In vitro and in vivo functions of T cells produced in complemented thymi of chimeric mice generated by blastocyst complementation.

Blastocyst complementation is an intriguing way of generating humanized animals for organ preparation in regenerative medicine and establishing novel models for drug development. Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4+ and CD8+ T cells proliferated and produced IFNγ, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFNγ-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs/cells in interspecific chimeras will be important in the near future.

Role of Anti-Fractalkine Antibody in Suppression of Joint Destruction by Inhibiting Migration of Osteoclast Precursors to the Synovium in Experimental Arthritis.

OBJECTIVE: To elucidate the role of the fractalkine (FKN)/CX3 CR1 pathway in joint destruction in rheumatoid arthritis. METHODS: We examined the effect of treatment with anti-mouse FKN (anti-mFKN) monoclonal antibody (mAb) on joint destruction and the migration of osteoclast precursors (OCPs) into the joint, using the collagen-induced arthritis (CIA) model. DBA/1 mice were immunized with bovine type II collagen to induce arthritis, and then treated with anti-mFKN mAb. Disease severity was monitored by arthritis score, and joint destruction was evaluated by soft x-ray and histologic analyses. Plasma levels of joint destruction markers were assessed by enzyme-linked immunosorbent assay. FKN expression on endothelial cells was detected by immunohistochemistry. Bone marrow-derived OCPs were labeled with fluorescein and transferred to mice with CIA, and the migration of the OCPs to the joints was then analyzed. RESULTS: Both prophylactic and therapeutic treatment with anti-mFKN mAb significantly decreased the arthritis and soft x-ray scores. Plasma levels of cartilage oligomeric matrix protein and matrix metalloproteinase 3 decreased after treatment with anti-mFKN mAb. Histologic analysis revealed that anti-mFKN mAb inhibited synovitis, pannus formation, and cartilage destruction, as well as suppressed bone damage, with a marked reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. Anti-mFKN mAb strongly inhibited the migration of bone marrow-derived OCPs into the affected synovium. CONCLUSION: Anti-mFKN mAb notably ameliorates arthritis and joint destruction in the CIA model, as well as inhibits migration of OCPs into the synovium. These results suggest that inhibition of the FKN/CX3 CR1 pathway could be a novel strategy for treatment of both synovitis and joint destruction in rheumatoid arthritis.

Temporal identity transition from Purkinje cell progenitors to GABAergic interneuron progenitors in the cerebellum.

In the cerebellum, all GABAergic neurons are generated from the Ptf1a-expressing ventricular zone (Ptf1a domain). However, the machinery to produce different types of GABAergic neurons remains elusive. Here we show temporal regulation of distinct GABAergic neuron progenitors in the cerebellum. Within the Ptf1a domain at early stages, we find two subpopulations; dorsally and ventrally located progenitors that express Olig2 and Gsx1, respectively. Lineage tracing reveals the former are exclusively Purkinje cell progenitors (PCPs) and the latter Pax2-positive interneuron progenitors (PIPs). As development proceeds, PCPs gradually become PIPs starting from ventral to dorsal. In gain- and loss-of-function mutants for Gsx1 and Olig1/2, we observe abnormal transitioning from PCPs to PIPs at inappropriate developmental stages. Our findings suggest that the temporal identity transition of cerebellar GABAergic neuron progenitors from PCPs to PIPs is negatively regulated by Olig2 and positively by Gsx1, and contributes to understanding temporal control of neuronal progenitor identities.

The c-Ski family member and transcriptional regulator Corl2/Skor2 promotes early differentiation of cerebellar Purkinje cells.

