Early Developmental Time Points Research Articles

Age of first words predicts cognitive ability and adaptive skills in children with ASD.

Acquiring useful language by age 5 has been identified as a strong predictor of positive outcomes in individuals with Autism Spectrum Disorders (ASD). This study examined the relationship between age of language acquisition and later functioning in children with ASD (n = 119). First word acquisition at a range of ages was probed for its relationship to cognitive ability and adaptive behaviors at 52 months. Results indicated that although producing first words predicted better outcome at every age examined, producing first words by 24 months was a particularly strong predictor of better outcomes. This finding suggests that the historic criterion for positive prognosis (i.e., "useful language by age 5") can be updated to a more specific criterion with an earlier developmental time point.

Ontogenetic immune challenges shape adult personality in mallard ducks.

Consistent individual differences in behaviour are widespread in animals, but the proximate mechanisms driving these differences remain largely unresolved. Parasitism and immune challenges are hypothesized to shape the expression of animal personality traits, but few studies have examined the influence of neonatal immune status on the development of adult personality. We examined how non-pathogenic immune challenges, administered at different stages of development, affected two common measures of personality, activity and exploratory behaviour, as well as colour-dependent novel object exploration in adult male mallard ducks (Anas platyrhynchos). We found that individuals that were immune-challenged during the middle (immediately following the completion of somatic growth) and late (during the acquisition of nuptial plumage) stages of development were more active in novel environments as adults relative to developmentally unchallenged birds or those challenged at an earlier developmental time point. Additionally, individuals challenged during the middle stage of development preferred orange and avoided red objects more than those that were not immune-challenged during development. Our results demonstrate that, in accordance with our predictions, early-life immune system perturbations alter the expression of personality traits later in life, emphasizing the role that developmental plasticity plays in shaping adult personality, and lending support to recent theoretical models that suggest that parasite pressure may play an important role in animal personality development.

Abstract 4294: Using the zebrafish model to determine the role of the HACE1 tumor suppressor in apoptosis, cell proliferation and development

Abstract HACE1 is a novel tumor suppressor gene located at human chromosome 6q21. HACE1 possesses both ankyrin repeats and a catalytically active HECT domain. HACE1 is downregulated in a variety of human cancers including Wilms’ tumor, melanoma, breast cancer, lung cancer, lymphoma, and colon cancer, thus representing a potentially broadly applicable genetic target. While HACE1 is widely expressed in human tissues, its role in normal development remains unknown. The zebrafish has established itself as a robust model for studying vertebrate development and modeling human cancers by virtue of conserved genetics and direct evaluation of gene expression through imaging afforded by transparent embryos. A zebrafish hace1 homologue has been identified. Whole mount in situ hybridization (WISH) assays with probes to the ankyrin repeats and HECT domains demonstrate expression of zebrafish hace1 in heart, brain and kidney at early developmental time points (24 hours post-fertilization (hpf) – 7 days post-fertilization (dpf)), consistent with human expression. WISH studies in wild type embryos, employing probes to the zebrafish hace1 HECT domain and cmcl2 (heart) or cdh17 (kidney), show co-localization at early developmental time points. Moreover, morpholino knockdown of hace1 in cmcl2-GFP transgenic embryos reveals severely perturbed cardiac development and function beginning at 48 hpf, further implicating this gene in normal vertebrate cardiac development. We have also generated two transgenic zebrafish lines harboring either wild type or dominant negative mutated C876S (C876S DN) human HACE1 genes with a green fluorescent protein (GFP) tag under the control of the ubiquitous β-actin promoter. Compared to wild type embryos, we observed increased apoptosis in 28 hpf C876S DN by acridine orange and caspase 3 assays, both untreated and following irradiation-induced cellular damage. There was no statistically significant difference in cellular proliferation between wild type embryos and C876S DN by 5-bromo-2-deoxyuridine (BrdU) incorporation (p=0.18). These results suggest that loss of HACE1 function alone is not sufficient to lead to a dysregulated cellular proliferation and implicate additional genetic lesions, such as p53 in tumor formation. C876S DN transgenic zebrafish are being crossed with p53 mutated zebrafish in order to determine the combined effect of both mutations on apoptosis, cell proliferation, and tumor formation. Further characterization of the HACE1 transgenic zebrafish model will serve to better our understanding of the role of human HACE1 in normal development and tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4294. doi:10.1158/1538-7445.AM2011-4294

