An insight into spectral composition of light available for photosynthesis via remotely assessed absorption coefficient at leaf and canopy levels.

Non-invasive comparative analysis of the spectral composition of energy absorbed by crop species at leaf and plant levels was carried out using the absorption coefficient retrieved from leaf and plant reflectance as an informative metric. In leaves of three species with contrasting leaf structures and photosynthetic pathways (maize, soybean, and rice), the blue, green, and red fractions of leaf absorption coefficients were 48, 20, and 32%, respectively. The fraction of green light in the total budget of light absorbed at the plant level was higher than at the leaf level approaching the size of the red fraction (24% green vs. 25.5% red) and surpassing it inside the canopy. The plant absorption coefficient in the far-red region (700-750 nm) was significant reaching 7-10% of the absorption coefficient in green or red regions. The spectral composition of the absorbed light in the three species was virtually the same. Fractions of light in absorbed PAR remained almost invariant during growing season over a wide range of plant chlorophyll content. Fractions of absorption coefficient in the green, red, and far-red were in accord with published results of quantum yield for CO2 fixation on an absorbed light basis. The role of green and far-red light in photosynthesis was demonstrated in simple experiments in natural conditions. The results show the potential for using leaf and plant absorption coefficients retrieved from reflectance to quantify photosynthesis in each spectral range.

The femoral deformity in Blount's disease: a comparative study of infantile, juvenile and adolescent Blount's disease.

An associated femoral deformity in patients with Blount's disease is not commonly described in the literature. The objective of this study is to establish the presence and magnitude of deformity in the coronal plane of the distal femur in children of all ages with Blount's disease and compare this to a matched cohort of children from the same population. This was a retrospective review of patients from an academic hospital. A control group was established by matching for age and gender from a group of unaffected patients with mid to proximal third femur fractures or controls at the same hospital. Study participants were categorized by age at onset of deformity as follows: infantile (<4 years), juvenile (4-10 years) and adolescent (>10 years). The measurements of the anatomic lateral distal femoral angle (aLDFA) were recorded and the Wilcoxon rank-sum test was used to determine statistically significant differences in the LFDA between children with Blount's disease and those without. Seventy-two Black African children were included in the study with 118 affected limbs (27 infantile, 55 juvenile and 36 adolescent). The overall average aLDFA for all patients with Blount's disease was 87° (range 73-100°). Overall, children with Blount's disease had a higher aLDFA than children without (87° vs. 82°). There was a significant association between LDFA in the control group and children with Blount's disease in each of the three groups. This study found distal femoral varus deformity to be present in all groups of children with Blount's disease. In this study population, it was most significant in the infantile and adolescent groups when compared to a control group from the same population. Although further studies are required, the surgeon must always assess the distal femoral component in treating children with Blount's disease.

Assessment of the reliability and reproducibility of the Langenskiöld classification in Blount's disease.

The Langenskiöld classification is the most commonly utilized classification system for the radiological features of Blount's disease. Although there is only a single study found on the interobserver variability and none found on the intraobserver variability, it is commonly used for prognostication and guiding management decisions. The aim of this study was to determine the reliability and reproducibility of the Langenskiöld classification. A retrospective review of radiographs was done of patients treated for infantile and juvenile Blount's disease at Chris Hani Baragwanath Academic Hospital from 2006 to 2016. There were 70 radiographs of acceptable quality, which were reviewed and staged on two occasions according to the Langenskiöld classification by three orthopaedic consultants and three orthopaedic surgery senior residents. Pearson correlation coefficients, percentage agreements, and κ statistics were used to evaluate both the reliability and reproducibility. Of the 70 images staged, only two (2.9%) were staged the same by all six observers, and 20 (28.6%) images differed by a single stage. The consultants had 17 (24.3%) images staged the same whereas the residents had 12 (17.1%) images staged the same. The overall κ for all six observers showed a fair agreement of 0.24. Again, the consultants had a higher κ-value compared to residents of 0.25 and 0.24, respectively. The reproducibility amongst all observers was fair with a κ-value of 0.38. The consultants had a higher mean score of 0.48 compared to 0.26 for the residents. There was only a fair overall reliability and reproducibility amongst the six observers. We recommend the Langenskiöld classification be used with caution when being used for prognostication and management planning as well as when interpreting any research relying on this classification. Level of evidence: Level III, diagnostic study.

