Cell-autonomous retinoic acid receptor signaling has stage-specific effects on mouse enteric nervous system.

Retinoic acid (RA) signaling is essential for enteric nervous system (ENS) development, since vitamin A deficiency or mutations in RA signaling profoundly reduce bowel colonization by ENS precursors. These RA effects could occur because of RA activity within the ENS lineage or via RA activity in other cell types. To define cell-autonomous roles for retinoid signaling within the ENS lineage at distinct developmental time points, we activated a potent floxed dominant-negative RA receptor α (RarαDN) in the ENS using diverse CRE recombinase-expressing mouse lines. This strategy enabled us to block RA signaling at premigratory, migratory, and postmigratory stages for ENS precursors. We found that cell-autonomous loss of RA receptor (RAR) signaling dramatically affected ENS development. CRE activation of RarαDN expression at premigratory or migratory stages caused severe intestinal aganglionosis, but at later stages, RarαDN induced a broad range of phenotypes including hypoganglionosis, submucosal plexus loss, and abnormal neural differentiation. RNA sequencing highlighted distinct RA-regulated gene sets at different developmental stages. These studies show complicated context-dependent RA-mediated regulation of ENS development.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo.

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

NGF-TrkA Signaling by Sensory Nerves Coordinates the Vascularization and Ossification of Developing Endochondral Bone.

Developing tissues dictate the amount and type of innervation they require by secreting neurotrophins, which promote neuronal survival by activating distinct tyrosine kinase receptors. Here, we show that nerve growth factor (NGF) signaling through neurotrophic tyrosine kinase receptor type 1 (TrkA) directs innervation of the developing mouse femur to promote vascularization and osteoprogenitor lineage progression. At the start of primary ossification, TrkA-positive axons were observed at perichondrial bone surfaces, coincident with NGF expression in cells adjacent to centers of incipient ossification. Inactivation of TrkA signaling during embryogenesis in TrkA(F592A) mice impaired innervation, delayed vascular invasion of the primary and secondary ossification centers, decreased numbers of Osx-expressing osteoprogenitors, and decreased femoral length and volume. These same phenotypic abnormalities were observed in mice following tamoxifen-induced disruption of NGF in Col2-expressing perichondrial osteochondral progenitors. We conclude that NGF serves as a skeletal neurotrophin to promote sensory innervation of developing long bones, a process critical for normal primary and secondary ossification.

Tracking Quantum-Dot labeled neurotropic factors transport along primary neuronal axons in compartmental microfluidic chambers.

Neurons are highly polarized cells, with very long axons. Neurotrophic factors like the neuronal growth factor (NGF) are secreted from neuronal targets to promote neuron survival and proper function. These neurotrophic factors must undergo retrograde axonal transport towards the cell body, wherein they initiate signaling pathways important for neurons' various functions and overall health. This process of long-distance axonal signaling is conducted by the dynein motor protein, which transmits signaling endosomes of ligand-receptor complexes retrogradely along microtubule tracks. Here we describe step by step the use of polydimethylsiloxane (PDMS) compartmentalized microfluidic chambers for tracking axonal transport of trophic factors, with a focus on labeled NGF. We describe in detail how to fabricate the molds, assemble the PDMS platform, plate neurons and image, as well as analyze NGF transport along the axon. This method is useful for studying molecular communication mechanisms within the neuron's different compartments as well as between the neuron and its diverse microenvironments, both in health and under pathological conditions.

Cellular and subcellular imaging of motor protein-based behavior in embryonic rat brain.

Development of the cerebral cortex is a very dynamic process, involving a series of complex morphogenetic events. Following division of progenitor cells in the ventricular zone, neurons undergo a series of morphological changes and migrate outward toward the cortical plate, where they differentiate and integrate into functional circuits. Errors at several of stages during neurogenesis and migration cause a variety of severe cortical malformations. A number of disease genes encode factors associated with the cytoskeleton, which plays a crucial role throughout cortical development. Methods for regulating gene expression coupled with imaging of subcellular structures have provided important insight into the mechanisms governing normal and abnormal brain development. We describe here a series of protocols for imaging motor protein-dependent processes in real time in the developing rat brain.

Studying endosomes in cultured neurons by live-cell imaging.

Endosomes play critical roles on regulating surface receptor levels as well as signaling cascades in all cell types, including neurons. Endocytosis and endosomal trafficking is routinely studied after fixation, but live imaging is increasingly being used to capture the dynamic nature of endosomes and is allowing increasingly sophisticated glimpses into trafficking processes in live neurons. In this chapter, we describe the basics of neuronal primary cultures, methods for expressing fluorescent proteins, and live imaging of cargos and endosomal regulators.

