Defining the ultrastructure of the hematopoietic stem cell niche by correlative light and electron microscopy
- University of Illinois at Chicago, United States
- University of California, San Diego, United States
- University of Wisconsin-Madison, United States
Abstract
The blood system is supported by hematopoietic stem and progenitor cells (HSPCs) found in a specialized microenvironment called the niche. Many different niche cell types support HSPCs, however how they interact and their ultrastructure has been difficult to define. Here we show that single endogenous HSPCs can be tracked by light microscopy, then identified by serial block-face scanning electron microscopy (SBEM) at multiscale levels. Using the zebrafish larval kidney marrow (KM) niche as a model, we followed single fluorescently-labeled HSPCs by light sheet microscopy, then confirmed their exact location in a 3D SBEM dataset. We found a variety of different configurations of HSPCs and surrounding niche cells, suggesting there could be functional heterogeneity in sites of HSPC lodgement. Our approach also allowed us to identify dopamine beta-hydroxylase (dbh) positive ganglion cells as a previously uncharacterized functional cell type in the HSPC niche. By integrating multiple imaging modalities, we could resolve the ultrastructure of single rare cells deep in live tissue and define all contacts between an HSPC and its surrounding niche cell types.
Data availability
SBEM datasets have been deposited in the National Center for Microscopy and Imaging Research (NCMIR) publicly accessible resource database Cell Image Library (CIL). There are six SBEM datasets (accession numbers: CIL:54845, CIL:54846, CIL:54847, CIL:54848, CIL:54849, CIL:54850) that are accessible as group with the following link: http://cellimagelibrary.org/groups/54850. CIL accession numbers are referenced in Table 1. Newly generated plasmids have been deposited in Addgene (#188944 and #188945).
Article and author information
Author details
Funding
National Heart, Lung, and Blood Institute (R01HL142998)
- Owen J Tamplin
National Institute of Diabetes and Digestive and Kidney Diseases (K01DK103908)
- Owen J Tamplin
American Heart Association (19POST34380221)
- Sobhika Agarwala
National Institute of Neurological Disorders and Stroke (1U24NS120055-01)
- Mark H Ellisman
National Institute of General Medical Sciences (R24 GM137200)
- Mark H Ellisman
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Cristina Lo Celso, Imperial College London, United Kingdom
Ethics
Animal experimentation: All experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committees at the University of Illinois at Chicago (Protocol ACC 19-051) and the University of Wisconsin-Madison (Protocol M006348).
Version history
- Received: November 12, 2020
- Preprint posted: November 13, 2020 (view preprint)
- Accepted: July 4, 2022
- Accepted Manuscript published: August 9, 2022 (version 1)
- Version of Record published: August 19, 2022 (version 2)
Copyright
© 2022, Agarwala 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.
Metrics
-
- 2,433
- views
-
- 682
- downloads
-
- 2
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Defining the ultrastructure of the hematopoietic stem cell niche by correlative light and electron microscopy
eLife 11:e64835.
https://doi.org/10.7554/eLife.64835
Further reading
-
- Developmental Biology
- Evolutionary Biology
Despite rapid evolution across eutherian mammals, the X-linked MIR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (SLITRK2 and FMR1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked MIR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernible defects, but simultaneous ablation of five clusters containing 19 members of the MIR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility, and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked MIR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the MIR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.
-
- Developmental Biology
We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism:
SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.
