Journal: bioRxiv

Article Title: Distributed neural computation and the evolution of the first brains

doi: 10.1101/2025.10.03.680388

Figure Lengend Snippet: a) Simplified phylogeny of animals shows that acoel brains are likely intermediate between cnidarian diffuse nets and the centralized brains of typical bilaterians. b) Photograph of juvenile Hofstenia miamia . c) Staining with voltage dye reveals a superficial network of dense neuropil (blue arrow) that extends into a sparser posterior nerve net (green arrow). d) Close-up view of neuropil stained sparsely with tubulin dye (orange) reveals that the neuropil (orange) contains many neurites running in parallel, with cellular clusters (cyan) interspersed between neurite bundles. Sensory neurons (likely clusters of H1 cells; bright orange) are set within many of these patches. e) Cross-section of brain stained with a Par3 antibody reveals that the brain has two layers: superficial neuropil, and deeper cell bodies that project outward. f) Staining with an ERK antibody (z-projected segmentation overlaid) shows that brain interneurons can be multipolar, with a central cell body generating multiple neurites. g) Cross-section of brain stained with an antibody against β-catenin reveals another sensory neuron class (possibly H2 ) with two projections that innervate brain neuropil. h) Electron microscopy cross-section shows the fine organization of the brain, confirming the relative configuration of tissue types within the head. The superficial neuropil (previously ‘layer 1’) is visible immediately beneath the skin, while neural cell bodies (previously ‘layer 2’) lie deeper in the tissue, internal to body wall muscle (green). Together, these layers compose the brain. i) Electron microscopy close-up of the brain shows dense neuropil; the box is a 6.7×6.7µm square. j) Segmenting neural projections within the highlighted box in (i) reveals over 400 neurites in a single section of neuropil. k) Segmentation of cellular clusters within neuropil allows quantification of brain structure and its variability. l) Quantifying the numbers of cellular clusters across brains reveals that, although cluster numbers increase with age (i.e. days after hatching) and size (i.e. head width, a good proxy for overall body size ), worms vary widely in how many clusters they possess. Linear regression p<0.0001, n=49. Scale bars: 200µm (c), 50µm (d,e), 20µm (f,g), 10µm (h).

Article Snippet: Primary antibodies used: Par3 (St. John’s Laboratory #STJ94951, 1:200), pERK (Cell Signaling Technologies #4370T, 1:200) , FMRFamide (EMDMillipore #AB15348, 1:1000) .

Techniques: Staining, Electron Microscopy

Journal: Developmental cell

Article Title: ?1 integrin establishes endothelial cell polarity and arteriolar lumen formation via a Par3-dependent mechanism

doi: 10.1016/j.devcel.2009.12.006

Figure Lengend Snippet: Luminal occlusion and loss of polarity is an arterial phenomenon. (A) Microarray analysis of endothelial cells (ECs), sorted based on β1 integrin protein expression (from E16.5 β1f/f;Cre+ embryos), exhibited increased levels of adhesion related genes, and a decrease in the polarity gene Par3. (B) Loss of β1 integrin protein (green) results in mis-localization of PECAM-1 (red) from laterally placed cell-cell contacts to global cell surface expression (arrows). TOPRO-3 nuclear stain in blue. (C) To confirm loss of β1 integrin and Par3 on a protein level, ECs sorted in the same manner were evaluated by Western blot. Sorted β1+ and β1- endothelial cells from β1f/f;Cre+ mice, and β1+ ECs from β1f/+;Cre+ mice were loaded equally by cell number, and demonstrated loss of β1 and Par3 protein (α-enolase loading control). (D) Normally polarized expression of VE-cadherin (top panel in red, SMA in green) at lateral cell-cell contacts (arrows) is dispersed and circumferentially expressed in cells occluding the lumen within β1f/f; Cre+ vessels (arrows). CD99 (bottom panels, green) demonstrates a polarized apical expression (arrowheads), and lateral co-localization with PECAM-1 (yellow, arrows), that after β1 integrin ablation redistributes to surround the cell at E15.5 (arrows), much like PECAM-1 (in red). (E) Luminal occlusion is distinctly noted in arteries (arrows), as delineated by PECAM-1 (top panels in black, bottom panels in red) and lack of EphB4 (bottom panels, green) expression. “A” denotes arteries, “V” veins, and “L” lymphatics. (F) The extent of occlusion can vary in mid-sized arteries (arrows) at E15.5 after endothelial β1 integrin deletion, but as compared to nearby veins is a distinctly arterial phenomenon. Lower panels are high magnification of vessels in upper panels. PECAM-1 in black. (G) Vessels were evaluated at E15.5, quantified for luminal patency, and confirmed that the phenotype is exclusively arterial (n=5 each, *p< .001). (H) Par3 protein expression is preferentially expressed in embryonic arteries, as evidenced in β1f/f;Cre- at E15.5 (Par3 in red, β1 integrin in green, TOPRO-3 nuclear stain in blue). Panels (a) – (c) are higher magnification of A – V pair above. (H-a,b) Par3 co-localizes with β1 integrin (yellow) in the basal aspect of the arterial endothelial layer (arrows), but is also prominent in the surrounding smooth muscle cell layer (in red, arrowheads). (H-c) Veins also express Par3 in conjunction with β1 (yellow, arrows), but to a lesser extent than arterial vessels. (I) To evaluate Par3 expression in vessel subtypes, dorsal aortas (A) and inferior vena cavas (V) of 4 week old and adult animals were flushed with Laemmli buffer and evaluated by Western blot. Primary human endothelial cells were evaluated for Par3 expression among vessel subtypes (human aortic endothelial cells – HAECs, human umbilical vein ECs - HUVECs, and human saphenous vein ECs – HSVECs). GAPDH and α-enolase loading controls. (J) Normal basal expression of Par3 (red) with co-expression of β1 integrin (in green, co-localization in yellow) in β1f/f; Cre- vessels (arrows) is aggregated and mis-localized in β1f/f; Cre+ vessels (arrows), with complete absence in β1 deleted cells within the vessel lumen (arrowheads). TOPRO-3 nuclear stain in blue. (B, D-F, H, J) Scale bars as labeled for each row. See also Figure S3.