Pharyngeal arch deficiencies affect taste bud development in the circumvallate papilla with aberrant glossopharyngeal nerve formation.

BACKGROUND: The pharyngeal arches (PAs) generate cranial organs including the tongue. The taste placodes, formed in particular locations on the embryonic tongue surface, differentiate into taste buds harbored in distinct gustatory papillae. The developing tongue also has a complex supply of cranial nerves through each PA. However, the relationship between the PAs and taste bud development is not fully understood. RESULTS: Ripply3 homozygous mutant mice, which have impaired third/fourth PAs, display a hypoplastic circumvallate papilla and lack taste buds, although the taste placode is normally formed. Formation of the glossopharyngeal ganglia is defective and innervation toward the posterior tongue is completely missing in Ripply3 mutant embryos at E12.5. Moreover, the distribution of neuroblasts derived from the epibranchial placode is severely, but not completely, atenuated, and the neural crest cells are diminished in the third PA region of Ripply3 mutant embryos at E9.5-E10.5. In Tbx1 homozygous mutant embryos, which exhibit another type of deficiency in PA development, the hypoplastic circumvallate papilla is observed along with abnormal formation of the glossopharyngeal ganglia and severely impaired innervation. CONCLUSIONS: PA deficiencies affect multiple aspects of taste bud development, including formation of the cranial ganglia and innervation to the posterior tongue.

Unlocking epigenetic codes in neurogenesis.

During embryonic and adult neurogenesis, neuronal stem cells follow a highly conserved path of differentiation to give rise to functional neurons at various developmental stages. Epigenetic regulation--including DNA modifications, histone modifications, and noncoding regulatory RNAs, such as microRNA (miRNA) and long noncoding RNA (lncRNA)--plays a pivotal role in embryonic and adult neurogenesis. Here we review the latest in our understanding of the epigenetic regulation in neurogenesis, with a particular focus on newly identified cytosine modifications and their dynamics, along with our perspective for future studies.

Specification of dopaminergic subsets involves interplay of En1 and Pitx3.

Mesodiencephalic dopaminergic (mdDA) neurons control locomotion and emotion and are affected in multiple psychiatric and neurodegenerative diseases, including Parkinson's disease (PD). The homeodomain transcription factor Pitx3 is pivotal in mdDA neuron development and loss of Pitx3 results in programming deficits in a rostrolateral subpopulation of mdDA neurons destined to form the substantia nigra pars compacta (SNc), reminiscent of the specific cell loss observed in PD. We show here that in adult mice in which the gene encoding a second homeoprotein, engrailed 1 (En1), has been deleted, dramatic loss of mdDA neurons and striatal innervation defects were observed, partially reminiscent of defects observed in Pitx3(-/-) mice. We then continue to reveal developmental crosstalk between En1 and Pitx3 through genome-wide expression analysis. During development, both En1 and Pitx3 are required to induce expression of mdDA genes in the rostrolateral subset destined to form the SNc. By contrast, Pitx3 and En1 reciprocally regulate a separate gene cluster, which includes Cck, demarcating a caudal mdDA subset in wild-type embryos. Whereas En1 is crucial for induction of this caudal phenotype, Pitx3 antagonizes it rostrolaterally. The combinatorial action of En1 and Pitx3 is potentially realized through at least three levels of molecular interaction: (1) influencing each other's expression level, (2) releasing histone deacetylase-mediated repression of Nurr1 target genes and (3) modulating En1 activity through Pitx3-driven activation of En1 modulatory proteins. These findings show how two crucial mediators of mdDA neuronal development, En1 and Pitx3, interact in dopaminergic subset specification, the importance of which is exemplified by the specific vulnerability of the SNc found in PD.

An autocrine Wnt5a-Ror signaling loop mediates sympathetic target innervation.

During nervous system development, axon branching at nerve terminals is an essential step in the formation of functional connections between neurons and target cells. It is known that target tissues exert control of terminal arborization through secretion of trophic factors. However, whether the in-growing axons themselves produce diffusible cues to instruct target innervation remains unclear. Here, we use conditional mutant mice to show that Wnt5a derived from sympathetic neurons is required for their target innervation in vivo. Conditional deletion of Wnt5a resulted in specific deficits in the extension and arborization of sympathetic fibers in their final target fields, while no defects were observed in the overall tissue patterning, proliferation, migration or differentiation of neuronal progenitors. Using compartmentalized neuronal cultures, we further demonstrate that the Ror receptor tyrosine kinases are required locally in sympathetic axons to mediate Wnt5a-dependent branching. Thus, our study suggests an autocrine Wnt5a-Ror signaling pathway that directs sympathetic axon branching during target innervation.

