Zygotic expression of Exostosin1 (Ext1) is required for BMP signaling and establishment of dorsal-ventral pattern in Xenopus.

Exostosin 1 (EXT1) is a glycosyltransferase that contributes to the biosynthesis of heparan sulfate proteoglycans (HSPG). Loss of ext1 function leads to the human genetic disorder hereditary multiple exostoses (HME) and inhibits development in mouse, zebrafish and Drosophila. In Xenopus, loss of maternal EXT1 leads to impaired wnt11 signaling, resulting in a loss of dorsal embryonic development (Tao et al., 2005), but the functions of zygotic ext1 have not been elucidated. In this study, morpholino oligonucleotides were used to generate a zygotic partial loss of function for ext1, in order to evaluate the requirements for ext1 function in gastrulation and paracrine signaling. Transcriptional profiling was carried out by microarray. Validation and subsequent analyses of gene expression were performed using Q-RT-PCR and in situ hybridization. Western blots were used to assess paracrine signaling pathway activity. Introduction of ext1 MO led to gastrulation defects, which were partially rescued by co-injection of ext1 mRNA. Microarray-based comparisons of gene expression in control vs. Ext1 MO embryos identified several developmentally significant genes that are dependent upon Ext1 function, including brachyury (Xbra). In addition, decreased Ext1 was shown to reduce the level of Wnt8 and BMP4 signaling and disrupt ventral-specific gene expression. Ext1 function is required for maintenance of normal levels of BMP and wnt, as well as their target genes. In addition, expression of xbra and the establishment of ventral mesoderm depend upon normal levels of Ext1. These findings suggest that ext1-dependent synthesis of HSPG is critical for wnt and BMP signaling, mesodermal identity, and ventral pattern.

Deep ancestry of mammalian X chromosome revealed by comparison with the basal tetrapod Xenopus tropicalis.

BACKGROUND: The X and Y sex chromosomes are conspicuous features of placental mammal genomes. Mammalian sex chromosomes arose from an ordinary pair of autosomes after the proto-Y acquired a male-determining gene and degenerated due to suppression of X-Y recombination. Analysis of earlier steps in X chromosome evolution has been hampered by the long interval between the origins of teleost and amniote lineages as well as scarcity of X chromosome orthologs in incomplete avian genome assemblies. RESULTS: This study clarifies the genesis and remodelling of the Eutherian X chromosome by using a combination of sequence analysis, meiotic map information, and cytogenetic localization to compare amniote genome organization with that of the amphibian Xenopus tropicalis. Nearly all orthologs of human X genes localize to X. tropicalis chromosomes 2 and 8, consistent with an ancestral X-conserved region and a single X-added region precursor. This finding contradicts a previous hypothesis of three evolutionary strata in this region. Homologies between human, opossum, chicken and frog chromosomes suggest a single X-added region predecessor in therian mammals, corresponding to opossum chromosomes 4 and 7. A more ancient X-added ancestral region, currently extant as a major part of chicken chromosome 1, is likely to have been present in the progenitor of synapsids and sauropsids. Analysis of X chromosome gene content emphasizes conservation of single protein coding genes and the role of tandem arrays in formation of novel genes. CONCLUSIONS: Chromosomal regions orthologous to Therian X chromosomes have been located in the genome of the frog X. tropicalis. These X chromosome ancestral components experienced a series of fusion and breakage events to give rise to avian autosomes and mammalian sex chromosomes. The early branching tetrapod X. tropicalis' simple diploid genome and robust synteny to amniotes greatly enhances studies of vertebrate chromosome evolution.

Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis.

BACKGROUND: The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. RESULTS: Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. CONCLUSIONS: In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.

A genetic map of Xenopus tropicalis.

We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132cM in length, and 4 smaller linkage groups between 7 and 40cM. The total effective size of the map is 1658cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.

Compound heterozygous loss of Ext1 and Ext2 is sufficient for formation of multiple exostoses in mouse ribs and long bones.

