Review and experimental evaluation of the embryonic development and evolutionary history of flipper development and hyperphalangy in dolphins (Cetacea: Mammalia).

Cetaceans are the only mammals to have evolved hyperphalangy, an increase in the number of phalanges beyond the mammalian plesiomorphic condition of three phalanges per digit. In this study, cetaceans were used as a novel model to review previous studies of mammalian hyperphalangy and contribute new experimental evidence as to the molecular origins of this phenotype in embryos of the pantropical spotted dolphin (Stenella attenuata). Results show embryos of dolphins, mice, and pigs share similar spatiotemporal patterns of signaling proteins known to shape limbs of mammals (e.g., FGF8, BMP2/4, WNT, GREM). However, fetal dolphins differ in that their interdigital tissues are retained, instead of undergoing apoptosis, and that multiple waves of interdigital signals likely contribute to the patterning of supernumerary joints and phalanges in adjacent digits. Integration of fossil and experimental evidence suggests that the presence of interdigital webbing within the fossils of semi-aquatic cetaceans, recovered from the Eocene Epoch (49Ma), was probably the result of BMP-antagonists counteracting interdigital apoptosis during embryonic limb development. Modifications to signals originating in these interdigital tissues likely contributed to the origin of an incipient form of hyperphalangy in obligatorily aquatic cetaceans about 35Ma. Finally, an extreme form of hyperphalangy, with six or more phalanges per digit, evolved independently in rorqual whales (Balaenopteridae) and delphinids, and was probably associated with a wave of signaling within the interdigital tissues.

The role of metallothioneins, selenium and transfer to offspring in mercury detoxification in Franciscana dolphins (Pontoporia blainvillei).

The concentrations of mercury (Hg), selenium (Se) and metallothioneins (MT) were evaluated in fetuses, calves, juveniles and adults of the endangered coastal Franciscana dolphin (Pontoporia blainvillei) from Argentina. Mercury concentrations varied among analyzed tissues (liver, kidney, muscle and brain), with liver showing the higher concentrations in all specimens. An age-dependent accumulation was found in liver, kidney and brain. No significant relationship between Hg and MT concentrations was found for all tissues analyzed. Hepatic Hg molar concentrations were positively correlated with those of Se, indicating a great affinity between these two elements. Furthermore, dark granules of HgSe were observed in Kupffer cells in the liver by electron microscopy, suggesting the role of this macrophage in the detoxification of Hg. A transfer of Hg through placenta was proved. The presence of Hg in brain in all age classes did not show concentrations associated with neurotoxicity.

Toxic heritage: Maternal transfer of pyrethroid insecticides and sunscreen agents in dolphins from Brazil.

Pyrethroids (PYR) and UV filters (UVF) were investigated in tissues of paired mother-fetus dolphins from Brazilian coast in order to investigate the possibility of maternal transfer of these emerging contaminants. Comparison of PYR and UVF concentrations in maternal and fetal blubber revealed Franciscana transferred efficiently both contaminants to fetuses (F/M > 1) and Guiana dolphin transferred efficiently PYR to fetuses (F/M > 1) different than UVF (F/M < 1). PYR and UVF concentrations in fetuses were the highest-ever reported in biota (up to 6640 and 11,530 ng/g lw, respectively). Muscle was the organ with the highest PYR and UVF concentrations (p < 0.001), suggesting that these two classes of emerging contaminants may have more affinity for proteins than for lipids. The high PYR and UVF concentrations found in fetuses demonstrate these compounds are efficiently transferred through placenta. This study is the first to report maternal transfer of pyrethroids and UV filters in marine mammals.

Characterization of alpha-fetoprotein in fetal striped dolphin (Stenella coeruleoalba): purification of protein product and molecular cloning of the corresponding transcript.

Alpha-fetoprotein (AFP) is a fetal glycoprotein that is known as a biomarker for monitoring pregnancy in many mammalian species. However, characterization of AFP has not yet been undertaken in any cetacean species. Here, we purified AFP from the serum of fetal striped dolphin by chemical precipitation followed by a combination of immunoadsorbent column chromatography and gel filtration. The molecular masses of native and denatured dolphin AFP were estimated to be ∼78,000 Da by gel filtration and ∼68,000 Da by SDS-PAGE, respectively, representing typical masses reported for mammalian AFPs. In fetal serum, only the AFP band (∼68,000 Da) appeared to be immunoreactive to an antiserum against purified dolphin AFP, indicating sufficient specificity for the development of an AFP immunoassay. Full-length cDNA encoding for the dolphin AFP was cloned from fetal liver and revealed an open reading frame comprising 610 amino acid residues, which included a putative signal peptide of 18 amino acid residues. This was followed by a sequence identical to the N-terminus of purified AFP. The deduced amino acid sequence of dolphin AFP showed more than 80% identity to those of other mammalian AFPs. To our knowledge, the present report represents the first identification and characterization of AFP from any cetacean species.

