Mice carrying the Agouti allele black-and-tan (ta) normally have a sharp boundary between dorsal black hair and yellow ventral hair; the Hde mutation raises the pigmentation boundary, producing an apparent dorsal-to-ventral transformation. We identify a 216 kb deletion in Hde that removes all but the first exon of the Tbx15 gene, whose embryonic expression in developing mesenchyme correlates with pigmentary and skeletal malformations observed in Hde/Hde animals. Construction of a targeted allele of Tbx15 confirmed that the Hde phenotype was caused by Tbx15 loss of function. Early embryonic expression of Tbx15 in dorsal mesenchyme is complementary to Agouti expression in ventral mesenchyme; in the absence of Tbx15, expression of Agouti in both embryos and postnatal animals is displaced dorsally. Transplantation experiments demonstrate that positional identity of the skin with regard to dorsoventral pigmentation differences is acquired by E12.5, which is shortly after early embryonic expression of Tbx15. Fate-mapping studies show that the dorsoventral pigmentation boundary is not in register with a previously identified dermal cell lineage boundary, but rather with the limb dorsoventral boundary. Embryonic expression of Tbx15 in dorsolateral mesenchyme provides an instructional cue required to establish the future positional identity of dorsal dermis. These findings represent a novel role for T-box gene action in embryonic development, identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals, and provide insight into the mechanisms that underlie region-specific differences in body morphology.Many members of the animal kingdom display coat or skin color differences along their dorsoventral axis. To determine the mechanisms that control regional differences in pigmentation, we have studied how a classical mouse mutation,  Greg Barsh and colleagues show that a member of the well-known family of T-box genes helps to form an important pigmentation boundary in mice A fundamental question in developmental biology is how adjacent regions of the vertebrate body acquire differences in their appearance or morphology. Mechanisms that establish the general body plan make use of a relatively small number of signaling pathways shared among all animals  allele and for the yellow markings around the feet, ears, or head, i.e., tan points or head spots, of certain dog breeds.One of the most obvious aspects of regional color variation in vertebrates is a dark dorsal surface juxtaposed to a light ventral surface, apparent in the color of skin, scales, feathers, or hair, in which the boundary between dorsal and ventral compartments is often sharp and lies in register with the limbs. In rodents and probably other mammals, this dorsoventral difference in hair color is brought about by differences in pigment type as determined by allelic variation of the uti gene . Secreteuti gene , Agouti uti gene  and can uti gene ; thus, iAgouti mRNA isoforms that differ by virtue of their transcriptional initiation site and 5′ untranslated exons. A “hair cycle-specific” transcript is expressed in both dorsal and ventral skin for 2–3 days during early hair growth, while a “ventral-specific” transcript is expressed throughout the entire period of active hair growth, but only in ventral skin  and beyond and may play a role in pigment cell differentiation  exhibit dorsal hair follicles that are noticeably more developed than ventral hair follicles, along with a gradual dorsoventral decrease in dermal thickness F. Howeveta/ta mice are usually attributed to pigment-type differences caused by ventral-specific expression of Agouti, but animals homozygous for a null allele of Agouti, extreme nonagouti (ea), have ventral hairs that contain less melanin than dorsal hairs, giving a slightly paler appearance to the ventral coat  and downregulation of tyrosinase activity induced by Agouti. Melanin content of individual hairs is likely to be influenced both by the number of pigment cells and their follicular environment. Regardless, dorsoventral differences in hair pigment content of ea/ea mice persist throughout multiple hair cycles into adulthood, similar to hair length (but unlike skin thickness). Thus, at least three characteristics distinguish dorsal from ventral skin: differences in pigment-type synthesis (depending on Agouti genotype), differences in hair length, and differences in melanin content.Differences between dorsal and ventral pigmentation of ral coat G. Using /ae mice G. By condroopy ear is a recessive mutation on mouse Chromosome 3; the original allele described more than 40 years ago by Hde, is available through The Jackson Laboratory . External craniofacial malformations are the most obvious characteristic of Hde/Hde animals, including widely spaced eyes, small palpebral fissures, a broad nasal area, and a shortened skull held in an elevated position, which presumably causes or contributes to the abnormal position of the ears.Named after its effects on craniofacial morphology, droopy ear because the original allele was described to affect pigment pattern in a way that suggests a possible dorsal to ventral transformation: “On a genetic background (ta and WA) which causes the belly hair to be lighter than the back hair, the belly hair comes up farther round the sides of the body and face” . However, ea/ea and ea/ea; Hde/Hde neonates are clearly distinguishable in the first few days after birth, when a dorsoventral gradient of melanogenic activity is apparent under the skin  contig and additional markers to a 1.4 Mb region that contained eight genes, including Tbx15 . However, the deletion removes 534 of the 602 amino acids encoded by Tbx15 (including the T-box DNA-binding domain), Hde/+ animals are grossly normal, and the phenotype of Hde/Hde animals is identical to that described for the original allele. In addition, other than M6pr-ps, no other genes or transcripts have been annotated to the 216 kb deletion.Using sequence information from  as Tbx8  and thenas Tbx15 . By comp M6pr-ps B and 3C. M6pr-ps . By NortTbx15 by gene targeting in embryonic stem cells. The targeted allele, LacZTbx15, carries an IRES-LacZ-neo cDNA cassette that disrupts the open reading frame at codon 154 early in the T-box domain  generated an independent mutation of x domain D. Animalmals see . These oTbx15 as first detectable at E9.5 in the limb buds, progressing to the branchial arches, flanks, and craniofacial regions through E12.5. To investigate this pattern in more detail, we hybridized a Tbx15 mRNA probe to a series of transverse sections at E12.5 and observed expression in multiple mesenchymal tissues of the head, trunk, and developing limbs . However, at E14.5, the normal ventral-to-dorsal gradient of Agouti expression appeared to extend more dorsally in Hde/Hde embryos , and dorsal skin gave rise to black hair (n = 4). Transplantation of flank skin gave rise to a patch of yellow hair juxtaposed against a patch of black hair in 85% of the successful grafts (n = 13); the remaining two flank grafts produced solely black or yellow hair. In no case did we observe intermingling of black and yellow hairs. As predicted from the experiments using tissue sections ; furthermore, positional identity is maintained throughout later stages of skin development, even though expression of Tbx15 broadens to include ventral as well as dorsal skin.We found that E12.5 is the earliest time at which embryonic ventral skin is able to produce hair when transplanted to the testis. Of the grafts that produced hair, ventral skin gave rise to yellow hair , Wnt7a, and Lmx1b are part of a network whose restricted domains of expression help to establish dorsoventral identity , T box–containing genes have been identified as developmental regulators in a wide spectrum of tissues and multicellular organisms , limb mesenchyme (Tbx15 and Tbx18), and craniofacial mesenchyme   . These ooglossia . Despitesenchyme , the conTbx15, Tbx15 that lies on Chromosome 1p11.1 as a candidate for acromegaloid facial appearance (AFA) syndrome, for which there is a weak positive LOD score to Chromosome 1p  excluded allelism of Alx3 and Hde, and a full description of the Tbx15 skeletal phenotype will be published elsewhere.Two of us (S. Kuijper and F. Meijlink) became interested in the elopment  and the oopy ear . In spitTbx15 in pigment patterning was motivated by the effects of Agouti in postnatal animals. However, Agouti is also expressed in the embryo, where it provides a convenient marker of ventral dermis identity. Because an expanded domain of embryonic Agouti expression in Hde/Hde animals is detectable by E14.5, the effects of Tbx15 on dorsoventral patterning must occur prior to this time. Among other T-box genes whose developmental actions are at least partially understood, two general themes have emerged, one focused on the ability to specify alternative fates for an undifferentiated group of precursor cells and another focused on the ability to support proliferative expansion of a cell population whose fate is already determined .In embryonic mesenchyme, expression of elopment . HoweverTbx15 also affects regional distribution of hair color in the limbs, with areas that would normally produce black hair giving rise to yellow hair instead. However, neither normal patterns of pigment-type synthesis in the limb nor their disruption in Hde/Hde mice correspond to obvious developmental compartments. Furthermore, losses of function for En1 or Wnt7a, which cause a partial transformation of the distal limb from dorsum to ventrum .  