Ohlebusch et al., 2020 - Investigation of alpl expression and Tnap-activity in zebrafish implies conserved functions during skeletal and neuronal development. Scientific Reports   10:13321 Full text @ Sci. Rep.

Figure 1

(A) Comparison of alpl orthologues between human, mouse and zebrafish. (B) Conservation of synteny between human, mouse, and zebrafish. Neighboring genes of the ALPL (Chr. 1), Alpl (Chr. 4), and alpl (Chr. 11) genes display similar order among the three organisms, which indicates a high level of conservation between species. The zebrafish alpl gene is not duplicated within the genome. The direction of the arrows indicates the orientation of the respective gene in the genome. Grey arrows and names indicate genes only detectable in single species. Genomic data was obtained from the ENSEMBL (genome assembly: GRCh38.p13, GRCm38.p6, GRCz11) and Genomicus v98.01 databases.

Figure 2

alpl expression during zebrafish embryogenesis. (A) and (B) Spatio-temporal expression pattern of alpl in zebrafish embryos at early segmentation (3–12-somites, A) until long-pec stages (48 hpf, B). (C) Detail images of head regions and of sections stained via ISH for alpl at long-pec stadium. Dashed white lines indicate level of sections shown at higher magnifications (1–4). Scale bar: 100 µm. Ao, dorsal aortic root; Cb, cerebellum; Dc, diencephalon; He, heart tube/heart; Le, lens; Mc, mesencephalon; MHB, midbrain-hindbrain boundary; Ne, nephros; No, notochord; Nt, neural tube; Pf, pectoral fin; Rc, rhombencephalon; Re, retina; Som, somites; Tb, tail bud.

Figure 3

Expression pattern of alpl and activity of Tnap in adult zebrafish tissues. (A) alpl expression in different organs of adult zebrafish at the mRNA level determined with qPCR targeting exon 5/6. RQ: relative quotient (here calculated in comparison to the expression level of muscle samples). Statistics were calculated using two-sided student t-test, paired; ***p ≤ 0.001, **p < 0.01, *p < 0.05, n.s., not significant; ref, reference. (B) Enzymatic AP-activity in different organs of adult zebrafish measured with a CSPD-assay after treatment with different concentrations of the specific inhibitor levamisole. **p < 0.01, *p < 0.05. Normalization was performed using values for non-treated muscle samples. n = 3, technical replicates.

EXPRESSION / LABELING:
Gene:
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Anatomical Terms:
Stage: Adult

Figure 4

alpl expression pattern in whole adult zebrafish brains. (A) Scheme of different brain regions used for qPCR. (B) alpl expression at the mRNA levels determined by qPCR targeting exon 5/6 in different regions of adult zebrafish brain. Statistics were calculated using two-sided student t-test, paired; **p < 0.01, *p < 0.05, n.s., not significant; ref, reference. (C) ISH visualized expression domains of alpl in whole adult brains (marked by black and white arrows). Signals were detected in the telencephalon, tectum opticum and hypothalamus. Vibratome cross sections of single regions showed tissue-internal alpl expression. Control samples were hybridized with sense probe instead of antisense probe. Scale bar: 250 µm (whole brain images); 100 µm (close up view and sections). CCe, corpus cerebelli; Dl/Dd, lateral/dorsal zone of telencephalon; Hd, dorsal zone of the periventricular hypothalamus; IL, inferior lobe of hypothalamus; LR, lateral recess of diencephalic ventricle; Mo, medulla oblongata; PG, preglomerular area; PGZ, peripheral growth zone (layer 1 and 2); PiT, posterior inferotemporal cortex; Tel, telencephalon; TeO, tectum opticum; TH, tuberal hypothalamus; Val, ventral anterior thalamic nucleus; lateral part; Vam, ventral anterior thalamic nucleus; medial part.

