Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer’s disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases. Alzheimer’s disease (AD) is the most common form of dementia and the sixth leading cause of death in the US. Estimates indicate that ~5 million people in the US currently have the disease and that number is expected to increase up to ~16 million by 2050. Current therapeutic options are limited to symptomatic treatments highlighting the urgent need to develop and evaluate novel disease-modifying approaches. Identification of sensitive and specific AD biomarkers will be critical for the development of these therapeutics.Over the past two decades, extensive effort has focused on the identification and development of AD biomarkers specifically linked to disease pathology  was used to enrich the relatively low abundance tau protein prior to western-blot analysis. Equal volumes of pooled control and AD CSF samples were fractionated and then western-blots analyzed with antibodies specific for different regions of the protein; a summary of the antibodies used is shown in Fractions were also analyzed for the presence of C-terminal fragments using the rabbit polyclonal antibody K9JA, which binds to the microtubule repeat and C-terminal flanking region of tau (aa 243-441). K9JA exhibited robust staining of full length tau441 standard ; howeverTo more accurately quantify CSF tau levels, a set of novel tau and ptau ELISAs were developed . Assays The HT7-BT2, HT7-Tau5, Tau12-BT2 and Tau12-HT7 tau assays demonstrated ~100-fold dynamic range of quantitation using tau 441 standard, with the LLQ ranging from 1.6 pg/ml to 7.8 pg/ml . Signal The HT7-77G7 assay is specific for tau species containing more C-terminal sequences (aa 159-335). The dynamic range and LLQ observed (16 pg/ml) were similar to the other tau ELISAs ; howeverAll three ptau assays, HT7-AT270, Tau12-AT270 and HT7-PHF6, demonstrated ~100-fold dynamic range of quantitation using synthetic ptau standards, with LLQs ranging from 2 pg/ml to 7.8 pg/ml . In the To evaluate the discriminatory power of the assays, tau and ptau levels were measured in a cohort of 20 AD and 20 age-matched control CSF samples (20x20 sample set); demographic information included in 2 = 0.87-0.95)  were 2- to 4-fold lower, while tau species containing additional C-terminal sequence  were 3-fold lower . Levels 87-0.95) . On the 87-0.95) , consist87-0.95) . A signaOverall, CSF tau levels were found to be significantly higher in AD samples compared to controls in all but the HT7-77G7 assay ; Table 32 =0.67-0.72). By comparison, the HT7-PHF6 and Tau12-AT270 correlations were less robust (r2 =0.18-0.37) and in some cases not significant.The 20x20 samples were also analyzed using the ptau ELISAs. All three ptau ELISAs exhibited significantly higher levels in AD compared to control samples, with increases ranging from 1.4-fold to 2.4-fold ; Table 3To further compare the discriminatory power of the different tau and ptau assays, all possible combinations of tau and ptau ratios were calculated for each individual CSF sample and then these values used to evaluate differences and discrimination between AD and control samples . This anIn this study, tau profiles and the relative differences in tau and ptau levels between AD and age-matched control CSF samples were investigated using a combination of qualitative and quantitative biochemical assays. A sensitive western-blotting method was used to demonstrate that tau is present in CSF as a series of N-terminal and mid-domain fragments. Results from a set of novel ELISAs specific for different overlapping regions of tau demonstrated that the ability of CSF tau and ptau to differentiate AD from control is dependent on the tau species measured. These endpoints provide novel tools to investigate CSF tau in AD and other neurodegenerative disorders.A number of previous studies have reported on the characterization of tau in CSF using western-blotting-based techniques; however, results from these studies are mixed and incongruent ,37,40,41To address these limitations, RP-HPLC was utilized to enrich and concentrate tau from a large volume of pooled, denatured CSF thereby enabling analysis of the relatively low levels of tau by western-blotting. N-terminal and mid-domain tau fragments were detected in both AD and control CSF, ranging in size from <20 kDa to ~40 kDa. In contrast, C-terminal-containing fragments were not detected using the K9JA polyclonal antibody. Furthermore, full-length tau was not detected with any of the antibodies tested. The lack of detectable C-terminal fragments was surprising given reports indicating that MTBR-containing fragments are present in CSF ,44,45. WTau is a putative substrate for various proteases such as calpain, caspases, cathepsins and thrombin  differences in levels between AD and control. These findings are consistent with the high degree of correlation between assays. Despite the similarities, however, subtle differences in tau assay performance were also noted. Most interesting was the fact that the two assays specific for tau species containing N-terminal sequences, aa 9-163 (Tau12-HT7) and aa 9-198 (Tau12-BT2), exhibited the highest degree of differentiation . In comparison, differentiation was less robust (p = 0.0031) with HT7-BT2, specific for aa 159-198. This difference could be partly linked to the sensitivity of BT2 to phosphorylation at S199  as levelAnother interesting finding was the lack of a quantifiable CSF signal in the HT7-77G7 assay. This was not an assay artifact as complete recovery of a tau spike in CSF matrix was observed. Given the relatively robust signal measured in the HT7-Tau5 assay (aa 159-225), the lack of signal in the HT7-77G7 assay (aa 159-335) suggests extensive cleavage of tau between the Tau5 (aa 218-225) and 77G7 (aa 316-335) epitopes, further supporting the idea that CSF tau is fragmented. The HT7-77G7 result is also consistent with the inability to detect full-length tau by western-blotting. This was further confirmed using another ELISA, Tau12-DC39, specific for tau containing aa 9-441 . As compOf the ptau ELISAs evaluated, the HT7-AT270 (aa 159-p181) assay exhibited the highest level of discrimination, though only slightly better than HT7-PHF6 (aa 159-p231). This finding is consistent with data reporting that p181, p231 and p199 were equivalent in their ability to discriminate AD from controls . InteresIn summary, our results indicate that tau is present in control and AD CSF as a mixture of fragments. Results from our novel tau and ptau assays provide evidence that the discrimination of AD from control is dependent on the subset of tau species measured and that development of more robust AD biomarkers may be possible. One limitation of the study is the relatively small sample set used. Ultimate confirmation of these findings will require analysis of larger cohorts of AD and control samples to ensure robust statistical analysis. Additional assays and reagents will also be needed to fully investigate any C-terminal fragments or aggregates present but not detected by the methods employed here. Finally, these results could have implications in the development of CSF tau and ptau biomarkers for other neurodegenerative diseases, including other tauopathies where disease-associated changes in tau and ptau have not been consistently observed.The study was approved by the regional ethics committee at the University of Gothenburg.Pooled control and pooled AD CSF samples were generated by board-certified laboratory technicians at the Clinical Neurochemistry Laboratory, the Sahlgrenska University Hospital in Mölndal, Sweden. Samples were categorized as control or AD on the basis of CSF tau, ptau and Aβ42 cut-points that are 90% sensitive and specific for AD ) [Individual AD and age-matched CSF samples were purchased from