Computer-aided Three-dimensional Reconstruction Research Articles

Three-dimensional morphology and crystallographic characteristics of the low-temperature bainitic microstructure in high-carbon steel

The three-dimensional morphology of low-temperature bainite in high-carbon steel was reconstructed by serial sectioning and computer-aided three-dimensional reconstruction techniques. Combined with laser scanning confocal microscope and electron backscatter diffraction analysis, the microstructural evolution and crystallographic orientation of low-temperature bainite were studied. The results show that the three-dimensional morphology of the low-temperature bainite sheaves is a plate-like structure. During the isothermal transformation, some new bainite plates stop growing after impinging on preformed bainite plates. However, some new bainite plates resume growth on the opposite surface of the impingement zone after briefly stopping. This is attributed to the accumulation of strain energy in the collision zone caused by impingement between bainite plates, which provides the driving force for new nucleation, thus stimulating new nucleation on the opposite surface of the collision zone. The bainite formed on the opposite surface maintains the same crystallographic orientation as the “twinned bainite”. Finally, the bainite plates form interlocked microstructures in the prior austenite grains via impingement, nucleation, and intersection.

Advanced Three-Dimensional Technologies in Craniofacial Reconstruction.

After studying this article, the participant should be able to: 1. Describe the evolution of three-dimensional computer-aided reconstruction and its current applications in craniofacial surgery. 2. Recapitulate virtual surgical planning, or computer-assisted surgical simulation, workflow in craniofacial surgery. 3. Summarize the principles of computer-aided design techniques, such as mirror-imaging and postoperative verification of results. 4. Report the capabilities of computer-aided manufacturing, such as rapid prototyping of three-dimensional models and patient-specific custom implants. 5. Evaluate the advantages and disadvantages of using three-dimensional technology in craniofacial surgery. 6. Critique evidence on advanced three-dimensional technology in craniofacial surgery and identify opportunities for future investigation. Increasingly used in craniofacial surgery, virtual surgical planning is applied to analyze and simulate surgical interventions. Computer-aided design and manufacturing generates models, cutting guides, and custom implants for use in craniofacial surgery. Three-dimensional computer-aided reconstruction may improve results, increase safety, enhance efficiency, augment surgical education, and aid surgeons' ability to execute complex craniofacial operations. Subtopics include image analysis, surgical planning, virtual simulation, custom guides, model or implant generation, and verification of results. Clinical settings for the use of modern three-dimensional technologies include acquired and congenital conditions in both the acute and the elective settings. The aim of these techniques is to achieve superior functional and aesthetic outcomes compared to conventional surgery. Surgeons should understand this evolving technology, its indications, limitations, and future direction to use it optimally for patient care. This article summarizes advanced three-dimensional techniques in craniofacial surgery with cases highlighting clinical concepts.

Multimodal image fusion technology for diagnosis and treatment of the skull base-infratemporal tumors

To explore the value of incorporated multimodal image fusion technology with computer-aided design of the skull base-infratemporal tumor treatment. A retrospective study was carried out to enroll seventeen patients with skull base-infratemporal tumors treated at Peking University Hospital of Stomatology from February 2011 to September 2018. Plain CT, enhanced CT and MRI data were imported into the iPlan 3.0 software (BrainLab navigation system), and the image fusion was performed for each patient preoperatively. Then the three-dimensional images of the tumor, vital vessels and craniofacial bones were reconstructed to prepare virtual operation design. We evaluated the application of multimodal image fusion technology that had been incorporated with computer-aided planning during the navigation-guided biopsy or surgery, through the analysis of the biopsy and operation data and regular follow-up postoperatively. The mean age of 17 patients (7 males and 10 females) was 46 years. Primary tumors occurred in 11 cases, and recurrent tumors in 6 cases. The size of the 17 tumors ranged from 2.9 cm to 9 cm, and the mean size was 4.35 cm. There were 7 cases with skull base bone destruction and/or intracranial extension, and 10 cases with tumors adjacent to the skull base. High-quality multimodal fused images were obtained in all the 17 cases. The spatial-position relationships of the tumors, adjacent craniomaxillofacial bones and vital vessels labeled with different colors were displayed well on the generated fusion images. The multimodal image fusion technology that incorporated with computer-aided three-dimensional reconstruction and then applied in navigation-guided biopsy or surgery showed that, preoperative analysis and virtual operation design functioned with good results, especially in cases with small tumor size, recurrence or illdefined borders in the skull base-infratemporal region. Operation was carried out in 16 cases after preoperative diagnosis and assessment, and 1 case was performed by navigation-guided biopsy only. The proportions of navigation-guided surgery and biopsy were 70.6% (12/17) and 17.6% (3/17) individually. The positive rate of pathologic diagnosis using navigation-guided biopsy was 100% (3/3). All the navigation-guided biopsies or operations were carried out successfully. Complications included 1 case of cerebrospinal fluid leak from a recurred meningioma patient postoperatively, and 1 case of facial paralysis resulting from parotid-gland deep lobe tumor. Most (14/15) tumors got complete removal with safe boundary through intra-operative navigation verification and post-operative imaging confirmation, except for one case of subtotal resection to avoid the injury of cavernous sinus. The pathological results of the tumors could be classified to mesenchymal (10), adenogenous (3), neurogenic (3) or epithelial (1) resources. The follow-up time ranged from 3 to 94 months, with the median follow-up time of 9 months. Taking full advantages of individualized multimodal images, could help analyze the three-dimensional spatial position relationship of tumors, vital vessels and craniofacial bones properly, and then complete the virtual operation design well. The incorporated multimodal image fusion technology with navigation technology may improve the accuracy and safety of core needle biopsy and surgical treatment of skull base-infratemporal tumors.

