Developmental programming and lineage branching of early human telencephalon.

The dorsal and ventral human telencephalons contain different neuronal subtypes, including glutamatergic, GABAergic, and cholinergic neurons, and how these neurons are generated during early development is not well understood. Using scRNA-seq and stringent validations, we reveal here a developmental roadmap for human telencephalic neurons. Both dorsal and ventral telencephalic radial glial cells (RGs) differentiate into neurons via dividing intermediate progenitor cells (IPCs_div) and early postmitotic neuroblasts (eNBs). The transcription factor ASCL1 plays a key role in promoting fate transition from RGs to IPCs_div in both regions. RGs from the regionalized neuroectoderm show heterogeneity, with restricted glutamatergic, GABAergic, and cholinergic differentiation potencies. During neurogenesis, IPCs_div gradually exit the cell cycle and branch into sister eNBs to generate distinct neuronal subtypes. Our findings highlight a general RGs-IPCs_div-eNBs developmental scheme for human telencephalic progenitors and support that the major neuronal fates of human telencephalon are predetermined during dorsoventral regionalization with neuronal diversity being further shaped during neurogenesis and neural circuit integration.

Proliferative Capacity of Adult Mouse Brain.

We studied cell proliferation in the postnatal mouse brain between the ages of 2 and 30 months and identified four compartments with different densities of proliferating cells. The first identified compartment corresponds to the postnatal pallial neurogenic (PPN) zone in the telencephalon; the second to the subpallial postnatal neurogenic (SPPN) zone in the telencephalon; the third to the white matter bundles in the telencephalon; and the fourth to all brain parts outside of the other three compartments. We estimated that about 3.4 million new cells, including 0.8 million in the subgranular zone (SGZ) in the hippocampus, are produced in the PPN zone. About 21 million new cells, including 10 million in the subependymal zone (SEZ) in the lateral walls of the lateral ventricle and 2.7 million in the rostral migratory stream (RMS), are produced in the SPPN zone. The third and fourth compartments together produced about 31 million new cells. The analysis of cell proliferation in neurogenic zones shows that postnatal neurogenesis is the direct continuation of developmental neurogenesis in the telencephalon and that adult neurogenesis has characteristics of the late developmental process. As a developmental process, adult neurogenesis supports only compensatory regeneration, which is very inefficient.

Organization of radial glia reveals growth pattern in the telencephalon of a percomorph fish Astatotilapia burtoni.

In the brain of teleost fish, radial glial cells are the main astroglial cell type. To understand how radial glia structures are adapting to continuous growth of the brain, we studied the astroglial cells in the telencephalon of the cichlid fish Astatotilapia burtoni in small fry to large specimens. These animals grow to a standard length of 10-12 cm in this fish species, corresponding to a more than 100-fold increase in brain volume. Focusing on the telencephalon where glial cells are arranged radially in the everted (dorsal) pallium, immunocytochemistry for glial markers revealed an aberrant pattern of radial glial fibers in the central division of the dorsal pallium (DC, i.e., DC4 and DC5). The main glial processes curved around these nuclei, especially in the posterior part of the telencephalon. This was verified in tissue-cleared brains stained for glial markers. We further analyzed the growth of radial glia by immunocytochemically applied stem cell (proliferating cell nuclear antigen [PCNA], Sox2) and differentiation marker (doublecortin) and found that these markers were expressed at the ventricular surface consistent with a stacking growth pattern. In addition, we detected doublecortin and Sox2 positive cells in deeper nuclei of DC areas. Our data suggest that radial glial cells give rise to migrating cells providing new neurons and glia to deeper pallial regions. This results in expansion of the central pallial areas and displacement of existing radial glial. In summary, we show that radial glial cells can adapt to morphological growth processes in the adult fish brain and contribute to this growth.

Rab11fip5 regulates telencephalon development via ephrinB1 recycling.

