Late-life shift in caloric intake affects fly metabolism and longevity.

The prevalence of obesity is increasing in older adults and contributes to age-related decline. Caloric restriction (CR) alleviates obesity phenotypes and delays the onset of age-related changes. However, how late in life organisms benefit from switching from a high-(H) to a low-calorie (L) diet is unclear. We transferred male flies from a H to a L (HL) diet or vice versa (LH) at different times during life. Both shifts immediately change fly rate of aging even when applied late in life. HL shift rapidly reduces fly mortality rate to briefly lower rate than in flies on a constant L diet, and extends lifespan. Transcriptomic analysis uncovers that flies aged on H diet have acquired increased stress response, which may have temporal advantage over flies aged on L diet and leads to rapid decrease in mortality rate after HL switch. Conversely, a LH shift increases mortality rate, which is temporarily higher than in flies aged on a H diet, and shortens lifespan. Unexpectedly, more abundant transcriptomic changes accompanied LH shift, including increase in ribosome biogenesis, stress response and growth. These changes reflect protection from sudden release of ROS, energy storage, and use of energy to growth, which all likely contribute to higher mortality rate. As the beneficial effects of CR on physiology and lifespan are conserved across many organisms, our study provides framework to study underlying mechanisms of CR interventions that counteract the detrimental effects of H diets and reduce rate of aging even when initiated later in life.

Integrated single-cell transcriptomic and epigenetic study of cell state transition and lineage commitment in embryonic mouse cerebellum.

Recent studies using single-cell RNA-sequencing have revealed cellular heterogeneity in the developing mammalian cerebellum, yet the regulatory logic underlying this cellular diversity remains to be elucidated. Using integrated single-cell RNA and ATAC analyses, we resolved developmental trajectories of cerebellar progenitors and identified putative trans- and cis-elements that control cell state transition. We reverse engineered gene regulatory networks (GRNs) of each cerebellar cell type. Through in silico simulations and in vivo experiments, we validated the efficacy of GRN analyses and uncovered the molecular control of a posterior transitory zone (PTZ), a distinct progenitor zone residing immediately anterior to the morphologically defined rhombic lip (RL). We showed that perturbing cell fate specification in the PTZ and RL causes posterior cerebellar vermis hypoplasia, the most common cerebellar birth defect in humans. Our study provides a foundation for comprehensive studies of developmental programs of the mammalian cerebellum.

LISA2: Learning Complex Single-Cell Trajectory and Expression Trends.

Single-cell transcriptional and epigenomics profiles have been applied in a variety of tissues and diseases for discovering new cell types, differentiation trajectories, and gene regulatory networks. Many methods such as Monocle 2/3, URD, and STREAM have been developed for tree-based trajectory building. Here, we propose a fast and flexible trajectory learning method, LISA2, for single-cell data analysis. This new method has two distinctive features: (1) LISA2 utilizes specified leaves and root to reduce the complexity for building the developmental trajectory, especially for some special cases such as rare cell populations and adjacent terminal cell states; and (2) LISA2 is applicable for both transcriptomics and epigenomics data. LISA2 visualizes complex trajectories using 3D Landmark ISOmetric feature MAPping (L-ISOMAP). We apply LISA2 to simulation and real datasets in cerebellum, diencephalon, and hematopoietic stem cells including both single-cell transcriptomics data and single-cell assay for transposase-accessible chromatin data. LISA2 is efficient in estimating single-cell trajectory and expression trends for different kinds of molecular state of cells.

An adherent-cell depletion technique to generate human neural progenitors and neurons.

Existing methodologies to produce human neural stem cells and neurons from embryonic stem cells frequently involve multistep processes and the use of complex and expensive media components, cytokines or small molecules. Here, we report a simple technique to generate human neuroepithelial progenitors and neurons by periodic mechanical dissection and adherent-cell depletion on regular cell-culture grade plastic surfaces. This neural induction technique does not employ growth factors, small molecules or peptide inhibitors, apart from those present in serum-free supplements. Suggestive of their central nervous system origin, we found that neural progenitors formed by this technique expressed radial glia markers, and, when differentiated, expressed TUBB3, RBFOX3 (NeuN) and serotonin, but not markers for peripheral neurons. With these data, we postulate that incorporation of periodic mechanical stimuli and plastic surface-mediated cell selection could improve and streamline existing human neuron production protocols.

Defining developmental diversification of diencephalon neurons through single cell gene expression profiling.