Early development of the human dentition revisited.

In this review, classical data on the early steps in human odontogenesis are summarized and updated with specific insights into the development of the upper and lower embryonic jaws to help in understanding some oral pathologies. The initial step of human odontogenesis is classically characterized by two parallel horseshoe‐shaped epithelial laminae. These originate from the oral epithelium and an ingrowth into the jaw mesenchyme: the internal dental lamina gives rise to deciduous tooth primordia, while the external vestibular lamina represents the developmental base of the oral vestibule. However, a more complex situation was revealed by recent studies combining analyses of the dental and adjacent oral epithelia on histological sections and computer‐aided three‐dimensional (3D) reconstructions during the 2nd month of human embryonic development. The dental epithelium forms a mound, where swellings appear later, corresponding to the individual primordia of deciduous teeth. External to the developing deciduous dentition, the 3D reconstructions do not show any continuous vestibular lamina but instead a complex of discontinuous epithelial bulges and ridges. The patterns of these epithelial structures and their relationship to the dental epithelium differ not only between the upper and lower jaws but also between the lip and cheek segments in each jaw. Knowledge of early odontogenesis may help in understanding some oral pathologies. For example, the human lateral incisor has a dual origin: it arises in the area of fusion between the medial nasal and maxillary facial processes and involves material from these two regions. Such a dual origin at the site of fusion of facial processes represents a predisposition to developmental vulnerability for the upper lateral incisor, resulting in its frequent anomalies (absence, hypoplasia, duplication), especially in patients with a cleft lip and/or jaw. Other pathologies, such as a minute supernumerary tooth, desmoplastic ameloblastoma or extraosseous odontogenic cysts are located external to the upper or lower dentition, and might be derived from structures that transiently appear during early development of the oral vestibule in humans.

Morphometric characteristics of cells in the alveolar region of mammalian lungs.

Morphometric procedures have been used to study the characteristics of cells in the alveolar region of the lungs of rats, dogs, baboons, and humans. Compared with the other species, human lungs were found to contain greater numbers of macrophages and to have larger alveolar type II, endothelial, and interstitial cells. The thickness of the interstitium and the pulmonary capillary endothelium were also significantly greater in the human lungs. These differences in human lung anatomy may be due to increased exposure to airborne pollutants and to tobacco smoke. Despite the above differences and the fact that there are large variations in size and functional characteristics of the lungs of these mammals, an overall striking similarity in characteristics of individual lung cells was found. The distribution of cells in alveolar tissue was remarkably constant between species as was the average volume and surface area of most cell types. Computer-aided three-dimensional reconstruction techniques were used to determine the spatial relationship of organelles in individual alveolar type II cells from rats. A three-dimensional reconstruction of cells permits quantification of number, size, surface area, and volume of subcellular organelles and correlations of their three-dimensional spatial relationships.

