Mechanisms underlying the direct programming of mouse embryonic fibroblasts to thymic epithelial cells by FOXN1.

Thymic epithelial cells (TECs) are a critical functional component of the thymus's ability to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpressing the transcription factor FOXN1 initiates transdifferentiation of fibroblasts into TEC-like cells (iTECs) that support T cell differentiation in culture or after transplant. In this study, we characterized iTEC programming at the cellular and molecular level to determine how it proceeds and identified mechanisms that can be targeted for improving this process. These data showed that iTEC programming consisted of discrete gene expression changes that differed early and late in the process, and that iTECs upregulated markers of both cortical and medullary TEC (cTEC and mTEC) lineages. We demonstrated that promoting proliferation enhanced iTEC generation, and that Notch inhibition allowed induction of mTEC differentiation. Finally, we showed that MHCII expression was the major difference between iTECs and fetal TECs. MHCII expression was improved by co-culturing iTECs with fetal double-positive T-cells. This study supports future efforts to improve iTEC generation for both research and translational uses.

Foxn1 overexpression promotes thymic epithelial progenitor cell proliferation and mTEC maintenance, but does not prevent thymic involution.

The transcription factor FOXN1 is essential for fetal thymic epithelial cell (TEC) differentiation and proliferation. Postnatally, Foxn1 levels vary widely between TEC subsets, from low/undetectable in putative TEC progenitors to highest in differentiated TEC subsets. Correct Foxn1 expression is required to maintain the postnatal microenvironment; premature downregulation of Foxn1 causes a rapid involution-like phenotype, and transgenic overexpression can cause thymic hyperplasia and/or delayed involution. We investigated a K5.Foxn1 transgene that drives overexpression in mouse TECs, but causes neither hyperplasia nor delay or prevention of aging-related involution. Similarly, this transgene cannot rescue thymus size in Foxn1lacZ/lacZ mice, which undergo premature involution as a result of reduced Foxn1 levels. However, TEC differentiation and cortico-medullary organization are maintained with aging in both K5.Foxn1 and Foxn1lacZ/lacZ mice. Analysis of candidate TEC markers showed co-expression of progenitor and differentiation markers as well as increased proliferation in Plet1+ TECs associated with Foxn1 expression. These results demonstrate that the functions of FOXN1 in promoting TEC proliferation and differentiation are separable and context dependent, and suggest that modulating Foxn1 levels can regulate the balance of proliferation and differentiation in TEC progenitors.

The untapped potential of the GENSAT mice-A valuable resource for developmental biology.

Gene Expression Nervous System Atlas (GENSAT) transgenic mice express EGFP, tdTomato, or Cre recombinase in a wide range of cell types. The mice and the bacterial artificial chromosome transgenes are available from repositories (MMRRC or CHORI), thereby making these resources readily available to the research community. This resource of 1,386 transgenic lines was developed and validated for neuroscience research. However, GENSAT mice have many potential applications in other contexts including studies of development outside of the CNS. The cell type-specific expression of fluorescent proteins in these mice has been used to identify cells in living embryos, in living embryo explants, and in stem or progenitor cell populations in postnatal tissues. The large number of fluorescent protein driver lines generated by GENSAT greatly expands the range of cell type markers that can be used for live cell sorting. In addition, the GENSAT project has generated 278 new Cre driver lines. This review provides an overview of the GENSAT lines and information for identifying lines that may be useful for a particular application. I also provide a review of the few published cases in which GENSAT mice have been used for studies of embryonic development or analysis of stem/progenitor cells in nonneural tissues. genesis 54:245-256, 2016. © 2016 Wiley Periodicals, Inc.

SDF and GABA interact to regulate axophilic migration of GnRH neurons.