Figure 5

alpl expression pattern (combined with PCNA IF) and Tnap-activity in sections of the adult telencephalon. (A) Sagittal and cross sections of the telencephalic area of an adult zebrafish brain stained with alpl ISH and nuclear staining with DAPI. Signals were detected in the dorsolateral zone of the telencephalon (Dl) and in the ventricular zones, among others the telencephalic ventricle (TelV). The cutting planes of the different sections are indicated through the black lines and marked with numbers. Scale bars: 50 µm or 100 µm (B) ELF 97-staining showed sites of AP-activity within the telencephalon at sites of alpl expression. Scale bars: 100 µm. (C) Cross sections of the telencephalon of an adult zebrafish brain co-stained with alpl ISH (red) and IF for proliferation marker PCNA (green). Blue fluorescence depicts DAPI nuclear staining and gives structural information. C1, C2 and C3 are high resolution images of the respective regions of interest. The alpl ISH signals were indicated with white arrows, positive PCNA IF signals were marked with green arrowheads, and merged signals for both were highlighted using a yellow arrowheads. Scale bars: 50 µm or 100 µm. Ao, dorsal aortic root; Dc/Dd/Dl, central/dorsal/lateral zone of dorsal telencephalon; OB, olfactory bulb; Tel, telencephalon; TeO, Tectum opticum; TeIV, telencephalic ventricles.

Figure 6

alpl expression pattern (combined with PCNA IF) and Tnap-activity in sections of the adult mesencephalon, diencephalon, and hypothalamus. The cutting planes of the different sections were indicated in schematic overviews through black lines and marked with numbers. (A) Cross sections of adult zebrafish brain stained with alpl ISH (red) and nuclear staining using DAPI (blue) in the region of the mesencephalon, diencephalon, and hypothalamus. Scale bars: 50 µm (high resolution photos) or 100 µm (overview). The alpl ISH signals were detected, among others, in ventricular areas (DiV, TeV), the dorsomedial optic tract (DOT), and the hypothalamus (HC). (B) ELF 97-staining showed sites of AP-activity within the torus longitudinalis (TL), the hypothalamus (TH/IL), and the medulla oblongata (Mo). (C) Cross sections of adult zebrafish brain co-stained with alpl ISH (red), PCNA IF (green), and DAPI (blue) in the region of the mesencephalon. Scale bars: 50 µm or 100 µm. Merged alpl ISH, PCNA IF, and DAPI signals were shown. The respective localizations of the region of interests, depicted in the high resolution pictures, was indicated with white boxes. The alpl ISH signals were marked with a white arrows, PCNA signals with a green arrowheads, and cells that were positive for alpl and PCNA were indicated with yellow arrowheads. Bv, blood vessel; CC/CCe, cerebellum; Dc/Dd/Dl, central/dorsal/lateral zone of telencephalon; Dil, inferior lobe; DiV, diencephalic ventricle; DOT, dorsomedial optic tract; Hc/Hd, caudal/dorsal zone of periventricular hypothalamus; Mo, Medulla oblongata; TeV, telencephalic ventricles; TeO, Tectum opticum; TH/IL, hypothalamus; TL, torus longitudinalis; A/VM, anterior/ ventromedial thalamic nucleus; Val/Vam, valvula cerebelli.

Figure 7

Inhibition of Tnap-activity leads to alterations in axon and mineralization. (A) Incubation of zebrafish embryos in different concentrations of levamisole altered normal development at 24 and 48 hpf depending on its concentration and resulted in cardiovascular defects (red arrow indicates enlarged pericardial sac). (B) Investigation of axonal growth by acetylated tubulin staining detected reduced growth within the optic tectum at 48 hpf. Scale bars: 25 µm. Red arrows point to differences in neural development under levamisole treatment. (C) Blocking of Tnap-activity resulted in hampered mineralization and influenced craniofacial development at 120 hpf. Alcian-blue stains the cartilage and Alizarin-red the mineralized structures. Fluorescence signals of Alizarin-red are additionally depicted in the upper right corner. Scale bars: 100 µm. Black arrows display the morphological changes of the cartilage and red arrows the missing mineralized structures. Cb, ceratobranchial cartilage; Ch, ceratohylal cartilage; Ep, ethmoid plate; Hs, hysosympletic; Ih, interhylal; M, Meckel’s cartilage; No, notochord; Ov, otic vesicle; Pc, parachordal cartilage; Pq, palatoquadrate cartilage; Tr, trabecular cartilage.

Figure S4 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Figure S14 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Acknowledgments:
ZFIN wishes to thank the journal Scientific Reports for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Sci. Rep.