RNA In Embryos Research Articles

Tuning of RNA editing by ADAR is required in Drosophila

RNA editing increases during development in more than 20 transcripts encoding proteins involved in rapid synaptic neurotransmission in Drosophila central nervous system and muscle. Adar (adenosine deaminase acting on RNA) mutant flies expressing only genome-encoded, unedited isoforms of ion-channel subunits are viable but show severe locomotion defects. The Adar transcript itself is edited in adult wild-type flies to generate an isoform with a serine to glycine substitution close to the ADAR active site. We show that editing restricts ADAR function since the edited isoform of ADAR is less active in vitro and in vivo than the genome-encoded, unedited isoform. Ubiquitous expression in embryos and larvae of an Adar transcript that is resistant to editing is lethal. Expression of this transcript in embryonic muscle is also lethal, with above-normal, adult-like levels of editing at sites in a transcript encoding a muscle voltage-gated calcium channel.

Identification of germ plasm-enriched mRNAs in Drosophila melanogaster by the cDNA microarray technique

The development of embryonic germ cells in Drosophila depends on the germ plasm, the most posterior part of the ooplasm. The germ plasm is devoted to the formation of future germ cells and is known to contain all the factors that are necessary to induce germ cell fate. Besides having a characteristic organelle and protein distribution, the germ plasm also contains a large number of localized RNA species that have been shown to play crucial roles in germ cell determination. To identify germ plasm-enriched, localized transcripts, we used a two-step method composed of cDNA microarray (containing 3200 annotated Drosophila cDNAs) and in situ RNA hybridization techniques. We compared germ plasm deficient, normal and ectopic germ plasm conditions in the cDNA microarray experiments. RNA species whose concentration increased when ectopic germ plasm was present and decreased when the germ plasm was missing were selected. These candidates were then subjected to a second screen which compared the distribution of the given RNA in wild type embryos and in eggs with ectopic germ plasm. Finally, 17 RNA species were found to be enriched in the germ plasm. Based on these data, we estimate that around 1% of the Drosophila genes encode for germ plasm-enriched, localized transcripts. We conclude that this combination of microarray and in situ hybridization techniques is a simple but powerful experimental design for the genome-wide identification of genes coding for germ plasm localized transcripts.

Transcriptional profiling using a novel cDNA array identifies differential gene expression during porcine embryo elongation

A novel porcine cDNA array, containing 1,015 PCR products selected for embryonic expression, was used for transcriptional profiling of conceptuses at four stages of peri-implantation development. Total conceptus RNA from small spherical, large spherical, tubular, and filamentous stages was amplified, converted to cDNA, and hybridized to membranes. Initially, normalized signal intensities obtained using cDNA from total RNA or from amplified RNA were compared. Uniform distribution of P-values associated with t-tests conducted for each gene indicated no evidence that amplification introduced bias. Analysis of data obtained by using amplified targets and the novel array identified genes differentially expressed across stages. Such genes were identified by testing for significant stage effects in gene-specific mixed models. A total of nine genes were declared differentially expressed. Six of the nine genes had P-values less than 0.001, and a false discovery rate of approximately 17% was associated with this significance threshold. Two out of six genes were significant when using the Bonferroni method to control the probability of one or more false positives. The other three genes had P-values between 0.001 and 0.01 and exhibited differences greater than twofold between stages. All four genes selected for confirmation (steroidogenic acute regulatory protein, interleukin 1 beta, transforming growth factor beta 3, and thymosin beta 10) were shown to be differentially expressed by using quantitative real time RT-PCR. Our study shows that RNA amplification is useful for transcriptional profiling with limiting porcine embryonic RNA, and that this novel targeted array can detect differential gene expression during trophoblastic elongation. Finally, our results contribute to an increased understanding of the temporal patterns of expression of known genes controlling conceptus development, as well as identify novel genes also differentially regulated during implantation.