Stromal derived growth factor (SDF-1) and gamma-aminobutyric acid (GABA) are two extracellular cues that regulate the rate of neuronal migration during development and may act synergistically. The molecular mechanisms of this interaction are still unclear. Gonadotropin releasing hormone-1 (GnRH) neurons are essential for vertebrate reproduction. During development, these neurons emerge from the nasal placode and migrate through the cribriform plate into the brain. Both SDF-1 and GABA have been shown to regulate the rate of GnRH neuronal migration by accelerating and slowing migration, respectively. As such, this system was used to explore the mechanism by which these molecules act to produce coordinated cell movement during development. In the present study, GABA and SDF-1 are shown to exert opposite effects on the speed of cell movement by activating depolarizing or hyperpolarizing signaling pathways, GABA via changes in chloride and SDF-1 via changes in potassium. GABA and SDF-1 were also found to act synergistically to promote linear rather than random movement. The simultaneous activation of these signaling pathways, therefore, results in tight control of cellular speed and improved directionality along the migratory pathway of GnRH neurons.

Mouse and zebrafish Hoxa3 orthologues have nonequivalent in vivo protein function.

Hox genes play evolutionarily conserved roles in specifying axial position during embryogenesis. A prevailing paradigm is that changes in Hox gene expression drive evolution of metazoan body plans. Conservation of Hox function across species, and among paralogous Hox genes within a species, supports a model of functional equivalence. In this report, we demonstrate that zebrafish hoxa3a (zfhoxa3a) expressed from the mouse Hoxa3 locus can substitute for mouse Hoxa3 in some tissues, but has distinct or null phenotypes in others. We further show, by using an allele encoding a chimeric protein, that this difference maps primarily to the zfhoxa3a C-terminal domain. Our data imply that the mouse and zebrafish proteins have diverged considerably since their last common ancestor, and that the major difference between them resides in the C-terminal domain. Our data further show that Hox protein function can evolve independently in different cell types or for specific functions. The inability of zfhoxa3a to perform all of the normal roles of mouse Hoxa3 illustrates that Hox orthologues are not always functionally interchangeable.

Textpresso site-specific recombinases: A text-mining server for the recombinase literature including Cre mice and conditional alleles.

Textpresso Site Specific Recombinases (http://ssrc.genetics.uga.edu/) is a text-mining web server for searching a database of more than 9,000 full-text publications. The papers and abstracts in this database represent a wide range of topics related to site-specific recombinase (SSR) research tools. Included in the database are most of the papers that report the characterization or use of mouse strains that express Cre recombinase as well as papers that describe or analyze mouse lines that carry conditional (floxed) alleles or SSR-activated transgenes/knockins. The database also includes reports describing SSR-based cloning methods such as the Gateway or the Creator systems, papers reporting the development or use of SSR-based tools in systems such as Drosophila, bacteria, parasites, stem cells, yeast, plants, zebrafish, and Xenopus as well as publications that describe the biochemistry, genetics, or molecular structure of the SSRs themselves. Textpresso Site Specific Recombinases is the only comprehensive text-mining resource available for the literature describing the biology and technical applications of SSRs.

Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants.

The formation of stem cell-derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body-derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of beta-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apoptosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide.

Cell death and survival of neural progenitor (NP) cells are determined by signals that are largely unknown. We have analyzed pro-apoptotic signaling in individual NP cells that have been derived from mouse embryonic stem cells. NP formation was concomitant with elevated apoptosis and increased expression of ceramide and prostate apoptosis response 4 (PAR-4). Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis. Apoptotic cells also stained for proliferating cell nuclear antigen (a nuclear mitosis marker protein), but not for nestin (a marker for NP cells). In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis. The other cell was nestin(+), but PAR-4(-), and was not apoptotic. Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

Embryology of the Parathyroid Glands.

The parathyroid glands are essential for regulating calcium homeostasis in the body. The genetic programs that control parathyroid fate specification, morphogenesis, differentiation, and survival are only beginning to be delineated, but are all centered around a key transcription factor, GCM2. Mutations in the Gcm2 gene as well as in several other genes involved in parathyroid organogenesis have been found to cause parathyroid disorders in humans. Therefore, understanding the normal development of the parathyroid will provide insight into the origins of parathyroid disorders.

Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus.

BACKGROUND: Thymic epithelial cells (TECs) promote thymocyte maturation and are required for the early stages of thymocyte development and for positive selection. However, investigation of the mechanisms by which TECs perform these functions has been inhibited by the lack of genetic tools. Since the Foxn1 gene is expressed in all presumptive TECs from the early stages of thymus organogenesis and broadly in the adult thymus, it is an ideal locus for driving gene expression in differentiating and mature TECs. RESULTS: We generated two knock-in alleles of Foxn1 by inserting IRES-Cre or IRES-lacZ cassettes into the 3' UTR of the Foxn1 locus. We simultaneously electroporated the two targeting vectors to generate the two independent alleles in the same experiment, demonstrating the feasibility of multiplex gene targeting at this locus. Our analysis shows that the knockin alleles drive expression of Cre or lacZ in all TECs in the fetal thymus. Furthermore, the knockin alleles express Cre or lacZ in a Foxn1-like pattern without disrupting Foxn1 function as determined by phenotype analysis of Foxn1 knockin/Foxn1 null compound heterozygotes. CONCLUSION: These data show that multiplex gene targeting into the 3' UTR of the Foxn1 locus is an efficient method to express any gene of interest in TECs from the earliest stage of thymus organogenesis. The resulting alleles will make possible new molecular and genetic studies of TEC differentiation and function. We also discuss evidence indicating that gene targeting into the 3' UTR is a technique that may be broadly applicable for the generation of genetically neutral driver strains.

The mouse vesicular inhibitory amino acid transporter gene: expression during embryogenesis, analysis of its core promoter in neural stem cells and a reconsideration of its alternate splicing.

The vesicular inhibitory amino acid transporter, VIAAT (also known as vesicular GABA transporter VGAT) transports GABA or glycine into synaptic vesicles. To initiate an analysis of the expression and regulation of VIAAT during neurogenesis we have cloned and characterized the mouse Viaat gene. We find that the mouse Viaat coding sequence is encoded by two exons spanning 5.3 kb. A survey of expression by whole mount in situ hybridization of mouse embryos indicates that Viaat is activated early in neuron differentiation and is expressed widely within the developing CNS; however, we did not detect expression in the superficial non-neural structures that express the GABA synthase Gad1. Analysis of the Viaat promoter indicates that a minimal promoter region containing a CG rich sequence is sufficient for efficient expression in neural stem and precursor cells. Our analysis of the Viaat sequence and splicing does not support the existence of two Viaat isoforms as previously proposed [Ebihara et al., Brain Res. Mol Brain Res. 110 (2003), 126-139]. Instead, the alternative isoform Viaat-a appears to be due to PCR artifacts that have occurred independently in multiple labs.

Directed neuronal differentiation of human embryonic stem cells.

BACKGROUND: We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons.HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII). RESULTS: A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7-10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons. CONCLUSION: This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.

Using the Textpresso Site-Specific Recombinases Web server to identify Cre expressing mouse strains and floxed alleles.

Effective tools for searching the biomedical literature are essential for identifying reagents or mouse strains as well as for effective experimental design and informed interpretation of experimental results. We have built the Textpresso Site Specific Recombinases (Textpresso SSR) Web server to enable researchers who use mice to perform in-depth searches of a rapidly growing and complex part of the mouse literature. Our Textpresso Web server provides an interface for searching the full text of most of the peer-reviewed publications that report the characterization or use of mouse strains that express Cre or Flp recombinase. The database also contains most of the publications that describe the characterization or analysis of strains carrying conditional alleles or transgenes that can be inactivated or activated by site-specific recombinases such as Cre or Flp. Textpresso SSR complements the existing online databases that catalog Cre and Flp expression patterns by providing a unique online interface for the in-depth text mining of the site specific recombinase literature.

A focused in situ hybridization screen identifies candidate transcriptional regulators of thymic epithelial cell development and function.

