RESEARCH ARTICLE

Prenatal Development of Crocodylus niloticus niloticus Laurenti, 1768 MIROSLAV PETERKA1,2, JEAN YVES SIRE3 MARIA HOVORAKOVA1, JAN PROCHAZKA1,4, LUC FOUGEIROL5 RENATA PETERKOVA1, AND LAURENT VIRIOT6 1

Department of Teratology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic 2 Department of Anthropology, Faculty of Science, Charles University, Prague, Czech Republic 3 UMR 7138, Universite´ Pierre et Marie Curie-Paris, Paris, France 4 Department of Developmental Biology, Faculty of Science, Charles University, Prague, Czech Republic 5 La Ferme aux Crocodiles, Les Blachettes, Pierrelatte, France 6 Institut de Ge´nomique Fonctionnelle de Lyon, UMR 5242 CNRS INRA UCBL ENS, Ecole Normale Supe´rieure de Lyon, Lyon, France

ABSTRACT

J. Exp. Zool. (Mol. Dev. Evol.) 314B, 2010

Prenatal development in crocodilians represents a very interesting model for comparative studies. As the speed of prenatal development of crocodilians varies depending on incubation conditions, the staging of embryos and fetuses is a very important prerequisite for data correlation. To establish a background for future developmental studies on Crocodylus niloticus, we characterized its prenatal development in a collection comprising 169 animals during embryonic/incubation days 9–70. The characteristics included external morphology, head morphometry, and wet body weight determined before fixation. We documented the external morphology of prenatal Nile crocodiles in a large collection of photographs and described landmarks during the morphogenesis of the head, face and limbs. In the development of the facial processes (medial nasal, lateral nasal, maxillary), three phases could be distinguished: union, separation, reunion. At the free jaw margin, a regular series of prominences was present. The outer aspect of a prominence gave rise to a labial scale, the inner aspect to a tooth. In contrast to mammals (humans and mice), the hindlimbs of C. niloticus developed faster than the forelimbs. We also determined changes in basic measures of the head and of the wet body weight. Both morphological and morphometric characteristics showed an apparent inter-individual variability among animals of the same age. This variability decreased among animals of a similar body weight (irrespective of their age). Body weight can be considered as the most representative and complex parameter for crocodile staging reflecting the overall growth of a whole embryo/fetus. J. Exp. Zool. (Mol. Dev. Evol.) 314B, 2010. & 2010 Wiley-Liss, Inc. How to cite this article: Peterka M, Yves Sire J, Hovorakova M, Prochazka J, Fougeirol L, Peterkova R, Viriot L. 2010. Prenatal development of Crocodylus niloticus niloticus Laurenti, 1768. J. Exp. Zool. (Mol. Dev. Evol.) 314B:[page range].

Crocodile prenatal development is highly dependent on environmental parameters. The most important conditions are the incubation temperature, humidity, the type of substrate, the amount of contact between the eggs and the substrate, and gas exchange (Miller, ’85). In nature, the optimal temperature and other conditions are accomplished by the deposition of eggs into

Grant Sponsor: GACR; Grant number: 304/07/0223; Grant Sponsor: Ministry of Education, Youth and Sports of the Czech Republic; Grant number: MSM0021620843. Correspondence to: Miroslav Peterka, Department of Teratology, Institute of Experimental Medicine, Academy of Sciences CR, Videnska 1083, 142 20 Prague 4, Czech Republic. E-mail: [email protected] Received 21 July 2009; Revised 23 November 2009; Accepted 24 November 2009 Published online in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/jez.b.21335

& 2010 WILEY-LISS, INC.