FGF-20 and DKK1 are transcriptional targets of β-catenin and FGF-20 is implicated in cancer and development

beta-catenin is the major effector of the canonical Wnt signaling pathway. Mutations in components of the pathway that stabilize beta-catenin result in augmented gene transcription and play a major role in many human cancers. We employed microarrays to identify transcriptional targets of deregulated beta-catenin in a human epithelial cell line (293) engineered to produce mutant beta-catenin and in ovarian endometrioid adenocarcinomas characterized with respect to mutations affecting the Wnt/beta-catenin pathway. Two genes strongly induced in both systems-FGF20 and DKK1-were studied in detail. Elevated levels of FGF20 RNA were also observed in adenomas from mice carrying the Apc(Min)allele. Both XFGF20 and Xdkk-1 are expressed early in Xenopus embryogenesis under the control of the Wnt signaling pathway. Furthermore, FGF20 and DKK1 appear to be direct targets for beta-catenin/TCF transcriptional regulation via LEF/TCF-binding sites. Finally, by using small inhibitory RNAs specific for FGF20, we show that continued expression of FGF20 is necessary for maintenance of the anchorage-independent growth state in RK3E cells transformed by beta-catenin, implying that FGF-20 may be a critical element in oncogenesis induced by the Wnt signaling pathway.

The Zebrafish Hypochromic Mutant [iItalic]Shiraz[/iItalic] Encodes a Novel Mitochondrial Glutaredoxin That Establishes a Link between Heme and Fe/S Production.

Iron is required in the mitochondria both to produce heme, which is used for hemoglobin synthesis, and to make iron-sulfur (Fe/S) clusters, which confer electron transfer or catalytic functions to proteins. Cellular iron utilization and Fe/S cluster production are thought to occur independently, yet the processes are coordinated through currently uncharacterized pathways. The shiraz (sir) zebrafish mutant manifests a hypochromic, microcytic anemia. Positional cloning of sir discovered a deletion at the locus that included the zebrafish orthologue to glutaredoxin 5 (grx5), a gene required in yeast for Fe/S cluster assembly. We found that grx5 is highly expressed in the developing blood and fetal liver of both zebrafish and mouse embryos. Antisense-mediated morpholino knockdown of grx5 prevented hemoglobin production, and overexpression of zebrafish, yeast, mouse, or human grx5 RNA in sir embryos completely rescued hemoglobin production, indicating that grx5 is the gene responsible for the sir phenotype. Expression of zebrafish grx5 was found to rescue Fe/S protein production in the yeast Δgrx5 strain, demonstrating that the role of grx5 in Fe/S cluster assembly is conserved among eukaryotes. The surprising finding that mutating a gene necessary for Fe/S cluster assembly caused a lack of hemoglobin synthesis suggested that we had discovered a connection between these pathways. In vertebrates, iron regulatory protein 1 (IRP1) acts as a sensor of intracellular iron levels and controls cellular iron homeostasis via posttranscriptional regulation of iron uptake, storage, and utilization genes. For instance, IRP1 binds to the 5′ iron response element (IRE) in the aminolevulinate synthase 2 (ALAS2) mRNA, blocking translation when cellular iron is low. However, when cellular iron is replete, IRP1 binds a Fe/S cluster and its RNA-binding activity is abolished. We hypothesized that the loss of Fe/S cluster assembly in sir would activate IRP1 and block ALAS2 synthesis, resulting in hypochromia. In support of this model, overexpression of ALAS2 RNA without the 5′ IRE rescued sir hypochromia, while overexpression of ALAS2 including the IRE did not facilitate rescue. Furthermore, antisense morpholino knockdowns of IRP1 caused rescue of hemoglobin synthesis in sir embryos. The combination of these data indicate that hemoglobin production in the differentiating red cell is monitored through Fe-S cluster assembly as a mechanism to gauge iron levels and accordingly direct heme synthesis. This finding illustrates a crucial role for the mitochondrial Fe/S cluster assembly machinery during hemoglobin production, and has broad implications for the role of such genes in human hypochromic anemias.