Rab11 family-interacting protein 5 (Rab11fip5) is an adaptor protein that binds to the small GTPase Rab11, which has an important function in endosome recycling and trafficking of cellular proteins to the plasma membrane. Rab11fip5 is involved in many cellular processes, such as cytoskeleton rearrangement, iron uptake and exocytosis in neuroendocrine cells, and is also known as a candidate gene for autism-spectrum disorder. However, the role of Rab11fip5 during early embryonic development is not clearly understood. In this study, we identified Rab11fip5 as a protein that interacts with ephrinB1, a transmembrane ligand for Eph receptors. The PDZ binding motif in ephrinB1 and the Rab-binding domain in Rab11fip5 are necessary for their interaction in a complex. EphrinB1 and Rab11fip5 display overlapping expression in the telencephalon of developing amphibian embryos. The loss of Rab11fip5 function causes a reduction in telencephalon size and a decrease in the expression level of ephrinB1. Moreover, morpholino oligonucleotide-mediated knockdown of Rab11fip5 decreases cell proliferation in the telencephalon. The overexpression of ephrinB1 rescues these defects, suggesting that ephrinB1 recycling by the Rab11/Rab11fip5 complex is crucial for proper telencephalon development.

Reoccurring neural stem cell divisions in the adult zebrafish telencephalon are sufficient for the emergence of aggregated spatiotemporal patterns.

Regulation of quiescence and cell cycle entry is pivotal for the maintenance of stem cell populations. Regulatory mechanisms, however, are poorly understood. In particular, it is unclear how the activity of single stem cells is coordinated within the population or if cells divide in a purely random fashion. We addressed this issue by analyzing division events in an adult neural stem cell (NSC) population of the zebrafish telencephalon. Spatial statistics and mathematical modeling of over 80,000 NSCs in 36 brain hemispheres revealed weakly aggregated, nonrandom division patterns in space and time. Analyzing divisions at 2 time points allowed us to infer cell cycle and S-phase lengths computationally. Interestingly, we observed rapid cell cycle reentries in roughly 15% of newly born NSCs. In agent-based simulations of NSC populations, this redividing activity sufficed to induce aggregated spatiotemporal division patterns that matched the ones observed experimentally. In contrast, omitting redivisions leads to a random spatiotemporal distribution of dividing cells. Spatiotemporal aggregation of dividing stem cells can thus emerge solely from the cells' history.

PDK1 regulates the survival of the developing cortical interneurons.

Inhibitory interneurons are critical for maintaining the excitatory/inhibitory balance. During the development cortical interneurons originate from the ganglionic eminence and arrive at the dorsal cortex through two tangential migration routes. However, the mechanisms underlying the development of cortical interneurons remain unclear. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) has been shown to be involved in a variety of biological processes, including cell proliferation and migration, and plays an important role in the neurogenesis of cortical excitatory neurons. However, the function of PDK1 in interneurons is still unclear. Here, we reported that the disruption of Pdk1 in the subpallium achieved by crossing the Dlx5/6-Cre-IRES-EGFP line with Pdk1fl/fl mice led to the severely increased apoptosis of immature interneurons, subsequently resulting in a remarkable reduction in cortical interneurons. However, the tangential migration, progenitor pools and cell proliferation were not affected by the disruption of Pdk1. We further found the activity of AKT-GSK3ß signaling pathway was decreased after Pdk1 deletion, suggesting it might be involved in the regulation of the survival of cortical interneurons. These results provide new insights into the function of PDK1 in the development of the telencephalon.

Characterization of neurogenic niches in the telencephalon of juvenile and adult sharks.

Neurogenesis is a multistep process by which progenitor cells become terminally differentiated neurons. Adult neurogenesis has gathered increasing interest with the aim of developing new cell-based treatments for neurodegenerative diseases in humans. Active sites of adult neurogenesis exist from fish to mammals, although in the adult mammalian brain the number and extension of neurogenic areas is considerably reduced in comparison to non-mammalian vertebrates and they become mostly reduced to the telencephalon. Much of our understanding in this field is based in studies on mammals and zebrafish, a modern bony fish. The use of the cartilaginous fish Scyliorhinus canicula (representative of basal gnathostomes) as a model expands the comparative framework to a species that shows highly neurogenic activity in the adult brain. In this work, we studied the proliferation pattern in the telencephalon of juvenile and adult specimens of S. canicula using antibodies against the proliferation marker proliferating cell nuclear antigen (PCNA). We have characterized proliferating niches using stem cell markers (Sex determining region Y-box 2), glial markers (glial fibrillary acidic protein, brain lipid binding protein and glutamine synthase), intermediate progenitor cell markers (Dlx2 and Tbr2) and markers for migrating neuroblasts (Doublecortin). Based in the expression pattern of these markers, we demonstrate the existence of different cell subtypes within the PCNA immunoreactive zones including non-glial stem cells, glial progenitors, intermediate progenitor-like cells and migratory neuroblasts, which were widely distributed in the ventricular zone of the pallium, suggesting that the main progenitor types that constitute the neurogenic niche in mammals are already present in cartilaginous fishes.

