HSD2 neurons in the hindbrain drive sodium appetite.

Sodium-depleted animals develop an appetite for aversive concentrations of sodium. Here we show that chemogenetic activation of aldosterone-sensitive neurons that express 11ß-hydroxysteroid dehydrogenase type 2 (HSD2) in the nucleus of the solitary tract is sufficient to drive consumption of sodium-containing solutions in mice, independently of thirst or hunger. These HSD2-positive neurons are necessary for full expression of sodium appetite and have distinct downstream targets that are activated during sodium depletion.

Role of estradiol in intrinsic hindbrain AMPK regulation of hypothalamic AMPK, metabolic neuropeptide, and norepinephrine activity and food intake in the female rat.

This study addressed the hypothesis that dorsomedial hindbrain adenosine 5'-monophosphate-activated protein kinase (AMPK) imposes inherent estradiol-dependent control of hypothalamic AMPK, neuropeptide, and norepinephrine (NE) activity and feeding in the female rat. Estradiol (E)- or oil (O)-implanted ovariectomized rats were injected with the AMPK inhibitor compound c (Cc) or vehicle into the caudal fourth ventricle (CV4) prior to micropunch-dissection of individual hypothalamic metabolic loci or assessment of food intake. Cc decreased hindbrain dorsal vagal complex phosphoAMPK (pAMPK) in both E and O; tissue ATP levels were reduced by this treatment in O only. In E/Cc, pAMPK expression was diminished in the lateral hypothalamic area (LHA) and ventromedial (VMH) and paraventricular (PVH) nuclei; only PVH pAMPK was suppressed by this treatment in O/Cc. Cc decreased PVH corticotropin-releasing hormone and arcuate (ARH) proopiomelanocortin (POMC) and neuropeptide Y in O, but suppressed only POMC in E. O/Cc exhibited both augmented (PVH, VMH) and decreased (LHA, ARH) hypothalamic NE content, whereas Cc treatment of E elevated preoptic and dorsomedial hypothalamic nucleus NE. Cc completely or incompletely repressed feeding in E versus O, respectively. Results implicate dorsomedial hindbrain AMPK in physiological stimulus-induced feeding in females. Excepting POMC, hypothalamic neuropeptide responses to this sensor may be contingent on estrogen. Estradiol likely designates hypothalamic targets of altered NE signaling due to hindbrain AMPK activation. Divergent changes in NE content of hypothalamic loci in O/Cc uniquely demonstrate sensor-induced bimodal catecholamine signaling to those sites.

NMDA receptors on zebrafish Mauthner cells require CaMKII-α for normal development.

Calcium/calmodulin dependent protein kinase 2 (CaMKII) is a multifunctional protein that is highly enriched in the synapse. It plays important roles in neuronal functions such as synaptic plasticity, synaptogenesis, and neural development. Gene duplication in zebrafish has resulted in the occurrence of seven CaMKII genes (camk2a, camk2b1, camk2b2, camk2g1, camk2g2, camk2d1, and camk2d2) that are developmentally expressed. In this study, we used single cell, real-time quantitative PCR to investigate the expression of CaMKII genes in individual Mauthner cells (M-cells) of 2 days post fertilization (dpf) zebrafish embryos. We found that out of seven different CaMKII genes, only the mRNA for CaMKII-α was expressed in the M-cell at detectable levels, while all other isoforms were undetectable. Morpholino knockdown of CaMKII-α had no significant effect on AMPA synaptic currents (mEPSCs) but decreased the amplitude of NMDA mEPSCs. NMDA events exhibited a biexponential decay with τfast ≈ 30 ms and τslow ≈ 300 ms. Knockdown of CaMKII-α specifically reduced the amplitude of the slow component of the NMDA-mediated currents (mEPSCs), without affecting the fast component, the frequency, or the kinetics of the mEPSCs. Immunolabelling of the M-cell showed increased dendritic arborizations in the morphants compared with controls, and knockdown of CaMKII-α altered locomotor behaviors of touch responses. These results suggest that CaMKII-α is present in embryonic M-cells and that it plays a role in the normal development of excitatory synapses. Our findings pave the way for determining the function of specific CaMKII isoforms during the early stages of M-cell development.

The time-course of hindbrain neuronal activity varies according to location during either intraperitoneal or subcutaneous tumor growth in rats: single Fos and dual Fos/dopamine ß-hydroxylase immunohistochemistry.