Precision Med . Written and verbal consents were obtained from participants at screening and enrollment. For all patients, participants were > 55 years of age, in good general health having no other neurological, psychiatric or major medical diagnosis that could contribute significantly to cognitive impairment or dementia. For AD, patients were selected based upon a probable diagnosis of AD using NINCDS-ADRA criteria, a Hachinski score <  4 and with an MMSE between 14-26. Control subjects were classified as healthy, but no cognitive testing was performed. The complete demographic and individual data for these samples are shown in Human CSF was denatured in guanidine-HCl  to a final concentration of 6 M guanidine-HCl. 24 ml injections of the denatured CSF (6 ml CSF + 18 ml guanidine-HCl) were fractionated with an Agilent 1100 series HPLC running at 1.5 ml/min over a Poros R1/10 protein column  heated to 65°C. 30 x 2 ml fractions were collected for each sample using an water/acetonitrile gradient (0-60% acetonitrile over 35 min) in the presence of 0.1% (volume/volume) trifluoroacetic acid. Fractions were dried to completion in a SpeedVac Explorer  overnight. Dried fractions were stored at -20°C until analysis. A similar strategy was used previously to extract and enrich Aβ peptides from human plasma and CSF and to eliminate matrix interference ,51.Dried HPLC fractions were resuspended in tricine sample buffer, and separated on Novex 10-20% tricine gels according to manufacturer’s directions. Gels were transferred onto 0.45 µm polyvinylidene difluoride (PVDF) membrane  in CAPS transfer buffer (pH 11.0) at room temperature for 90 min. Membranes were blocked in TBST  Tween-20) with 1% BSA  for 1 hr. Membranes were probed with HT7 (Pierce), Tau12 , KJ9A , and mouse IgG1 monoclonal isotype control  conjugated to HRP  in TBST with 1% BSA for 16 hrs at room temperature. Probed membranes were developed using SuperSignal West Femto Maximum Sensitivity Substrate .(1-42) (Aβ42), tau and p-tau (181) according to the manufacturer’s instructions . Briefly, suspension array bead sets were incubated with reference standards, QC samples, human CSF along with biotinylated reporter overnight. Excess unbound material was removed via vacuum filtration, followed by incubation with streptavidin-phycoerythrin (PE) for 60 min. The antibody/peptide complexes were detected via PE fluorescence as measured by a Bio-Rad BioPlex instrument running Luminex xPonent software. Analyte levels were quantified using an unweighted 4-parameter logistic (4PL) curve fit generated from the reference standards using BioPlex manager 5.0 softwareINNO BIO AlzBio 3 was used to measure CSF AβTau ELISAs were developed using the following mouse monoclonal antibodies for capture; antibody information listed in Tau ELISAs were run as follows. High binding black 96 well plates  were coated by the addition of 2.5 µg/ml  or 5 µg/ml (Tau12) capture antibodies which were diluted in Tris buffered saline (TBS), pH 8. Plate sealers were attached then the plates were incubated at 37°C for 1 hr. Plates were washed with TBST (TBS containing 0.05% Tween-20) before blocking nonspecific binding sites with 3% bovine serum albumin  (w/v) in TBS. Plate sealers were attached and the plates were incubated at room temperature for 2-4 hrs while shaking. Plates were washed with TBST before the addition of 50 µl per well diluted human CSF and human tau441 standard curves which were each prepared in a final assay buffer concentration of 1% BSA (w/v) and 0.05% Tween-20 (v/v) in Tris buffered saline (TBS), pH 8. Plate sealers were attached, then assay plates containing human CSF and standard curves were incubated overnight at 4°C while shaking. Alkaline phosphatase (AP) conjugated BT2, HT7, Tau5 or 77G7 antibodies were diluted into assay buffer before being added to the assay plate (50 µl per well) to co-incubate with human CSF and htau441 standard curves for 1 hr at room temperature while shaking. Plates were washed with TBST before being developed using alkaline phosphatase substrate . Luminescence counts were measured using a Packard TopCount . Log-transformed luminescence counts from individual samples were interpolated to concentration using a second-order polynomial fit to the respective standards . CSF tau levels were plotted after correction for dilution factor in the respective assays. Assay lower limit of quantitation (LLQ) was set based on the lowest calibrator demonstrating acceptable total error (bias + precision of < 30%).A pooled CSF sample from AD patients and an age-matched pooled control CSF sample were 2-fold serially diluted from 2- to 256-fold in a final assay buffer concentration of 1% BSA (w/v) and 0.05% tween-20 (v/v) in Tris buffered saline (TBS), pH 8 before an aliquot of each was spiked with recombinant human tau441 protein  at a final concentration of 100 pg/ml. Diluted CSF samples with and without tau441 spike were assayed in the HT7-77G7 ELISA as described above.Human CSF was analyzed in three different ptau assays: HT7-AT270 (p181) and HT7-PHF6 (p231) and Tau12-AT270 (p181). The HT7-AT270 and HT7-PHF6 ELISAs utilized HT7 (amino acids 159-163), while the Tau12-AT270 assay used Tau12 (amino acids 9-18), as the capture antibody, and the respective analytes were detected using alkaline phosphatase (AP) conjugated AT270 pT181 tau antibody  or PHF6 pT231 specific monoclonal antibody ; antibody information listed in pTau ELISAs were run as follows. Black high-binding plates  were coated with 2.5 µg/ml of capture antibodies HT7 or Tau12 in carbonate-bicarbonate buffer at pH 9.4 . After overnight incubation at 4°C, plates were washed with PBS and nonspecific binding sites were blocked using 3% BSA in PBS buffer for at least 4 hrs at 4°C. Standards and CSF samples (50 µL) were added to plates, followed by 50 µL of AP conjugated AT270 (pThr181) and PHF6 (pThr231) tau antibodies for the respective assays. After overnight incubation at 4°C, plates were washed with PBS (containing 0.05% tween) and developed using alkaline phosphatase substrate . Luminescence counts were measured using Envision . Log-transformed luminescence counts from individual samples were interpolated to concentration using a 3rd order polynomial fit to the respective standards  CSF ptau levels were plotted after correction for dilution factor in the respective assays. Assay lower limit of quantitation (LLQ) was set based on the lowest calibrator demonstrating acceptable total error (bias + precision of < 30%).Statistical calculations were performed using GraphPad Prism 5.00 . Differences in biomarker levels between AD and control samples were examined using unpaired two-tailed Student’s t-test. Data was log-transformed prior to t-test comparisons to correct for non-Gaussian distributions as determined by the D’Agostino & Pearson normality test. Results were considered significant for p-values < 0.01Figure S1Tau peptides used for 77G7 antibody epitope mapping.A set of 29 overlapping peptides spanning the length of human tau 441 were generated, coupled to beads and used to map the epitope of tau antibody 77G7 using a Luminex-based multiplex assay.(DOCX)Click here for additional data file.Figure S2Mapping the tau epitope of antibody 77G7.Left panel) 77G7 exhibited binding to human Tau 441 and the C-terminal human tau fragment aa 231-441 but not to the tau fragments aa 1-125 or aa 126-230; 77G7 also bound to the other tau isoforms (data not shown). Mid-domain tau antibody HT7 exhibited binding to tau 441 and fragment aa 126-230 as expected. Binding was not observed with the anti-IL6 control antibody. Right panel) A set of 29 overlapping peptides spanning the length of human tau 441 were generated, coupled to beads and used in a Luminex-based multiplex assay to screen 77G7, HT7 and anti-IL6 control. 