Gene Expression In Early Development Research Articles

235 THE INVESTIGATION OF mRNA EXPRESSION OF SEVERAL CHROMATIN REMODELLING GENES DURING BOVINE PREIMPLANTATION DEVELOPMENT

The efficiency of obtaining live calves from somatic cell nuclear transfer remains quite low. One factor implicated in this failure is inadequate chromatin remodelling of the donor nucleus. Five Polycomb group (PcG) genes were investigated as potential remodelling factors in bovine oocytes and preimplantation embryos. These genes (Cbx6, Eed, Edr1, Yy1, and Zfp144) are involved in transcriptional activation and cell cycle regulation. We hypothesize that inadequate expression may cause the developmental abnormalities seen following cloning. This study is aimed at characterizing normal expression, prior to comparative studies with cloned embryos. Three single abattoir-derived in vitro-matured (IVM) oocytes or in vitro-produced (IVP) embryos from each of the following stages: 2-cell, 4-cell, 8-cell, 16–32 cell, morula, Day 7 blastocyst, and Day 8 hatched blastocyst, were studied. Messenger RNA was isolated from individual samples with Dynabeads (Dynal, Inc., Lake Success, NY, USA) and then cDNA was created and amplified with a SMART cDNA synthesis kit (BD Biosciences Clontech, Palo Alto, CA, USA). Products were diluted 1:10 and used to amplify target genes by PCR. PCR products were sequenced for confirmation of identity. All amplified embryo samples expressed the housekeeping genes Poly(A) polymerase and actin. Similarly, all embryos up to the 8-cell stage expressed GDF9, an oocyte-specific gene, while IFN-tau, involved in maternal recognition of pregnancy, was expressed in one morula and all blastocyst samples. Cbx6 was not expressed at any stage. The other four genes were all expressed fairly consistently throughout the pre-implantation period. Eed transcript was detected in all samples, with the exception of one oocyte, one 4-cell embryo, and one 8-cell embryo. Edr1 transcript was detected in all samples except for two oocytes, one 16–32-cell embryo, and one Day 7 blastocyst. Yy1 was expressed in all but one oocyte, one 2-cell embryo, two 4-cell embryos, and one Day 7 blastocyst. Finally, Zfp144 transcript was detected in one oocyte and in all embryos until the 16–32 stage, and then was not detected until seen in one Day 7 and all Day 8 blastocysts. Cbx6, yet to be fully characterized in any species, is also known as the neuronal pentraxin receptor, and is involved in transport and clearance of synaptic debris. It is known to have the characteristic chromobox domain of the Cbx family, of which several family members play a role during pre-implantation development. Eed, Edr1, Yy1, and Zfp144 are expressed throughout the pre-implantation period, although levels of Zfp144 mRNA appear to drop during the embryonic genome activation and then reappear by the late blastocyst stage. This consistent expression may suggest a role for the proteins in chromatin remodelling or modulating patterns of gene expression in early development. Further studies are required to determine if these factors are expressed incorrectly in cloned embryos, potentially providing a link to the abnormalities observed post nuclear transfer.

Thyroid Hormone-Dependent Gene Expression in Differentiated Embryonic Stem Cells and Embryonal Carcinoma Cells: Identification of Novel Thyroid Hormone Target Genes by Deoxyribonucleic Acid Microarray Analysis

T3 is required for normal early development, but relatively few T3-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonic carcinoma (EC) cells have reduced T3 nuclear binding capacity and only modestly express the known T3 target genes, neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), in response to T3. Full T3 induction in transient transfection of EC cells was restored with cotransfection of a TR expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem (ES) cells compared with expression in cells with deficient (EC) or mutant TR (TRalpha P398H mutant ES cells), to identify T3 target genes. T3 stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only approximately 60 genes (1%) met criteria for direct T3 stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T3 target genes, neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T3 responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T3, may mediate thyroid hormone actions in early development.

High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.

