ZeBraInspector, a platform for the automated segmentation and analysis of body and brain volumes in whole 5 days post-fertilization zebrafish following simultaneous visualization with identical orientations.

In recent years, the zebrafish has become a well-established laboratory model. We describe here the ZeBraInspector (ZBI) platform for high-content 3D imaging (HCI) of 5 days post-fertilization zebrafish eleuthero-embryos (EEs). This platform includes a mounting method based on 3D-printed stamps to create a grid of wells in an agarose cast, facilitating batch acquisitions with a fast-confocal laser scanning microscope. We describe reference labeling in cleared fish with a fluorescent lipophilic dye. Based on this labeling, the ZBI software registers. EE 3D images, making it possible to visualize numerous identically oriented EEs on a single screen, and to compare their morphologies and any fluorescent patterns at a glance. High-resolution 2D snapshots can be extracted. ZBI software is therefore useful for diverse high-content analyses (HCAs). Following automated segmentation of the lipophilic dye signal, the ZBI software performs volumetric analyses on whole EEs and their nervous system white matter. Through two examples, we illustrate the power of these analyses for obtaining statistically significant results from a small number of samples: the characterization of a phenotype associated with a neurodevelopmental mutation, and of the defects caused by treatments with a toxic anti-cancer compound.

Mesencephalic origin of the inferior lobe in zebrafish.

BACKGROUND: Although the overall brain organization is shared in vertebrates, there are significant differences within subregions among different groups, notably between Sarcopterygii (lobe-finned fish) and Actinopterygii (ray-finned fish). Recent comparative studies focusing on the ventricular morphology have revealed a large diversity of the hypothalamus. Here, we study the development of the inferior lobe (IL), a prominent structure forming a bump on the ventral surface of the teleost brain. Based on its position, IL has been thought to be part of the hypothalamus (therefore forebrain). RESULTS: Taking advantage of genetic lineage-tracing techniques in zebrafish, we reveal that cells originating from her5-expressing progenitors in the midbrain-hindbrain boundary (MHB) participate in the formation of a large part of the IL. 3D visualization demonstrated how IL develops in relation to the ventricular system. We found that IL is constituted by two developmental components: the periventricular zone of hypothalamic origin and the external zone of mesencephalic origin. The mesencephalic external zone grows progressively until adulthood by adding new cells throughout development. CONCLUSION: Our results disprove a homology between the IL and the mammalian lateral hypothalamus. We suggest that the IL is likely to be involved in multimodal sensory integration rather than feeding motivation. The teleost brain is not a simpler version of the mammalian brain, and our study highlights the evolutionary plasticity of the brain which gives rise to novel structures.

Postembryonic Fish Brain Proliferation Zones Exhibit Neuroepithelial-Type Gene Expression Profile.

In mammals, neuroepithelial cells play an essential role in embryonic neurogenesis, whereas glial stem cells are the principal source of neurons at postembryonic stages. By contrast, neuroepithelial-like stem/progenitor (NE) cells have been shown to be present throughout life in teleosts. We used three-dimensional (3D) reconstructions of cleared transgenic wdr12:GFP medaka brains to demonstrate that this cell type is widespread in juvenile and to identify new regions containing NE cells. We established the gene expression profile of optic tectum (OT) NE cells by cell sorting followed by RNA-seq. Our results demonstrate that most OT NE cells are indeed active stem cells and that some of them exhibit long G2 phases. We identified several novel pathways (e.g., DNA repair pathways) potentially involved in NE cell homeostasis. In situ hybridization studies showed that all NE populations in the postembryonic medaka brain have a similar molecular signature. Our findings highlight the importance of NE progenitors in medaka and improve our understanding of NE-cell biology. These cells are potentially useful not only for neural stem cell studies but also for improving the characterization of neurodevelopmental diseases, such as microcephaly. Stem Cells 2017;35:1505-1518.

Development of hypothalamic serotoninergic neurons requires Fgf signalling via the ETS-domain transcription factor Etv5b.