77G7 exhibited binding to peptide 22 (aa 316-335) and much lower level binding to peptide 25 but none of the other peptides (right panel and data not shown). HT7 exhibited binding to peptide 11 (aa 150-170) but none of the other peptides (right panel and data not shown) as expected while the control anti-IL6 antibody did not react with any of the peptides tested.(DOCX)Click here for additional data file.Figure S3Verification of signal specificity in tau ELISAs by immunodepletion.Pooled CSF was immunodepleted with tau antibody HT7 (IP) or treated with protein A/G beads alone (control). Samples were analyzed in tau ELISAs A) HT7-BT2, B) HT7-Tau5, C) Tau12-BT2 and D) Tau12-HT7. Data represents mean ± SEM from 3-4 determinations. Dashed lines indicate the assay LLQ corrected for CSF dilution.(DOCX)Click here for additional data file.Figure S4Spike recovery in tau ELISAs.Pooled CSF samples were treated with tau 441 spikes ranging from 10-800 pg/ml. Spiked samples and a matching untreated control were analyzed in tau ELISAs A) HT7-BT2, B) HT7-Tau5, C) Tau12-BT2 and D) Tau12-HT7 and spike recovery determined (%). Data represents mean ± SEM from 3 determinations. Dashed lines indicate 100% spike recovery.(DOCX)Click here for additional data file.Figure S5Verification of signal specificity in ptau ELISAs by immunodepletion and peptide competition.Pooled CSF samples from healthy control subjects (black bars) or AD patients (red bars) were immunodepleted with tau antibody HT7 (IP) or protein A/G beads alone (control). CSF samples were also treated with pT181 or pT231 peptides for competition analysis. Samples were analyzed in ptau ELISAs A) HT7-AT270, B) HT7-PHF6, and C) Tau12-AT270. Data represents mean ± SEM from 3 determinations. Dashed lines indicate the assay LLQ corrected for CSF dilution.(DOCX)Click here for additional data file.Figure S6Spike recovery in ptau ELISAs.Pooled CSF samples were treated with pT181 or pT231 spikes ranging from 12.5-200 pg/ml. Spiked samples and a matching untreated controls were analyzed in ptau ELISAs A) HT7-AT270, B) HT7-PHF6, and C) Tau12-AT270 and spike recovery determined (%). Data represents mean ± SEM from 3 determinations. Dashed lines indicate 100% spike recovery.(DOCX)Click here for additional data file.Figure S7Analysis of tau and ptau levels in 20 AD and 20 control CSF samples.A set of 20 AD and 20 age-matched normal control CSF samples were analyzed using INNO-BIA AlzBio3. Statistics based on 2-tailed Student’s t test comparison of log-transformed data (tau and ptau) or untransformed data (Aβ42). * p < 0.05; ** p< 0.01; *** p < 0.001.(DOCX)Click here for additional data file.Methods S1Epitope mapping and immunodepletion and spike recovery.(DOCX)Click here for additional data file.Table S1Analysis of tau assay ratios in 20x20 sample set.(DOCX)Click here for additional data file.Table S2Tau ELISA correlations.(DOCX)Click here for additional data file.Table S3Analysis of ptau assay ratios in 20 x 20 sample set.(DOCX)Click here for additional data file.Table S4Complete demographic information for 20 x 20 sample set.(DOCX)Click here for additional data file.Table S5Individual data for 20 x 20 sample set.(DOCX)Click here for additional data file.


Prox1-EGFP reporter mouse model and optical projection tomography technology to characterize both the temporal and spatial development of the lymphatic vessel network in mouse testes and ovaries. We find that lymphangiogenesis in the testis is initiated during late gestation, but in contrast to other organs, lymphatic vessels remain confined to the testis cap and, unlike blood vessels, do not infiltrate the entire organ. Conversely, lymphatic vessels invade the ovarian tissue, beginning postnatally, and sprouting from preexisting lymphatic vessels at the extraovarian rete. The ovary develops a rich network of lymphatic vessels, extending from the medulla into the surrounding cortex adjacent to developing follicles. This study reveals distinct patterns of lymphangiogenesis in the testes and ovaries and will serve as the basis for the identification of the divergent molecular pathways that control morphogenesis and the function of the lymphatic vasculature in these two organs.The lymphatic vasculature forms a specialized part of the circulatory system, being essential for maintaining tissue fluid homeostasis and for transport of hormones, macromolecules, and immune cells. Although lymphatic vessels are assumed to play an important role in most tissues, their morphogenesis and function in the gonads remains poorly understood. Here we have exploited a lymphatic-specific  The lymphatic vasculature consists of an extensive network of small, blind-ended capillaries, larger vessels and collecting ducts The lymphatic vasculature originates from the anterior cardinal vein during embryonic development; around 9.5 days post coitum (dpc) in the mouse. Initially, a subset of venous endothelial cells expresses two transcription factors, SRY-box containing gene 18 (SOX18) and the nuclear receptor COUP-TFII , which directly induce the expression of Prospero-related homeobox 1 (PROX1), a factor crucial for lymphatic development In mammals, testes and ovaries are also connected to the lymphatic network, and it is assumed that, as in other tissues, lymphatic vessels drain extracellular fluids and macromolecules from the gonadal interstitium to support normal physiology and function. Lymphatics of mammalian gonads were recognized more than 40 years ago, and have since been characterized in diverse animal species including primates, bull, goat, pig, rabbit and rodents post coitum (dpc), where they presumably grow from a pre-existing network of vessels at the gonad-mesonephros border In mice, superficial lymphatic vessels have been identified in both testes and ovaries during late gestation, around 17 days It is important to consider reported differences between species when analyzing the lymphatic vasculature of the gonads. A recent study suggested that the adult testis of smaller rodents does not possess lymphatic vessels within the interstitial space and that the vessels are restricted to the testis cap; the tunica albuginea post natum (dpn) Although there appeared to be no major differences in ovarian lymphatics between mammalian species, emerging evidence strongly suggests sex-specific differences both with regards to lymphangiogenic initiation and vessel distribution within the gonads. Superficial lymphatic vessels of the ovary were suggested during fetal life Prox1 is regarded as a hallmark of lymphangiogenesis The visualization of lymphatic development has commonly relied on markers such as LYVE1 and podoplanin (PDPN). However, these proteins are also expressed by cell types other than lymphatic endothelial cells, including embryonic venous endothelial cells Prox1-EGFP reporter mouse (Tg(Prox1-EGFP)221Gsat/Mmcd) that faithfully recapitulates endogenous PROX1 expression In this study, we took advantage of a Prox1-GFP BAC transgenic mice (Tg(Prox1-EGFP)221Gsat/Mmcd) were provided by Dr Y.K. Hong  Prox1-GFP) males were mated with wild-type females, both on an outbred CD1 background to create heterozygous Tg(Prox1-GFP) offspring for analyses. Non-transgenic offspring were collected from wild-type CD1 matings. Developmental stage was assigned as noon of the day on which the mating plug was observed corresponding to 0.5 dpc. Gonadal sex was determined by morphological assessment. Protocols and use of animals in the described experiments were approved by the Animal Ethics Committee of the University of Queensland, in accordance with the Queensland Animal Care and Protection Act (2001).Prox1-EGFP was performed on freshly dissected (untreated) tissues in phosphate buffered saline (PBS) or by confocal microscopy following dehydration of PFA-fixed samples in methanol and clearing in benzyl alcohol:benzyl benzoate as described for optical projection tomography. Section