of pigment cells in ventral epidermis [Loss of  ventrum  or ventr ventrum , respectpidermis .)Tbx15 shares certain features with control of dorsoventral limb patterning by Lmx1b, a LIM domain transcription factor that acts downstream of Wnt7a and En1  or the limb (Lmx1b)  . In the ectoderm . Wnt7a, ectoderm , whose eectoderm . As descTbx15 in dorsal trunk mesenchyme, which then establishes dorsal positional identity of those cells as manifested by differences in Agouti expression, pigment-cell development, and hair growth. Because the ventral limit of Tbx15 expression corresponds to the dorsal limit of En1 expression and because the normal position of the pigmentation boundary lies approximately in register with the limb-bud outgrowths, we depict the position of a putative dorsoventral boundary in trunk ectoderm as coincident with the limb dorsoventral boundary. This model is consistent with studies in the chick, where distinct dorsal and ventral lineage compartments exist for ectoderm in both the limb   , 2000.Tbx15 in ventral mesenchyme should give rise to a dorsalized pigmentation phenotype and could be tested with gain-of-function approaches. However, Tbx15 expression is very dynamic and is restricted to dorsal mesoderm only from E11.5 to E13.5. It is possible that Tbx15 influences skin patterning in a very narrow window of development; alternatively, establishment of dorsal identity by Tbx15 may require another as-yet-unidentified factor that is only present in the mesenchyme underlying dorsal ectoderm.Our model predicts that ectopic expression of Msx1, whose expression marks a subgroup of somite-derived mesenchymal cells that contribute to dermis in a narrow stripe along the paraspinal region , mice carrying the R26R lacZ reporter allele , and C57BL/6J (B6) e/aea mice . The Hde mutation arose in the 1960s in Harwell, probably on the BN strain background . We obtained Hde on a B6/EiC3H background, introduced the ta allele from the BTBR strain, and have maintained the line as a mixed Hde/+ × Hde/+ intercross stock with periodic outcrossing to BTBR or B6. For timed matings, the morning of the plug was considered E0.5. Postnatally, the day of birth was considered to be P0.5.All mice were obtained originally from The Jackson Laboratory , except the BA strain , For measurements of hair length and color, the entire interlimb region of skin was first dissected with a single incision at the dorsal midline and preserved with powdered sodium bicarbonate. Slices 2–2.5 mm in width were then prepared parallel to the dorsoventral axis, hair length boundaries determined from electronic images with Adobe Photoshop , and measurements obtained using ImageJ . This approach samples awls and auchenes, because they are much thicker and therefore visually more predominant than zigzag underhairs. To assess dorsoventral variation in hair-type distribution, several hundred hairs were plucked from the middorsum or midventrum of 8-wk-old male BA strain animals, then sorted and categorized using a dissection microscope. No attempt was made to distinguish between awls and auchenes.For skin histology, 12 μm sections from paraffin-embedded tissue were stained with hematoxylin and eosin. For DOPA staining, the dermis and epidermis were split after 3 h of incubation in 2 M sodium bromide at 37°C (this preparation causes most hair follicles to remain with the dermis), individually fixed for 1 h, then rinsed and stained with 0.1% L-DOPA , 0.1 M sodium phosphate buffer (pH 6.8) for 5 h at 37°C in the dark, changing the staining solution after 1 h. The samples were then fixed overnight, dehydrated, and mounted. This method is sufficient to stain interfollicular melanocytes without creating a high background. The fixative used was always 4% paraformaldehyde.Hde was generated from an intersubspecific intercross between Hde/Hde and CAST/Ei mice. We initially localized Hde to a 1 cM interval between D3Mit233 and D3Mit11. F2 animals carrying recombinant chromosomes between these markers whose genotype at de was indeterminate (Hde/+ or +/+) were progeny-tested by crossing to Hde/Hde animals. Further genetic mapping established a minimal region of 0.1 cM between D3Mit213 and 16.MMHAP32FLF1; these markers were used to initiate construction of a physical map with BAC genomic clones . End sequence from those BACs was used to develop SSCP markers M1 to M3, as depicted in http://genome.ucsc.edu); the 1.4 Mb interval between M1 and M4 contains eight genes: four hydroxysteroid dehydrogenase isomerases, Hsd3b3, Hsd3b2, Hsd3b6, and Hsd3b1; an hydroacid oxidase, Hao3; a tryptophanyl-tRNA synthetase, Wars2; a T-box gene, Tbx15; and a novel gene, 4931427F14Rik. In the genome sequence, M1 primers correspond to AGGCCTCCAATAAGTTCTGGGTACC and AAGCTTGCTCTCTGGATTCCATTTGTAG, the M2 reverse primer corresponds to AAGCTTGGCTTTAGTCCCAGTGGGC, and the M3 primers correspond to CCTCCAGGAAGAATCTACTAGGCAC and AATGAAAAAAAAAAAAGTAAGATTGAAAG. Minor differences among the sequences of the primers we obtained from the BAC ends and the public genome sequence may represent strain differences or sequencing errors on the BAC DNA.A high-resolution map for Hde deletion using primers GGAGCAGATCCAATTGCTTT, TCCATAGCCCATCTTCACAA, and CATGTCCACTTCTGCTTCCA. This PCR assay produces a 392 bp product from the Hde chromosome and a 595 bp product from the nonmutant chromosome.A multiplex genotyping assay was developed to genotype for the Tbx15 was constructed using the same approach described in IRES-LacZ-neo cassette with 5′ and 3′ homology arms of 3.5 kb and 1.8 kb was inserted into a unique BamHI site that lies 479 nucleotides downstream of the transcriptional initiation site (relative to the mRNA sequence) in exon 3. Positive ES clones were injected into B6 blastocysts, and chimeric founders crossed to either B6 mice or to Hde/+ animals.A targeted allele of Hde/+ mice. Embryos E13.5 or younger were fixed for 24 h; those older than E13.5 and postnatal skin were fixed for 36–48 h prior to embedding. The Tbx15 probe was generated by RT–PCR using primers GGCGGCTAAAATGAGTGAAC and TGCCTGCTTTGGTGATGAT (corresponds to exons 1 and 2), and the En1 probe was generated by PCR from genomic DNA using primers ACGCACCAGGAAGCTAAAGA and AGCAACGAAAACGAAACTGG (located in the last exon). The Agouti probe corresponds to the protein-coding sequence.In situ hybridization was carried out on 12-μm paraffin sections using digoxigenin-labeled RNA probes  according to standard protocols . Embryosta/ta × B6-a/a)F1 embryos at E12.5 were dissected in sterile Tyrode's solution, and embryonic skin was divided into dorsal, flank, and ventral pieces, each 1–2 mm2 in size, as shown in  accession numbers discussed in this paper are for 4931427F14Rik (AK016477), Agouti gene (L06451), Alx3 (U96109), Alx4 (AF001465), En1(L12703), M6pr-ps (X64069), Tbx14 (AF013282), Tbx15 (AF041822), Tbx18 (AF306666), and Tbx22 (NM_145224).The GenBank (http://www.ncbi.nlm.nih.gov/omim/) accession numbers discussed in this paper are for acromegaloid facial appearance (MIM 102150), frontofacionasal syndrome (MIM 229400) and human syndrome X-linked cleft palate and ankyloglossia (MIM 303400).The OMIM (


This study evaluated the psychometric properties of the ADDQoL-Teen, an innovative individualised, patient-centred questionnaire measuring perceived impact of diabetes mellitus on quality of life (QoL) of teenagers. Respondents rate all 30 life domains for frequency, and personally applicable domains for 'bother'. Two overview items measure present QoL and diabetes-dependent QoL. ADDQoL-Teen design was based on the ADDQoL (for adults with diabetes).Interviews and discussion groups were conducted with 23 teenagers aged 13–16 years, during work to design the ADDQoL-Teen. The new questionnaire was then completed by 152 young people, (mean age 16.4 ± 2.4 years), attending diabetes clinics at six UK centres.lie in bed (-3.68 ± 3.41), interrupting activities (-3.5 ± 3.23), worry about the future (-3.45 ± 3.28), career (-3.43 ± 3.15) and sweets (-3.24 ± 3.24), (maximum range -9 to +3). Analysis of the overview items showed that although 72.5% considered that their present QoL was good or brilliant, 61.8% felt that having diabetes had a negative impact on QoL, but 35.6% reported no impact and 2.6% reported a positive impact on QoL.Five domains detracted from the measure's reliability and factor structure, four of which were analysed separately and one deleted. The 25-domain ADDQoL-Teen had high internal consistency reliability  and could be summed into an overall Average Weighted Impact score. There were two subscales: a 10-item Impact-Self subscale  and a 15-item Impact-Other subscale . Both subscales had good internal consistency reliability, [Cronbach's alpha coefficients of 0.82 (N = 142) and 0.88 (N = 138) respectively]. Domains reported as most severely (and negatively) impacted by diabetes were (mean weighted impact ± SD): The ADDQoL-Teen is a new measure of perceived impact of diabetes and its treatment on QoL of teenagers. It will help healthcare professionals and parents consider QoL issues as well as medical outcomes when caring for young people with diabetes. It may be used in clinical trials and for routine clinical monitoring in a context of continuing evaluation. Increasing numbers of children are being diagnosed with diabetes mellitus  and, oncAdults may not be able to assess a child's point of view adequately, so children themselves should rate their own QoL wherever possible -5, and aThis paper describes the design and subsequent psychometric validation of a new teenager-centred, individualised measure of the impact of diabetes on the QoL of teenagers, the ADDQoL-Teen. The ADDQoL-Teen follows the philosophy underpinning the individualised ADDQoL measure for adults, but ideas in the teenager version are more specific and concrete than the broader, more abstract concepts of the adult version.Four hospitals in the Greater London area participated in the research to design the questionnaire, following Ethical Committee approval. To help identify QoL issues for inclusion in the ADDQoL-Teen measure, clinic sessions were observed, health professionals consulted, and a literature review undertaken. Semi-structured interviews using open-ended questions were then conducted with 10 teenagers with diabetes, and discussions took place with 13 teenagers in small groups of 2–4 teenagers each. The views of 23 young people, aged 13–16 years, were obtained in all. The groups were single-sex as teenagers might be inhibited from talking freely about sensitive issues with members of the opposite sex present.This qualitative research identified important QoL issues that formed the content of 30 items in the new ADDQoL-Teen questionnaire. The items were designed to reflect the teenagers' own perceptions of life with diabetes and measure their individual feelings about the importance of the issues in their everyday lives, rather than being based on researchers' or professionals' opinions. The questionnaire items, response choices and format were based on comments from the teenagers to ensure that the items were child-centred and had face validity for the teenagers themselves. The design of a questionnaire for teenagers was part of a wider study to design child-centred questionnaires for children with diabetes in three age ranges including 5–8 years (ADDQoL-Junior) and 9–12 years (ADDQoL-Junior Plus) .frequency ('a' stem) with which diabetes impacts on each aspect of life, and then how much that particular domain bothers them ('b' stem). The majority of stem 'a'/frequency items are in the format: Do you ever ..... because of your diabetes? and stem 'b'/bother items in the format: Does it bother you when ..... because of your diabetes? . Stem 'b'/bother has response options scoring from -3, -2, -1, 0  and a positive response option  scoring 1.In order to produce an individualised questionnaire, the ADDQoL measure of the impact of diabetes on QoL of adults  measuresextra things, 13: out of fix, and 30: holidays) that have a positive sense. The 'b'/bother stem is scored differently for these three positive items. For example, the responses to item 7b (How do you feel about having extra things because of your diabetes?) are scored 3, 2, 1, 0, -1 (from I like having them very much......I don't like having them).The ADDQoL-Teen questionnaire has 30 items dealing with specific life domains, in which the wording of item stems and response choices is appropriate to teenagers. Table present QoL and QB: diabetes-dependent QoL. QA  is scored 3, 2, 1, -1, -2 respectively. QB  is scored -3, -2, -1, 0, 1. There is a free comments section at the end of the questionnaire where respondents are asked if there is anything else they would like to say about their life with diabetes.Finally there are two overview/global items: QA: Negative items: The 'a'/frequency ratings in categories scoring 1, 2, and 3 are multiplied by the