Molecular correlates of hypothalamic development in songbird ontogeny in comparison with the telencephalon.

Development of the songbird brain provides an excellent experimental model for understanding the regulation of sex differences in ontogeny. Considering the regulatory role of the hypothalamus in endocrine, in particular reproductive, physiology, we measured the structural (volume) and molecular correlates of hypothalamic development during ontogeny of male and female zebra finches. We quantified by relative quantitative polymerase chain reaction (rqPCR) the expression of 14 genes related to thyroid and steroid hormones actions as well as 12 genes related to brain plasticity at four specific time points during ontogeny and compared these expression patterns with the expression of the same genes as detected by transcriptomics in the telencephalon. These two different methodological approaches detected specific changes with age and demonstrated that in a substantial number of cases changes observed in both brain regions are nearly identical. Other genes however had a tissue-specific developmental pattern. Sex differences or interactions of sex by age were detected in the expression of a subset of genes, more in hypothalamus than telencephalon. These results correlate with multiple known aspects of the developmental and reproductive physiology but also raise a number of new functional questions.

Mosaic Heterochrony in Neural Progenitors Sustains Accelerated Brain Growth and Neurogenesis in the Juvenile Killifish N. furzeri.

Although developmental mechanisms driving an increase in brain size during vertebrate evolution are actively studied, we know less about evolutionary strategies allowing accelerated brain growth. In zebrafish and other vertebrates studied to date, apical radial glia (RG) constitute the primary neurogenic progenitor population throughout life [1]; thus, RG activity is a determining factor of growth speed. Here, we ask whether enhanced RG activity is the mechanism selected to drive explosive growth, in adaptation to an ephemeral habitat. In post-hatching larvae of the turquoise killifish, which display drastic developmental acceleration, we show that the dorsal telencephalon (pallium) grows three times faster than in zebrafish. Rather than resulting from enhanced RG activity, we demonstrate that pallial growth is the product of a second type of progenitors (that we term NGPs for non-glial progenitors) that actively sustains neurogenesis and germinal zone self-renewal. Intriguingly, NGPs appear to retain, at larval stages, features of early embryonic progenitors. In parallel, RGs enter premature quiescence and express markers of astroglial function. Altogether, we propose that mosaic heterochrony within the neural progenitor population might permit rapid pallial growth by safeguarding both continued neurogenesis and astroglial function.

Conserved and divergent expression dynamics during early patterning of the telencephalon in mouse and chick embryos.

The mammalian and the avian telencephalon are nearly indistinguishable at early embryonic vesicle stages but differ substantially in form and function at their adult stage. We sequenced and analyzed RNA populations present in mouse and chick during the early stages of embryonic telencephalon to understand conserved and lineage-specific developmental differences. We found approximately 3000 genes that orchestrate telencephalon development. Many chromatin-associated epigenetic and transcription regulators show high expression in both species and some show species-specific expression dynamics. Interestingly, previous studies associated them to autism, intellectual disabilities, and mental retardation supporting a causal link between their impaired functions during telencephalon development and brain dysfunction. Strikingly, the conserved up-regulated genes were differentially enriched in ontologies related to development or functions of the adult brain. Moreover, a differential enrichment of distinct repertoires of transcription factor binding motifs in their upstream promoter regions suggest a species-specific regulation of the various gene groups identified. Overall, our results reveal that the ontogenetic divergences between the mouse and chick telencephalon result from subtle differences in the regulation of common patterning signaling cascades and regulatory networks unique to each species at their very early stages of development.

Self-organizing neuruloids model developmental aspects of Huntington's disease in the ectodermal compartment.

