Effect of progressive mandibular advancement on pharyngeal airway size in anesthetized adults.

BACKGROUND: General anesthesia in adult humans is associated with narrowing or complete closure of the pharyngeal airway. The purpose of this study was to determine the effect of progressive mandibular advancement on pharyngeal airway size in normal adults during intravenous infusion of propofol for anesthesia. METHODS: Magnetic resonance imaging was performed in nine normal adults during wakefulness and during propofol anesthesia. A commercially available intraoral appliance was used to manually advance the mandible. Images were obtained during wakefulness without the appliance and during anesthesia with the participants wearing the appliance under three conditions: without mandibular advancement, advancement to 50% maximum voluntary advancement, and maximum advancement. Using computer software, airway area and maximum anteroposterior and lateral airway diameters were measured on the axial images at the level of the soft palate, uvula, tip of the epiglottis, and base of the epiglottis. RESULTS: Airway area across all four airway levels decreased during anesthesia without mandibular advancement compared with airway area during wakefulness (P < 0.007). Across all levels, airway area at 50% advancement during anesthesia was less than that at centric occlusion during wakefulness (P = 0.06), but airway area with maximum advancement during anesthesia was similar to that during wakefulness (P = 0.64). In general, anteroposterior and lateral airway diameters during anesthesia without mandibular advancement were decreased compared with wakefulness and were restored to their wakefulness values with 50% and/or maximal advancement. CONCLUSIONS: Maximum mandibular advancement during propofol anesthesia is required to restore the pharyngeal airway to its size during wakefulness in normal adults.

Aboral ectoderm-specific expression and regulation of the LpS1 genes of {\it Lytechinus pictus\/}

The Spec genes serve as molecular markers for examining the ontogeny of the aboral ectoderm lineage of sea urchin embryos. These genes are activated at late-cleavage stage only in cells contributing to the aboral ectoderm of Strongylocentrotus purpuratus and encode 14,000-17,000 Da calcium-binding proteins. A comparative analysis was undertaken to better understand the mechanisms underlying the activation and function of the Spec genes by investigating Spec homologues from Lytechinus pictus, a distantly related sea urchin. Spec antibodies cross-reacted with 34,000 Da proteins in L. pictus embryos that displayed a similar ontogenetic pattern to that of Spec proteins. One cDNA clone, LpS1, was isolated by hybridization to a synthetic oligonucleotide corresponding to a calcium-binding domain or EF-hand. The LpS1 mRNA has developmental properties similar to those of the Spec mRNAs. LpS1 encodes a 34,000 Da protein containing eight EF-hand domains, which share structural homology with the Spec EF-hands; however, little else in the protein sequence is conserved, implying that calcium-binding is important for Spec protein function. Genomic DNA blot analysis showed two LpS1 genes, LpS1$\alpha$ and LpS1$\beta$, in L. pictus. Partial gene structures for both LpS1$\alpha$ and $\beta$ were constructed based on genomic clones isolated from an L. pictus genomic library. These revealed internal duplications of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Sequencing analysis showed there was little in common among the 5$\sp\prime$-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF.^ A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5$\sp\prime$-flanking DNA from the LpS1$\beta$ gene were sufficient for correct temporal and spatial expression of reporter genes in sea urchin embryos. Deletions at the 5$\sp\prime$ end to 511, 368, or 108bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase (CAT) activity and disrupted the restricted activation of the lac Z gene in aboral ectoderm cells.^ A full-length Spec1 protein and a truncated LpS1 protein were induced and partially purified from an in vitro expression system. (Abstract shortened with permission of author.) ^

Regulation of Spec genes in aboral ectoderm cells of sea urchin {\it Strongylocentrotus purpuratus}