corresponding 'b'/bother ratings to give a weighted score from -9 to +3 (maximum negative to maximum positive impact of diabetes on a domain). Zero scores on the 'a'/frequency rating are ignored as these items are not applicable to the individual and no 'b'/bother rating is made. The overall ADDQoL-Teen Average Weighted Impact score (ADDQoL-Teen AWI) can be calculated by summing weighted impact scores for all applicable domains before dividing by the number of domains applicable to the individual teenager. ADDQoL Teen AWI varies from -9 to +3, the maximum negative to maximum positive weighted impact of diabetes on overall QoL.Positive items: items 7, 13, and 30 have weighted scores from -3 to +9 (maximum negative to maximum positive impact of diabetes on that domain). The weighting procedure for positive items is similar to that for negative items.Overview items: QA and QB are not included in the calculation of AWI, but analysed individually.In order to determine the psychometric properties of the 30-item ADDQoL-Teen, at least 150 completed questionnaires were needed, as factor analyses ideally require five or more respondents per item , and alsor the patient had moved from paediatric to adult care in the previous year. Moving from paediatric to adult care was defined as moving out of the care of the paediatric team. Depending on the size of the caseload in each research area, the nurse either included all patients or a random sample that fitted the criteria in the sampling frame. Ethical Committee approval was obtained for the study to be conducted at all the centres.The criteria for inclusion were: the patient was expected to move from paediatric to adult care in the following year No – I do not response option  would normally be included in factor and reliability analyses, as the SPSS statistical package treats N/A responses as missing. Furthermore, if the SPSS default of listwise deletion of missing data is used, all cases that have any missing values across all 30 items are lost to analysis. Results of reliability and factor analyses are therefore reported below with SPSS set to pairwise deletion of missing data, and N/A responses to read as zero, to avoid considerable loss of data. This procedure has been fully described for the original development of the ADDQoL for adults  and the slope (0.916) also close to 1, .No – I do not (N/A) response option (Table miss events (82%), 14: sleep away (80%) and 30: holidays (72%). Thus the great majority of respondents did not consider that they missed any events, or sleeping over at a friend's house as a result of their diabetes, nor had their diabetes resulted in going away on British Diabetic Association  holidays or weekends or making new friends. The areas of greatest importance/frequency of feeling (Response: Yes – a lot in the 'a'/frequency stem) were: 5: insulin (69%), 9: finger tests (49%), 28: social life (26%), and 2: sweets (25%). The areas where the highest percentage of young people considered they were very much bothered were: 12: unwell (25%), 6: bleed, 8: interrupt do and 19: high BG ,  was used by up to 19% of respondents: 19% liked going to the diabetes clinic, 7% liked finger prick blood tests, 5% liked taking insulin, and 3% liked other people fussing or worrying about them because of their diabetes .Frequency analyses of domains indicated that from 1 to 82% of respondents used the on Table . The itelie in bed (-3.68), 8: interrupt do (-3.5), 20: worry future (-3.45), 21: career (-3.43) and 2: sweets (-3.24) , 18: low BG (-1.71), 25: responsibility (-1.83) and 10: control (-1.85). Diabetes had the most positive impact on 30: holidays (2.4) (noting that this item was only applicable to 28% of respondents) and 7: extra things (1.78). Overview items found that although the majority (72.5%) considered that their present QoL was good or brilliant (mean 1.79), 61.8% felt that having diabetes had a negative impact on QoL (mean -0.83), but 35.6% considered it had no impact on QoL, and 2.6% that the disorder had a positive impact on QoL  for 16: uture -3.5, 21: caextra things, 13: out of fix, 30: holidays) had unsatisfactory loadings, (<0.4), in a forced 1-factor analysis of the 30-item scale. It was decided to omit them from summation of AWI and, as further reliability and factor analyses did not indicate that they formed a subscale, to analyse each of them as separate items. Item 29: clinic visits had a relatively low corrected item-total correlation (0.218), reduced the reliability of the whole scale, and had an unsatisfactory forced 1-factor loading (<0.2). Indeed a high percentage reported that they were not bothered by attending clinic (57%) or that they liked it (19%). Item 29 can also be analysed separately. However, item 14: sleep away had an unsatisfactory forced 1-factor loading (<0.4), and although it contributed to the overall scale reliability, a very high percentage (80%) regarded the domain as N/A and, of those for whom it was applicable, only 8% found that it impacted a lot or a fair bit on their QoL. As the domain was covered by 17: miss events, it was decided to delete item 14 from the scale.Preliminary factor and reliability analyses were conducted to determine the number of items in the scale that could be summed into the overall ADDQoL-Teen AWI score. Full results are not provided, but these analyses resulted in the decision not to include five items  in the summation of an overall scale AWI, for the following reasons. The three positive items . All corrected item-total correlations were >0.37, i.e. well above the acceptable minimum. None of the 25 items would increase the alpha coefficient if deleted from the scale (Table lie in bed) loaded at ≥0.4 . Factor 2 was concerned with consumption of food and drink . Items concerning the effects of diabetes and its treatment on the individual loaded on the remaining five factors in a pattern that was difficult to interpret and with some items double loading. The scree plot indicated two factors.An unforced Principal Components Analysis with Varimax rotation found seven factors (not shown). Items referring to other people/the external world loaded on Factor 1 , and 28: social life. Item 27: go toilet double loaded, loading slightly higher (0.358) on this factor than on Factor 2 (0.329). Factor 2 contained items connected with diabetes, its treatment and effects on the individual, e.g. 5: insulin, 6: bleed, 9: finger tests, and 11: moody. Item 25: responsibility double loaded, slightly higher on Factor 2 (0.295) than on Factor 1 (0.293). The factors accounted for 20.7% and 17.0% of the variance respectively.A forced 2-factor analysis gave the clearest factor structure Table . Factor responsibility double loaded slightly higher on Factor 2 than on Factor 1, and it was decided to retain this item in the Impact-Self subscale because taking responsibility for diabetes and its treatment will rest increasingly on the individual child as he/she grows older.The best solution seemed to be that the 25-item scale had two subscales, one relating to the effects of diabetes and its treatment on interactions with others and the external world  and the second to effects on the individual . Item 25: others fuss, miss events, career, different, not allowed, family life, play sport and social life, on the Impact-Other subscale, clearly relate to interactions with the others and the external world. The consumption of food and drink very often occurs in a social context. Frequent visits to the toilet (27: go toilet) or having to stop an activity to inject insulin (8: interrupt do) may cause embarrassment socially as well as being annoying for the individual. The association of the other items on this scale with the external world is also explicable: item 10: control refers to the individual taking control of diabetes, with less help from other people; and having to get up early in the morning to test/inject may be a major issue, particularly for teenagers, again making the young person with diabetes feel different from others (16: lie in bed).Domains of insulin, bleed, finger tests, moody, unwell, wake nights, high BG and low BG. As pointed out above, taking responsibility for treatment may have greater impact on the individual child with increasing age (item 25), at the same time the child with diabetes may worry about his/her own future (item 20).Eight of the ten items of the Impact-Self subscale clearly relate to the effects of diabetes and its treatment on the individual (domains of lie in bed) would increase alpha if deleted, and then only by 0.001. Similarly the 10-item Impact-Self subscale also had very satisfactory reliability . All corrected item-total correlations were satisfactory (>0.38) and no item would increase alpha if deleted. These analyses confirmed the reliability of the subscales, and gave support for summing the subscale items into their respective subscale total scores.Internal consistency reliability of the 15-item Impact-Other subscale was very satisfactory , but falling short of the optimal alpha of 0.9. All corrected item-total correlations were satisfactory (>0.38) and only one item  are required of measures that are being used to compare an individual's scores across time  and for Analysis of the data for the whole sample found that mean overall ADDQoL-Teen AWI was -2.39 ± 1.68, mean Impact-Other was -2.44 ± 1.86 and mean Impact-Self was -2.31 ± 1.86, (maximum possible range -9 to 3) implying that young people perceived that diabetes had a negative impact on their QoL, on interactions with others and the external world, and on themselves.bleed reached significance. Female respondents showed significantly greater perceived negative impact of having bleeding or bruising at site of insulin injection (-3.01 ± 2.92) than did males (-1.65 ± 2.21) . Sex differences also approached significance for 20: worry future (p = 0.011), and 22: different (p = 0.043) and, considering the three positive items as another family of variables, for 7: extra things (p = 0.026). Compared with males, females showed a tendency towards greater perceived negative impact of diabetes on feeling different from peers, worries about the future, but greater positive impact on getting extra things because of their diabetes.There were no significant sex differences in ADDQoL-Teen AWI and subscale scores after a Bonferroni correction requiring significance of p = 0.017 or less for that family of variables. However, the sex difference in Impact-Self approached significance (p = 0.028) on a Mann-Whitney test, with female respondents tending to show greater perceived negative impact of diabetes on self-related factors (-2.6 ± 1.85) than did male respondents (-1.99 ± 1.84). Considering the 25 ADDQoL-Teen items as another group (with Bonferroni correction requiring minimum significance of p = 0.002), sex differences in 6: diabetes-dependent QoL (rho = 0.49), and a smaller correlation, as expected, with overview item QA: present QoL (rho = 0.34).Small but significant positive correlations with age were found for AWI and the two subscales Table  indicatiworry future (-3.53 ± 3.36), 16: lie in bed (-3.51 ± 3.32), 8: interrupt do (-3.48 ± 3.33), and 23: not allowed (-3.4 ± 3.12). A forced 1-factor analysis of the scores of the 124 teenagers in the 13–18 age range on all 30 items, found support for excluding the same five items from the scale as described above for the whole sample . All 25 ADDQoL-Teen items loaded >0.4 on a forced 1-factor analysis except 10: control and 25: responsibility .The mean age of those in the 13–18 year age group was 15.82 ± 1.47, a little less than that of the whole sample (16.36). Mean weighted impact scores of the younger group were very similar to those of the full sample  on Impact-Other in the 13–18 age group. 