Neuronal activity in the nucleus of the solitary tract, ventrolateral medulla, area postrema, and parabrachial nucleus was studied in rats with intraperitoneal or subcutaneous tumors on the 7th, 14th, 21st, and 28th day after injection of fibrosarcoma tumor cells. We found that the number of Fos and dopamine ß-hydroxylase immunopositive neurons differs between animals with intraperitoneal and subcutaneous tumors and also between tumor-bearing rats at different times following injection. Our data indicate that responses of the brainstem structures to peripheral tumor growth depend on the localization as well as the stage of the tumor growth.

AT2receptors recruit c-Src, SHP-1 and FAK upon activation by Ang II in PND15 rat hindbrain.

The functional role of AT(2) receptors is unclear and it activates unconventional signaling pathways, which in general do not involve a classical activation of a G-protein. In the present study, we aimed to investigate the transduction mechanism of AT(2) Ang II receptors in PND15 rat hindbrain membrane preparations, which represents a physiological developmental condition. To determine whether Ang II AT(2) receptors induced association to SHP-1 in rat hindbrain, co-immunoprecipitation assays were performed. Stimulation of Ang II AT(2) receptors induced both a transient tyr-phosphorylation and activation of SHP-1. The possible participation of c-Src in Ang II-mediated SHP-1 activation, we demonstrated by recruitment of c-Src in immunocomplexes obtained with anti AT(2) or anti-SHP-1 antibodies. The association of SHP-1 to c-Src was inhibited by PD123319 and the c-Src inhibitor PP2. Similarly, SHP-1 activity determined in AT(2)-immunocomplexes was inhibited by PD123319 and the c-Src inhibitor PP2. Following stimulation with Ang II, AT(2) receptors recruit c-Src, which was responsible for SHP-1 tyr-phosphorylation and activation. Since AT(2) receptors are involved in neuron migration, we tested the presence of FAK in immunocomplexes. Surprisingly, AT(2)-immunocomplexes contained mainly the 85kDa fragment of FAK. Besides, p125FAK associated to SHP-1. In summary, we demonstrated the presence of an active signal transduction mechanism in PND15 rat hindbrain, a developmental stage critical for cerebellar development. In this model, we showed a complex containing AT(2)/SHP-1/c-Src/p85FAK, suggesting a potential role of Ang II AT(2) receptors in cerebellar development and neuronal differentiation.

In vitro leptin treatment of rainbow trout hypothalamus and hindbrain affects glucosensing and gene expression of neuropeptides involved in food intake regulation.

The aim of the present study was to evaluate in hypothalamus and hindbrain of rainbow trout in vitro the effect of leptin treatment on glucosensing capacity and the expression of orexigenic and anorexigenic peptides involved in the control of food intake. In a first experiment, the response of parameters involved in glucosensing (GK, PK and GSase activities; GK expression and glucose; glycogen and DHAP levels) and the expression of orexigenic (NPY) and anorexigenic (POMC, CART, CRF) peptides was assessed in hypothalami and hindbrain incubated for 1h with 2, 4 or 8mM d-glucose alone (controls) or with 10nM leptin, or with 10nM leptin plus inhibitors of leptin signaling pathways (50nM wortmannin and 500nM AG490). Leptin treatment increased levels in parameters involved in glucosensing. Leptin treatment decreased NPY mRNA levels in hypothalamus without affecting the expression of the other peptides assessed. Leptin effects were reverted in the presence of inhibitors for all parameters assessed suggesting the involvement of JAK/STAT and IRS-PI(3)K pathways. In a second experiment, we observed time-dependent (1-3h) and dose (10, 20 and 50nM)- effects of leptin treatment in decreasing NPY mRNA levels without affecting expression of the other peptides assessed. Considering the orexigenic action of NPY in fish, it seems that the anorexic effect of leptin can be mediated by reduced expression of NPY occurring in hypothalamus, and that change can be related to the activation of the glucosensing system occurring simultaneously.

Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II.

BACKGROUND: In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A). It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear. RESULTS: In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK) and myosin light chain kinase (MLCK), which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points. CONCLUSIONS: Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.

Genetic disruption of CYP26B1 severely affects development of neural crest derived head structures, but does not compromise hindbrain patterning.