The Spec genes of the sea urchin Stronylocentrotus purpuratus serves as an excellent model for studying cell type-specific gene expression during early embryogenesis. The Spec1/Spec2 genes encode cytosolic calcium-binding proteins related to the calmodulin/troponin C/myosin light chain superfamily. Members of the Spec gene family are activated shortly after the sixth cleavage as the lineage-specific founder cells giving rise to aboral ectoderm are established, and the accumulation of the Spec mRNAs is limited exclusively to aboral ectoderm cell lineages. In this dissertation, the transcriptional regulation of the Spec genes was studied. Sequence comparisons of the Spec gene 5$\sp\prime$ flanking regions showed that a DNA block of approximately 800 bp from the 3$\sp\prime$ end of the first exon to the 5$\sp\prime$ end of a repetitive DNA element, termed RSR, was highly conserved. In Spec2a, the conserved region was a continuous stretch of DNA, but in Spec1 and Spec2c, DNA insertions interrupt the conserved sequence block and alter the relative placement of the RSR element and other 5$\sp\prime$ flanking DNA. Thus, drastic rearrangements have occurred within the putative control regions of the Spec genes. In vivo expression experiments using the sea urchin embryo gene-transfer system showed that while the 5$\sp\prime$ flanking regions of all three Spec genes conferred proper temporal activation to the reporter CAT gene, only the Spec2a 5$\sp\prime$ flanking region could restrict lacZ gene expression to aboral ectoderm cells. However, the Spec2a conserved region alone was not sufficient to confer proper spatial expression, suggesting that negative spatial elements are also associated with the proper activation of Spec2a. A major positive regulatory region, defined as the RSR enhancer, was identified between base pairs $-$631 and $-$443 on Spec2a. The RSR enhancer was essential for maximal activity and conferred preferential aboral ectoderm expression to a lacZ reporter gene. DNaseI footprinting and band-shift analysis of the RSR enhancer revealed multiple DNA-elements. One of the elements, an A/T-rich sequence called the A/T palindrome was studied in detail. This element binds a single 45-kDa nuclear protein, the A/T palindrome binding protein (A/TBP), whose DNA-binding specificity suggests a possible relationship with the bicoid-class homeodomain proteins. Mutated A/T palindromes are incapable of binding the 45-kDa protein and lower promoter activity by 8-fold. DNA-binding activity for A/TBP is low in unfertilized eggs, increases by the 16-cell stage and continues rising in blastulae. These data suggest that A/TBP plays a major role in the activation of the Spec2a gene in aboral ectoderm cells. ^

TH and XFKBP12 interact and regulate proliferation and differentiation in neural ectoderm through the TOR signaling pathway

During development, embryos must carefully integrate the processes of cell proliferation and differentiation. TH has been identified in Xenopus laevis as a gene product that functions in regulating differentiation of the neural ectoderm through its effect on cell proliferation. However, the mechanism and molecular pathway through which TH functions are not known. We identified the Xenopus FK506 binding protein homolog (XFKBP12) as a protein that interacted with TH in a yeast two-hybrid screen with TH as the bait. The direct and specific interaction between TH and XFKBP12 was supported by several tests including CO-IP, drug competence assay and mutagenesis analysis. To investigate the function of XFKBP12 during embryogenesis, we created an XFKBP12 loss of function embryo using antisense morpholino oligonucleotides (MO). XFKBP12 MO injected embryos displayed similar phenotypes as TH depleted embryos. We also demonstrated that both TH and XFKBP12 functioned through the TOR signaling pathway which is a target for cancer therapies. The interaction between TH and XFKBP 12 was required to regulate the proliferation of neural cells. Therefore, our study indicates that TH represents the endogenous ligand of XFKBP12 and together they coordinate neural cell proliferation and differentiation through the conserved rapamycin sensitive TOR pathway. Thus, understanding how this pathway functions in development will not only provide us important insights into the relationship between proliferation and differentiation, but help design rational cancer therapies targeting this pathway. ^

Functional analysis of the sea urchin {\it orthodenticle\/} -related gene, {\it SPOTX\/}, in aboral ectoderm-specific gene activation and aboral ectoderm cell fate specification