27: go toilet loads >0.4 on Impact-Other in the 13–18 age group, but double loads with the wider age range.Regression analysis found no significant difference between the forced 1-factor loadings for the subset of 13–18 year olds and those for whole sample (N = 152). The correlation of 0.954 was close to 1, the constant 0.027) was close to zero  and the slope (1.04) was also close to 1, . This high correlation indicated that data from the whole sample could substitute for that from the narrower age range. Table  was closcontrol would marginally increase alpha if deleted (0.9133). All corrected item-total correlations were satisfactory. The scale was found to be reliable at 0.9 with up to two items missing and reliable at 0.8 with up to 10 items missing. The 15-item Impact-Other subscale had good internal consistency reliability  and was reliable at 0.8 with up to four items missing. The 10-item Impact-Self subscale was reliable  if no items were missing. All corrected subscale item-total correlations were satisfactory.Cronbach's alpha of the whole 25-item scale was 0.9132, (N = 106) and only 10: Note: Cronbach's alpha for the sample in the 13–18 age range was only marginally lower (by 0.005) than that for the narrow 13–16 age range , again indicating that the addition of respondents aged 17–18 years is not harmful to the questionnaire's reliability.The free comments section at the end of the ADDQoL-Teen was used by 49 young people in all. The majority of respondents' comments emphasised a response that they had already made to a questionnaire item. The following areas were mentioned by at least four individuals and are not directly covered in the questionnaire. Consideration will be given to adding further items to cover these new areas in the future:• The effect of diabetes on patient's lives, and having to organise/plan life around diabetes and its treatment (nine respondents).• Other people, including healthcare professionals, not understanding diabetes and its effects on the young person's life (five respondents).• Concerns about weight, and difficulty losing weight (four respondents).th birthday. However, each of these four 17 year olds commented on a different aspect of life with diabetes not already covered by the questionnaire . If the questionnaire was not suitable for those aged 17–18, and was missing important domains for these older respondents, it is very likely that a greater number of older respondents would have taken the opportunity to comment at this point. We can be reassured therefore that the questionnaire is suitable for the older age group (17–18 years), even though the measure was not specifically piloted with them.Although 17–18 year olds were not included in the focus groups at the questionnaire design stage, analysis of the free comments showed that only five of the 49 respondents offering comments fell outside the narrower age range of 13–16 years: four young people were aged 17, and one was within a few days of their 13The ADDQoL-Teen is a new child-centred, individualised questionnaire measuring the impact of diabetes and its treatment on the QoL of teenagers. The items not only reflect the concerns of teenagers with this condition, as expressed in interviews and focus groups, but also use teenagers' wording where possible. Twenty-five of the life domains form a scale with excellent internal consistency reliability. Summation of the weighted impact scores from the applicable items into a single score, the ADDQoL-Teen AWI, gives a measure of the Average Weighted Impact of diabetes on the QoL of the individual. There are two subscales: the 15-item Impact-Other subscale, measuring the impact of diabetes and its treatment on interactions with others and the external world, and the 10-item Impact-Self subscale, measuring the impact of diabetes and its treatment on the individual. Both subscales have good internal consistency reliability. The two overview items (QA and QB) provide global measures of an individual's present QoL, and the perceived impact of diabetes and its treatment on their QoL respectively and, as expected, QB has a higher correlation with AWI than QA, as both QB and AWI measure impact of diabetes on QoL. Of the original 30 items, one item, concerning sleeping away from home, was deleted from the scale as it detracted from scale reliability and factor structure, and was not applicable to the great majority of respondents. Four items, three of which concerned potential positive aspects of diabetes such as getting extra things like snacks or treats, either did not load well with the 25 items in the single scale, or detracted from reliability, but can be analysed individually.good or brilliant, young people perceived overall negative impact of diabetes on QoL (AWI), including negative impact on interactions with others and the external world (Impact-Other), and on themselves (Impact-Self). However, interesting information can also be gleaned by analysing frequencies of individual domains. Domains reported as most severely (and negatively) impacted by diabetes were lie in bed, interrupt do, worry future, career and sweets. These show the particular concerns of young people about not being able to stay in bed in the morning like many of their contemporaries, owing to the demands of the diabetes treatment regimen, and the way that this treatment regimen interrupts their normal day-to-day activities. Respondents were also looking to the future and were concerned about their career prospects, getting married, having children, and their longer-term health. The impact of diabetes on consumption of carbohydrates was most notable in relation to eating sweets. The usefulness of the questionnaire's bi-polar scale was indicated by the numbers of individuals who chose a positive response: almost a fifth of respondents liked attending their diabetes clinic, and perhaps a surprising number liked taking insulin or doing finger prick blood tests (5% and 7% respectively). It was also interesting to note that concerns about having a low blood glucose level had the least negative impact on QoL of any of the domains, although this aspect of diabetes is of major concern to healthcare professionals.Despite the majority describing their present QoL as diabetes-dependent QoL was too low (rho = 0.49) to allow the single overview item to replace the 25-item scale for most purposes.Some sex differences were found. Girls and young women showed significantly greater perceived impact of experiencing bleeding or bruising at the site of insulin injection, and there was a non-significant tendency for females to show greater perceived negative impact than males with respect to feeling different from peers, and worries about the future, but greater positive impact on getting extra things because of their diabetes. With increasing age, correlations indicated reduced perceived negative impact of diabetes on overall QoL (AWI), on relationships with others and the external world (Impact-Other) and on self-related factors (Impact-Self). Present QoL also improved with increasing age. The moderate correlation between ADDQoL-Teen AWI and the overview item QB: Content validity was also good: relatively few respondents mentioned new domains in the free comments section at the end of the questionnaire. However, consideration will be given in the future to adding further items to cover new areas: organising life around diabetes, other people's understanding of the condition, and concerns about excess weight.The teenagers involved in interviews and focus groups during work to design the questionnaire lived in and around London. However, the respondents in the questionnaire study were from six areas in Britain, and there were clear indications of acceptability to all in terms of very high completion rates, and that neutral, non-regional vocabulary had been chosen. In order to have sufficient data for the psychometric analyses it was necessary to use the data for all 152 respondents, even those who fell outside the age range for which the questionnaire was originally designed (13–16 years). Nevertheless, completion rates indicated the acceptability of the questionnaire to a much wider age range than that for which it was originally intended. This is a valuable outcome, as there is considerable variability in age range at paediatric, adolescent, transition and adult diabetes clinics between different diabetes services in the UK. Indeed the mean age of respondents from one of the centres in the present study, a paediatric clinic, was 19.4 years.The questionnaire can also be recommended for 13–18 year olds, as analyses performed on the subset of data for this age group found results very similar to those for the full data set. Although the sample size 124) in the 13–18 year age range was less than optimal, the factor structure was clear and very similar to that of the wider age range, and the full 25-item scale and two subscales also had very good internal consistency reliability. There appeared to be some slight differences in mean weighted impact scores between the two groups  are faced with the day-to-day responsibility for the management of diabetes, and any improvements in QoL, whilst welcome in themselves, may also mean that these young patients will be more likely to follow the planned treatment regimen which will, in turn, help improve control of blood glucose levels and contribute to a reduction of long-term complications of diabetes.Although physiological measures are used to monitor the treatment of children and young people with diabetes, there are no child-centred, individualised psychological instruments currently in use in paediatric clinics that measure the The internal consistency reliability and some aspects of the validity of the new child-centred, individualised ADDQoL-Teen have been established for young people with diabetes, and the measure may be recommended for use with individual patients. The new questionnaire should help health professionals to consider psychological issues as well as medical outcomes when caring for young people with diabetes. The instrument is also expected to be useful in evaluating new treatments and educational interventions for diabetes in clinical trials.CB and RJH designed the ADDQoL-Teen, with RJH conducting the interviews and focus groups with teenagers that informed the design of the measure. JD and NJHM conceived and designed the interview study and JD conducted the interviews and collated the questionnaires. CVM carried out the psychometric and statistical analyses of questionnaire data and drafted the manuscript. CB contributed to the interpretation of psychometric analyses, decision-making regarding item selection, and manuscript preparation. All authors read and approved the final manuscript.For access to and a licence to use the ADDQoL-Teen, contact the copyright holder, Clare Bradley PhD, Professor of Health Psychology, Health Psychology Research, Royal Holloway, University of London, Egham, Surrey, TW20 0EX.c.bradley@rhul.ac.ukEmail:


S) mutant mice.The progressive nature of Wallerian degeneration has long been controversial. Conflicting reports that distal stumps of injured axons degenerate anterogradely, retrogradely, or simultaneously are based on statistical observations at discontinuous locations within the nerve, without observing any single axon at two distant points. As axon degeneration is asynchronous, there are clear advantages to longitudinal studies of individual degenerating axons. We recently validated the study of Wallerian degeneration using yellow fluorescent protein (YFP) in a small, representative population of axons, which greatly improves longitudinal imaging. Here, we apply this method to study the progressive nature of Wallerian degeneration in both wild-type and slow Wallerian degeneration  among a majority of fully intact or degenerated axons 37–42 h after transection and 40–44 h after crush injury. Axons exist in this state only transiently, probably for less than one hour. Surprisingly, axons degenerated anterogradely after transection but retrogradely after a crush, but in both cases a sharp boundary separated intact and fragmented regions of individual axons, indicating that Wallerian degeneration progresses as a wave sequentially affecting adjacent regions of the axon. In contrast, most or all WldS axons, once they finally degenerate, do so by a fundamentally different mechanism, indicated by differences in the rate, direction and abruptness of progression, and by different early morphological signs of degeneration. These observations suggest that WldS axons undergo a slow anterograde decay as axonal components are gradually depleted, and do not simply follow the degeneration pathway of wild-type axons at a slower rate.We conclude that Wallerian degeneration progresses rapidly along individual wild-type axons after a heterogeneous latent phase. The speed of progression and its ability to travel in either direction challenges earlier models in which clearance of trophic or regulatory factors by axonal transport triggers degeneration. Wld S) mutant mouse, in which this active process seems to be turned off. Accordingly, this mutant shows a tenfold delay in Wallerian degeneration and synapse breakdown after experimental nerve injury [WldS that arose by spontaneous mutation [WldS mutation on mouse chromosome 4 comprises a stable 85-kb tandem triplication [S mice express a novel chimeric protein (WldS protein) in neuronal nuclei that has full Nmnat 1 activity but seemingly no Ube4b function since the expressed N-terminus lacks ability for multi-ubiquitination within the ubiquitin-proteasome system (UPS) [S protein could be responsible for the phenotype through a nuclear process that has an indirect effect on the axon although recent results foster that the WldS mechanism is likely to involve a gain of function of NAD synthesis [S protein remains a possibility [Wallerian degeneration, the characteristic degeneration sequence of nerve fibres separated from their cell bodies, was described by Waller in 1850 . Followie injury . The dele injury . The undmutation ,10. Genelication ,12 encodem (UPS) ,14, the ynthesis . Howeversibility ,17.S is currently under intense investigation. The neuroprotective mutation alleviates diverse PNS axon disorders, including dysmyelination and dying back neuropathy in P0-/- mutants [pmn mutants [S was reported to attenuate pathology in acute CNS lesions caused by stroke [gad) mice, a CNS axonal spheroid pathology [From a clinical point of view not only traumatic disorders such as nerve, spinal cord or head injury result in Wallerian degeneration  but it i mutants , motor n mutants  and axon mutants -28. Morey stroke , Parkinsy stroke , and in athology . A betteDespite research extending over more than 150 years and its frequent use as tool to detect interneuronal connections in the CNS since the time of Cajal, fundamental issues of Wallerian degeneration remain unresolved and controversial even on a purely morphological level. Among these is the spatiotemporal pattern of Wallerian degeneration along the separated nerve stump. Understanding the exact pattern of spread should provide additional insights into the mechanisms of axon death and may indicate strategies to alter Wallerian degeneration in neurological disease. Shortly after the pioneering investigations of Waller, and in the following decades, there has been much debate as to whether degeneration occurs in an anterograde direction, a retrograde direction or simultaneously along the separated nerve stump axons .4L5L6 radiculotomy. Contrary to these views, Lunn and colleagues [The controversy over the directionality of Wallerian degeneration has arisen partly because appropriate methods to follow axons over considerable distances did not exist until recently but also because the course of Wallerian degeneration varies with many experimental factors. Thus, spatiotemporal evolution of Wallerian degeneration depends on the laboratory animals used ,34-36, olleagues  showed blleagues . In celllleagues . Taking lleagues  tracked lleagues ,53.In view of the contradictions and anomalies in the previous literature, we have reassessed the directionality of Wallerian degeneration using a recently introduced technique to visualize individual fluorescent axons over cm-long distances during degeneration . This waS nerves that could yield important clues for understanding classic Wallerian degeneration. Therefore, we also report a detailed characterisation of injury-induced axon degeneration in slow Wallerian degeneration mutant mice in order to determine whether axons degenerate with a similar spatial evolution to that in wild-type mice, but in "slow motion", or whether the process is fundamentally distinct. We report a series of differences between axon degeneration in wild-type and WldS mice, suggesting that irreversible injury in axons where Wallerian degeneration is blocked eventually leads to a different pathway of degeneration.To our knowledge there have been no reports about the spatiotemporal pattern of the much-delayed axon degeneration in peripheral WldIn preliminary experiments we used conventional light and electron microscopy to investigate whether Wallerian degeneration is progressive in wild-type mouse peripheral nerves. We were never able to find any significant gradients of degeneration along injured nerves that were processed with these traditional methods (data not shown). We then looked for signs of progression in localised observations of degenerating YFP-H nerves because fragmentation of YFP-labelled axons from these mice corresponds to granular disintegration of axoplasm as well as myelin ovoid formation and YFP positive axons represent the whole myelinated axon population . Axonal The failure to observe any gradient of degeneration in the above experiment does not mean that Wallerian degeneration is not progressive. It could propagate so rapidly that it was not detectable by this method, or a gradient might not be detectable because of the considerable statistical noise of the highly asynchronous process. In order to investigate these possibilities we turned to confocal tracing of individual axons in long wholemount YFP-H nerve segments. At 37 h after transection we found 2.0 % of fluorescent axons with extensive proximal fragmentation and intact distal regions indicating an anterograde gradient of Wallerian degeneration in these axons Fig. . Around In contrast, all partially fragmented axons after crush injury at all investigated timepoints were fragmented in distal tibial nerve but not at the proximal end of the distal stump Fig. . Once agSummarising all these quantification results of cut and crush lesions at time points where partially fragmented fibres were observed, on average 94.8 % of all axons were either completely intact or fragmented and 5.2 % showed a gradient of Wallerian degeneration, whose orientation depended on the lesion type.To study further the gradients of axonal fragmentation both after cut and crush injury Fig. , 4 we quConcerning the first question we found that Wallerian degeneration progresses as a wave, with the wave front defining the point to which fragmentation had spread along the axon. In partially fragmented axons separated from the cell body by transection or proximally compressed by crush lesion, axon regions with no features of degeneration abruptly change into segment lengths with marked breakdown within a transition zone of less than one millimeter Fig. , 6. A shIn order to determine whether the anterograde and retrograde fragmentation wave runs at the same velocity along the axon we next estimated the rate of progression. As the average axon length measured was 24 mm and the majority of axons must have entirely fragmented between 41 h and 42 h after transection  Fig. , the minAdd Fig 1.pdf). These data are consistent with the axonal membrane remaining intact after 30 sec nerve crush, unlike that of a transected nerve.We then tested whether our crush lesions interrupted axon continuity as in nerve transection, because a failure to do so could underlie the different direction of propagation in crushed nerves (see Discussion). In fluorescent wholemount preparations of crushed nerve segments we found continuous longitudinal YFP signals across the crush site, and in teased fibre bundles of osmificated nerve segments after crush injury, a majority of fibres remained continuous across the crush site  and subsequent multiple axonal swellings . This feature was far more prominent in crushed WldS axons than in the transection experiment where we observed end bulbs just occasionally. Further distally these swellings disappear with remaining axonal constrictions and breaks  representing incomplete fragmentation. As in transected nerves, distal parts of the crushed WldS axon were free of degeneration signs . Compared to transection lesion, more axons at 15 days remained entirely intact (15.4 %)  while more distal regions of the same axons were again incompletely fragmented  and further distal still lacked any degeneration signs . The proximal end bulbs and localised swellings were larger than at 15 days , possibly due to continued accumulation of retrogradely transported material.We then followed up the earlier EM experiments using crushed nerves from Wldges Fig. . An exam %) Fig. . By 20 dS axons is directional with an exclusively anterograde pattern both after transection and crush injury. This pattern of degeneration is qualitatively different from that in wild-type mice, which shows asynchronous, bidirectional fragmentation and degeneration.In summary, quantification 20 days after crush lesion revealed that all axons showed anterograde gradients of complete or incomplete fragmentation Fig. . Thus, tS axons. In contrast to our observations in wild-type mice, there was no clearly delineated boundary or wave front separating degenerated and fully