Wolfgang Driever

The growing brain faces the challenge to establish stable spatially defined neural proliferation zones that accomplish both neural stem cell (NSC) maintenance and dynamic neurogenesis. We investigated the relative impact of Notch signaling, Notch-dependent, and Notch-independent HES/Her transcription factors on target gene expression in the larval zebrafish brain. Both genetic evidence and transcriptional network analysis reveal a pivotal role of Her6, a Notch-independent HES1 homolog, in NSC control. Her6 at G1, and Notch signaling at G2, regulate distinct cell cycle control genes, favoring arrest. Her6 and Notch activity combined differentially control delta , jagged , lfng and notch3 expression to potentially shift Notch signaling mechanisms from Delta-driven lateral inhibition to Jagged-Notch3-mediated lateral induction. Indeed, we detect in situ spatial expression profiles consistent with lateral induction in high her6 expressing regions of the larval and adult brain. Her6-dependent lateral induction may spatially organize coherent populations of long-term NSCs in active proliferation zones.

Neural proliferation zones drive strong and persistent growth of the developing vertebrate brain. How Notch signaling and HES/Her transcription factors in vivo maintain stable neural stem cell (NSC) populations during brain growth is not well understood. Genetic evidence revealed Her6, a Notch-independent homolog of mammalian HES1, to be essential for maintenance of zebrafish NSCs. We recombined mNeonGreen into the her6 locus and analyzed temporal Her6-mNG fluorescence profiles in vivo . Her6 is expressed dynamically in all NSCs, with a majority revealing stable oscillation, albeit from cell to cell across a wide frequency range. Her6 oscillates cell-autonomously, unaffected by inhibition of Notch, and is robustly reinitiated after experimental perturbations. Mathematical modelling predicts, and we indeed find in vivo , that Her6 oscillations prevail only when both transcript and protein degradation rate constants are equal. These intrinsic control parameters may have evolved for the Her6 oscillator to contribute to robust maintenance of NSCs.

Dopamine and noradrenaline have conserved roles in control of physiology and behaviors of vertebrates. However, vertebrate genetic models completely devoid of catecholamines are not available. We generated genetically catecholamine-free zebrafish larvae by combining mutations in all three genes involved in L-DOPA biosynthesis: the tyrosine hydroxylase genes th and th2 , and tyrosinase tyr . Here we show that catecholamine-deficient 5 day-old zebrafish larvae are viable and develop an anatomically normal nervous system, including the full complement of catecholaminergic neuron somata and projections. In contrast, selected physiological functions that depend on catecholamines are impaired, including hatching and heart rate regulation upon temperature challenges. Spontaneous locomotion and optomotor behaviors are also impaired. Despite the changes observed, it is surprising that larvae develop a largely normal behavioral repertoire. Our model will be useful to investigate the activities of additional neurotransmitters expressed in catecholaminergic neurons, and how physiology and neural circuit function are regulated in the absence of catecholamines.

SMAC is a mitochondrial intermembrane space protein, which is released during apoptosis and whose known function is antagonism of inhibitor of apoptosis proteins in the cytosol, to facilitate caspase activation. Recent data suggest that SMAC can also be released by sub-lethal signals in the apoptosis pathway, in the absence of cell death. We here explored potential functions of SMAC in non-apoptotic cells. We found that a portion of SMAC is spontaneously released into the cytosol in the absence of apoptosis, regulated by the BCL-2-family proteins BAX and BAK and the fission GTPase DRP1. In cancer cell lines, SMAC was required for the activation of caspases in lethal and non-lethal conditions, while this contribution to caspase-activation was much smaller in non-malignant fibroblast lines. In cells with high levels of cytosolic SMAC, SMAC deficiency reduced in vitro migration, invasion and anchorage-independent growth as well as metastasis in a xenograft model in zebrafish. SMAC-deficient cells further showed a reduced activity in interferon signaling, associated with reduced cytosolic presence of mitochondrial DNA and activation of the stimulator of interferon genes (STING), and SMAC expression levels correlated with interferon-induced genes in cancer data sets. We further found that SMAC can regulate mitochondrial morphology and integrity. Finally, high gene-expression of SMAC was associated with poor prognosis in patients of several cancer types. These results identify SMAC as a regulator of inflammation and growth behavior of cancer cells. They further report a mitochondrial function of SMAC and demonstrate a role of SMAC in human cancer biology across several cancer entities.

