A versatile transcription factor: Multiple roles of orthopedia a (otpa) beyond its restricted localization in dopaminergic systems of developing and adult zebrafish (Danio rerio) brains.

Many transcription factors boost neural development and differentiation in specific directions and serve for identifying similar or homologous structures across species. The expression of Orthopedia (Otp) is critical for the development of certain cell groups along the vertebrate neuraxis, for example, the medial amygdala or hypothalamic neurosecretory neurons. Therefore, the primary focus of the present study is the distribution of Orthopedia a (Otpa) in the larval and adult zebrafish (Danio rerio) brain. Since Otpa is also critical for the development of zebrafish basal diencephalic dopaminergic cells, colocalization of Otpa with the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) is studied. Cellular colocalization of Otpa and dopamine is only seen in magnocellular neurons of the periventricular posterior tubercular nucleus and in the posterior tuberal nucleus. Otpa-positive cells occur in many additional structures along the zebrafish neuraxis, from the secondary prosencephalon down to the hindbrain. Furthermore, Otpa expression is studied in shh-GFP and islet1-GFP transgenic zebrafish. Otpa-positive cells only express shh in dopaminergic magnocellular periventricular posterior tubercular cells, and only colocalize with islet1-GFP in the ventral zone and prerecess caudal periventricular hypothalamic zone and the perilemniscal nucleus. The scarcity of cellular colocalization of Otpa in islet1-GFP cells indicates that the Shh-islet1 neurogenetic pathway is not active in most Otpa-expressing domains. Our analysis reveals detailed correspondences between mouse and zebrafish forebrain territories including the zebrafish intermediate nucleus of the ventral telencephalon and the mouse medial amygdala. The zebrafish preoptic Otpa-positive domain represents the neuropeptidergic supraopto-paraventricular region of all tetrapods. Otpa domains in the zebrafish basal plate hypothalamus suggest that the ventral periventricular hypothalamic zone corresponds to the otp-expressing basal hypothalamic tuberal field in the mouse. Furthermore, the mouse otp domain in the mammillary hypothalamus compares partly to our Otpa-positive domain in the prerecess caudal periventricular hypothalamic zone (Hc-a).

Adult islet1 Expression Outlines Ventralized Derivatives Along Zebrafish Neuraxis.

Signals issued by dorsal roof and ventral floor plates, respectively, underlie the major patterning process of dorsalization and ventralization during vertebrate neural tube development. The ventrally produced morphogen Sonic hedgehog (SHH) is crucial for vertebrate hindbrain and spinal motor neuron development. One diagnostic gene for motor neurons is the LIM/homeodomain gene islet1, which has additional ventral expression domains extending into mid- and forebrain. In order to corroborate motor neuron development and, in particular, to improve on the identification of poorly documented zebrafish forebrain islet1 populations, we studied adult brains of transgenic islet1-GFP zebrafish (3 and 6 months). This molecular neuroanatomical analysis was supported by immunostaining these brains for tyrosine hydroxylase (TH) or choline acetyltransferase (ChAT), respectively, revealing zebrafish catecholaminergic and cholinergic neurons. The present analysis of ChAT and islet1-GFP label confirms ongoing adult expression of islet1 in zebrafish (basal plate) midbrain, hindbrain, and spinal motor neurons. In contrast, non-motor cholinergic systems lack islet1 expression. Additional presumed basal plate islet1 positive systems are described in detail, aided by TH staining which is particularly informative in the diencephalon. Finally, alar plate zebrafish forebrain systems with islet1 expression are described (i.e., thalamus, preoptic region, and subpallium). We conclude that adult zebrafish continue to express islet1 in the same brain systems as in the larva. Further, pending functional confirmation we hypothesize that the larval expression of sonic hedgehog (shh) might causally underlie much of adult islet1 expression because it explains findings beyond ventrally located systems, for example regarding shh expression in the zona limitans intrathalamica and correlated islet1-GFP expression in the thalamus.