Serotonin is a monoamine neurotransmitter that is involved in numerous physiological functions and its dysregulation is implicated in various psychiatric diseases. In all non-placental vertebrates, serotoninergic (5-HT) neurons are present in several regions of the brain, including the hypothalamus. In placental mammals, however, 5-HT neurons are located in the raphe nuclei only. In all species, though, 5-HT neurons constitute a functionally and molecularly heterogeneous population. How the non-raphe 5-HT populations are developmentally encoded is unknown. Using the zebrafish model we show that, in contrast to the raphe populations, hypothalamic 5-HT neurons are generated independently of the ETS-domain transcription factor Pet1 (Fev). By applying a combination of pharmacological tools and gene knockdown and/or overexpression experiments, we demonstrate that Fgf signalling acts via another ETS-domain transcription factor, Etv5b (Erm), to induce hypothalamic 5-HT neurons. We provide evidence that Etv5b exerts its effects by regulating cell cycle parameters in 5-HT progenitors. Our results highlight a novel role for Etv5b in neuronal development and provide support for the existence of a developmental heterogeneity among 5-HT neurons in their requirement for ETS-domain transcription factors.

Plasticity of Dopaminergic Phenotype and Locomotion in Larval Zebrafish Induced by Brain Excitability Changes during the Embryonic Period.

During the embryonic period, neuronal communication starts before the establishment of the synapses with alternative forms of neuronal excitability, called here embryonic neural excitability (ENE). ENE has been shown to modulate the unfolding of development transcriptional programs, but the global consequences for developing organisms are not all understood. Here, we monitored calcium (Ca2+) transients in the telencephalon of zebrafish embryos as a proxy for ENE to assess the efficacy of transient pharmacological treatments to either increase or decrease ENE. Increasing or decreasing ENE at the end of the embryonic period promoted an increase or a decrease in the numbers of dopamine (DA) neurons, respectively. This plasticity of dopaminergic specification occurs in the subpallium (SP) of zebrafish larvae at 6 d postfertilization (dpf), within a relatively stable population of vMAT2-positive cells. Nondopaminergic vMAT2-positive cells hence constitute an unanticipated biological marker for a reserve pool of DA neurons that can be recruited by ENE. Modulating ENE also affected larval locomotion several days after the end of the treatments. In particular, the increase of ENE from 2 to 3 dpf promoted hyperlocomotion of larvae at 6 dpf, reminiscent of zebrafish endophenotypes reported for attention deficit hyperactivity disorders (ADHDs). These results provide a convenient framework for identifying environmental factors that could disturb ENE as well as to study the molecular mechanisms linking ENE to neurotransmitter specification.

Molecular and Cellular Analysis of the Repair of Zebrafish Optic Tectum Meninges Following Laser Injury.

We studied cell recruitment following optic tectum (OT) injury in zebrafish (Danio rerio), which has a remarkable ability to regenerate many of its organs, including the brain. The OT is the largest dorsal layered structure in the zebrafish brain. In juveniles, it is an ideal structure for imaging and dissection. We investigated the recruited cells within the juvenile OT during regeneration in a Pdgfrß-Gal4:UAS-EGFP line in which pericytes, vascular, circulating, and meningeal cells are labeled, together with neurons and progenitors. We first performed high-resolution confocal microscopy and single-cell RNA-sequencing (scRNAseq) on EGFP-positive cells. We then tested three types of injury with very different outcomes (needle (mean depth in the OT of 200 µm); deep-laser (depth: 100 to 200 µm depth); surface-laser (depth: 0 to 100 µm)). Laser had the additional advantage of better mimicking of ischemic cerebral accidents. No massive recruitment of EGFP-positive cells was observed following laser injury deep in the OT. This type of injury does not perturb the meninx/brain-blood barrier (BBB). We also performed laser injuries at the surface of the OT, which in contrast create a breach in the meninges. Surprisingly, one day after such injury, we observed the migration to the injury site of various EGFP-positive cell types at the surface of the OT. The migrating cells included midline roof cells, which activated the PI3K-AKT pathway; fibroblast-like cells expressing numerous collagen genes and most prominently in 3D imaging; and a large number of arachnoid cells that probably migrate to the injury site through the activation of cilia motility genes, most likely being direct targets of the FOXJ1a gene. This study, combining high-content imaging and scRNAseq in physiological and pathological conditions, sheds light on meninges repair mechanisms in zebrafish that probably also operate in mammalian meninges.