SequentialShhexpression in the development of the mouse upper functional incisor

The mouse incisor is a frequently used model in studies of the molecular control of organ development. The appropriate interpretation of data on normogenesis is essential for understanding the data obtained in mutant mice. For this reason, we performed a very detailed investigation of the development of the upper incisor in wild-type mice from embryonic day (ED) 11.5 till 14.5. A combination of histology, whole mount in situ hybridization, computer-aided three-dimensional reconstructions, and fluorescent microscopy, has been used. Several sonic hedgehog (Shh) expression domains have been detected in the upper incisor region during early prenatal development. At ED11.5-13.5, there was a single Shh positive domain present in the anterior part of left or right upper jaw arches, corresponding to the epithelial thickening. More posteriorly, a new Shh expression domain appeared in the incisor bud in the developmentally more advanced ED13.5 embryos. At ED14.5, only this posterior Shh expression in the incisor germ remained detectable. This study brings new insights into the early development of the upper incisor in mice and completes the data on normal mouse incisor development. The temporal-spatial pattern of Shh expression reflects the development of two tooth generations, being detectable in two successive, antero-posteriorly located areas in the prospective incisor region in the upper jaw. The first, anterior and superficial Shh expression domain reflects the rudimentary tooth development suppressed during evolution. Only the subsequent, posterior and deeper Shh expression region, appearing at ED13.5, correlates with the prospective upper functional incisor in wild-type mice.

The architecture of Norway spruce ectomycorrhizae: three-dimensional models of cortical cells, fungal biomass, and interface for potential nutrient exchange

Gathering realistic data on actual fungal biomass in ectomycorrhized fine root systems is still a matter of concern. Thus far, observations on architecture of ectomycorrhizae (ECMs) have been limited to analyses of two-dimensional (2-D) images of tissue sections. This unavoidably causes stereometrical problems that lead to inadequate assumptions about actual size of cells and their arrangement within ECM's functional compartments. Based on extensive morphological investigations of field samples, we modeled the architectural components of an average-sized Norway spruce ECM. In addition to our comprehensive and detailed quantitative data on cell sizes, we studied actual shape and size, in vivo arrangement, and potential nutrient exchange area of plant cortical cells (CCs) using computer-aided three-dimensional (3-D) reconstructions based on semithin serial sections. We extrapolated a factual fungal biomass in ECMs (Hartig net (HN) included) of 1.71t ha(-1) FW (0.36t ha(-1) DW) for the top 5cm of soil for an autochthonous, montane, optimum Norway spruce stand in the Tyrolean Alps. The corresponding potential nutrient exchange area in ECMs including main axes of ECM systems, which is defined as the sum of interfaces between plant CCs and the HN, amounts to at least 3.2 × 10(5) m(2) ha(-1). This is the first study that determines the contribution of the HN to the total fungal biomass in ECMs as well as the quantification of its contact area. Our results may stimulate future research on fungal below-ground processes and their impact on the global carbon cycle.

Loss of organisation in CYLD defective tumours is associated with diminished DKK2 expression.

Patients carrying heterozygous germline truncating mutations in the CYLD gene develop multiple primary hair follicle related tumours. A highly organised tumour, termed cylindroma, and a highly disorganised tumour, termed spiradenoma, may both develop in the same patient. Furthermore, histological features of both tumour types have been described within the same tumour specimen. We used three-dimensional computer-aided reconstruction of these tumours to explore growth patterns of cylindromas and spiradenomas.

Development of the olfactory and vomeronasal organs in Discoglossus pictus (Discoglossidae, Anura)

Using histological techniques and computer-aided three-dimensional reconstructions of histological serial sections, we studied the development of the olfactory and vomeronasal organs in the discoglossid frog Discoglossus pictus. The olfactory epithelium in larval D. pictus represents one continuous unit of tissue not divided into two separate portions. However, a small pouch of olfactory epithelium (the "ventromedial diverticulum") is embedded into the roof of the buccal cavity, anteromedial to the internal naris. The lateral appendix is present in D. pictus through the entire larval period and disappears during the onset of metamorphosis. The disappearance of the lateral appendix at this time suggests that it is a typical larval organ related to aquatic life. The vomeronasal organ develops during hindlimb development, which is comparatively late for anurans. The development of the vomeronasal organ in D. pictus follows the same general developmental pattern recognized for neobatrachians. As with most anurans, the vomeronasal glands appear later than the vomeronasal organ. After metamorphosis, the olfactory organ of adult D. pictus is composed of a series of three interconnected chambers: the cavum principale, cavum medium, and cavum inferius. We suggest that the ventromedial diverticulum at the anterior border of the internal naris of larval D. pictus might be homologous with the ventral olfactory epithelium of bufonids and with the similar diverticulum of Alytes.