IF experiments were performed on 7 µm sagittal sections of paraffin-embedded tissues pre-fixed in 4% paraformaldehyde (PFA) in PBS as previously described Prox1-EGFP mice, as well as fetal gonadal samples represented inWhole-mount immunofluorescence (IF) imaging of Prox1-EGFP mice were subjected to whole-mount IF as previously described Pre-fixed ovaries (4% PFA in PBS) from adult Primary antibodies used were: rabbit anti-DDX4 , rabbit anti-LYVE1 , chicken anti-GFP , rabbit anti-HSD3B1 1∶200; , rabbit Prox1-EGFP fetuses from 12.5 dpc onwards. Gonad-mesonephros complex pairs of at least one litter of no less than five embryos of each sex at each developmental stage were analysed by fluorescent microscopy. Representative XY samples between 15.5 and 19.5 dpc are shown in Prox1-driven EGFP expression was detected before 14.5 dpc (data not shown), after which weak EGFP signal was detected in the mesonephros. At 16.5 dpc, strong EGFP signal was visible along the spermatic cord, likely following the pampiniform plexus of veins before ending at the rete testis with few vessels sprouting laterally. At 17.5 dpc, extensive branching of superficial EGFP-positive vessels was observed, originating from the spermatic cord/rete testis before fanning out over the testis cap. At 18.5 dpc, the superficial EGFP-positive vessels were spread across the testis surface. However, at this stage it was more difficult to visualize the vessels due to increasing EGFP signal from the testis soma, apparently from within the testis cords.To characterize the onset of lymphatic vessel growth in the developing testes, we visualized EGFP expression in XY gonad-mesonephros complexes of Prox1-EGFP expression in the developing testis and that EGFP-positive vessels are lymphatic, we performed whole-mount IF on Prox1-EGFP gonad-mesonephros complexes from 16.5, 17.5 and 18.5 dpc fetuses, using antibodies to Neuropilin 2 (NRP2) which is expressed in lymphatic vessels Prox1-EGFP gonadal samples was confirmed by confocal microscopy  expression . At thisstis cap . Prominestis cap , arrow, stis cap . Notablystis cap .Prox1-EGFP testes. Using whole-mount microscopy, we observed a dense network of lymphatic vessels on the testis surface emanating from the spermatic cord  detected lymphatic vessels only in the tunica albuginea  expression  revealed pronounced lymphatic vessels in the extraovarian rete, but no EGFP-positive vessels within the ovary proper -positive oocytes were devoid of, but in close proximity to lymphatic vessels   vessels . In part vessels . On the  vessels . Interespression , arrow wProx1-EGFP adult ovaries. These experiments revealed a surprisingly extensive lymphatic network within the ovary proper -axis Contrary to the testis, the adult ovary contains a rich network of internal lymphatic vessels, both within the medulla and also the cortex. Although follicles are devoid of lymphatics, the extent to which the cortical space is invaded by a mesh of larger lymphatic vessels was surprising . PreviouVegfc, Vegfd and Vegfr3Vegfa has emerged as one factor involved in dimorphic blood vascularization Clearly, significant sexual dimorphism exists in the timing of initial lymphangiogenesis of the gonads. In testes, lymphatic vessels first appear at around 17 dpc, 2–3 days prior to birth. In female mice however, lymphatic vessels do not invade the ovary proper until after birth at around 10 dpn. In the ovary, lymphatic growth has been linked to hormone signalling corresponding to folliculogenesis, also corresponding with an up-regulation of the pro-lymphatic genes Figure S1Testicular, but not ovarian lymphangiogenesis is initiated during late gestation in mice, as visualized with Prox1-EGFP transgenic gonad-mesonephros complexes. A) Until 16.5 dpc, no EGFP-positive lymphatic vessels are observed in the developing testis, but are observed in the adjacent mesonephros from around 15.5 dpc. At 17.5 dpc a more extensive lymphatic network is visible along the spermatic cord and in the mesonephros, ultimately spanning out over the testis cap. From 18.5 dpc, Prox1-EGFP signal also appears from inside the testis proper, making visualization of superficial vessels difficult. T = testis; M = mesonephros. B) Comparable to XY development, the mesonephros of XX fetuses are observed to contain EGFP-positive vessels during fetal life, also prominent in the Müllerian duct between 15.5–16.5 dpc. No EGFP-positive lymphatic vessels are observed in the ovary proper during fetal life, but a rich vascular network is seen developing along the ovarian ligaments from 17.5 dpc. O = ovary; M = mesonephros; MD = Müllerian duct. C) At 19.5 dpc, no EGFP-positive vessels are observed inside the ovary, as visualized by section IF co-stained with the granulosa cell marker FOXL2. A prominent lymphatic vessel is detected in the extraovarian rete (arrow), and oocytes in the ovarian cortex are also expressing EGFP (aterisks). O = ovary; EoR = extraovarian rete; scale bar = 50 µm.(TIF)Click here for additional data file.Video S1OPT reconstruction showing Prox1-EGFP localization in 17.5 dpc mouse testis as contrasted against ENG-positive blood vessels. Whole-mount IF for EGFP and ENG was performed on Prox1-EGFP transgenic testis-mesonephros complexes. 3D-rendering show distinct organisation of the lymphatic network as it emanates from the spermatic cord to the rete testis, then sprouting out over the testis cap. No EGFP-positive lymphatic vessels are observable within the testis.(M4V)Click here for additional data file.Video S2OPT reconstruction showing Prox1-EGFP localization in adult (9-week) mouse ovary contrasted against ENG-positive blood vessels and LYVE1-positive lymphatic capillaries. Whole-mount IF for EGFP, ENG and LYVE1 was performed on Prox1-EGFP transgenic ovaries. 3D-rendering show distinct organisation of the lymphatic network as it emanates from the rete ovarii, a region also expressing LYVE1-positive capillaries, then into the medulla. An extensive network of Prox1-EGFP-positive, LYVE1-negative lymphatic vessels is observable throughout the ovary, including the cortex.(M4V)Click here for additional data file.


Sauropods are often imagined to have held their heads high atop necks that ascended in a sweeping curve that was formed either intrinsically because of the shape of their vertebrae, or behaviorally by lifting the head, or both. Their necks are also popularly depicted in life with poses suggesting avian flexibility. The grounds for such interpretations are examined in terms of vertebral osteology, inferences about missing soft tissues, intervertebral flexibility, and behavior. Osteologically, the pronounced opisthocoely and conformal central and zygapophyseal articular surfaces strongly constrain the reconstruction of the cervical vertebral column. The sauropod cervico-dorsal vertebral column is essentially straight, in contrast to the curvature exhibited in those extant vertebrates that naturally hold their heads above rising necks. Regarding flexibility, extant vertebrates with homologous articular geometries preserve a degree of zygapophyseal overlap at the limits of deflection, a constraint that is further restricted by soft tissues. Sauropod necks, if similarly constrained, were capable of sweeping out large feeding surfaces, yet much less capable of retracting the head to explore the enclosed volume in an avian manner. Behaviorally, modern vertebrates generally assume characteristic neck postures which are close to the intrinsic curvature of the undeflected neck. With the exception of some vertebrates that can retract their heads to balance above their shoulders at rest , the undeflected neck generally predicts the default head height at rest and during locomotion. Sauropod necks were but one remarkable aspect of an altogether remarkable vertebrate. The necks of many sauropod taxa comprised a larger