First description of corpus luteum with central cavity during pregnancy in Sotaia guianensis (Van Bénéden, 1864) - Case report / Primeira descrição de corpo lúteo cavitário gestacional em Sotalia guianensis (Van Bénéden, 1864) - Relato de Caso

O objetivo foi descrever pela primeira vez o corpo lúteo cavitário gestacional em Sotalia guianensis, espécie de golfinhocosteiro que se distribui ao longo da costa Atlântica Ocidental. Os ovários de um espécime com 196cm de comprimentocorporal, com 9,2 meses de gestação, capturado acidentalmente durante atividade pesqueira na costa norte do estado doRio de Janeiro. O material foi avaliado morfologicamente e processado para cortes em parafina, corado pela hematoxilina eeosina para microscopia. O ovário direito apresentava forma amendoada, com sulcos em uma lateral, três folículos e umcorpo lúteo polar em involução. O ovário esquerdo pesava sete vezes mais que o direito. A superfície era enrugada, com umfolículo, seis corpos lúteos e quatro albicans. O maior corpo lúteo era protruso, com diâmetro médio de 3cm e uma cavidadecentral de 1cm de diâmetro, revestida por paredes regulares e espessas, sugerindo ser um corpo lúteo em desenvolvimento.Os outros corpos lúteos eram maciços e estavam em regressão. A microscopia do corpo lúteo não demonstrou envolvimentopatológico. Esta descrição, ímpar na espécie, representa uma contribuição ao conhecimento da biologia (reprodutiva) da S.guianensis, que consta como "Dados Deficientes" na lista vermelha da União Internacional para Conservação da Natureza.

This is the first study to describe the corpus luteum with central cavity during pregnancy in Sotalia guianensis, a coastal dolphin species distributed along the Atlantic Ocean coast. The material analyzed was a pair of ovaries of a 196-cm-long female was 9.2 months pregnant, accidentally captured on the north coast of the state of Rio de Janeiro, Brazil. Both ovaries were measured and inspected for anatomic structures. After, ovaries were process in paraffin-embedded and stained with hematoxylin and eosin for microscopy. The left ovary had a wrinkled outside, one follicle, six corpora lutea and four corpora albicans. The largest corpus luteum was protruded, 3 cm long in mean diameter with one central cavity. It was lined by uniform thick walls and peripheral vascularization, which suggests that the corpus luteum is under development. The left ovary was deformed due to the presence of corpus luteum, which accounted for the seven-fold higher weight as compared to the right ovary. The histology of the corpus luteum did not evince any pathologic status of the material. This description, the first ever made for the species, offers a contribution to the existing knowledge about the reproduction biology of S. guianensis, which is a member of the "Insufficient Data" Red List prepared by the World Conservation Union.

Volumetric neuroimaging of the atlantic white-sided dolphin (Lagenorhynchus acutus) brain from in situ magnetic resonance images.

The structure and development of the brain are extremely difficult to study in free-ranging marine mammals. Here, we report measurements of total white matter (WM), total gray matter (GM), cerebellum (WM and GM), hippocampus, and corpus callosum made from magnetic resonance (MR) images of fresh, postmortem brains of the Atlantic white-sided dolphin (Lagenorhynchus acutus) imaged in situ (i.e., the brain intact within the skull, with the head still attached to the body). WM:GM volume ratios of the entire brain increased from fetus to adult, illustrating the increase in myelination during ontogeny. The cerebellum (WM and GM combined) of subadult and adult dolphins ranged from 13.8 to 15.0% of total brain size, much larger than that of primates. The corpus callosum mid-sagittal area to brain mass ratios (CCA/BM) ranged from 0.088 to 0.137, smaller than in most mammals. Dolphin hippocampal volumes were smaller than those of carnivores, ungulates, and humans, consistent with previous qualitative results assessed from histological studies of the bottlenose dolphin brain. These quantitative measurements of white matter, gray matter, corpus callosum, and hippocampus are the first to be determined from MR images for any cetacean species. We establish here an approach for accurately determining the size of brain structures from in situ MR images of stranded, dead dolphins. This approach can be used not only for comparative and developmental studies of marine mammal brains but also for investigation of the potential impacts of natural and anthropogenic chemicals on neurodevelopment and neuroanatomy in exposed marine mammal populations.

