Electronmicrographs of Type I cells.

<p>(A) Region of the niche near the vascular cavity (vc) showing Type I cells lining the edge of the cavity. Note the microvilli (mv) projecting into the cavity. <i>Zonulae adherens</i> join adjacent cells (double arrow head). (B) Type I niche cells near the emergence of the streams have elongated nuclei containing heterochromatin and euchromatin. A nucleolus (n) is apparent. Connective tissue (ct) is adjacent to the dorsal surface of the niche facing the accessory lobe (AL); a cell with features of a crustacean hemocyte (asterisk; hyaline cell, e.g., <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039267#pone.0039267-HoseJ1" target="_blank">[56]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039267#pone.0039267-ChavesdaSilva1" target="_blank">[57]</a>) is located within this tissue, although a blood vessel is not apparent. (C) Type I cells contain an abundance of mitochondrial profiles (white arrow), rough endoplasmic reticulum (black arrow), polysomes (arrowhead) and vesicles. (D) Cytoplasm of type I cells in high magnification: Observe the numerous mitochondria (arrow) and microtubules, seen in detail in the insert, lower left. (E) Another view of the apical surface of a Type I cell, showing a <i>zonula adherens</i> between adjacent cells, as well as microvilli projecting into the lumen of the vascular cavity (vc). The insert (upper right) shows a higher magnification of a <i>zonula adherens</i>. (F) Type I cells united by a septate junction. Scale bars: (A) 0.5 µm; (B) 5 µm; (C) 1 µm; (D, E) 0.5 µm; Inserts in (D) 0.1 µm and (E) 0.5 µm; (F) 0.2 µm.</p

Ultrastructure of the neurogenic niche cells.

<p>(A) Type I cells have nuclei that are frequently elongated; these contain both heterochromatin and euchromatin, and a nucleolus (n) is often seen. Mitochondrial profiles (white arrow) and rough endoplasmic reticulum (double arrowhead) are abundant. In the upper part of the image note the processes (arrows) projecting into the vascular cavity (vc).The vascular cavity is colorized yellow to highlight the processes of the niche cells projecting into this area. (B) Type II cells have a nucleus containing abundant euchromatin and sparse heterochromatin. Note mitochondrial profiles (arrow) and many vesicles in the cytoplasm. (C) Type III cells display a nucleus composed of clumps of heterochromatin. Their cytoplasm contains vesicles, some of them with membranes in their interior (black arrow), as well as a Golgi apparatus (outlined in C and magnified in the insert). (D) Type IV cells are found in pairs, suggestive of cell division, however, no shared plasma membrane between the two nuclei is visible. In the cytoplasm observe mitochondrial profiles (white arrow), dilated vesicles and cisternae of the endoplasmic reticulum (double arrowhead). Scale bars: (A–D) 2 µm. Insert in (C), 0.5 µm.</p

Electron micrographs of the vascular cavity.

<p>(A) Type I cells lining the vascular cavity (vc), which contains granular electron dense material. Note the microvilli (mv) projecting to the lumen of the cavity. Also observe the <i>zonulae adherens</i> joining adjacent cells (double arrow head). (B) A higher magnification showing the rim of the vascular cavity in another area. Note again the electron dense material with a globular organization, sometimes aggregated in clumps (black arrow) in the lumen of the vascular cavity. Clumps are better seen in (C). (D) High magnification of microvilli of a type I cell containing many microfilaments (arrow). (E, F) Higher magnifications of the round blobs composing these clumps. Scale bars: (A) 2 µm; (B) 1 µm; (C) 0.2 µm; (D, E) 0.2 µm; (F) 0.1 µm.</p

Stacked confocal scans of a 100 µm sagittal section in a <i>P. clarkii</i> brain show the neurogenic niche (A–D), stained for GS (blue), protruding from the ventral surface (arrow, red outline) of the accessory lobe (AL).

