The relationship between consecutive pregnancies in Thoroughbred mares. Does the location of one pregnancy affect the location of the next, is this affected by mare age and foal heat to conception interval or related to pregnancy success.

Delayed uterine involution is a major cause of early reproductive failure in mares. Involution is affected by mare age, and foaling to covering interval. Involution rates vary between the previously non-gravid horn (PNGH), which recovers the quicker, and the previously gravid horn (PGH). Location of a pregnancy and its likely success may, therefore, be affected by its location relative to the previous pregnancy. This study aimed to determine: (i) the location of concepti in consecutive pregnancies; (ii) whether this varies with mare age or foaling to conception interval; (iii) whether location in relation to the previous pregnancy affects success. 1383 Thoroughbred mares were monitored by ultrasonic scanning during oestrus and early pregnancy. Significantly (p<0.01) more pregnancies were located in the PNGH (79.2%) than the PGH (20.8%). The number of pregnancies in PGH significantly increased with mare age (p<0.01) and foaling to conception interval (p<0.05). Significantly (p<0.001) more pregnancies located in the PGH (16.5%) failed, than those in the PNGH (4.6%). It can be concluded that most pregnancies locate in the PNGH where their chances of success are greatest. The larger number of pregnancies locating in the PGH in older mares and those with shorter foaling to conception intervals may in part account for the higher conceptus mortality rates in such mares. Hence breeding older mares on alternate years and maximising foaling to conception interval may improve reproductive success. Alternatively termination of pregnancies located in the PGH followed by timely recovering may be justifiable as might ET in older mares covered close to foaling.

Do feathered dinosaurs exist? Testing the hypothesis on neontological and paleontological evidence.

The origin of birds and avian flight from within the archosaurian radiation has been among the most contentious issues in paleobiology. Although there is general agreement that birds are related to theropod dinosaurs at some level, debate centers on whether birds are derived directly from highly derived theropods, the current dogma, or from an earlier common ancestor lacking suites of derived anatomical characters. Recent discoveries from the Early Cretaceous of China have highlighted the debate, with claims of the discovery of all stages of feather evolution and ancestral birds (theropod dinosaurs), although the deposits are at least 25 million years younger than those containing the earliest known bird Archaeopteryx. In the first part of the study we examine the fossil evidence relating to alleged feather progenitors, commonly referred to as protofeathers, in these putative ancestors of birds. Our findings show no evidence for the existence of protofeathers and consequently no evidence in support of the follicular theory of the morphogenesis of the feather. Rather, based on histological studies of the integument of modern reptiles, which show complex patterns of the collagen fibers of the dermis, we conclude that "protofeathers" are probably the remains of collagenous fiber "meshworks" that reinforced the dinosaur integument. These "meshworks" of the skin frequently formed aberrant patterns resembling feathers as a consequence of decomposition. Our findings also draw support from new paleontological evidence. We describe integumental structures, very similar to "protofeathers," preserved within the rib area of a Psittacosaurus specimen from Nanjing, China, an ornithopod dinosaur unconnected with the ancestry of birds. These integumental structures show a strong resemblance to the collagenous fiber systems in the dermis of many animals. We also report the presence of scales in the forearm of the theropod ornithomimid (bird mimic) dinosaur, Pelecanimimus, from Spain. In the second part of the study we examine evidence relating to the most critical character thought to link birds to derived theropods, a tridactyl hand composed of digits 1-2-3. We maintain the evidence supports interpretation of bird wing digit identity as 2,3,4, which appears different from that in theropod dinosaurs. The phylogenetic significance of Chinese microraptors is also discussed, with respect to bird origins and flight origins. We suggest that a possible solution to the disparate data is that Aves plus bird-like maniraptoran theropods (e.g., microraptors and others) may be a separate clade, distinctive from the main lineage of Theropoda, a remnant of the early avian radiation, exhibiting all stages of flight and flightlessness.

Developmental basis of limb evolution.

