Genetic Inducible Fate Mapping Research Articles

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.

Lineage tracing and genetic ablation of ADAM12+ perivascular cells identify a major source of profibrotic cells during acute tissue injury

Profibrotic cells that develop upon injury generate permanent scar tissue and impair organ recovery, though their origin and fate are unclear. Here we show that transient expression of ADAM12 (a disintegrin and metalloprotease 12) identifies a distinct proinflammatory subset of platelet-derived growth factor receptor-α-positive stromal cells that are activated upon acute injury in the muscle and dermis. By inducible genetic fate mapping, we demonstrate in vivo that injury-induced ADAM12(+) cells are specific progenitors of a major fraction of collagen-overproducing cells generated during scarring, which are progressively eliminated during healing. Genetic ablation of ADAM12(+) cells, or knockdown of ADAM12, is sufficient to limit generation of profibrotic cells and interstitial collagen accumulation. ADAM12(+) cells induced upon injury are developmentally distinct from muscle and skin lineage cells and are derived from fetal ADAM12(+) cells programmed during vascular wall development. Thus, our data identify injury-activated profibrotic progenitors residing in the perivascular space that can be targeted through ADAM12 to limit tissue scarring.

Dynamic Changes in Interneuron Morphophysiological Properties Mark the Maturation of Hippocampal Network Activity

During early postnatal development, neuronal networks successively produce various forms of spontaneous patterned activity that provide key signals for circuit maturation. Initially, in both rodent hippocampus and neocortex, coordinated activity emerges in the form of synchronous plateau assemblies (SPAs) that are initiated by sparse groups of gap-junction-coupled oscillating neurons. Subsequently, SPAs are replaced by synapse-driven giant depolarizing potentials (GDPs). Whether these sequential changes in mechanistically distinct network activities correlate with modifications in single-cell properties is unknown. To determine this, we studied the morphophysiological fate of single SPA cells as a function of development. We focused on CA3 GABAergic interneurons, which are centrally involved in generating GDPs in the hippocampus. As the network matures, GABAergic neurons are engaged more in GDPs and less in SPAs. Using inducible genetic fate mapping, we show that the individual involvement of GABAergic neurons in SPAs is correlated to their temporal origin. In addition, we demonstrate that the SPA-to-GDP transition is paralleled by a remarkable maturation in the morphophysiological properties of GABAergic neurons. Compared with those involved in GDPs, interneurons participating in SPAs possess immature intrinsic properties, receive synaptic inputs spanning a wide amplitude range, and display large somata as well as membrane protrusions. Thus, a developmental switch in the morphophysiological properties of GABAergic interneurons as they progress from SPAs to GDPs marks the emergence of synapse-driven network oscillations.

Abstract SY37-03: Targeting developmental pathways in colon cancer cells and stem cells

Abstract The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base. In the absence of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Interestingly, both stem cell compartments can serve as cell of origin for the development of colorectal cancer in mice. The Pathways that regulate self-renewal, commitment as well as the interplay between these two populations remain to be clearly defined but are likely to involve Wnt Notch Hedgehog. The Notch, Wnt and Hedgehog signaling pathways play critical roles during embryonic development. These factors modulate proliferation or differentiation of numerous cell types and are also involved in the regulation of the self-renewal and/or differentiation of embryonic and adult stem cells. It is however becoming increasingly clear that these pathways are involved in tumorigenesis when reactivated in adult tissues through mutations or overexpression of pathway components. Aberrant reactivation of the Hedgehog (Hh) pathway in adult tissues can lead for example to the development of cancers such as basal cell carcinoma (BCC) and medulloblastoma is often the result of inactivating mutations in PATCHED (PTCH) or activating SMOOTHENED (SMO) mutations. Notch1 mutations are found in most T-cell acute lymphoblastic leukemias and activation of the Wnt pathway, often through APC (adenomatous polyposis coli) gene mutations, is observed in a majority of colon adenocarcinomas. However all 3 pathways play a role in normal gut homeostasis and may be involved in some aspect of colon tumorigenesis. In addition to the development of agents targeting these pathways in tumors where they are mutated we will discuss the effect on inhibiting Wnt, Notch and Hedgehog signaling on stem cells in the normal intestine and in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY37-03. doi:1538-7445.AM2012-SY37-03