Zebrafish represents an excellent model to study the function of vertebrate genes (e.g., well-developed genetics, large number of mutants, and genomic sequencing in progress), inasmuch as we have tools to manipulate gene expression. Recent use of injected morpholinos in eggs provides a good method to " knockdown " gene expression in early development (Nasevicius and Ekker, 2000), and the "caged" RNA injected in eggs allows to overexpress a gene in a specific set of cells (Ando et al., 2001). However, a method to specifically modify gene expression in the juvenile or in the adult is still missing. Such a method would be a very powerful tool to understand gene function in differentiated tissues. We describe here an electroporation-based approach, which allows gene transfer in adult tissues. Its efficiency was assessed using a GFP (green fluorescent protein) dependent assay. We then used this method to disrupt the Fgf signalling pathway during the process of regeneration.

A conserved role of a DEAD box helicase in mRNA masking.

Clam p82 is a member of the cytoplasmic polyadenylation element-binding protein (CPEB) family of RNA-binding proteins and serves dual functions in regulating gene expression in early development. In the oocyte, p82/CPEB is a translational repressor, whereas in the activated egg, it acts as a polyadenylation factor. Coimmunoprecipitations were performed with p82 antibodies in clam oocyte and egg lysates to identify stage-regulated accessory factors. p47 coprecipitates with p82 from oocyte lysates in an RNA-dependent manner and is absent from egg lysate p92-bound material. Clam p47 is a member of the RCK/p54 family of DEAD box RNA helicases. Xp54, the Xenopus homolog, with bona fide helicase activity, is an abundant and integral component of stored mRNP in oocytes (Ladomery et al., 1997). In oocytes, clam p47 and p82/CPEB are found in large cytoplasmic mRNP complexes. Whereas the helicase level is constant during embryogenesis, in contrast to CPEB, clam p47 translocates to nuclei at the two-cell stage. To address the role of this class of helicase in masking, Xp54 was tethered via 3' UTR MS2-binding sites to firefly luciferase, following microinjection of fusion protein and nonadenylated reporter mRNAs into Xenopus oocytes. Tethered helicase repressed luciferase translation three- to fivefold and, strikingly, mutations in two helicase motifs (DEAD--> DQAD and HRIGR-->HRIGQ), activated translation three- to fourfold, relative to MS2. These data suggest that this helicase family represses translation of maternal mRNA in early development, and that its activity may be attenuated during meiotic maturation, prior to cytoplasmic polyadenylation.

A Dictyostelium nuclear phosphatidylinositol phosphate kinase required for developmental gene expression.

The generation of diacylglycerol (DAG) in response to receptor stimulation is a well-documented signalling mechanism that leads to activation of protein kinase C (PKC). Putative alternative effectors contain sequences that interact with DAGs, but the mechanisms of signal transduction are unknown. We have identified a Dictyostelium gene encoding a novel protein which contains a domain with high identity to the DAG-binding domain of PKC. It does not encode a PKC homologue as the conservation does not extend outside this region. We confirm that the proposed DAG-binding domain is sufficient to mediate interaction of a fusion protein with vesicles containing DAG. The protein also shows significant homology to mammalian phosphatidylinositol phosphate (PIP) kinases and we show that this domain has PIP kinase activity. The protein, PIPkinA, is enriched in the nucleus and abrogation of gene function by homologous recombination inhibits early developmental gene expression, blocking development at an early stage. Thus, we have identified a PIP kinase from Dictyostelium which is required for development, is a candidate effector for DAG and has the potential to synthesize nuclear PIP(2).

Identification and characterization of spdR mutations that bypass the BsgA protease-dependent regulation of developmental gene expression in Myxococcus xanthus.