Specific gene silencing using small interfering RNAs in fish embryos

Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. Here we demonstrate a siRNA-mediated gene silencing technique in rainbow trout embryos. We found that siRNAs effectively suppressed the transient expression of episomally located foreign GFP genes at an early developmental stage and inhibited the expression of GFP genes in stable transgenic trout embryos. Similar gene silencing was observed with an siRNA against the endogenous tyrosinase A gene. siRNAs interfered with the expression of maternally inherited mRNA. siRNAs did not affect non-relevant gene expression and siRNAs with a 4 base mismatch did not affect target gene expression. siRNA gene silencing is therefore highly sequence-specific. Our findings are the first evidence that siRNA-mediated gene silencing is effective in fish. This technique could be a powerful tool for studying gene function during embryonic development in aquacultural fish species, zebrafish, and medaka.

A cellular retinoic acid-binding protein from zebrafish ( Danio rerio): cDNA sequence, phylogenetic analysis, mRNA expression, and gene linkage mapping

We report the sequence of a cDNA clone coding for a cellular retinoic acid-binding protein (CRABP) in zebrafish. The encoded polypeptide is 142 amino acids in length with an estimated molecular mass of 15.8 kDa and a calculated isoelectric point of 5.2. The zebrafish CRABP exhibits highest sequence identity to the pufferfish CRABPIIa (83%) and CRABPIIb (79%), and human CRABPII (74%) than to any other member of the intracellular lipid-binding protein (ILBP) family. A phylogenetic tree for different members of the ILBP multigene family including fatty acid-binding proteins (FABPs), cellular retinol-binding proteins (CRBPs) and CRABPs shows that the cloned zebrafish cDNA encodes a protein that clusters with CRABPs from other species and not with CRBPs and FABPs. Reverse-transcription polymerase chain reactions (RT-PCR), using oligonucleotide primers specific to the zebrafish CRABP cDNA made from total RNA of embryos collected at various developmental stages, did not detect the CRABP mRNA until 12 h post-fertilization. In adult zebrafish, CRABP mRNA was detected by RT-PCR in total RNA extracted from muscle, testes and skin, barely detectable in heart, ovary and brain and undetectable in liver, kidney and intestine. Quantitative RT-PCR (qRT-PCR) revealed a similar tissue-specific distribution for zebrafish CRABP mRNA with highest levels of CRABP mRNA in muscle followed by testes, skin, ovary and much lower levels in heart. Radiation hybrid mapping assigned the CRABP gene to linkage group 16 in the zebrafish genome. Comparison of the mapped zebrafish CRABP and human CRABPII genes revealed that zebrafish linkage group 16 has a syntenic relationship with human chromosome 1. Based on phylogenetic analysis and the syntenic relationship to the CRABPII gene in human, the zebrafish cDNA clone appears to code for a type II CRABP.

RhoA is highly up-regulated in the process of early heart development of the chick and important for normal embryogenesis.

We have used molecular techniques, combined with classic embryological methods, to identify up-regulated genes associated with early heart development. One of the cDNAs identified and isolated by screening a chick lambda cDNA library was the small guanosine triphosphatase RhoA. RhoA has at least three different length mRNA species, each varying in the length of the 3' untranslated region. In situ hybridisation and immunocytochemistry analysis of RhoA expression show marked up-regulation in the heart-forming region. In other systems, RhoA signalling has been shown to be important for both gene expression and morphology. To investigate the function of RhoA in early heart development, we used small interfering RNAs (siRNA) in early chick embryos. Disruption of RhoA expression by siRNA treatment resulted in lack of heart tube fusion and abnormal head development. These data indicate that RhoA is important for normal embryogenesis.