Purkinje cells (PCs) provide the primary output from the cerebellar cortex, which controls movement and posture, and loss of PCs causes severe cerebellar dysfunction. The mechanisms underlying cell fate determination and early differentiation of PC remain largely unknown. Here we show that the c-Ski family member and transcriptional regulator Corl2 is required for correct differentiation of PCs. In Corl2 knock-out embryos, initial PC specification appeared largely normal, but in a subset of presumptive PCs generated near the ventral border of the PC domain, cell fate choice was compromised and cells showed a mixed identity expressing the interneuron marker Pax2 as well. Additionally, selection and maintenance of the transmitter phenotype was compromised in most developing PCs in the mutants. During later differentiation steps, induction of PC marker genes was significantly suppressed, suggesting that maturation was delayed in the absence of Corl2. Consistently, defects in migration, cell polarization and dendrite formation were observed in mutant PCs, although their axonal trajectories appeared normal. These phenotypes closely resembled those of mutants for Rora, an essential regulator of PC differentiation. However, Rora expression was not significantly changed in the Corl2 mutants, indicating that Corl2 does not simply act upstream of Rora to promote PC differentiation. ChIP experiments revealed that Corl2 bound to the promoter regions of several PC-selective genes, which are also known to be direct downstream targets of RORα. Altogether, our results identified a novel regulatory program of PC differentiation involving Corl2, which might cooperate with the RORα pathway.

The basic helix-loop-helix transcription factor Nato3 controls neurogenic activity in mesencephalic floor plate cells.

Floor plate (FP) cells, the ventral midline cells of the developing neural tube, have long been thought to be non-neurogenic organizer cells that control neuronal patterning and axonal guidance. Recent studies have revealed that mesencephalic FP (mesFP) cells have neurogenic activity and generate dopaminergic neurons. However, the mechanisms underlying the control of neurogenic potential in FP cells are not yet fully understood. Here we identified the bHLH factor Nato3 as an FP-specific transcription factor. In Nato3-null mutant mice, FP cells in the spinal cord were correctly specified, but could not properly mature. By contrast, in the developing mesencephalon, loss of Nato3 did not affect FP differentiation, but led to loss of neurogenic activity in the medial subpopulation of mesFP cells by suppressing proneural gene expression and inducing cell cycle arrest. As a consequence, the number of midbrain dopaminergic neurons generated was decreased in mutants. We also found that Hes1, which is known to be required for non-dividing organizer cell development in the neural tube, was aberrantly upregulated in the mesFP cells of Nato3 mutants. Consistently, forced expression of Nato3 repressed Hes1 expression and consequently induced premature neurogenesis. Finally, we showed that forced expression of Hes1 in mesFP cells induced cell cycle arrest and downregulation of proneural factors. Taken together, these results suggest that Nato3 confers neurogenic potential on mesFP cells by suppressing classical non-neurogenic FP cell differentiation, at least in part, through repressing Hes1.

Lmx1a and Lmx1b cooperate with Foxa2 to coordinate the specification of dopaminergic neurons and control of floor plate cell differentiation in the developing mesencephalon.

Mesencephalic dopaminergic (mesDA) neurons control movement and behavior, and their loss causes severe neurological disorders, such as Parkinson's disease. Recent studies have revealed that mesDA neurons originate from mesencephalic floor plate (FP) cells, which had been thought of as non-neurogenic organizer cells regulating regional patterning and axonal projections. Otx2 and its FP-specific downstream factor Lmx1a have been shown to be sufficient to confer neurogenic activity on FP cells and determine a mesDA fate. However, the mechanism underlying how these factors control mesDA development and how FP cells and mesDA neurons are coordinately specified are still largely unknown. In the present study, we obtained evidence that Lmx1a and Lmx1b cooperate with Foxa2 to specify mesDA neuron identity by gain-of-function approaches using transgenic mice. Lmx1a/b appeared to select a mesDA fate by suppressing red nucleus fate in the context of Foxa2-positive progenitors, at least in part, through repressing the Sim1-Lhx1 and Ngn1 pathways that inhibit proper mesDA differentiation. We also found that, in the mesencephalon, FP cell fate is primarily determined by Foxa2 with a supportive action of Lmx1a/b through repressing Nkx6.1, which inhibits FP cell differentiation. Thus, FP and mesDA identities are determined by distinct specification pathways, both of which are controlled by the same combination of transcription factors, Lmx1a/b and Foxa2, and, as a consequence, mesDA neurons are generated from mesencephalic FP cells.

Purkinje cells originate from cerebellar ventricular zone progenitors positive for Neph3 and E-cadherin.

