Takuma Kumamoto

Foxg1 Coordinates the Switch from Nonradially to Radially Migrating Glutamatergic Subtypes in the Neocortex through Spatiotemporal Repression

The specification of neuronal subtypes in the cerebral cortex proceeds in a temporal manner; however, the regulation of the transitions between the sequentially generated subtypes is poorly understood. Here, we report that the forkhead box transcription factor Foxg1 coordinates the production of neocortical projection neurons through the global repression of a default gene program. The delayed activation of Foxg1 was necessary and sufficient to induce deep-layer neurogenesis, followed by a sequential wave of upper-layer neurogenesis. A genome-wide analysis revealed that Foxg1 binds to mammalian-specific noncoding sequences to repress over 12 transcription factors expressed in early progenitors, including Ebf2/3, Dmrt3, Dmrta1, and Eya2. These findings reveal an unexpected prolonged competence of progenitors to initiate corticogenesis at a progressed stage during development and identify Foxg1 as a critical initiator of neocorticogenesis through spatiotemporal repression, a system that balances the production of nonradially and radially migrating glutamatergic subtypes during mammalian cortical expansion.

The timing of upper-layer neurogenesis is conferred by sequential derepression and negative feedback from deep-layer neurons.

The prevailing view of upper-layer (UL) neurogenesis in the cerebral cortex is that progenitor cells undergo successive rounds of asymmetric cell division that restrict the competence and production of UL neurons later in development. However, the recent discovery of UL fate-committed early progenitors raises an alternative perspective concerning their ontogeny. To investigate the emergence of UL progenitors, we manipulated the timing and extent of cortical neurogenesis in vivo in mice. We demonstrated that UL competence is tightly linked to deep-layer (DL) neurogenesis and that this sequence is determined primarily through derepression of Fezf2 by Foxg1 within a closed transcriptional cascade. We further demonstrated that the sequential acquisition of UL competence requires negative feedback, which is propagated from postmitotic DL neurons. Thus, neocortical progenitors integrate intrinsic and extrinsic cues to generate UL neurons through a system that controls the sequence of DL and UL neurogenesis and to scale the production of intracortical projection neurons based on the availability of their subcortical projection neuron counterparts during cortical development and evolution.

Neuronal subtype specification in establishing mammalian neocortical circuits

The functional integrity of the neocortical circuit relies on the precise production of diverse neuron populations and their assembly during development. In recent years, extensive progress has been made in the understanding of the mechanisms that control differentiation of each neuronal type within the neocortex. In this review, we address how the elaborate neocortical cytoarchitecture is established from a simple neuroepithelium based on recent studies examining the spatiotemporal mechanisms of neuronal subtype specification. We further discuss the critical events that underlie the conversion of the stem amniotes cerebrum to a mammalian-type neocortex, and extend these key findings in the light of mammalian evolution to understand how the neocortex in humans evolved from common ancestral mammals.

Evolutionary conservation and conversion of Foxg1 function in brain development

Among the forkhead box protein family, Foxg1 is a unique transcription factor that plays pleiotropic and non‐redundant roles in vertebrate brain development. The emergence of the telencephalon at the rostral end of the neural tube and its subsequent expansion that is mediated by Foxg1 was a key reason for the vertebrate brain to acquire higher order information processing, where Foxg1 is repetitively used in the sequential events of telencephalic development to control multi‐steps of brain circuit formation ranging from cell cycle control to neuronal differentiation in a clade‐ and species‐specific manner. The objective of this review is to discuss how the evolutionary changes in cis‐ and trans‐regulatory network that is mediated by a single transcription factor has contributed to determining the fundamental vertebrate brain structure and its divergent roles in instructing species‐specific neuronal circuitry and functional specialization.

Dual-color deep-tissue three-photon microscopy with a multiband infrared laser

Multiphoton microscopy combined with genetically encoded fluorescent indicators is a central tool in biology. Three-photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 µm opens up new opportunities for deep-tissue imaging. However, novel strategies are needed to enable in-depth multicolor fluorescence imaging and fully develop such an imaging approach. Here, we report on a novel multiband SWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 µm that has characteristics optimized for 3P microscopy: sub-70 fs duration, 1.25 MHz repetition rate, and µJ-range pulse energy. In turn, we achieve simultaneous 3P excitation of green fluorescent protein (GFP) and red fluorescent proteins (mRFP, mCherry, tdTomato) along with third-harmonic generation. We demonstrate in-depth dual-color 3P imaging in a fixed mouse brain, chick embryo spinal cord, and live adult zebrafish brain, with an improved signal-to-background ratio compared to multicolor two-photon imaging. This development opens the way towards multiparametric imaging deep within scattering tissues.Microscopy: Looking deeper with three photonsResearchers in France are using a novel infra-red light source to examine both fixed and living tissue samples in deeper detail than previously possible with a technique called three-photon microscopy. The absorption of three photons at different infra-red frequencies stimulates subsequent emission of light from fluorescent molecules in the sample. Detecting the fluorescence by microscopy reveals the location and interactions of the molecules concerned. Emmanuel Beaurepaire and Frederic Duon at the University of Paris-Saclay, developed a procedure to emit ultra-short pulses of infra-red laser light with optimal characteristics for three-photon microscopy. They demonstrated their innovation by studying fluorescent proteins in brain and nerve tissue taken from mice and chicks, and also in live zebrafish brain. The procedure offers opportunities to study molecular structures and interactions more effectively than previously possible with three-photon microscopy.

A Sensitive and Versatile In Situ Hybridization Protocol for Gene Expression Analysis in Developing Amniote Brains

The detection of specific RNA molecules in embryonic tissues has wide research applications including studying gene expression dynamics in brain development and evolution. Recent advances in sequencing technologies have introduced new animal models to explore the molecular principles underlying the assembly and diversification of brain circuits between different amniote species. Here, we provide a step-by-step protocol for a versatile in situ hybridization method that is immediately applicable to a range of amniote embryos including zebra finch and Madagascar ground gecko, two new model organisms that have rapidly emerged for comparative brain studies over recent years. The sensitive detection of transcripts from low to high abundance expression range using the same platform enables direct comparison of gene of interest among different amniotes, providing high-resolution spatiotemporal information of gene expression to dissect the molecular principles underlying brain evolution.

Foxg1 regulates the onset of projection neuron production in the neocortex

P3-e25 Lineage tracing of Nkx2.2-expressing cells by novel genetically-defined lineage tracing method in chick spinal cord Hitoshi Goto 1,2 , Katsuhiko Ono 1,2,3, Hirohide Takebayashi 2,3,4, Hidekiyo Harada 5, Harukazu Nakamura 5, Kazuhiro Ikenaka 2,3 1 Department of Biology, Kyoto Prefectual University of Medicine, Kyoto 2 Div. of Neurobiol. and Bioinformatics, National Institute for Physiological Sciences, Okazaki 3 Sokendai, Hayama 4 Department of Morphological Neural Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 5 Dep. of Molecular Neurobiology, Graduate School of Life Sciences and Institute of Development, Aging and Cancer, Tohoku University, Miyagi