Vertebrate Head Research Articles

Tracing the Evolutionary Origin of Chordate Somites in the Hemichordate Ptychodera flava.

Metameric somites are a novel character of chordates with unclear evolutionary origins. In the early branching chordate amphioxus, anterior somites are derived from the paraxial mesodermal cells that bud off the archenteron (i.e., enterocoely) at the end of gastrulation. Development of the anterior somites requires fibroblast growth factor (FGF) signaling, and distinct somite compartments express orthologs of vertebrate nonaxial mesodermal markers. Thus, it has been proposed that the amphioxus anterior somites are homologous to the vertebrate head mesoderm, paraxial mesoderm, and lateral plate mesoderm. To trace the evolutionary origin of somites, it is essential to study the chordates' closest sister group, Ambulacraria, which includes hemichordates and echinoderms. The anterior coeloms of hemichordate and sea urchin embryos (respectively called protocoel and coelomic pouches) are also formed by enterocoely and require FGF signals for specification and/or differentiation. In this study, we applied RNA-seq to comprehensively screen for regulatory genes associated with the mesoderm-derived protocoel of the hemichordate Ptychodera flava. We also used a candidate gene approach to identify P. flava orthologs of chordate somite markers. In situ hybridization results showed that many of these candidate genes are expressed in distinct or overlapping regions of the protocoel, which indicates that molecular compartments exist in the hemichordate anterior coelom. Given that the hemichordate protocoel and amphioxus anterior somites share a similar ontogenic process (enterocoely), induction signal (FGF), and characteristic expression of orthologous genes, we propose that these two anterior coeloms are indeed homologous. In the lineage leading to the emergence of chordates, somites likely evolved from enterocoelic, FGF-dependent, and molecularly compartmentalized anterior coeloms of the deuterostome last common ancestor.

Cell type and regulatory analysis in amphioxus illuminates evolutionary origin of the vertebrate head

To shed light on the enigmatic origin of the vertebrate head, our study employs an integrated approach that combines single-cell transcriptomics, perturbations in signaling pathways, and cis-regulatory analysis in amphioxus. As a representative of a basal lineage within the chordate phylum, amphioxus retains many characteristics thought to have been present in the common chordate ancestor. Through cell type characterization, we identify the presence of prechordal plate-like, pre-migratory, and migratory neural crest-like cell populations in the developing amphioxus embryo. Functional analysis establishes conserved roles of the Nodal and Hedgehog signaling pathways in prechordal plate-like populations, and of the Wnt signaling pathway in neural crest-like populations’ development. Furthermore, our trans-species transgenic experiments highlight similarities in the regulatory environments that drive neural crest-like and prechordal plate-like developmental programs in both vertebrates and amphioxus. Our findings provide evidence that the key features of vertebrate head development can be traced back to the common ancestor of all chordates.

Developmental sequence of the chondrocranium in the obligate carnivorous larvae of Lepidobatrachus laevis (Amphibia: Ceratophryidae).

The vertebrate head and its skull represent a significant innovation that has played a key role in the evolutionary and ecological success of vertebrates. For a global and integral understanding of the evolution of the head skeleton, it is essential to have reliable information on the development of chondrocranium in a wide range of vertebrate species. Therefore, we studied the cranial chondrogenesis of the larva of the Budgett frog, Lepidobatrachus laevis (Ceratophryidae, Neobatrachia). We studied the development using several methods, including histological preparation of transverse sections of the chondrocranium, morphological analysis of three different states of development (mesenchymal aggregation, differentiation, and chondrification), and three-dimensional digital reconstructions. As a result, we observed that the Anlage of the chondrocranium at Gosner stage 19 is laterally compressed, that is, it is initially higher than wide. It gradually flattens, enlarges, and differentiates until reaching a very wide and flat shape at Gosner stage 26. Furthermore, we show that the chondrocranial development of L. laevis takes place in a mosaic pattern, which differs to nontetrapod vertebrates in which an anterior to posterior gradient of chondrification is observed. We identified 19 developmental stages in L. laevis according to the chondrification state of its cranial structures. The first element reaching the differentiation-into-chondroblast stage is the hypobranchial plate of the branchial basket, and chondrification, that is, final differentiation, occurs simultaneously in several structures of the neurocranium and viscerocranium. We hypothesize that the rapid chondrification of L. laevis' chondrocranium is an adaptation to the semi-arid climate conditions from its type locality, El Gran Chaco in South America. Due to the only temporary availability of suitable water accumulations this apparent accelerated development would make sense to ensure the tadpoles are froglets by the time the water body disappears. Here, we provide novel information on chondrocranial development in L. laevis. We compare our results to the developmental sequences, previously known from other vertebrate taxa, particularly within the amphibian phylogeny, in an attempt to understand developmental variability and the evolutionary history of the vertebrate head skeleton.