Multiple Hereditary Exostoses (MHE) syndrome is caused by haploinsufficiency in Golgi-associated heparan sulfate polymerases EXT1 or EXT2 and is characterized by formation of exostoses next to growing long bones and other skeletal elements. Recent mouse studies have indicated that formation of stereotypic exostoses requires a complete loss of Ext expression, suggesting that a similar local loss of EXT function may underlie exostosis formation in patients. To further test this possibility and gain greater insights into pathogenic mechanisms, we created heterozygous Ext1(+/-) and compound Ext1(+/-)/Ext2(+/-) mice. Like Ext2(+/-) mice described previously (Stickens et al. Development 132:5055), Ext1(+/-) mice displayed rib-associated exostosis-like outgrowths only. However, compound heterozygous mice had nearly twice as many outgrowths and, more importantly, displayed stereotypic growth plate-like exostoses along their long bones. Ext1(+/-)Ext2(+/-) exostoses contained very low levels of immuno-detectable heparan sulfate, and Ext1(+/-)Ext2(+/-) chondrocytes, endothelial cells and fibroblasts in vitro produced shortened heparan sulfate chains compared to controls and responded less vigorously to exogenous factors such as FGF-18. We also found that rib outgrowths formed in Ext1(f/+)Col2Cre and Ext1(f/+)Dermo1Cre mice, suggesting that ectopic skeletal tissue can be induced by conditional Ext ablation in local chondrogenic and/or perichondrial cells. The study indicates that formation of stereotypic exostoses requires a significant, but not complete, loss of Ext expression and that exostosis incidence and phenotype are intimately sensitive to, and inversely related to, Ext expression. The data also indicate that the nature and organization of ectopic tissue may be influenced by site-specific anatomical cues and mechanisms.

Helium/oxygen-driven albuterol nebulization in the management of children with status asthmaticus: a randomized, placebo-controlled trial.

OBJECTIVES: We investigated the effect of heliox-powered albuterol therapy on hospital length of stay and clinical status in children with moderate to severe status asthmaticus. DESIGN: Prospective, randomized, placebo-controlled trial. SETTING: Twenty-five-bed pediatric intensive care unit at an academic children's medical center. PATIENTS: Forty-two children (2-21 yrs of age) with moderate to severe status asthmaticus. INTERVENTIONS: Patients were randomized to receive either heliox-powered nebulized albuterol or air/oxygen-powered nebulized albuterol (placebo) until they were transitioned to albuterol delivered by a metered dose inhaler. MEASUREMENTS AND MAIN RESULTS: Clinical asthma scores were recorded on enrollment and every 4 hrs thereafter. Patients in the heliox group (n = 22) and the control group (n = 20) had similar ages (mean +/- sem: 88 +/- 9.9 vs. 98 +/- 11.1 months, respectively; p = .51), time to study enrollment (618 +/- 70.4 vs. 597 +/- 84.1 mins, respectively; p = .72), and clinical asthma scores at study entry (5.9 +/- 0.2 vs. 5.7 +/- 0.3, respectively; p = .72). There were no significant differences between groups in time to eligibility to hospital discharge (66.2 +/- 8.7 vs. 63.4 +/- 8.6 hrs, respectively; p = .61), time to clinical asthma score <3 (22 +/- 2.8 vs. 21.2 +/- 5.3 hrs, respectively; p = .27), or time to eligibility for intensive care unit discharge (34.4 +/- 6.8 vs. 33.3 +/- 8.2 hrs, respectively; p = .64). There were no significant differences in adverse events between groups. CONCLUSIONS: Despite the previously demonstrated effects of heliox on improved aerosol particle delivery into the distal airways, heliox-powered nebulized albuterol therapy for children admitted to the hospital with moderate to severe status asthmaticus does not shorten hospital length of stay or hasten rates of clinical improvement when compared with air/oxygen-powered nebulized albuterol.

The genome of the Western clawed frog Xenopus tropicalis.

The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

Remobilization of Tol2 transposons in Xenopus tropicalis.

BACKGROUND: The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency. RESULTS: To test whether Tol2 transposons integrated in the Xenopus tropicalis genome are substrates for remobilization, we injected in vitro transcribed Tol2 mRNA into one-cell embryos harbouring a single copy of a Tol2 transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single Tol2 transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the Tol2 transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis. CONCLUSIONS: In this study, we demonstrate that single copy Tol2 transposons integrated into the Xenopus tropicalis genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.

Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6.