Regeneration of Amputated Zebrafish Fin Rays from De Novo Osteoblasts

Determining the cellular source of new skeletal elements is critical for understanding appendage regeneration in amphibians and fish. Recent lineage-tracing studies indicated that zebrafish fin ray bone regenerates through the dedifferentiation and proliferation of spared osteoblasts, with limited if any contribution from other cell types. Here, we examined the requirement for this mechanism by using genetic ablation techniques to destroy virtually all skeletal osteoblasts in adult zebrafish fins. Animals survived this injury and restored the osteoblast population within 2 weeks. Moreover, amputated fins depleted of osteoblasts regenerated new fin ray structures at rates indistinguishable from fins possessing a resident osteoblast population. Inducible genetic fate mapping confirmed that new bone cells do not arise from dedifferentiated osteoblasts under these conditions. Our findings demonstrate diversity in the cellular origins of appendage bone and reveal that de novo osteoblasts can fully support the regeneration of amputated zebrafish fins.

Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions

BackgroundThe hypothalamus is a brain region with essential functions for homeostasis and energy metabolism, and alterations of its development can contribute to pathological conditions in the adult, like hypertension, diabetes or obesity. However, due to the anatomical complexity of the hypothalamus, its development is not well understood. Sonic hedgehog (Shh) is a key developmental regulator gene expressed in a dynamic pattern in hypothalamic progenitor cells. To obtain insight into hypothalamic organization, we used genetic inducible fate mapping (GIFM) to map the lineages derived from Shh-expressing progenitor domains onto the four rostrocaudally arranged hypothalamic regions: preoptic, anterior, tuberal and mammillary.ResultsShh-expressing progenitors labeled at an early stage (before embryonic day (E)9.5) contribute neurons and astrocytes to a large caudal area including the mammillary and posterior tuberal regions as well as tanycytes (specialized median eminence glia). Progenitors labeled at later stages (after E9.5) give rise to neurons and astrocytes of the entire tuberal region and in particular the ventromedial nucleus, but not to cells in the mammillary region and median eminence. At this stage, an additional Shh-expressing domain appears in the preoptic area and contributes mostly astrocytes to the hypothalamus. Shh-expressing progenitors do not contribute to the anterior region at any stage. Finally, we show a gradual shift from neurogenesis to gliogenesis, so that progenitors expressing Shh after E12.5 generate almost exclusively hypothalamic astrocytes.ConclusionsWe define a fate map of the hypothalamus, based on the dynamic expression of Shh in the hypothalamic progenitor zones. We provide evidence that the large neurogenic Shh-expressing progenitor domains of the ventral diencephalon are continuous with those of the midbrain. We demonstrate that the four classical transverse zones of the hypothalamus have clearly defined progenitor domains and that there is little or no cell mixing between the tuberal and anterior or the preoptic and anterior hypothalamus. Finally, we show that, in the tuberal hypothalamus, neurons destined for every mediolateral level are produced during a period of days, in conflict with the current 'three-wave' model of hypothalamic neurogenesis. Our work sets the stage for a deeper developmental analysis of this complex and important brain region.

Wnt1 expression temporally allocates upper rhombic lip progenitors and defines their terminal cell fate in the cerebellum