An amphioxus neurula stage cell atlas supports a complex scenario for the emergence of vertebrate head mesoderm

The emergence of new structures can often be linked to the evolution of novel cell types that follows the rewiring of developmental gene regulatory subnetworks. Vertebrates are characterized by a complex body plan compared to the other chordate clades and the question remains of whether and how the emergence of vertebrate morphological innovations can be related to the appearance of new embryonic cell populations. We previously proposed, by studying mesoderm development in the cephalochordate amphioxus, a scenario for the evolution of the vertebrate head mesoderm. To further test this scenario at the cell population level, we used scRNA-seq to construct a cell atlas of the amphioxus neurula, stage at which the main mesodermal compartments are specified. Our data allowed us to validate the presence of a prechordal-plate like territory in amphioxus. Additionally, the transcriptomic profile of somite cell populations supports the homology between specific territories of amphioxus somites and vertebrate cranial/pharyngeal and lateral plate mesoderm. Finally, our work provides evidence that the appearance of the specific mesodermal structures of the vertebrate head was associated to both segregation of pre-existing cell populations, and co-option of new genes for the control of myogenesis.

The chondrocranium of the tadpole of Hymenochirus boettgeri (Anura: Pipidae) and the sequence and timing of its development

The evolutionary development of the vertebrate head has played a critical role in shaping the diversity of body plans and ecological roles within vertebrate species. This evolutionary journey is characterised by the emergence of the head compartment during the Cambrian radiation, the evolution of jaws during the Silurian, and the subsequent diversification of vertebrate groups. The cranium, consisting of the neurocranium, dermatocranium, and viscerocranium, plays a central role in craniofacial support. Research on the development of the cranium has been carried out in several vertebrate species, providing valuable insights into their unique patterns of cartilage development. While most vertebrates show typical sequences of chondrocranial development, anurans divert from these patterns. Our study focused on the chondrocranial development of Congo dwarf frog Hymenochirus boettgeri (Anura: Pipidae) tadpoles, providing a detailed sequential analysis from mesenchymal elements to the premetamorphic chondrocranium. The chondrocranium of these tadpoles showed several reductions as a consequence of their miniaturization and their unique suction feeding lifestyle. We found a mosaic-like development of anuran mandibular and hyoid arches, and we also discussed the intricate patterns of suprarostral cartilage development and deviations in branchial basket development compared to other anuran species. Our study also contrasts anuran neurocranial development with that of other osteichthyan species, highlighting unique features such as the similar direction of development of anterior and posterior neurocranial components. Overall, our research contributes to our understanding of chondrocranial development in anurans, highlighting both conserved and unique features in H. boettgeri tadpoles. Our findings enrich our knowledge of vertebrate cranial development and provide insights into the evolutionary history of anuran chondrocranial features.

Ultrastructure of the lamprey head mesoderm reveals evolution of the vertebrate head

The cranial muscle is a critical component in the vertebrate head for a predatory lifestyle. However, its evolutionary origin and possible segmental nature during embryogenesis have been controversial. In jawed vertebrates, the presence of pre-otic segments similar to trunk somites has been claimed based on developmental observations. However, evaluating such arguments has been hampered by the paucity of research on jawless vertebrates. Here, we discovered different cellular arrangements in the head mesoderm in lamprey embryos (Lethenteron camtschaticum) using serial block-face scanning electron and laser scanning microscopies. These cell populations were morphologically and molecularly different from somites. Furthermore, genetic comparison among deuterostomes revealed that mesodermal gene expression domains were segregated antero-posteriorly in vertebrates, whereas such segregation was not recognized in invertebrate deuterostome embryos. These findings indicate that the vertebrate head mesoderm evolved from the anteroposterior repatterning of an ancient mesoderm and developmentally diversified before the split of jawless and jawed vertebrates.