Neuroanatomy of Halobiotus crispae (Eutardigrada: Hypsibiidae): Tardigrade brain structure supports the clade panarthropoda

The position of Tardigrada in the animal tree of life is a subject that has received much attention, but still remains controversial. Whereas some think tardigrades should be categorized as cycloneuralians, most authors argue in favor of a phylogenetic position within Panarthropoda as a sister group to Arthropoda or Arthropoda + Onychophora. Thus far, neither molecular nor morphological investigations have provided conclusive results as to the tardigrade sister group relationships. In this article, we present a detailed description of the nervous system of the eutardigrade Halobiotus crispae, using immunostainings, confocal laser scanning microscopy, and computer-aided three-dimensional reconstructions supported by transmission electron microscopy. We report details regarding the structure of the brain as well as the ganglia of the ventral nerve cord. In contrast to the newest investigation, we find transverse commissures in the ventral ganglia, and our data suggest that the brain is partitioned into at least three lobes. Additionally, we can confirm the existence of a subpharyngeal ganglion previously called subesophagal ganglion. According to our results, the original suggestion of a brain comprised of at least three parts cannot be rejected, and the data presented supports a sister group relationship of Tardigrada to 1) Arthropoda or 2) Onychophora or 3) Arthropoda + Onychophora.

The formation mechanisms of interlocked microstructures in low-carbon high-strength steel weld metals

Microstructural features and the formation mechanisms of interlocked microstructures of acicular ferrite in a low-carbon high-strength steel weld metal were investigated by means of computer-aided three-dimensional reconstruction technique and electron backscattered diffraction analysis. Multiple nucleation on inclusions, sympathetic nucleation or repeated nucleation, hard impingement, mutual intersection, and fixed orientation relationships of acicular ferrite grains were observed. They were all responsible for the formation of interlocked microstructures in the weld metal. During the process of isothermal transformation, the pre-formed acicular ferrite laths or plates partitioned austenite grains into many small and separate regions, and the growth of later formed acicular ferrite grains was confined in these small regions. Thus, the crystallographic grain size became smaller with the increasing holding time. ► Acicular ferrite is formed by multiple nucleation and sympathetic nucleation. ► Hard impingement and intersection of ferrite grains occur at later stages. ► The pre-formed ferrite laths partition austenite grains into smaller regions. ► The growth of later formed ferrite grains is confined in the smaller regions.

Distinctive Left-Sided Distribution of Adrenergic-Derived Cells in the Adult Mouse Heart

Adrenaline and noradrenaline are produced within the heart from neuronal and non-neuronal sources. These adrenergic hormones have profound effects on cardiovascular development and function, yet relatively little information is available about the specific tissue distribution of adrenergic cells within the adult heart. The purpose of the present study was to define the anatomical localization of cells derived from an adrenergic lineage within the adult heart. To accomplish this, we performed genetic fate-mapping experiments where mice with the cre-recombinase (Cre) gene inserted into the phenylethanolamine-n-methyltransferase (Pnmt) locus were cross-mated with homozygous Rosa26 reporter (R26R) mice. Because Pnmt serves as a marker gene for adrenergic cells, offspring from these matings express the β-galactosidase (βGAL) reporter gene in cells of an adrenergic lineage. βGAL expression was found throughout the adult mouse heart, but was predominantly (89%) located in the left atrium (LA) and ventricle (LV) (p<0.001 compared to RA and RV), where many of these cells appeared to have cardiomyocyte-like morphological and structural characteristics. The staining pattern in the LA was diffuse, but the LV free wall displayed intermittent non-random staining that extended from the apex to the base of the heart, including heavy staining of the anterior papillary muscle along its perimeter. Three-dimensional computer-aided reconstruction of XGAL+ staining revealed distribution throughout the LA and LV, with specific finger-like projections apparent near the mid and apical regions of the LV free wall. These data indicate that adrenergic-derived cells display distinctive left-sided distribution patterns in the adult mouse heart.