Can developmental processes account for vertebrate limb homology, the overall similarity of definitive limb structure despite differences in different taxa which often relate to evolutionary adaptations? Relevant evidence is from molecular studies, from 'cut & paste' experimental embryology and from classical descriptive accounts of embryology and structure. There is striking evidence of a similar pattern of homologous regulatory gene expression (eg Shh, and Hox A & D genes) in tetrapod limb buds, and both similarity and differences when these are compared with expression patterns in a teleost fish paired fin bud. But these findings are as yet from too few tetrapod species (chick and mouse) to permit a 'molecular bauplan' for the limb to be proposed with any certainty. Further, the identification of similar networks of regulatory genes common to non-homologous developmental systems limits possibilities for finding a basis for classical structural homology in terms of expression of system-specific genes or gene networks. An integrated approach is needed, combining evidence from the fin-limb transition, and from study of the patterns and processes of amphibian and avian limb embryology, and this points towards a conserved developmental bauplan for the pentadactyl skeleton of the type earlier proposed by Alberch. Key features include the digital arch, restriction of digit number to a maximum of 5 and stereotyped connections between prechondrogenic condensations. But this is a dynamic and not rigidly fixed bauplan. It has no single set of skeletal elements (except proximally), since the position of joint formation in the prechondrogenic condensations is not stereotyped. Urodele amphibians in particular demonstrate heterochronic differences in the timing of events. Heterochrony may underlie some of the important changes in the pentadactyl pattern during evolution.

Developmental basis of limb homology in urodeles: heterochronic evidence from the primitive hynobiid family.

The vertebrate limb is a classic example of homology, long assumed to be underpinned by a developmental 'bauplan' of the type proposed in the Shubin/Alberch branching and segmenting model. In the anuran/amniote pattern skeletogenesis proceeds in a proximodistal direction with digits forming from the posterior to the anterior. But in free-living larvae of 'advanced' urodeles, the pattern of skeletogenesis is distinctly different with digits 1 and 2 and the basal commune developing early, in an anterior/distal position. This different pattern is cited as evidence for a diphyletic theory of tetrapod evolution. Reassessing this problem, we analysed the pattern of early skeletogenesis of three genera (Salamandrella, Ranodon, Onychodactylus) of the 'basal' family of hynobiids, using immunofluorescence to localize chondroitin-6-SO4 in Salamandrella. Here the developmental sequence was more proximodistal (intermedium preceding basal commune; early formation of the digital arch). This pattern, also found in direct developing urodeles such as Bolitoglossa subpalmata, resembled that in anurans/amniotes. Uniquely amongst tetrapods, urodeles use their developing limbs for locomotion. We attribute the unusual pattern in 'advanced' urodeles to adaptive modification of the developing limb. Differences in the pattern between 'basic' and 'advanced' urodeles and between urodeles and anuran/amniotes are interpreted as heterochronic within an overall single tetrapod developmental bauplan.

Posterior half amputation of the chick wing bud: the response of the developing vasculature, and subsequent wound healing.

Experimental analyses examining pattern formation in the developing chick limb have concentrated on the skeleton, muscles and nerves, and have rarely considered blood vessels. To investigate the relationship between the vasculature and limb development, posterior amputations were performed on 3.5-4 day chick limb-buds. It has been shown that the removal of the posterior half alters the developmental fate of the anterior tissue: it becomes necrotic and fails to differentiate into the complement of skeletal parts predicted by fate maps. The possibility that this developmental failure results from interference with the future arterial supply was examined by Indian ink injection between 3-48 h after operation. Scanning electron microscopy (SEM) and resin histology were used to examine the wound repair at similar post-operative intervals. Results from the Indian ink injections showed that within 6 h of operation a collateral circulation was established by means of a branch from the truncated primary subclavian artery. The capillary density in the operated limbs appeared normal when compared to the contralateral limb. The results support the view that the poor developmental performance of the anterior half is due to removal of the zone of polarizing activity (ZPA) rather than to experimentally-induced alteration to the vascular supply. Histological and SEM examination of the wound healing process showed that epithelialization of the cut surface occurred within 24 h, and that the peridermal cells of the bilayered ectoderm appeared to initiate the regrowth. The wound site was not visible 48 h after operation, showing that wound healing at these developmental ages occurs quickly, with no scar tissue formation.(ABSTRACT TRUNCATED AT 250 WORDS)

The fate map of the chick forelimb-bud and its bearing on hypothesized developmental control mechanisms.