The BsgA protease of Myxococcus xanthus is an intracellular protease closely related to the Lon protease of Escherichia coli. BsgA is required for normal levels of developmentally induced gene expression. In this report, we describe the identification of mutations that suppress the developmental defect of bsgA mutants. These mutations localized to the spdR gene (suppressor protease deficiency regulator) that appears to play a role in the regulation of early developmental gene expression. Mutations in spdR fully restored the ability of a bsgA mutant to form fruiting bodies and spores and, with one exception, restored the expression of several development-specific lacZ fusions. spdR mutants exhibited characteristic phenotypic properties including increased expression of the development-specific tps gene during vegetative growth, formation of fruiting bodies and spores on semi-rich nutrient medium and completion of starvation-induced development in a shorter time period than wild-type strains. The spdR locus was cloned and sequenced and found to encode a member of the NtrC family of two-component transcriptional regulators. One interpretation of these data is that SpdR acts, directly or otherwise, to regulate developmental gene expression negatively and that the BsgA protease is required to relieve this inhibitory effect at the onset of development. However, Western immunoblot analysis indicated that SpdR is present at a relatively constant level during growth and early development in both wild-type and BsgA protease-deficient cells. This finding suggests that BsgA does not function to degrade SpdR at the onset of development.

Ca2+Is Required for Phosphorylation of Clam p82/CPEBin Vitro:Implications for Dual and Independent Roles of MAP and Cdc2 Kinases

During early development gene expression is controlled principally at the translational level. Oocytes of the surf clamSpisula solidissimacontain large stockpiles of maternal mRNAs which are translationally dormant or masked until meiotic maturation. Fertilisation of the oocyte leads to rapid polysomal recruitment of the abundant cyclin and ribonucleotide reductase mRNAs at about the time they undergo cytoplasmic polyadenylation. Clam p82, a 3′ UTR RNA-binding protein, and a member of the CPEB (cytoplasmic polyadenylation element binding protein) family, functions as a translational masking factor in oocytes and as a polyadenylation factor in fertilised eggs. In meiotically maturing clam oocytes, p82/CPEB is rapidly phosphorylated on multiple residues to a 92-kDa apparent size, prior to its degradation during the first cell cleavage. Here we examine the protein kinase(s) that phosphorylates clam p82/CPEB using a clam oocyte activation cell-free system that responds to elevated pH, mirroring the pH rise that accompanies fertilisation. We show that p82/CPEB phosphorylation requires Ca2+(<100 μM) in addition to raised pH. Examination of the calcium dependency combined with the use of specific inhibitors implicates the combined and independent actions of cdc2 and MAP kinases in p82/CPEB phosphorylation. Calcium is necessary for both the activation and the maintenance of MAP kinase, whose activity is transientin vitro,asin vivo.While cdc2 kinase plays a role in the maintenance of MAP kinase activity, it is not required for the activation of MAP kinase. We propose a model of clam p82/CPEB phosphorylation in which MAP kinase initially phosphorylates clam p82/CPEB, at a minor subset of sites that does not alter its migration, and cdc2 kinase is necessary for the second wave of phosphorylation that results in the large mobility size shift of clam p82/CPEB. The possible roles of phosphorylation for the function and regulation of p82/CPEB are discussed.

The Roles of 5′-HS2, 5′-HS3, and the γ-Globin TATA, CACCC, and Stage Selector Elements in Suppression of β-Globin Expression in Early Development

The roles of HS2 and HS3 from the human beta-globin locus control region and of the TATA, CACCC, and stage selector elements of the gamma-globin promoter, in competitive inhibition of beta-globin gene expression in early development, were tested using stable transfections of HEL and K562 cells. Cells with an HS3gamma beta construct demonstrate that HS3 exhibits enhancing activity, but compared with HS2, this site participates less consistently in the inhibition of embryonic/fetal beta-globin expression. In cells with HS3HS2gamma beta constructs, the two HS sites act in concert to more effectively enhance gamma-globin gene expression and to drive stage-specific expression of the gamma- and beta-globin genes. A gamma-globin gene with a -161 promoter can competitively inhibit beta-globin gene expression. HS3HS2gamma beta constructs were used to determine the effects of gamma-globin promoter mutations within this region on competition. The CACCC and TATA elements, but not the stage selector element, inhibit inappropriate embryonic/fetal stage expression of the beta-globin gene. The mutation in the gamma-globin TATA element results in the use of two major alternative transcription start sites. The data suggest that proteins binding to the gamma-globin CACCC and TATA elements interact with those binding to HS2 and/or HS3 to preclude beta-globin transcription in early development.

The clam 3' UTR masking element-binding protein p82 is a member of the CPEB family.