Measurement of guinea pig basilar membrane using computer-aided three-dimensional reconstruction system

Cochleas are known to have the ability to analyze a frequency widely, and this ability seems to be owed mostly to the basilar membrane (BM) configuration. However, the relationship between the cochlear frequency-position map and the BM configuration is not clear. Therefore, in this paper, the internal structures of a guinea pig cochlea, especially the BM configuration, were reconstructed and measured using a computer-aided three-dimensional (3-D) reconstruction system. Then, an attempt was made to examine the influence of the BM configuration on the cochlear frequency-position map. The measurement results indicate that the width of the BM increased and its thickness decreased with an increase in the distance from the basal turn towards the apical turn. Theoretical consideration reveals that the wide frequency-position of the cochlea is achieved by not only the BM configuration change along the length of the cochlea but also the change of the Young's modulus of the BM along the length of the cochlea.

Transition from cylindroma to spiradenoma in CYLD‐defective tumours is associated with reduced DKK2 expression

Patients carrying heterozygous germline truncating mutations in the CYLD gene develop multiple primary hair follicle-related tumours. A highly patterned tumour, termed cylindroma, and a highly disorganized tumour, termed spiradenoma, may both develop in the same patient. Furthermore, histological features of both tumour types have been described within the same tumour specimen. We used three-dimensional computer-aided reconstruction of these tumours to demonstrate contiguous growth of cylindromas into spiradenomas, thus suggesting a transition between the two tumour types. To explore factors that may influence cutaneous tumour patterning, genome-wide transcriptomic analysis of 32 CYLD-defective tumours was performed. Overexpression of the Wnt/β-catenin signalling pathway was observed relative to normal perilesional tissue. Morphometric analysis was used to investigate the relationship between Wnt pathway-related gene expression and tumour organization. This revealed an association between reduced Dickkopf 2 (DKK2-a negative regulator of the Wnt/β-catenin signalling pathway) expression and loss of tumour patterning. Reduced DKK2 expression was associated with methylation of the DKK2 gene promoter in the majority of tumour samples assayed. RNA interference-mediated silencing of DKK2 expression in cylindroma primary cell cultures caused an increase in colony formation, cell viability, and anchorage-independent growth. Using these data, we propose a model where epigenetic programming may influence tumour patterning in patients with CYLD mutations.

Shh expression in a rudimentary tooth offers new insights into development of the mouse incisor

For teeth as for any organ, knowledge of normal development is essential for the proper interpretation of developmental anomalies in mutant mice. It is generally accepted that tooth formation is initiated with a single signaling center that, in the incisor region, is exclusively related to the development of the functional adult incisor. Here, using a unique combination of computer-aided three-dimensional reconstructions and whole mount in situ hybridization of mandibles from finely staged wild-type mouse embryos, we demonstrate that several Sonic hedgehog (Shh) expression domains sequentially appear in the lower incisor region during early development. In contrast to the single Shh expression domain that is widely assumed to be present in each lower incisor area at ED12.5-13.5, we identified two spatially distinct regions of Shh expression that appear in an anterior-posterior sequence during this period. The initial anterior, more superficially located Shh expression region represented the rudimentary (so-called deciduous) incisor, whereas only the later posterior deeper situated region corresponded to the prospective functional incisor. In the more advanced embryos, only this posterior Shh expression in the incisor bud was detectable as a precursor of the enamel knot. This study offers a new interpretation of published molecular data on the mouse incisor from initiation through ED13.5. We suggest that, as with Shh expression, other molecular data that have been ascribed to the progressive development of the mouse functional incisor at early stages, in fact, correspond to a rudimentary incisor whose development is aborted.

Morphological evidences in circumvallate papilla and von Ebners' gland development in mice

In rodents, the circumvallate papilla (CVP), with its underlying minor salivary gland, the von Ebners' gland (VEG), is located on the dorsal surface of the posterior tongue. Detailed morphological processes to form the proper structure of CVP and VEG have not been properly elucidated. In particular, the specific localization patterns of taste buds in CVP and the branching formation of VEG have not yet been elucidated. To understand the developmental mechanisms underlying CVP and VEG formation, detailed histological observations of CVP and VEG were examined using a three-dimensional computer-aided reconstruction method with serial histological sections and pan-Cytokeratins immunostainings. In addition, to define the developmental processes in CVP and VEG formation, we examined nerve innervations and cell proliferation using microinjections of AM1-43 and immunostainings with various markers, including phosphoinositide 3-kinase, Ki-67, PGP9.5, and Ulex europaeus agglutinin 1 (UEA1). Results revealed specific morphogenesis of CVP and VEG with nerve innervations patterns, evaluated by the coincided localization patterns of AM1-43 and UEA1. Based on these morphological and immunohistochemical results, we suggest that nerve innervations and cell proliferations play important roles in the positioning of taste buds in CVP and branching morphogenesis of VEG in tongue development.