Carbon particles and isotopic quail grafts were used as markers to study the salient features of the fate map of the chick forelimb between stages 20 and 27. The grafting technique confirmed the reliability of the carbon method: they both revealed striking asymmetries in which apical mesodermal tissue was progressively displaced in a proximal direction (as would be expected on the basis of growth by net apical addition of tissue) but also in a preaxial direction, while postaxial tissue became elongated in the direction of limb outgrowth. Ectoderm showed a similar preaxial-postaxial asymmetry but became displaced from initially underlying mesoderm. In marked contrast to mesoderm, distal ectoderm remained at a constant distance from the apical ectodermal ridge (or became incorporated into it), thus implying that the ectodermal sheet is anchored distally and grows by uniform stretching proximally. Within the ectoderm itself, the outer peridermal layer is displaced distally relative to the underlying epidermal basal layer. Peripheral mesoderm showed patterns of displacement which were intermediate between those of ectoderm and chondrogenic core mesoderm. It is argued that such morphogenetic phenomena are integral components of developmental mechanisms of significance in the control of pattern generation. Implications of the interpretation and use of the fate map in relation to theories of limb development, particularly those based on mechanisms defined in terms of limb axes, are reviewed.

Immunohistological and ultrastructural study of the developing tendons of the avian foot.

The aim of the present report is to provide a detailed description of the morphogenesis and initial differentiation of the long tendons of the chick foot, the long autopodial tendons (LAT), from day 6 to day 11 of development. The fine structure of the developing LAT was studied by light and transmission electron microscopy. The characterization by immunofluorescent techniques of the extracellular matrix was performed using laser scanning confocal (tenascin, elastin, fibrillin, emilin, collagen type I, II, III, IV and VI) or routine fluorescence (tenascin, 13F4) microscopy. In addition, cell proliferation in pretendinous blastemas was analyzed by the detection of BrdU incorporation by immunofluorescence. The light microscopic analysis permitted the identification of different stages during LAT morphogenesis. The first stage is the formation of a thick ectoderm-mesenchyme interface along the digital rays, followed by the differentiation of the "mesenchyme lamina", an extracellular matrix tendon precursor, and ending with the formation and differentiation of the cellular condensation that forms the tendon blastema around this lamina. The immunofluorescence study revealed the presence and arrangement of the different molecules analyzed. Tenascin and collagen type VI are precocious markers of the developing tendons and remain present during the whole process of tendon formation. Collagen type I becomes mainly restricted to the developing tendons from day 7.5. Collagens type II and IV are never detected in the developing tendons, while a faint labeling for collagen type III is first detected at day 7. The analysis of the distribution of the elastic matrix components in the developing tendons is a major contribution of our study. Elastin was detected in the periphery of the tendons from day 8 and also in fibrils anchoring the tendons to the skeletal elements. At the same stage, emilin strongly stains the core of the tendon rods, while fibrillin is detected a little later. Our study indicates the existence of an ectoderm-mesoderm interaction at the first stage of the tendon formation. In addition, our results show the different spatial and temporal pattern of distribution of extracellular matrix molecules in developing tendons. Of special importance are the findings concerning the tendinous elastic matrix and its possible role in tendon maturation and stabilization.

Forensic odontology, part 5. Child abuse issues.

Child abuse, child maltreatment, non-accidental injury and child homicide: all terms that are hard to believe exist in the 21st civilised century, but non-accidental injury of children is a major problem, crossing all socioeconomic, ethnic and educational groups, and is happening all over the world. Available statistics on child abuse and deaths related to abuse are frightening, and as many cases are not reported, actual numbers are likely to be much higher. This paper aims to increase understanding of child abuse issues and encourage the dental team to be alert to the possibility of abuse, recognise the physical injuries and make referrals to the appropriate agency if necessary. In child abuse cases physical injuries to the head and facial area are common while other types of abuse are less visible but are damaging to a vulnerable child in other ways. Keeping children safe is a shared responsibility and a top priority for all of us.

Forensic odontology, part 2. Major disasters.