An important question in developmental biology is how embryonic cell types are derived from a fertilized egg. To address this question, this thesis investigates the mechanisms by which the aboral ectoderm-specific Spec2a gene is spatially and temporally regulated during sea urchin embryogenesis. The Spec2a gene of the sea urchin Strongylocentratus purpuratus has served as a valuable maker to understand the basis of lineage-specific gene activation and the role of transcription factors in cell fate specification. The hypothesis is that transcription factors responsible for cell type-specific gene activation are key components in the initial cell specification step. The Spec2a gene, which encodes a small cytosolic calcium-binding protein, is expressed exclusively in aboral ectoderm cell lineages. The 1516-bp control region of the Spec2a gene contains a 188-bp enhancer element required for temporal activation and aboral ectoderm/mesenchyme cell expression, while an unidentified element upstream of the enhancer represses expression in mesenchyme cells. Using an enhancer activation assay, combined with site-directed mutagenesis, I showed that three TAATCC/T sites within the enhancer are responsible for enhancer activity. Mutagenizing these sites and a fourth one just upstream abolished all activity from the Spec2a control region. A 77-bp DNA fragment from the Spec2a enhancer containing two of the TAATCC/T sites is sufficient for aboral ectoderm/mesenchyme cell expression. A cDNA encoding SpOtx, an orthodenticle-related protein, was cloned from S. purpuratus and shown to bind with high affinity to the TAATCC/T sequences within the Spec2a control region. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells, suggesting that SpOtx might play a role in the initial temporal activation of the Spec2a gene and most likely has additional functions in the developing embryo. To reveal the broader biological functions of SpOtx, I injected SpOtx mRNA into living sea urchin eggs to determine what effects overexpressing the SpOtx protein might have on embryo development. SpOtx mRNA-injected embryos displayed dramatic alterations in development. Instead of developing into pluteus larvae with 15 different cell types, uniform epithelia balls were formed. These balls consisted of a thin layer of squamous cells with short cilia highly reminiscent of aboral ectoderm. Immunohistochemical staining and RT-PCR demonstrated that the SpOtx-injected embryoids expressed aboral ectoderm markers uniformly, but showed very weak or no expression of markers for non-aboral ectoderm cell types. These data strongly suggested that overexpression of SpOtx redirected the normal fate of non-aboral ectoderm cells to that of aboral ectoderm. These results show that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate. ^

Evolution of cis-regulatory elements in a repetitive sequence adjacent to a sea urchin aboral ectoderm -specific gene

The creation, preservation, and degeneration of cis-regulatory elements controlling developmental gene expression are fundamental genome-level evolutionary processes about which little is known. In this study, critical differences in cis-regulatory elements controlling the expression of the sea urchin aboral ectoderm-specific spec genes were identified and explored. In genomes of species within the Strongylocentrotidae family, multiple copies of a repetitive sequence element termed RSR were present, but RSRs were not detected in genomes of species outside Strongylocentrotidae. RSRs are invariably associated with spec genes, and in Strongylocentrotus purpuratus, the spec2a RSR functioned as a transcriptional enhancer displaying greater activity than RSRs from the spec1 or spec2c paralogs. Single base-pair differences at two cis-regulatory elements within the spec2a RSR greatly increased the binding affinities of four transcription factors: SpCCAAT-binding factor at one element and SpOtx, SpGoosecoid, and SpGATA-E at another. The cis-regulatory elements to which SpCCAAT-binding factor, SpOtx, SpGoosecoid, and SpGATA-E bound were recent evolutionary acquisitions that could act either to activate or repress transcription, depending on the cell type. These elements were found in the spec2a RSR ortholog in Strongylocentrotus pallidus but not in the RSR orthologs of Strongylocentrotus droebachiensis or Hemicentrotus pulcherrimus. These results indicate that spec genes exhibit a dynamic pattern of cis-regulatory element evolution while stabilizing selection preserves their aboral ectoderm expression domain. ^

Molecular basis of Corynebacterium diphtheriae virulence and infection in the Caenorhabditis elegans model host

Corynebacterium diphtheriae is the causative agent of cutaneous and pharyngeal diphtheria in humans. While lethality is certainly caused by diphtheria toxin, corynebacterial colonization may primarily require proteinaceous fibers called pili, which mediate adherence to specific tissues. The type strain of C. diphtheriae possesses three distinct pilus structures, namely the SpaA, SpaD, and SpaH-type pili, which are encoded by three distinct pilus gene clusters. The pilus is assembled onto the bacterial peptidoglycan by a specific transpeptidase enzyme called sortase. Although the SpaA pili are shown to be specific for pharyngeal cells in vitro, little is known about functions of the three pili in bacterial pathogenesis. This is mainly due to lack of in vivo models of corynebacterial infection. As an alternative to mouse models as mice do not have functional receptors for diphtheria toxin, in this study I use Caenorhabditis elegans as a model host for C. diphtheriae. A simple C. elegans model would be beneficial in determining the specific role of each pilus-type and the literature suggests that C. elegans infection model can be used to study a variety of bacterial species giving insight into bacterial virulence and host-pathogen interactions. My study examines the hypothesis that pili and toxin are major virulent determinants of C. diphtheriae in the C. elegans model host.

