provided insight into cell-specific injury signatures. Our study provides a deep, cell-based view of early injury-associated molecular events in AKI and documents a versatile, genetic tool to monitor cell-specific and temporal-specific biological processes in disease modeling.

Reprogramming within hours following nuclear transfer into mouse but not human zygotes.

Fertilized mouse zygotes can reprogram somatic cells to a pluripotent state. Human zygotes might therefore be useful for producing patient-derived pluripotent stem cells. However, logistical, legal and social considerations have limited the availability of human eggs for research. Here we show that a significant number of normal fertilized eggs (zygotes) can be obtained for reprogramming studies. Using these zygotes, we found that when the zygotic genome was replaced with that of a somatic cell, development progressed normally throughout the cleavage stages, but then arrested before the morula stage. This arrest was associated with a failure to activate transcription in the transferred somatic genome. In contrast to human zygotes, mouse zygotes reprogrammed the somatic cell genome to a pluripotent state within hours after transfer. Our results suggest that there may be a previously unappreciated barrier to successful human nuclear transfer, and that future studies could focus on the requirements for genome activation.

Transgene insertion induced dominant male sterility and rescue of male fertility using round spermatid injection.

Transgene insertions in the mouse often cause mutations at chromosomal loci. Analysis of insertion mutations that cause male sterility may lead to the identification of novel molecular mechanisms implicated in male fertility. Here we show a line of transgenic mice with dominant inheritance of male sterility (DMS) that was found amid several lines that were normally fertile. Transgene-positive males from this line invariably were sterile, whereas transgenic females and transgene-negative male littermates were fertile. Histologic analysis and TUNEL staining for apoptotic cells in DMS testis showed spermatogenesis arrest at metaphase of meiosis I (M-I), accompanied by massive apoptosis of spermatocytes. Meiosis I arrest was incomplete, however, as small numbers of spermatids and spermatozoa were found. Both round spermatids and spermatozoa were evaluated for their permissiveness in the assisted reproductive technologies intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI). Surprisingly, ROSI but not ICSI gave live offspring, suggesting that mature sperm had deteriorated by the time of recovery from the epididymis. Mapping the transgene insertion by fluorescence in situ hybridization revealed a site on chromosome 14 D3-E1. Two candidate genes, GFR alpha 2 and GnRH, that were previously mapped to that region and the functions of which in spermatogenesis are well established were not altered in DMS. As a consequence, positional cloning of the DMS locus will be essential to identify new molecules potentially involved in arrest at M-I. Furthermore, mice carrying this genetic trait might be useful for studies of assisted reproductive technologies and male contraceptives.