We have only to look back over the last 12 months to realise that time and time again, an incident occurs where there are mass fatalities. These incidents have instant and long-lasting impact on families, communities and sometimes whole countries. The aim of this paper is to emphasise the need for an efficient and sensitive response to assist in the identification of victims of such incidents and the necessity for trained team responses. Many countries now have Disaster Victim Identification (DVI) teams that are multi-disciplinary, and plans and protocols in place in readiness. The paper can only hope to give a brief overview of the disaster situation for the reader: whole books have been written on this topic. The forensic odontologist has a major role in disaster incidents when there are accurate and available antemortem dental records.

Forensic odontology, Part 1. Dental identification.

This series is based upon fact, experience, and some personal views of the author and gives a brief glimpse of forensic odontological issues with regard to the identification of human remains (to include mass fatality incidents), biting injuries and child abuse. The aim of the first paper is to give the reader greater understanding of the role of the forensic odontologist in the identification of human remains, and emphasise the importance of keeping good quality, accurate and comprehensive dental records. Identification of the deceased greatly assists families and friends at this difficult time, as well as aiding law enforcement agencies; getting it wrong is devastating to families and unacceptable. The dental identification process must be carefully undertaken and relies upon the comparison of information from the antemortem record with findings from the postmortem examination, and the efficiency of this process is dependent on the quality and availability of the dental record. As dental team members it is our responsibility to keep and maintain accurate records of our patients. The resilience of the dental structures to postmortem assault, denture labelling, and teeth as a source of DNA, all contribute to making identification successful. Dental identification is widely used, not only in the single fatality situation, but also in mass fatality incidents and cases of missing persons.

Forensic odontology, part 3. The Australian bushfires - Victoria state, February 2009.

This paper aims to demonstrate the stages in the disaster victim identification of those who lost their lives in the Australian bushfires that raged across the state of Victoria in February 2009. Communities were damaged or destroyed leaving families distressed and homeless, and as the number of deaths increased the Disaster Victim Identification (DVI) teams were activated, with plans evolving to deal with this emergency. The identification process was challenging due to many factors, such as the dangers and difficulties involved in body recovery and the charring and commingling of remains. It would take several months of careful work to identify the dead using a multidisciplinary approach. The impact of this incident will have long-lasting consequences for the families and communities involved. At the time of writing all but one of the 173 victims had been identified, mostly by dental methods: quite remarkable when only small fragments of the dental structures remained in many cases. This article is based on the author's personal experience working to assist the organised and experienced Australian Dental DVI Team.

Forensic odontology, part 4. Human bite marks.

The aim of this paper is to give a brief overview of bite mark analysis: its usefulness and limitations. The study and analysis of such injuries is challenging and complex. The correct protocols for collection, management, preservation, analysis and interpretation of this evidence should be employed if useful information is to be obtained for the courts. It is now possible, with advances in digital technology, to produce more accurate and reproducible comparison techniques which go some way to preventing and reducing problems such as photographic distortions. Research needs to be continued to increase our knowledge of the behaviour of skin when bitten. However, when presented with a high quality bite mark showing good dental detail, and a limited, accessible number of potential biters, it can be extremely useful in establishing a link between the bitten person and the biter or excluding the innocent.

Disaster dentistry.

This article gives a brief glimpse of some of the issues involved with dental identification of fatalities in disaster situations. It is based on the personal views and experiences of the author as a forensic dentist in the aftermath of the Asian tsunami and the Sharm el Sheikh bombings.

Birth and death of cells in limb development: a mapping study.

Cell death and cell proliferation are basic cellular processes that need to be precisely controlled during embryonic development. The developing vertebrate limb illustrates particularly well how correct morphogenesis depends on the appropriate spatial and temporal balance between cell death and cell proliferation. Precise knowledge of the patterns of cell proliferation and cell death during limb development is required to understand how their modifications may contribute to the generation of the great diversity of limb phenotypes that result from spontaneous mutations or induced genetic manipulations. We have performed a comprehensive analysis of the patterns of cell death, assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL), and cell proliferation, assayed by anti-phosphorylated histone H3 immunohistochemistry, in consecutive sections of forelimbs and hindlimbs covering an extensive period of chick and mouse limb development. Our results confirm and expand previous reports and show common and specific areas of cell death for each species. Mitotic cells were found scattered in a uniform distribution across the early limb bud, with the exception of the areas of cell death in which mitotic cells were scarce. At later stages, mitotic cells were seen more abundantly in the digital tips. The aim of the present study was to satisfy the need for organized data sets describing these processes, which will allow the side-by-side comparison between the two major model organisms of limb development, i.e., the mouse and the chick.

