Developmental and Neurotoxicity of Acrylamide to Zebrafish.

Acrylamide is a commonly used industrial chemical that is known to be neurotoxic to mammals. However, its developmental toxicity is rarely assessed in mammalian models because of the cost and complexity involved. We used zebrafish to assess the neurotoxicity, developmental and behavioral toxicity of acrylamide. At 6 h post fertilization, zebrafish embryos were exposed to four concentrations of acrylamide (10, 30, 100, or 300 mg/L) in a medium for 114 h. Acrylamide caused developmental toxicity characterized by yolk retention, scoliosis, swim bladder deficiency, and curvature of the body. Acrylamide also impaired locomotor activity, which was measured as swimming speed and distance traveled. In addition, treatment with 100 mg/L acrylamide shortened the width of the brain and spinal cord, indicating neuronal toxicity. In summary, acrylamide induces developmental toxicity and neurotoxicity in zebrafish. This can be used to study acrylamide neurotoxicity in a rapid and cost-efficient manner.

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease.

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

CFTR deficiency causes cardiac dysplasia during zebrafish embryogenesis and is associated with dilated cardiomyopathy.

Mutations in the CFTR gene cause cystic fibrosis (CF) with myocardial dysfunction. However, it remains unknown whether CF-related heart disease is a secondary effect of pulmonary disease, or an intrinsic primary defect in the heart. Here, we used zebrafish, which lack lung tissue, to investigate the role of CFTR in cardiogenesis. Our findings demonstrated that the loss of CFTR impairs cardiac development from the cardiac progenitor stage, resulting in cardiac looping defects, a dilated atrium, pericardial edema, and a decrease in heart rate. Furthermore, we found that cardiac development was perturbed in wild-type embryos treated with a gating-specific CFTR channel inhibitor, CFTRinh-172, at the blastula stage of development, but not at later stages. Gene expression analysis of blastulas indicated that transcript levels, including mRNAs associated with cardiovascular diseases, were significantly altered in embryos derived from cftr mutants relative to controls. To evaluate the role of CFTR in human heart failure, we performed a genetic association study on individuals with dilated cardiomyopathy and found that the I556V mutation in CFTR, which causes a channel defect, was associated with the disease. Similar to other well-studied channel-defective CFTR mutants, CFTR I556V mRNA failed to restore cardiac dysplasia in mutant embryos. The present study revealed an important role for the CFTR ion channel in regulating cardiac development during early embryogenesis, supporting the hypothesis that CF-related heart disease results from an intrinsic primary defect in the heart.

Isoniazid causes heart looping disorder in zebrafish embryos by the induction of oxidative stress.

BACKGROUND: The cardiotoxicity of isoniazid on zebrafish embryos and its underlying mechanism is unclear. METHODS: Here, we exposed zebrafish embryos at 4 h post-fertilization to different levels of isoniazid and recorded the morphology and number of malformed and dead embryos under the microscope. RESULTS: The high concentration of isoniazid group showed more malformed and dead embryos than the low concentration of isoniazid group and control group. The morphology of the heart and its alteration were visualized using transgenic zebrafish (cmlc2: GFP) and confirmed by in situ hybridization. The negative effects of isoniazid on the developing heart were characterized by lower heart rate and more heart looping disorders. Mechanistically, PCR showed decreased expression of heart-specific transcription factors when exposed to isoniazid. Oxidative stress was induced by isoniazid in cardiomyocytes, mediated by decreased activities of catalase and superoxide dismutase, which were rescued by scavengers of reactive oxygen species. CONCLUSION: In conclusion, this study demonstrated that isoniazid led to heart looping disturbance by the downregulation of cardiac-specific transcription factors and induction of cardiomyocyte apoptosis.

Chemical screens in a zebrafish model of CHARGE syndrome identifies small molecules that ameliorate disease-like phenotypes in embryo.