The oldest three-dimensionally preserved vertebrate neurocranium

The neurocranium is an integral part of the vertebrate head, itself a major evolutionary innovation1,2. However, its early history remains poorly understood, with great dissimilarity in form between the two living vertebrate groups: gnathostomes (jawed vertebrates) and cyclostomes (hagfishes and lampreys)2,3. The 100 Myr gap separating the Cambrian appearance of vertebrates4–6 from the earliest three-dimensionally preserved vertebrate neurocrania7 further obscures the origins of modern states. Here we use computed tomography to describe the cranial anatomy of an Ordovician stem-group gnathostome: Eriptychius americanus from the Harding Sandstone of Colorado, USA8. A fossilized head of Eriptychius preserves a symmetrical set of cartilages that we interpret as the preorbital neurocranium, enclosing the fronts of laterally placed orbits, terminally located mouth, olfactory bulbs and pineal organ. This suggests that, in the earliest gnathostomes, the neurocranium filled out the space between the dermal skeleton and brain, like in galeaspids, osteostracans and placoderms and unlike in cyclostomes2. However, these cartilages are not fused into a single neurocranial unit, suggesting that this is a derived gnathostome trait. Eriptychius fills a major temporal and phylogenetic gap in our understanding of the evolution of the gnathostome head, revealing a neurocranium with an anatomy unlike that of any previously described vertebrate.

DLX gene expression in the developing chick pharyngeal arches and relationship to endothelin signaling and avian jaw patterning.

A hinged jaw that articulates with the skull base is a striking feature of the vertebrate head and has been greatly modified between, and within, vertebrate classes. Genes belonging to the DLX homeobox family are conserved mediators of local signaling pathways that distinguish the dorsal and ventral aspects of the first pharyngeal arch. Specifically, a subset of DLX genes are expressed in the cranial neural crest-derived mandibular ectomesenchyme in response to ventral endothelin signaling, an important step that confers the first arch with maxillary and mandibular identities. Downstream targets of DLX genes then execute the morphogenetic processes that lead to functional jaws. Identifying lineage-specific variations in DLX gene expression and the regulatory networks downstream of DLX action is necessary to understand how different kinds of jaws evolved. Here, we describe and compare the expression of all six DLX genes in the chick pharyngeal arches, focusing on the period of active patterning in the first arch. Disruption of endothelin signaling results in the down-regulation of ventral-specific DLX genes and confirms their functional role in avian jaw patterning. This expression resource will be important for comparative embryology and for identifying synexpression groups of DLX-regulated genes in the chick.

Linking Vertebrate Gene Duplications to the New Head Hypothesis.

Vertebrates have diverse morphologies and various anatomical novelties that set them apart from their closest invertebrate relatives. A conspicuous head housing a large brain, paired sense organs, and protected by a skeleton of cartilage and bone is unique to vertebrates and is a defining feature of this taxon. Gans and Northcutt (1980s) proposed that the evolution of this "new head" was dependent on two key developmental innovations: neural crest cells (NCCs) and ectodermal placodes. NCCs are migratory embryonic cells that form bone, cartilage, and neurons in the new head. Based on genome size, Ohno (1970s) proposed a separate hypothesis, stating that vertebrate genome content was quadrupled via two rounds (2R) of whole genome duplications (WGDs), and the surplus of genetic material potentiated vertebrate morphological diversification. While both hypotheses offer explanations for vertebrate success, it is unclear if, and how, the "new head" and "2R" hypotheses are linked. Here, we consider both hypotheses and evaluate the experimental evidence connecting the two. Overall, evidence suggests that while the origin of the NC GRN predates the vertebrate WGDs, these genomic events may have potentiated the evolution of distinct genetic subnetworks in different neural crest subpopulations. We describe the general composition of the NC GRN and posit that its increased developmental modularity facilitated the independent evolution of NC derivatives and the diversification of the vertebrate head skeleton. Lastly, we discuss experimental strategies needed to test whether gene duplications drove the diversification of neural crest derivatives and the "new head".