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis Kim T. Cardenas The thymus and parathyroid glands originate from organ-specific domains of 3rd pharyngeal pouch (PP) endoderm. At embryonic day 11.5 (E11.5), the ventral thymus and dorsal parathyroid domains can be identified by Foxn1 and Gcm2 expression respectively. Neural crest cells, (NCCs) play a role in regulating patterning of 3rd PP endoderm. In addition, pharyngeal endoderm influences fate determination via secretion of Sonic hedgehog (Shh), a morphogen required for Gcm2 expression and generation of the parathyroid domain. Gcm2 is a downstream target of the transcription factor Tbx1, which in turn is positively regulated by Shh. Although initially expressed throughout pharyngeal pouch endoderm, Tbx1 expression is excluded from the thymus-specific domain of the 3rd PP by E10.5, but persists in the parathyroid domain. Based on these observations, we hypothesized that Tbx1 expression is non-permissive for thymus fate specification and that enforced expression of Tbx1 in the fetal thymus would impair thymus development. To test this hypothesis, we generated knock-in mice containing a Cre-inducible allele that allows for tissue-specific Tbx1 expression. Expression of the R26iTbx1 allele in fetal and adult thymus using Foxn1Cre resulted in severe thymus hypoplasia throughout ontogeny that persisted in the adult. Thymic epithelial cell (TEC) development was impaired as determined by immunohistochemical and FACS analysis of various differentiation markers. The relative level of Foxn1 expression in fetal TECs was significantly reduced. TECs in R26iTbx1/+ thymi assumed an almost universal expression of Plet-1, a marker associated with a TEC stem/progenitor cell fate. In addition, embryonic R26iTbx1/+ mice develop a perithymic mesechymal capsule that appears expanded compared to control littermates. Interestingly, thymi from neonatal and adult R26iTbx1/+ but not R26+/+ mice were encased in adipose tissue. This thymic phenotype also correlated with a decrease in thymocyte cellularity and aberrant thymocyte differentiation. The results to date support the conclusion that enforced expression of Tbx1 in TECs antagonizes their differentiation and prevents normal organogenesis via both direct and indirect effects.

Gene expression in sea urchin development: Study of {\it LpS1\/} gene regulation and cloning and characterization of the {\it SpKrox}-{\it 1\/} gene

Cell differentiation are associated with activation of cell lineage-specific genes. The $LpS{\it 1}\beta$ gene of Lytechinus pictus is activated at the late cleavage stage. $LpS{\it 1}\beta$ transcripts accumulate exclusively in aboral ectoderm lineages. Previous studies demonstrated two G-string DNA-elements, proximal and distal G-strings, which bind to an ectoderm-enriched nuclear factor. In order to define the cis-elements which control positive expression of the $LpS{\it 1}\beta$ gene, the regulatory region from $-$108 to +17 bp of the $LpS{\it 1}\beta$ gene promoter was characterized. The ectoderm G-string factor binds to a G/C-rich region larger than the G-string itself and the binding of the G-string factor requires sequences immediately downstream from the G-string. These downstream sequences are essential for full promoter activity. In addition, only 108 bp of $LpS{\it 1}\beta\ 5\sp\prime$ flanking DNA drives $LpS{\it 1}\beta$ gene expression in aboral ectoderm/mesenchyme cells. Therefore, for positive control of $LpS{\it 1}\beta$ gene expression, two regions of 5$\sp\prime$ flanking DNA are required: region I from base pairs $-$762 to $-$511, and region II, which includes the G/C-rich element, from base pairs $-$108 to $-$61. A mesenchyme cell repressor element is located within region I.^ DNA-binding proteins play key roles in determination of cell differentiation. The zinc finger domain is a DNA-binding domain present in many transcription factors. Based on homologies in zinc fingers, a zinc finger-encoding gene, SpKrox-1, was cloned from S. purpuratus. The putative SpKrox-1 protein has all structural characteristics of a transcription factor: four zinc fingers for DNA binding; acidic domain for transactivation; basic domain for nuclear targeting; and leucine zipper for dimerization. SpKrox-1 RNA transcripts showed a transient expression pattern which correlates largely with early embryonic development. The spatial expression of SpKrox-1 mRNA was distributed throughout the gastrula and larva ectodermal wall. However, SpKrox-1 was not expressed in pigment cells. The SpKrox-1 gene is thus a marker of a subset of SMCs or ectoderm cells. The structural features, and the transient temporal and restricted spatial expression patterns suggest that SpKrox-1 plays a role in a specific developmental event. ^