intact regions. In order to quantify this, we counted the number of axonal constrictions and breaks along the length of YFP positive WldS axons following injury. At all post-lesion time-points randomly chosen axons showed gradually decreasing signs of degeneration (constrictions or interruptions) along their length , giving a maximum velocity of WldS degeneration progression of 11 mm / 5 days = 2.2 mm/day. This is similar to the reported velocity of slow axonal transport (0.1–3.0 mm/day) [S peripheral nerves are summarized schematically in Fig. In the above experiments we noted the gradual change from degenerated regions to intact regions in Wldgth Fig. . This maion Fig. . This qu mm/day) -59. All S mutant mice undergo a fundamentally distinct process rather than simply following the same pathway in slow motion.We have shown that the fragmentation of axons undergoing Wallerian degeneration in a mixed wild-type peripheral nerve is a rapid, asynchronous and progressive process. By using a recently developed method to visualise individual axons over cm-long distances, and by targeting a short critical period during which nearly all axons degenerate, we have made the first observations in vivo of partially fragmented individual axons and thus determined the directionality and the wave-like nature of Wallerian degeneration, as well as estimating its velocity. Furthermore, we have shown that nerves of WldLesioned wild-type axons remain morphologically normal for a latency period of ca. 36–44 hours, which depends on lesion type and individual axonal properties ,44,46,60We have shown that Wallerian degeneration in wild-type nerves is a wave-like process that can travel in either direction along the axon, depending on lesion type. 29 partially fragmented axons were observed, and all showed a sharp boundary between fragmented and non-fragmented zones, such that all axon regions up to the wavefront were degenerated and all regions beyond it remained intact. Fragmentation had reached different points along the nerve in different individual axons Fig. , reflectS protein. The fastest reported components of axonal transport move at around 14–25 mm/h, but it is reasonable to expect that some minor, thus far undetected, components may move faster [We discuss here two models to explain the wave-like propagation of Wallerian degeneration in wild-type nerves: one based on fast axonal transport and the other based on calcium influx . Thus, the proximal end of the distal stump may be less vulnerable than after transection, and fragmentation may begin instead at the distal end because this is the hardest part to supply with everything the axon needs to survive.There are several possible reasons why degeneration may begin proximally in a transected nerve but distally in a crushed nerve. The proximal end of a transected distal stump is especially vulnerable because of the exposure of the axoplasm to the external ionic environment, and because all extra-axonal structures that normally support the axon have been totally disrupted, e.g., blood vessels, Schwann cells, extracellular matrix, perineurium. One of these factors may cause a calcium entry wave to begin at this point. In contrast, intact endoneural blood vessels can be found close to a nerve crush ,78, the S nerves that are incompatible with delayed axon degeneration following a similar mechanism to Wallerian degeneration in wild-type nerves, only slower. The spread of degeneration along WldS nerves is around 100-fold slower, axon degeneration is more synchronous, at least relative to how long it takes to occur, it progresses in a proximal to distal direction in crushed nerves as well as transected nerves, there is a continuous gradient of degeneration along the length of the axon rather than an abrupt change at a boundary, and the first sign of axon degeneration is a constriction rather than a complete interruption. We therefore propose that the ultimate degeneration of axons in WldS mice be termed "slow anterograde axon decay" rather than Wallerian degeneration as summarised schematically in Fig. We observed a series of differences in the pattern of Wallerian degeneration in WldS axons eventually undergo a passive process of atrophy, rather than an active process of self-destruction similar to apoptosis that appears to take place in wild-type axons [S (data not shown). Even the fact that some proteins are synthesised locally in axons [S axons, as it remains unclear which proteins are made there and in what quantities.Based on these differences, we propose that injured Wldpe axons ,15,16,86in axons ,87-89 maS axons: one based on slow axonal transport and the other on a temperature gradient along the limb. The gradual nature of axonal atrophy in WldS makes it difficult to be precise about the rate at which it progresses along the axon, but it is certainly not incompatible with the velocity of slow axonal transport of 0.1–3.0 mm/day [S nerves, leading to localised neurofilament-depleted constrictions and terminal and intermediate swellings containing disorganised neurofilaments [S axons, and additionally report a gradient of such features along the nerve. A net anterograde movement of cytoskeletal proteins could therefore underlie the anterograde gradient of axonal atrophy in WldS axons injured for many days.We discuss two models to explain the slow anterograde progression of degeneration along Wld0 mm/day -58,90. Cilaments ,92. We hS nerve. A decrease in temperature has been shown to delay degeneration both in wild-type and WldS axons after injury [S axons there is a proximal to distal gradient of degeneration regardless of lesion type and the change from intact to degenerated is a gradual one. Thus, a temperature gradient could play a more important role here.Alternatively, a proximal-distal decreasing gradient of temperature along the limb could underlie the observed difference in degeneration rate at different points in the Wldr injury ,74,93-96S is in marked contrast to the presynaptic nerve terminal at the neuromuscular junction, which is the first structure to degenerate in both wild-type and WldS nerves [S heterozygotes . This supports the hypothesis of compartmentalised degeneration mechanisms of axons and synapses [The extremely long survival of distal tibial nerve following injury in WldS nerves ,8,46. Insynapses  and suggWldS gene and the direction of degeneration is also shared [in vivo after nerve lesion or in disease, once methods for in vivo imaging of single axons become more refined and more readily available, e.g., Cell ViZio, Mauna Kea Technologies [Finally, the methods we report here could now be applied to study spontaneous nerve degeneration in 'dying-back' disease. The 'dying-back' model also predicts the transitory existence of partially degenerated axons, but as in Wallerian degeneration such axons have never been directly observed and there is no indication of the speed of 'dying-back' of individual axons ,99. Thero shared . Thus, io shared ,100, wheo shared  and it inologies .S nerves is also progressive, but differences in the topographic pattern and morphology of degeneration indicate a fundamentally different process from that in wild-type nerves. We propose that WldS axons ultimately undergo atrophy, in a passive process similar to that which Wallerian degeneration was once thought to be.In summary, we report the first direct observation of partially degenerated single axons in lesioned nerves undergoing Wallerian degeneration, indicating that Wallerian degeneration propagates in wild-type nerves as a wave whose speed is at least as fast as the highest reported rate of fast axonal transport. It could be faster still in mouse sciatic and tibial nerve. The direction of degeneration is proximal to distal after a cut, but the reverse after a crush. For now the mechanism remains unknown, but these observations will ultimately need to be explained in any comprehensive model of the Wallerian degeneration mechanism. Injury-induced axon degeneration in WldWldS mutation, the transgenic WldS mutation (tg-WldS) [S/YFP-H mice were used purely for reasons of convenience in their breeding. They express similar levels of WldS protein as homozygous natural mutant WldS mice and display a similar retarded time-course of axon degeneration. They additionally express YFP in approximately 3% of myelinated motor and sensory fibres in the PNS, equal to the original YFP-H line obtained from the Jackson Laboratories.Crossbreeding and genotyping of the YFP-H line  and triptg-WldS)  and the tg-WldS) . Triple Six- to 10-week-old mice from the YFP-H line and triple heterozygote mice for the second part of the study were anaesthetised by intraperitoneal injection of Ketanest  and Rompun , sciatic nerves were transected or crushed firmly close to the Foramen intrapiriforme and the wound was closed with a single suture. Complete nerve transection was performed with conventional surgical scissors and crush lesion was achieved with fine watchmaker's forceps  for 30 seconds. The continuity of the sciatic nerve was always preserved after crush lesion as checked in situ. For light and electron microscopy we removed distal ~2.5 centimeter long nerve stumps after 15 (transection only), 20, 25 and 30 days in triple heterozygote mice following intracardial perfusion. The first 2 millimeters of the distal nerve stump were discarded to reduce artifacts, and the next 2 mm from the most proximal and distal end of the peripheral nerve stump was prepared for Durcupan embedding and electron microscopy.For conventional fluorescence microscopy and confocal tracing of individual YFP labelled wild-type axons after 34 h, 37 h, 40 h, 41 h, 42 h and 48 h following cut lesion and 37 h, 40 h, 42 h, 43 h, 44 h and 48 h following crush lesion the operated YFP-H mice were sacrificed by cervical dislocation and nerve segments prepared as follows.S axons from triple heterozygote mice we dissected sciatic-tibial nerve segments after 12.5 d (transection only), 15d, 17.5 d (transection only), 20 d and 22.5 d (transection only). For each investigated time-point 2 – 3 YFP-H or triple heterozygote mice were operated.For confocal tracing of individual YFP labelled WldSciatic nerves of mice from the YFP-H line were crushed as described in the paragraph above and the short nerve segment containing the crush site immediately removed after lesion, freed from surrounding connective tissue and subsequently either prepared for wholemount fluorescence embedding as described previously  or for tAfter sternotomy under deep anaesthesia mice were killed by cardiac puncture and instantly intracardially perfused first with a solution containing 10 000 i.e./l heparin  and 1 % procainhydrochloride in 0.1 M PBS for 30 s and then with fresh half-strength Karnovsky's fixative .Nerve samples for light and electron microscopy were embedded in Durcupan and further processed for examination with a Zeiss Axiophot light microscope and Zeiss EM 902 electron microscope as described previously .The percentage of preserved myelinated axons in proximal and distal ends of peripheral nerve stumps from triple heterozygote mice after transection and crush injury was determined as described previously ,54.S axons running through the