proportion of the presacral axial skeleton than found in any extant non-avian, with individual vertebrae representing extremes of pneumaticity, elongation, and size. The combination of relatively tiny head, elongate neck, and enormous body has posed fascinating questions regarding sauropods, including how they fed, moved, and simply how they appeared in life.Since only their fossilized bones remain, and usually incomplete and imperfectly preserved at that, even their skeletal reconstructions have been subject to differences in interpretation and sometimes artistic liberties. The overall bauplan of these great giants remains controversial (see below), let alone how they might have held their heads and used their necks in life. Settling the essential questions of sauropod feeding habits and the role of their remarkable necks in feeding will be challenging, since the origin and role of the long neck of the giraffe remains controversial despite their being available for direct observation, as living and behaving animals. Even if alive today, some contention could be expected regarding how the sauropod got its long, long neck. But given only their fossils, much must be inferred and little can be observed directly. This review attempts to summarize what can be concluded about sauropod neck articulation based on correlations between function and  form. The methodology is necessarily inferential and incremental, accumulating a coherent explanation that, while highly incomplete and speculative, is at least consonant with what can be derived from other sources.By analogy, imagine that giraffes were extinct, and known only by their desiccated bones, with some cervical columns remaining in apparently close articulation but in opisthotonic pose Unlike other sciences where the subjects under study are extant and their behaviors  can be observed directly, in paleontology, when the subjects are extinct their behaviors can only be inferred. The process of scientific inference regarding extinct behavior is therefore indirect and conjectural, and many of the presumptions about how these animals lived out their lives rely on tacit intuition and the selection of modern examples that appear to support a given conjecture.Since their first discovery, sauropods have long been the subject of speculation, e.g., that they had a sprawling stance Note that the above speculations are suggestions not only about potential function  but about behavior, and unless outright refuted as physically impossible, are subsequently adopted – or not – based on their appeal and support by analogical reasoning (see below). There is also a tendency to propose conjectures that are not scientifically testable  yet seem compelling, popular, and make for a satisfactory story Recently, however, there has been increasing use of a method to challenge the mythology using observations of modern vertebrates and certain bridging assumptions to ‘ground-truth’ proposals about sauropod pose, flexibility, and behavior. The application of this methodology to examine the mythology is the subject of this review.Conjectures about sauropod neck function, physiology, and feeding behavior are invariably based on skeletal reconstructions by illustrations or mounts and while such reconstructions are very familiar and seemingly authoritative, they often amount to hypotheses or conjectures incorporating significant artistic interpretation (see below). Some effort will be devoted to this issue, since sauropod reconstructions, whether physical or pictorial, are often used uncritically, as will be discussed. The relationships between osteological form and biological function in general, and of vertebral articular geometry and joint articulation in particular are becoming increasingly understood, and recent studies are confirming that vertebral osteology can tell us something about pose and flexibility. The vertebrate neck is not merely a chain of bones and joints, but a system, and observable correlates between structure and function in the necks of extant vertebrates are becoming better understood, thus permitting more principled application towards interpreting sauropod neck function.“… the truth will out. Nature's phenomena will agree or they'll disagree with your theory. Although you may gain some temporary fame and excitement, you will not gain a good reputation as a scientist if you haven't tried to be very careful…” There seems a universal tendency to confer greater trust upon simple parsimonious explanations of natural phenomena, those that capture some essence in few words and to hold broadly without exception. It has been suggested that, based on how extant amniotes hold their heads in alert rest, that sauropods assumed a posture with the neck is maximally extended (dorsiflexed) at the base and the head maximally ventriflexed The necessarily speculative nature of theorizing in paleontology is unfortunately susceptible to the social phenomenon of ‘group polarization’, as speculations are adopted and repeated by secondary sources Regarding sauropod necks, for instance, while historically they have long been depicted with a wide range of combinations neck curvature (from straight to sharply reflex-curved) and slope at mid-neck  Analysis of neck curvature in modern vertebrates provides a means to estimate neck curvature in sauropods. The starting point is to distinguish intrinsic curvature as that which remains in a vertebral column when all intervertebral joints are in an undeflected state. Osteological mounts of extant vertebrates are valuable resources illustrating intrinsic curvature and how it arises from their particular osteology. When the vertebral columns of extant birds, reptiles and mammals are assembled with the successive vertebrae spaced according to their separations in life, and with each joint undeflected, the columns assume a familiar and characteristic intrinsic curve associated with that taxon in an osteologically neutral pose (ONP). The process of estimating intrinsic curvature is of course neither absolute nor exact, nor is it immune to artistic bias and measurement error, in particular as regards speculation about the thickness of the intervertebral separation for extinct vertebrates.Despite their common depiction with rising curvature at the base of the neck, reconstructions of the undeflected neck in sauropods in the cervico-dorsal vertebral columns suggest they were straight where the neck transitions into the anterior dorsals To depolarize the dichotomy between sauropod necks as straight-and-stiff-and-horizontal versus curved-and-flexible-and-upright requires replication and independent confirmation, and convergence of contributions from multiple directions. This will allow for more substantive pursuits, such as seeking deeper explanations for their extreme specializations. But proposals about sauropod neck curvature, pose, head height and so forth have been confounded and conflicted in the literature, and progress will likely require that they be understood in basically the following order:intrinsic curvature of the vertebral column in the undeflected state.intervertebral flexibility.habitual pose for a variety of activities, including feeding, locomotion, and alert rest.characteristic motions involved in browsing, drinking, display, surveillance.vertical and lateral reach, feeding envelopes versus reachability volumes.Just as the sauropod neck is becomes better understood in terms of topics (1–3), the post-cervical skeleton is as well, permitting refined estimations of the motions and characteristics of the vertebrate as whole. Importantly, studies of sauropod forelimbs and pectoral girdles Estimates of head elevation as based on osteology alone is generally lower than that suggested by an analysis of forces and moments Inferences about sauropod necks  rapidly lead from the hard evidence, the