Anatomía cardíaca de Pontoporia blainvillei / Cardiac anatomy of the Pontoporia blainvillei

El objetivo de este estudio fue describir la conformación interna y externa y la irrigación del corazón del delfín franciscana de la Plata (Pontoporia blainvillei) para lo cual fueron disecados 12 corazones. El pericardio se fijaba caudalmente al diafragma, ventralmente al músculo transverso torácico y, lateralmente, a las costillas y músculos intercostales. El corazón era aplastado dorsoventralmente, su base era craneal, su ápex era compartido por ambos ventrículos. El ventrículo izquierdo era de pared gruesa, mientras que la del ventrículo derecho más delgada y depresible. La cara ventral correspondía a la cara auricular y la cara dorsal a la cara atrial de la Nomina Anatomica Veterinaria, 2005 (NAV). En la conformación interior del atrio derecho destacaba el gran desarrollo de la cresta terminal y de la fosa oval. Dentro del ventrículo derecho, los músculos papilares se distribuían en subbarterioso, magnus y parvi. Una trabécula septomarginal se extendía entre los músculos papilares subarterioso y magnus. También fueron observadas trabéculas menores del mismo tipo, situadas más ventralmente a la anterior. Las venas pulmonares se unían en un tronco común antes de llegar al atrio izquierdo. El ventrículo izquierdo tenía dos músculos papilares bien desarrollados. La irrigación cardíaca arterial mostraba un predominio de la arteria coronaria izquierda y se notaba la presencia de anastomosis intercoronarias.

The aim of this study is to describe the external and internal conformation and the arterial irrigation of La Plata Dolphin's heart (Pontoporia blainvillei). Twelve animals obtained from nets of fishing ships were used and their hearts were studied by means of simple dissection. The pericardium was fixed caudally to the diaphragm, ventrally to the thoracic transverse muscle and laterally to the ribs and intercostal muscles. The heart weighted about 115 grams, was flattened dorsoventrally, its base was cranial, its apex was shared by both ventricles. The ventral face corresponds to the auricular face of the Nomina Anatomica Veterinaria (NAV), and the dorsal face corresponds to the atrial face. In the internal conformation of the right atrium the great development of the crista terminalis and the fossa ovalis stood out. Inside the right ventricle the papillaris muscles were observed distributed in subarteriosus, magnus and parvi. The dextra septomarginal trabecules were multiple and situated between the papillaris muscles. In the left ventricle they had two well developed papillaris muscles. The left coronary artery is the most important vessel in the arterial irrigation of the cardiac territory.

Developmental basis for hind-limb loss in dolphins and origin of the cetacean bodyplan.

Among mammals, modern cetaceans (whales, dolphins, and porpoises) are unusual in the absence of hind limbs. However, cetacean embryos do initiate hind-limb bud development. In dolphins, the bud arrests and degenerates around the fifth gestational week. Initial limb outgrowth in amniotes is maintained by two signaling centers, the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA). Our data indicate that the cetacean hind-limb bud forms an AER and that this structure expresses Fgf8 initially, but that neither the AER nor Fgf8 expression is maintained. Moreover, Sonic hedgehog (Shh), which mediates the signaling activity of the ZPA, is absent from the dolphin hind-limb bud. We find that failure to establish a ZPA is associated with the absence of Hand2, an upstream regulator of Shh. Interpreting our results in the context of both the cetacean fossil record and the known functions of Shh suggests that reduction of Shh expression may have occurred approximately 41 million years ago and led to the loss of distal limb elements. The total loss of Shh expression may account for the further loss of hind-limb elements that occurred near the origin of the modern suborders of cetaceans approximately 34 million years ago. Integration of paleontological and developmental data suggests that hind-limb size was reduced by gradually operating microevolutionary changes. Long after locomotor function was totally lost, modulation of developmental control genes eliminated most of the hind-limb skeleton. Hence, macroevolutionary changes in gene expression did not drive the initial reduction in hind-limb size.

The ontogenetic changes in the thermal properties of blubber from Atlantic bottlenose dolphin Tursiops truncatus.

