Genetic Inducible Fate Mapping Research Articles

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.

Sonic hedgehog is required in pulmonary endoderm for atrial septation

The genesis of the septal structures of the mammalian heart is central to understanding the ontogeny of congenital heart disease and the evolution of cardiac organogenesis. We found that Hedgehog (Hh) signaling marked a subset of cardiac progenitors specific to the atrial septum and the pulmonary trunk in the mouse. Using genetic inducible fate mapping with Gli1(CreERT2), we marked Hh-receiving progenitors in anterior and posterior second heart field splanchnic mesoderm between E8 and E10. In the inflow tract, Hh-receiving progenitors migrated from the posterior second heart field through the dorsal mesocardium to form the atrial septum, including both the primary atrial septum and dorsal mesenchymal protrusion (DMP). In the outflow tract, Hh-receiving progenitors migrated from the anterior second heart field to populate the pulmonary trunk. Abrogation of Hh signaling during atrial septal progenitor specification resulted in atrial and atrioventricular septal defects and hypoplasia of the developing DMP. Hedgehog signaling appeared necessary and sufficient for atrial septal progenitor fate: Hh-receiving cells rendered unresponsive to the Hh ligand migrated into the atrium in normal numbers but populated the atrial free wall rather than the atrial septum. Conversely, constitutive activation of Hh signaling caused inappropriate enlargement of the atrial septum. The close proximity of posterior second heart field cardiac progenitors to pulmonary endoderm suggested a pulmonary source for the Hh ligand. We found that Shh is required in the pulmonary endoderm for atrial septation. Therefore, Hh signaling from distinct pulmonary and pharyngeal endoderm is required for inflow and outflow septation, respectively. These data suggest a model in which respiratory endoderm patterns the morphogenesis of cardiac structural components required for efficient cardiopulmonary circulation.

Characterization of Nkx6-2-Derived Neocortical Interneuron Lineages

Ventral telencephalic progenitors expressing the homeodomain transcription factor Nkx6-2 have been shown to give rise to a multitude of cortical interneuron subtypes usually associated with origin in either the medial ganglionic eminence or the caudal ganglionic eminence. The function of Nkx6-2 in directing the fate of those progenitors has, however, not been thoroughly analyzed. We used a combination of genetic inducible fate mapping and in vivo loss-of-function to analyze the requirement of Nkx6-2 in determining the fate of cortical interneurons. We have found that interneuron subtypes are born with a characteristic temporal pattern. Furthermore, we extend the characterization of interneurons from the Nkx6-2 lineage through the application of electrophysiological methods. Analysis of these populations in Nkx6-2 null mice suggests that there is a small and partially penetrant loss of delayed non-fast spiking somatostatin/calretinin double positive cortical interneurons in the absence of Nkx6-2 gene function.

Embryonic origins of ZebrinII parasagittal stripes and establishment of topographic Purkinje cell projections

The establishment of neural circuits involves both the precise positioning of cells within brain regions and projection of axons to specific target cells. In the cerebellum (Cb), the medial–lateral (M-L) and anterior–posterior (A-P) position of each Purkinje cell (PC) and the topography of its axon can be defined with respect to two coordinate systems within the Cb; one based on the pattern of lobules and the other on PC gene expression in parasagittal clusters in the embryo (e.g. Pcp2) and stripes in the adult (e.g. ZebrinII). The relationship between the embryonic clusters of molecularly defined PCs and particular adult PC stripes is not clear. Using a mouse genetic inducible fate mapping (GIFM) approach and a Pcp2-CreER-IRES-hAP transgene, we marked three bilateral clusters of PC clusters with myristolated green fluorescent protein (mGfp) on approximately embryonic day (E) 15 and followed their fate into adulthood. We found that these three clusters contributed specifically to ZebrinII-expressing PCs, including nine of the adult stripes. This result suggests that embryonic PCs maintain a particular molecular identity, and that each embryonic cluster can contribute PCs to more than one adult M-L stripe. Each PC projects a primary axon to one of the deep cerebellar nuclei (DCN) or the vestibular nuclei in the brainstem in an organized fashion that relates to the position of the PCs along the M-L axis. We characterized when PC axons from the three M-L clusters acquire topographic projections. Using a combination of GIFM to mark the PC clusters with mGfp and staining for human placental alkaline phosphatase (hAP) in Pcp2-CreER-IRES-hAP transgenic embryos we found that axons from each embryonic PC cluster intermingled with neurons within particular DCN or projected out of the Cb toward the vestibular nuclei by E14.5. These studies show that PC molecular patterning, efferent circuitry, and DCN nucleogenesis occur simultaneously, suggesting a link between these processes.