A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development

The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-βPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development.

Patterning of the Vertebrate Head in Time and Space by BMP Signaling.

How head patterning is regulated in vertebrates is yet to be understood. In this study, we show that frog embryos injected with Noggin at different blastula and gastrula stages had their head development sequentially arrested at different positions. When timed BMP inhibition was applied to BMP-overexpressing embryos, the expression of five genes: xcg-1 (a marker of the cement gland, which is the front-most structure in the frog embryo), six3 (a forebrain marker), otx2 (a forebrain and mid-brain marker), gbx2 (an anterior hindbrain marker), and hoxd1 (a posterior hindbrain marker) were sequentially fixed. These results suggest that the vertebrate head is patterned from anterior to posterior in a progressive fashion and may involve timed actions of the BMP signaling.

Cardiac competence of the paraxial head mesoderm fades concomitant with a shift towards the head skeletal muscle programme

The vertebrate head mesoderm provides the heart, the great vessels, some smooth and most head skeletal muscle, in addition to parts of the skull. It has been speculated that the ability to generate cardiac and smooth muscle is the evolutionary ground-state of the tissue. However, whether indeed the entire head mesoderm has generic cardiac competence, how long this may last, and what happens as cardiac competence fades, is not clear.Bone morphogenetic proteins (Bmps) are known to promote cardiogenesis. Using 41 different marker genes in the chicken embryo, we show that the paraxial head mesoderm that normally does not engage in cardiogenesis has the ability to respond to Bmp for a long time. However, Bmp signals are interpreted differently at different time points. Up to early head fold stages, the paraxial head mesoderm is able to read Bmps as signal to engage in the cardiac programme; the ability to upregulate smooth muscle markers is retained slightly longer. Notably, as cardiac competence fades, Bmp promotes the head skeletal muscle programme instead. The switch from cardiac to skeletal muscle competence is Wnt-independent as Wnt caudalises the head mesoderm and also suppresses Msc-inducing Bmp provided by the prechordal plate, thus suppressing both the cardiac and the head skeletal muscle programmes.Our study for the first time suggests a specific transition state in the embryo when cardiac competence is replaced by skeletal muscle competence. It sets the stage to unravel the cardiac-skeletal muscle antagonism that is known to partially collapse in heart failure.

Cartilage diversification and modularity drove the evolution of the ancestral vertebrate head skeleton

The vertebrate head skeleton has evolved a myriad of forms since their divergence from invertebrate chordates. The connection between novel gene expression and cell types is therefore of importance in this process. The transformation of the jawed vertebrate (gnathostome) head skeleton from oral cirri to jointed jaw elements required a diversity of cartilages as well as changes in the patterning of these tissues. Although lampreys are a sister clade to gnathostomes, they display skeletal diversity with distinct gene expression and histologies, a useful model for addressing joint evolution. Specifically, the lamprey tissue known as mucocartilage has noted similarities with the jointed elements of the mandibular arch in jawed vertebrates. We thus asked whether the cells in lamprey mucocartilage and gnathostome joint tissue could be considered homologous. To do this, we characterized new genes that are involved in gnathostome joint formation and characterized the histochemical properties of lamprey skeletal types. We find that most of these genes are minimally found in mucocartilage and are likely later innovations, but we do identify new activity for gdf5/6/7b in both hyaline and mucocartilage, supporting its role as a chondrogenic regulator. Contrary to previous works, our histological assays do not find any perichondrial fibroblasts surrounding mucocartilage, suggesting that mucocartilage is non-skeletogenic tissue that is partially chondrified. Interestingly, we also identify new histochemical features of the lamprey otic capsule that diverge from normal hyaline. Paired with our new insights into lamprey mucocartilage, we propose a broader framework for skeletal evolution in which an ancestral soxD/E and gdf5/6/7 network directs mesenchyme along a spectrum of cartilage-like features.