In Atlantic bottlenose dolphins Tursiops truncatus, both the thickness and lipid content of blubber vary across ontogeny and across individuals of differing reproductive and nutritional status. This study investigates how these changes in blubber morphology and composition influence its thermal properties. Thermal conductivity (W m(-1) deg.(-1), where deg. is degrees C) and thermal insulation (m(2) deg. W(-1)) of dolphin blubber were measured in individuals across an ontogenetic series (fetus through adult, N=36), pregnant females (N=4) and emaciated animals (N=5). These thermal properties were determined by the simultaneous use of two common experimental approaches, the heat flux disc method and the standard material method. Thickness, lipid and water content were measured for each blubber sample. Thermal conductivity and insulation varied significantly across ontogeny. Blubber from fetuses through sub-adults was less conductive (range=0.11-0.13+/-0.02 W m(-1) deg.(-1)) than that of adults (mean=0.18 W m(-1) deg.(-1)). The conductivity of blubber from pregnant females was similar to non-adult categories, while that of emaciated animals was significantly higher (0.24 +/- 0.04 W m deg.(-1)) than all other categories. Blubber from sub-adults and pregnant females had the highest insulation values while fetuses and emaciated animals had the lowest. In nutritionally dependent life history categories, changes in blubber's thermal insulation were characterized by stable blubber quality (i.e. conductivity) and increased blubber quantity (i.e. thickness). In nutritionally independent animals, blubber quantity remained stable while blubber quality varied. A final, unexpected observation was that heat flux measurements at the deep blubber surface were significantly higher than that at the superficial surface, a pattern not observed in control materials. This apparent ability to absorb heat, coupled with blubber's fatty acid composition, suggest that dolphin integument may function as a phase change material.

Postural role of lateral axial muscles in developing bottlenose dolphins (Tursiops truncatus).

Foetal dolphins (Tursiops truncatus) are bent ventrolaterally, such that the tailflukes and lower jaw are juxtaposed. The lateral flexibility required en utero may compromise the efficiency of the dorsoventral oscillations required of the swimming neonate. The m. intertransversarius caudae dorsalis (IT) is the most laterally placed epaxial muscle. Bilateral contractions of the IT could limit lateral deformations of the flexible tailstock of the early neonate. We test the hypothesis that the IT is functioning as a postural muscle in neonates by examining its morphological, histological and biochemical properties. The neonatal IT has a relatively large cross-sectional area and bending moment, as well as a large proportion of slow-twitch fibres and elevated myoglobin concentrations. Our results demonstrate that the IT is functionally capable of performing this specific postural function in neonatal dolphins. In later life-history stages, when postural control is no longer needed, the IT serves to fine-tune the position of the tailstock during locomotion. The changing function of the adult IT is concomitant with changes in morphology and biochemistry, and most notably, with an increase in the proportion of fast-twitch fibres. We suggest that these changes reflect strong selective pressure to improve locomotor abilities by limiting lateral deformations during this critical life-history stage.

Heart development in the spotted dolphin (Stenella attenuata).

Marine mammals show many deviations from typical mammalian characteristics due to their high degree of specialization to the aquatic environment. In Cetaceans, some of the features of limbs and dentition resemble very ancestral patterns. In some species, hearts with a clearly bifid apex (a feature normally present during mammalian embryogenesis prior to completion of ventricular septation) have been described. However, there is a scant amount of data regarding heart development in Cetaceans, and it is not clear whether the bifid apex is the rule or the exception. We examined samples from a unique collection of embryonic dolphin specimens macroscopically and histologically to learn more about normal cardiac development in the spotted dolphin. It was found that during the dolphin's 280 days of gestation, the heart completes septation at about 35 days. However, substantial trabecular compaction, which normally occurs in chicks, mice, and humans at around that time period, was delayed until day 60, when coronary circulation became established. At that time, the apex still appeared bifid, similarly to early fetal mouse or rat hearts. By day 80, however, the heart gained a compacted, characteristic shape, with a single apex. It thus appears that the bifid apex in the adult Cetacean heart is probably particular to certain species, and its significance remains unclear.

The lateral enamel lamina--component of tooth primordia in selected mammalian species.