2 nests made from plant detritus or other suitable substrates at suitable locations. In crocodilians, incubation conditions (temperature, humidity, gaseous environment) play an important role in determining developmental speed (for review see Deeming and Ferguson, ’90). A higher temperature accelerates prenatal growth and development. Consequently, the prehatching period is much shorter—e.g., 85 days at 341C compared with 110 days at 281C (Hutton, ’87; Webb and Cooper-Preston, ’89; Whitehead et al., ’90). Various incubation temperatures also lead to differences in the pattern of skin coloration (Murray et al., ’90). Experiments performed by Deeming and Ferguson (’89) in Alligator mississippiensis showed that hatchlings from eggs incubated at 331C had darker black stripes than hatchlings incubated at 301C. Temperature-dependent sex determination has been observed in all crocodilian groups (Deeming and Ferguson, ’89; Murray et al., ’90; Whitehead et al., ’90). Bardsley et al. (’95) tried to find mathematical models for the growth of A. mississippiensis embryos developing at 301C (female producing) and 331C (male producing). Most of the longitudinal measures and embryonic stages increased by a timescaling factor of 1.2 from 30 to 331C. This suggests that a common temperature dependent rate-limiting step controls all the growth features in the alligator. Prenatal development in crocodilians represents a very interesting model for comparative embryological studies. Such studies could help to elucidate the developmental mechanisms implicated in the reptile–mammal transition during evolution. In the field of craniofacial research, the A. mississippiensis has been presented as a very convenient developmental model (Ferguson, ’81). As the speed of development of crocodilians is very sensitive to developmental conditions, mainly temperature, the staging of embryos and fetuses is a very important prerequisite for correlating data between different developmental studies. A detailed morphological description of the prehatching developmental stages is available in A. mississippiensis (Parker, 1883; Clarke, 1891; Resse, ’08; Ferguson, ’85) and correlated with standard incubation conditions in an artificial incubator (26, 28, 30, 32, 34, or 361C; 100% humidity) (Ferguson and Joanen, ’82). The basic morphological and morphometrical aspects have been described during the prenatal development of Crocodylus porosus and compared between two incubation temperatures (27.5 and 29.51C, 30.5 and 31.51C); the embryos and fetuses were harvested from incubation day 2 to 86 (Magnusson and Taylor, ’80). A comparison of prenatal development has been made on the basis of morphometric data correlated with morphological stages in A. mississippiensis, C. porosus, and C. johnsoni (Deeming and Ferguson, ’90). Iungman et al. (2008) calibrated the developmental series of the broad-snouted caiman, Caiman latirostris, against an established series for A. mississippiensis. For C. niloticus, detailed drawings and a description of the embryonic and fetal stages are available (Voeltzkow, ’02). However, Voeltzkow (’02) harvested the eggs from wild nests and incubated J. Exp. Zool. (Mol. Dev. Evol.)

PETERKA ET AL. them in boxes with sand in his private house without an incubator. Therefore, detailed information is not available about the incubation conditions. We aimed to establish a background for future developmental studies on C. niloticus: we documented and characterized the prenatal development of the Nile crocodile on the basis of external morphological criteria and head measures. These parameters were correlated with the day of incubation and a wet body weight determined before fixation.

MATERIAL AND METHODS The collection comprised 169 embryos and fetuses of the C. niloticus (Table 1) harvested in the Pierrelatte farm in the south of France during the years 1999–2001. From April to July, female crocodiles were laying from 7 to 34 eggs (average 25 eggs), which were buried in the sand. The eggs were collected a few hours after laying, and put into a box with wet vermiculite, which was then placed into an incubator. Only one incubator was used. The temperature was monitored by a thermometer in the incubator, and was maintained at 321C during the whole period of incubation. In this crocodile species, natural incubation period takes from 85 to 95 days. The harvesting of embryos and fetuses was performed daily on incubation days 7–70. Immediately after harvesting, the wet body weight was determined to characterize the biological stage during prenatal development (Peterka et al., 2002). The body weight in milligrams (mg) was evaluated in all 169 embryos/fetuses harvested at embryonic day (ED) 7–70 (Table 1). The values were plotted in a graph to document the dependence between the body weight and the age of the animals (Fig. 1). Then the specimens were placed in a fixative: Bouin–Hollande solution, 70% ethanol, or 4% paraformaldehyde. They were randomly selected for different fixative solutions. The external morphology and head morphometry were only investigated in the group of Bouin–Hollande-fixed animals (these animals are labeled gray in Table 1). The rest of the animals were used for other purposes. The 74 Bouin–Hollande-fixed specimens were photographed using a Leica MZ6 stereo-loupe connected to a Leica DC480 camera (Leica Microsystems BmbH, Wetzlar, Germany). Head morphometry was performed in 62 animals (12 animals could not be measured because of artificial damage or deformation of the head). The Leica stereo-loupe was used equipped with an ocular micrometer (all distances were recalculated to millimeters). We measured: the total length of the head, the lengths of the maxilla and mandible, the distance between the snout tip and the middle of the eye, the maximum width of the head, and the width of the snout. These measures were plotted in graphs in dependence on the day of incubation or the body weight of the measured crocodiles. Data were statistically evaluated using correlation

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Table 1. Wet body weight (in mg) of crocodile embryos and fetuses harvested at embryonic day 7–70, incubation temperature 32oC. Days Weight Days Weight Days Weight Days Weight 7 8 8 9 9 9 9 10 10 10 10 10 10 11 11 12 12 13 13 13 13 14 14 15 15 16 16 17 17 17 18 18 18 18 19 19 19 19 19 19 20 20 20

