Ectopic over expression of kiss1 may compensate for the loss of kiss2.

Kisspeptin (KISS) is a neuropeptide which plays a central role in the regulation of the hypothalamic-pituitary-gonadal axis, and is essential for sexual maturation and fertility in mammals. Unlike mammals, which possess only one KISS gene, two paralogous genes, kiss1 and kiss2, have been identified in zebrafish and other non-mammalian vertebrates. Previous studies suggest that Kiss2, but not Kiss1, is the reproduction relevant form amongst the two. To better understand the role of each of these isoforms in reproduction, a loss of function approach was applied. Two genetic manipulation techniques-clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN)-were used to generate kiss1 and kiss2 knockout (KO) zebrafish lines, respectively. Examination of these KO lines showed that reproductive capability was not impaired, confirming earlier observations. Further analysis revealed that KO of kiss2 caused a significant increase in expression levels of kiss1, kiss2r and tac3a, while KO of kiss1 had no effect on the expression of any of the examined genes. In situ hybridization analysis revealed that kiss1 mRNA is expressed only in the habenula in wild type brains, while in kiss2 KO fish, kiss1 mRNA-expressing cells were identified also in the ventral telencephalon, the ventral part of the entopeduncular nucleus, and the dorsal and ventral hypothalamus. Interestingly, these regions are known to express kiss2r, and the ventral hypothalamus normally expresses kiss2. These results suggest that a compensatory mechanism, involving ectopic kiss1 expression, takes place in the kiss2 KO fish, which may substitute for Kiss2 activity.

Knockdown of unc119c results in visual impairment and early-onset retinal dystrophy in zebrafish.

PURPOSE: UNC119 proteins are involved in G protein trafficking in mouse retinal photoreceptors and Caenorhabditis elegans olfactory neurons. An Unc119 null allele is associated with cone-rod dystrophy in mouse, but the mechanism leading to disease is not understood. We studied the role of Unc119 paralogs and Arl3l2 in zebrafish vision and retinal organization resulting from unc119c and arl3l2 knockdown. METHODS: Zebrafish unc119c was amplified by PCR from retina and pineal gland cDNA. Its expression pattern in the eye and pineal gland was determined by whole-mount in-situ hybridization. unc119c and arl3l2 were knocked down using morpholino-modified oligonucleotides (MO). Their visual function was assessed with a quantitative optomotor assay on 6 days post-fertilization larvae. Retinal morphology was analyzed using immunohistochemistry with anti-cone arrestin (zpr-1) and anti-cone transducin-α (GNAT2) antibodies. RESULTS: The zebrafish genome contains four genes encoding unc119 paralogs located on different chromosomes. The exon/intron arrangements of these genes are identical. Three Unc119 paralogs are expressed in the zebrafish retina, termed Unc119a-c. Based on sequence similarity, Unc119a and Unc119b are orthologs of mammalian UNC119a and UNC119b, respectively. A third, Unc119c, is unique and not present in mammals. Whole mount in-situ hybridization revealed that unc119a and unc119b RNA are ubiquitously expressed in the CNS, and unc119c is specifically expressed in photoreceptive tissues (pineal gland and retina). A Unc119 interactant, Arl3l2 also localizes to the pineal gland and the retina. As measured by the optomotor response, unc119c and arl3l2 knockdown resulted in significantly lower vision compared to wild-type zebrafish larvae and control morpholino (MO). Immunohistological analysis with anti-cone transducin and anti-cone arrestin (zpr-1) indicates that knockdown of unc119c leads to photoreceptor degeneration mostly affecting cones. CONCLUSIONS: Our results suggest that Unc119c is the only Unc119 paralog that is highly specific to the retina in zebrafish. Unc119c and Arl3l2 proteins are important for the function of cones.

The Dlx5 and Foxg1 transcription factors, linked via miRNA-9 and -200, are required for the development of the olfactory and GnRH system.

During neuronal development and maturation, microRNAs (miRs) play diverse functions ranging from early patterning, proliferation and commitment to differentiation, survival, homeostasis, activity and plasticity of more mature and adult neurons. The role of miRs in the differentiation of olfactory receptor neurons (ORNs) is emerging from the conditional inactivation of Dicer in immature ORN, and the depletion of all mature miRs in this system. Here, we identify specific miRs involved in olfactory development, by focusing on mice null for Dlx5, a homeogene essential for both ORN differentiation and axon guidance and connectivity. Analysis of miR expression in Dlx5(-/-) olfactory epithelium pointed to reduced levels of miR-9, miR-376a and four miRs of the -200 class in the absence of Dlx5. To functionally examine the role of these miRs, we depleted miR-9 and miR-200 class in reporter zebrafish embryos and observed delayed ORN differentiation, altered axonal trajectory/targeting, and altered genesis and position of olfactory-associated GnRH neurons, i.e. a phenotype known as Kallmann syndrome in humans. miR-9 and miR-200-class negatively control Foxg1 mRNA, a fork-head transcription factor essential for development of the olfactory epithelium and of the forebrain, known to maintain progenitors in a stem state. Increased levels of z-foxg1 mRNA resulted in delayed ORN differentiation and altered axon trajectory, in zebrafish embryos. This work describes for the first time the role of specific miR (-9 and -200) in olfactory/GnRH development, and uncovers a Dlx5-Foxg1 regulation whose alteration affects receptor neuron differentiation, axonal targeting, GnRH neuron development, the hallmarks of the Kallmann syndrome.

Profiling, Bioinformatic, and Functional Data on the Developing Olfactory/GnRH System Reveal Cellular and Molecular Pathways Essential for This Process and Potentially Relevant for the Kallmann Syndrome.

During embryonic development, immature neurons in the olfactory epithelium (OE) extend axons through the nasal mesenchyme, to contact projection neurons in the olfactory bulb. Axon navigation is accompanied by migration of the GnRH+ neurons, which enter the anterior forebrain and home in the septo-hypothalamic area. This process can be interrupted at various points and lead to the onset of the Kallmann syndrome (KS), a disorder characterized by anosmia and central hypogonadotropic hypogonadism. Several genes has been identified in human and mice that cause KS or a KS-like phenotype. In mice a set of transcription factors appears to be required for olfactory connectivity and GnRH neuron migration; thus we explored the transcriptional network underlying this developmental process by profiling the OE and the adjacent mesenchyme at three embryonic ages. We also profiled the OE from embryos null for Dlx5, a homeogene that causes a KS-like phenotype when deleted. We identified 20 interesting genes belonging to the following categories: (1) transmembrane adhesion/receptor, (2) axon-glia interaction, (3) scaffold/adapter for signaling, (4) synaptic proteins. We tested some of them in zebrafish embryos: the depletion of five (of six) Dlx5 targets affected axonal extension and targeting, while three (of three) affected GnRH neuron position and neurite organization. Thus, we confirmed the importance of cell-cell and cell-matrix interactions and identified new molecules needed for olfactory connection and GnRH neuron migration. Using available and newly generated data, we predicted/prioritized putative KS-disease genes, by building conserved co-expression networks with all known disease genes in human and mouse. The results show the overall validity of approaches based on high-throughput data and predictive bioinformatics to identify genes potentially relevant for the molecular pathogenesis of KS. A number of candidate will be discussed, that should be tested in future mutation screens.