The embryonic diencephalon forms integration centers and relay stations in the forebrain. Anecdotal expression studies suggest that the diencephalon contains multiple developmental compartments and subdivisions. Here, we utilized single cell RNA sequencing to profile transcriptomes of dissociated cells from the diencephalon of E12.5 mouse embryos. We identified the divergence of different progenitors, intermediate progenitors, and emerging neurons. By mapping the identified cell groups to their spatial origins, we characterized the molecular features of cell types and cell states arising from various diencephalic domains. Furthermore, we reconstructed the developmental trajectory of distinct cell lineages, and thereby identified the genetic cascades and gene regulatory networks underlying the progression of the cell cycle, neurogenesis and cellular diversification. The analysis provides new insights into the molecular mechanisms underlying the amplification of intermediate progenitor cells in the thalamus. The single cell-resolved trajectories not only confirm a close relationship between the rostral thalamus and prethalamus, but also uncover an unexpected close relationship between the caudal thalamus, epithalamus and rostral pretectum. Our data provide a useful resource for systematic studies of cell heterogeneity and differentiation kinetics within the diencephalon.

The Molecular Pathway Regulating Bergmann Glia and Folia Generation in the Cerebellum.

Evolution of complex behaviors in higher vertebrates and primates require the development of sophisticated neuronal circuitry and the expansion of brain surface area to accommodate the vast number of neuronal and glial populations. To achieve these goals, the neocortex in primates and the cerebellum in amniotes have developed specialized types of basal progenitors to aid the folding of their cortices. In the cerebellum, Bergmann glia constitute such a basal progenitor population, having a distinctive morphology and playing a critical role in cerebellar corticogenesis. Here, we review recent studies on the induction of Bergmann glia and their crucial role in mediating folding of the cerebellar cortex. These studies uncover a key function of FGF-ERK-ETV signaling cascade in the transformation of Bergmann glia from radial glia in the ventricular zone. Remarkably, in the neocortex, the same signaling axis operates to facilitate the transformation of ventricular radial glia into basal radial glia, a Bergmann glia-like basal progenitor population, which have been implicated in the establishment of neocortical gyri. These new findings draw a striking similarity in the function and ontogeny of the two basal progenitor populations born in distinct brain compartments.

Regulation of self-renewing neural progenitors by FGF/ERK signaling controls formation of the inferior colliculus.

The embryonic tectum displays an anteroposterior gradient in development and produces the superior colliculus and inferior colliculus. Studies suggest that partition of the tectum is controlled by different strengths and durations of FGF signals originated from the so-called isthmic organizer at the mid/hindbrain junction; however, the underlying mechanism is unclear. We show that deleting Ptpn11, which links FGF with the ERK pathway, prevents inferior colliculus formation by depleting a previously uncharacterized stem cell zone. The stem-zone loss is attributed to shortening of S phase and acceleration of cell cycle exit and neurogenesis. Expression of a constitutively active Mek1 (Mek1DD), the known ERK activator, restores the tectal stem zone and the inferior colliculus without Ptpn11. By contrast, Mek1DD expression fails to rescue the tectal stem zone and the inferior colliculus in the absence of Fgf8 and the isthmic organizer, indicating that FGF and Mek1DD initiate qualitatively and/or quantitatively distinctive signaling. Together, our data show that the formation of the inferior colliculus relies on the provision of new cells from the tectal stem zone. Furthermore, distinctive ERK signaling mediates Fgf8 in the control of cell survival, tissue polarity and cytogenetic gradient during the development of the tectum.

Gbx2 is essential for maintaining thalamic neuron identity and repressing habenular characters in the developing thalamus.

The thalamus and habenula, two important nodes of the forebrain circuitry, are derived from a single developmental compartment, called prosomere 2, in the diencephalon. Habenular and thalamic neurons display distinct molecular identity, neurochemistry, and connectivity. Furthermore, their progenitors exhibit distinctive neurogenic patterns with a marked delay in the onset of neurogenesis in the thalamus. However, the progenitors in prosomere 2 express many common developmental regulators and the mechanism underlying the specification and differentiation of these two populations of neurons remains unknown. Gbx2, coding for a homeodomain transcription factor, is initially expressed in thalamic neuronal precursors that have just exited the cell cycle, and its expression is maintained in many mature thalamic neurons in adults. Deletion of Gbx2 severely disrupts histogenesis of the thalamus and abolishes thalamocortical projections in mice. Here, by using genome-wide transcriptional profiling, we show that Gbx2 promotes thalamic but inhibits habenular molecular characters. Remarkably, although Gbx2 is expressed in postmitotic neuronal precursors, deletion of Gbx2 changes gene expression and cell proliferation in dividing progenitors in the developing thalamus. These defects are partially rescued by the mosaic presence of wild-type cells, demonstrating a cell non-autonomous role of Gbx2 in regulating the development of thalamic progenitors. Our results suggest that Gbx2 is essential for the acquisition of the thalamic neuronal identity by repressing habenular identity through a feedback signaling from postmitotic neurons to progenitors.

