In focus in HCB

In this first Editorial for 2021, and our 50th overall (!), we highlight four Original Articles describing (1) the intracellular localization of the guanine nucleotide exchange factor EGFP-fused DENND1B in several different cell lines by live-cell confocal microscopy); (2) the phenotypic and functional characterization of cardiac macrophages during heart development; (3) the increased level of the redox protein thioredoxin 1 (Trx1) in the bone and bone marrow following ischemi-reperfusion injury; and (4) description of a new protocol for double whole-mount ISH combined with immunoperoxidase staining for analyzing cellular gene expression patterns during forelimb development in avian embryos. We wish you good reading!

A connecdenn family member forms gathered line structure

The DENND1 family is comprised of three members, DENND1A-C, which function as guanine-nucleotide exchanging factors for the small G protein Rab35. This function is located in the N-terminus of the highly conserved DENN domain, whereas the clathrin-binding and the adaptor protein-2-interaction motif are part of the C-terminus (Marat et al. 2011). DENND1B/connecdenn 2 is known to be involved in clathrin-mediated endocytosis and fast recycling of megalin (Shah et al. 2013). In the current work, Park et al. (2021) investigated the intracellular localization of EGFP-fused DENND1B in several different cell lines by live-cell confocal microscopy. In the renal epithelial cell line BS-C-1, DENND1B exhibited two patterns: straight-line stress fiber-like structures, and a hitherto unknown gathered line structure. The gathered line structures were located at the bottom of spreading lamellipodia and as shown by optogenetic experiments, local activation of Rac1 may be important for their formation in this subcellular locale. They disappeared at the retracting site during cell movement in EGF-stimulated BS-C-1 cells indicating a relationship to cell migration. A complex relationship of the gathered line structures with cytoskeletal elements was revealed. F-actin bundles were observed to surround clusters of gathered lines but were not in direct contact with them (Fig. 1).

Fig. 1

a BS-C-1 cells transfected with pEGFP-DENND1B (green) and processed for F-actin staining with Alexa Fluor 594-phalloidin (red). The fluorescence signals of Alexa Fluor 594-phalloidin overlapped with those of EGFP-DENND1B in the straight-line structure (arrowheads), but not in gathered line structures (open arrows). From Park et al. (2021)

However, cytochalasin D treatment resulted in the disappearance of the gathered line structures indicating that the surrounding F-actin bundles may be indirectly important for their stability. The gathered lines were also partially associated with microtubules. When microtubules were depolymerized by nocodazole, gathered lines were disrupted indicating an involvement of microtubules in their formation and/or maintenance. Since the DENND1B-localized gathered line structures could not be observed in cells coexpressing DENND1B and Rab35, it was specualated the formation and/or the recruitment of DENND1B to the gathered line structures was hampered by Rab35. Last but not least, by the expression of DENND1B truncation mutants, the localization of DENND1B to gathered line structures was shown to be dependent on the aa 279–692 region located in the C-terminus of DENND1B protein.

Getting to the heart of embryonic cardiac macrophages

Macrophages have often been considered as immune system-derived monocytes, circulating throughout the body and directed towards specific areas of need when required. However, more recently, mounting evidence indicates that macrophage phenotypes are tissue-specific, and the origin of tissue macrophages has become a topic of wide-spread interest in cell biology (Epelman et al. 2014). Gula et al. (2021) have now performed a series of experiments designed to characterize the phenotypes and functions of cardiac macrophages during development of the murine heart. They utilized a variety of experimental techniques including multi-label immunofluorescence for cell phenotype determination on frozen sections and tissue whole-mount preparations imaged by both conventional wide-field and confocal microscopy, flow cytometry on isolated embryonic cardiac cells, and RT-PCR on sorted single cell suspensions. For the immunofluorescence studies, fetal hearts from days E11–E18 were used, and for the flow cytometry and RT-PCR fetal hearts from E14 and E17. The phenotype of cardiac macrophages was determined by staining with antibodies for CD45, CD68, CD64, F4/80, CD11b, CD206 and Lyve-1 (Fig. 2).