PINCH2 is a new five LIM domain protein, homologous to PINCHand localized to focal adhesions☆

PINCH is a five LIM domain protein involved in the regulation of integrin-mediated cell adhesion. It has been shown that PINCH interacts with integrin-linked kinase and Nck2. Here we describe a new isoform of PINCH, which we call PINCH2. Therefore, we rename PINCH to PINCH1. PINCH2 has an overall similarity of 92% to PINCH1 and contains five LIM domains like PINCH1. While protein and gene structure of the PINCH homologues are very similar and well conserved during evolution, we observed differential expression pattern of the mRNAs. Based on northern hybridization of mouse embryo RNA, PINCH1 is already detectable at E8.5. It is highly expressed during later stages of development and in all adult mouse tissues analyzed, with the highest levels in heart, lung, bladder, skin, and uterus. In contrast, significant PINCH2 expression starts at E14.5. In adult mice it is widely expressed, similar to PINCH1, but absent from spleen and thymus. In situ hybridization confirmed the Northern data and showed differential expression of PINCH1 and PINCH2 in embryonic intestine. Finally, we demonstrate that PINCH2 localizes to focal adhesions in NIH 3T3 cells and to Z-disks in primary rat cardiomyocytes.

Mouse Mix gene is activated early during differentiation of ES and F9 stem cells and induces endoderm in frog embryos.

In frog and zebrafish, the Mix/Bix family of paired type homeodomain proteins play key roles in specification and differentiation of mesendoderm. However, in mouse, only a single Mix gene (mMix) has been identified to date and its function is unknown. We have analyzed the expression of mouse Mix RNA and protein in embryos, embryoid bodies formed from embryonic stem cells and F9 teratocarcinoma cells, as well as several differentiated cell types. Expression in embryoid bodies in culture mirrors that in embryos, where Mix is transcribed transiently in primitive (visceral) endoderm (VE) and in nascent mesoderm. In F9 cells induced by retinoic acid to differentiate to VE, mMix is coordinately expressed with three other endodermal transcription factors, well before AFP, and its protein product is localized to the nucleus. In a subpopulation of nascent mesodermal cells from embryonic stem cell embryoid bodies, mMix is coexpressed with Brachyury. Intriguingly, mMix mRNA is detected in a population (T+Flk1+) of cells which may contain hemangioblasts, before the onset of hematopoiesis and activation of hematopoietic markers. In vitro and in vivo, mMix expression in nascent mesoderm is rapidly down-regulated and becomes undetectable in differentiated cell types. In the region of the developing gut, mMix expression is confined to the mesoderm of mid- and hindgut but is absent from definitive endoderm. Injection of mouse mMix RNA into early frog embryos results in axial truncation of developing tadpoles and, in animal cap assays, mMix alone is sufficient to activate expression of several endodermal (but not mesodermal) markers. Although these observations do not exclude a possible cell-autonomous function for mMix in mesendodermal progenitor cells, they do suggest an additional, non-cell autonomous role in nascent mesoderm in the formation and/or patterning of adjacent definitive endoderm.

Structural organization of the sea urchin DNA (cytosine-5)-methyltransferase gene and characterization of five alternative spliced transcripts

Sea urchin DNA (cytosine-5)-methyltransferase ( Dnmt1) that is responsible for maintenance of DNA methylation patterns clearly shares similarity with various Dnmt1s identified in vertebrates. In this study, we determined the structure of the sea urchin Dnmt1 gene by screening a genomic library of the sea urchin Paracentrotus lividus with the complementary DNA (cDNA) as probe. Analysis of the positive clones demonstrated that the Dnmt1 gene consists of 34 exons and 33 introns spanning a distance of 35 kb. All exon-intron junction sequences agree with the GT/AG consensus with the exception of the 3′ acceptor site of intron 8 where CT replaces AG consensus. The differences in the total number of exons between sea urchin and mouse genes reside mainly in the N-terminal region of the protein (exons 5–7 of the sea urchin, exons 5–12 of the mouse) where there is very low similarity in the amino acid sequence. By reverse transcription-polymerase chain reaction using oligonucleotides spanning different regions of the cDNA we carried out a comprehensive analysis of alternative splicing of the Dnmt1 messenger RNA (mRNA) in sea urchin embryos at different stages of development. We demonstrated the presence of at least five alternative spliced mRNAs that are regulated during development and are translated in truncated or deleted proteins.