Harnessing the potential of human embryonic stem cells to mimic normal and aberrant development with standardized models is a pressing challenge. Here we use micropattern technology to recapitulate early human neurulation in large numbers of nearly identical structures called neuruloids. Dual-SMAD inhibition followed by bone morphogenic protein 4 stimulation induced self-organization of neuruloids harboring neural progenitors, neural crest, sensory placode and epidermis. Single-cell transcriptomics unveiled the precise identities and timing of fate specification. Investigation of the molecular mechanism of neuruloid self-organization revealed a pulse of pSMAD1 at the edge that induced epidermis, whose juxtaposition to central neural fates specifies neural crest and placodes, modulated by fibroblast growth factor and Wnt. Neuruloids provide a unique opportunity to study the developmental aspects of human diseases. Using isogenic Huntington's disease human embryonic stem cells and deep neural network analysis, we show how specific phenotypic signatures arise in our model of early human development as a consequence of mutant huntingtin protein, outlining an approach for phenotypic drug screening.

Characteristic expressions of the natriuretic peptide family in the telencephalon of juvenile chick.

Elucidation of the genes regulating the critical (sensitive) period of imprinting behavior may shed light on the mechanism underlying neural plasticity in early childhood learning. We focused on the family of natriuretic peptides (NPs) as candidates involved in the regulation of the critical period. In avians, several structurally related molecules comprised the NP family, including renal NP (RNP), B-type NP (BNP) and C-type NP (CNP1, CNP3 and CNPP). To understand the functional roles of NPs in neural plastic changes, we aimed to characterize NPs and their receptors in chick brain. We found that CNP3 mRNA was expressed in several regions in the telencephalon, including the visual Wulst (VW, considered as mammalian visual cortex) and amygdala. CNP1 mRNA was expressed throughout the telencephalon. Using real-time PCR, the gene expression levels of NPs and their receptors (NPR1 and NPR2) were studied during and after the critical period of imprinting (post-hatching day [P]1 and P7). CNP3 mRNA was found to show higher expression in the VW of P1 chicks than in VW of P7 chicks. Moreover, the ability of these peptides to stimulate chicken NPR1 or NPR2 was tested in HEK293 cells expressing either of the receptors. The activation of NPR1 was stronger with CNP3 than with other subtypes of CNP. In the VW, CNP3-expressing cells were negative for NPR1, but they resided in the vicinity of NPR1-expressing cells. These results suggest that CNP3 and its receptor NPR1 in the VW may have functional roles in the early learning.

Sfrp1 Modulates Cell-signaling Events Underlying Telencephalic Patterning, Growth and Differentiation.

The mammalian dorsal telencephalic neuroepithelium develops-from medial to lateral-into the choroid plaque, cortical hem, hippocampal primordium and isocortex under the influence of Bmp, Wnt and Notch signaling. Correct telencephalic development requires a tight coordination of the extent/duration of these signals, but the identification of possible molecular coordinators is still limited. Here, we postulated that Secreted Frizzled Related Protein 1 (Sfrp1), a multifunctional regulator of Bmp, Wnt and Notch signaling strongly expressed during early telencephalic development, may represent 1 of such molecules. We report that in E10.5-E12.5 Sfrp1-/- embryos, the hem and hippocampal domains are reduced in size whereas the prospective neocortex is medially extended. These changes are associated with a significant reduction of the medio-lateral telencephalic expression of Axin2, a read-out of Wnt/ßcatenin signaling activation. Furthermore, in the absence of Sfrp1, Notch signaling is increased, cortical progenitor cell cycle is shorter, with expanded progenitor pools and enhanced generation of early-born neurons. Hence, in postnatal Sfrp1-/- animals the anterior hippocampus is reduced and the neocortex is shorter in the antero-posterior and medio-lateral axis but is thicker. We propose that, by controlling Wnt and Notch signaling in opposite directions, Sfrp1 promotes hippocampal patterning and balances medio-lateral and antero-posterior cortex expansion.

Expression of radial glial markers (GFAP, BLBP and GS) during telencephalic development in the catshark (Scyliorhinus canicula).