Molecular regulatory mechanisms and embryonic tissue specification in sea urchin development

To understand how a eukaryote achieves differential transcription of genes in precise spatial patterns, the molecular details of tissue specific expression of the Strongylocentrotus purpuratus Spec2a gene were investigated by functional studies of the cis-regulatory components in the upstream enhancer. Regional activation of Spec2a in the aboral ectoderm is conferred by a combination of activators and repressors. The positive regulators include previously identified SpOtx and a trans-regulatory factor binding at the CCAAT site in the Spec2a enhancer. The nuclear protein binding to the CCAAT box was determined to be the heterotrimeric CCAAT binding factor (SpCBF). SpCBF also mediates general activation in the ectoderm. The negative regulators consist of an oral ectoderm repressor (OER), an endoderm repressor (ENR), and an S. Purpuratus goosecoid homologue (SpGsc). OER functions to prevent expression in the oral ectoderm, while ENR is required to repress endoderm expression. SpGsc antagonizes the SpOtx function by competing for binding at SpOtx target genes in oral ectoderm, where it functions as an active repressor. Thus, SpOtx and SpGsc perform collectively to establish and maintain the oral-aboral axis. Finally, purification of ENR and OER proteins from sea urchin blastula stage nuclear extracts was performed using site-specific DNA-affmity chromatography. ^

Molecular and genetic analyses of Fgf signaling during cardiac outflow tract development

Epithelial-mesenchymal tissue interactions regulate the development of derivatives of the caudal pharyngeal arches (PAs) to govern the ultimate morphogenesis of the aortic arch and outflow tract (OFT) of the heart. Disruption of these signaling pathways is thought to contribute to the pathology of a significant proportion of congenital cardiovascular defects in humans. In this study, I tested whether Fibroblast Growth Factor 15 (Fgf15), a secreted signaling molecule expressed within the PAs, is an extracellular mediator of tissue interactions during PA and OFT development. Analyses of Fgf15−/− mouse embryonic hearts revealed abnormalities primarily localized to the OFT, correlating with aberrant cardiac neural crest cell behavior. The T-box-containing transcription factor Tbx1 has been implicated in the cardiovascular defects associated with the human 22q11 Deletion Syndromes, and regulates the expression of other Fgf family members within the mouse PAs. However, expression and genetic interaction studies incorporating mice deficient for Tbx1, its upstream regulator, Sonic Hedgehog (Shh), or its putative downstream effector, Fgf8, indicated that Fgf15 functions during OFT development in a manner independent of these factors. Rather, analyses of compound mutant mice indicated that Fgf15 and Fgf9, an additional Fgf family member expressed within the PAs, genetically interact, providing insight into the factors acting in conjunction with Fgf15 during OFT development. Finally, in an effort to further characterize this Fgf15-mediated developmental pathway, promoter deletion analyses were employed to isolate a 415bp sequence 7.1Kb 5′ to the Fgf15 transcription start site both necessary and sufficient to drive reporter gene expression within the epithelium of the PAs. Sequence comparisons among multiple mammalian species facilitated the identification of evolutionarily conserved potential trans-acting factor binding sites within this fragment. Subsequent studies will investigate the molecular pathway(s) through which Fgf15 functions via identification of factors that bind to this element to govern Fgf15 gene expression. Furthermore, targeted deletion of this element will establish the developmental requirement for pharyngeal epithelium-derived Fgf15 signaling function. Taken as a whole, these data demonstrate that Fgf15 is a component of a novel, Tbx1-independent molecular pathway, functioning within the PAs in a manner cooperative with Fgf9, required for proper development of the cardiac OFT. ^