CHARGE syndrome is an autosomal dominant congenital disorder caused primarily by mutations in the CHD7 gene. Using a small molecule screen in a zebrafish model of CHARGE syndrome, we identified 4 compounds that rescue embryos from disease-like phenotypes. Our screen yielded DAPT, a Notch signaling inhibitor that could ameliorate the craniofacial, cranial neuronal and myelination defects in chd7 morphant zebrafish embryos. We discovered that Procainamide, an inhibitor of DNA methyltransferase 1, was able to recover the pattern of expression of isl2a, a cranial neuronal marker while also reducing the effect on craniofacial cartilage and myelination. M344, an inhibitor of Histone deacetylases had a strong recovery effect on craniofacial cartilage defects and could also modestly revert the myelination defects in zebrafish embryos. CHIC-35, a SIRT1 inhibitor partially restored the expression of isl2a in cranial neurons while causing a partial reversion of myelination and craniofacial cartilage defects. Our results suggest that a modular approach to phenotypic rescue in multi-organ syndromes might be a more successful approach to treat these disorders. Our findings also open up the possibility of using these compounds for other disorders with shared phenotypes.

Blood flow distribution in embryonic common snapping turtles Chelydra serpentina (Reptilia; Chelonia) during acute hypoxia and α-adrenergic regulation.

Embryonic turtles have four distinct vascular beds that separately perfuse the developing embryo's body and the extra-embryonic yolk sac, amnion and chorioallantoic membrane (CAM). The mechanisms enabling differential regulation of blood flow through these separate beds, in order to meet the varying demands of the embryo during development, is of current interest. The present investigation followed the changes in blood flow distribution during an acute exposure to hypoxia and after α-adrenergic blockade. We monitored heart rate (fH), mean arterial pressure (Pm), and determined relative blood flow distribution (%Q̇sys) using colored microspheres. At 70% and 90% of the incubation period hypoxia elicited a bradycardia without changing Pm while %Q̇sys was altered only at 70%, increasing to the CAM and liver. Blockade of α-adrenergic responses with phentolamine did not change fH or Pm but increased %Q̇sys to the shell. These results show the capacity of embryos to redistribute cardiac output during acute hypoxia, however α-adrenergic receptors seemed to play a relatively small role in embryonic cardiovascular regulation.

Combined Danio rerio embryo morbidity, mortality and photomotor response assay: A tool for developmental risk assessment from chronic cyanoHAB exposure.

Freshwater harmful algal blooms produce a broad array of bioactive compounds, with variable polarity. Acute exposure to cyanotoxins can impact the liver, nervous system, gastrointestinal tract, skin, and immune function. Increasing evidence suggests chronic effects from low-level exposures of cyanotoxins and other associated bioactive metabolites of cyanobacterial origin. These sundry compounds persist in drinking and recreational waters and challenge resource managers in detection and removal. A systematic approach to assess the developmental toxicity of cyanobacterial metabolite standards was employed utilizing a robust and high throughput developmental Danio rerio embryo platform that incorporated a neurobehavioral endpoint, photomotor response. Subsequently, we applied the platform to cyanobacterial bloom surface water samples taken from temperate recreational beaches and tropical lake subsistence drinking water sources as a model approach. Dechorionated Danio rerio embryos were statically immersed beginning at four to six hours post fertilization at environmentally relevant concentrations, and then assessed at 24 h and 5 days for morbidity, morphological changes, and photomotor response. At least one assessed endpoint deviated significantly for exposed embryos for 22 out of 25 metabolites examined. Notably, the alkaloid lyngbyatoxin-a resulted in profound, dose-dependent morbidity and mortality beginning at 5 µg/L. In addition, hydrophobic components of extracts from beach monitoring resulted in potent morbidity and mortality despite only trace cyanotoxins detected. The hydrophilic extracts with several order of magnitude higher concentrations of microcystins resulted in no morbidity or mortality. Developmental photomotor response was consistently altered in environmental bloom samples, independent of the presence or concentration of toxins detected in extracts. While limited with respect to more polar compounds, this novel screening approach complements specific fingerprinting of acutely toxic metabolites with robust assessment of developmental toxicity, critical for chronic exposure scenarios.

Developmental effects of trichloroacetate in Zebrafish embryos: Association with the production of superoxide anion.