Cyp26b1 encodes a cytochrome-P450 enzyme that catabolizes retinoic acid (RA), a vitamin A derived signaling molecule. We have examined Cyp26b1(-/-) mice and report that mutants exhibit numerous abnormalities in cranial neural crest cell derived tissues. At embryonic day (E) 18.5 Cyp26b1(-/-) animals exhibit a truncated mandible, abnormal tooth buds, reduced ossification of calvaria, and are missing structures of the maxilla and nasal process. Some of these abnormalities may be due to defects in formation of Meckel's cartilage, which is truncated with an unfused distal region at E14.5 in mutant animals. Despite the severe malformations, we did not detect any abnormalities in rhombomere segmentation, or in patterning and migration of anterior hindbrain derived neural crest cells. Abnormal migration of neural crest cells toward the posterior branchial arches was observed, which may underlie defects in larynx and hyoid development. These data suggest different periods of sensitivity of anterior and posterior hindbrain neural crest derivatives to elevated levels of RA in the absence of CYP26B1.

Characterization of primary neurospheres generated from mouse ventral rostral hindbrain.

Serotonergic (5-HT) neurons of the reticular formation play a key role in the modulation of behavior, and their dysfunction is associated with severe neurological and psychiatric disorders, such as depression and schizophrenia. However, the molecular mechanisms underlying the differentiation of the progenitor cells and the specification of the 5-HT phenotype are not fully understood. A primary neurosphere cell-culture system from mouse ventral rostral hindbrain at embryonic day 12 was therefore established. The generated primary neurospheres comprised progenitor cells and fully differentiated neurons. Bromodeoxyuridine incorporation experiments in combination with immunocytochemistry for neural markers revealed the proliferation capacity of the neural multipotent hindbrain progenitors within neurospheres and their ability to differentiate toward the neuronal lineage and serotonergic phenotype. Gene expression analysis by reverse transcription with the polymerase chain reaction showed that the neurospheres were regionally specified, as reflected by the expression of the transcription factors Gata2 and Pet1. Treatment of dissociated primary neurospheres with exogenous Shh significantly increased the number of 5-HT-immunopositive cells compared with controls, whereas neutralization of endogenous Shh significantly decreased the number of 5-HT neurons. Thus, the primary neurosphere culture system presented here allows the expansion of hindbrain progenitor cells and the experimental control of their differentiation toward the serotonergic phenotype. This culture system is therefore a useful model for in vitro studies dealing with the development of 5-HT neurons.

Dorsal hindbrain 5'-adenosine monophosphate-activated protein kinase as an intracellular mediator of energy balance.

The fuel-sensing enzyme AMP-activated protein kinase (AMPK) has been implicated in central nervous system control of energy balance. Hypothalamic AMPK activity is increased by food deprivation, and this elevation is inhibited by refeeding or by leptin treatment. The contribution of extrahypothalamic AMPK activity in energy balance control has not been addressed. Here, we investigate the effects of physiological state on the AMPK activity in hindbrain nucleus tractus solitarius (NTS) neurons because treatments that reduce energy availability in these neurons trigger behavioral, endocrine, and autonomic responses to restore energy balance. Food-deprived rats showed significantly increased AMPK activity in both NTS- and hypothalamus-enriched lysates compared with those that were ad libitum fed. Pharmacological inhibition of AMPK activity in medial NTS neurons, by intraparenchymal injection of compound C, suppressed food intake and body weight gain compared with vehicle. Fourth ventricle (4th i.c.v.) compound C delivery increased heart rate and spontaneous activity in free-moving rats. Suppression of AMPK activity has been implicated in leptin's anorectic action in the hypothalamus. Given the role of leptin signaling in food intake inhibition within the medial NTS, we also examined whether stimulation of hindbrain AMPK by 4th i.c.v. administration of 5-aminoimidazole-4-carboxamide-riboside (AICAR), an AMP-mimicking promoter of AMPK activity, could attenuate the inhibition of food intake by 4th i.c.v. leptin. The intake-suppressive effects of leptin (at 2 and 4 h) were completely reversed by AICAR. We conclude that 1) hindbrain AMPK activity contributes to energy balance control through regulation of food intake and energy expenditure, 2) leptin's intake-reducing effects in the NTS are mediated by AMPK, and 3) central nervous system AMPK controls whole-body homeostasis at anatomically distributed sites across the neuraxis.

The 48-kDa alternative translation isoform of PP2A:B56epsilon is required for Wnt signaling during midbrain-hindbrain boundary formation.