Calpain 1 and Calpastatin expression is developmentally regulated in rat brain

Calpains and caspases are cysteine endopeptidases which share many similar substrates. Caspases are essential for caspase-dependent apoptotic death where calpains may play an augmentive role, while calpains are strongly implicated in necrotic cell death morphologies. Previous studies have demonstrated a down-regulation in the expression of many components of the caspase-dependent cell death pathway during CNS development. We therefore sought to determine if there is a corresponding upregulation of calpains. The major CNS calpains are the μ-and m-isoforms, composed of the unique 80 kDa calpain 1 and 2 subunits, respectively, and the shared 28 kDa small subunit. In rat brain, relative protein and mRNA levels of calpain 1, calpain 2, caspase 3, and the endogenous calpain inhibitor-calpastatin, were evaluated using western blot and real-time RT-PCR. The developmental time points examined ranged from embryonic day 18 until postnatal day 90. Calpain 1 and calpastatin protein and mRNA levels were low at early developmental time points and increased dramatically by P30. Conversely, caspase-3 expression was greatest at E18, and was rapidly downregulated by P30. Calpain 2 protein and mRNA levels were relatively constant throughout the E18-P90 age range examined. The inverse relationship of calpain 1 and caspase 3 levels during CNS development is consistent with the shift from caspase-dependent to caspase-independent cell death mechanisms following CNS injury in neonatal vs. adult rat brain.

Effect of parental exposure to trenbolone and the brominated flame retardant BDE-47 on fertility in rainbow trout (Oncorhynchus mykiss)

We exposed sexually maturing male rainbow trout (Oncorhynchus mykiss) to BDE-47 (a polybrominated diphenyl ether) and female rainbow trout to trenbolone (an anabolic steroid). Male trout were orally exposed for 17 days to 55 microg/kg/day BDE-47 and female trout continuously exposed for 60-77 days to a measured trenbolone water concentration of 35 ng/L. After the exposure, eggs and semen were collected and in vitro fertilization trials performed using a sperm:egg ratio of 300,000:1. In the BDE-47 study, eggs from control females were fertilized with semen from exposed males, while in the trenbolone study, eggs from exposed females were fertilized with semen from control males. All treatments were evaluated at two-three early developmental time-points representing first cleavage (0.5 day), embryonic keel (9 days), and eyed stages (19 days), respectively. The results indicated that BDE-47 exposure did not alter fertility as embryonic survival was similar between control and exposed groups. Trenbolone exposure also did not alter embryo survival. However, in the embryos fertilized with eggs from trenbolone exposed females, a noticeable delay in developmental progress was observed. On day 19 when eye development is normally complete, the majority of the embryos either lacked eyes or displayed under-developed eyes, in contrast to control embryos. This finding suggests steroidal androgen exposure in sexually maturing female rainbow trout can impact developmental timing of F1 offspring.

Genetic identification of a novel NeuroD1 function in the early differentiation of islet α, PP and ε cells

Nkx2.2 and NeuroD1 are vital for proper differentiation of pancreatic islet cell types. Nkx2.2-null mice fail to form β cells, have reduced numbers of α and PP cells and display an increase in ghrelin-producing ε cells. NeuroD1-null mice display a reduction of α and β cells after embryonic day (e) 17.5. To begin to determine the relative contributions of Nkx2.2 and NeuroD1 in islet development, we generated Nkx2.2−/−;NeuroD1−/− double knockout (DKO) mice. As expected, the DKO mice fail to form β cells, similar to the Nkx2.2-null mice, suggesting that the Nkx2.2 phenotype may be dominant over the NeuroD1 phenotype in the β cells. Surprisingly, however, the α, PP and ε phenotypes of the Nkx2.2-null mice are partially rescued by the simultaneous elimination of NeuroD1, even at early developmental time points when NeuroD1 null mice alone do not display a phenotype. Our results indicate that Nkx2.2 and NeuroD1 interact to regulate pancreatic islet cell fates, and this epistatic relationship is cell-type dependent. Furthermore, this study reveals a previously unappreciated early function of NeuroD1 in regulating the specification of α, PP and ε cells.

Presynaptic Unsilencing: Searching for a Mechanism

Nascent synaptic networks have a high incidence of silent synapses. In this issue of Neuron, Shen et al. show that a brief burst of action potentials rapidly awaken silent synapses by increasing the availability of synaptic vesicles for fusion through BDNF-triggered presynaptic actin remodeling mediated by the small GTPase Cdc42.

TrkB Has a Cell-Autonomous Role in the Establishment of Hippocampal Schaffer Collateral Synapses

Neurotrophin signaling has been implicated in the processes of synapse formation and plasticity. To gain additional insight into the mechanism of BDNF and TrkB influence on synapse formation and synaptic plasticity, we generated a conditional knock-out for TrkB using the cre/loxp system. Using three different cre-expressing transgenic mice, three unique spatial and temporal configurations of TrkB deletion were obtained with regard to the hippocampal Schaffer collateral synapse. We compare synapse formation in mutants in which TrkB is ablated either in presynaptic or in both presynaptic and postsynaptic cells at early developmental or postdevelopmental time points. Our results indicate a requirement for TrkB at both the presynaptic and postsynaptic sites during development. In the absence of TrkB, synapse numbers were significantly reduced. In vivo ablation of TrkB after synapse formation did not affect synapse numbers. In primary hippocampal cultures, deletion of TrkB in only the postsynaptic cell, before synapse formation, also resulted in deficits of synapse formation. We conclude that TrkB signaling has a cell-autonomous role required for normal development of both presynaptic and postsynaptic components of the Schaffer collateral synapse.