During early development gene expression is controlled principally at the translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs that are translationally dormant or masked until meiotic maturation. Activation of the oocyte by fertilization leads to translational activation of the abundant cyclin and ribonucleotide reductase mRNAs at a time when they undergo cytoplasmic polyadenylation. In vitro unmasking assays have defined U-rich regions located approximately centrally in the 3' UTRs of these mRNAs as translational masking elements. A clam oocyte protein of 82 kDa, p82, which selectively binds the masking elements, has been proposed to act as a translational repressor. Importantly, mRNA-specific unmasking in vitro occurs in the absence of poly(A) extension. Here we show that clam p82 is related to Xenopus CPEB, an RNA-binding protein that interacts with the U-rich cytoplasmic polyadenylation elements (CPEs) of maternal mRNAs and promotes their polyadenylation. Cloned clam p82/CPEB shows extensive homology to Xenopus CPEB and related polypeptides from mouse, goldfish, Drosophila and Caenorhabditis elegans, particularly in their RNA-binding C-terminal halves. Two short N-terminal islands of sequence, of unknown function, are common to vertebrate CPEBs and clam p82. p82 undergoes rapid phosphorylation either directly or indirectly by cdc2 kinase after fertilization in meiotically maturing clam oocytes, prior to its degradation during the first cell cleavage. Phosphorylation precedes and, according to inhibitor studies, may be required for translational activation of maternal mRNA. These data suggest that clam p82 may be a functional homolog of Xenopus CPEB.

A test for adaptive change in DNA sequences controlling transcription.

Spatial and temporal differences in gene expression in early development result from the interaction of transcription factors with enhancer and silencer sequences in DNA. The evolution of the developmental process thus involves changes in the DNA sequences that bind transcription factors. Here we advocate a non-parametric statistical test-comparing levels of polymorphism and fixed substitutions between species -to look for evidence of adaptive evolution in sequences controlling gene expression. The test is illustrated by DNA sequence changes in the proximal part of the 'zebra' elements in the fushi terazu gene of the Drosophila melanogaster species group, which yield significant evidence for adaptive substitutions. (This is despite highly significant evidence that all parts of the sequence have been subject to strong selective constraint). The test can be applied generally to investigate adaptive evolution in the control of gene expression.

Flow cytometric study of differentiating cultures of Bacillus subtilis.

We report on 1) the development of a flow cytometry-based technique for detecting beta-galactosidase in differentiating cultures of Bacillus subtilis and 2) the application of this technique in the study of early developmental gene expression. The problems associated with generating detectable signals (despite the small size of B. subtilis cells) have been overcome using the fluorogenic substrate 5-octanolyaminofluorescein di-beta-D-galactopyranoside (C8-FDG). Additionally, to control for background fluorescence during the staining process, we included a control population in the C8-FDG staining mixture that consists of cells devoid of the lacZ gene prestained with another dye, PKH26. The distinct emission spectra of C8-fluorescein and PKH26 allow nonspecific C8-FDG staining in this control population to be monitored using two-color analysis. This technique has been applied in the study of developmental gene expression in sporulating cultures of B. subtilis, and it has been found that such cultures are heterogeneous, comprising two cell populations. One population is induced for expression of early sporulation genes, which is determined using lacZ fusions, whereas the other remains uninduced. These results have allowed us to understand better the patterns of gene expression exhibited by wild-type and mutant cultures early during the development process of spore formation.

Multiple signal transduction pathways regulate discoidin I gene expression in Dictyostelium discoideum