Prognostication in Developmental Dysplasia of the Hip Using the Ossific Nucleus Center Edge Angle.

BACKGROUND: The aim of this paper was to determine the prognostic potential of the ossific nucleus center edge angle (ONCEA) in patients below 5 years of age treated for developmental dysplasia of the hip (DDH) to predict final outcome and the need for a secondary procedure. METHODS: The interobserver and intraobserver reliability was calculated using the intraclass correlation coefficient for measurement of the ONCEA. The ONCEA was divided a priori into 3 groups: group A≥10 degrees, group B -9 to 9 degrees, and group C ≤-10 degrees. Final outcome was measured using the McKay score and Severin classification. The presence of osteonecrosis was recorded at final follow-up. RESULTS: One hundred one patients with 133 dislocated hips underwent closed or open reduction for DDH. Mean age at presentation was 19 months with a mean age at final follow-up of 12.4 years. A significant difference was shown in a comparison of the 3 ONCEA groups using the McKay score, Severin classification, and need for a secondary procedure. Eighty seven of the 101 patients underwent ONCEA reliability measurements. The ONCEA was shown to have a mean intrarater reliability of 0.89, and a mean interrater reliability of 0.77. CONCLUSIONS: The ONCEA is a reliable measurement in predicting medium-term outcome of the hip post reduction in children under the age of 5 years with DDH and might be useful as a predictor for a secondary procedure before the age of 5 years. LEVEL OF EVIDENCE: Level III-prognostic case control study. CLINICAL RELEVANCE: This case control study shows the importance of measuring the ONCEA within 6 months of removing the final cast after reduction of a dislocated hip and its implications for further management and outcome.

Vitamin D Status in Blount Disease.

INTRODUCTION: Blount disease can be defined as idiopathic proximal tibial vara. Several etiologies including the mechanical theory have been described. Obesity is the only causative factor proven to be associated with Blount disease. The aim of this study is to assess if there is an association of vitamin D deficiency and Blount disease. METHODS: This a retrospective study of preoperative and postoperative patients with Blount disease who were screened for vitamin D deficiency. Patients with genu varum due to confirmed vitamin D deficiency and rickets were excluded. The study patients had the following blood tests done: calcium, phosphate, alkaline phosphatase, parathyroid, and 25-hydroxyvitamin D (25(OH)D) hormones. RESULTS: We recruited 50 patients. The mean age of these patients was 10.4 years (SD±3.88) with average body mass index of 28.7 kg/m (±10.2). Thirty (60%) patients were diagnosed with infantile, 4 (8%) juvenile, and 16 (32%) adolescent Blount disease. Eight (16%) patients were found to be vitamin D deplete (25(OH)D levels <50 nmol/L). Of these, 8 patients, 6 were insufficient (25(OH)D levels between 30 and 50 nmol/L) and the other 2 were deficient (25(OH)D levels <30 nmol/L). CONCLUSIONS: This study showed that the prevalence of vitamin D deficiency in children with Blount disease was similar to that of healthy children living in Johannesburg. There is no evidence that vitamin D deficiency is a factor in causing Blount disease. LEVEL OF EVIDENCE: Level III-retrospective study.

Effects of water-saving irrigation practices and drought resistant rice variety on greenhouse gas emissions from a no-till paddy in the central lowlands of China.

As pressure on water resources increases, alternative practices to conserve water in paddies have been developed. Few studies have simultaneously examined the effectiveness of different water regimes on conserving water, mitigating greenhouse gases (GHG), and maintaining yields in rice production. This study, which was conducted during the drought of 2013, examined all three factors using a split-plot experiment with two rice varieties in a no-till paddy managed under three different water regimes: 1) continuous flooding (CF), 2) flooded and wet intermittent irrigation (FWI), and 3) flooded and dry intermittent irrigation (FDI). The Methane (CH4) and nitrous oxide (N2O) emissions were measured using static chamber-gas measurements, and the carbon dioxide (CO2) emissions were monitored using a soil CO2 flux system (LI-8100). Compared with CF, FWI and FDI irrigation strategies reduced CH4 emissions by 60% and 83%, respectively. In contrast, CO2 and N2O fluxes increased by 65% and 9%, respectively, under FWI watering regime and by 104% and 11%, respectively, under FDI managed plots. Although CO2 and N2O emissions increased, the global warming potential (GWP) and greenhouse gas intensity (GHGI) of all three GHG decreased by up to 25% and 29% (p<0.01), respectively, using water-saving irrigation strategies. The rice variety also affected yields and GHG emissions in response to different water regimes. The drought-resistance rice variety (HY3) was observed to maintain yields, conserve water, and reduce GHG under the FWI irrigation management compared with the typical variety (FYY299) planted in the region. The FYY299 only had significantly lower GWP and GHGI when the yield was reduced under FDI water regime. In conclusion, FWI irrigation strategy could be an effective option for simultaneously saving water and mitigating GWP without reducing rice yields using drought-resistant rice varieties, such as HY3.

Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division.

In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

Epidemiology of acute osteoarticular sepsis in a setting with a high prevalence of pediatric HIV infection.

BACKGROUND: There is limited information on osteoarticular infections in human immunodeficiency virus (HIV)-infected children. The purpose of this study was to determine the effect of HIV on the epidemiology of osteoarticular infections in a setting with a high prevalence of pediatric HIV infection. METHODS: A retrospective evaluation of children presenting with acute septic arthritis or osteomyelitis from June 2005 to July 2009 was undertaken. Standard departmental protocols for the management of osteoarticular infections, including testing for HIV, were practised. RESULTS: A total of 102 cases of acute septic arthritis or osteomyelitis were identified during the study period. These included 22 (21.6%) episodes in HIV-infected children, 66 (64.7%) in HIV-non-infected children, and 14 (13.7%) cases in whom the HIV status was unknown. The median age of children was 30.6 months (range, 9.2 to 82.9 mo) and did not differ by HIV status. Streptococcus pneumoniae was identified in 8 of 12 (66.7%) HIV-infected children compared with 3 (9.7%) of 31 HIV-non-infected children (P<0.001). Conversely, fewer episodes in HIV-infected children (4.8%) were associated with Staphylococcus aureus compared with HIV-non-infected children (24.6%; P=0.06). No patients died. Twelve cases required repeated surgical procedures. CONCLUSIONS: Empirical management of osteoarticular infections in settings with a high prevalence of HIV-infected children or children known to be HIV infected needs to be tailored based on a higher proportion of episodes being due to S. pneumoniae in HIV-infected children. CLINICAL RELEVANCE: Our results suggest that HIV-infected children with osteoarticular infections should be started on broader spectrum antibiotics before culture results are available. LEVEL OF EVIDENCE: Level IV, diagnostic study.

Genome sequence of Haloplasma contractile, an unusual contractile bacterium from a deep-sea anoxic brine lake.

We present the draft genome of Haloplasma contractile, isolated from a deep-sea brine and representing a new order between Firmicutes and Mollicutes. Its complex morphology with contractile protrusions might be strongly influenced by the presence of seven MreB/Mbl homologs, which appears to be the highest copy number ever reported.

Nodal-mediated epigenesis requires dynamin-mediated endocytosis.

Nodal proteins are diffusible morphogens that drive pattern formation via short-range feedback activation coupled to long-range Lefty-mediated inhibition. In the sea urchin embryo, specification of the secondary (oral-aboral) axis occurs via zygotic expression of nodal, which is localized to the prospective oral ectoderm at early blastula stage. In mid-blastula stage embryos treated with low micromolar nickel or zinc, nodal expression expands progressively beyond the confines of this localized domain to encompass the entire equatorial circumference of the embryo, producing radialized embryos lacking an oral-aboral axis. RNAseq analysis of embryos treated with nickel, zinc, or cadmium (which does not radialize embryos) showed that several genes involved in endocytosis were similarly perturbed by nickel and zinc but not cadmium. Inhibiting dynamin, a GTPase required for receptor-mediated endocytosis, phenocopies the effects of nickel and zinc, suggesting that dynamin-mediated endocytosis is required as a sink to limit the range of Nodal signaling.

The evolution of Runx genes II. The C-terminal Groucho recruitment motif is present in both eumetazoans and homoscleromorphs but absent in a haplosclerid demosponge.