T cell development involves TRAF3IP3-mediated ERK signaling in the Golgi.

Generation of T lymphocytes in the thymus is guided by signal transduction from the T cell receptor (TCR), but the underlying mechanism is incompletely understood. Here we have identified a Golgi-associated factor, TRAF3-interacting protein 3 (TRAF3IP3), as a crucial mediator of thymocyte development. TRAF3IP3 deficiency in mice attenuates the generation of mature thymocytes caused by impaired thymocyte-positive selection. TRAF3IP3 mediates TCR-stimulated activation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) and its upstream kinase mitogen/extracellular signal-regulated kinase (MEK). Interestingly, TRAF3IP3 exerts this signaling function through recruiting MEK to the Golgi and, thereby, facilitating the interaction of MEK with its activator BRAF. Transgenic expression of a constitutively active MEK rescues the T cell development block in Traf3ip3 knockout mice. These findings establish TRAF3IP3 as a novel regulator of T cell development and suggest a Golgi-specific ERK signaling mechanism that regulates thymocyte development.

The cochlear sensory epithelium derives from Wnt responsive cells in the dorsomedial otic cup.

Wnt1 and Wnt3a secreted from the dorsal neural tube were previously shown to regulate a gene expression program in the dorsal otic vesicle that is necessary for vestibular morphogenesis (Riccomagno et al., 2005. Genes Dev. 19, 1612-1623). Unexpectedly, Wnt1(-/-); Wnt3a(-/-) embryos also displayed a pronounced defect in the outgrowth of the ventrally derived cochlear duct. To determine how Wnt signaling in the dorsal otocyst contributes to cochlear development we performed a series of genetic fate mapping experiments using two independent Wnt responsive driver strains (TopCreER and Gbx2(CreER)) that when crossed to inducible responder lines (Rosa(lacZ) or Rosa(zsGreen)) permanently labeled dorsomedial otic progenitors and their derivatives. Tamoxifen time course experiments revealed that most vestibular structures showed some degree of labeling when recombination was induced between E7.75 and E12.5, consistent with continuous Wnt signaling activity in this tissue. Remarkably, a population of Wnt responsive cells in the dorsal otocyst was also found to contribute to the sensory epithelium of the cochlear duct, including auditory hair and support cells. Similar results were observed with both TopCreER and Gbx2(CreER) strains. The ventral displacement of Wnt responsive cells followed a spatiotemporal sequence that initiated in the anterior otic cup at, or immediately prior to, the 17-somite stage (E9) and then spread progressively to the posterior pole of the otic vesicle by the 25-somite stage (E9.5). These lineage-tracing experiments identify the earliest known origin of auditory sensory progenitors within a population of Wnt responsive cells in the dorsomedial otic cup.

Shp2-dependent ERK signaling is essential for induction of Bergmann glia and foliation of the cerebellum.

Folding of the cortex and the persistence of radial glia (RG)-like cells called Bergmann glia (BG) are hallmarks of the mammalian cerebellum. Similar to basal RG in the embryonic neocortex, BG maintain only basal processes and continuously express neural stem cell markers. Past studies had focused on the function of BG in granule cell migration and how granule cell progenitors (GCP) regulate cerebellar foliation. The molecular control of BG generation and its role in cerebellar foliation are less understood. Here, we have analyzed the function of the protein tyrosine phosphatase Shp2 in mice by deleting its gene Ptpn11 in the entire cerebellum or selectively in the GCP lineage. Deleting Ptpn11 in the entire cerebellum by En1-cre blocks transformation of RG into BG but preserves other major cerebellar cell types. In the absence of BG, inward invagination of GCP persists but is uncoupled from the folding of the Purkinje cell layer and the basement membrane, leading to disorganized lamination and an absence of cerebellar folia. In contrast, removing Ptpn11 in the GCP lineage by Atoh1-cre has no effect on cerebellar development, indicating that Shp2 is not cell autonomously required in GCP. Furthermore, we demonstrate that Ptpn11 interacts with Fgf8 and is essential for ERK activation in RG and nascent BG. Finally, expressing constitutively active MEK1 rescues BG formation and cerebellar foliation in Shp2-deficient cerebella. Our results demonstrate an essential role of Shp2 in BG specification via fibroblast growth factor/extracellular signal-regulated protein kinase signaling, and reveal a crucial function of BG in organizing cerebellar foliation.