The expression of the discoidin I genes in Dictyostelium discoideum is regulated by the concerted action of the extracellular factors cyclic adenosine monophosphate (cAMP), folate, prestarvation factor (PSF) and conditioned media factor (CMF). However, the pathways by which these signals are transduced and the interactions between the pathways have been unexplored so far. We have analysed wild-type and mutant cells with defined lesions in signal transduction to elucidate these regulatory processes, and shown that different pathways are used for the down-regulation and induction of these genes. The cAMP receptor cARI is required for the cAMP-mediated down-regulation of discoidin I gene expression but not for the induction of discoidin I expression during development. Surprisingly, induction of the discoidin I genes requires Gα2, the G-protein subunit which is generally believed to couple to cARI, to control the expression of cAMP-inducible genes. Thus, our data suggest that Gα2 interacts with different receptors to regulate gene expression in early development. Furthermore, the analysis shows that discoidin induction in bacterially grown cells occurs in two sequential steps. The first is a low basal induction which occurs in late log-phase growth prior to starvation. PSF can induce the basal level, and the induction is independent of Gα2. The developmental induction following starvation is much stronger, dependent on Gα2 and probably signaled by CMF, which is secreted at that time. We also demonstrate that the growth history of the cells has a major influence on the pattern of gene expression and on the choice of the signal transduction pathway that is used to induce the discoidin I genes. Overall, these studies reveal complex regulatory interactions between the stimulatory and inhibitory signaling pathways controlling the expression of the discoidin I gene family.

An analysis of Xenopus tyrosine kinase genes and their expression in early development.

Xenopus laevis and X. borealis were screened for tyrosine kinase genes using the polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR and 34 X. laevis and 23 X. borealis tyrosine genes were identified. Eighteen of the genes represented novel tyrosine kinase family members. The rest could be classified into known tyrosine kinase subfamilies, of which however only three have been previously identified in Xenopus. Eight clones, including bFGFR (xFGFR1) and potential Trk, Ins R., Fak, Fyn, and Abl homologs, were used to probe temporal and spatial gene expression in early development. Quantitative RT-PCR and whole-mount and in situ hybridization showed that most of these mRNAs were present throughout development and were broadly distributed, mainly in ectodermal and mesodermal derived tissues. At the blastula stage, bFGFR mRNA was detected within the ectoderm and a gradient of expression was noted within the invaginating mesoderm. The unexpected promiscuous expression of many tyrosine kinase genes in early development is discussed.

Gene expression in single cells of Bacillus subtilis: evidence that a threshold mechanism controls the initiation of sporulation

Early during endospore formation in the bacterium Bacillus subtilis, two distinct cell types are formed. The initiation of this developmental pathway requires several physiological conditions (e.g., nutrient deprivation) and is controlled by the Spo0A transcription factor. We have found that in a culture of sporulating cells, there are two subpopulations, one that has initiated the developmental program and activated the expression of early developmental genes and one in which early developmental gene expression remains uninduced. We measured the expression of developmental (spo) genes in single cells of B. subtilis by using spo-lacZ fusions. Cells containing a spo-lacZ fusion were stained with a dye that fluoresces upon hydrolysis by beta-galactosidase, and the fluorescence in individual cells was measured with a flow cytometer. For Spo+ cells, we found that the proportion of the population expressing early developmental genes correlates well with the fraction of the population that eventually produces spores. In addition, mutations that cause a decrease in the amount of activated (phosphorylated) Spo0A transcription factor cause a decrease in the size of the subpopulation expressing early developmental genes that are directly activated by Spo0A approximately P. Again, the size of the subpopulation correlates well with the fraction of cells that produce spores. These results indicate that a threshold level of activated Spo0A (Spo0A approximately P) or of a component of the phosphorylation pathway must accumulate to induce sporulation gene expression and that most of the cells that are able to induce the expression of early genes that are directly activated by Spo0A approximately P go on to produce mature spores.(ABSTRACT TRUNCATED AT 250 WORDS)

Coupling between gene expression and DNA synthesis early during development in Bacillus subtilis.

Endospore formation in the bacterium Bacillus subtilis involves generation of two cell types, each with different developmental fates. Each cell type contains an active chromosome, and treatments that inhibit DNA synthesis at the beginning of development inhibit spore formation. We describe experiments demonstrating that gene expression early during sporulation is coupled to DNA synthesis. Expression of several genes that are induced early during sporulation, before the formation of two cell types, is inhibited when DNA synthesis is inhibited. Genes that are affected require the transcription factor encoded by spo0A for normal induction. Spo0A protein is normally activated early in development by a multicomponent phosphorylation pathway, or phospho-relay. Altered function mutations in spo0A that bypass the need for the phospho-relay allow early sporulation gene expression, even when DNA synthesis is inhibited. These results indicate that inhibition of DNA synthesis prevents activation of the Spo0A transcription factor by inhibiting a step in the phospho-relay. It seems likely that coupling early developmental gene expression to DNA synthesis is a general mechanism to prevent inappropriate or unnecessary gene expression.