Alternative translation is an underappreciated post-transcriptional regulation mechanism. Although only a small number of genes are found to be alternatively translated, most genes undergoing alternative translation play important roles in tumorigenesis and development. Protein phosphatase 2A (PP2A) is involved in many cellular events during tumorigenesis and development. The specificity, localization, and activity of PP2A are regulated by B regulatory subunits. B56epsilon, a member of the B56 regulatory subunit family, is involved in multiple signaling pathways and regulates a number of developmental processes. Here we report that B56epsilon is alternatively translated, leading to the production of a full-length form and a shorter isoform that lacks the N-terminal 76 amino acid residues of the full-length form. Alternative translation of B56epsilon occurs through a cap-dependent mechanism. We provide evidence that the shorter isoform is required for Wnt signaling and regulates the midbrain/hindbrain boundary formation during Xenopus embryonic development. This demonstrates that the shorter isoform of B56epsilon has important biological functions. Furthermore, we show that the N-terminal sequence of B56epsilon, which is not present in the shorter isoform, contains a nuclear localization signal, whereas the C terminus of B56epsilon contains a nuclear export signal. The shorter isoform, which lacks the N-terminal nuclear localization signal, is restricted to the cytoplasm. In contrast, the full-length form can be localized to the nucleus in a cell type-specific manner. The finding that B56epsilon is alternatively translated adds a new level of regulation to PP2A holoenzymes.

Complementary expression of HSPG 6-O-endosulfatases and 6-O-sulfotransferase in the hindbrain of Xenopus laevis.

Heparan sulfate proteoglycans (HSPGs) are abundant cell surface molecules, consisting of glycosaminoglycan (GAG) chains bound to a protein core. There is high diversity in the sulfation pattern within each GAG chain, creating specific binding sites for many proteins including cell signalling factors, whose activities and distribution are modified by their association with HSPGs (Danesin et al., 2006; Freeman et al., 2008). Here, we describe the distinct expression of three enzymes which modify the 6-O-sulfation state of HSPGs: two 6-O-endosulfatases (Sulf1 and Sulf2), and a 6-O-sulfotransferase (6OST-1). We use in situ hybridisation to determine the spatial distribution of transcripts during tailbud stages of Xenopus laevis development, with a particular focus on neural regions where the 6-O-sulfatases are expressed ventrally and the 6-O-sulfotransferase is restricted dorsally. The complementary expression of these enzymes in the hindbrain and neural tube suggest a role for specific HSPG structure in dorsoventral patterning, possibly through modifying the activity or distribution of signalling molecules such as BMP, Sonic hedgehog, Wnt and/or FGF.

How degrading: Cyp26s in hindbrain development.

The vitamin A derivative retinoic acid performs many functions in vertebrate development and is thought to act as a diffusible morphogen that patterns the anterior-posterior axis of the hindbrain. Recent work in several systems has led to insights into how the spatial distribution of retinoic acid is regulated. These have shown local control of synthesis and degradation, and computational models suggest that degradation by the Cyp26 enzymes plays a critical role in the formation of a morphogen gradient as well as its ability to compensate for fluctuations in RA levels.

Dietary carbohydrates induce changes in glucosensing capacity and food intake of rainbow trout.

We hypothesize that variations in dietary carbohydrate levels produce changes in glucosensor parameters in previously characterized glucosensing areas (hypothalamus and hindbrain) related with the regulation of food intake of a carnivorous fish species like rainbow trout. Therefore, we fed trout with standard, carbohydrate-free (CF) or high-carbohydrate (HC) diets for 10 days to assess changes in glucosensing system and food intake. Fish fed CF diet displayed hypoglycemia and increased food intake. Fish fed a HC diet displayed hyperglycemia and decreased food intake. Changes in food intake due to dietary carbohydrates were accompanied in hypothalamus and hindbrain of fish fed with HC diet by changes in parameters involved in glucosensing, such as increased glucose, glucose 6-phosphate, and glycogen levels and increased glucokinase (GK), glycogen synthase, and pyruvate kinase activities as well as increased GK and GLUT2 expression. All those results address for the first time in fish, despite the relative intolerance to glucose of carnivorous species, that dietary carbohydrates are important regulators of the glucosensing system in carnivorous fish, suggesting that the information generated by this system can be associated with the changes observed in food intake.

Involvement of lactate in glucose metabolism and glucosensing function in selected tissues of rainbow trout.

