Serial-section electronmicroscopy (ssEM) is themethod of choice for studyingmacroscopic biological samples at extremely high resolution in three dimensions. In the nervous system, nanometer-scale images are necessary to reconstruct dense neural wiring diagrams in the brain, so called connectomes. In order to use this data, consisting of up to 108 individual EM images, it must be assembled into a volume, requiring seamless 2D stitching from each physical section followed by 3D alignment of the stitched sections. The high throughput of ssEM necessitates 2D stitching to be done at the pace of imaging, which currently produces tens of terabytes per day. To achieve this, we present a modular volume assembly software pipeline ASAP (Assembly Stitching and Alignment Pipeline) that is scalable to datasets containing petabytes of data and parallelized to work in a distributed computational environment. The pipeline is built on top of the Render (27) services used in the volume assembly of the brain of adult Drosophilamelanogaster (30). It achieves high throughput by operating on themeta-data and transformations of each image stored in a database, thus eliminating the need to render intermediate output. ASAP ismodular, allowing for easy incorporation of new algorithms without significant changes in the workflow. The entire software pipeline includes a complete set of tools for stitching, automated quality control, 3D section alignment, and final rendering of the assembled volume to disk. ASAP has been deployed for continuous stitching of several large-scale datasets of the mouse visual cortex and human brain samples including one cubic millimeter of mouse visual cortex (28; 8) at speeds that exceed imaging. The pipeline also has multi-channel processing capabilities and can be applied to fluorescence and multi-modal datasets like array tomography.
The current manuscript describes is a software infrastructure resource that is being made publicly available. The manuscript is not a data generation manuscript. Nevertheless, one of the datasets used is already publicly available on https://www.microns-explorer.org/cortical-mm3#em-imagery with available imagery and segmentation (https://tinyurl.com/cortical-mm3).Moreover cloud-volume (https://github.com/seung-lab/cloud-volume) can be used to programmatically download EM imagery from either Amazon or Google with the cloud paths described below. The imagery was reconstructed in two portions, referred to internally by their nicknames 'minnie65' and 'minnie35' reflecting their relative portions of the total data. The two portions are aligned across an interruption in sectioning.minnie65:AWS Bucket: precomputed://https://bossdb-open-data.s3.amazonaws.com/iarpa_microns/minnie/minnie65/emGoogle Bucket: precomputed://https://storage.googleapis.com/iarpa_microns/minnie/minnie65/emminnie35:AWS Bucket: precomputed://https://bossdb-open-data.s3.amazonaws.com/iarpa_microns/minnie/minnie35/emGoogle Bucket: precomputed://https://storage.googleapis.com/iarpa_microns/minnie/minnie35/emWe have also made available in Dryad raw data of the remaining datasets https://doi.org/10.5061/dryad.qjq2bvqhr
ASAP-TEM-sampleDryad Digital Repository, doi:10.5061/dryad.qjq2bvqhr.
MICrONS multi-area datasethttps://doi.org/10.1101/2021.07.28.454025.
- Gayathri Mahalingam
- Russel Torres
- Daniel Kapner
- Tim Fliss
- Shamishtaa Seshamani
- Rob Young
- Samuel Kinn
- JoAnn Buchanan
- Marc M Takeno
- Wenjing Yin
- Daniel J Bumbarger
- R Clay Reid
- Forrest Collman
- Nuno Macarico da Costa
The funders had no role in study design and interpretation, or the decision to submit the work for publication.
Animal experimentation: All procedures were carried out in accordance with Institutional Animal Care and Use Committee approval at the Allen Institute for Brain Science with protocol numbers 1503, 1801 and 1808
Human subjects: Human surgical specimen was obtained from local hospital in collaboration with local neurosurgeon. The sample collection was approved by the Western Institutional Review Board (Protocol # SNI 0405). Patient provided informed consent and experimental procedures were approved by hospital institute review boards before commencing the study.
- Albert Cardona, University of Cambridge, United Kingdom
- Received: December 20, 2021
- Accepted: July 10, 2022
- Accepted Manuscript published: July 26, 2022 (version 1)
© 2022, Mahalingam et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Neonatal cerebral hypoxia-ischemia (HI) is the leading cause of death and disability in newborns with the only current treatment being hypothermia. An increased understanding of the pathways that facilitate tissue repair after HI may aid the development of better treatments. Here, we study the role of lactate receptor HCAR1 in tissue repair after neonatal HI in mice. We show that HCAR1 knockout mice have reduced tissue regeneration compared with wildtype mice. Furthermore, proliferation of neural progenitor cells and glial cells, as well as microglial activation was impaired. Transcriptome analysis showed a strong transcriptional response to HI in the subventricular zone of wildtype mice involving about 7300 genes. In contrast, the HCAR1 knockout mice showed a modest response, involving about 750 genes. Notably, fundamental processes in tissue repair such as cell cycle and innate immunity were dysregulated in HCAR1 knockout. Our data suggest that HCAR1 is a key transcriptional regulator of pathways that promote tissue regeneration after HI.
Btg3-associated nuclear protein (Banp) was originally identified as a nuclear matrix-associated region (MAR)-binding protein and it functions as a tumor suppressor. At the molecular level, Banp regulates transcription of metabolic genes via a CGCG-containing motif called the Banp motif. However, its physiological roles in embryonic development are unknown. Here, we report that Banp is indispensable for the DNA damage response and chromosome segregation during mitosis. Zebrafish banp mutants show mitotic cell accumulation and apoptosis in developing retina. We found that DNA replication stress and tp53-dependent DNA damage responses were activated to induce apoptosis in banp mutants, suggesting that Banp is required for regulation of DNA replication and DNA damage repair. Furthermore, consistent with mitotic cell accumulation, chromosome segregation was not smoothly processed from prometaphase to anaphase in banp morphants, leading to a prolonged M-phase. Our RNA- and ATAC-sequencing identified 31 candidates for direct Banp target genes that carry the Banp motif. Interestingly, a DNA replication fork regulator, wrnip1, and two chromosome segregation regulators, cenpt and ncapg, are included in this list. Thus, Banp directly regulates transcription of wrnip1 for recovery from DNA replication stress, and cenpt and ncapg for chromosome segregation during mitosis. Our findings provide the first in vivo evidence that Banp is required for cell-cycle progression and cell survival by regulating DNA damage responses and chromosome segregation during mitosis.