The effect of isothermal holding on the microstructures and mechanical properties of a low carbon alloy steel

A low carbon high-strength steel was isothermally treated at 900°C for varying times and was immediately quenched into ice water. The microstructures and grain refinement were characterized by the techniques of serial sectioning and computer-aided three-dimensional reconstruction. The microstructures in the specimens that were isothermally maintained for short periods of time consisted of predominantly martensite and much lower bainite. The lower bainite was lath-like, plate-like, rod-like and lenticular in shape. Most were lath-like or plate-like. The lower bainite laths or plates divided the prior austenite grains into many smaller regions, and the martensite that transformed at lower temperatures was thus confined in the smaller zones and thus had a smaller packet size. The specimens that were isothermally held for short periods thus had higher strength, compared with the directly quenched specimens.

Three-Dimensional Morphology and Growth Behavior of Ferrite Allotriomorphs Formed at Grain Boundary Edges

AbstractThree-dimensional morphology of ferrite allotriomorphs nucleated at grain boundary edges in low carbon steel was revealed by means of serial sectioning in conjunction with computer-aided three-dimensional reconstruction techniques. The typical morphology of them was of an elongated triangular pyramid. The length, width and thickness of ferrite allotriomorphs were measured on the basis of three-dimensional reconstructed images. The aspect ratio (length/width) of them was 4. 5 on an average and scattered from 3 to 6. The measured sizes were smaller than calculated ones, which was probably attributed to solute drag-like effect of manganese in the alloy.

The Morphology of the Rat Vibrissal Follicle-Sinus Complex Revealed by Three-Dimensional Computer-Aided Reconstruction

The vibrissal follicle-sinus complex (FSC) is a sensory receptor of the mammalian integumentary system that is located around the mouth. The purpose of the present study was to identify the actual 3-dimensional structure of the rat vibrissal FSC. Rat skin tissue was serially sectioned at a thickness of 10 µm and then stained with Masson’s trichrome. The serial sections were reconstructed 3-dimensionally using Reconstruct software. The rat vibrissal follicle is a spindle-shaped structure that is embedded within a blood sinus and enveloped within a thick collagenous capsule. The vibrissal FSC is innervated by the deep vibrissal and superficial vibrissal nerves. The deep vibrissal nerve, travelling in the basal-to-apical direction, penetrates the thick collagenous capsule of the vibrissal FSC. The sinus system can be divided into a superior portion, known as the ring sinus, and an inferior portion, known as the cavernous sinus. The ring sinus contains a C-shaped structure, the ringwulst, which is suspended from the mesenchymal sheath of the follicle. Collagenous trabeculae can be seen in the cavernous sinus but not in the ring sinus. The ring sinus encircles the follicle obliquely and asymmetrically. The ringwulst encircles the follicle incompletely, in a C-shaped fashion. This study has demonstrated the previously underappreciated 3-dimensional structure of the vibrissal FSC, which differs from previously reported descriptions, and provides data that will enhance the understanding of vibrissal function.

Development of the ethmoidal structures of the endocranium in Discoglossus pictus (Anura: Discoglossidae)

We use histological techniques and computer-aided three-dimensional reconstructions made from serial histological sections to describe the ontogeny of the ethmoidal endocranium of discoglossid frog Discoglossus pictus. We identify a pattern of development for the suprarostral cartilage that differs from previous findings and probably represents the ancestral anuran pattern. The nasal cartilages, including the inferior prenasal cartilage, are de novo adult structures. The only larva-derived structures of the adult nasal capsules are the posterior aspects of the solum nasi and septum nasi. We also identify patterns of development for the ethmoid plate and postnasal wall that occur during early in ontogenesis. These patterns are associated with development events during metamorphic climax. The pattern of timing of chondrification of the anterior nasal cartilages more closely coincides with that of the neobatrachian species than that recorded for the pelobatid frog Spea. In addition, this study supports a sister taxon relationship between Discoglossus and Alytes.