fossil material, to speculation. Even the reassembly of the undeflected vertebral column requires understanding how they were connected by soft tissues, and yet their intervertebral joints are not known and can only be inferred. Inferences about habitual neck posture and movement are even more derivative – and necessarily more speculative – as they build upon assumptions about intervertebral flexibility, which must build upon assumptions about the intervertebral joints, and so forth.0) for some extinct taxon t0. Since P(t0) cannot be observed directly, an indirect argument is offered that involves an ‘osteological correlate’ O that reliably co-occurs with P in extant taxa and which is reliably preserved in the fossil record. O and P should be “causally associated” The Extant Phylogenetic Bracket (EPB) 0The co-occurrence of O and P is examined for the so-called extant phylogenetic bracket (EBP), namely the set of taxa that comprise the closest-related outgroup to the extinct taxon t0 might be offered as evidence that this taxon also exhibited property P: Given the physical correlate holds for extant taxa, the presence of the physical evidence O in a fossil of an extinct taxon tIf the osteological correlate O is exhibited by all taxa in the EPB, then a so-called ‘Type I’ inference is supported for the extinct taxon, i.e., While some inference about the extinct taxon is well supported by observations on extant counterparts, the inference necessarily remains a speculation – just a “more constrained speculation” An invocation of EPB is particularly weak if, in addition to neglecting osteological correlates, draws conclusions based on only a limited sampling of extant taxa that exhibit the given property: The undeflected state of an intervertebral joint, geometrically defined by centering the associated pre- and postzygapophyses.A vector constructed from cotyle center to condyle center, used to quantify curvature along a vertebral column .The angular difference between successive vertebral axes. Intervertebral curvature is zero when the axes are geometrically collinear .The curvature of a vertebral column in ONP, as distinguished from curvature induced by joint deflection .The set of all achievable combinations of mediolateral and dorsoventral flexion. As applied to a vertebral column: the set of poses , also termed a ‘reachability envelope’.Contact between vertebrae that limits angular deflection at a vertebral joint and provides load-bearing bracing against disarticulation. Osteological stops may be present independently for dorsiflexion and for mediolateral flexion, or not at all.During intervertebral joint flexion, displacement between pre- and postzygapophyses is limited by the surrounding ligamentous synovial capsule, which draws taut prior to their disarticulation, preserving a ‘safety factor’, a minimal overlap . The ZSF provides a not-to-exceed limit on joint deflection, which is further restricted by soft tissues and behaviorally (see below).To support speculation that some extinct taxon had some unpreserved property  based on 1) observation of that property in closely-related living forms, the EPB, and 2) an ‘osteological correlate’. Presence of that correlated physical evidence in the extinct taxon supports inference that the unpreserved property was also present (see below).The first two of the above five tasks concern estimation of intrinsic curvature and the extremes of what the joint geometry might allow – basic geometric (kinematic) aspects of sauropod neck shape and flexibility, and do not concern estimating their habitual poses, postural preferences, or behavioral tendencies. The success with which neck curvature and flexibility is replicated through the manipulation of the dry bones of extant vertebrates might be used to gauge the feasibility of estimating sauropod neck curvature and flexibility Quantification of intrinsic curvature and joint flexibility requires first establishing the undeflected state of the intervertebral joints. The osteologically neutral pose or ONP A vertebral column in ONP reveals the characteristic curve of the undeflected neck, which provides an important guide to how that neck is utilized Intervertebral joint flexion, in general, involves one vertebra rotating about an instantaneous or ‘momentary’ center of rotation relative to the other Wedge-shaped intervertebral disks contribute to the intrinsic lordotic or kyphotic spinal curvature of amphiplatyan vertebral columns, such as mammalian vertebrae and particularly apparent in the human lumbar spine Numerous articulated sauropod cervical vertebral columns have been found with their central condyles deeply inserted within cotyles . Since both the central articulation and the zygapophyses are diarthrodial, with free sliding motion within their capsules, angular deflection at the centrum results in predominantly a translation or gliding motion of parallel articular surfaces, which is especially apparent as the postzygapophyses sliding across prezygapophyses. That translation must be arrested at some point otherwise disarticulation will occur. It is expected that sauropods, like modern vertebrates, arrested motion prior to disarticulation, preserving a residual overlap or zygapophyseal safety factor (ZSF).Intervertebral joints flex dorsoventrally, mediolaterally, and in combination  to define a range of motion (ROM). Manipulation of dissections of turkey cervical columns A conservative ZSF of 0.5 was used to estimate the relative neck flexibility in two diplodocids In some vertebrates, in addition to limiting deflection by the ligamentous synovial capsule surrounding the zygapophyses, intervertebral joint flection may be limited by physical contact between vertebrae, e.g., between the postzygapophyses of one vertebra against the neural spine of the more caudal vertebra It has been suggested that dorsiflexion in sauropods was also limited by osteological stops, given their presence in Camelus bactrianus, Giraffa Camelopardalis and Struthio camelus While some extant cervical vertebrae are braced ontologically as they reach the limits of dorsal or lateral deflection, many extant vertebrates do not exhibit apparent morphological adaptations (pers. obs.). Nor does osteological bracing against excessive dorsiflexion appear present in sauropods . Each successive pair of vertebral axes  defines The normal division of human spine into regions of intrinsically lordotic or kyphotic spinal curvature arises partly by wedge-shaped intervertebral disks as mentioned, and partly by the vertebral osteology . The osteological contribution can be subtle but accumulative, as in the slight wedge shape of the vertebrae in the lumbar spine Many birds also have ventral OIC cranially, which together with the rise at the base of the neck, creates a sigmoid curve with an inflection in curvature mid-neck . ReptileCaution is needed to distinguish between behaviorally-induced curvature and that which is intrinsic to the osteology, particularly when attempting to draw broad generalizations about default behavioral postures Speculation regarding the relationship between ONP and characteristic poses of the neck during rest and locomotion for sauropods seems of less importance to understanding their biology than how they used their necks for feeding. The relationship between ONP and the characteristic pose for feeding in modern herbivores is straightforward only for grazers, while many low browsers will take advantage of vegetation that requires raising the head above ONP, and high browsers will very frequently feed by ventriflexion far below ONP Given the rarity and inaccessibility of physical mounts of dinosaur skeletons, skeletal drawings and composites have traditionally used to reconstruct and subsequently analyze sauropod vertebral columns. More recently, 3D digital models are being used in preference