Evolutionary developmental biology of the tetrapod limb.

New insights into the origin of the tetrapod limb, and its early development and patterning, are emerging from a variety of fields. A wide diversity of approaches was reported at the BSDB Spring Symposium on 'The Evolution of Developmental Mechanisms' (Edinburgh, 1994); here I review the contributions these various approaches have made to understanding the evolutionary developmental biology of the tetrapod limb. The fields covered included palaeontology, descriptive embryology, experimental embryological analysis of interactions within developing limbs plus description and manipulation of homeobox gene expression in early limb buds. Concepts are equally varied, sometimes conflicting, sometimes overlapping. Some concern the limb 'archetype' (can the palaeontologists and morphologists still define this with precision? how far is there a limb developmental bauplan?); others are based on identification of epigenetic factors (eg secondary inductions), as generating pattern; while yet others assume a direct gene-morphology relationship. But all the contributors ask the same compelling question: can we explain both the similarity (homology) and variety of tetrapod limbs (and the fins of the Crossopterygians) in terms of developmental mechanisms?

Experimental analysis of the role of the ZPA in the development of the wing buds of wingless (ws) mutant embryos.

Phenotypically wingless (ws) chick embryos have wing buds characterized by the spreading of mesenchymal cell death in an anterior-to-posterior direction beginning at stage 19. It has been argued that this may reflect the absence of a functional polarizing zone (ZPA). When tested by preaxial grafting into normal wing buds (stages 20-21), wingless ZPAs (stage 18-19) had duplicating properties identical with those of normal ZPAs. Equally, normal chick or quail ZPA (stages 20-22) grafted into the posterior margin of wingless wing buds (stages 18-20) failed to inhibit the pattern of cell death or to evoke any improvement in their developmental performance. The wingless (ws) condition is not, therefore, due to a ZPA deficiency. Possible explanations are the prior programming for cell death of the wingless mesenchyme, or somitic deficiency, but it appears more likely that the mutant limb mesenchyme fails to transmit or respond to factor(s) produced by the ZPA.

The distribution of the polarizing zone (ZPA) in the legbud of the chick embryo.

The stage-21 to 22 legbud polarizing zone (ZPA) was mapped by transplanting small blocks of posterior marginal mesenchyme preaxially into stage-20 to -22 chick wing buds and assessing the degree of duplication of the wing digital skeleton produced in the host. Blocks taken from the posterior flank, from the angle between posterior flank and the proximal base of the limb bud, and from the most anterior distal position chosen (under the AER), all had very low activity. Blocks taken from the posterior margin of the legbud, plus the next distal block under the posterior part of the AER, all had high activity. We consider that barrier and amputation results on wing and legbud, when interpreted in the light of maps of the ZPA in both limb buds, are consistent with the hypothesis that both leg and wing have their growth and anteroposterior axis of pattern formation controlled by the ZPA.

The effect of the zone of polarizing activity (ZPA) on the anterior half of the chick wing bud.

Removal of the posterior half of the chick wing bud between stages 17-22 results in failure of the anterior distal tissue to survive and differentiate. This observation has been interpreted in terms of a requirement by the anterior half of a factor supplied by the posterior half of the limb containing the zone of polarizing activity (ZPA). This relationship has been tested by grafting ZPA tissue to the posterior surface of the anterior half after posterior half removal. Grafts made proximally on the cut surface did not significantly improve survival and development, nor did the ZPA prevent the expansion of the cell death in the ANZ beyond its normal boundaries into the distal mesenchyme. However, when grafted distally the ZPA inhibited cell death in the apical mesenchyme and caused the anterior mesenchyme to change its normal prospective fate (radius and digit 2). In all these cases, in addition to digit 2, digit 3 and frequently also digit 4 differentiated. The anterior half went on to develop a full set of digits and zeugopod parts in almost 50% of cases, although no skeleton resulting from this regulation of the anterior half had totally size regulated. These results demonstrate a developmental 'rescue' effect by the ZPA, and further support the view that the ZPA has a central and unique function in normal limb bud development, controlling survival and differentiation of the mesenchyme along the anteroposterior axis.