Fatty acid and lipid biosynthetic genes are expressed at constant molar ratios but different absolute levels during embryogenesis.

In plants, fatty acid and complex lipid synthesis requires the correct spatial and temporal activity of many gene products. Quantitative northern analysis showed that mRNA for the biotin carboxylase subunit of heteromeric acetyl-coenzyme A carboxylase, fatty acid synthase components (3-oxoacyl-acyl carrier protein [ACP] reductase, enoyl-ACP reductase, and acyl-ACP thioesterase), and stearoyl-ACP desaturase accumulate in a coordinate manner during Brassica napus embryogenesis. The mRNAs were present in a constant molar stoichiometric ratio. Transcript abundance of mRNAs for the catalytic proteins was found to be similar, whereas the number of ACP transcripts was approximately 7-fold higher. The peak of mRNA accumulation of all products was between 20 and 29 d after flowering; by 42 d after flowering, the steady-state levels of all transcripts fell to about 5% of their peak levels, which suggests that the mRNAs have similar stability and kinetics of synthesis. Biotin carboxylase was found to accumulate to a maximum of 59 fmol mg(-1) total RNA in embryos, which is in general agreement with the value of 170 fmol mg(-1) determined for Arabidopsis siliques (J.S. Ke, T.N. Wen, B.J. Nikolau, E.S. Wurtele [2000] Plant Physiol 122: 1057-1071). Embryos accumulated between 3- and 15-fold more transcripts per unit total RNA than young leaf tissue; the lower quantity of leaf 3-oxoacyl-ACP reductase mRNA was confirmed by reverse transcriptase-polymerase chain reaction. This is in conflict with analysis of B. napus transcripts using an Arabidopsis microarray (T. Girke, J. Todd, S. Ruuska, J. White, C. Benning, J. Ohlrogge [2000] Plant Physiol 124: 1570-1581) where similar leaf to seed levels of fatty acid synthase component mRNAs were reported.

Optical projection tomography as a tool for 3D microscopy and gene expression studies.

Current techniques for three-dimensional (3D) optical microscopy (deconvolution, confocal microscopy, and optical coherence tomography) generate 3D data by "optically sectioning" the specimen. This places severe constraints on the maximum thickness of a specimen that can be imaged. We have developed a microscopy technique that uses optical projection tomography (OPT) to produce high-resolution 3D images of both fluorescent and nonfluorescent biological specimens with a thickness of up to 15 millimeters. OPT microscopy allows the rapid mapping of the tissue distribution of RNA and protein expression in intact embryos or organ systems and can therefore be instrumental in studies of developmental biology or gene function.

C-Jun ARE Targets mRNA Deadenylation by an EDEN-BP (Embryo Deadenylation Element-binding Protein)-dependent Pathway

In mammalian cells, certain mRNAs encoding cytokines or proto-oncogenes are especially unstable, because of the presence of a particular sequence element in their 3'-untranslated region named ARE (A/U-rich element). AREs cause this instability by provoking the rapid shortening of the poly(A) tail of the mRNA. The deadenylation of mRNAs mediated by AREs containing repeats of the AUUUA motif (class I/II AREs) is conserved in Xenopus embryos. Here, we first extend these observations by showing that c-Jun ARE, a representative of class III (non-AUUUA) AREs, also provokes the deadenylation of a reporter RNA in Xenopus embryos. Next, by immunodepletion and immunoneutralization experiments, we show that, in Xenopus, the rapid deadenylation of RNAs that contain the c-Jun ARE, but not an AUUUA ARE, requires EDEN-BP. This RNA-binding protein was previously shown to provoke the rapid deadenylation of certain Xenopus maternal RNAs. Finally, we show that CUG-BP, the human homologue of EDEN-BP, specifically binds to c-Jun ARE. Together, these results identify CUG-BP as a plausible deadenylation factor responsible for the post-transcriptional control of c-Jun proto-oncogene mRNA in mammalian cells.