Differential BMP signaling controls formation and differentiation of multipotent preplacodal ectoderm progenitors from human embryonic stem cells.

Sensory and endoneurocrine tissues as diverse as the lens, the olfactory epithelium, the inner ear, the cranial sensory ganglia, and the anterior pituitary arise from a common pool of progenitors in the preplacodal ectoderm (PPE). Around late gastrulation, the PPE forms at the border surrounding the anterior neural plate, and expresses a unique set of evolutionarily conserved transcription regulators including Six1, Eya 1 and Eya2. Here, we describe the first report to generate and characterize the SIX1(+) PPE cells from human embryonic stem (ES) cells by adherent differentiation. Before forming PPE cells, differentiating cultures first expressed the non-neural ectoderm specific transcriptional factors TFAP2A, GATA2, GATA3, DLX3, and DLX5, which are crucial in establishing the PPE competence. We demonstrated that bone morphogenetic protein (BMP) activity plays a transient but essential role in inducing expression of these PPE competence factors and eventually the PPE cells. Interestingly, we found that attenuating BMP signaling after establishing the competence state induces anterior placode precursors. By manipulating BMP and hedgehog signaling pathways, we further differentiate these precursors into restricted lineages including the lens placode and the oral ectoderm (pituitary precursor) cells. Finally, we also show that sensory neurons can be generated from human PPE cells, demonstrating the multipotency of the human ES-derived PPE cells.

Gbx2 regulates thalamocortical axon guidance by modifying the LIM and Robo codes.

Combinatorial expression of transcription factors forms transcriptional codes to confer neuronal identities and connectivity. However, how these intrinsic factors orchestrate the spatiotemporal expression of guidance molecules to dictate the responsiveness of axons to guidance cues is less understood. Thalamocortical axons (TCAs) represent the major input to the neocortex and modulate cognitive functions, consciousness and alertness. TCAs travel a long distance and make multiple target choices en route to the cortex. The homeodomain transcription factor Gbx2 is essential for TCA development, as loss of Gbx2 abolishes TCAs in mice. Using a novel TCA-specific reporter, we have discovered that thalamic axons are mostly misrouted to the ventral midbrain and dorsal midline of the diencephalon in Gbx2-deficient mice. Furthermore, conditionally deleting Gbx2 at different embryonic stages has revealed a sustained role of Gbx2 in regulating TCA navigation and targeting. Using explant culture and mosaic analyses, we demonstrate that Gbx2 controls the intrinsic responsiveness of TCAs to guidance cues. The guidance defects of Gbx2-deficient TCAs are associated with abnormal expression of guidance receptors Robo1 and Robo2. Finally, we demonstrate that Gbx2 controls Robo expression by regulating LIM-domain transcription factors through three different mechanisms: Gbx2 and Lhx2 compete for binding to the Lmo3 promoter and exert opposing effects on its transcription; repressing Lmo3 by Gbx2 is essential for Lhx2 activity to induce Robo2; and Gbx2 represses Lhx9 transcription, which in turn induces Robo1. Our findings illustrate the transcriptional control of differential expression of Robo1 and Robo2, which may play an important role in establishing the topography of TCAs.

Gbx2 plays an essential but transient role in the formation of thalamic nuclei.

Unlike the laminar arrangement of neurons in the neocortex, thalamic neurons aggregate to form about dozens of nuclei, many of which make topographic connections with specific areas in the neocortex. The molecular mechanisms underlying the formation of thalamic nuclei remain largely unknown. Homeodomain transcription factor Gbx2 is specifically expressed in the developing thalamus. Deleting Gbx2 leads to severe disruption of the histogenesis of the thalamus in mice, demonstrating an essential role of Gbx2 in this brain structure. Using inducible genetic fate mapping, we have previously shown that the neuronal precursors for different sets of thalamic nuclei have distinctive onset and duration of Gbx2 expression, suggesting that the dynamic expression of Gbx2 plays an important role in the specification and differentiation of thalamic nuclei. Here, we showed that the Gbx2 lineage exclusively gives rise to neurons but not glia in the thalamus. We performed conditional deletion to examine the temporal requirements of Gbx2 in the developing thalamus in mice. Corresponding to the dynamic and differential expression of Gbx2 in various thalamic nucleus groups, deleting Gbx2 at different embryonic stages disrupts formation of distinct sets of thalamic nuclei. Interestingly, different thalamic nuclei have remarkably different requirements of Gbx2 for the survival of thalamic neurons. Furthermore, although Gbx2 expression persists in many thalamic nuclei until adulthood, only the initial expression of Gbx2 following neurogenesis is crucial for the differentiation of thalamic nuclei. Our results indicate that the dynamic expression of Gbx2 may act as an important determinant in coupling with other developmental programs to generate distinct thalamic nuclei.