Development of A-type allatostatin immunoreactivity in antennal lobe neurons of the sphinx moth Manduca sexta

The antennal lobe (AL) of the sphinx moth Manduca sexta is a well-established model system for studying mechanisms of neuronal development. To understand whether neuropeptides are suited to playing a role during AL development, we have studied the cellular localization and temporal expression pattern of neuropeptides of the A-type allatostatin family. Based on morphology and developmental appearance, we distinguished four types of AST-A-immunoreactive cell types. The majority of the cells were local interneurons of the AL (type Ia) which acquired AST-A immunostaining in a complex pattern consisting of three rising (RI-RIII) and two declining phases (DI, DII). Type Ib neurons consisted of two local neurons with large cell bodies not appearing before 7/8 days after pupal ecdysis (P7/P8). Types II and III neurons accounted for single centrifugal neurons, with type II neurons present in the larva and disappearing in the early pupa. The type III neuron did not appear before P7/P8. RI and RII coincided with the rises of the ecdysteroid hemolymph titer. Artificially shifting the pupal 20-hydroxyecdysone (20E) peak to an earlier developmental time point resulted in the precocious appearance of AST-A immunostaining in types Ia, Ib, and III neurons. This result supports the hypothesis that the pupal rise in 20E plays a role in AST-A expression during AL development. Because of their early appearance in newly forming glomeruli, AST-A-immunoreactive fibers could be involved in glomerulus formation. Diffuse AST-A labeling during early AL development is discussed as a possible signal providing information for ingrowing olfactory receptor neurons.

Up-regulation of novel intermediate filament proteins in primary fiber cells: An indicator of all vertebrate lens fiber differentiation?

The early embryonic development and expression patterns of the eye lens specific cytoskeletal proteins, CP49 and CP95, were determined for the chick and were found to be similar in both human and mouse. These proteins, as well as their homologs in other species, are obligate polymerization partners which form unique filamentous structures termed “beaded filaments.” CP49 and CP95 appeared as protein products after 3 days of embryonic development in the chick during the elongation of primary fiber cells. Although limited data were obtained for human embryos at these early developmental timepoints, they were consistent with the interpretation that the up-regulation of these lens specific proteins began only after the initiation of lens vesicle closure. In situ hybridization with the mouse lens confirmed that message levels for beaded filament proteins were greatly elevated in differentiating primary fiber cells. Nuclease protection assays established that mRNA levels for CP49 remained relatively constant while CP95 mRNA levels increased once the process of secondary fiber formation was under way. Although present in relatively low abundance, the mRNA for a unique splice variant of CP49, CP49INS, was also detected early in embryonic development and into adulthood. Peptide-specific antibodies directed against unique predicted sequences were able to confirm the protein expression of CP49INS in both embryonic and adult chick lens cells. These data present the first detailed study of the expression of CP49 and CP95 during early lens development. They suggest that the up-regulated expression of CP49 and CP95 could serve as pan-specific markers for all vertebrate lens fiber development. Anat Rec 258:25–33, 2000. © 2000 Wiley-Liss, Inc.

Troponin I isoform expression is developmentally regulated in differentiating embryonic stem cell-derived cardiac myocytes.

We studied troponin I (TnI) isoform expression in the mouse embryonic stem (ES) cell model of cardiogenesis as an essential first step to understanding the relationship between TnI isoform transitions and myofibrillar function. Cultures of differentiating ES cells were grown on coverslips to permit microscopic inspection of foci of spontaneously contracting cardiac myocytes developing in culture. TnI expression was followed over time to test whether the cardiac myocytes undergo the developmental pattern of expression characteristic of vertebrate cardiogenesis, in which slow skeletal TnI (ssTnI) is expressed initially, followed by induction of cardiac (cTnI) isoform expression. Cardiac TnI expression was examined using the cardiac-specific, monoclonal TI-1 antibody (Ab) while all striated muscle ThI isoforms were detected using the monoclonal TI-4 Ab. Cardiac-specific TnI expression was detected in only 8% (8/96) of foci contracting less than 5 days while TI-4 positive staining was present in 95% (71/73) of foci. These results indicate that other striated muscle TnI isoforms were being expressed in most of the TI-4 positive staining foci. The proportion of contracting foci expressing the cardiac isoform increased steadily over time, such that 100% of foci contracting more than 20 days (13/13) stained positive with the TI-1 Ab. Dual labeling experiments with both TI-1 and TI-4 anti-TnI Abs in the same culture confirmed that within each foci, the area expressing cTnI increased with the days of spontaneous contraction. Western blot analysis of micro-dissected ES cell derived cardiac myocytes confirmed that TI-4 immunostaining at early developmental time points represented ssTnI, and not the fast skeletal TnI isoform. We conclude that ES cell-derived cardiac myocytes display the developmental induction of cardiac TnI expression characteristic of vertebrate cardiac development. Thus, this model should be useful for studying the regulation and functional significance of TnI isoform expression during in vitro cardiogenesis.