BACKGROUND: The Runt DNA binding domain (Runx) defines a metazoan family of sequence-specific transcription factors with essential roles in animal ontogeny and stem cell based development. Depending on cis-regulatory context, Runx proteins mediate either transcriptional activation or repression. In many contexts Runx-mediated repression is carried out by Groucho/TLE, recruited to the transcriptional complex via a C-terminal WRPY sequence motif that is found encoded in all heretofore known Runx genes. FINDINGS: Full-length Runx genes were identified in the recently sequenced genomes of phylogenetically diverse metazoans, including placozoans and sponges, the most basally branching members of that clade. No sequences with significant similarity to the Runt domain were found in the genome of the choanoflagellate Monosiga brevicollis, confirming that Runx is a metazoan apomorphy. A contig assembled from genomic sequences of the haplosclerid demosponge Amphimedon queenslandica was used to construct a model of the single Runx gene from that species, AmqRunx, the veracity of which was confirmed by expressed sequences. The encoded sequence of the Runx protein OscRunx from the homoscleromorph sponge Oscarella carmella was also obtained from assembled ESTs. Remarkably, a syntenic linkage between Runx and Supt3h, previously reported in vertebrates, is conserved in A. queenslandica. Whereas OscRunx encodes a C-terminal Groucho-recruitment motif, AmqRunx does not, although a Groucho homologue is found in the A. queenslandica genome. CONCLUSION: Our results are consistent with the hypothesis that sponges are paraphyletic, and suggest that Runx-WRPY mediated recruitment of Groucho to cis-regulatory sequences originated in the ancestors of eumetazoans following their divergence from demosponges.

Oral-aboral axis specification in the sea urchin embryo III. Role of mitochondrial redox signaling via H2O2.

In sea urchin embryos, specification of the secondary (oral-aboral) axis occurs via nodal, expression of which is entirely zygotic and localized to prospective oral ectoderm at blastula stage. The initial source of this spatial anisotropy is not known. Previous studies have shown that oral-aboral (OA) polarity correlates with a mitochondrial gradient, and that nodal activity is dependent both on mitochondrial respiration and p38 stress-activated protein kinase. Here we show that the spatial pattern of nodal activity also correlates with the mitochondrial gradient, and that the latter correlates with inhomogeneous levels of intracellular reactive oxygen species. To test whether mitochondrial H(2)O(2) functions as a redox signal to activate nodal, zygotes were injected with mRNA encoding either mitochondrially-targeted catalase, which quenches mitochondrial H(2)O(2) and down-regulates p38, or superoxide dismutase, which augments mitochondrial H(2)O(2) and up-regulates p38. Whereas the former treatment inhibits the initial activation of nodal and entrains OA polarity toward aboral when confined to half of the embryo via 2-cell stage blastomere injections, the latter does not produce the opposite effects. We conclude that mitochondrial H(2)O(2) is rate-limiting for the initial activation of nodal, but that additional rate-limiting factors, likely also involving mitochondria, contribute to the asymmetry in nodal expression.

Runx expression is mitogenic and mutually linked to Wnt activity in blastula-stage sea urchin embryos.

BACKGROUND: The Runt homology domain (Runx) defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR) studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP) indicates that Runx target sites within 5' sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP) reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545-558, 2005) is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK)-3. CONCLUSIONS/SIGNIFICANCE: These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

Cis-regulatory control of the nodal gene, initiator of the sea urchin oral ectoderm gene network.

Expression of the nodal gene initiates the gene regulatory network which establishes the transcriptional specification of the oral ectoderm in the sea urchin embryo. This gene encodes a TGFbeta ligand, and in Strongylocentrotus purpuratus its transcription is activated in the presumptive oral ectoderm at about the 30-cell stage. Thereafter Nodal signaling occurs among all cells of the oral ectoderm territory, and nodal expression is required for expression of oral ectoderm regulatory genes. The cis-regulatory system of the nodal gene transduces anisotropically distributed cytoplasmic cues that distinguish the future oral and aboral domains of the early embryo. Here we establish the genomic basis for the initiation and maintenance of nodal gene expression in the oral ectoderm. Functional cis-regulatory control modules of the nodal gene were identified by interspecific sequence conservation. A 5' cis-regulatory module functions both to initiate expression of the nodal gene and to maintain its expression by means of feedback input from the Nodal signal transduction system. These functions are mediated respectively by target sites for bZIP transcription factors, and by SMAD target sites. At least one SMAD site is also needed for the initiation of expression. An intron module also contains SMAD sites which respond to Nodal feedback, and in addition acts to repress vegetal expression. These observations explain the main features of nodal expression in the oral ectoderm: since the activity of bZIP factors is redox sensitive, and the initial polarization of oral vs. aboral fate is manifested in a redox differential, the bZIP sites account for the activation of nodal on the oral side; and since the immediate early signal transduction response factors for Nodal are SMAD factors, the SMAD sites account for the feedback maintenance of nodal gene expression.