Patterning and compartment formation in the diencephalon.

The diencephalon gives rise to structures that play an important role in connecting the anterior forebrain with the rest of the central nervous system. The thalamus is the major diencephalic derivative that functions as a relay station between the cortex and other lower order sensory systems. Almost two decades ago, neuromeric/prosomeric models were proposed describing the subdivision and potential segmentation of the diencephalon. Unlike the laminar structure of the cortex, the diencephalon is progressively divided into distinct functional compartments consisting principally of thalamus, epithalamus, pretectum, and hypothalamus. Neurons generated within these domains further aggregate to form clusters called nuclei, which form specific structural and functional units. We review the recent advances in understanding the genetic mechanisms that are involved in the patterning and compartment formation of the diencephalon.

Numerous isoforms of Fgf8 reflect its multiple roles in the developing brain.

Soluble growth factors play an important role in the coordination and integration of cell proliferation, differentiation, fate determination, and morphogenesis during development of multicellular organisms. Fibroblast growth factors (FGFs) are a large family of polypeptide growth factors that are present in organisms ranging from nematodes to humans. RNA alternative splicing of FGFs and their receptors further enhances the complexity of this ligand-receptor system. The mouse Fgf8 gene produces eight splice variants, which encode isoform proteins with different N-termini and distinct receptor-binding affinity and biological activity. In this article, we review the roles of Fgf8 in vertebrate development and summarize the recent findings on the in vivo function of different Fgf8 splice variants. We propose that multiple Fgf8 isoform proteins act in concert to regulate the overall function of Fgf8 and account for the diverse and essential role of Fgf8 during vertebrate development.

Gbx2 and Fgf8 are sequentially required for formation of the midbrain-hindbrain compartment boundary.

In vertebrates, the common expression border of two homeobox genes, Otx2 and Gbx2, demarcates the prospective midbrain-hindbrain border (MHB) in the neural plate at the end of gastrulation. The presence of a compartment boundary at the MHB has been demonstrated, but the mechanism and timing of its formation remain unclear. We show by genetic inducible fate mapping using a Gbx2(CreER) knock-in mouse line that descendants of Gbx2(+) cells as early as embryonic day (E) 7.5 do not cross the MHB. Without Gbx2, hindbrain-born cells abnormally populate the entire midbrain, demonstrating that Gbx2 is essential for specifying hindbrain fate. Gbx2(+) and Otx2(+) cells segregate from each other, suggesting that mutually exclusive expression of Otx2 and Gbx2 in midbrain and hindbrain progenitors is responsible for cell sorting in establishing the MHB. The MHB organizer gene Fgf8, which is expressed as a sharp transverse band immediately posterior to the lineage boundary at the MHB, is crucial in maintaining the lineage-restricted boundary after E7.5. Partial deletion of Fgf8 disrupts MHB lineage separation. Activation of FGF pathways has a cell-autonomous effect on cell sorting in midbrain progenitors. Therefore, Fgf8 from the MHB may signal the nearby mesencephalic cells to impart distinct cell surface characteristics or induce local cell-cell signaling, which consequently prevents cell movements across the MHB. Our findings reveal the distinct function of Gbx2 and Fgf8 in a stepwise process in the development of the compartment boundary at the MHB and that Fgf8, in addition to its organizer function, plays a crucial role in maintaining the lineage boundary at the MHB by restricting cell movement.

The mouse homeobox gene Gbx2 is required for the development of cholinergic interneurons in the striatum.