27 12 32 22 40 104 111 78 132 132 138 143 167 149 168 162 240 133 286 137 210 357 390 300 456 429 538 489 511 680 463 621 699 703 411 555 575 580 605 725 668 669 785

20 20 20 21 21 22 22 22 22 23 23 23 23 24 24 24 24 24 24 24 24 24 24 24 24 24 25 25 25 25 25 25 25 25 25 25 25 25 26 26 26 26 27

814 1,085 1,222 965 1,335 1,020 1,020 1,422 1,467 940 1,079 1,662 1,722 628 628 905 1,061 1,195 1,287 1,314 1,363 1,413 1,413 1,564 1,596 1,993 1,021 1,021 1,199 1,489 1,680 1,803 1,872 1,994 1,994 2,074 2,075 2,009 1,569 1,660 1,876 2,032 1,662

27 27 27 28 28 28 28 28 28 28 28 29 29 29 30 30 30 30 30 30 30 31 31 31 31 32 32 32 32 32 33 33 33 34 34 35 35 35 35 35 36 36 37

1,687 2,202 2,470 1,456 1,456 2,227 2,588 2,588 2,714 2,853 3,182 2,793 3,009 3,072 483 1,656 2,398 2,398 3,241 3,448 3,354 1,551 2,545 3,115 3,683 2,967 3,243 4,222 4,222 4,571 2,838 3,297 3,696 3,570 5,047 2,800 3,637 3,659 4,162 4,228 5,709 5,801 3,528

37 37 38 38 38 39 39 39 40 40 40 40 40 41 41 43 45 45 45 45 45 45 50 50 50 52 52 53 53 53 53 53 55 55 55 66 66 70 70 70

5,158 7,169 3,073 5,513 5,533 3,565 7,227 7,600 3,990 5,793 6,620 6,691 8,350 7,373 7,580 11,362 9,009 11,318 11,899 12,690 12,690 12,820 13,834 17,072 22,049 12,487 12,487 20,703 20,957 22,329 22,329 24,367 23,951 23,718 24,853 27,525 27,823 34,471 37,255 39,325

Gray area indicates the specimens morphologically and morphometrically analyzed in this study.

coefficients. The correlation coefficients were compared according to Williams (’59). Basic external morphological characteristics were monitored in all Bouin–Hollande-fixed specimens. Because of the variability between developmental stages at each ED (see below), a representative animal was selected at each available ED to form a developmental series with progressively increasing age (ED) and body weight (mg). Table 2 presents the developmental events that have been first observed in this series of representative specimens. The head of a fetus (ED 53) was routinely embedded in paraffin, and sections (7 um) were made in a plane parallel to the lateral face of the right maxilla. The sections were colored by the Azan method (Heidenhein‘s aniline blue staining, Mallory ’42), which stains enamel orange (Orange G) and dentin blue (Aniline Blue).

RESULTS Body Weight of Embryos and Fetuses The lowest body weight was found in the youngest available specimens: one embryo at ED 7 (body weight 27 mg) and two embryos at ED 8 (12 and 32 mg). The oldest fetus at ED 70 showed the maximum body weight 39,325 mg (Table 1). The body weight increase in the prehatching crocodiles was expressed by an exponential curve during ED 7–70. A mild increase in weight was seen ED 35, followed by a rapid progressive increase in body weight during the rest of the incubation period (Table 1, Fig. 1). Before ED 20, there was relatively little variability in body weight among embryos harvested on a specific day of incubation. However, this variability increased with the increasing age of the crocodiles, being accompanied by an apparent variability in body size and differentiation of their external morphology. This implies that specimens of the same chronological age (harvested after the same period of incubation) could show marked differences in their external features (Fig. 2). The extent of morphogenetic variation increased with the age of the animals. In general, the external morphodifferentiation correlated much better with the body weight of the crocodiles than with their chronological age (Fig. 2). For example, the head dimensions during the incubation period correlated much better with the body weight of the animals (irrespective of their age), than with the chronological age of the animals (irrespective of their body weight). All the above data documented the importance of body weight as a parameter characterizing the developmental stage of crocodile embryos/fetuses. For this reason, both the incubation day (ED) and the body weight (mg) of crocodile embryos/fetuses will be specified in the following text. External Morphology The external morphology was investigated in a sample of crocodiles fixed in a Bouin–Hollande fluid (Table 1). In the youngest investigated embryo at ED 9 (22 mg), the head, trunk, limb-buds, and tail were already distinct (Fig. 3). The eye placodes and nasal J. Exp. Zool. (Mol. Dev. Evol.)