Organotypic slice culture of GFP-expressing mouse embryos for real-time imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system(1), allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques(2). The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period(2). Representative results using the tauGFP mouse line are demonstrated.

Neuronal degeneration in autonomic nervous system of Dystonia musculorum mice.

BACKGROUND: Dystonia musculorum (dt) is an autosomal recessive hereditary neuropathy with a characteristic uncoordinated movement and is caused by a defect in the bullous pemphigoid antigen 1 (BPAG1) gene. The neural isoform of BPAG1 is expressed in various neurons, including those in the central and peripheral nerve systems of mice. However, most previous studies on neuronal degeneration in BPAG1-deficient mice focused on peripheral sensory neurons and only limited investigation of the autonomic system has been conducted. METHODS: In this study, patterns of nerve innervation in cutaneous and iridial tissues were examined using general neuronal marker protein gene product 9.5 via immunohistochemistry. To perform quantitative analysis of the autonomic neuronal number, neurons within the lumbar sympathetic and parasympathetic ciliary ganglia were calculated. In addition, autonomic neurons were cultured from embryonic dt/dt mutants to elucidate degenerative patterns in vitro. Distribution patterns of neuronal intermediate filaments in cultured autonomic neurons were thoroughly studied under immunocytochemistry and conventional electron microscopy. RESULTS: Our immunohistochemistry results indicate that peripheral sensory nerves and autonomic innervation of sweat glands and irises dominated degeneration in dt/dt mice. Quantitative results confirmed that the number of neurons was significantly decreased in the lumbar sympathetic ganglia as well as in the parasympathetic ciliary ganglia of dt/dt mice compared with those of wild-type mice. We also observed that the neuronal intermediate filaments were aggregated abnormally in cultured autonomic neurons from dt/dt embryos. CONCLUSIONS: These results suggest that a deficiency in the cytoskeletal linker BPAG1 is responsible for dominant sensory nerve degeneration and severe autonomic degeneration in dt/dt mice. Additionally, abnormally aggregated neuronal intermediate filaments may participate in neuronal death of cultured autonomic neurons from dt/dt mutants.

Neural crest stem cell multipotency requires Foxd3 to maintain neural potential and repress mesenchymal fates.

Neural crest (NC) progenitors generate a wide array of cell types, yet molecules controlling NC multipotency and self-renewal and factors mediating cell-intrinsic distinctions between multipotent versus fate-restricted progenitors are poorly understood. Our earlier work demonstrated that Foxd3 is required for maintenance of NC progenitors in the embryo. Here, we show that Foxd3 mediates a fate restriction choice for multipotent NC progenitors with loss of Foxd3 biasing NC toward a mesenchymal fate. Neural derivatives of NC were lost in Foxd3 mutant mouse embryos, whereas abnormally fated NC-derived vascular smooth muscle cells were ectopically located in the aorta. Cranial NC defects were associated with precocious differentiation towards osteoblast and chondrocyte cell fates, and individual mutant NC from different anteroposterior regions underwent fate changes, losing neural and increasing myofibroblast potential. Our results demonstrate that neural potential can be separated from NC multipotency by the action of a single gene, and establish novel parallels between NC and other progenitor populations that depend on this functionally conserved stem cell protein to regulate self-renewal and multipotency.

ATX expression and LPA signalling are vital for the development of the nervous system.

Autotaxin (ATX) is a secreted glycoprotein widely present in biological fluids, originally isolated from the supernatant of melanoma cells as an autocrine motility stimulation factor. Its enzymatic product, lysophosphatidic acid (LPA), is a phospholipid mediator that evokes growth-factor-like responses in almost all cell types through G-protein coupled receptors. To assess the role of ATX and LPA signalling in pathophysiology, a conditional knockout mouse was created. Ubiquitous, obligatory deletion resulted to embryonic lethality most likely due to aberrant vascular branching morphogenesis and chorio-allantoic fusion. Moreover, the observed phenotype was shown to be entirely depended on embryonic, but not extraembryonic or maternal ATX expression. In addition, E9.5 ATX null mutants exhibited a failure of neural tube closure, most likely independent of the circulatory failure, which correlated with decreased cell proliferation and increased cell death. More importantly, neurite outgrowth in embryo explants was severely compromised in mutant embryos but could be rescued upon the addition of LPA, thus confirming a role for ATX and LPA signalling in the development of the nervous system. Finally, expression profiling of mutant embryos revealed attenuated embryonic expression of HIF-1a in the absence of ATX, suggesting a novel effector pathway of ATX/LPA.

The first appearance of sympathetic ganglia in human embryos at stage 13.