Embryonic ethanol exposure disrupts craniofacial neuromuscular integration in zebrafish larvae.

Forming a vertebrate head involves the meticulous integration of multiple tissue types during development. Prenatal alcohol exposure is known to cause a variety of birth defects, especially to tissues in the vertebrate head. However, a systematic analysis of coordinated defects across tissues in the head is lacking. Here, we delineate the effects of ethanol on individual tissue types and their integration during craniofacial development. We found that exposure to 1% ethanol induced ectopic cranial muscle and nerve defects with only slight effects on skeletal pattern. Ectopic muscles were, however, unaccompanied by ectopic tendons and could be partially rescued by anesthetizing the larvae before muscle fibers appeared. This finding suggests that the ectopic muscles result from fiber detachment and are not due to an underlying muscle patterning defect. Interestingly, immobilization did not rescue the nerve defects, thus ethanol has an independent effect on each tissue even though they are linked in developmental time and space. Time-course experiments demonstrated an increase in nerve defects with ethanol exposure between 48hpf-4dpf. Time-lapse imaging confirmed the absence of nerve pathfinding or misrouting defects until 48hpf. These results indicate that ethanol-induced nerve defects occur at the time of muscle innervation and after musculoskeletal patterning. Further, we investigated the effect of ethanol on the neuromuscular junctions of the craniofacial muscles and found a reduced number of postsynaptic receptors with no significant effect on the presynaptic terminals. Our study shows that craniofacial soft tissues are particularly susceptible to ethanol-induced damage and that these defects appear independent from one another. Thus, the effects of ethanol on the vertebrate head appear highly pleiotropic.

Colec12 and Trail signaling confine cranial neural crest cell trajectories and promote collective cell migration.

Collective and discrete neural crest cell (NCC) migratory streams are crucial to vertebrate head patterning. However, the factors that confine NCC trajectories and promote collective cell migration remain unclear. Computational simulations predicted that confinement is required only along the initial one-third of the cranial NCC migratory pathway. This guided our study of Colec12 (Collectin-12, a transmembrane scavenger receptor C-type lectin) and Trail (tumor necrosis factor-related apoptosis-inducing ligand, CD253) which we show expressed in chick cranial NCC-free zones. NCC trajectories are confined by Colec12 or Trail protein stripes in vitro and show significant and distinct changes in cell morphology and dynamic migratory characteristics when cocultured with either protein. Gain- or loss-of-function of either factor or in combination enhanced NCC confinement or diverted cell trajectories as observed in vivo with three-dimensional confocal microscopy, respectively, resulting in disrupted collective migration. These data provide evidence for Colec12 and Trail as novel NCC microenvironmental factors playing a role to confine cranial NCC trajectories and promote collective cell migration.

Ontogeny of the trigeminal system and associated structures in Alligator mississippiensis.

From the appearance of the vertebrate head, the trigeminal system has played a role in behavioral and ecological adaptation. The trigeminal nerve is the primary cranial somatosensory nerve, also innervating the jaw muscles. In crocodylians, the trigeminal nerve plays a role in modulating the high bite force and unique integumentary sensation. In association with these behaviors, crocodylians are known for large trigeminal nerves, a high volume of trigeminal-innervated musculature, and densely packed, specialized sensory receptors. These innovations also occurred in concert with a restructuring of the lateral braincase wall. These morphologies have previously been investigated in phylogenetic and evolutionary contexts, but an ontogenetic, whole-system investigation of trigeminal tissue and associated musculature, cartilage, and bone is lacking, as is an understanding of developmental timing of morphologies significant to hypotheses of homology. Here, we use contrast-enhanced computed tomographyimaging to provide description and analysis of the trigeminal system in an ontogenetic series of Alligator mississippiensis from embryonic to adult form. We explore growth rates and allometric relationships of structures and discuss the significance to hypotheses of homology. We find a high growth rate and allometric trajectory of the trigeminal nerve in comparison to other cranial nerves, likely associated with the large volume of trigeminal musculature and high densities of sensory receptors. We identify a similar trend in the pterygoideus dorsalis muscle, the highest contributor to bite force. We narrow ontogenetic timing of features related to the trigeminal topological paradigm and the undeveloped epipterygoid. Overall, we provide a basis for understanding trigeminal development in crocodylians, which upon comparison across reptiles will reveal ontogenetic origins of morphological variation.