Article Snippet: Separately, tissue sections also underwent Tyramide Signal Amplification System with Alexa Fluor secondary antibodies (Invitrogen) per manufacturer’s instructions with β1 integrin 1:500 (Chemicon), PECAM-1 1:500 (raised in rabbit, a generous gift from Josephine Enciso and Joseph Madri, Yale University, CT), Par3 1:300 (Upstate Biotechnology), Rab7 1:500 (Cell Signalling), and TOPRO-3 (Invitrogen) nuclear stain.

Techniques: Microarray, Expressing, Staining, Western Blot, Control, Labeling

Journal: Developmental cell

Article Title: ?1 integrin establishes endothelial cell polarity and arteriolar lumen formation via a Par3-dependent mechanism

doi: 10.1016/j.devcel.2009.12.006

Figure Lengend Snippet: Postnatal retinal β1 integrin deletion results in Par3 loss and abnormal endothelial cell polarity. (A) Wild-type (WT) retinal endothelial cell protein evaluated by Western blot demonstrates Par3 levels peak at P6-P9. (B) Par3 (red) is expressed in WT retinal vessels (P10) with β1 integrin (green, arrows). TOPRO-3 nuclear stain in blue. (C) Postnatal β1 integrin ablation induced by tamoxifen injection (from P2 to P7) in the β1f/n; iCre+ retina, results in large cyst-like outgrowths from the vasculature at P9 (arrows in right panel). Isolectin B4 (IsoB4) in red. (D) Postnatal tamoxifen induction when traced using a LacZ R26R Cre reporter line (R26R; iCre+) labels a small subset of retinal endothelia at P7, as compared to constitutive expression (R26R; Cre+). Arteries (A) and veins (V) labeled respectively. (E) After postnatal induction retinal endothelial cells were isolated (at P9) and evaluated by Western blot. β1 integrin protein is notably decreased in β1f/n; iCre+, as is Par3 protein levels (right column). (F) When β1f/n; iCre+ crossed to a EYFP R26R reporter undergoes β1 ablation, the β1 deleted retinal ECs (EYFP+ in green, arrows) become abnormally located in the vasculature in cyst-like structures (labeled by IsoB4 in red) and demonstrate loss of β1 protein (blue). (B-D, F) Scale bars as included for each row. See also Figure S4.

Article Snippet: Separately, tissue sections also underwent Tyramide Signal Amplification System with Alexa Fluor secondary antibodies (Invitrogen) per manufacturer’s instructions with β1 integrin 1:500 (Chemicon), PECAM-1 1:500 (raised in rabbit, a generous gift from Josephine Enciso and Joseph Madri, Yale University, CT), Par3 1:300 (Upstate Biotechnology), Rab7 1:500 (Cell Signalling), and TOPRO-3 (Invitrogen) nuclear stain.