The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus.

A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model.

The sea urchin kinome: a first look.

This paper reports a preliminary in silico analysis of the sea urchin kinome. The predicted protein kinases in the sea urchin genome were identified, annotated and classified, according to both function and kinase domain taxonomy. The results show that the sea urchin kinome, consisting of 353 protein kinases, is closer to the Drosophila kinome (239) than the human kinome (518) with respect to total kinase number. However, the diversity of sea urchin kinases is surprisingly similar to humans, since the urchin kinome is missing only 4 of 186 human subfamilies, while Drosophila lacks 24. Thus, the sea urchin kinome combines the simplicity of a non-duplicated genome with the diversity of function and signaling previously considered to be vertebrate-specific. More than half of the sea urchin kinases are involved with signal transduction, and approximately 88% of the signaling kinases are expressed in the developing embryo. These results support the strength of this nonchordate deuterostome as a pivotal developmental and evolutionary model organism.

The genomic underpinnings of apoptosis in Strongylocentrotus purpuratus.

Programmed cell death through apoptosis is a pan-metazoan character involving intermolecular signaling networks that have undergone substantial lineage-specific evolution. A survey of apoptosis-related proteins encoded in the sea urchin genome provides insight into this evolution while revealing some interesting novelties, which we highlight here. First, in addition to a typical CARD-carrying Apaf-1 homologue, sea urchins have at least two novel Apaf-1-like proteins that are each linked to a death domain, suggesting that echinoderms have evolved unique apoptotic signaling pathways. Second, sea urchins have an unusually large number of caspases. While the set of effector caspases (caspases-3/7 and caspase-6) in sea urchins is similar to that found in other basal deuterostomes, signal-responsive initiator caspase subfamilies (caspases-8/10 and 9, which are respectively linked to DED and CARD adaptor domains) have undergone echinoderm-specific expansions. In addition, there are two groups of divergent caspases, one distantly related to the vertebrate interleukin converting enzyme (ICE)-like subfamily, and a large clan that does not cluster with any of the vertebrate caspases. Third, the complexity of proteins containing an anti-apoptotic BIR domain and of Bcl-2 family members approaches that of vertebrates, and is greater than that found in protostome model systems such as Drosophila or Caenorhabditis elegans. Finally, the presence of Death receptor homologues, previously known only in vertebrates, in both Strongylocentrotus purpuratus and Nematostella vectensis suggests that this family of apoptotic signaling proteins evolved early in animals and was subsequently lost in the nematode and arthropod lineage(s). Our results suggest that cell survival is contingent upon a diverse array of signals in sea urchins, more comparable in complexity to vertebrates than to arthropods or nematodes, but also with unique features that may relate to specific requirements imposed by the biphasic life cycle and/or immunological idiosyncrasies of this organism.

CBFbeta is a facultative Runx partner in the sea urchin embryo.

BACKGROUND: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. RESULTS: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFbeta is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFbeta does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFbeta retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1. CONCLUSION: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation.

Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo.

BACKGROUND: Runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. To discover fundamental Runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin Runx protein SpRunt-1. Previously we showed that embryos depleted of SpRunt-1 arrest development at early gastrula stage and underexpress the conventional protein kinase C SpPKC1. RESULTS: We report here that SpRunt-1 deficiency leads to ectopic cell proliferation and extensive apoptosis. Suppression of the apoptosis by pharmacological inhibition of caspase-3 prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of SpRunt-1 are secondary to the apoptosis. Inhibition or knockdown of SpPKC1 also causes apoptosis, while cell survival is rescued in SpRunt-1 morphant embryos coinjected with SpPKC1 mRNA, suggesting that the apoptosis associated with SpRunt-1 deficiency is caused by the deficit in SpPKC1 expression. Chromatin immunoprecipitation indicates that SpRunt-1 interacts physically with SpPKC1 in vivo, and cis-regulatory analysis shows that this interaction activates SpPKC1 transcription. CONCLUSIONS: Our results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis.