Journal: Developmental biology
Article Title: Rbm8a deficiency causes hematopoietic defects by modulating Wnt/PCP signaling
doi: 10.1016/j.ydbio.2025.08.021
Figure Lengend Snippet: ( A ) Schematic of basic hematopoietic lineage and marker relationships. Created with Biorender.com (Subscription: Individual, Agreement number: IZ25WQERBS). AGM: aorta-gonadal-mesonephros area; ICM: intermediate cell mass; PBI: posterior blood island; CHT: caudal hematopoietic territory; VDA: ventral dorsal aorta. ( B – F ) Expression patterns for individual marker genes in wild type, morpholino-injected, rbm8a - and vangl2 -mutant zebrafish embryos at stage-matched 18 somites. Schematic of zebrafish embryo in the last panel in B-H shows where marker expression is expected and analyzed. ( B ) myoD as marker for somitic muscles shows comparable expression across conditions, with vangl2 mutants depicting the short, deteriorating tail typical for these mutants (asterisk). ( C ) The early endothelial marker sox7 is reduced in the trunk upon rbm8a perturbation (arrowheads) and seemingly normal in vangl2 mutants (asterisk). ( D ) The endothelial marker kdrl is decreased similarly as sox7 . ( E ) The erythroid marker gata1 is reduced in all rbm8a -perturbed embryo conditions (arrowheads) and only retained in posterior-most LPM cells of vangl2 mutants (asterisk). ( F ) The hematopoietic progenitor marker gfi1aa is reduced in all rbm8a -perturbed embryo conditions (arrowheads) and barely detectable in vangl2 -mutant embryos (asterisk). ( G ) In contrast, gfi1b is unchanged in all conditions. ( H ) The hematopoietic progenitor marker runx1 in rbm8a morphants and mutants is reduced or absent in the trunk and posterior blood island (PBI) (embryo midline, future dorsal aorta, arrowheads); vangl2 mutants also show reduced trunk expression (arrowhead) yet retain PBI runx1 expression (asterisk). ( I ) mRNA in situ hybridization-based quantification n of gata1 , gfi1aa , and gfi1b expression levels; individual data points (normalized signal intensity/embryo) shown with mean and standard deviation, significance calculated by Mann-Whitney test: gata1 ISH signal intensity in wild type vs. MO-rbm8aATG p < 0.0001, gfi1aa ISH signal intensity in wild type vs. MO-rbm8aATG p = 0,031, gfi1b ISH signal intensity in wild type vs. MO-rbm8aATG p = 0.7727 (not significant). ( J-M ) mRNA in situ hybridization-based quantification of runx1 in select areas (aorta-gonadal-mesonephros area (AGM): embryo midline, future dorsal aorta), posterior blood island (PBI), neurons). ( J ) Representative scale of runx1 signal ranked from low to high in rbm8a wild type siblings. The numbers on the bottom represent the average scores for AGM, PBI and neurons, respectively. ( K ) Signal in AGM. ( L ) Signal in PBI. ( M ) Signal in dorsal neurons. Individual data points (average of 4 scores per embryo) shown with mean and standard deviation, significance calculated by Mann-Whitney test: runx1a signal intensity in AGM ( K ) in wild type vs. rbm8a Δ5/ + p = 0.0048, wild type vs. rbm8a Δ5/Δ5 p < 0.0001, rbm8a Δ5/ + vs. rbm8a Δ5/Δ5 p = 0.0009; PBI ( L ) in wild type vs. rbm8a Δ5/ + p = 0.0077, wild type vs. rbm8a Δ5/Δ5 p < 0.0001, rbm8a Δ5/ + vs. rbm8a Δ5/Δ5 p = 0.0007; neurons ( M ) wild type vs. rbm8a Δ5/ + p = 0.1604 (not significant), wild type vs. rbm8a Δ5/Δ5 p = 0.005, rbm8a Δ5/ + vs. rbm8a Δ5/Δ5 p = 0.0481. Violin plot in background depicts population of the not averaged scores. Numbers (n) = average/total (see for details). Scale bar in ( B ): 100 μm, applies to all panels in B-H.
Article Snippet: The following primer or plasmids to generate templates for ISH probes were used: kdrl (using pBS-kdrl (a kind gift from Dr. Leonard I. Zon), linearized with EcoRI (R0101S, NEB), T7 polymerase for IVT) ( ); sox7 (using primer fwd 5 ′- CGACCAAAACTCCCTTCCG-3 ′ and primer rev 5 ′- AAAA TAATACGACTCACTATAG GGTTGTTGTAGTAGGCTGC-3 ′, T7 polymerase for IVT), gata1 (using primer f wd 5 ′- TGGGAAAGACAGTCCCAGG-3 ′ and primer rev 5 ′- AAAAA TAATACGACTCACTATAG GGCCTTCACACTAGTGTGGG-3 ′, T7 polymerase for IVT); runx1 (using pBS-runx1a , linearized with HindIII (R0104S, NEB), T7 polymerase for IVT) ( ) (a kind gift from Dr. Teresa V. Bowman); gfi1aa (using primer fwd 5 ′- TTATCATCAGCCCCGTTACC-3 ′ and primer rev 5 ′- AAAA TAATACGACTCACTATAG GGAATGGACGGCTTTATGTTGC-3 ′, T7 polymerase for IVT) ( ); gfi1b (using primer fwd 5 ′- ACCAACCTCAAACGAGAGC-3 ′ and primer rev 5 ′- AAAA TAATACGACTCACTATAG GGATTGTCCATCAACTTCTGTC-3 ′, T7 polymerase for IVT) , myoD (linearized with BamHI, T7 polymerase for IVT) , dlx3b (in pBS, linearized with SalI, T7 polymerase for IVT) , tbxta (in pBS-KS, linearized with XhoI, T7 polymerase for IVT) , and hgg1 (linearized with EcoRI, T7 polymerase for IVT) ( ).
Techniques: Marker, Expressing, Injection, Mutagenesis, Muscles, In Situ Hybridization, Standard Deviation, MANN-WHITNEY
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