RESUMO
Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in-situ expression maps of hundreds of genes. We exploit direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high throughput and large multiplexing. We validate the method across a number of species, and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental and tissue biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to high-throughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.
RESUMO
Embryonic development is a complex and dynamic process that unfolds over time and involves the production and diversification of increasing numbers of cells. The impact of developmental time on the formation of the central nervous system is well documented, with evidence showing that time plays a crucial role in establishing the identity of neuronal subtypes. However, the study of how time translates into genetic instructions driving cell fate is limited by the scarcity of suitable experimental tools. We introduce BirthSeq, a new method for isolating and analyzing cells based on their birth date. This innovative technique allows for in vivo labeling of cells, isolation via fluorescence-activated cell sorting, and analysis using high-throughput techniques. We calibrated the BirthSeq method for developmental organs across three vertebrate species (mouse, chick and gecko), and utilized it for single-cell RNA sequencing and novel spatially resolved transcriptomic approaches in mouse and chick, respectively. Overall, BirthSeq provides a versatile tool for studying virtually any tissue in different vertebrate organisms, aiding developmental biology research by targeting cells and their temporal cues.
Assuntos
Análise de Célula Única , Animais , Camundongos , Análise de Célula Única/métodos , Embrião de Galinha , Lagartos/genética , Lagartos/embriologia , Desenvolvimento Embrionário/genética , Transcriptoma/genética , Citometria de Fluxo/métodos , Vertebrados/genética , Separação Celular/métodos , Galinhas , Análise de Sequência de RNA/métodosRESUMO
The cerebellum is a conserved structure of vertebrate brains that develops at the most anterior region of the alar rhombencephalon. All vertebrates display a cerebellum, making it one of the most highly conserved structures of the brain. Although it greatly varies at the morphological level, several lines of research point to strong conservation of its internal neural circuitry. To test the conservation of the cerebellar circuit, we compared the developmental history of the neurons comprising this circuit in three amniote species: mouse, chick, and gecko. We specifically researched the developmental time of generation of the main neuronal types of the cerebellar cortex. This developmental trajectory is known for the mammalian cell types but barely understood for sauropsid species. We show that the neurogenesis of the GABAergic lineage proceeds following the same chronological sequence in the three species compared: Purkinje cells are the first ones generated in the cerebellar cortex, followed by Golgi interneurons of the granule cell layer, and lately by the interneurons of the molecular layer. In the cerebellar glutamatergic lineage, we observed the same conservation of neurogenesis throughout amniotes, and the same vastly prolonged neurogenesis of granule cells, extending much further than for any other brain region. Together these data show that the cerebellar circuitry develops following a tightly conserved chronological sequence of neurogenesis, which is responsible for the preservation of the cerebellum and its function. Our data reinforce the developmental perspective of homology, whereby similarities in neurons and circuits are likely due to similarities in developmental sequence.
Assuntos
Cerebelo , Neurogênese , Animais , Cerebelo/anatomia & histologia , Mamíferos , Camundongos , Neurogênese/fisiologia , Neurônios/metabolismo , Células de PurkinjeRESUMO
The neocortex (NCx) generates at the dorsal region of the pallium in the forebrain. Several adjacent structures also contribute with neurons to NCx. Ventral pallium (VP) is considered to generate several populations of neurons that arrive through tangential migration to the NCx. Amongst them are the Cajal-Retzius cells and some transient pyramidal neurons. However, the specific site and timing of generation, trajectory of migration and actual contribution to the pyramidal population remains elusive. Here, we investigate the spatio-temporal origin of neuronal populations from VP in an in vivo model, using a transposase mediated in utero electroporation method in embryonic mouse. From E11 to E14 cells born at the lateral corner of the neocortical neuroepithelium including the VP migrated ventro-laterally to settle all areas of the ventral telencephalon. Specifically, neurons migrated into amygdala (Ag), olfactory cortices, and claustrum (Cl). However, we found no evidence for any neurons migrating tangentially toward the NCx, regardless the antero-posterior level and developmental time of the electroporation. Our results challenge the described ventral-pallial origin of the transient pyramidal neuron population. In order to find the exact origin of cortical neurons that were previously Dbx1-fate mapped we used the promoter region of the murine Dbx1 locus to selectively target Dbx1-expressing progenitors and label their lineage. We found these progenitors in low numbers in all pallial areas, and not only in the ventral pallial ventricular zone. Our findings on the local cortical origin of the Dbx1-derived pyramidal neurons reconcile the observation of Dbx1-derived neurons in the cortex without evidence of dorsal tangential migration from VP and provide a new framework for the origin of the transient Dbx1-derived pyramidal neuron population. We conclude that these neurons are born locally within the dorsal pallial neuroepithelium.