excised stumps over their whole length was achieved as described previously [In both YFP-H and triple heterozygote mice after cut or crush lesion the entire nerve distances from the proximal sciatic to the distal tibial nerve were carefully excised, the perineurium and the branch of the commune fibular nerve removed and the remaining ~2.5 centimeter long stumps treated for wholemount fluorescence preparation using Vectashield Mounting Medium (Vector Laboratories) as described previously . For roueviously . Single Imaging of restricted transition zones dividing intact and fragmented axon regions in YFP-H nerves was performed under highest possible resolution with a Zeiss LSM 510 META confocal system (LSM Software Release 3.2) coupled to a Zeiss Axiovert 200 microscope.S axons in wholemount peripheral nerve stumps (2–3 nerve preparations for each timepoint) after transection and crush injury were equally traced individually with the BioRad Radiance 2000 laser scanning confocal microscope under high resolution, and divided into two groups: axons without any features of axonal disintegration were counted and assigned to the group "intact". Fibres that showed degeneration signs like constrictions or interruptions of the longitudinal YFP signal at the proximal site were associated into the group "fragmented with anterograde gradient". Means and standard deviations for all experiments were calculated using Microsoft Excel.For the first part of the study focusing on sciatic/tibial nerves from the YFP-H line in each wholemount preparation (2 – 3 nerve preparations per time point) YFP labelled wild-type axons running continuously through the excised peripheral nerve stump after cut and crush injury were traced individually with the laser scanning confocal microscope under high resolution and divided into four groups: axons without any features of axonal disintegration were assigned to the group "intact". Axons that showed fragmentation of the longitudinal YFP signal over the whole length of the fibre were assigned to the group "entirely fragmented". Axons without any sign of fragmentation at one end but clear axon breakdown at the other end were assigned either into the group "fragmented with anterograde gradient" or "fragmented with retrograde gradient" depending whether the fragmentation appeared close to the lesion point or at the most distal point of the tibial nerve segment. For the second part of the study dealing with triple heterozygote mice YFP labelled WldS axons was measured during the tracing process with the confocal laser scanning microscope and the distribution of axonal interruptions (both in wild-type and WldS axons) and/or constrictions (only in WldS axons) was graphed against the distance in mm from the cut or crush point. Means and standard deviations were calculated using Microsoft Excel.The length of degenerated YFP labelled wild-type and WldAll animal experiments were carried out under appropriate German licences: Tierschutzgenehmigung K 13, 11/00 and Anzeige K30/99.MPC (corresponding author) and BB jointly conceived of the design for this study, interpreted the experimental results and wrote the manuscript; BB carried out preliminary light and electron microscopy experiments, confocal imaging of YFP labelled axons, axon quantification and statistical analysis; RA: collaborated on the set up of the experiments, improvement of histological techniques and interpretation of results, performed teased fibre experiments, assisted in writing the manuscript; DW and DSG: provided excellent technical assistance in many parts of the experiments, performed sciatic nerve cut lesions and carried out routine work; KA: contributed to the discussion of the experimental results, preliminary light and electron microscopy work was done in his lab; RRR: contributed to the interpretation of the experiments and writing of the manuscript; confocal microscopy of YFP axons was mostly done in his lab; MPC: performed sciatic nerve crush lesions and partially confocal imaging, supervised all aspects of the study and main work was done in his lab.All authors read and approved the final manuscript.Many nerve fibres are unbroken directly after a 30 second crush as shown in YFP-H wholemount nerves and osmificated teased fibre preparations.A-C: Sciatic nerves crushed for 30 seconds at maximum pressure and then immediately fixed and imaged by osmium staining  or YFP fluorescence (C). D: YFP-H nerve crushed for 5 seconds at maximum pressure. Nerves in (A) and (B) were partially teased apart after staining, leading to accidental breakage of a few fibres. However, the majority of fibres clearly cross the crush site unbroken. YFP signal (identifying the axoplasm) at the crush site in (C-D) is weak, probably due to squeezing of the axoplasm longitudinally out of the crush site, or quenching of fluorescence by the crushed tissue, or both. To compensate, the photographs are deliberately overexposed. There is no sign of YFP positive axoplasm escaping laterally into the extracellular space, as would be expected if the axolemma were broken. Instead, some YFP positive axons clearly cross the crush site unbroken. In (C) the overexposure would prevent any broken axons from being identified, but in (D) the signal from most or all axons fades gradually as the axon enters the crush site, rather than stopping abruptly as one would expect if the axon were broken. Scale bars: 20 μm  and 50 μm .Click here for fileS axons proceeds anterogradely beginning with the formation of end bulbs and axonal swellings at the most proximal end and accompanying proximal axonal atrophy.Degeneration of individual crushed Wld Confocal composite picture showing seven consecutive lengths (from top to bottom in overview) of the proximo-distal course of an individual YFP labelled WldS axon within a peripheral triple heterozygote nerve stump 15 days after crush injury displaying an anterograde gradient of axon degeneration. This axon exhibits an end bulb at the most proximal end (red arrow in overview) and subsequent multiple axonal swellings delimited by constrictions (red asterisks) (inset 1). Further distally these swellings disappear with remaining constrictions (red asterisks) and sporadic breaks (white arrow) (inset 2). Distal parts of the crushed WldS axon are free of degeneration signs (inset 3 and 4). Scale bar: YFP fluorescence has been pseudo-coloured yellow with the applied confocal imaging software (Biorad LaserSharp 2000). 500 μmClick here for fileS axons finally includes complete proximal fragmentation.Anterograde degeneration with formation of massive end bulbs of compressed Wld Confocal composite picture showing six consecutive lengths (from top to bottom in overview) of the proximo-distal course of an individual YFP labelled WldS axon within a peripheral triple heterozygote nerve stump 20 days after crush injury demonstrating a clearer anterograde progression of axon degeneration than in Add Fig 2.pdf. Likewise after 15 days following crush injury this picture shows a massive end bulb at the most proximal end of the distal stump (red arrow in overview) and subsequent multiple axonal swellings and constrictions. Complete axonal fragmentation is evident in inset 1  and gradually gives way to incomplete breakup with axonal constrictions (red asterisks) (inset 2). Distal parts of the crushed WldS axon lack degeneration signs (inset 3 and 4). YFP fluorescence has been pseudo-coloured yellow with the applied confocal imaging software (Biorad LaserSharp 2000). Scale bar: 500 μmClick here for file


During endochondral bone formation, the hypertrophy of chondrocytes is accompanied by selective expression of several genes including type X collagen and alkaline phosphatase. This expression is stimulated by inducers including BMPs and ascorbate. A 316 base pair region of the type X collagen (Col X) promoter has been previously characterized as the site required for BMP regulation. The intent of this study was to examine the role of Mitogen Activated Protein (MAP) and related kinase pathways in the regulation of Col X transcription and alkaline phosphatase activity in pre-hypertrophic chick chondrocytes.Using a luciferase reporter regulated by the BMP-responsive region of the type X collagen promoter, we show that promoter activity is increased by inhibition of extra-cellular signal regulated kinases 1 or 2 (ERK1/2). In contrast the ability of BMP-2 to induce alkaline phosphatase activity is little affected by ERK1/2 inhibition. The previously demonstrated stimulatory affect of p38 on Col X was shown to act specifically at the BMP responsive region of the promoter. The inhibitory effect of the ERK1/2 pathway and stimulatory effect of the p38 pathway on the Col X promoter were confirmed by the use of mutant kinases. Inhibition of upstream kinases: protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase pathways increased basal Col X activity but had no effect on the BMP-2 induced increase. In contrast, ascorbate had no effect on the BMP-2 responsive region of the Col X promoter nor did it alter the increase in promoter activity induced by ERK1/2 inhibition. The previously shown increase in alkaline phosphatase activity induced by ascorbate was not affected by any kinase inhibitors examined. However some reduction in the alkaline phosphatase activity induced by the combination of BMP-2 and ascorbate was observed with ERK1/2 inhibition.Our results demonstrate that ERK1/2 plays a negative role while p38 plays a positive role in the BMP-2 activated transcription of type X collagen. This regulation occurs specifically at the BMP-2 responsive promoter region of Col X. Ascorbate does not modulate Col X at this region indicating that BMP-2 and ascorbate exert their action on chondrocyte hypertrophy via different transcriptional pathways. MAP kinases seem to have only a modest effect on alkaline phosphatase when activity is induced by the combination of both BMP-2 and ascorbate. During skeletal development and growth, bone formation occurs either by intramembraneous or endochondral bone formation. In endochondral bone formation, which occurs at the growth plates of long bones, cartilage is formed first, then the chondrocytes undergo a proliferative phase followed by hypertrophy, changes in gene expression, and matrix calcification, after which the cartilage is replaced by bone. Although generally referred to as chondrocyte hypertrophy, cell enlargement is just one manifestation of the more complex process of chondrocyte maturation, which can be considered an end-stage of chondrocyte differentiation. It is important to define the mechanisms that induce chondrocyte maturation, not only to understand bone development, but also to help prevent hypertrophy and ossification during cartilage tissue engineering.Hypertrophic chondrocytes are characterized by their increased levels of alkaline phosphatase (ALP), reduced levels of type II and IX collagens, and the emergence of type X collagen (Col X), which is a specific marker of hypertrophy ,2. AscorIn osteoblastic cells, BMPs and ascorbate have been shown to operate via mechanisms that at least partly involve mitogen activated protein kinases (MAP kinases). For example, ascorbate promotes extracellular matrix