Fig. 2

Confocal images from whole-mount preparations of embryonic rat heart. a Lyve-1 + single stained cells located at myocardial surface. b Merged confocal images showing cells co-staining with Lyve-1 and CD68 or CD45. la, left atrium; lv left ventricle; rv, right ventricle; ot, outflow track. From Gula et al. (2021)

Taken together, the results provided the following details concerning embryonic cardiac macrophages: (1) by immunofluorescence they were founded in greatest abundance in the subepicardial space, as opposed to adult animals where they are located throughout the entire myocardial wall; and (2) their migration into the heart followed pathways of developing blood vessels and lymphatic vessels; (3) by flow cytometry, their heterogeneity was highlighted, and three newly defined subpopulations were identified: CD64low, CD64highCD206, and CD64highCD206+; and (4) by RT-PCR analysis differences were observed between the various macrophage phenotypes and embryonic stage regarding genes involved in angiogenesis, extracellular matrix remodeling, and lymphangiogenesis. Given this characterization, the authors suggest that cardiac tissue macrophages may be involved in diverse functions during cardiac development, as well as in pathological processes.

Reactive oxygen species in a remote organ response to myocardial infarction

Oxidative stress, associated with production of reactive oxygen species (ROS) is known to be involved in a myriad of disease and pathological situations via the stimulation of inflammatory processes (van der Vliet and Janssen-Heininger 2014). Indeed, ROS have been shown to be generated by subsequent reperfusion following myocardial ischemic events. Hydrogen peroxide as a diffusible ROS is known to effect redox signaling in sites distant from their tissue origin (Lismont et al. 2019). Moreover, leukocytes originating in the bone marrow appear to be the main cell types infiltrating the myocardium during reperfusion, and themselves are sensitive to alterations in redox status. This has led Godoy et al. (2021) to investigate the redox regulatory system proteins of the thioredoxin (Trxs; regulate protein thiol groups) and peroxiredoxins (Prxs; regulate hydrogen peroxide) families in myocardium, kidney, bone and bone marrow from rats following myocardial infarction-induced reperfusion. For their immunohistochemistry, Western blotting and ELISA experiments they used very carefully validated antibodies, with the data available in the “Redox Atlas of the Mouse” webpage (

Fig. 3

a Immunohistochemical localization of Trx1 in rat femur in sham-operated and myocardial infarcted animals. Obl, osteoblasts; Mk, megakaryocytes; HPC, other hemaopoietic cells; BLC, bone lining cells; PC, zone of proliferating cartilage; HC, zone of hypertrophic cartilage; nu, nucleus. From Godoy et al. (2021)

Their results showed that following reperfusion and compared to sham-operated animals, (1) Trx1 levels were increased in the heart and femur; (2) in the femur and lumbar vertebrae, the Trx1 elevation was found to be associated with bone-lining cells, osteoblasts, and megakaryocytes (Fig. 3); (3) the increased Trx1 expression in heart and bone was aslo demonstrated by RT-PCR; and (4) elevation of Trx1 serum levels; and (5) treatment of animals with the glutathione precursor N-acetyl cysteine resulted in a reduction of Trx1 immunoreactivity in bone marrow precursor hematopoietic cells; and (6) no significant alterations in the expression of the other Trx and Prx family proteins was observed. The authors propose a model illustrating the mechanism of Trx1 upregulation in the bone marrow following myocardial infarction, illustrating the remote organ effect of ischemia–reperfusion injury.

Identification of avian cell lineages sharing markers by double ISH and IHC

The chemokine receptor CXCR4 is involved in the control of cell migration during limb and cloacal muscle formation (Hunger et al. 2012) as well as of sympathetic ganglia progenitor cells, (Kasemeier-Kulesa et al. 2010). The migratory path of the mesodermally and neural crest-derived cells occurs in close spatial relationship and in addition the two cell lineages have markers in common. Thus, the in-situ identification of the two cell lineages poses some difficulties. In their present work, Yahya et al. (2021) report a protocol for double whole-mount ISH combined with immunoperoxidase staining for analyzing gene expression pattern in mesodermal and neural crest cells during forelimb development in avian embryos (Fig. 4).

Fig. 4

Combined double whole-mount ISH and immunostaining for CXCR4, Sox10, Ap2α, and HNK1 in stage HH18-19 and HH 22–23 embryos. CXCR4 appears in blue, Sox10 and Ap2α in red, and HNK1 in brown. From Yahya et al. (2021)

Specifically, they analyzed the expression of CXCR4, Myf5, Pax3, Sox10, Ap2α and Slug as well as Nkx2.2, HNK1 and desmin. For ISH, digoxigenin- and FITC-conjugated riboprobes were used and together with immunoperoxidase IHC this permitted the simultaneous detection of specific mRNAs and the respective proteins. As an example of the application of the protocol, the expression pattern of mesodermal and neural crest cells during forelimb development in embryos ranging from HH18 to HH25 stage was analyzed.


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Taatjes, D.J., Roth, J. In focus in HCB. Histochem Cell Biol 155, 1–8 (2021).

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