<p>Serotonergic labeling (5-HT) in the olfactory lobe (OL) and AL (A–D, green) reveals fibers from the Dorsal Giant Neuron (DGN) innervating these lobes (A and Aii; white arrows). 5-HT labeling of individual glomeruli (dotted outlines in Aii) and DGN fibers (Aii, arrows) are clearly shown in this section of the AL. Magnifications of this section, including images deeper into the tissue (Ai, B, C) display the three separate laser channels representing the nuclear stain, propidium iodide (PI, red), serotonin (5-HT, green) and glutamine synthetase (GS, blue); PI and GS labeling together reveal the multi-layered aspect of the niche when the brain is sectioned sagittally (B, C, D). In C and D, white arrows indicate the GS-labeled cytoplasm (blue) of the niche cells, demonstrating that the deepest, most dorsal niche layer (outline in D) does not label for GS (dotted line in C, lower panels). The PI labeling cells also delineates the trail of cells, composing a blood vessel that extends from within the AL into the niche itself (Ai, white square; B,white square; Bi, arrow). Higher magnifications show a serotonin immunoreative “crown” of terminals within the outer, ventral layer of the niche (C, asterisk); a weakly immunoreactive serotonergic fiber(s) (arrowheads) is observed connecting to this serrated-like region. Scale bar: (A) 200 µm; (Ai, Aii, B) 20 µm; (D) 10 µm; (Bi, C) 5 µm.</p

Neurogenic niche in the adult brain of the crayfish <i>P. clarkii</i>.

<p>(A) Schematic diagram of the crayfish brain showing cell clusters 9 and 10 (circled), and the olfactory and accessory lobes. (B) The lateral proliferation zone (LPZ) in cluster 10 and the medial proliferation zone (MPZ) in cluster 9, connected with each other on the ventral surface of the deutocerebrum by the migratory streams and the neurogenic niche, labeled with an antibody against glutamine synthetase (blue). BrdU (green) labels cells in the niche, streams and proliferation zones. The nuclear marker propidium iodide (red) labels cell body regions, clusters 9 and 10. (C) Model summarizing a view of events leading to the production of new olfactory interneurons in adult crayfish. Neuronal precursor (1^st-generation) cells reside within a neurogenic niche where they undergo mitosis. Their daughters (2^nd-generation precursors) migrate along tracts created by the fibers of the niche cells, towards either the lateral proliferation zone (LPZ) or medial proliferation zone (MPZ). At least one more division will occur in the LPZ and MPZ before the progeny (third and subsequent generations) differentiate into neurons. (D) The connection between the cavity in the center of the niche and the vasculature was demonstrated by injecting dextran tetramethylrhodamine (3,000 MW) into the dorsal cerebral artery. The niche sits on top of a blood vessel (arrows). The cavity (asterisk), outlined by anti-Elav (green) labeling, is filled with the dextran dye (red). Propidium iodide (blue) labeling of the nuclei of niche cells is also shown. Inset: shows vasculature of the olfactory (OL) and accessory (AL) lobes labeled with dextran dye. (Images adapted from Beltz et al., 2011). Scale bars: (B) 100 µm; (D) 25 µm; insert in D, 100 µm.</p

Alex Fluor® 568 hydrazide dye injection of Type I niche cells reveals their bipolar morphology.

<p>Each Type I cell has a long process that stretches to either cluster 10 or cluster 9. (A) Montage showing the long process of a dye-injected cell, projecting towards cluster 10. Arrowheads point to the fascicle of Type I cell processes, labeled for GS, that form the migratory stream, and a short process that extends to the vascular cavity (B). Arrow in (B) points to the cell body, where the dye was injected. The injection of dye into Type I cells results in deposition of the dye in the vascular cavity (B and C). (C) A dye fill of a different Type I cell in which the niche is labeled for GS and stained with DAPI, shows the vascular cavity containing Alexa Fluor®, surrounded by the niche cells. (D–G) Additional ultrastructural details of the cytoplasm of Type I cells. (D) Vesicles (arrows). (E) Note rough endoplasmic reticulum (arrowheads) and some clumps of ribosomes (arrows). (F) Mitochondria (mt). (G) Golgi apparatus (arrow). Examples are colorized green. Scale bar: (B, C) 10 µm; (D) 2 µm; (E) 1 µm; (F) 0.5 µm. (G) 0.5 µm.</p

The niche and surrounding tissues.

<p>(A) Semi-thin section showing the niche and the central vascular cavity (vc). The connective tissue (ct) below the niche (colorized purple) has many cells (arrows) similar to that seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039267#pone-0039267-g007" target="_blank">Figure 7</a> B (asterisk), with features resembling hemocytes. (B) A stacked confocal image of the neurogenic niche labeled with glutamine synthetase (GS, blue) and stained for DAPI (cyan) and the lectin ACL (green); separated emission channels are shown in Bi (GS), Bii (DAPI), Biii (ACL). ACL delineates the vasculature lying underneath (as in A) and enveloping the niche (compare also with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039267#pone-0039267-g004" target="_blank">Figure 4</a> D, E). Biv is the composite of the GS and ACL channels only, showing the close apposition of the two labels, but no overlap. A cell (arrow in B) without cytoplasmic GS labeling and with the morphological features of hemocytes is detected in the vasculature/connective tissue (ACL). (C) A confocal image (serotonin, green; GS, blue; PI, red) reveals a cell (arrow) with the morphological characteristics of a hemocyte (as in 8A, B) in the most dorsal layer of the niche, which also (as in 8B) is not delineated by GS labeling (Ci, single channel for GS). Cells lining the vasculature (perivascular cells) do contain GS label. Scale bar: (A) 10 µm; (B) 20 µm; (C) 10 µm.</p

Combined confocal images (B, C) of PI-labeled cells in the niche (A) and Nomarski optics showing bubble-like material in the vascular cavity (higher magnification in C; rectangle in A and B).

<p>These structures of unknown origin are also observed in EM sections (e.g., 9B–C, E–F) that show the material to be composed of electron dense particles. Scale bar: (A) 40 µm.</p

Confocal images of a sagittal niche section (A–C) with separate channels displayed in A (5-HT, green; GS, blue; PI, red; arrow pointing to the niche in A), reveal more dorsal aspects of the niche (B and C) and how cells are organized in regions just below the niche.

<p>Corresponding semi-thin (E) and ultra-thin sections (F–G) through regions indicated in B reveal perivascular cells lining a blood vessel. (D, E) A semi-thin section in two magnifications. Note the vascular cavity (vc) and the lumen of two vessels, one below and one above the niche (colorized in pink). Within one of them a granular hemocyte is evident (white arrow). Observe the thin layer of connective tissue limiting the outermost part of the niche (black arrow, D), and in the innermost region, the loose connective tissue (ct) where there is a vessel. (F, G) Electron micrographs of a vessel dorsal to the niche in two magnifications, showing the perivascular cells (arrowheads) lining the vessel, with nuclei composed of heterochromatin apposed to the nuclear envelope, and euchromatin, which constitutes the major part of the nucleoplasm. Also, a nucleolus (n) is seen in one of the nuclei. (G) The cytoplasm of the perivascular cells (arrowhead) showing mitochondrial profiles (white arrow and insert [higher magnification of area limited by the white square]) and many endoplasmic reticulum cisternae (black arrow). Accessory lobe (AL in D and F). Scale bars: (A) 50 µm; (B and C) 6 µm; (D) 35 µm; (E) 20 µm; (F) 5 µm; (G) 2 µm; Insert in (G), 1 µm.</p

Scanning electron micrographs of the brain showing the niche and migratory streams.

<p> (A) The neurogenic niche is seen as a swelling in the central part of the migratory stream on the surface of the accessory lobe (AL). The dotted line delineates the posterior edge of the niche and proximal part of the streams. Note the vascular cavity (arrow) centrally in the niche. (B) The migratory stream on the surface of the accessory lobe (AL) (the dotted line marks the anterior edge of the stream) and the two clusters of cell bodies, cluster 9 (CL 9) and cluster 10 (CL 10). Note that anterior is to the left in the image, and medial is at the top. The niche is not observed, but appears to be obscured by connective tissue. (C) Higher magnification of (B). The migratory stream broadens as it approaches cell cluster 9 (the stream is colorized yellow). The depression (arrow) marks the position of a cell and its processes. Scale bars: (A, B) 50 µm; (C) 20 µm.</p