The aim of this study was to obtain evidence in rainbow trout for a role of lactate in glucose homeostasis as well as in the function of glucosensing tissues. In a first set of experiments, trout were injected, either (1) intraperitoneally (N=8) with 5 ml kg(-1) of Cortland saline alone (control) or saline containing l-(+)-lactate (22.5 mg kg(-1) or 45 mg kg(-1)), oxamate (22.5 mg kg(-1)) or d-glucose (500 mg kg(-1)), or (2) intracerebroventricularly (N=11) with 1 microl 100 g(-1) body mass of Cortland saline alone (control) or containing d-glucose (400 microg microl(-1)) or l-(+)-lactate (400 microg microl(-1)), with samples being obtained 6 h after treatment. In a second set of experiments, hypothalamus, hindbrain and Brockmann bodies were incubated in vitro for 1 h at 15 degrees C in modified Hanks' medium containing 2, 4 or 8 mmol l(-1) l-(+)-lactate alone (control) or with 50 mmol l(-1) oxamate, 1 mmol l(-1) DIDS, 1 mmol l(-1) dichloroacetate, 10 mmol l(-1) 2-deoxy-d-glucose, 1 mmol l(-1) alpha-cyano-4-hydroxy cinnamate or 10 mmol l(-1) d-glucose. The response of parameters assessed (metabolite levels, enzyme activities and glucokinase expression) in tissues provided evidence for (1) a role for lactate in the regulation of glucose homeostasis through changes not only in brain regions but also in liver energy metabolism, which are further reflected in changes in plasma levels of metabolites; (2) the possible presence in trout brain of an astrocyte-neuron lactate shuttle similar to that found in mammals; and (3) the lack of capacity of lactate to mimic in vitro (but not in vivo) glucose effects in fish glucosensing regions.

Zebrafish colgate/hdac1 functions in the non-canonical Wnt pathway during axial extension and in Wnt-independent branchiomotor neuron migration.

Vertebrate gastrulation involves the coordinated movements of populations of cells. These movements include cellular rearrangements in which cells polarize along their medio-lateral axes leading to cell intercalations that result in elongation of the body axis. Molecular analysis of this process has implicated the non-canonical Wnt/Frizzled signaling pathway that is similar to the planar cell polarity pathway (PCP) in Drosophila. Here we describe a zebrafish mutant, colgate (col), which displays defects in the extension of the body axis and the migration of branchiomotor neurons. Activation of the non-canonical Wnt/PCP pathway in these mutant embryos by overexpressing DeltaNdishevelled, rho kinase2 and van gogh-like protein 2 (vangl2) rescues the extension defects suggesting that col acts as a positive regulator of the non-canonical Wnt/PCP pathway. Further, we show that col normally regulates the caudal migration of nVII facial hindbrain branchiomotor neurons and that the mutant phenotype can be rescued by misexpression of vangl2 independent of the Wnt/PCP pathway. We cloned the col locus and found that it encodes histone deacetylase1 (hdac1). Our previous results and studies by others have implicated hdac1 in repressing the canonical Wnt pathway. Here, we demonstrate novel roles for zebrafish hdac1 in activating non-canonical Wnt/PCP signaling underlying axial extension and in promoting Wnt-independent caudal migration of a subset of hindbrain branchiomotor neurons.

In vitro evidences for glucosensing capacity and mechanisms in hypothalamus, hindbrain, and Brockmann bodies of rainbow trout.

We aimed to support in vitro the glucosensing capacity observed in vivo in rainbow trout hypothalamus, hindbrain, and Brockmann bodies (BB) and to obtain preliminary evidence of the mechanisms involved. The response of parameters involved in the glucosensing capacity [hexokinase, hexokinase IV (glucokinase), and pyruvate kinase activities and glucose and glycogen levels] was assessed in these tissues incubated for 1 h with 2, 4, or 8 mM D-glucose alone (control) or with specific agonists/inhibitors of the steps involved in glucosensing capacity in mammals. These agents were a competitor for glucose phosphorylation (15 mM mannose), sulfonylurea receptor-1 effectors (500 microM tolbutamide or diazoxide), glycolytic intermediates (15 mM glycerol, lactate, or pyruvate), and inhibitors of glucose transport (10 microM cytochalasin B), glycolysis [20 mM 2-deoxy-D-glucose (2-DG)], and L-type calcium channel (1 microM nifedipine). Control incubations of the three tissues displayed increased glucose and glycogen levels and glucokinase activities in response to increased medium glucose, thus supporting our previous in vivo studies. Furthermore, critical components of the glucosensing mammalian machinery are apparently functioning in the three tissues. The responses in brain regions to all substances tested (except 2-DG and nifedipine) were similar to those observed in mammals, suggesting a similar glucosensing machinery. In contrast, in BB, only the effects of 2-DG, lactate, pyruvate, diazoxide, and nifedipine were similar to those of mammalian beta-cells, suggesting that some of the components of the piscine glucosensing model are different than those of mammals. Such differences may relate to the importance of amino acids rather than glucose signaling in the trout BB.