Expression of early developmental genes in Dictyostelium discoideum is initiated during exponential growth by an autocrine-dependent mechanism

Throughout growth, Dictyostelium cells continuously produce an autocrine factor, PSF, that accumulates in proportion to cell density. Production of PSF declines rapidly when cells are shifted to starvation conditions, and the properties of PSF are distinct from those of regulatory factors produced by starving cells. During late exponential growth, PSF induces expression of several early developmental genes, including those for proteins important in cAMP signaling and cell aggregation. Examples are the aggregation stage cAMP receptor (cAR1), the aggregation-specific form of cyclic nucleotide phosphodiesterase, and gp24 (contact sites B). Through PSF, growing cells detect environmental conditions (cell number high, food approaching depletion) that are appropriate for production of the gene products needed to initiate aggregation and development.

Genetic analysis of oogenesis and the role of maternal gene expression in early development.

Oogenesis is a complex process of cytodifferentiation, involving a variety of cell types, that results in the production of the female gamete. In higher organisms, this gamete contributes nearly all the cytoplasm composing the developing zygote. The developmental significance of this cytoplasmic contribution has been revealed by a large body of experimental evidence indicating that the maternal genome plays a primary role in the embryo-genesis of most organisms—at least until gastrulation, the stage at which overt cellular differentiation generally begins (reviewed by Davidson, 1976).

Histone gene expression in early development of Xenopus laevis: Analysis of histone mRNA in oocytes and embryos by blot-hybridization and cell-free translation

This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis, and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3. Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.

Accumulation of individual histone mRNAs during embryogenesis of the sea urchin Strongylocentrotus purpuratus

We have determined that the stockpile of maternal histone mRNAs for both H2B and H3 in eggs of the sea urchin Strongylocentrotus purpuratus corresponds to 0.8–1 million molecules per egg. This value is a somewhat smaller quantity than had previously been estimated. On the other hand, the increase brought about by embryonic mRNA synthesis is 10- to 12-fold, rather than 4-fold. Further analysis of the time course of accumulation of these mRNAs suggests that histone gene expression in early development involves a complex interaction between activation of stockpiled maternal messengers, fluctuating rates of histone gene transcription, and alterations of histone mRNA stability. The maternal pool of H1 mRNA is considerably smaller (80,000 molecules). During cleavage, it partially catches up and peaks at about half the value for H2B and H3. We also examined previtellogenic oocytes for the presence of histone mRNAs by the same methods and were unable to detect any. Thus the stockpile of maternal histone mRNAs must accumulate after the previtellogenic stage of oogenesis.

RNA Synthesis and the Ribosomal Cistrons in Early Molluscan Development

A review of RNA synthesis patterns in molluscs points to two conclusions. First, there is no common pattern which could be characteristic of “mosaic” development. Second, among bivalves the pattern of gene expression in early development is the same as in sea urchin embryos. DNA-like RNA predominates during cleavage, including a prominent, short-lived species (about 9S) which enters light polysomes. Ribosomal RNA (rRNA) synthesis cannot be detected prior to gastrulation. New gene transcripts appear and are required for development beyond gastrulation. Among gastropods, there is variation regarding the need for embryonic gene expression for gastrulation and subsequent differentiation, and the time of onset of rRNA synthesis. The synthesis of poly(A) + RNA has been demonstrated in snail veligers. The principle limiting factor in rRNA synthesis during cleavage of molluscan and other embryos is probably the length of interphase. The time of activation of rRNA synthesis beyond this stage may be influenced by the magnitude of the rRNA stockpile. The oocytes of two species of dam with similar requirements for rRNA and with equal time available for oogenesis differ in the degree of amplification of the rRNA cistrons, suggesting that they may also differ in the redundancy or rate of transcription of those cistrons.