BACKGROUND: Thymic epithelial cells (TECs) are necessary for normal T cell development. Currently, one transcription factor, Foxn1 is known to be necessary for the progression of fetal TEC differentiation. However, some aspects of fetal TEC differentiation occur in Foxn1 mutants, suggesting the existence of additional transcriptional regulators of TEC differentiation. The goal of this study was to identify some of the additional candidate transcription factors that may be involved in the specification and/or differentiation of TECs during fetal development. METHODOLOGY/PRINCIPAL FINDINGS: We identified candidate fetal TEC transcriptional regulators via data and text mining. From our data mining we selected the transcription factors Foxg1, Isl1, Gata3, Nkx2-5, Nkx2-6 and Sox2 for further studies. Whole mount in situ hybridizations confirmed the expression of these transcription factors within subdomains of the third pharyngeal pouch from E9.5-E10.5. By E11.5 days Foxg1 and Isl1 transcripts were the only mRNAs from this group of genes detected exclusively within the thymus domain of the third pouch. Based on this initial in situ hybridization analysis, we focused on defining the expression of Foxg1 and Isl1 during multiple stages of thymus development and TEC differentiation. We found that Foxg1 and Isl1 are specifically expressed in differentiating TECs during fetal and postnatal stages of thymus development. In addition, we found differential expression of Islet1 and Foxn1 within the fetal and postnatal TEC population. CONCLUSIONS/SIGNIFICANCE: Our studies have identified two developmental transcription factors that are excellent candidate regulators of thymic epithelial cell specification and differentiation during fetal development. Our results suggest that Foxg1 and Isl1 may play a role in the regulation of TEC differentiation during fetal and postnatal stages. Our results also demonstrate heterogeneity of TECs marked by the differential expression of transcription factors, potentially providing new insights into the regulation of TEC differentiation.

Whole mount in situ hybridization of E8.5 to E11.5 mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

An evolving NGF-Hoxd1 signaling pathway mediates development of divergent neural circuits in vertebrates.

Species are endowed with unique sensory capabilities that are encoded by divergent neural circuits. One potential explanation for how divergent circuits have evolved is that conserved extrinsic signals are differentially interpreted by developing neurons of different species to yield unique patterns of axonal connections. Although nerve growth factor (NGF) controls survival, maturation and axonal projections of nociceptors of different vertebrates, whether the NGF signal is differentially transduced in different species to yield unique features of nociceptor circuits is unclear. We identified a species-specific signaling module induced by NGF and mediated by a rapidly evolving Hox transcription factor, Hoxd1. NGF promoted robust expression of Hoxd1 in mice, but not chickens, both in vivo and in vitro. Mice lacking Hoxd1 displayed altered nociceptor circuitry that resembles that normally found in chicks. Conversely, ectopic expression of Hoxd1 in developing chick nociceptors promoted a pattern of axonal projections reminiscent of the mouse. Thus, conserved growth factors control divergent neuronal transcriptional events that mediate interspecies differences in neural circuits and the behaviors that they control.

Transcriptional regulation of thymus organogenesis and thymic epithelial cell differentiation.

Transcriptional regulatory networks are the central regulatory mechanisms that control organ identity, patterning, and differentiation. In the case of the thymus, several key transcription factors have been identified that are critical for various aspects of thymus organogenesis and thymic epithelial cell (TEC) differentiation. The thymus forms from the third pharyngeal pouch endoderm during embryogenesis. Organ development progresses from initial thymus cell fate specification, through multiple stages of TEC differentiation and cortical (cTEC) and medullary (mTEC) formation. Transcription factors have been identified for each of these stages: a Hoxa3-dependent cascade at initial fate specification, Foxn1 for early (and later) TEC differentiation, and NF-kappaB for mTEC differentiation. As important as these factors are, their interrelationships are not understood, and many more transcription factors are likely required for complete thymus organogenesis to occur. In this chapter, we review the literature on these known genes, as well as identify gaps in our knowledge for future studies.