Mechanisms coupling heart function and cardiac morphogenesis can be accessed in lower vertebrate embryos that can survive to swimming tadpole stages on diffused oxygen. Forward genetic screens in Xenopus tropicalis have identified more than 80 mutations affecting diverse developmental processes, including cardiac morphogenesis and function. In the first positional cloning of a mutation in X. tropicalis, we show that non-contractile hearts in muzak (muz) embryos are caused by a premature stop codon in the cardiac myosin heavy chain gene myh6. The mutation deletes the coiled-coil domain responsible for polymerization into thick filaments, severely disrupting the cardiomyocyte cytoskeleton. Despite the lack of contractile activity and absence of a major structural protein, early stages of cardiac morphogenesis including looping and chamber formation are grossly normal. Muz hearts subsequently develop dilated chambers with compressed endocardium and fail to form identifiable cardiac valves and trabeculae.

Rapid gynogenetic mapping of Xenopus tropicalis mutations to chromosomes.

Pilot forward genetic screens in Xenopus tropicalis have isolated over 60 recessive mutations. Here we present a simple method for mapping mutations to chromosomes using gynogenesis and centromeric markers. When coupled with available genomic resources, gross mapping facilitates evaluation of candidate genes as well as higher resolution linkage studies. Using gynogenesis, we have mapped the genetic locations of the 10 X. tropicalis centromeres, and performed fluorescence in situ hybridization to validate these locations cytologically. We demonstrate the use of this very small set of centromeric markers to map mutations efficiently to specific chromosomes. Developmental Dynamics 238:1398-1406, 2009. (c) 2009 Wiley-Liss, Inc.

Ventilator-associated pneumonia in the pediatric intensive care unit: characterizing the problem and implementing a sustainable solution.

OBJECTIVES: To characterize ventilator-associated pneumonia (VAP) in our pediatric intensive care unit (PICU), implement an evidence-based pediatric VAP prevention bundle, and reduce VAP rates. STUDY DESIGN: The setting is a 25-bed PICU in a 475-bed free-standing pediatric academic medical center. VAP was diagnosed according to Centers for Disease Control and National Nosocomial Infections Surveillance System definitions. A pediatric VAP prevention bundle was established and implemented. Baseline VAP rates were compared with implementation and post-bundle-implementation periods. RESULTS: VAP is significantly associated with increased PICU length of stay, mechanical ventilator days, and mortality rates (length of stay VAP 19.5+/-15.0 vs non-VAP 7.5+/-9.2, P< .001; ventilator days VAP 16.3+/-14.7 vs non-VAP 5.3+/-8.4, P< .001; mortality VAP 19.1% vs non-VAP 7.2%, P= .01). The VAP rate was reduced from 5.6 (baseline) to 0.3 infections per 1000 ventilator days after bundle implementation; P< .0001. Subglottic/tracheal stenosis, trauma, and tracheostomy are significantly associated with VAP. CONCLUSIONS: PICU VAP is associated with increased morbidity and mortality rates. A multidisciplinary improvement team can implement a sustainable pediatric-specific VAP prevention bundle, resulting in VAP rate reduction.

Identification of a mutation in the Clock1 gene affecting zebrafish circadian rhythms.

As part of an ongoing program to identify genes involved in maintaining circadian rhythms of zebrafish, 6,500 mutagenized genomes were screened for dominant mutants affecting circadian locomotor activity. Molecular analysis of one of these mutant lines, Clk1(dg3), revealed an I254N mutation in the PAS domain of the Clock1 protein. This isoleucine is tightly conserved in the Clock genes of several different species, and the I254N was not seen in any of the wild-type zebrafish population tested. Analysis of circadian activity rhythms as well as melatonin rhythms in homozygotes revealed the biological clock runs with a shortened period. The effect of this Clock1 mutation was characterized in vitro using a cell culture system where it appears to enhance the transactivation ability of the I254N Clock1 protein compared with that of the normal gene product.

Distribution of polymorphic and non-polymorphic microsatellite repeats in Xenopus tropicalis.