GABAergic Purkinje cells (PCs) provide the primary output from the cerebellar cortex, which controls movement and posture. Although the mechanisms of PC differentiation have been well studied, the precise origin and initial specification mechanism of PCs remain to be clarified. Here, we identified a cerebellar and spinal cord GABAergic progenitor-selective cell surface marker, Neph3, which is a direct downstream target gene of Ptf1a, an essential regulator of GABAergic neuron development. Using FACS, Neph3(+) GABAergic progenitors were sorted from the embryonic cerebellum, and the cell fate of this population was mapped by culturing in vitro. We found that most of the Neph3(+) populations sorted from the mouse E12.5 cerebellum were fated to differentiate into PCs while the remaining small fraction of Neph3(+) cells were progenitors for Pax2(+) interneurons, which are likely to be deep cerebellar nuclei GABAergic neurons. These results were confirmed by short-term in vivo lineage-tracing experiments using transgenic mice expressing Neph3 promoter-driven GFP. In addition, we identified E-cadherin as a marker selectively expressed by a dorsally localized subset of cerebellar Neph3(+) cells. Sorting experiments revealed that the Neph3(+) E-cadherin(high) population in the embryonic cerebellum defined PC progenitors while progenitors for Pax2(+) interneurons were enriched in the Neph3(+) E-cadherin(low) population. Taken together, our results identify two spatially demarcated subregions that generate distinct cerebellar GABAergic subtypes and reveal the origin of PCs in the ventricular zone of the cerebellar primordium.

Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells.

Directed differentiation and purification of mesencephalic dopaminergic (mesDA) neurons from stem cells are crucial issues for realizing safe and efficient cell transplantation therapies for Parkinson's disease. Although recent studies have identified the factors that regulate mesDA neuron development, the mechanisms underlying mesDA neuron specification are not fully understood. Recently, it has been suggested that mesencephalic floor plate (FP) cells acquire neural progenitor characteristics to generate mesDA neurons. Here, we directly examined this in a fate mapping experiment using fluorescence-activated cell sorting (FACS) with an FP cell-specific surface marker, and demonstrate that mesencephalic FP cells have neurogenic activity and generate mesDA neurons in vitro. By contrast, sorted caudal FP cells have no neurogenic potential, as previously thought. Analysis of dreher mutant mice carrying a mutation in the Lmx1a locus and transgenic mice ectopically expressing Otx2 in caudal FP cells demonstrated that Otx2 determines anterior identity that confers neurogenic activity to FP cells and specifies a mesDA fate, at least in part through the induction of Lmx1a. We further show that FACS can isolate mesDA progenitors, a suitable transplantation material, from embryonic stem cell-derived neural cells. Our data provide insights into the mechanisms of specification and generation of mesDA neurons, and illustrate a useful cell replacement approach for Parkinson's disease.

Helt determines GABAergic over glutamatergic neuronal fate by repressing Ngn genes in the developing mesencephalon.

The mechanism underlying the determination of neurotransmitter phenotype in the developing mesencephalon, particularly GABAergic versus glutamatergic fate, remains largely unknown. Here, we show in mice that the basic helix-loop-helix transcriptional repressor gene Helt (also known as Megane and Heslike) functions as a selector gene that determines GABAergic over glutamatergic fate in the mesencephalon. Helt was coincidently expressed in all the progenitor domains for mesencephalic GABAergic neurons. In the mesencephalon of Helt-deficient embryos, GABAergic neurons were mostly absent and glutamatergic neurons emerged instead. Conversely, ectopically expressed Helt suppressed glutamatergic formation and induced GABAergic neurogenesis. However, the Helt mutants showed normal progenitor domain formation. In consequence, postmitotic expression of the homeodomain factor Nkx2.2, which was specifically expressed by GABAergic populations in wild-type embryos, was maintained despite the transmitter phenotype conversion from GABAergic to glutamatergic in the Helt mutants, suggesting that Helt is not involved in neuronal identity specification. Furthermore, we identified proneural genes Ngn1 and Ngn2, which were selectively expressed in glutamatergic progenitors in the developing mesencephalon and had the ability to confer the glutamatergic fate, as downstream target genes of Helt. These results suggest that Helt determines GABAergic over glutamatergic fate, at least in part, by repressing Ngn (Neurog) genes and that basic helix-loop-helix transcription factor networks involving Helt and Ngns are commonly used in the mesencephalon for determination of the GABAergic versus glutamatergic transmitter phenotype.