Fetuses used for histological analysis.

<p>The fetal age was estimated based on the relation between crown-rump length and time of gestation described by Schnorr and Kressin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127691#pone.0127691.ref024" target="_blank">24</a>]. They were genotyped for the 212 bp insertion-deletion variant (<i>indel</i>) associated with polledness in beef and dual-purpose breeds and for the Holstein polled mutation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127691#pone.0127691.ref017" target="_blank">17</a>].</p><p>Fetuses used for histological analysis.</p

Schematic illustration of developmental stages of the horn bud and frontal skin in wild type fetuses.

<p>Horn bud: Multiple layers of keratinocytes are present between 2 and 6 months of gestation and hair follicles are lacking below the horn bud between 2 and 4 months. Note the appearance of thick nerve bundles below the horn bud at 3 months of gestation. Hair follicles and sebaceous glands are present below the horn bud at 4–5 months. Frontal skin: Note thin epidermis at all time-points. Sebaceous glands are present at about 5–6 months of gestation. Note absence of thick nerve bundles in the dermis. Macroscopic pictures: The black arrow depicts the region of the horn bud. Note presence of hair follicles at 6–7 months of gestation. Red = nerve fibres, yellow = sebaceous glands.</p

Features of horn buds and frontal skin from wild type and polled fetuses (gd 172 and 177).

<p>(A): Horn bud from a wild type fetus with multiple layers of vacuolated keratinocytes. Note presence of thick nerve bundles in the dermis below the horn bud (black stars). (B): Frontal skin from a wild type fetus. Note absence of thick nerve bundles in the dermis. (C): Macroscopic picture of a horn bud from a wild type fetus. Note indentation of skin (black arrow). Region of the horn bud (D) and frontal skin (E) from a polled fetus. Note absence of thick nerve bundles in the dermis. (F): Macroscopic picture of a polled fetus without indentation of the skin. Haematoxylin and eosin. Gd = gestation days, ep = epidermis, de = dermis.</p

Histological analysis of horn buds and frontal skin from wild type fetuses (gd 70 and 83).

<p>(A) and (B): Horn buds with multiple layers of vacuolated keratinocytes. (C) and (D): Frontal skin. Haematoxylin and eosin. Gd = gestation days, ep = epidermis, de = dermis.</p

Histological analysis of horn buds and frontal skin from wild type fetuses (gd 212, 230 and 268).

<p>(A-C): Horn buds. Note differentiated epidermis and thick nerve bundles in the dermis below the horn bud (black stars). Insets show well-differentiated keratinocytes in the epidermis and sebaceous glands (black arrows). (D-F): Frontal skin. Note the absence of thick nerve bundles in the dermis. Insets show immature keratinocytes in (D) and (E) and well-differentiated keratinocytes in (F). Note sebaceous glands in the insets (black arrows). Haematoxylin and eosin. Insets: Scale bar represents 200μm. Gd = gestation days, ep = epidermis, de = dermis.</p

Neuron specific enolase staining from a wild type fetus (gd 268).

<p>(A) and (B): Horn bud. (B): Magnification of (A). Note positive staining of the thick nerve bundles in the dermis. (C) and (D): Frontal skin. (D): Magnification of (C). Note positive staining of nerve fibres of normal size in the dermis. Gd = gestation days, ep = epidermis, de = dermis.</p

Glycerol uptake and utilisation is perturbed in <i>aqp</i>-null <i>T</i>. <i>b</i>. <i>brucei</i>.

<p>(A) ATP levels were assessed in the strains indicated after incubation in 5 mM glucose or glycerol. Readings were taken in triplicate and normalised to substrate only. * indicates significantly different (<i>P</i><0.001) to wild-type (WT) using an ANOVA test in GraphPad Prism. Error bars, SD. (B) Radiolabelled glycerol uptake was assessed in the strains indicated. Readings were taken in quadruplicate. * indicates significant difference (<i>P</i><0.05) using a Student’s <i>t</i>-test. Error bars, SD.</p

<i>T</i>. <i>b</i>. <i>brucei</i> tolerates the loss of all three <i>AQPs</i>.