Non-thalamic origin of zebrafish sensory nuclei implies convergent evolution of visual pathways in amniotes and teleosts.

Ascending visual projections similar to the mammalian thalamocortical pathway are found in a wide range of vertebrate species, but their homology is debated. To get better insights into their evolutionary origin, we examined the developmental origin of a thalamic-like sensory structure of teleosts, the preglomerular complex (PG), focusing on the visual projection neurons. Similarly to the tectofugal thalamic nuclei in amniotes, the lateral nucleus of PG receives tectal information and projects to the pallium. However, our cell lineage study in zebrafish reveals that the majority of PG cells are derived from the midbrain, unlike the amniote thalamus. We also demonstrate that the PG projection neurons develop gradually until late juvenile stages. Our data suggest that teleost PG, as a whole, is not homologous to the amniote thalamus. Thus, the thalamocortical-like projections evolved from a non-forebrain cell population, which indicates a surprising degree of variation in the vertebrate sensory systems.

X-FaCT: Xenopus-Fast Clearing Technique.

Accessibility and imaging of cell compartments in big specimens are crucial for cellular biological research but also a matter of contention. Confocal imaging and tissue clearing on whole organs allow for 3D imaging of cellular structures after being subjected to in-toto immunohistochemistry. Lately, the passive CLARITY technique (PACT) has been adapted to clear and immunolabel large specimens or individual organs of several aquatic species. We recently demonstrated tissue clearing on one-week old tadpole brain (Fini et al., Sci Rep 7:43786, 2017). We here describe a further simplified version with clearing of small tissue samples (thickness inferior to 500 µm)) carried out by immersion in a fructose-based high-refractive index solution (fbHRI). By refining steps of the protocol, we were able to reduce the overall procedure time by two thirds. This offers the advantages of reducing the time of experimentation to a week and minimizes procedure-induced tissue deformations. This protocol can be easily adapted to be performed on thick section. We present an example of immunohistochemistry performed on NF45 Xenopus laevis brains with anti-pH 3 (phosphorylated histone H3) antibody used to stain chromatin condensation commonly associated with proliferation.

Comparative analysis of monoaminergic cerebrospinal fluid-contacting cells in Osteichthyes (bony vertebrates).

Cerebrospinal fluid-contacting (CSF-c) cells containing monoamines such as dopamine (DA) and serotonin (5-HT) occur in the periventricular zones of the hypothalamic region of most vertebrates except for placental mammals. Here we compare the organization of the CSF-c cells in chicken, Xenopus, and zebrafish, by analyzing the expression of synthetic enzymes of DA and 5-HT, respectively, tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), and draw an evolutionary scenario for this cell population. Due to the lack of TH immunoreactivity in this region, the hypothalamic CSF-c cells have been thought to take up DA from the ventricle instead of synthesizing it. We demonstrate that a second TH gene (TH2) is expressed in the CSF-c cells of all the three species, suggesting that these cells do indeed synthetize DA. Furthermore, we found that many CSF-c cells coexpress TH2 and TPH1 and contain both DA and 5-HT, a dual neurotransmitter phenotype hitherto undescribed in the brain of any vertebrate. The similarities of CSF-c cells in chicken, Xenopus, and zebrafish suggest that these characteristics are inherited from the common ancestor of the Osteichthyes. A significant difference between tetrapods and teleosts is that teleosts possess an additional CSF-c cell population around the posterior recess (PR) that has emerged in specific groups of Actinopterygii. Our comparative analysis reveals that the hypothalamus in mammals and teleosts has evolved in a divergent manner: placental mammals have lost the monoaminergic CSF-c cells, while teleosts have increased their relative number.

Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos.

Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development.

High-resolution 3D imaging of whole organ after clearing: taking a new look at the zebrafish testis.