To assess the developmental toxicity of trichloroacetate (TCA), zebrafish embryos were exposed to 8 to 48 mM of TCA and evaluated for developmental milestones from 8- to 144-hour postfertilization (hpf). All developmental toxicities are reported in this paper. Embryos were found to have developed edema in response to 16 to 48 mM of TCA exposure at 32- to 80-hpf, experienced delay in hatching success in response to 24 to 48 mM at 80-hpf. Lordosis was observed in developing embryos exposed to 40 to 48 mM at 55- to 144-hpf. The observed toxic effects of TCA exposure were found to be concentration and exposure period independent. Effects were found to be associated with increases in superoxide anion production, but these increases were also found to be concentration and time independent. TCA resulted in concentration-dependent increases in embryonic lethality at 144-hpf, with an LC50 determined to be 29.7 mM.

Developmental Exposure to Low Concentrations of Organophosphate Flame Retardants Causes Life-Long Behavioral Alterations in Zebrafish.

As the older class of brominated flame retardants (BFRs) are phased out of commercial use because of findings of neurotoxicity with developmental exposure, a newer class of flame retardants have been introduced, the organophosphate flame retardants (OPFRs). Presently, little is known about the potential for developmental neurotoxicity or the behavioral consequences of OPFR exposure. Our aim was to characterize the life-long neurobehavioral effects of 4 widely used OPFRs using the zebrafish model. Zebrafish embryos were exposed to 0.1% DMSO (vehicle control); or one of the following treatments; isopropylated phenyl phosphate (IPP) (0.01, 0.03, 0.1, 0.3 µM); butylphenyl diphenyl phosphate (BPDP) (0.003, 0.03, 0.3, 3 µM); 2-ethylhexyl diphenyl phosphate (EHDP) (0.03, 0.3, 1 µM); isodecyl diphenyl phosphate (IDDP) (0.1, 0.3, 1, 10 µM) from 0- to 5-days postfertilization. On Day 6, the larvae were tested for motility under alternating dark and light conditions. Finally, at 5-7 months of age the exposed fish and controls were tested on a battery of behavioral tests to assess emotional function, sensorimotor response, social interaction and predator evasion. These tests showed chemical-specific short-term effects of altered motility in larvae in all of the tested compounds, and long-term impairment of anxiety-related behavior in adults following IPP, BPDP, or EHDP exposures. Our results show that OPFRs may not be a safe alternative to the phased-out BFRs and may cause behavioral impacts throughout the lifespan. Further research should evaluate the risk to mammalian experimental models and humans.

Developmental ethanol exposure impairs locomotor movement in Japanese medaka (Oryzias latipes) larvae targeting epigenome.

Evidence indicated ethanol exposure during development disrupts brain functions that induces fetal alcohol spectrum disorder (FASD) phenotypes with behavioral abnormalities. We aimed to investigate whether prenatal ethanol exposure has any potential impact on behavior of a FASD fish model. Fertilized Japanese medaka (Oryzias latipes) eggs were exposed to 100-300 mM ethanol or 2 mM 5-azacytidine (5-azaC), 0-2 day post fertilization (dpf), in embryo-rearing medium (ERM). Survived embryos were maintained in clean ERM and used either for gene expression analysis on 2- and 6-dpf or allowed to hatch for behavioral study. Photomotor response of 3-4 day post hatch larvae were tracked for 3 h with light-dark transitions. It was observed that larval swimming was phototactic; enhanced in presence of light, declined in dark. Phototactic response was also observed in larvae prenatally exposed to ethanol or 5-azaC; however, the total distance swum by these larvae compared to controls declined. Further analysis indicated that, in light phases, total swimming activity and average swimming speed were reduced in larvae prenatally exposed to ethanol (300 mM) or 5-azaC. Expression analysis of baz1a and baz2a in embryos indicated developmental regulation. Ethanol (100-300 mM) or 5-azaC (2 mM) were able to modulate downregulation of both baz1a and baz2a mRNAs only in 6 dpf embryos of 300 mM ethanol and 5-azaC (2 mM) groups. These studies indicated that prenatal exposure to ethanol or 5-azaC was able to disrupt movements and thus swimming behavior in FASD phenotypes probably due to delayed remodeling of genome and epigenome.