Behavioural and neuronal basis of olfactory imprinting and kin recognition in larval fish.

Imprinting is a specific form of long-term memory of a cue acquired during a sensitive phase of development. To ensure that organisms memorize the right cue, the learning process must happen during a specific short time period, mostly soon after hatching, which should end before irrelevant or misleading signals are encountered. A well-known case of olfactory imprinting in the aquatic environment is that of the anadromous Atlantic and Pacific salmon, which prefer the olfactory cues of natal rivers to which they return after migrating several years in the open ocean. Recent research has shown that olfactory imprinting and olfactory guided navigation in the marine realm are far more common than previously assumed. Here, we present evidence for the involvement of olfactory imprinting in the navigation behaviour of coral reef fish, which prefer their home reef odour over that of other reefs. Two main olfactory imprinting processes can be differentiated: (1) imprinting on environmental cues and (2) imprinting on chemical compounds released by kin, which is based on genetic relatedness among conspecifics. While the first process allows for plasticity, so that organisms can imprint on a variety of chemical signals, the latter seems to be restricted to specific genetically determined kin signals. We focus on the second, elucidating the behavioural and neuronal basis of the imprinting process on kin cues using larval zebrafish (Danio rerio) as a model. Our data suggest that the process of imprinting is not confined to the central nervous system but also triggers some changes in the olfactory epithelium.

Identification of accessory olfactory system and medial amygdala in the zebrafish.

Zebrafish larvae imprint on visual and olfactory cues of their kin on day 5 and 6 postfertilization, respectively. Only imprinted (but not non-imprinted) larvae show strongly activated crypt (and some microvillous) cells demonstrated by pERK levels after subsequent exposure to kin odor. Here, we investigate the olfactory bulb of zebrafish larvae for activated neurons located at the sole glomerulus mdG2 which receives crypt cell input. Imprinted larvae show a significantly increased activation of olfactory bulb cells compared to non-imprinted larvae after exposure to kin odor. Surprisingly, pERK activated Orthopedia-positive cell numbers in the intermediate ventral telencephalic nucleus were higher in non-imprinted, kin odor stimulated larvae compared to control and to kin-odor stimulated imprinted larvae and control. Moreover, DiI tracing experiments in adult zebrafish show a neuronal circuit from crypt/microvillous olfactory sensory neurons via dorsomedial olfactory bulb and intermediate ventral telencephalic nucleus (thus, arguably the teleostean medial amygdala) to tuberal hypothalamus, demonstrating for the first time an accessory olfactory system in teleosts.

Eppur Si Muove: Evidence for an External Granular Layer and Possibly Transit Amplification in the Teleostean Cerebellum.

The secreted signaling factor Sonic Hedgehog (Shh) acts in the floor plate of the developing vertebrate CNS to promote motoneuron development. In addition, shh has dorsal expression domains in the amniote alar plate (i.e., in isocortex, superior colliculus, and cerebellum). For example, shh expressing Purkinje cells act in transit amplification of external granular layer (EGL) cells of the developing cerebellum. Our previous studies had indicated the presence of an EGL in anamniote zebrafish, but a possible role of shh in the zebrafish cerebellar plate remained elusive. Therefore, we used an existing zebrafish transgenic line Tg(2.4shha-ABC-GFP)sb15; Shkumatava et al., 2004) to show this gene activity and its cellular localization in the larval zebrafish brain. Clearly, GFP expressing cells occur in larval alar zebrafish brain domains, i.e., optic tectum and cerebellum. Analysis of critical cerebellar cell markers on this transgenic background and a PH3 assay for mitotic cells reveals that Purkinje cells and eurydendroid cells are completely non-overlapping postmitotic cell populations. Furthermore, shh-GFP cells never express Zebrin II or parvalbumin, nor calretinin. They are thus neither Purkinje cells nor calretinin positive migrating rhombic lip derived cells. The shh-GFP cells also never correspond to PH3 positive cells of the ventral cerebellar proliferative zone or the upper rhombic lip-derived EGL. From this marker analysis and the location of shh-GFP cells sandwiched between calretinin positive rhombic lip derived cells and parvalbumin positive Purkinje cells, we conclude that shh-GFP expressing cells qualify as previously reported olig2 positive eurydendroid cells, which are homologous to the amniote deep cerebellar nuclei. We confirm this using double transgenic progeny of shh-GFP and olig2-dsRed zebrafish. Thus, these zebrafish eurydendroid cells may have the same role in transit amplification as Purkinje cells do in amniotes.