Zebrafish testis has become a powerful model for reproductive biology of teleostean fishes and other vertebrates and encompasses multiple applications in applied and basic research. Many studies have focused on 2D images, which is time consuming and implies extrapolation of results. Three-dimensional imaging of whole organs recently became an important challenge to better understand their architecture and allow cell enumeration. Several protocols have thus been developed to enhance sample transparency, a limiting step for imaging large biological samples. However, none of these methods has been applied to the zebrafish testis. We tested five clearing protocols to determine if some of them could be applied with only small modifications to the testis. We compared clearing efficiency at both macroscopic and microscopic levels. CUBIC and PACT were suitable for an efficient transparency, an optimal optical penetration, the GFP fluorescence preservation and avoiding meaningful tissue deformation. Finally, we succeeded in whole testis 3D capture at a cellular resolution with both CUBIC and PACT, which will be valuable in a standard workflow to investigate the 3D architecture of the testis and its cellular content. This paves the way for further development of high content phenotyping studies in several fields including development, genetic or toxicology.

zPACT: Tissue Clearing and Immunohistochemistry on Juvenile Zebrafish Brain.

In studies of brain function, it is essential to understand the underlying neuro-architecture. Very young zebrafish larvae are widely used for neuroarchitecture studies, due to their size and natural transparency. However, this model system has several limitations, due to the immaturity, high rates of development and limited behavioral repertoire of the animals used. We describe here a modified version of the passive clearing technique (PACT) ( Chung et al., 2013 ; Tomer et al., 2014 ; Yang et al., 2014 ; Treweek et al., 2015) , which facilitates neuroanatomical studies on large specimens of aquatic species. This method was initially developed for zebrafish (Danio rerio) ( Frétaud et al., 2017 ; Mayrhofer et al., 2017 ; Xavier et al., 2017 ), but has also been successfully tested on other fish, such as medaka (Oryzias latipes) ( Dambroise et al., 2017 ), Mexican cave fish (Astyanax mexicaus) and African zebra mbuna (Metriaclima zebra), and on other aquatic species, such as Xenopus spp. (Xenopus laevis, Xenopus tropicalis) ( Fini et al., 2017 ) . This protocol, based on the CLARITY method developed and modified by Deisseroth's laboratory and others ( Chung et al., 2013 ; Tomer et al., 2014 ; Yang et al., 2014 ), was adapted for use in aquatic species, including zebrafish in particular (zPACT). This protocol is designed to render zebrafish specimens optically transparent while preserving the overall architecture of the tissue, through crosslinking in a polyacrylamide/formaldehyde mesh. Most of the lipids present in the specimen are then removed by SDS treatment, to homogenize the refractive index of the specimen by eliminating light scattering at the water/lipid interface, which causes opacity. The final clearing step, consists of the incubation of the specimen in a fructose-based mounting medium (derived from SeeDB) ( Ke et al., 2013 ) , with a refractive index matching that of the objective lens of the microscope. The combination of this technique with the use of genetically modified zebrafish in which green fluorescent protein (GFP) is expressed in specific cell populations provides opportunities to describe anatomical details not visible with other techniques.

A novel brain tumour model in zebrafish reveals the role of YAP activation in MAPK- and PI3K-induced malignant growth.

Somatic mutations activating MAPK and PI3K signalling play a pivotal role in both tumours and brain developmental disorders. We developed a zebrafish model of brain tumours based on somatic expression of oncogenes that activate MAPK and PI3K signalling in neural progenitor cells and found that HRASV12 was the most effective in inducing both heterotopia and invasive tumours. Tumours, but not heterotopias, require persistent activation of phospho (p)-ERK and express a gene signature similar to the mesenchymal glioblastoma subtype, with a strong YAP component. Application of an eight-gene signature to human brain tumours establishes that YAP activation distinguishes between mesenchymal glioblastoma and low grade glioma in a wide The Cancer Genome Atlas (TCGA) sample set including gliomas and glioblastomas (GBMs). This suggests that the activation of YAP might be an important event in brain tumour development, promoting malignant versus benign brain lesions. Indeed, co-expression of dominant-active YAP (YAPS5A) and HRASV12 abolishes the development of heterotopias and leads to the sole development of aggressive tumours. Thus, we have developed a model proving that neurodevelopmental disorders and brain tumours might originate from the same activation of oncogenes through somatic mutations, and established that YAP activation is a hallmark of malignant brain tumours.

