Dynamic temporal requirement of Wnt1 in midbrain dopamine neuron development.

Wnt1-expressing progenitors generate midbrain dopamine (MbDA) and cerebellum (Cb) neurons in distinct temporal windows and from spatially discrete progenitor domains. It has been shown that Wnt1 and Lmx1a participate in a cross-regulatory loop that is utilized during MbDA neuron development. However, Wnt1 expression dynamically changes over time and precedes that of Lmx1a. The spatial and temporal requirements of Wnt1 in development and specifically its requirement for MbDA neurons remain to be determined. To address these issues, we generated a conditional Wnt1 allele and temporally deleted Wnt1 coupled with genetic lineage analysis. Using this approach, we show that patterning of the midbrain (Mb) and Cb by Wnt1 occurs between the one-somite and the six- to eight-somite stages and is solely dependent on Wnt1 function in the Mb, but not in the Cb. Interestingly, an En1-derived domain persists after the early deletion of Wnt1 and mutant cells express OTX2. However, the En1-derived Wnt1-mutant domain does not contain LMX1a-expressing progenitors, and MbDA neurons are depleted. Thus, we demonstrate an early requirement of Wnt1 for all MbDA neurons. Subsequently, we deleted Wnt1 in the ventral Mb and show a continued late requirement for Wnt1 in MbDA neuron development, but not in LMX1a-expressing progenitors. Specifically, Wnt1 deletion disrupts the birthdating of MbDA neurons and causes a depletion of MbDA neurons positioned medially and a concomitant expansion of MbDA neurons positioned laterally during embryogenesis. Collectively, our analyses resolve the spatial and temporal function of Wnt1 in Mb and Cb patterning and in MbDA neuron development in vivo.

Molecular organization and timing of Wnt1 expression define cohorts of midbrain dopamine neuron progenitors in vivo.

Midbrain dopamine (MbDA) neurons are functionally heterogeneous and modulate complex functions through precisely organized anatomical groups. MbDA neurons are generated from Wnt1-expressing progenitors located in the ventral mesencephalon (vMes) during embryogenesis. However, it is unclear whether the progenitor pool is partitioned into distinct cohorts based on molecular identity and whether the timing of gene expression uniquely identifies subtypes of MbDA neurons. In this study we show that Wnt1-expressing MbDA progenitors from embryonic day (E)8.5-12.5 have dynamic molecular identities that correlate with specific spatial locations in the vMes. We also tested the hypothesis that the timing of Wnt1 expression in progenitors is related to the distribution of anatomically distinct cohorts of adult MbDA neurons using genetic inducible fate mapping (GIFM). We demonstrate that the Wnt1 lineage contributes to specific cohorts of MbDA neurons during a 7-day epoch and that the contribution to MbDA neurons predominates over other ventral Mb domains. In addition, we show that calbindin-, GIRK2-, and calretinin-expressing MbDA neuron subtypes are derived from Wnt1-expressing progenitors marked over a broad temporal window. Through GIFM and quantitative analysis we demonstrate that the Wnt1 lineage does not undergo progressive lineage restriction, which eliminates a restricted competence model of generating MbDA diversity. Interestingly, we uncover that two significant peaks of Wnt1 lineage contribution to MbDA neurons occur at E9.5 and E11.5. Collectively, our findings delineate the temporal window of MbDA neuron generation and show that lineage and timing predicts the terminal distribution pattern of MbDA neurons.

Adult-born hippocampal neurons are more numerous, faster maturing, and more involved in behavior in rats than in mice.

Neurons are born throughout adulthood in the hippocampus and show enhanced plasticity compared with mature neurons. However, there are conflicting reports on whether or not young neurons contribute to performance in behavioral tasks, and there is no clear relationship between the timing of maturation of young neurons and the duration of neurogenesis reduction in studies showing behavioral deficits. We asked whether these discrepancies could reflect differences in the properties of young neurons in mice and rats. We report that young neurons in adult rats show a mature neuronal marker profile and activity-induced immediate early gene expression 1-2 weeks earlier than those in mice. They are also twice as likely to escape cell death, and are 10 times more likely to be recruited into learning circuits. This comparison holds true in two different strains of mice, both of which show high rates of neurogenesis relative to other background strains. Differences in adult neurogenesis are not limited to the hippocampus, as the density of new neocortical neurons was 5 times greater in rats than in mice. Finally, in a test of function, we find that the contribution of young neurons to fear memory is much greater in rats than in mice. These results reveal substantial differences in new neuron plasticity and function between these two commonly studied rodent species.

Comparative analysis of conditional reporter alleles in the developing embryo and embryonic nervous system.

A long-standing problem in development is understanding how progenitor cells transiently expressing genes contribute to complex anatomical and functional structures. In the developing nervous system an additional level of complexity arises when considering how cells of distinct lineages relate to newly established neural circuits. To address these problems, we used both cumulative marking with Cre/loxP and Genetic Inducible Fate Mapping (GIFM), which permanently and heritably marks small populations of progenitors and their descendants with fine temporal control using CreER/loxP. A key component used in both approaches is a conditional phenotyping allele that has the potential to be expressed in all cell types, but is quiescent because of a loxP flanked Stop sequence, which precedes a reporter allele. Upon recombination, the resulting phenotyping allele is 'turned on' and then constitutively expressed. Thus, the reporter functions as a high fidelity genetic lineage tracer in vivo. Currently there is an array of reporter alleles that can be used in marking strategies, but their recombination efficiency and applicability to a wide array of tissues has not been thoroughly described. To assess the recombination/marking potential of the reporters, we utilized CreER(T) under the control of a Wnt1 transgene (Wnt1-CreER(T)) as well as a cumulative, non-inducible En1(Cre) knock-in line in combination with three different reporters: R26R (LacZ reporter), Z/EG (EGFP reporter), and Tau-Lox-STOP-Lox-mGFP-IRES-NLS-LacZ (membrane-targeted GFP/nuclear LacZ reporter). We marked the Wnt1 lineage using each of the three reporters at embryonic day (E) 8.5 followed by analysis at E10.0, E12.5, and in the adult. We also compared cumulative marking of cells with a history of En1 expression at the same stages. We evaluated the reporters by whole-mount and section analysis and ascertained the strengths and weaknesses of each of the reporters. Comparative analysis with the reporters elucidated complexities of how the Wnt1 and En1 lineages contribute to developing embryos and to axonal projection patterns of neurons derived from these lineages.

A practical approach to genetic inducible fate mapping: a visual guide to mark and track cells in vivo.

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage. GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreER(T)). CreER(T) contains a modified estrogen receptor ligand binding domain which renders CreER(T) sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreER(T), which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP. Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreER(T) release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreER(T). We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders. This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreER(T);mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.