Crypt cells are involved in kin recognition in larval zebrafish.

Zebrafish larvae imprint on visual and olfactory kin cues at day 5 and 6 postfertilization, respectively, resulting in kin recognition later in life. Exposure to non-kin cues prevents imprinting and kin recognition. Imprinting depends on MHC class II related signals and only larvae sharing MHC class II alleles can imprint on each other. Here, we analyzed which type of olfactory sensory neuron (OSN) detects kin odor. The single teleost olfactory epithelium harbors ciliated OSNs carrying OR and TAAR gene family receptors (mammals: main olfactory epithelium) and microvillous OSNs with V1R and V2R gene family receptors (mammals: vomeronasal organ). Additionally, teleosts exhibit crypt cells which possess microvilli and cilia. We used the activity marker pERK (phosphorylated extracellular signal regulated kinase) after stimulating 9 day old zebrafish larvae with either non-kin conspecific or food odor. While food odor activated both ciliated and microvillous OSNs, only the latter were activated by conspecific odor, crypt cells showed no activation to both stimuli. Then, we tested imprinted and non-imprinted larvae (full siblings) for kin odor detection. We provide the first direct evidence that crypt cells, and likely a subpopulation of microvillous OSNs, but not ciliated OSNs, play a role in detecting a kin odor related signal.

Combinatorial analysis of calcium-binding proteins in larval and adult zebrafish primary olfactory system identifies differential olfactory bulb glomerular projection fields.

In the zebrafish (Danio rerio) olfactory epithelium, the calcium-binding proteins (CBPs) calretinin and S100/S100-like protein are mainly expressed in ciliated or crypt olfactory sensory neurons (OSNs), respectively. In contrast parvalbumin and calbindin1 have not been investigated. We present a combinatorial immunohistological analysis of all four CBPs, including their expression in OSNs and their axonal projections to the olfactory bulb in larval and adult zebrafish. A major expression of calretinin and S100 in ciliated and crypt cells, respectively, with some expression of S100 in microvillous cells is confirmed. Parvalbumin and calbindin1 are strongly expressed in ciliated and microvillous cells, but not in crypt cells. Moreover, detailed combinatorial double-label experiments indicate that there are eight subpopulations of zebrafish OSNs: S100-positive crypt cells (negative for all other three CBPs), parvalbumin only, S100 and parvalbumin, parvalbumin and calbindin1, and parvalbumin and calbindin1 and calretinin-positive microvillous OSNs, as well as a major parvalbumin and calbindin1 and calretinin, and minor parvalbumin and calbindin1 and calretinin-only-positive ciliated OSN populations. CBP-positive projections to olfactory bulb are consistent with previous reports of ciliated OSNs projecting to dorsal and ventromedial glomerular fields and microvillous OSNs to ventrolateral glomerular fields. We newly describe parvalbumin-positive fibers to the mediodorsal field which is calretinin free, with its anterior part showing additionally calbindin1-positive fibers, but absence thereof in the posterior part, indicating an origin from microvillous OSNs in both parts. One singular glomerulus (mdG2) exhibits S100 and parvalbumin-positive fibers, apparently originating from all crypt cells plus some microvillous OSNs. Arguments for various olfactory labeled lines are discussed.