The lateral enamel lamina (LEL) is a part of the enamel organ, which is probably not involved in tooth formation. It represents, besides the "stalk" of the tooth primordium, a second interconnection between enamel organ and oral epithelium or vestibular lamina. We detected the LEL in the sheep (Ovis aries), the dolphin (Stenella attenuata), and the vole (Microtus agrestis) by light microscopy and computer-aided three-dimensional reconstruction. The LEL could be found in cap to bell stage tooth primordia, most clearly in slowly developing tooth germs. LEL-like structures have been furthermore described or depicted in tooth germs of the mouse, the elk (Alces alces), the dugong (Dugong dugong), the elephant (Loxodonta africana), and the human. Probably it is a part of all mammalian tooth primordia that undergoes regression during morphogenesis of the enamel organ. As a reducing structure, it should be considered in studies of tooth development.

Time, pattern, and heterochrony: a study of hyperphalangy in the dolphin embryo flipper.

The forelimb of whales and dolphins is a flipper that shows hyperphalangy (numerous finger bones). Hyperphalangy is also present in marine reptiles, including ichthyosaurs and plesiosaurs. The developmental basis of hyper-phalangy is unclear. Kükenthal suggested that phalanx anlagen split into three pieces during cetacean development, thereby multiplying the ancestral number. Alternatively, Holder suggested that apical ectodermal ridge (AER)-directed limb outgrowth might be prolonged by a timing shift (heterochrony), leading to terminal addition of extra phalanges. We prepared a series of whole mounted and serially sectioned embryonic flipper buds of the spotted dolphin Stenella attenuata. This cetacean shows marked hyperphalangy on digits II and III. We confirm previous reports that the proximodistal laying down of phalanges is prolonged in digits II and III. Histology showed that the apical ectoderm was thickened into a cap. There was a weak ridge-like structure in some embryos. The cap or ridge formed part of a bud-like mass that persisted on digits II and III at stages when it had disappeared from other digits. Thus the dolphin differs from other mammals in showing a second period of limb outgrowth during which localized hyperphalangy develops. New phalanges only formed at the tip of the digits. These findings are consistent with a model in which heterochrony leads to the terminal addition of new phalanges. Our results are more easily reconciled with the progress zone model than one in which the AER is involved in the expansion of a prepattern. We suggest that patterning mechanisms with a temporal component (i.e., the "progress zone" mechanism) are potential targets for heterochrony during limb evolution.

Rotation of middle ear ossicles during cetacean development.

Cetacean middle ears are unique among mammals in that they have an elongated tympanic membrane, a greatly reduced manubrium mallei, and an incudal crus longum that is shorter than the crus breve. Elongation of the tympanic membrane and reduction of the manubrium is thought to be related to an evolutionary rotation of the incus and malleus out of the plane of the tympanic membrane. We examined if rotation also occurs during ontogeny by comparing the middle ears of two species of dolphins (Delphinus delphis, Stenella attenuata) at different stages of development. We observed that: the incus has the body and crural proportions as in terrestrial mammals early in development; the incudomallear complex rotates approximately 90 degrees following ossification; the tympanic membrane is not elongated until relatively late in development. Therefore, some of the unique characteristics of the cetacean middle ear develop as modifications of an initially terrestrial-like morphology.

Magnetic resonance imaging and three-dimensional reconstructions of the brain of a fetal common dolphin, Delphinus delphis.

To demonstrate the kinds of data that can be obtained non-destructively and non-invasively from preserved museum specimens using modern imaging technology the head region of a whole body fetal specimen of the common dolphin, Delphinus delphis, aged 8-9 months post-conception, was scanned using Magnetic Resonance Imaging (MRI). Series of scans were obtained in coronal, sagittal and horizontal planes. A digital three-dimensional reconstruction of the whole brain was prepared from the coronal series of scans. Sectional areas and three-dimensional volumes were obtained of the cerebral hemispheres and of the brainstem-plus-cerebellum. Neuroanatomical features identified in the scans include the major sulci of the cerebral hemispheres, well-differentiated regions of gray and white matter, the mesencephalic, pontine, and cervical flexures, the "foreshortened" appearance of the forebrain, and the large auditory inferior colliculi. These findings show that numerous features of the fetal common dolphin brain can be visualized and analyzed from MRI scans.

Embryology of dolphins. Staging and ageing of embryos and fetuses of some cetaceans.