Sequence Analysis of the S3 Gene from a Turkey Reovirus

The deduced σ2 protein sequence from the S3 gene segment of a novel turkey reovirus, designated NC98, isolated from the bursa of birds exhibiting poult enteritis and mortality syndrome was determined. The isolate, serologically distinct from other avian reoviruses, was isolated in turkey embryo kidney cells and RNA was purified for cDNA synthesis. Oligonucleotide primers were designed based on conserved avian S3 nucleotide sequence data. The NC98 S3 open reading frame comprised 1101 base pairs and encoded 366 amino acids with a predicated molecular mass of 40.5 kDa. Although the S3 nucleotide sequence from several chicken isolates share at least 86% identity, they share only 64% with the NC98 turkey isolate. Interestingly, the S3 nucleotide sequence from a muscovy duck reovirus shares 55% identity with NC98 and 53% identity with chicken isolates. As observed in other avian reovirus σ2 protein sequences, a zinc-binding motif and double-stranded RNA binding domain were found within the predicted amino acid sequence of NC98. Phylogenetic analysis of the deduced σ2 sequence demonstrated that NC98 separated as a distinct virus relative to other avian strains. The results of this study indicate that NC98 is a novel turkey reovirus that shares limited genomic sequence identity to isolates of chicken and duck origin and should be considered a separate virus species within subgroup 2 of the Orthoreovirus genus.

Expression of retinol-binding protein messenger RNA and retinoic acid receptors in preattachment bovine embryos.

In cattle, retinoic acid (RA) has been indirectly associated with developmental potential of the embryo. RA is transported by retinol-binding protein (RBP) and actions of RA are mediated by several subtypes of nuclear retinoic acid receptors (RAR). Bovine embryos, produced in vitro from oocytes harvested from ovaries collected at a local abattoir, were frozen in liquid nitrogen at the oocyte, 2-, 4-, 8-, 16 to 20-cell, morula, blastocyst, and hatched blastocyst stages. Employing reverse transcription polymerase chain reaction (RT-PCR) we investigated mRNA expression for RBP, RARalpha, RARbeta, RARgamma, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Total RNA was extracted from 25 pooled embryos at each stage and RT-PCR analysis was repeated thrice. GAPDH transcript was detected in all stages. Transcripts for RBP, RARalpha, and RARgamma were also detected in all stages from the oocyte through to the hatched blastocyst. Expression of RARbeta was not detected at any stage. Whole-mount immunohistochemistry was performed with intact and hatched blastocysts using polyclonal antibodies against RARalpha and RARgamma2 to investigate if these embryonic mRNAs were translated to the mature protein. Strong immunostaining was observed for both RARalpha and RARgamma2 in the inner cell mass and trophectoderm of intact and hatched blastocysts. Expression of mRNA for RBP, RARalpha, RARgamma, and of the RARalpha and RARgamma2 receptor proteins in the bovine embryo suggests that RA is likely to directly regulate gene expression during preimplantation development in that species.

Isolation and Characterization of Vsx1, a Novel Mouse CVC paired-like Homeobox Gene Expressed during Embryogenesis and in the Retina

Gastrula stage mouse embryo RNA was screened by degenerate RT-PCR to yield a novel paired-like homeobox gene. The open reading frame encoded by the cDNA was most similar to human VSX1. Mouse Vsx1 encodes a protein of 363 amino acid residues that contains a CVC domain that was originally identified as a conserved motif among mouse CHX10, goldfish VSX-1 and C. elegans CEH-10. Linkage analysis showed that mouse Vsx1 mapped to the distal region of chromosome 2. RT-PCR analysis detected mouse Vsx1 transcripts from gastrulation and post-gastrulation stage mouse embryos, suggesting a role for Vsx1 during mouse embryogenesis. Analysis of the eyes of mouse chimeras generated with embryonic stem cells in which a lacZ reporter was targeted to the Vsx1 locus suggested that Vsx1 is expressed in the inner nuclear layer of the retina.