The oxidizing enzyme CYP26a1 tightly regulates the availability of retinoic acid in the gastrulating mouse embryo to ensure proper head development and vasculogenesis.

Retinoic acid (RA) has been implicated as one of the signals providing a posterior character to the developing vertebrate central nervous system. Embryonic RA first appears in the posterior region of the gastrulating embryo up to the node level, where it may signal within the adjacent epiblast and/or newly induced neural plate to induce a hindbrain and spinal cord fate. Conversely, rostral head development requires forebrain-inducing signals produced by the anterior visceral endoderm and/or prechordal mesoderm, and there is evidence that RA receptors must be in an unliganded state to ensure proper head development. As RA is a diffusible lipophilic molecule, some mechanism(s) must therefore have evolved to prevent activation of RA targets in anterior regions of the embryo. This might result from RA catabolism mediated by the CYP26A1 oxidizing enzyme, which is transiently expressed in anteriormost embryonic tissues; however, previous analysis of Cyp26a1(-/-) mouse mutants did not clearly support this hypothesis. Here we show that Cyp26a1(-/-) null mutants undergo head truncations when exposed to maternally-derived RA, at doses that do not affect wild-type head development. These anomalies are linked to a widespread ectopic RA signaling activity in rostral head tissues of CYP26A1-deficient embryos. Thus, CYP26A1 is required in the anterior region of the gastrulating mouse embryo to prevent teratological effects that may result from RA signaling. We also report a novel role of CYP26A1 during early development of the intra- and extra-embryonic vascular networks.

Calretinin immunoreactivity in the brain of the zebrafish, Danio rerio: distribution and comparison with some neuropeptides and neurotransmitter-synthesizing enzymes. II. Midbrain, hindbrain, and rostral spinal cord.

The distribution of calretinin (CR) in the brainstem and rostral spinal cord of the adult zebrafish was studied by using immunocytochemical techniques. For analysis of some brainstem nuclei and regions, CR distribution was compared with that of complementary markers (choline acetyltransferase, glutamic acid decarboxylase, tyrosine hydroxylase, neuropeptide Y). The results reveal that CR is a marker of various neuronal populations distributed throughout the brainstem, including numerous cells in the optic tectum, torus semicircularis, secondary gustatory nucleus, reticular formation, somatomotor column, gustatory lobes, octavolateral area, and inferior olive, as well as of characteristic tracts of fibers and neuropil. These results indicate that CR may prove useful for characterizing a number of neuronal subpopulations in zebrafish. Comparison of the distribution of CR observed in the brainstem of zebrafish with that reported in an advanced teleost (the gray mullet) revealed a number of similarities, and also some interesting differences. Our results indicate that many brainstem neuronal populations have maintained the CR phenotype in widely divergent teleost lines, so CR studies may prove very useful for comparative analysis.

Histone deacetylase 1 is essential for oligodendrocyte specification in the zebrafish CNS.

Histone deacetylases are critical components of transcriptional silencing mechanisms that regulate embryonic development. Recent work has shown that histone deacetylase 1 (hdac1) is required for neuronal specification during zebrafish CNS development. We show here that specification of oligodendrocytes, the myelinating cells of the CNS, also fails to occur in the hdac1 mutant hindbrain, but persistence of neural progenitors in the hindbrain ventricular zone, which express pax6a and sox2, is independent of hdac1 activity. Commitment of ventral neural progenitors to the oligodendrocyte fate is thought to require co-ordinate, hedgehog-dependent expression of olig2 and nkx2.2a in these cells, leading to expression of sox10 and subsequent differentiation of oligodendrocytes. Remarkably, transcription of olig2 is extinguished in ventral neural progenitors of the hdac1 mutant hindbrain, whereas expression of nkx2.2a is up-regulated in these cells, and sox10 expression is suppressed. Our results identify hdac1 as a novel, essential component of the mechanism that allocates neural progenitors to the oligodendrocyte fate, by attenuating expression of a subset of neural progenitor genes and rendering olig2 expression responsive to Hedgehog signalling.