The results of our bioinformatics analysis have found over 91,000 di-, tri-, and tetranucleotide microsatellites in our survey of 25% of the X. tropicalis genome, suggesting there may be over 360,000 within the entire genome. Within the X. tropicalis genome, dinucleotide (78.7%) microsatellites vastly out numbered tri- and tetranucleotide microsatellites. Similarly, AT-rich repeats are overwhelmingly dominant. The four AT-only motifs (AT, AAT, AAAT, and AATT) account for 51,858 out of 91,304 microsatellites found. Individually, AT microsatellites were the most common repeat found, representing over half of all di-, tri-, and tetranucleotide microsatellites. This contrasts with data from other studies, which show that AC is the most frequent microsatellite in vertebrate genomes (Toth et al. 2000). In addition, we have determined the rate of polymorphism for 5,128 non-redundant microsatellites, embedded in unique sequences. Interestingly, this subgroup of microsatellites was determined to have significantly longer repeats than genomic microsatellites as a whole. In addition, microsatellite loci with tandem repeat lengths more than 30 bp exhibited a significantly higher degree of polymorphism than other loci. Pairwise comparisons show that tetranucleotide microsatellites have the highest polymorphic rates. In addition, AAT and ATC showed significant higher polymorphism than other trinucleotide microsatellites, while AGAT and AAAG were significantly more polymorphic than other tetranucleotide microsatellites.

EXT1 regulates chondrocyte proliferation and differentiation during endochondral bone development.

Multiple Hereditary Exostoses (MHE) is an autosomal dominant skeletal disorder most frequently caused by mutations in the EXT1 gene. MHE affects proper development of endochondral bones, such that all affected individuals present with exostoses adjacent to the growth plate of long bones, while some individuals exhibit additional bone deformities. EXT1 functions as a heparan sulfate (HS) co-polymerase, and when defective causes improper elongation of glycosaminoglycan side chains on core proteins of HS proteoglycans. Although analysis of heterozygous EXT1-deficient mice has failed to reveal any significant gross morphological variations in skeletal development, significant alterations in molecular signaling occur in the developing long bones. Our results indicate that defects in EXT1 and the resulting reduction in HS lead to enhanced Indian Hedgehog diffusion causing an increase in chondrocyte proliferation and delayed hypertrophic differentiation.

Isolation and phenogenetics of a novel circadian rhythm mutant in zebrafish.

Widespread use of zebrafish (Danio rerio) in genetic analysis of embryonic development has led to rapid advances in the technology required to generate, map and clone mutated genes. To identify genes involved in the generation and regulation of vertebrate circadian rhythmicity, we screened for dominant mutations that affect the circadian periodicity of larval zebrafish locomotor behavior. In a screen of 6,500 genomes, we recovered 8 homozygous viable, semi-dominant mutants, and describe one of them here. The circadian period of the lager and lime (lag(dg2)) mutant is shortened by 0.7 h in heterozygotes,and 1.3 h in homozygotes. This mutation also shortens the period of the melatonin production rhythm measured from cultured pineal glands, indicating that the mutant gene product affects circadian rhythmicity at the tissue level, as well as at the behavioral level. This mutation also alters the sensitivity of pineal circadian period to temperature, but does not affect phase shifting responses to light. Linkage mapping with microsatellite markers indicates that the lag mutation is on chromosome 7. A zebrafish homolog of period1(per1) is the only known clock gene homolog that maps near the lag locus. However, all sequence variants found in per1 cDNA from lag(dg2) mutants are also present in wild type lines, and we were unable to detect any defect in per1 mRNA splicing, so this mutation may identify a novel clock gene.

Extubation failure in pediatric intensive care: a multiple-center study of risk factors and outcomes.

OBJECTIVE: To determine a contemporary failed extubation rate, risk factors, and consequences of extubation failure in pediatric intensive care units (PICUs). Three hypotheses were investigated: a) Extubation failure is in part disease specific; b) preexisting respiratory conditions predispose to extubation failure; and c) admission acuity scoring does not affect extubation failure. DESIGN: Twelve-month prospective, observational, clinical study. SETTING: Sixteen diverse PICUs in the United States. PATIENTS: Patients were 2,794 patients from the newborn period to 18 yrs of age experiencing a planned extubation trial. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A descriptive statistical analysis was performed, and outcome differences of the failed extubation population were determined. The extubation failure rate was 6.2% (174 of 2,794; 95% confidence interval, 5.3-7.1). Patient features associated with extubation failure (p <.05) included age < or =24 months; dysgenetic condition; syndromic condition; chronic respiratory disorder; chronic neurologic condition; medical or surgical airway condition; chronic noninvasive positive pressure ventilation; the need to replace the endotracheal tube on admission to the PICU; and the use of racemic epinephrine, steroids, helium-oxygen therapy (heliox), or noninvasive positive pressure ventilation within 24 hrs of extubation. Patients failing extubation had longer pre-extubation intubation time (failed, 148.7 hrs, SD +/- 207.8 vs. success, 107.9 hrs, SD +/- 171.3; p <.001), longer PICU length of stay (17.5 days, SD +/- 15.6 vs. 7.6 days, SD +/- 11.1; p <.001), and a higher mortality rate than patients not failing extubation (4.0% vs. 0.8%; p <.001). Failure was found to be in part disease specific, and preexisting respiratory conditions were found to predispose to failure whereas admission acuity did not. CONCLUSION: A variety of patient features are associated with an increase in extubation failure rate, and serious outcome consequences characterize the extubation failure population in PICUs.