Hedgehog signaling plays a cell-autonomous role in maximizing cardiac developmental potential

Elucidation of the complete roster of signals required for myocardial specification is crucial to the future of cardiac regenerative medicine. Prior studies have implicated the Hedgehog (Hh) signaling pathway in the regulation of multiple aspects of heart development. However, our understanding of the contribution of Hh signaling to the initial specification of myocardial progenitor cells remains incomplete. Here, we show that Hh signaling promotes cardiomyocyte formation in zebrafish. Reduced Hh signaling creates a cardiomyocyte deficit, and increased Hh signaling creates a surplus. Through fate-mapping, we find that Hh signaling is required at early stages to ensure specification of the proper number of myocardial progenitors. Genetic inducible fate mapping in mouse indicates that myocardial progenitors respond directly to Hh signals, and transplantation experiments in zebrafish demonstrate that Hh signaling acts cell autonomously to promote the contribution of cells to the myocardium. Thus, Hh signaling plays an essential early role in defining the optimal number of cardiomyocytes, making it an attractive target for manipulation of multipotent progenitor cells.

Genetic approaches identify adult pituitary stem cells

Adult tissues undergo continuous cell turnover in response to stress, damage, or physiological demand. New differentiated cells are generated from dedicated or facultative stem cells or from self-renewing differentiated cells. Here we describe a different stem cell strategy for tissue maintenance, distinct from that observed for dedicated or facultative stem cells. We report the presence of nestin-expressing adult stem cells in the perilumenal region of the mature anterior pituitary and, using genetic inducible fate mapping, demonstrate that they serve to generate subsets of all six terminally differentiated endocrine cell types of the pituitary gland. These stem cells, while not playing a significant role in organogenesis, undergo postnatal expansion and start producing differentiated progeny, which colonize the organ that initially entirely consisted of differentiated cells derived from embryonic precursors. This generates a mosaic organ with two phenotypically similar subsets of endocrine cells that have different origins and different life histories. These parallel but distinct lineages of differentiated cells in the gland may help the maturing organism adapt to changes in the metabolic regulatory landscape.

The Distinct Temporal Origins of Olfactory Bulb Interneuron Subtypes

Olfactory bulb (OB) interneurons are a heterogeneous population produced beginning in embryogenesis and continuing through adulthood. Understanding how this diversity arises will provide insight into how OB microcircuitry is established as well as adult neurogenesis. Particular spatial domains have been shown to contribute specific interneuron subtypes. However, the temporal profile by which OB interneuron subtypes are produced is unknown. Using inducible genetic fate mapping of Dlx1/2 precursors, we analyzed the production of seven OB interneuron subtypes and found that the generation of each subpopulation has a unique temporal signature. Within the glomerular layer, the production of tyrosine hydroxylase-positive interneurons is maximal during early embryogenesis and decreases thereafter. In contrast, the generation of calbindin interneurons is maximal during late embryogenesis and declines postnatally, whereas calretinin (CR) cell production is low during embryogenesis and increases postnatally. Parvalbumin interneurons within the external plexiform layer are produced only perinatally, whereas the generation of 5T4-positive granule cells in the mitral cell layer does not change significantly over time. CR-positive granule cells are not produced at early embryonic time points, but constitute a large percentage of the granule cells born after birth. Blanes cells in contrast are produced in greatest number during embryogenesis. Together we provide the first comprehensive analysis of the temporal generation of OB interneuron subtypes and demonstrate that the timing by which these populations are produced is tightly orchestrated.

Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers

BackgroundThe cerebellum has a striking morphology consisting of folia separated by fissures of different lengths. Since folia in mammals likely serve as a broad platform on which the anterior-posterior organization of the sensory-motor circuits of the cerebellum are built, it is important to understand how such complex morphology arises.ResultsUsing a combination of genetic inducible fate mapping, high-resolution cellular analysis and mutant studies in mouse, we demonstrate that a key event in initiation of foliation is the acquisition of a distinct cytoarchitecture in the regions that will become the base of each fissure. We term these regions 'anchoring centers'. We show that the first manifestation of anchoring centers when the cerebellar outer surface is smooth is an increase in proliferation and inward thickening of the granule cell precursors, which likely causes an associated slight invagination of the Purkinje cell layer. Thereafter, granule cell precursors within anchoring centers become distinctly elongated along the axis of the forming fissure. As the outer cerebellar surface begins to fold inwards, Bergmann glial fibers radiate in towards the base of the immature fissure in a fan shape. Once the anchoring center is formed, outgrowth of folia seems to proceed in a self-sustaining manner driven by granule cell migration along Bergmann glial fibers. Finally, by analyzing a cerebellum foliation mutant (Engrailed 2), we demonstrate that changing the timing of anchoring center formation leads to predictable changes in the shape and size of the surrounding folia.ConclusionWe present a new cellular model of the initial formation of cerebellar fissures with granule cells providing the driving physical force. Both the precise timing of the appearance of anchoring centers at the prospective base of each fissure and the subsequent coordinated action of granule cells and Bergmann glial fibers within the anchoring centers dictates the shape of the folia.

Genetic inducible fate mapping in mouse: Establishing genetic lineages and defining genetic neuroanatomy in the nervous system

A fascinating aspect of developmental biology is how organs are assembled in three dimensions over time. Fundamental to understanding organogenesis is the ability to determine when and where specific cell types are generated, the lineage of each cell, and how cells move to reside in their final position. Numerous methods have been developed to mark and follow the fate of cells in various model organisms used by developmental biologists, but most are not readily applicable to mouse embryos in utero because they involve physical marking of cells through injection of tracers. The advent of sophisticated transgenic and gene targeting techniques, combined with the use of site-specific recombinases, has revolutionized fate mapping studies in mouse. Furthermore, using genetic fate mapping to mark cells has opened up the possibility of addressing fundamental questions that cannot be studied with traditional methods of fate mapping in other organisms. Specifically, genetic fate mapping allows both the relationship between embryonic gene expression and cell fate (genetic lineage) to be determined, as well as the link between gene expression domains and anatomy (genetic anatomy) to be established. In this review, we present the ever-evolving development of genetic fate mapping techniques in mouse, especially the recent advance of Genetic Inducible Fate Mapping. We then review recent studies in the nervous system (focusing on the anterior hindbrain) as well as in the limb and with adult stem cells to highlight fundamental developmental processes that can be discovered using genetic fate mapping approaches. We end with a look toward the future at a powerful new approach that combines genetic fate mapping with cellular phenotyping alleles to study cell morphology, physiology, and function using examples from the nervous system.

Morphogenetic and Cellular Movements that Shape the Mouse Cerebellum

We used the cerebellum as a model to study the morphogenetic and cellular processes underlying the formation of elaborate brain structures from a simple neural tube, using an inducible genetic fate mapping approach in mouse. We demonstrate how a 90 degrees rotation between embryonic days 9 and 12 converts the rostral-caudal axis of dorsal rhombomere 1 into the medial-lateral axis of the wing-like bilateral cerebellar primordium. With the appropriate use of promoters, we marked specific medial-lateral domains of the cerebellar primordium and derived a positional fate map of the murine cerebellum. We show that the adult medial cerebellum is produced by expansion, rather than fusion, of the thin medial primordium. Furthermore, ventricular-derived cells maintain their original medial-lateral coordinates into the adult, whereas rhombic lip-derived granule cells undergo lateral to medial posterior transverse migrations during foliation. Thus, we show that progressive changes in the axes of the cerebellum underlie its genesis.