The cerebellum (Cb) controls movement related physiology using a diverse array of morphologically and biochemically distinct neurons. During development, the Cb is derived from rhombomere 1 (r1), an embryonic compartment patterned by a signaling center referred to as the isthmus organizer. The secreted glycoprotein WNT1 is expressed in the midbrain primordia (mesencephalon, mes) and at the posterior limit of the mes. WNT1 plays a pivotal role in maintaining the isthmus organizer and mutations in Wnt1 produce severe Cb defects that are generally attributed to aberrant organizer activity. Interestingly, Wnt1 is also expressed at the most posterior limit of dorsal r1, in a region known as the upper rhombic lip (URL). However, the distribution and molecular identity of Wnt1 expressing progenitors have not been carefully described in r1. We used Wnt1-Venus transgenic mice to generate a molecular map of Wnt1 expressing progenitors in relation to other well characterized Cb biomarkers such as MATH1 (ATOH1), LMX1a and OTX2. Our analysis validated Wnt1 expression in the URL and revealed molecularly-defined developmental zones in r1. We then used genetic inducible fate mapping (GIFM) to link transient Wnt1 expression in r1 to terminal cell fates in the mature Cb. Wnt1 expressing progenitors primarily contributed to neurons in deep cerebellar nuclei, granule cells, and unipolar brush cells in distinct but overlapping temporal windows and sparsely contributed to inhibitory neurons and Bergmann glia. We further demonstrate that the Wnt1 lineage does not follow a competency model of progressive lineage restriction to generate the Cb or the functionally related precerebellar system. Instead, progenitors initiate Wnt1 expression de novo to give rise to each Cb cell type and precerebellar nuclei. We also used GIFM to determine how the temporal control of Wnt1 expression is related to molecular identity and cell migration in Cb development. Our findings provide new insight into how lineage and timing establish cell diversity within the Cb system.

A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable

The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base. However, the relative functions of these two pools and their interrelationship are not understood. Here we specifically ablated Lgr5-expressing cells in mice using a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for the elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis, and that in the absence of these cells, Bmi1-expressing cells can serve as an alternative stem cell pool. These data provide the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions.

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.

Timing of Sonic hedgehog and Gli1 expression segregates midbrain dopamine neurons

The ventral midbrain (vMb) is organized into distinct anatomical domains and contains cohorts of functionally distinct subtypes of midbrain dopamine (mDA) neurons. We tested the hypothesis that genetic history and timing of gene expression within mDA neuron progenitors impart spatial diversity. Using genetic inducible fate mapping to mark the Sonic hedgehog (Shh) and Gli1 lineages at varying embryonic stages, we performed a quantitative and qualitative comparison of the two lineages' contribution to the mDA neuron domains. Dynamic changes in Shh and Gli1 expression in the vMb primordia delineated their spatial contribution to the embryonic day 12.5 vMb: Both lineages first contributed to the medial domain, but subsequently the Gli1 lineage exclusively contributed to the lateral vMb while the Shh lineage expanded more broadly across the vMb. The contribution of both lineages to the differentiated mDA neuron domain was initially biased anteriorly and became more uniform across the anterior/posterior vMb throughout development. Our findings demonstrate that the early Shh and Gli1 lineages specify mDA neurons of the substantia nigra pars compacta while the late Shh and Gli1 lineages maintain their progenitor state longer in the posterior vMb to extend the production of mDA neurons in the ventral tegmental area. Together, our study demonstrates that the timing of gene expression along with the genetic lineage (Shh or Gli1) within the neural progenitors segregate mDA neurons into distinct spatial domains.

Ascl1 Genetics Reveals Insights into Cerebellum Local Circuit Assembly

Two recently generated targeted mouse alleles of the neurogenic gene Ascl1 were used to characterize cerebellum circuit formation. First, genetic inducible fate mapping (GIFM) with an Ascl1(CreER) allele was found to specifically mark all glial and neuron cell types that arise from the ventricular zone (vz). Moreover, each cell type has a unique temporal profile of marking with Ascl1(CreER) GIFM. Of great utility, Purkinje cells (Pcs), an early cohort of Bergmann glia, and four classes of GABAergic interneurons can be genetically birth dated during embryogenesis using Ascl1(CreER) GIFM. Astrocytes and oligodendrocytes, in contrast, express Ascl1(CreER) throughout their proliferative phase in the white matter. Interestingly, the final position each neuron type acquires differs depending on when it expresses Ascl1. Interneurons (including candelabrum) attain a more outside position the later they express Ascl1, whereas Pcs have distinct settling patterns each day they express Ascl1. Second, using a conditional Ascl1 allele, we discovered that Ascl1 is differentially required for generation of most vz-derived cells. Mice lacking Ascl1 in the cerebellum have a major decrease in three types of interneurons with a tendency toward a loss of later-born interneurons, as well as an imbalance of oligodendrocytes and astrocytes. Double-mutant analysis indicates that a related helix-loop-helix protein, Ptf1a, functions with Ascl1 in generating interneurons and Pcs. By fate mapping vz-derived cells in Ascl1 mutants, we further discovered that Ascl1 plays a specific role during the time period when Pcs are generated in restricting vz progenitors from becoming rhombic lip progenitors.