Light induction of a vertebrate clock gene involves signaling through blue-light receptors and MAP kinases.

The signaling pathways that couple light photoreception to entrainment of the circadian clock have yet to be deciphered. Two prominent groups of candidates for the circadian photoreceptors are opsins (e.g., melanopsin) and blue-light photoreceptors (e.g., cryptochromes). We have previously showed that the zebrafish is an ideal model organism in which to study circadian regulation and light response in peripheral tissues. Here, we used the light-responsive zebrafish cell line Z3 to dissect the response of the clock gene zPer2 to light. We show that the MAPK (mitogen-activated protein kinase) pathway is essential for this response, although other signaling pathways may also play a role. Moreover, action spectrum analyses of zPer2 transcriptional response to monochromatic light demonstrate the involvement of a blue-light photoreceptor. The Cry1b and Cry3 cryptochromes constitute attractive candidates as photoreceptors in this setting. Our results establish a link between blue-light photoreceptors, probably cryptochromes, and the MAPK pathway to elicit light-induced transcriptional activation of clock genes.

Analysis of novel and recurrent mutations responsible for the tricho-rhino-phalangeal syndromes.

The tricho-rhino-phalangeal syndromes (TRPS type I, II, and III) are autosomal dominant disorders sharing the following characteristics: slowly growing and sparse scalp hair, medially thick and laterally thin eyebrows, bulbous tip of the nose, long flat philtrum, thin upper lip with vermilion border, and protruding ears. In addition, individuals with TRPS generally share skeletal and bone anomalies, including shortening of the phalanges and metacarpals (mild to severe brachydactyly), cone-shaped epiphyses, hip dysplasia, and short stature. The etiology of the different types of TRPS can result from either single base pair mutations, or the complete deletion of the TRPS1 gene, which encodes a zinc-finger transcription factor located on chromosomal band 8q24.1. We have identified nine heterozygous mutations, five novel and four recurrent, in unrelated families diagnosed with TRPS. The five novel mutations identified show 1- or 2-bp deletions and a single base substitution, whereas all of the recurrent mutations are single base substitutions. Seven of the nine mutations result in a premature stop codon, leading to a truncated, nonfunctional TRPS1 protein. The final two mutations are missense mutations in the GATA DNA binding zinc finger, which is believed to be important for the protein's normal function.

Maternal stores of α subtype histone mRNAs are not required for normal early development of sea urchin embryos.

The ability to specifically delete the store of maternal α-subtype histone mRNAs stored in the egg pronucleus has allowed us to examine the role of this major fraction of the maternal mRNA in the early development of the sea urchinStrongylocentrotus purpuratus. The egg nucleus was removed by centrifugation, and the resulting enucleate half eggs were fertilized. These haploid andromerogones lacked any stored α-subtype histone mRNAs. However, when grown in parallel with control embryos, they showed identical cleavage cycles, cell numbers, and patterns of cell differentiation. Measurements of the amount of α-histone mRNA in these andromerogones showed that there was no premature synthesis of α-histone mRNAs to compensate for the deleted maternal pool. Instead embryonic synthesis was normal in timing of initiation and duration. the ability of these embryos to develop into highly differentiated larvae without their maternal α-subtype histone mRNA pool suggests that this pool is not a critical component of early development per se. This suggestion is strengthened by the observation that the primitive sea urchinEucidaris tribuloides naturally lacks this maternal histone mRNA store. Evolutionary implications are discussed.