BioProject

The hypothalamus coordinates neuroendocrine functions in vertebrates, including circadian rhythm, metabolism, and appetite. More...

The hypothalamus coordinates neuroendocrine functions in vertebrates, including circadian rhythm, metabolism, and appetite. To explore its evolutionary origin, we attempt to create integrated transcription/connectome brain maps for swimming tadpoles of the ascidian, Ciona intestinalis, which serves as an approximation of the ancestral proto-vertebrate. This map features several cell types related to different regions of the vertebrate hypothalamus, including coronet cells, magnocellular neurons and the arcuate nucleus. Coronet cells express melanopsin and share additional properties with the saccus vasculosus, a specialized region of the hypothalamus that mediates photoperiodism in non-tropical fishes such as salmon. Comparative transcriptome analyses identified orthologous cell types for mechanosensory switch neurons, and VP+ and VPR+ relay neurons in different regions of the mouse hypothalamus. These observations provide evidence that the hypothalamus predates the evolution of the vertebrate brain. We discuss the possibility that switch neurons, coronet cells, and FoxP+/VPR relay neurons comprise a behavioral circuit that helps trigger metamorphosis of Ciona larvae in response to twilight. Overall design: After fertilization using gametes from the same individual, one-cell stage embryos were electroporated with Dmrt1>H2B:GFP and Dmrt1>LacZ (control condition) or Dmrt1>H2B:GFP and Dmrt1>NeuroD (NeuroD overexpression condition). At the late tailbud stage I, morphologically normal embryos were selected, and at late tailbud II, GFP+ embryos were dissociated as previously described in Cao et al, 2019. The dissociation was performed in duplicate. Single-cell suspensions were loaded onto The Chromium Controller (10x Genomics). For all of the samples, cells were lysed, cDNAs were barcoded and amplified with Chromium Single Cell 3′ Library and Gel Bead Kit v3 (10x Genomics) following the instructions of the manufacturer. Illumina sequencing libraries were prepared from the cDNA samples using the Nextera DNA library prep kit (Illumina). All of the libraries were sequenced on Illumina NovaSeq 6000 with S1 reagent kit (300 cycles, paired-end) following standard Illumina protocols. Raw sequencing reads were filtered by Illumina NovaSeq Control Software and only pass-filtered reads were used for further analysis. Reads that aligned to phix (using Bowtie version 1.1.1) were removed, as were reads that failed Illumina’s default chastity filter. We then combined the FASTQ files from each lane and separated the samples using the barcode sequences allowing one mismatch (using barcode_splitter version 0.18.2). In the read 2 of each sample, the rightmost bases after to position 100 were trimmed to keep only high-quality reads. Using 10x CellRanger version 3.0.2, the count pipeline was run with default settings on the trimmed FASTQ files to generate gene–barcode matrices for each sample. The reference sequence of Ciona intestinalis was obtained from the Ghost database (KH model 2012). The integration of scRNA-Seq data of NeuroD overexpressed embryos and the control embryos were performed with Seurat v3.0 Less...