Techniques: Western Blot, Staining, Injection, Expressing, Labeling, Isolation

Journal: Developmental cell

Article Title: ?1 integrin establishes endothelial cell polarity and arteriolar lumen formation via a Par3-dependent mechanism

doi: 10.1016/j.devcel.2009.12.006

Figure Lengend Snippet: Par3 partially rescues lumen occlusion and cyst formation in endothelial β1 integrin ablation. (A-F) Postnatal animals (β1f/n; iCre+) were induced with tamoxifen (P2-P7) and retinal vasculature evaluated from P9-12. A subset was then rescued with Par3 lentiviral ocular delivery 48hrs prior to evaluation. (A) The large cysts observed with postnatal β1 integrin ablation were resolved significantly with lentiviral Par3 replacement, but a few endothelial cells (ECs) still displayed smaller atypical aggregates (arrows). Isolectin B4 (IsoB4) in red. (B) When β1f/n; iCre+ crossed to EYFP R26R (in green) retinas were rescued with FLAG-tagged Par3 lentivirus (blue), a rescued vessel demonstrates a patent lumen (arrowheads). In contrast, ECs that underwent β1 deletion (green) but were not rescued remain abnormally shaped with an occluded lumen (arrows). Asterisk denotes EYFP+ red blood cell. (C) On analysis of retinal semithin sections, β1 ablation resulted in vessel occlusion (arrowheads) that was partially rescued with Par3 protein (arrows). Boxed areas are magnified on right. (D) Retinal vessels were quantified for percentage of deletion (left) by assessing β-gal positive endothelial cells within the abdominal muscle - tamoxifen group (dark grey), tamoxifen + Par3 rescue group (light grey). The number of occluded vessels was quantified and compared to percent deletion in a ratio (right). The β1f/n; iCre+ tamoxifen group (dark grey) demonstrates that the occlusion phenotype is in direct proportion to the amount of deletion. There is a significant (*) decrease in the ratio of occluded vessels (%) to percent deletion with Par3 rescue (light grey). Data shown as mean +/- SEM, n=7 each group, p value = 0.025. (E) On a per animal basis, the percentage of deletion varies (circles), but averages at approximately 30% for both groups; while the percentage of occlusion (triangles) is dramatically reduced from 27% in the non-rescue to 16% in the rescued group. (F) Higher magnification depicting the vessel occlusion (or patency in the rescue) that was quantified in the retina (arrow). (A-C, F) Scale bars as labeled for each row. See also Figure S6.

Article Snippet: Separately, tissue sections also underwent Tyramide Signal Amplification System with Alexa Fluor secondary antibodies (Invitrogen) per manufacturer’s instructions with β1 integrin 1:500 (Chemicon), PECAM-1 1:500 (raised in rabbit, a generous gift from Josephine Enciso and Joseph Madri, Yale University, CT), Par3 1:300 (Upstate Biotechnology), Rab7 1:500 (Cell Signalling), and TOPRO-3 (Invitrogen) nuclear stain.

Techniques: Labeling

Journal: Developmental cell

Article Title: ?1 integrin establishes endothelial cell polarity and arteriolar lumen formation via a Par3-dependent mechanism

doi: 10.1016/j.devcel.2009.12.006

Figure Lengend Snippet: Arrest of lumen formation and excess of cytosolic vacuoles in β1 deleted endothelial cells (ECs). (A) Semithin (left) and EM (right) analysis of luminal occlusion in E15.5 β1f/f;Cre+ animals demonstrates accumulation of multiple vesicles/vacuoles within the cell cytoplasm (arrows). (B) Evaluation of Rab7 (red) in context of β1 integrin (green) demonstrates Rab7 in the basal aspect of the endothelium and at the smooth muscle cell (SMC)/ EC junction with β1 integrin (yellow), but minimal expression within the endothelial layer (arrowhead). Upon β1 deletion, a dramatic increase of Rab7 expression in β1 ablated ECs was noted (right panel, arrowheads) with some maintenance of co-expression with β1 integrin in the SMC layer (arrow). (C) Schema depicts a working model of the cascade of events that drive lumen formation. Activation of β1 integrin instructs Par3 expression which then allows for polarization of the endothelium. Vesicular fusion to the apical cell membrane results in redistribution of junctional and adhesion proteins (in red, arrows), change in cell shape from cuboidal to squamous, and acquisition of a vascular lumen.

Article Snippet: Separately, tissue sections also underwent Tyramide Signal Amplification System with Alexa Fluor secondary antibodies (Invitrogen) per manufacturer’s instructions with β1 integrin 1:500 (Chemicon), PECAM-1 1:500 (raised in rabbit, a generous gift from Josephine Enciso and Joseph Madri, Yale University, CT), Par3 1:300 (Upstate Biotechnology), Rab7 1:500 (Cell Signalling), and TOPRO-3 (Invitrogen) nuclear stain.

Techniques: Expressing, Activation Assay, Membrane