to relying on 2D artwork Skeletal illustrations have been relied upon to both summarize the bauplan of a given taxon of sauropod, and as source material on which to base estimates of head height and speculations regarding feeding, and so forth. Significant artistic liberties are sometimes noted The macronarian Camarasaurus, for example, is usually depicted to have had a sharply rising neck at the base, largely due to illustrations based on the juvenile C. lentus CM 11338. The original specimen was preserved in a severe opisthotonic posture, with the cervicals wrenched back and the zygapophyses displaced out of articulation . The skeWhile skeletal reconstructions may incorporate artistic liberties, some degree of independent verification is afforded by the detailed steel engravings or photographs of the individual vertebrae illustrations were published in the original descriptions by C. Gilmore, J.B. Hatcher, W. Janensch, W. Wiman and others. These illustrations can be scanned, composited, and placed into articulation to roughly reconstruct their ONP  [31]–[32The neck of the iconic Giraffatitan brancai mount  is reconWhile extreme in the case of the Berlin mount, it is not uncommon for neural spines to be restored in sauropod skeletal mounts as if they were in ONP. The Apatosaurus ajax at the Yale Peabody Museum (YPM VP 001980), for instance, has a gently-curved sigmoidal-shaped neck. Close inspection shows that the zygapophyses are centered, as if the vertebrae were in ONP. Still closer inspection, requiring a ladder to reach up and tap on the darkly-varnished plaster, reveals an artistic amalgam of real material and plaster (pers. obs.). The gracefully-ascending curve to the neck appears to have been conceived first, then the details of the restoration made to neatly fit that vision.The neck of Camarasaurus lentus USNM 13786-310D  was presThe Denver Museum of Nature and Science Diplodocus longus DMNS 1494 is also mounted with an upward bend in the neck at its base . Again, 2D skeletal illustrations provide only limited insight into vertebral articulation, but have been resorted to given limited access to, and manipulation of, original specimens, especially those that are mounted. Yet entire articulated vertebral columns can be manipulated in a virtual 3D space provided their morphology is converted to digital form. Fossil vertebrae can be digitized to capture their surface morphology and subsequent digital retrodeformation An alternative to digitization of original material is to begin with a set of deformable 3D models can be subsequently formed to closely resemble the morphology of the original specimens. Deformable 3D surfaces can be created using subdivision surface model techniques Through a laborious process of building then adjusting generic models of axial and appendicular elements to fit specimens, eventually entire articulated digital skeletons can be constructed , 22, 23 The following general inferences regarding sauropod vertebral joints appear supported by the EPB, with avia and reptilia as outgroups:Intervertebral central articulations were diarthrotic, with close intervertebral separations.Anterior cervical vertebrae were essentially straight (negligible OIC in ONP).Posterior cervical vertebra had slight ventral OIC in ONPIntervertebral joint flexibility was limited by synovial capsules surrounding the zygapophyses which draw taut prior to permitting disarticulation, preserving a ZSF.Cervical vertebrae were limited in dorsiflexion by the ZSF, not by osteological stops.Specific conclusions regarding sauropod necks would include the need to revise the reconstructions of brachiosaurids and camarasaurids based on 1–3, above. Regarding 4 and 5, above, the relatively ‘stiff’ necks of sauropods  and their kinematics suggest a coherent role for the sauropods neck with regard to feeding. While not yet explored in detail, the following provides a review that draws on the deep understanding of neck function in extant vertebrates towards better understanding the corresponding function of sauropod necks. As discussed below, some of the insight derives not by analogy with birds, but by how the analogy with birds fails, and yet resembles that of another, very distant group: browsing mammalian herbivores.While the relatively inflexibility of sauropod necks compared to birds is sometimes viewed with skepticism The cervical column may be regarded kinematically as a spline-like chain of fixed-length links between joints of limited angular mediolateral and dorsoventral flexibility Consider the consequences of varying vertebral length, count, and flexibility, either singly or in combination. First, increasing vertebral length alone increases reach linearly and the surface area of the reachability envelope quadratically. Increasing intervertebral flexibility, particularly caudally, also increases surface area roughly linearly . Next, while holding overall neck length constant, increasing vertebral count while trading off intervertebral flexibility and vertebral length off can produces a tradeoff, resulting in the same reachability envelope. But increasing vertebral count without proportionately reducing intervertebral flexibility greatly dramatically increases the kinematic redundancy of the neck A substantial literature on extant amniotes  shows that:Taylor, Wedel, and Naish “living animals do not habitually maintain their necks in ONP. Instead …C0: “the neck is maximally extended at the cervico-dorsal junctionC1: “and maximally flexed at the cranial-cervical junctionC2: “so that the mid-cervical region is near vertical.C3: “This is true even in apparently short-necked animals. …C4: “The fact that elevated, extended necks are widespread across Amniota means thatC5: “elevated necks should be assumed for sauropods in the absence of evidence to the contrary.C6: Elevated neck postures for sauropods are indicated by the extant phylogenetic brackets at the levels of Saurischia, Archiosauria, Diapsida, Reptilia, and Amniota.C7: “0 if a physical property O is identified, that is correlated with P in all members of the extant outgroups comprising the EPB, and O is present in traces of the extinct taxon: Recall that the EPB method supports a Type I inference about an unpreserved property P in some extinct taxon tRegarding the conjectured C0–C4 The behavioral claims C0–C4 While some mammals can and do rest in an alert state by retracting the head to balance it atop a subvertical column, the far more widely-adopted posture in quadrupeds is to cantilever the head and neck before the shoulders, in approximate ONP. The weight of the head and neck is then supported passively by means of suspension through some combination of dorsal musculature and ligaments in tension The osseous labyrinth containing the semicircular canals constitutes a potential osteological correlate for supporting inferences about the preferred or stereotypic head posture in extinct vertebrates The habitual orientation of the head relative to gravity is a behavior property, one that is fortunately correlated with the gravitational orientation of the LSC, and hence of potential use as an osteological correlate An EPB-supported inference of the gravitational orientation of the cranium in the extinct vertebrate could be inferred from 1) the observed orientation of the LSC within the cranium of an extinct vertebrate Proceeding caudally through the occiput , the gravitational slope of the neck at the atlas-axis is constrained as well. Three independent lines of evidence can thus be combined. The LSC data supports a postulated slope for the atlas-axis relative to horizontal, and post-cervical skeletal reconstructions suggest the gravitational slope of the anteriormost dorsal vertebrate , and in the middle, ONP studies of re-articulated cervical columns in the undeflected state, suggest the relative slopes at