<p>(A) The schematic maps indicate the <i>AQP1</i> and <i>AQP2-3</i> regions replaced by selectable markers as also indicated on the right. Δ indicates the regions deleted while the probes used for Southern blotting are shown above the maps. H, <i>Hpa</i>I; S, <i>Sac</i>II. (B) The Southern blots indicate deletion of the <i>AQP1</i> alleles in <i>aqp1</i> and three independent <i>aqp1-2-3</i> strains. Wild-type (WT) is shown for comparison. Genomic DNA was digested with <i>Hpa</i>I. (C) The Southern blots indicate deletion of the <i>AQP2-3</i> alleles in <i>aqp1-2-3</i> strains. WT is shown for comparison. Genomic DNA was digested with <i>Sac</i>II.</p

Respiratory inhibitor-sensitivity in <i>T</i>. <i>b</i>. <i>gambiense</i> isolates and AQP-mediated glycerol transport.

<p>(A) SHAM EC₅₀ values for the <i>T</i>. <i>b gambiense</i> strains are indicated +/- glycerol. The inset shows pentamidine EC₅₀ values. * indicates significantly different (<i>P</i><0.05) to STIB930 using an ANOVA test in GraphPad Prism. All pairwise comparisons +/- 10 mM glycerol also indicated significant (<i>P</i> <0.001) differences using a Student’s <i>t</i>-test. Error bars, SD. (B) Propyl gallate and (C) Ascofuranone EC₅₀ values. Other details as in A. (D) Model for glycerol transport by AQPs in <i>T</i>. <i>b</i>. <i>gambiense</i>. The weight of the arrows indicates relative impact on glycerol utilisation and efflux, with AQP2 being the major contributor; note that transport across both the plasma and glycosomal membranes contributes to glycerol utilisation and efflux, see the text for more details. The right-hand panel indicates the situation in melarsoprol-resistant (reduced melarsoprol uptake) and SHAM-sensitive (reduced glycerol efflux) clinical isolates where a chimeric AQP2/3 replaces AQP2 and AQP3.</p

<i>aqp</i>-null <i>T</i>. <i>b</i>. <i>brucei</i> display defective glycerol-efflux and respiratory inhibitor-sensitivity.

<p>(A) Bloodstream <i>T</i>. <i>brucei</i> express a SHAM-sensitive mitochondrial trypanosome alternative oxidase (TAO). Under aerobic conditions, TAO activity allows ATP production without glycerol production as indicated by the black lines (left-hand blue ‘cell’). SHAM blocks TAO-activity, leading to the anaerobic production of glycerol, which is toxic if not removed, as indicated by the black lines (right-hand blue ‘cell’). SHAM dose-response curves for wild-type (WT) and <i>aqp1-2-3</i> null-cells. EC₅₀ values are indicated. (B) In the presence of SHAM and glycerol, the glycerol inhibits glycerol kinase (GK), also preventing ATP-production by the anaerobic route (blue ‘cell’). SHAM dose-response curves as in A but in the presence of 10 mM glycerol. (C) Propyl gallate and octyl gallate dose-response curves for wild-type (WT) and <i>aqp1-2-3</i> null-cells. EC₅₀ values are indicated. (D) SHAM EC₅₀ values +/- 10 mM glycerol from A-B and also from <i>aqp2</i>, <i>aqp2-3</i> and <i>aqp1-2-3</i> cells re-expressing ^GFPAQP2. * indicates significantly different (<i>P</i><0.01) to WT using an ANOVA test in GraphPad Prism. Pairwise comparisons +/- glycerol, except in the case of the <i>aqp1-2-3</i> null, indicated significant (<i>P</i> <0.001) differences using a Student’s <i>t</i>-test. Error bars, SD. The images to the right show re-expression of ^GFPAQP2 in <i>aqp1-2-3</i> null-cells.</p