Ascl1 genetics reveals insights into cerebellum local circuit assembly

Two recently generated targeted mouse alleles of the neurogenic gene Ascl1 were used to characterize cerebellum circuit formation. First, genetic inducible fate mapping (GIFM) with an Ascl1(CreER) allele was found to specifically mark all glial and neuron cell types that arise from the ventricular zone (vz). Moreover, each cell type has a unique temporal profile of marking with Ascl1(CreER) GIFM. Of great utility, Purkinje cells (Pcs), an early cohort of Bergmann glia, and four classes of GABAergic interneurons can be genetically birth dated during embryogenesis using Ascl1(CreER) GIFM. Astrocytes and oligodendrocytes, in contrast, express Ascl1(CreER) throughout their proliferative phase in the white matter. Interestingly, the final position each neuron type acquires differs depending on when it expresses Ascl1. Interneurons (including candelabrum) attain a more outside position the later they express Ascl1, whereas Pcs have distinct settling patterns each day they express Ascl1. Second, using a conditional Ascl1 allele, we discovered that Ascl1 is differentially required for generation of most vz-derived cells. Mice lacking Ascl1 in the cerebellum have a major decrease in three types of interneurons with a tendency toward a loss of later-born interneurons, as well as an imbalance of oligodendrocytes and astrocytes. Double-mutant analysis indicates that a related helix-loop-helix protein, Ptf1a, functions with Ascl1 in generating interneurons and Pcs. By fate mapping vz-derived cells in Ascl1 mutants, we further discovered that Ascl1 plays a specific role during the time period when Pcs are generated in restricting vz progenitors from becoming rhombic lip progenitors.

The Lineage Contribution and Role of Gbx2 in Spinal Cord Development

BackgroundForging a relationship between progenitors with dynamically changing gene expression and their terminal fate is instructive for understanding the logic of how cell-type diversity is established. The mouse spinal cord is an ideal system to study these mechanisms in the context of developmental genetics and nervous system development. Here we focus on the Gastrulation homeobox 2 (Gbx2) transcription factor, which has not been explored in spinal cord development.Methodology/Principal FindingsWe determined the molecular identity of Gbx2-expressing spinal cord progenitors. We also utilized genetic inducible fate mapping to mark the Gbx2 lineage at different embryonic stages in vivo in mouse. Collectively, we uncover cell behaviors, cytoarchitectonic organization, and the terminal cell fate of the Gbx2 lineage. Notably, both ventral motor neurons and interneurons are derived from the Gbx2 lineage, but only during a short developmental period. Short-term fate mapping during mouse spinal cord development shows that Gbx2 expression is transient and is extinguished ventrally in a rostral to caudal gradient. Concomitantly, a permanent lineage restriction boundary ensures that spinal cord neurons derived from the Gbx2 lineage are confined to a dorsal compartment that is maintained in the adult and that this lineage generates inhibitory interneurons of the spinal cord. Using lineage tracing and molecular markers to follow Gbx2-mutant cells, we show that the loss of Gbx2 globally affects spinal cord patterning including the organization of interneuron progenitors. Finally, long-term lineage analysis reveals that the presence and timing of Gbx2 expression in interneuron progenitors results in the differential contribution to subtypes of terminally differentiated interneurons in the adult spinal cord.Conclusions/SignificanceWe illustrate the complex cellular nature of Gbx2 expression and lineage contribution to the mouse spinal cord. In a broader context, this study provides a direct link between spinal cord progenitors undergoing dynamic changes in molecular identity and terminal neuronal fate.

Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells

BackgroundWhile the diversity and spatio-temporal origin of olfactory bulb (OB) GABAergic interneurons has been studied in detail, much less is known about the subtypes of glutamatergic OB interneurons.ResultsWe studied the temporal generation and diversity of Neurog2-positive precursor progeny using an inducible genetic fate mapping approach. We show that all subtypes of glutamatergic neurons derive from Neurog2 positive progenitors during development of the OB. Projection neurons, that is, mitral and tufted cells, are produced at early embryonic stages, while a heterogeneous population of glutamatergic juxtaglomerular neurons are generated at later embryonic as well as at perinatal stages. While most juxtaglomerular neurons express the T-Box protein Tbr2, those generated later also express Tbr1. Based on morphological features, these juxtaglomerular cells can be identified as tufted interneurons and short axon cells, respectively. Finally, targeted electroporation experiments provide evidence that while the majority of OB glutamatergic neurons are generated from intrabulbar progenitors, a small portion of them originate from extrabulbar regions at perinatal ages.ConclusionsWe provide the first comprehensive analysis of the temporal and spatial generation of OB glutamatergic neurons and identify distinct populations of juxtaglomerular interneurons that differ in their antigenic properties and time of origin.

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.

Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing β-cells

The Notch-signaling pathway is known to be fundamental in controlling pancreas differentiation. We now report on using Cre-based fate mapping to indelibly label pancreatic Notch-responsive cells (PNCs) at larval stages and follow their fate in the adult pancreas. We show that the PNCs represent a population of progenitors that can differentiate to multiple lineages, including adult ductal cells, centroacinar cells (CACs) and endocrine cells. These endocrine cells include the insulin-producing β-cells. CACs are a functional component of the exocrine pancreas; however, our fate-mapping results indicate that CACs are more closely related to endocrine cells by lineage as they share a common progenitor. The majority of the exocrine pancreas consists of the secretory acinar cells; however, we only detect a very limited contribution of PNCs to acinar cells. To explain this observation we re-examined early events in pancreas formation. The pancreatic anlage that gives rise to the exocrine pancreas is located in the ventral gut endoderm (called the ventral bud). Ptf1a is a gene required for exocrine pancreas development and is first expressed as the ventral bud forms. We used transgenic marker lines to observe both the domain of cells expressing ptf1a and cells responding to Notch signaling. We do not detect any overlap in expression and demonstrate that the ventral bud consists of two cell populations: a ptf1-expressing domain and a Notch-responsive progenitor core. As pancreas organogenesis continues, the ventral bud derived PNCs align along the duct, remain multipotent and later in development differentiate to form secondary islets, ducts and CACs.

Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei

BackgroundThe ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express Sonic Hedgehog (Shh). However, Shh expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points.ResultsWe employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes.ConclusionsWe define a ventral midbrain precursor map based on the timing of Gli1 and Shh expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions.

The Mouse Homeobox GeneGbx2Is 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.

Genetic Fate Mapping Reveals That the Caudal Ganglionic Eminence Produces a Large and Diverse Population of Superficial Cortical Interneurons

By combining an inducible genetic fate mapping strategy with electrophysiological analysis, we have systematically characterized the populations of cortical GABAergic interneurons that originate from the caudal ganglionic eminence (CGE). Interestingly, compared with medial ganglionic eminence (MGE)-derived cortical interneuron populations, the initiation [embryonic day 12.5 (E12.5)] and peak production (E16.5) of interneurons from this embryonic structure occurs 3 d later in development. Moreover, unlike either pyramidal cells or MGE-derived cortical interneurons, CGE-derived interneurons do not integrate into the cortex in an inside-out manner but preferentially (75%) occupy superficial cortical layers independent of birthdate. In contrast to previous estimates, CGE-derived interneurons are both considerably greater in number (approximately 30% of all cortical interneurons) and diversity (comprised by at least nine distinct subtypes). Furthermore, we found that a large proportion of CGE-derived interneurons, including the neurogliaform subtype, express the glycoprotein Reelin. In fact, most CGE-derived cortical interneurons express either Reelin or vasoactive intestinal polypeptide. Thus, in conjunction with previous studies, we have now determined the spatial and temporal origins of the vast majority of cortical interneuron subtypes.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells <em>In Vivo</em>

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.