Imaging of viral neuroinvasion in the zebrafish reveals that Sindbis and chikungunya viruses favour different entry routes.

Alphaviruses, such as chikungunya virus (CHIKV) and Sindbis virus (SINV), are vector-borne pathogens that cause acute illnesses in humans and are sometimes associated with neuropathies, especially in infants and elderly patients. Little is known about their mechanism of entry into the central nervous system (CNS), even for SINV, which has been used extensively as a model for viral encephalopathies. We previously established a CHIKV infection model in the optically transparent zebrafish larva; here we describe a new SINV infection model in this host. We imaged in vivo the onset and progression of the infection caused by intravenous SINV inoculation. Similar to that described for CHIKV, infection in the periphery was detected early and was transient, whereas CNS infection started at later time points and was persistent or progressive. We then tested the possible mechanisms of neuroinvasion by CHIKV and SINV. Neither virus relied on macrophage-mediated transport to access the CNS. CHIKV, but not SINV, always infects endothelial cells of the brain vasculature. By contrast, axonal transport was much more efficient with SINV than CHIKV, both from the periphery to the CNS and between neural tissues. Thus, the preferred mechanisms of neuroinvasion by these two related viruses are distinct, providing a powerful imaging-friendly system to compare mechanisms and prevention methods of encephalopathies.

Structural Analysis of an Evolved Transketolase Reveals Divergent Binding Modes.

The S385Y/D469T/R520Q variant of E. coli transketolase was evolved previously with three successive smart libraries, each guided by different structural, bioinformatical or computational methods. Substrate-walking progressively shifted the target acceptor substrate from phosphorylated aldehydes, towards a non-phosphorylated polar aldehyde, a non-polar aliphatic aldehyde, and finally a non-polar aromatic aldehyde. Kinetic evaluations on three benzaldehyde derivatives, suggested that their active-site binding was differentially sensitive to the S385Y mutation. Docking into mutants generated in silico from the wild-type crystal structure was not wholly satisfactory, as errors accumulated with successive mutations, and hampered further smart-library designs. Here we report the crystal structure of the S385Y/D469T/R520Q variant, and molecular docking of three substrates. This now supports our original hypothesis that directed-evolution had generated an evolutionary intermediate with divergent binding modes for the three aromatic aldehydes tested. The new active site contained two binding pockets supporting π-π stacking interactions, sterically separated by the D469T mutation. While 3-formylbenzoic acid (3-FBA) preferred one pocket, and 4-FBA the other, the less well-accepted substrate 3-hydroxybenzaldehyde (3-HBA) was caught in limbo with equal preference for the two pockets. This work highlights the value of obtaining crystal structures of evolved enzyme variants, for continued and reliable use of smart library strategies.

A workflow to process 3D+time microscopy images of developing organisms and reconstruct their cell lineage.

The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology.

Transgenic Zebrafish Expressing mCherry in the Mitochondria of Dopaminergic Neurons.

Genetic mutations and environmental toxins are known to affect mitochondrial health and have been implicated in the progressive degeneration of dopaminergic neurons in Parkinson's disease. To visualize mitochondria in dopaminergic neurons of live zebrafish, we used the regulatory elements of the dopamine transporter (dat) gene to target a reporter, mCherry, after fusion with the mitochondrial localizing signal (MLS) of Tom20. Immunoblot analysis of mitochondrial and cytosolic fractions from Tg(dat:tom20 MLS-mCherry) larvae shows that mCherry is efficiently targeted to the mitochondria. Confocal imaging of live fish was carried out from 1 day postfertilization (dpf) to 9 dpf. We also colocalized dat mRNA expression with the mCherry protein in the olfactory bulb (OB), subpallium (SP), pretectum (Pr), diencephalic clusters 2 and 3 (DC2/3), caudal hypothalamus (Hc), locus coeruleus (LC), anterior preoptic area (POa), retinal amacrine cells (RAC), caudal hypothalamus (Hc), and preoptic area (PO). Treating Tg(dat:tom20 MLS-mCherry) larvae with the dopaminergic neurotoxin MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) at 2 or 3 dpf resulted in a decrease in mCherry fluorescence in the pretectum, olfactory bulb, subpallium, diencephalic clusters 2 and 3, and the caudal hypothalamus. Labeling of mitochondria in nigrostriatal dopaminergic neurons of zebrafish could allow their visualization in vivo following genetic or pharmacological manipulations.