Radial glial cells (RGCs) are the first cell populations of glial nature to appear during brain ontogeny. They act as primary progenitor (stem) cells as well as a scaffold for neuronal migration. The proliferative capacity of these cells, both in development and in adulthood, has been subject of interest during past decades. In contrast with mammals where RGCs are restricted to specific ventricular areas in the adult brain, RGCs are the predominant glial element in fishes. However, developmental studies on the RGCs of cartilaginous fishes are scant. We have studied the expression patterns of RGCs markers including glial fibrillary acidic protein (GFAP), brain lipid binding protein (BLBP), and glutamine synthase (GS) in the telencephalic hemispheres of catshark (Scyliorhinus canicula) from early embryos to post-hatch juveniles. GFAP, BLBP and GS are first detected, respectively, in early, intermediate and late embryos. Expression of these glial markers was observed in cells with radial glia morphology lining the telencephalic ventricles, as well as in their radial processes and endfeet at the pial surface and their expression continue in ependymal cells (or tanycytes) in early juveniles. In addition, BLBP- and GS-immunoreactive cells morphologically resembling oligodendrocytes were observed. In late embryos, most of the GFAP- and BLBP-positive RGCs also coexpress GS and show proliferative activity. Our results indicate the existence of different proliferating subpopulations of RGCs in the embryonic ventricular zone of catshark. Further investigations are needed to determine whether these proliferative RGCs could act as neurogenic and/or gliogenic precursors.

DOT1L promotes progenitor proliferation and primes neuronal layer identity in the developing cerebral cortex.

Cortical development is controlled by transcriptional programs, which are orchestrated by transcription factors. Yet, stable inheritance of spatio-temporal activity of factors influencing cell fate and localization in different layers is only partly understood. Here we find that deletion of Dot1l in the murine telencephalon leads to cortical layering defects, indicating DOT1L activity and chromatin methylation at H3K79 impact on the cell cycle, and influence transcriptional programs conferring upper layer identity in early progenitors. Specifically, DOT1L prevents premature differentiation by increasing expression of genes that regulate asymmetric cell division (Vangl2, Cenpj). Loss of DOT1L results in reduced numbers of progenitors expressing genes including SoxB1 gene family members. Loss of DOT1L also leads to altered cortical distribution of deep layer neurons that express either TBR1, CTIP2 or SOX5, and less activation of transcriptional programs that are characteristic for upper layer neurons (Satb2, Pou3f3, Cux2, SoxC family members). Data from three different mouse models suggest that DOT1L balances transcriptional programs necessary for proper neuronal composition and distribution in the six cortical layers. Furthermore, because loss of DOT1L in the pre-neurogenic phase of development impairs specifically generation of SATB2-expressing upper layer neurons, our data suggest that DOT1L primes upper layer identity in cortical progenitors.

CTCF Governs the Identity and Migration of MGE-Derived Cortical Interneurons.

The CCCTC-binding factor (CTCF) is a central regulator of chromatin topology recently linked to neurodevelopmental disorders such as intellectual disability, autism, and schizophrenia. The aim of this study was to identify novel roles of CTCF in the developing mouse brain. We provide evidence that CTCF is required for the expression of the LIM homeodomain factor LHX6 involved in fate determination of cortical interneurons (CINs) that originate in the medial ganglionic eminence (MGE). Conditional Ctcf ablation in the MGE of mice of either sex leads to delayed tangential migration, abnormal distribution of CIN in the neocortex, a marked reduction of CINs expressing parvalbumin and somatostatin (Sst), and an increased number of MGE-derived cells expressing Lhx8 and other markers of basal forebrain projection neurons. Likewise, Ctcf-null MGE cells transplanted into the cortex of wild-type hosts generate fewer Sst-expressing CINs and exhibit lamination defects that are efficiently rescued upon reexpression of LHX6. Collectively, these data indicate that CTCF regulates the dichotomy between Lhx6 and Lhx8 to achieve correct specification and migration of MGE-derived CINs.SIGNIFICANCE STATEMENT This work provides evidence that CCCTC-binding factor (CTCF) controls an early fate decision point in the generation of cortical interneurons mediated at least in part by Lhx6. Importantly, the abnormalities described could reflect early molecular and cellular events that contribute to human neurological disorders previously linked to CTCF, including schizophrenia, autism, and intellectual disability.

Disappearance of cerebrovascular laminin immunoreactivity as related to the maturation of astroglia in rat brain.