Polymorphic forms of expressed bovine interferon-tau genes: relative transcript abundance during early placental development, promoter sequences of genes and biological activity of protein products.

Multiple interferon (IFN)-tau genes exist in cattle, but it has remained unclear how many are expressed, the extent of their variation, and whether different genes exhibit similar patterns of expression and code for proteins with similar biological activities. A total of 118 complementary DNA (cDNA) were bi-directionally sequenced from reverse-transcribed bovine (bo) conceptus RNA over the period from blastocyst formation until day 25 of pregnancy. Fourteen different cDNAs, encoding eight different IFN-tau, were confirmed unique. All showed high sequence conservation (>98% nucleotide identity; >96% amino acid identity). The cDNA fell into three, recently evolved, phylogenetic groups (tau1, 2, and 3). Mean concentrations of IFN-tau messenger RNA were greater at day 17 and day 19 than at day 14 and day 25, with different genes showing comparable expression patterns, although there appeared to be a major bias in expression of two genes (for boIFN-tau1c and tau3a) in blastocysts. Genes representing members of the three boIFN-tau groups were cloned. Their promoter regions were conserved over regions considered important for transcriptional activation. Recombinant protein generated in Escherichia coli from representative genes in the three groups had similar but not identical antiviral activities. In summary, many IFN-tau genes, which are probably under similar transcriptional control, are expressed in bovine trophoblast during the peri-implantation period of development.

Dach1 mutant mice bear no gross abnormalities in eye, limb, and brain development and exhibit postnatal lethality.

Drosophila dachshund is necessary and sufficient for compound eye development and is required for normal leg and brain development. A mouse homologue of dachshund, Dach1, is expressed in the developing retina and limbs, suggesting functional conservation of this gene. We have generated a loss-of-function mutation in Dach1 that results in the abrogation of the wild-type RNA and protein expression pattern in embryos. Homozygous mutants survive to birth but exhibit postnatal lethality associated with a failure to suckle, cyanosis, and respiratory distress. The heart, lungs, kidneys, liver, and skeleton were examined to identify factors involved in postnatal lethality, but these organs appeared to be normal. In addition, blood chemistry tests failed to reveal differences that might explain the lethal phenotype. Gross examination and histological analyses of newborn eyes, limbs, and brains revealed no detectable abnormalities. Since Dach1 mutants die shortly after birth, it remains possible that Dach1 is required for postnatal development of these structures. Alternatively, an additional Dach homologue may functionally compensate for Dach1 loss of function.

Identification of New Genes Ndr2 and Ndr3 Which Are Related to Ndr1/RTP/Drg1 but Show Distinct Tissue Specificity and Response to N-myc

Ndr1 was isolated as a gene upregulated in N-myc mutant mouse embryos and is repressed by N-myc and c-myc. Consistent with Myc regulation, the same gene was also isolated as one sensitive to transformation (Drg1), and in addition as one induced under a few stress conditions (RTP). Two new genes, Ndr2 and Ndr3, were identified which encode proteins highly related to Ndr1/RTP/Drg1 and constitute the Ndr gene family. Ndr2 and Ndr3 are under spatio-temporal regulations distinct from Ndr1, and are not activated in N-myc mutants. When whole embryo RNA was analyzed, Ndr3 expression was already high at 9.5 days postcoitus (dpc), while expression of Ndr2 and Ndr1 became significant after 12.5 dpc and 13.5 dpc, respectively. At 14.5 dpc, expression of these genes partially overlaps, but many tissues are unique to one of them. For instance, Ndr1 is strongly expressed in the liver and gut epithelium, Ndr2 in the ventricular zone throughout the CNS, and Ndr3 in the spinal cord and the thymus rudiment. Genes of the Ndr family probably have tissue-dependent allotments of the possibly related functions.