their two ends. The three lines of evidence combine satisfactorily with the following caveats :Sauropod heads were held in alert rest with a relatively small inclination α of the LSC.Sauropod cranio-cervico joints are held undeflected in alert rest.Sauropod necks are suspended, with intervertebral joints in approximate ONP .Sauropod cervico-dorsal vertebrae are held in approximately ONP in alert rest.So progressing from the cranium through the cervical column caudally and into the cervico-dorsal transition, a consistent  global picture is emerging. But speculations about how sauropods held their head in alert rest, when not otherwise occupied, has perhaps less relevance to sauropod biology compared to how the animal used its neck for feeding, and secondarily, while engaged in locomotion.Starting with the bare bones, plus caveats about their intervertebral separations based on modern vertebrates with similar articulations, the cervical vertebral columns of sauropods, relieved of their opisthotonic pose , are revSauropod skeletal reconstructions indicate a range of slopes for the anterior dorsal columns. Variation in the resting height and gravitational orientation of the head can be attributed primarily to variations in body plan without postulating any mechanism  for creating an upward bend in the base of the neck. Thus despite having no intrinsic upward bend at the base of the neck ONP, the sauropod head could have been placed at a substantial elevation above the shoulders, or at or even below the shoulders, simply due to the slope of the anterior dorsal column. Even modest dorsiflexion at the base could then produce several meters of additional head elevation in those sauropods with especially long necks, and those with long necks and high resting height could also ventriflex to bring the head down to browse low (as well as drink water). The once-held distinction between low versus high browsers is not sharply defined.Upper bounds on neck mobility are predicted geometrically for extant vertebrates, and those criteria, applied to sauropod necks, predict less-than-avian flexibility, presuming sauropod necks did not disarticulate (more than once per lifetime). Extant vertebrates that do not have a sigmoid curve to the neck, sauropod necks were well-suited for directing the head to different locations on a ‘feeding envelope’ surface rather than to any point within the volume within that surface (think cow not swan). While intervertebral flexibility was comparable to most that of reptiles, and less than most birds, they more than made up for ‘stiff’ necks by their absolute length. While some sauropods literally went to extraordinary lengths to sweep out a ‘feeding surface’ swath in front of them by flexing their necks at full extension dorsoventrally and mediolaterally, their necks were neither pre-curved avian-style nor sufficiently flexible to fully explore the volume of space contained within that surface. Despite some uvam acerbam arguments that sauropods held their heads high  sauropod necks were incapable of the prerequisite ability to retract the head sufficiently to balance it's weight above the shoulders, adopted passive suspension of the head, like extant vertebrates that share this inability. But how sauropods held their head when inactive seems of lesser importance to understanding their feeding behavior .Several of the conclusions in this review seem negative, about what sauropod necks did not look like, and what they did not do, and which popularizations are not scientifically supported and should be abandoned. For instance, none were shaped like swan necks, and there is no support for the persistent suggestion they held their heads high habitually. Perhaps the most useful such negative is that sauropods were not unique – at least, there is no evidence to suggest that what is known about the articulation, suspension, and function of extant archosaur vertebral columns does not apply as well to the sauropods, despite their extremes.All is certainly not negative: there are many EPB-supported (or supportable) hypotheses to propose and to test, given the commonality between extinct and extant archosaurs – inferences which investigators have barely begun to be explore. The lateral semicircular canal evidence is compelling (and likely to become more so), as is the upper-bounds on flexibility implied by vertebrates sensibly preserving a safety factor of overlap at the zygapophyses. Osteological bracing (both its presence and absence) and its relationship to loads imposed upon necks at the limit of flexibility has only been noted in a few cases. The kinematic importance of a sigmoidal intrinsic curve to the neck has been well appreciated for birds, and that extends to extinct vertebrates that share a sigmoidal design. But the implications of a neck that is a simple monotonic arc, devoid of a built-in inflection point, has not been previously explored either in extant vertebrates or in sauropods, and yet clearly has relevance to browse-gathering efficiency and behavior. Preconceived notions and ill-supported presuppositions will be replaced increasingly by newly-conceived notions as methodology replaces mythology in the study of sauropods.The 3D models that appear in this review have been developed in Autodesk Maya Regarding the modeling of individual bones, conventional digital modeling employes two somewhat disparate choices: importing a polygonal mesh of vertices that form a piecewise planar approximation to a surface from sampled positions across the given object which, given sufficiently many samples, creates an apparently smooth replica of an actual specimens  that represent sufficient morphology to capture the major osteological features  sufficient to model a range of variation across taxa for appendicular elements, and across both taxa and position within a vertebral column for axial elements. To model a specific dorsal vertebral column, for example, a generic dorsal vertebra model is duplicated multiple times to represent the first, mid, and last vertebrae of a given specimen. Each instance is then individually sculpted to match the shape and dimensions of its original counterpart, based on archival material, photographs, and, when available, digitized surface scans, CT, or other point-sampled data of actual specimens. Vertebral osteology in archosaurs varies sufficiently smoothly that missing elements can be interpolated over small intervals. Interpolation of missing or severely damaged elements based on adjacent elements is justifiably criticized as being somewhat speculative, as in the case of the restoration of C13–C15 in the Carnegie Museum specimen Apatosaurus louisae CM 3018 Next, blend shape animation The skeleton is then rigged to become fully articulated , the digital joints can be exercised to explore the intervertebral range of motion along the axial skeleton, reachability envelopes, and so forth, as exemplified by the figures in this review.Figure S1(TIF)Click here for additional data file.Figure S2(TIF)Click here for additional data file.File S1(ZIP)Click here for additional data file.Movie S1(MP4)Click here for additional data file.Movie S2(MP4)Click here for additional data file.Movie S3(MP4)Click here for additional data file.Movie S4(MP4)Click here for additional data file.Movie S5(MP4)Click here for additional data file.Movie S6(MP4)Click here for additional data file.Movie S7(MP4)Click here for additional data file.Movie S8A turntable movie depicting the spinal cord (red) entering the foramen magnum of Kaatedocus siberi.(MP4)Click here for additional data file.Movie S9Animation of Camarasaurus from its death pose into a life pose near ONP.(MP4)Click here for additional data file.Movie S10(MP4)Click here for additional data file.Movie S11(MP4)Click here for additional data file.Movie S12(MP4)Click here for additional data file.