production which, in turn, activates the extracellular-signal regulated kinases, (ERK1/2 or p42/ p44) in an osteoblastic cell line . MAP kinIn general, MAP kinase pathways involving ERK1/2 stimulate proliferation, growth and differentiation, whereas those that stimulate p38 kinase lead to differentiation and apoptosis . In earlChick sternal chondrocytes are a popular model for the study of chondrocyte maturation because under normal development chondrocytes from the cephalic portion of the sternum undergo hypertrophy followed by mineralization and bone formation, whereas the caudal portion remains as cartilage ,8. In thStudies with the ERK1/2 inhibitor U0126 indicated that blocking ERK1/2 signaling increased the activity of the type X collagen promoter but had no effect on alkaline phosphatase (ALP) activity in chick cephalic sternal chondrocytes Fig. . In contALP activity was highly variable between cell isolates and is expressed here normalized to BMP-2 treated controls for the purpose of combining experiments, typical ALP values ranged from approximately 0.5–2 nmol/min/μg DNA in controls to between 4 and 12 nmol/min/μg DNA in BMP-2 treated cultures.The effects of altered ERK1/2 signaling on Col X promoter activity in chick sternal chondrocytes was further studied both by transfection with mutant kinases and by treatment with additional kinase inhibitors. Col X promoter activity was increased, both in the presence and absence of BMP-2, when the mitogen-stimulated ERK pathway was suppressed by transfecting chondrocytes with dominant negative ERK-2 Figs. . ConversAs seen with the ERK1/2 inhibitor U0126, treatment with the more specific ERK1/2 inhibitor PD098059 increased b2/640 Col X promoter activity, in the presence of BMP-2 Fig. . Dose reTransfection with dominant negative p38 caused a decrease in Col X promoter activity in BMP-2 treated cephalic chondrocytes, reducing activity to half of that seen in BMP-2 treated controls (p < 0.005) and eliminating the BMP-2 response Figs.  and 2B. Addition of either PI3 kinase inhibitor or PKC inhibitor resulted in similar stimulation of the collagen type X promoter. Calphostin C, a PKC inhibitor, increased activity in BMP-2 treated cells more than 2-fold, an effect similar to that seen with the ERK1/2 inhibitor PD98059 at 10 μM Fig. . SimilarTo assess the possible effects of protein kinase inhibitors on cell proliferation and survival, we measured relative numbers of live cells using a tetrazolium (MTS) assay. The results indicated that all cultures treated with inhibitors, with and without BMP-2 and/or ascorbate, had cell numbers within 10% of untreated controls (data not shown).We examined the effect of 75 μM ascorbate-2-phosphate on the activity the Col X promoter in cultures treated with kinase inhibitors. Col X promoter activity was unaffected by addition of ascorbate, and 4 μM of the ERK1/2 inhibitor U0126 increased promoter activity to comparable levels both with and without ascorbate Fig. .ALP activity in the absence of exogenous BMP was stimulated at least 2-fold in ascorbate-treated cultures without inhibitors, as previously reported, and this stimulation was not significantly affected by addition of either ERK1/2 or p38 inhibitors Fig. . In cultThe present studies demonstrate that ERK1/2 inhibition increases activity of the BMP responsive region of the type X collagen promoter. This indicates that ERK1/2 signaling interferes with the ability of BMP-induced signals to stimulate type X collagen transcription. Interestingly ERK1/2 has also been shown to inhibit type I collagen expression in an osteoblastic cell line  suggestiIn contrast to the stimulatory effects of inhibiting the ERK pathway, p38 inhibition blocked BMP-stimulated Col X promoter activity. Zhen et al.  and BeieWhile the classical pathway for BMP signaling is via activation of R-Smads, there is also evidence for BMP signaling via a TGF-activated kinase (TAK1) leading to p38 signaling -26. HoweInhibiting either protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3 kinase) increased type X collagen promoter activity both in BMP-2 treated cultures and controls. Both PKC and PI3 kinase have been reported to negatively regulate p38  and posiAlthough alkaline phosphatase expression, like type X collagen expression, increases during chondrocyte hypertrophy and is stimulated by BMPs, its regulation clearly differs from Col X in several respects. ERK1/2 inhibition has little effect on the ALP activity induced by BMP-2 or ascorbate acting alone and reduces the ability of BMP-2 to further stimulate ALP activity in ascorbate treated cultures. Inhibiting p38 does not have a clear effect on BMP-stimulated alkaline phosphatase activity in this model although it has been shown to reduce ALP activity in long-term (12–15 day) micromass cultures .Preliminary studies with cyclohexamide indicate that new protein synthesis is required for the up-regulation of alkaline phosphatase mRNA in response to BMP-2. We propose that these differences reflect a direct Smad-mediated effect of BMPs on type X collagen expression and an indirect effect on ALP expression.A mechanism which could account for the observed effects of ERK and p38 signaling on expression of type X collagen and ALP is presented in Fig. Evidence that BMP stimulation of type X collagen requires both activated R-Smads and Runx2 has been previously reported ,7. LittlAs well as its demonstrated role in Col X expression as found here and by ,31, therRetinoic acid ,8, anothElucidating the signaling pathways by which chondrocytes are driven to hypertrophy is necessary in order to better understand skeletal development, cartilage disease and improve the design of tissue engineered cartilage. We showed here that the ERK1/2 pathway inhibits type X collagen production by either directly or indirectly acting at the BMP responsive region of the promoter. p38 kinase signaling stimulates type X collagen transcription at the same promoter region, probably in conjunction with BMP-2 activated Smads. The factor upstream of p38 in this stimulatory pathway is unknown. Alkaline phosphatase activity is likely to be regulated in a different way from type X collagen since MAP kinases do not contribute in the same way to this pathway. Although ascorbate and BMPs both induce hypertrophy in chondrocytes ascorbate does not act at the same region of the Col X promoter as BMPs.The ERK1/2 inhibitor PD98059, which blocks the upstream kinase (MEK1) of ERK1/2, the p38 inhibitor SB203580, and the PKC inhibitor Calphostin C were obtained from Sigma . UO126, also a MEK1 inhibitor, was obtained from Biomol  and LY294002, a phosphatidylinositol 3- (PI3) kinase inhibitor from Cell Signaling Technology . Plasmids were kindly donated as follows: constitutively active MEK1 from Michael Webber (University of Virginia); dominant negative p38  from RogChondrocytes were cultured as previously described . Cephalirenilla luciferase reporter (0.4 μg/well) (Promega) which served as a transfection control [4 precipitation or Fugene transfection reagent at 6 μl/ml (Promega). Since preliminary experiments using green fluorescent protein showed that Fugene was more effective in terms of numbers of cells transfected, this method was used for the majority of the experiments; however, relative outcomes between controls and treated cells were not affected by the transfection method.On day 1 of secondary culture (24 hrs after plating) the cell layer was washed in HBSS and the media changed to DMEM supplemented with 10% FBS in place of NuSerum. Cells were co-transfected with pGL2 plasmid containing the b2/640 type X collagen promoter region attached to a firefly luciferase reporter (0.2–1 μg/well)  and pRL null plasmid attached to a  control . When apTransfection proceeded for 5 hrs after which the cell layer was rinsed twice in HBSS and cultured with serum free medium . Some wells were supplemented with 75 μM ascorbate-2-phosphate  or 30 ng/ml human recombinant BMP-2 . Where inhibitors were used they were added at this point and cells incubated for 1 hr before the addition of BMP-2. Cells were cultured for a further 48 hours, then lysed and assayed using a dual luciferase assay kit (Promega).2, 0.1 mM MgCl2 and 1% Triton X-100 for 30 mins at 37°C, followed by overnight storage at 4°C. A sample of the cell lysate was reacted with p-nitrophenyl phosphate substrate in 1.5 M Tris buffer pH 9 with 1 mM ZnCl2 and 1 mM MgCl2. Phosphatase activity was measured specrophotometrically at 410 nm with 1 absorbance unit equivalent to 64 nmol of product. For DNA analysis, cells were trypsinized and a subsample of cell suspension centrifuged, the cell pellet lysed with the CyQUANT lysis buffer and the fluorescent DNA dye added. The resulting solution was transferred to a 96 well plate and DNA assayed fluorometrically. The remaining cells were extracted for the alkaline phosphatase assay as above. Alkaline phosphatase enzyme levels were calculated as nmol p-nitrophenol product per minute normalized to MTS units or μg DNA.For alkaline phosphatase assays, cells were switched to serum free medium on day 1 of secondary culture, inhibitors were added and cells incubated for 1 hr before the addition of ascorbate or BMP-2, as described for the luciferase assay. Cells were cultured for a further 72 hrs and then rinsed twice in HBSS. Cells numbers were assayed either by DNA quantification  or by MTS tetrazolium salt assay of mitochondrial activity . When MTS was used, a 1:10 dilution of MTS was applied in phenol red-free media for 30–60 minutes, 200 μl of media plus MTS was transferred to a 96 well plate and assayed in a 'Multiskan ascent' plate reader . The cell layer was then washed twice in HBSS and extracted with 0.15 M Tris, pH 9 with 0.1 mM ZnClStatistics were performed using Minitab™ software. After expressing results as a ratio of experimental/control within each experiment, the data from at least 3 experiments were combined. The combined data was tested for normality using the Anderson-Darling test. Normally distributed or non-parametric data were tested for differences between treatments using two-sample Students t-test or the Mann-Whitney test respectively. Where groups of experiment means were compared a paired t-test was used.ALP – Alkaline PhosphataseBMP – Bone Morphogenetic Proteinbp – base pairsCA – constitutively activecbfa – core binding factor alpha 1Col X – Collagen type XDN – dominant negativeERK – extracellular signal regulated protein kinaseMAP – Mitogen activated proteinMEK – ERK kinaseMTS – 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner saltPI3 – phosphhatidylinositol 3PKC – Protein Kinase CRunx – Runt associated proteinTAK – Transforming growth factor beta activated kinaseThe author(s) declare that they have no competing interests.GCR was involved in experimental design, performed 90% of the experiments, analyzed the data and wrote the first draft of the manuscript. EBG isolated chondrocytes, performed preliminary experiments and contributed to trouble-shooting of the methods. GG-K, performed preliminary experiments and developed methodology. PSL was involved in experimental design and data analysis, wrote portions of and edited the whole manuscript and was the project supervisor.