A novel role for neural crest cells in thymus organogenesis

Classical ablation studies have shown that neural crest cells (NCC) are critical for thymus organogenesis, though their role in this process has never been determined. We have used a mouse model deficient in NCC near the thymus rudiment to investigate the role of NCC in thymus organogenesis. Splotch mice exhibit a lack of NCC migration due to mutation in the gene encoding the transcription factor Pax 3. Homozygous mutants, designated Pax3Sp/Sp, display a range of phenotypes including spina bifida, cardiac outflow tract deformities, and craniofacial deformities. Pax3Sp/Sp, mice have also been reported to have hypoplastic and abnormal thymi, which is consistent with the expected result based on the classical ablation studies. However, in contrast to the dogma, we find that the thymus lobes in Pax3Sp/Sp, mice are even larger in size than those of littermate controls, although they fail to migrate and are therefore ectopic. Differentiation of the thymic epithelial compartments occurs normally, including the ability to import hematopoietic precursors, until the embryos die at embryonic day E13.0. We also investigated the patterning of the third pharyngeal pouch which gives rise to both the thymus and the parathyroid. Using RNA probes to detect expression of transcription factors exclusively expressed in the ventral, thymus- or dorsal, parathyroidfated domains of the E11.5 third pouch, we show that the parathyroid domain is restricted and the thymus-fated domain is expanded in Pax3Sp/Sp, embryos. Furthermore, mixing of the boundary between these domains occurs at E12.0. These results necessitate reconsideration of the previously accepted role for NCC in thymus organogenesis. NCC are not required for outgrowth of the thymus up to E13.0, and most strikingly, we have discovered a novel role for NCC in establishing parathyroid versus thymus fate boundaries in the third pharyngeal pouch. ^

Functions of GCN5 and P /CAF acetyltransferases in mammalian development

Histone acetyltransferases are important chromatin modifiers that function as transcriptional co-activators. The identification of the transcriptional regulator GCN5 as the first nuclear histone acetyltransferase in yeast directly linked chromatin remodeling to transcriptional regulation. Although emerging evidence suggests that acetyltransferases participate in multiple cellular processes, their roles in mammalian development remain undefined. In this study, I have cloned and characterized the mouse homolog of GCN5 and a closely related protein P/CAF that interacts with p300/CBP. In contrast to yeast GCN5, but similar to P/CAF, mouse GCN5 possesses an additional N-terminal domain that confers the ability to acetylate nucleosomal histones. GCN5 and P/CAF exhibit identical substrate specificity and both interact with p300/CBP. Interestingly, expression levels of GCN5 and P/CAF display a complementary pattern in mouse embryos and in adult tissues, suggesting that they have distinct tissue or developmental stage specific roles. To define the in vivo function of GCN5 and P/CAF, I have generated mice that are nullizygous for GCN5 or P/CAF. P/CAF null mice are viable and fertile with no gross morphological defects, indicating that P/CAF is dispensable for development and p300/CBP function in vivo. In contrast, mice lacking GCN5 die between 10.5–11 days of gestation. GCN5 null mice are severely retarded but have anterior ectopic outgrowth. Molecular marker analyses reveal that early mesoderm is formed in GCN5 null mice but further differentiation into distinct mesodermal lineages is perturbed. While presomitic mesoderm and chodamesoderm are missing in GCN5 mutant mice, extraembryonic tissues and lateral mesoderm are unaffected. This is consistent with our finding that GCN5 expression is absent in the heart and extraembryonic tissues but is uniform throughout the rest of the embryo. Remarkably, GCN5 mutant mice exhibit an unusually high incidence of apoptosis in the embryonic ectoderm and mesoderm. Finally, mice doubly null for GCN5 and P/CAF die much earlier than mice harboring the GCN5 mutation alone, suggesting that P/CAF and GCN5 share some overlapping function during embryogenesis. This work is the first study to show that specific acetyltransferase is important for cell survival as well as mesoderm differentiation or maintenance during early mammalian development. ^