Mammalian forebrain cholinergic neurons are composed of local circuit neurons in the striatum and projection neurons in the basal forebrain. These neurons are known to arise from a common pool of progenitors that primarily resides in the medial ganglionic eminence (MGE). However, little is known about the genetic programs that differentiate these two types of cholinergic neurons. Using inducible genetic fate mapping, here we examined the developmental fate of cells that express the homeodomain transcription factor Gbx2 in the MGE. We show that the Gbx2 lineage-derived cells that undergo tangential migration exclusively give rise to almost all cholinergic interneurons in the striatum, whereas those undergoing radial migration mainly produce noncholinergic neurons in the basal forebrain. Deletion of Gbx2 throughout the mouse embryo or specifically in the MGE results in abnormal distribution and significant reduction of cholinergic neurons in the striatum. We show that early-born (before embryonic day 12.5) cholinergic interneurons preferentially populate the lateral aspect of the striatum and mature earlier than late-born (after embryonic day 12.5) neurons, which normally reside in the medial part of the striatum. In the absence of Gbx2, early-born striatal cholinergic precursors display abnormal neurite outgrowth and increased complexity, and abnormally contribute to the medial part of the caudate-putamen, whereas late-born striatal cholinergic interneurons are mostly missing. Together, our data demonstrate that Gbx2 is required for the development of striatal cholinergic interneurons, perhaps by regulating tangential migration of the striatal cholinergic precursors.

Fgf8b-containing spliceforms, but not Fgf8a, are essential for Fgf8 function during development of the midbrain and cerebellum.

The single Fgf8 gene in mice produces eight protein isoforms (Fgf8a-h) with different N-termini by alternative splicing. Gain-of-function studies have demonstrated that Fgf8a and Fgf8b have distinct activities in the developing midbrain and hindbrain (MHB) due to their different binding affinities with FGF receptors. Here we have performed loss-of-function analyses to determine the in vivo requirement for these two Fgf8 spliceforms during MHB development. We showed that deletion of Fgf8b-containing spliceforms (b, d, f and h) leads to loss of multiple key regulatory genes, including Fgf8 itself, in the MHB region. Therefore, specific inactivation of Fgf8b-containing spliceforms, similar to the loss of Fgf8, in MHB progenitors results in deletion of the midbrain, isthmus, and cerebellum. We also created a splice-site mutation abolishing Fgf8a-containing spliceforms (a, c, e, and g). Mice lacking Fgf8a-containing spliceforms exhibit growth retardation and postnatal lethality, and the phenotype is variable in different genetic backgrounds, suggesting that the Fgf8a-containing spliceforms may play a role in modulating the activity of Fgf8. Surprisingly, no discernable defect was detected in the midbrain and cerebellum of Fgf8a-deficient mice. To determine if Fgf17, which is expressed in the MHB region and possesses similar activities to Fgf8a based on gain-of-function studies, may compensate for the loss of Fgf8a, we generated Fgf17 and Fgf8a double mutant mice. Mice lacking both Fgf8a-containing spliceforms and Fgf17 display the same defect in the posterior midbrain and anterior cerebellum as Fgf17 mutant mice. Therefore, Fgf8b-containing spliceforms, but not Fgf8a, are essential for the function of Fgf8 during the development of the midbrain and cerebellum.

Misexpression of Gbx2 throughout the mesencephalon by a conditional gain-of-function transgene leads to deletion of the midbrain and cerebellum in mice.

The mouse homeobox gene, Gbx2, is expressed in discreet domains in the neural tube and plays a key role in forebrain and hindbrain development. Previous studies have demonstrated that mutual inhibition between Gbx2 and Otx2, which are respectively expressed in the anterior and posterior parts of the neural plate, positions the prospective midbrain-hindbrain junction. We describe here a conditional Gbx2 gain-of-function transgenic mouse line, Gbx2-GOF, which expresses Gbx2 and red fluorescence protein, mCherry, upon Cre-mediated recombination. In the absence of Cre, beta-galactosidase is broadly expressed in mouse embryos and adult brains carrying the transgene. By combining Gbx2-GOF and En1(Cre) knock-in allele, we activated expression of Gbx2 and mCherry throughout the mesencephalon (mes) and rhombomere 1 (r1). The ectopic expression of Gbx2 causes an anterior shift of the mes/r1 junction at embryonic day 10.5. Interestingly, we found that persistent expression of Gbx2 throughout the mes/r1 region largely abolishes expression of the isthmic organizer gene Fgf8, leading to deletion of the midbrain and cerebellum at later stages. Our data suggest that the juxtaposition of the expression domains of Gbx2 and Otx2 within the mes/r1 area is essential for the maintenance of Fgf8 expression. Furthermore, the Gbx2-GOF transgenic line is suitable for functional study of Gbx2 during development.