Embryonic hypoxia programmes postprandial cardiovascular function in adult common snapping turtles (Chelydra serpentina).

Reduced oxygen availability (hypoxia) is a potent stressor during embryonic development, altering the trajectory of trait maturation and organismal phenotype. We previously documented that chronic embryonic hypoxia has a lasting impact on the metabolic response to feeding in juvenile snapping turtles (Chelydra serpentina). Turtles exposed to hypoxia as embryos [10% O2 (H10)] exhibited an earlier and increased peak postprandial oxygen consumption rate, compared with control turtles [21% O2 (N21)]. In the current study, we measured central blood flow patterns to determine whether the elevated postprandial metabolic response in H10 turtles is linked to lasting impacts on convective transport. Five years after hatching, turtles were instrumented to quantify systemic ([Formula: see text]) and pulmonary ([Formula: see text]) blood flows and heart rate (fH) before and after a ∼5% body mass meal. In adult N21 and H10 turtles, fH was increased significantly by feeding. Although total stroke volume (VS,tot) remained at fasted values, this tachycardia contributed to an elevation in total cardiac output ([Formula: see text]). However, there was a postprandial reduction in a net left-right (L-R) shunt in N21 snapping turtles only. Relative to N21 turtles, H10 animals exhibited higher [Formula: see text] due to increased blood flow through the right systemic outflow vessels of the heart. This effect of hypoxic embryonic development, reducing a net L-R cardiac shunt, may support the increased postprandial metabolic rate we previously reported in H10 turtles, and is further demonstration of adult reptile cardiovascular physiology being programmed by embryonic hypoxia.

Two light-activated neuroendocrine circuits arising in the eye trigger physiological and morphological pigmentation.

Two biological processes regulate light-induced skin colour change. A fast 'physiological pigmentation change' (i.e. circadian variations or camouflage) involves alterations in the distribution of pigment containing granules in the cytoplasm of chromatophores, while a slower 'morphological pigmentation change' (i.e. seasonal variations) entails changes in the number of pigment cells or pigment type. Although linked processes, the neuroendocrine coordination triggering each response remains largely obscure. By evaluating both events in Xenopus laevis embryos, we show that morphological pigmentation initiates by inhibiting the activity of the classical retinal ganglion cells. Morphological pigmentation is always accompanied by physiological pigmentation, and a melatonin receptor antagonist prevents both responses. Physiological pigmentation also initiates in the eye, but with repression of melanopsin-expressing retinal ganglion cell activity that leads to secretion of alpha-melanocyte-stimulating hormone (α-MSH). Our findings suggest a model in which eye photoperception links physiological and morphological pigmentation by altering α-MSH and melatonin production, respectively.

Folate deficiency and over-supplementation causes impaired folate metabolism: Regulation and adaptation mechanisms in Caenorhabditis elegans.

SCOPE: Impaired folate metabolism increases the risk of birth defects, neurodegenerative and cardiovascular disease, osteoporosis and cancer. We used Caenorhabditis elegans to investigate impaired folate metabolism by RNA interference of key enzymes in the methionine synthase (MS) and thymidylate synthase (TS) cycle and by folate deficiency and over-supplementation feeding studies. METHODS AND RESULTS: Folate status is influenced by genetic variations (polymorphisms), folate deficiency and supplementation. Single RNAi of dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR) and MS revealed that gene regulation is largely affected in both folate cycles. Adaptation requires a close transcriptional connection between TS and MS cycle. Coupled DHFR and MS expression is required to balance both cycles, but seems to reduce the overall rate of folate conversion. Feeding studies showed that folate over-supplementation to functioning metabolism inactivates MS and MTHFR expression and enhances TS activity, which favors DNA synthesis over methylation reactions. Folate deficiency disrupted homeostasis by favoring TS cycle and led to malformation in C. elegans offspring. Embryos show aneuploidy and are nonviable lacking DNA repair during meiotic stage of diakinesis. CONCLUSION: Single gene silencing alters gene expression in both cycles and disrupts folate homeostasis. Folate over-supplementation and deficiency favors TS over MS cycle and causes prophase DNA damage.