Processes and patterns: Insights on cranial covariation from an Apert syndrome mouse model.

Major cell-to-cell signaling pathways, such as the fibroblast growth factors and their four receptors (FGF/FGFR), are conserved across a variety of animal forms. FGF/FGFRs are necessary to produce several "vertebrate-specific" structures, including the vertebrate head. Here, we examine the effects of the FGFR2 S252W mutation associated with Apert syndrome on patterns of cranial integration. Our data comprise micro-computed tomography images of newborn mouse skulls, bred to express the Fgfr2 S252W mutation exclusively in either neural crest or mesoderm-derived tissues, and mice that express the Fgfr2 S252W mutation ubiquitously. Procrustes-based methods and partial least squares analysis were used to analyze craniofacial integration patterns. We found that deviations in the direction and degree of integrated shape change across the mouse models used in our study were potentially driven by the modular variation generated by differing expression of the Fgfr2 mutation in cranial tissues. Our overall results demonstrate that covariation patterns can be biased by the spatial distribution and magnitude of variation produced by underlying developmental-genetic mechanisms that often impact the phenotype in disproportionate ways.

Functions of the FGF signalling pathway in cephalochordates provide insight into the evolution of the prechordal plate.

ABSTRACTThe fibroblast growth factor (FGF) signalling pathway plays various roles during vertebrate embryogenesis, from mesoderm formation to brain patterning. This diversity of functions relies on the fact that vertebrates possess the largest FGF gene complement among metazoans. In the cephalochordate amphioxus, which belongs to the chordate clade together with vertebrates and tunicates, we have previously shown that the main role of FGF during early development is the control of rostral somite formation. Inhibition of this signalling pathway induces the loss of these structures, resulting in an embryo without anterior segmented mesoderm, as in the vertebrate head. Here, by combining several approaches, we show that the anterior presumptive paraxial mesoderm cells acquire an anterior axial fate when FGF signal is inhibited and that they are later incorporated in the anterior notochord. Our analysis of notochord formation in wild type and in embryos in which FGF signalling is inhibited also reveals that amphioxus anterior notochord presents transient prechordal plate features. Altogether, our results give insight into how changes in FGF functions during chordate evolution might have participated to the emergence of the complex vertebrate head.

The impact of Drew Noden's work on our understanding of craniofacial musculoskeletal integration.

The classical anatomist Drew Noden spearheaded craniofacial research, laying the foundation for our modern molecular understanding of development, evolution, and disorders of the craniofacial skeleton. His work revealed the origin of cephalic musculature and the role of cranial neural crest (CNC) in early formation and patterning of the head musculoskeletal structures. Much of modern cranial tendon research advances a foundation of knowledge that Noden built using classical quail-chick transplantation experiments. This elegant avian chimeric system involves grafting of donor quail cells into host chick embryos to identify the cell types they can form and their interactions with the surrounding tissues. In this review, we will give a brief background of vertebrate head formation and the impact of CNC on the patterning, development, and evolution of the head musculoskeletal attachments. Using the zebrafish as a model system, we will discuss examples of modifications of craniofacial structures in evolution with a special focus on the role of tendon and ligaments. Lastly, we will discuss pathologies in craniofacial tendons and the importance of understanding the molecular and cellular dynamics during craniofacial tendon development in human disease.

The evolutionary development of the jaws and dentition

The development of the jaws and dentition during half a billion years of evolution is described in broad lines and two theories regarding the origin of the dentition are discussed in detail. It is highly likely our dentition developed from dermal ossification 'denticles' originally protecting, in particular, the head of primitive extinct vertebrates. Simple, at first tubercular, later conical teeth developed into complex tooth and molar forms, especially in mammals. Special attention is given to the development of molar cusps. The process of continuous replacement of the dentition is also discussed in more detail.