The primordia of the sympathetic trunk ganglia were traced on serial sections of 10 embryos at stage 13 (32 postovulatory days). It was found that in all embryos, these primordia were present in the thoracic level T4 to T9 and they appeared as scattered aggregates of cells lying dorsally and laterally to the dorsal aortae.

Efficient CPP-mediated Cre protein delivery to developing and adult CNS tissues.

BACKGROUND: Understanding and manipulating gene function in physiological conditions is a major objective for both fundamental and applied research. In contrast to other experimental settings, which use either purely genetic or gene delivery (viral or non-viral) strategies, we report here a strategy based on direct protein delivery to central nervous system (CNS) tissues. We fused Cre recombinase with cell-penetrating peptides and analyzed the intracellular biological activity of the resulting chimerical proteins when delivered into cells endowed with Cre-mediated reporter gene expression. RESULTS: We show that active Cre enzymatic conjugates are readily internalized and exert their enzymatic activity in the nucleus of adherent cultured cells. We then evaluated this strategy in organotypic cultures of neural tissue explants derived from reporter mice carrying reporter "floxed" alleles. The efficacy of two protocols was compared on explants, either by direct addition of an overlying drop of protein conjugate or by implantation of conjugate-coated beads. In both cases, delivery of Cre recombinase resulted in genomic recombination that, with the bead protocol, was restricted to discrete areas of embryonic and adult neural tissues. Furthermore, delivery to adult brain tissue resulted in the transduction of mature postmitotic populations of neurons. CONCLUSION: We provide tools for the spatially restricted genetic modification of cells in explant culture. This strategy allows to study lineage, migration, differentiation and death of neural cells. As a proof-of-concept applied to CNS tissue, direct delivery of Cre recombinase enabled the selective elimination of an interneuron subpopulation of the spinal cord, thereby providing a model to study early events of neurodegenerative processes. Thus our work opens new perspectives for both fundamental and applied cell targeting protocols using proteic cargoes which need to retain full bioactivity upon internalisation, as illustrated here with the oligomeric Cre recombinase.

The mammalian myotome: a muscle with no innervation.

The segmented muscular myotome is the first muscle to form in all vertebrates. In fish and amphibian embryos, the myotome becomes innervated very early and is essential for larval swimming. Its role in birds and mammals, however, is not clear. Using immunohistochemistry on sections and whole mounts of rat embryos, we demonstrate that the mammalian myotome differentiates and develops over a period of 3 days without being invaded by the outgrowing spinal nerves. In contrast, the limb muscle masses become filled with fine nerve branches from the first time that myocyte differentiation can be detected. Additionally, we show that the mammalian myotome does not express clustered acetylcholine receptors until after embryonic day 13.5, which corresponds to the beginning of its transformation into the adult epaxial muscles, showing that there are no functional myotomal neuromuscular junctions before this age. We suggest that the mammalian myotome has entirely lost the function of neurally controlled muscular contraction: its remaining functions are likely to be as a signaling tissue, as a structural scaffold, and as an incubator for myogenic precursors of the deep back, abdominal, and intercostal muscles.

HoxD cluster scanning deletions identify multiple defects leading to paralysis in the mouse mutant Ironside.

A spontaneous semidominant mutation (Ironside, Irn) was isolated in mice, leading to severe hindlimb paralysis following multiple deletions in cis at the HoxD locus. To understand its cellular and molecular etiology, we embarked on a comparative analysis using systematic HoxD cluster deletions, produced via targeted meiotic recombination (TAMERE). Different lines of mice were classified according to the severity of their paralyses, and subsequent analyses revealed that multiple causative factors were involved, alone or in combination, in the occurrence of this pathology. Among them are the loss of Hoxd10 function, the sum of remaining Hoxd gene activity, and the ectopic gain of function of the neighboring gene Evx2, all contributing to the mispositioning, the absence, or misidentification of specific lumbo-sacral pools of motoneurons, nerve root homeosis, and hindlimb innervation defects. These results highlight the importance of a systematic approach when studying such clustered gene families, and give insights into the function and regulation of Hox and Evx2 genes during early spinal cord development.

Human fetal neural stem cells in rat brain: effects of preculturing and transplantation.

The fate of human fetal stem/progenitor cells transplanted into rat brain depends on conditions of preculturing (long or short) and state and site of transplantation. Human nestin-positive stem cells cultured according to the short protocol did not migrate into hypoxic and normal brain after transplantation, but actively migrated in damaged spinal cord. After transplantation of long-cultured cells into the brain mainly committed neuroblasts and solitary nestin-positive cells migrated from the site of transplantation into the brain.