Periods of cardiovascular susceptibility to hypoxia in embryonic american alligators (Alligator mississippiensis).

During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20 and 40% of embryonic incubation, whereas somatic growth was affected between 70 and 90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50-70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70-90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia toward the end of incubation altered ß-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study, we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator.

Phenotypic plasticity in the common snapping turtle (Chelydra serpentina): long-term physiological effects of chronic hypoxia during embryonic development.

Studies of embryonic and hatchling reptiles have revealed marked plasticity in morphology, metabolism, and cardiovascular function following chronic hypoxic incubation. However, the long-term effects of chronic hypoxia have not yet been investigated in these animals. The aim of this study was to determine growth and postprandial O2 consumption (V̇o2), heart rate (fH), and mean arterial pressure (Pm, in kPa) of common snapping turtles (Chelydra serpentina) that were incubated as embryos in chronic hypoxia (10% O2, H10) or normoxia (21% O2, N21). We hypothesized that hypoxic development would modify posthatching body mass, metabolic rate, and cardiovascular physiology in juvenile snapping turtles. Yearling H10 turtles were significantly smaller than yearling N21 turtles, both of which were raised posthatching in normoxic, common garden conditions. Measurement of postprandial cardiovascular parameters and O2 consumption were conducted in size-matched three-year-old H10 and N21 turtles. Both before and 12 h after feeding, H10 turtles had a significantly lower fH compared with N21 turtles. In addition, V̇o2 was significantly elevated in H10 animals compared with N21 animals 12 h after feeding, and peak postprandial V̇o2 occurred earlier in H10 animals. Pm of three-year-old turtles was not affected by feeding or hypoxic embryonic incubation. Our findings demonstrate that physiological impacts of developmental hypoxia on embryonic reptiles continue into juvenile life.

High-throughput characterization of chemical-associated embryonic behavioral changes predicts teratogenic outcomes.

New strategies are needed to address the data gap between the bioactivity of chemicals in the environment versus existing hazard information. We address whether a high-throughput screening (HTS) system using a vertebrate organism (embryonic zebrafish) can characterize chemical-elicited behavioral responses at an early, 24 hours post-fertilization (hpf) stage that predict teratogenic consequences at a later developmental stage. The system was used to generate full concentration-response behavioral profiles at 24 hpf across 1060 ToxCast™ chemicals. Detailed, morphological evaluation of all individuals was performed as experimental follow-up at 5 days post-fertilization (dpf). Chemicals eliciting behavioral responses were also mapped against external HTS in vitro results to identify specific molecular targets and neurosignalling pathways. We found that, as an integrative measure of normal development, significant alterations in movement highlighted active chemicals representing several modes of action. These early behavioral responses were predictive for 17 specific developmental abnormalities and mortality measured at 5 dpf, often at lower (i.e., more potent) concentrations than those at which morphological effects were observed. Therefore, this system can provide rapid characterization of chemical-elicited behavioral responses at an early developmental stage that are predictive of observable adverse effects later in life.

[Myeloid and erythroid hematopoietic transcription factor expression decline after knockdown of lmna genes in zebrafish embryos].

Objective: To investigate the effect of lmna gene down-regulation on early hematopoietic development of zebrafish. Methods: Phosphorodiamidate morpholino oligomer (PMO) technology was used to downregulate lmna gene expression in Zebrafish. Zebrafish embryos injected phosphorodiamidate morpholino antisense oligonucleotide of lmna gene mRNA by microinjection at unicellular stage were taken as the experimental group, and those injected meaningless phosphorodiamidate morpholino antisense oligonucleotide were taken as the control. The embryos were collected at 18, 24, 30 and 36 hpf after the fertilization. The real-time fluorescent quantitative PCR (RT-PCR) and whole embryo in situ hybridization methods were used to detect the expression of myeloid hematopoietic transcription factor pu. 1 and erythroid hematopoietic transcription factor gata1 in zebrafish. Results: RT-PCR showed that the expressions of pu. 1 and gata1 decreased in the experimental group compared with the control group (all P<0.05). Whole embryo in situ hybridization showed that the blue-black positive hybridization signals of pu. 1 and gata1 in experimental group were shallow than those in the control group. Conclusion: Myeloid hematopoietic and erythroid hematopoietic of zebrafish are blocked with the downregulation of lmna gene.