The present paper provides novel findings on the temporo-spatial correlation of perivascular laminin immunoreactivity with the early postnatal astrocyte development. The cerebrovascular laminin immunoreactivity gradually disappears during development. The fusion of the glial and vascular basal laminae during development makes the laminin epitopes inaccessible for antibody molecules (Krum et al., 1991, Exp Neurol 111:151). The fusion is supposed to correlate with the maturation of the glio-vascular connections. Glial development was followed by immunostaining for GFAP (glial fibrillary acidic protein), S100 protein, glutamine synthetase as glial markers and for nestin to visualize the immature glial structures. Our investigation focused on the period from postnatal day (P)2 to P16, on the dorso-parietal pallium. In the wall of the telencephalon the laminin immunoreactivity disappeared between P5 and P10; in subcortical structures it persisted to P12 or even to P16. Its disappearance overlapped the period when GFAP-immunopositive astrocytes were taking the place of radial glia. Despite the parallel time courses, however, the spatial patterns of the two processes were just the opposite: disappearance of the laminin immunoreactivity progressed from the middle zone whereas the appearance of GFAP from the pial surface and the corpus callosum. Rather, the regression of the vascular laminin immunoreactivity followed the progression of the immunoreactivities of glutamine synthetase and S100 protein. Therefore, the regression really correlates with a 'maturation' of astrocytes which, however, affects other astrocyte functions rather than cytoskeleton.

Differential antiviral immunity to Japanese encephalitis virus in developing cortical organoids.

Japanese encephalitis (JE) caused by Japanese encephalitis virus (JEV) poses a serious threat to the world's public health yet without a cure. Certain JEV-infected neural cells express a subset of previously identified intrinsic antiviral interferon stimulated genes (ISGs), indicating brain cells retain autonomous antiviral immunity. However, whether this happens in composited brain remains unclear. Human pluripotent stem cell (hPSC)-derived organoids can model disorders caused by human endemic pathogens such as Zika virus, which may potentially address this question and facilitate the discovery of a cure for JE. We thus generated telencephalon organoid and infected them with JEV. We found JEV infection caused significant decline of cell proliferation and increase of cell death in brain organoid, resulting in smaller organoid spheres. JEV tended to infect astrocytes and neural progenitors, especially the population representing outer radial glial cells (oRGCs) of developing human brain. In addition, we revealed variable antiviral immunity in brain organoids of different stages of culture. In organoids of longer culture (older than 8 weeks), but not of early ones (less than 4 weeks), JEV infection caused typical activation of interferon signaling pathway. Preferential infection of oRGCs and differential antiviral response at various stages might explain the much more severe outcomes of JEV infection in the younger, which also provide clues to develop effective therapeutics of such diseases.

Simultaneous lineage tracing and cell-type identification using CRISPR-Cas9-induced genetic scars.

A key goal of developmental biology is to understand how a single cell is transformed into a full-grown organism comprising many different cell types. Single-cell RNA-sequencing (scRNA-seq) is commonly used to identify cell types in a tissue or organ. However, organizing the resulting taxonomy of cell types into lineage trees to understand the developmental origin of cells remains challenging. Here we present LINNAEUS (lineage tracing by nuclease-activated editing of ubiquitous sequences)-a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes, generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae, and in heart, liver, pancreas, and telencephalon of adult fish. LINNAEUS provides a systematic approach for tracing the origin of novel cell types, or known cell types under different conditions.

Gene expression of Dio2 (thyroid hormone converting enzyme) in telencephalon is linked with predisposed biological motion preference in domestic chicks.

Filial imprinting leads to the formation of social attachment if training is performed during a brief sensitive period after hatching. We found that thyroid hormone (3,5,3'-triiodothyronine, T3) acts as a critical determining factor of the sensitive period in domestic chicks. Imprinting upregulates gene expression of the converting enzyme (Dio2, type 2 iodothyronine deiodinase) in the telencephalon, leading to increased brain T3 content. If systemically applied, T3 facilitates imprinting in aged chicks even after the sensitive period is over. Imprinting is also associated with the rapid development of visual perception. Exposure to motion pictures induces a predisposed preference to Johansson's biological motion (BM), and those individuals with higher BM preference are more easily imprinted. Here, we examined whether Dio2 expression is also linked with BM predisposition. Chicks were trained by a rotating red block, and tested for imprinting (experiment 1) and BM preference (experiment 2). To examine the time courses of behavioural and physiological processes, Dio2 expression in telencephalon was compared among three groups: naïve control chicks, and chicks trained for a short (0.5 h) or long period (2 h). In experiment 1, higher Dio2 expression appeared in the 2-h group than in the 0.5-h/control groups, but it was not correlated with the individual imprinting score. In experiment 2, a significant positive correlation appeared between Dio2 expression and BM preference in 2-h-trained chicks. Memory priming by T3 is therefore functionally linked to BM preference induction, leading to successful imprinting to natural objects even when they are initially exposed to artificial objects.