Comparative embryology has been neglected in the past decades. In our opinion the decreased interest has been caused by the fact that the time factor has not sufficiently been respected. Therefore we used the Staging and Ageing Method (Sterba 1999) to determine the ontogenetic age of embryos and to state the time tables and time relations of various ontogenetic events. We examined 167 embryos and fetuses of four dolphin species (collection Dr. Senckenbergische Anatomie, Frankfurt am Main). The study of intrauterine development and growth while consistently respecting the time factor enabled us to determine the time course of organogenesis of all organ systems. The resulting comparison with other mammals detected the time lag in ossification, retardation of odontogeny, the time of the origin and development of the fluke, dorsal fin and flipper, etc. We believe the Staging and Ageing Method serves for a much more efficient evaluation of existing embryological collections which very often contain embryos of unknown age.

On the development of Cetacean extremities: II. Morphogenesis and histogenesis of the flippers in the spotted dolphin (Stenella attenuata).

Externally, the flippers of Cetacea resemble fish fins, but their internal structure is entirely mammalian. They show, however, some adaptative deviations from the typical pattern of the mammalian extremities, the most striking of which is an increased number of phalanges. The aim of this study is to describe the course of the development of flippers in the spotted dolphin (Stenella attenuata) and compare its features with other similar species from an evolutionary perspective. Early stages of flipper development were studied histologically. Differentiation of cartilaginous anlagens of the skeleton progresses proximodistally, condensation in digital rays being evident sooner than chondrogenesis in the carpal region. In one specimen, the temporary presence of cartilaginous rudiments of two carpal elements, which are not found in adults, was observed. At all examined stages, phalangeal number progressively increases up to (radial to ulnar) 3, 7, 7, 5, 3 in the most advanced stage. The reason for this condition is the specialised function of these limb-like structures. It is a classical example of convergence, in which mammalian extremities change their form to emulate the fin function. A similar condition is found in another group of originally terrestrial animals secondarily fully adapted to the aquatic mode of life-Ichyosauria (Reptilia).

On the development of Cetacean extremities: I. Hind limb rudimentation in the Spotted dolphin (Stenella attenuata).

The Cetacea are group of animals which have completely lost their hind limbs during the course of evolution as a result of their entirely aquatic mode of life. It is known, however, that during their embryonal period, the hind limb buds are temporarily present. The control mechanisms of this regression are not yet understood, and vestigial limbs can sometimes be found in adults. The aim of the present study is to describe the course of hind limb rudimentation during prenatal development of Stenella attenuata (Spotted dolphin) at tissue and cell levels and compare the results with other natural or experimentally induced amelias. Hind limb buds of dolphin embryos, CRL 10-30 mm, were examined histologically. Before total disappearance, they show histodifferentiation comparable with other mammals. Initially, they form the apical ectodermal ridge, which soon regresses. The mesenchyme undergoes the process of condensation to form anlagens of prospective skeletal elements. These condensations are surrounded by vascular plexuses. During the course of rudimentation, some mesenchymal cells die, while the others are incorporated into the body wall. Nerve ingrowth into rudimentary limb buds was also detected. The temporary presence of hind limb rudiments in cetacean embryos can be regarded as a good example of recapitulation of phylogenesis in ontogenesis.

Prenatal development of the integument in Delphinidae (Cetacea: Odontoceti).

The prenatal development of epidermis, dermis, and hypodermis was studied in embryos of different age of two delphinid species (Stenella attenuata, Delphinus delphis), using light and transmission electron microscopical methods. The delphinid embryo is covered by a multilayered tissue formed by four different epidermal generations (periderm, stratum intermedium-I, str. intermedium-II, str. spinosum) produced by the str. basale. The first layer appears at about 40-50 mm of body length, the second type (s.i.-I) about 60-160 mm, and the third type (s.i.-II) is present at 160-500 mm. The first spinosal cells are produced at 225-260 mm body length; thenceforth, the epidermis increases continuously in thickness. Epidermal ridge formation begins about 400-mm body length. The development of the dermis is characterized by the early production of thin connective tissue fibers (40-70-mm body length) and simultaneously the cutaneous muscle matures in structure. Vascular development intensifies between embryos of 150-225 mm, and collagen production increases markedly in fetuses of 225-260-mm length. These events are paralleled by an increase in dermal thickness. The first elastic fibers can be recognized in the skin from the abdomen at about 600-mm body length. The development of the hypodermis is marked by very rapid and constantly progressing growth, beginning about 60-mm body length. The first typical fat cells appear in animals of 360-400 mm. Regional differences are obvious for all skin layers with regard to the flippers, where structural maturation proceeds more rapidly than in dorsal or abdominal regions.