Identification of the optic recess region as a morphogenetic entity in the zebrafish forebrain.

Regionalization is a critical, highly conserved step in the development of the vertebrate brain. Discrepancies exist in how regionalization of the anterior vertebrate forebrain is conceived since the "preoptic area" is proposed to be a part of the telencephalon in tetrapods but not in teleost fish. To gain insight into this complex morphogenesis, formation of the anterior forebrain was analyzed in 3D over time in zebrafish embryos, combining visualization of proliferation and differentiation markers, with that of developmental genes. We found that the region containing the preoptic area behaves as a coherent morphogenetic entity, organized around the optic recess and located between telencephalon and hypothalamus. This optic recess region (ORR) makes clear borders with its neighbor areas and expresses a specific set of genes (dlx2a, sim1a and otpb). We thus propose that the anterior forebrain (secondary prosencephalon) in teleosts contains three morphogenetic entities (telencephalon, ORR and hypothalamus), instead of two (telencephalon and hypothalamus). The ORR in teleosts could correspond to "telencephalic stalk area" and "alar hypothalamus" in tetrapods, resolving current inconsistencies in the comparison of basal forebrain among vertebrates.

Dopaminergic Neurons Controlling Anterior Pituitary Functions: Anatomy and Ontogenesis in Zebrafish.

Dopaminergic (DA) neurons located in the preoptico-hypothalamic region of the brain exert a major neuroendocrine control on reproduction, growth, and homeostasis by regulating the secretion of anterior pituitary (or adenohypophysis) hormones. Here, using a retrograde tract tracing experiment, we identified the neurons playing this role in the zebrafish. The DA cells projecting directly to the anterior pituitary are localized in the most anteroventral part of the preoptic area, and we named them preoptico-hypophyseal DA (POHDA) neurons. During development, these neurons do not appear before 72 hours postfertilization (hpf) and are the last dopaminergic cell group to differentiate. We found that the number of neurons in this cell population continues to increase throughout life proportionally to the growth of the fish. 5-Bromo-2'-deoxyuridine incorporation analysis suggested that this increase is due to continuous neurogenesis and not due to a phenotypic change in already-existing neurons. Finally, expression profiles of several genes (foxg1a, dlx2a, and nr4a2a/b) were different in the POHDA compared with the adjacent suprachiasmatic DA neurons, suggesting that POHDA neurons develop as a distinct DA cell population in the preoptic area. This study offers some insights into the regional identity of the preoptic area and provides the first bases for future functional genetic studies on the development of DA neurons controlling anterior pituitary functions.

Single active-site mutants are sufficient to enhance serine:pyruvate α-transaminase activity in an ω-transaminase.

We have analyzed the natural evolution of transaminase structure and sequence between an α-transaminase serine-pyruvate aminotransferase and an ω-transaminase from Chromobacterium violaceum with < 20% sequence identity, and identified the active-site regions that are least conserved structurally. We also show that these structural changes correlate strongly with transaminase substrate specificity during evolution and therefore might normally be presumed to be essential determinants of substrate specificity. However, key residues are often conserved spatially during evolution and yet originate from within a different region of the sequence via structural reorganizations. In the present study, we also show that α-transaminase-type serine-pyruvate aminotransferase activity can be engineered into the CV2025 ω-transaminase scaffold with any one of many possible single-point mutations at three key positions, without the requirement for significant backbone remodeling, or repositioning of the residue from a different region of sequence. This finding has significant implications for enzyme redesign in which solutions to substrate specificity changes may be found more efficiently than is achieved by engineering in all sequence and structure determinants identified by correlation to substrate specificity.