Cleft palate is caused by CNS dysfunction in Gad1 and Viaat knockout mice.

BACKGROUND: Previous studies have shown that disruption of GABA signaling in mice via mutations in the Gad1, Gabrb3 or Viaat genes leads to the development of non-neural developmental defects such as cleft palate. Studies of the Gabrb3 and Gad1 mutant mice have suggested that GABA function could be required either in the central nervous system or in the palate itself for normal palatogenesis. METHODOLOGY/PRINCIPAL FINDINGS: To further examine the role of GABA signaling in palatogenesis we used three independent experimental approaches to test whether Gad1 or Viaat function is required in the fetal CNS for normal palate development. We used oral explant cultures to demonstrate that the Gad1 and Viaat mutant palates were able to undergo palatogenesis in culture, suggesting that there is no defect in the palate tissue itself in these mice. In a second series of experiments we found that the GABA(A) receptor agonist muscimol could rescue the cleft palate phenotype in Gad1 and Viaat mutant embryos. This suggested that normal multimeric GABA(A) receptors in the CNS were necessary for normal palatogenesis. In addition, we showed that CNS-specific inactivation of Gad1 was sufficient to disrupt palate development. CONCLUSIONS/SIGNIFICANCE: Our results are consistent with a role for Gad1 and Viaat in the central nervous system for normal development of the palate. We suggest that the alterations in GABA signaling lead to non-neural defects such as cleft palate as a secondary effect due to alterations in or elimination of fetal movements.

Gata3-deficient mice develop parathyroid abnormalities due to dysregulation of the parathyroid-specific transcription factor Gcm2.

Heterozygous mutations of GATA3, which encodes a dual zinc-finger transcription factor, cause hypoparathyroidism with sensorineural deafness and renal dysplasia. Here, we have investigated the role of GATA3 in parathyroid function by challenging Gata3+/- mice with a diet low in calcium and vitamin D so as to expose any defects in parathyroid function. This led to a higher mortality among Gata3+/- mice compared with Gata3+/+ mice. Compared with their wild-type littermates, Gata3+/- mice had lower plasma concentrations of calcium and parathyroid hormone (PTH) and smaller parathyroid glands with a reduced Ki-67 proliferation rate. At E11.5, Gata3+/- embryos had smaller parathyroid-thymus primordia with fewer cells expressing the parathyroid-specific gene glial cells missing 2 (Gcm2), the homolog of human GCMB. In contrast, E11.5 Gata3-/- embryos had no Gcm2 expression and by E12.5 had gross defects in the third and fourth pharyngeal pouches, including absent parathyroid-thymus primordia. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays showed that GATA3 binds specifically to a functional double-GATA motif within the GCMB promoter. Thus, GATA3 is critical for the differentiation and survival of parathyroid progenitor cells and, with GCM2/B, forms part of a transcriptional cascade in parathyroid development and function.

Ceramide regulates atypical PKCzeta/lambda-mediated cell polarity in primitive ectoderm cells. A novel function of sphingolipids in morphogenesis.

In mammals, the primitive ectoderm is an epithelium of polarized cells that differentiates into all embryonic tissues. Our study shows that in primitive ectoderm cells, the sphingolipid ceramide was elevated and co-distributed with the small GTPase Cdc42 and cortical F-actin at the apicolateral cell membrane. Pharmacological or RNA interference-mediated inhibition of ceramide biosynthesis enhanced apoptosis and impaired primitive ectoderm formation in embryoid bodies differentiated from mouse embryonic stem cells. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog. Ceramide depletion prevented plasma membrane translocation of PKCzeta/lambda, its interaction with Cdc42, and phosphorylation of GSK-3beta, a substrate of PKCzeta/lambda. Recombinant PKCzeta formed a complex with the polarity protein Par6 and Cdc42 when bound to ceramide containing lipid vesicles. Our data suggest a novel mechanism by which a ceramide-induced, apicolateral polarity complex with PKCzeta/lambda regulates primitive ectoderm cell polarity and morphogenesis.