The amygdala is a heterogeneous, multinuclear telencephalic structure critical for motivated and emotion‐related behaviors in vertebrates. In ray‐finned fish (Actinopterygii) like the teleost zebrafish, a telencephalic outward‐growing process called eversion makes defining amygdaloid territories particularly challenging. Teleosts are also peculiar in that they develop numerous dopaminergic (DA) neurons in the subpallium, while in tetrapods, such populations are less prominent or appear only transiently. To shed light on the organization of the amygdala in teleosts, we pursued an evolutionary developmental approach focusing on the topological origin of subpallial DA neurons. Specifically, we analyzed the distribution of tyrosine hydroxylase (Th) in conjunction with expression patterns of pax6a+b, isl1a, nkx2.1, lhx8a, otpa+b, and calb2a as markers of different telencephalic subdivisions in brains of 5‐ and 30‐day‐old zebrafish (Danio rerio, Teleostei). Our data show that the previously identified dorsalmost division of the ventral telencephalon (Vdd) needs to be subdivided into an anteroventral pax6a/b‐positive portion (Vdd1) and a posterodorsal pax6a/b‐negative portion (Vdd2). This pax6a‐negative Vdd2 portion develops into the extended medial amygdala (EMeA), including the DA population adjacent to the pallial–subpallial border. Our results also show that the EMeA DA neurons form a heterogeneous group of amygdaloid neurons because they differentially express calb2a and sst7. Our work sheds light on the early evolution and development of the amygdala and provides a foundation for functional analysis of the newly defined DA subtypes of the extended amygdala in zebrafish.

Dopamine and noradrenaline have conserved roles in control of physiology and behaviors of vertebrates. However, vertebrate genetic systems completely devoid of catecholamines are not available. We have generated a genetic zebrafish model completely devoid of catecholamines by combining mutations in all three genes involved in L-DOPA synthesis: the tyrosine hydroxylase genes th and th2 , and tyrosinase tyr . We found catecholamine-deficient zebrafish larvae to be viable and to develop an anatomically normal nervous system including catecholaminergic neuron somata and projections, albeit with reduced cell numbers detected in some clusters. In contrast, selected physiological functions that depend on catecholamines are impaired, including hatching and heart rate regulation upon temperature challenges. Spontaneous locomotion and optomotor behaviors are also impaired. Despite the changes observed, it is surprising that larvae develop a largely normal behavioral repertoire. Our model will be useful to investigate how physiology and neural circuit function are regulated in catecholamine deficient larvae.

The amygdala is a heterogenous multinuclear telencephalic structure critical for motivated and emotion-related behaviors in vertebrates. In ray-finned fish (actinopterygii) like the teleost zebrafish, a telencephalic outward growing process called eversion makes defining amygdaloid territories particularly challenging. Teleosts are also peculiar in that they develop prominent dopaminergic neuron groups in the subpallium, which are absent from tetrapods. To shed light on amygdala organization in teleosts, we pursued an evolutionary-developmental approach focusing on the topological origin of subpallial dopaminergic neurons. Specifically, we analyzed developmental expression patterns of Tyrosine hydroxylase in conjunction with pax6a+b , isl1a , nkx2.1 , lhx8a, calb2a as telencephalic topology markers in brains of 5- and 30-day-old zebrafish ( Danio rerio , Teleostei). Our results reveal, that the subpallial dopaminergic neurons develop within a pax6a negative dorsal subpallial domain (Vdd), which forms a primordial portion of the extended amygdala, including the medial amygdala and the anterior bed nucleus of the stria terminalis. Moreover, these dopaminergic neurons differentially coexpress calb2a and sst7 , indicating population heterogeneity and potentially functional diversity. Our data also show that the zebrafish extended amygdala is formed by the dorsal LGE-like Vdd, which is subdivided into a Vdd2 subdivision that may correspond to the extended medial amygdala, including the bed nucleus of the stria terminalis, and forms the pallial-subpallial border region, and a more ventral Vdd1 that is pax6a positive and that corresponds to the central amygdala. Our work contributes to understanding development and evolution of the amygdala, and provides a foundation for functional analysis of the newly defined dopaminergic subtypes of the extended amygdala.