Development of sympathetic cardiovascular control in embryonic, hatchling, and yearling female American alligator (Alligator mississippiensis).

We used arterial tyramine injections to study development of sympathetic actions on in vivo heart rate and blood pressure in embryonic, hatching and yearling female American alligators. Tyramine is a pharmacological tool for understanding comparative and developmental sympathetic regulation of cardiovascular function, and this indirect sympathomimetic agent causes endogenous neuronal catecholamine release, increasing blood pressure and heart rate. Arterial tyramine injection in hatchling and yearling alligators caused the typical vertebrate response - rise in heart rate and blood pressure. However, in embryonic alligators, tyramine caused a substantial and immediate bradycardia at both 70% and 90% of embryonic development. This embryonic bradycardia was accompanied by hypotension, followed by a sustained hypertension similar to the hatchling and juvenile responses. Pretreatment with atropine injection (cholinergic receptor blocker) eliminated the embryonic hypotensive bradycardia, and phentolamine pretreatment (α-adrenergic receptor blocker) eliminated the embryonic hypotensive and hypertensive responses but not the bradycardia. In addition, hexamethonium pretreatment (nicotinic receptor blocker) significantly blunted embryos' bradycardic tyramine response. However, pretreatment with 6-hydroxydopamine, a neurotoxin that destroys catecholaminergic terminals, did not eliminate the embryonic bradycardia. Tyramine likely stimulated a unique embryonic response - neurotransmitter release from preganglionic nerve terminals (blocked with hexamethonium) and an acetylcholine mediated bradycardia with a secondary norepinephrine-dependent sustained hypertension. In addition, tyramine appears to stimulate sympathetic nerve terminals directly, which contributed to the overall hypertension in the embryonic, hatchling and yearling animals. Data demonstrated that humoral catecholamine control of cardiovascular function was dominant over the immature parasympathetic nervous system in developing alligator embryos, and suggested that sympathetic and parasympathetic nerve terminals were present and developing in ovo but were not tonically active.

Survival of the stillest: predator avoidance in shark embryos.

Sharks use highly sensitive electroreceptors to detect the electric fields emitted by potential prey. However, it is not known whether prey animals are able to modulate their own bioelectrical signals to reduce predation risk. Here, we show that some shark (Chiloscyllium punctatum) embryos can detect predator-mimicking electric fields and respond by ceasing their respiratory gill movements. Despite being confined to the small space within the egg case, where they are vulnerable to predators, embryonic sharks are able to recognise dangerous stimuli and react with an innate avoidance response. Knowledge of such behaviours, may inform the development of effective shark repellents.

csrnp1a is necessary for the development of primitive hematopoiesis progenitors in zebrafish.

The CSRNP (cystein-serine-rich nuclear protein) transcription factors are conserved from Drosophila to human. Functional studies in mice, through knockout for each of their paralogs, have resulted insufficient to elucidate the function of this family of proteins in vertebrate development. Previously, we described the function of the zebrafish ortholog, Csnrp1/Axud1, showing its essential role in the survival and proliferation of cephalic progenitors. To extend our understanding of this family, we have studied the function of its paralog csrnp1a. Our results show that csrnp1a is expressed from 0 hpf, until larval stages, particularly in cephalic territories and in the intermediate cell mass (ICM). Using morpholinos in wild type and transgenic lines we observed that Csrnp1a knockdown generates a mild reduction in head size and a depletion of blood cells in circulation. This was combined with in situ hybridizations to analyze the expression of different mesodermal and primitive hematopoiesis markers. Morphant embryos have impaired blood formation without disruption of mesoderm specification, angiogenesis or heart development. The reduction of circulating blood cells occurs at the hematopoietic progenitor level, affecting both the erythroid and myeloid lineages. In addition, cell proliferation was also altered in hematopoietic anterior sites, specifically in spi1 expression domain. These and previous observations suggest an important role of Csnrps transcription factors in progenitor biology, both in the neural and hematopoietic linages.