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Figure 1. A graph showing the prenatal body weight of crocodile embryos and fetuses. The exponential curve shows the wet body weight of crocodiles at embryonic day 7–56. Because the weights of the fetuses older than ED 56 were too high, they have not been included in the graph.

Table 2. Basic external morphological characteristics in a series of crocodile embryos/fetuses ranked according to their increasing age (ED) and body weight (mg). ED/mg 9/22 10/132 13/133 14/357 16/429 17/489 20/668 21/1335 22/1467 24/1596 27/2470 34/5047

External morphological characteristics Nasal pits, facial outgrowths (medial nasal, lateral nasal, maxillary, mandibulary), eye placodes, prominent midbrain, forelimb and hindlimb buds The facial outgrowths became more prominent, limb buds elongated Medial and lateral nasal outgrowths are not yet fused with maxillary outgrowths The fusion of medial nasal and maxillary outgrowths starts, the anterior part of the fore and hindlimbs became paddleshaped The mandible growths forward, an elbow is not yet visible The formation of the upper jaw arch further progresses, the mandible further growths forward and its anterior limit approaches that one of the upper jaw, the elbow is visible in the forelimb The eye is still round, finger rays appear in both fore and hindlimbs Typical snout shape with a prominence on its tip, the eye shape becomes oval, knee-joint, finger rays and the membranes between them are apparent Scale anlagen are well distinct on the back, the anlage of supratympanic scale is prominent The prominence on the snout starts to be spiky, inter-finger membranes present Separate fingers on the forelimbs, phalanx of the fingers start to be distinct, claws anlagen appear The prominence on the snout gets flat From this stage, the basic external morphological characteristics are determined and the scales progressively differentiate

pits, as well as the ear anlagen, were clearly detectable. The embossment on the top of the head was formed by the prominent midbrain. During further development, the tail markedly elongated and curled (Fig. 3), and the limbs (Figs. 3 and 4) as well as the head and face (Figs. 5–8) progressively differentiated (see also Table 2).

observation—at ED 9 (22 mg) (Fig. 3). The limb-buds grew and elongated during further development, and the limbs progressively differentiated (Figs. 3 and 4).

Forelimbs and HindLimbs. The buds of the forelimbs and hindlimbs were already visible in the youngest embryo under

At ED 17 (489 mg), the prospective elbow and knee started to be

J. Exp. Zool. (Mol. Dev. Evol.)

In an embryo at ED 14 (357 mg), the length of the limb primordia increased and a paddle shaped autopodia became visible. distinct (Fig. 3) and became clearly apparent from ED 21 (Fig. 4).

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Figure 2. An example of a difference between embryos of the same age. Although both embryos were harvested at embryonic day (ED 20), they show an apparent difference in size, body weight, and morphodifferentiation. (A) An embryo harvested at ED20, body weight 668 mg. (B) An embryo harvested at ED20, body weight 1222 mg. Note, for example, the larger head and limbs and their more advanced differentiation in the heavier embryo. During early head development, the mandible growth is retarded compared with the upper jaw. This situation is still maintained in the developmentally less advanced embryo (A). At the later stage, the mandible growth deficit is no longer apparent (B). Bar—5mm.

Five or four fingers developed in a forelimb or hindlimb, respectively. The differentiation of the finger rays appeared to be more advanced in the hindlimbs (see Fig. 4, the fetus at ED 20 and ED 22). The fingers were interconnected by a membrane, which later disappeared in the forelimbs (about at ED 27), but remained preserved in the hindlimbs (see ED 53, Fig. 4). The phalanges started to be apparent at ED 27 and the distal phalanx widened. During further development, the nails differentiated. The nail-anlages were present on the first, second, and third fingers on each forelimb and hindlimb from ED 31 (Fig. 4). In the oldest fetus under observation (ED 53), five fingers were well differentiated on the forelimbs, with nails on the first, second, and third fingers. The hindlimb only had four fingers, the first, second, and third fingers had nails. Development of the Head and Face. The midbrain area was the most prominent on the embryonic head until the end of the first month.

In the face, three phases could be distinguished during the development of the medial nasal, lateral nasal, and maxillary facial outgrowths: (1) Initially, (Fig. 5, ED 9/22 mg), the primordia of the nasal and maxillary outgrowths on each right or left side of the face were interconnected, forming a common structure; (2) From this common base, the medial nasal, lateral nasal, and maxillary outgrowths bulged, being separated by a narrow groove (Fig. 5, ED 10/143 mg); (3) Subsequently, the groove between the right and left medial nasal outgrowths disappeared; the anterior limit of the maxillary outgrowth merged with the medial and lateral facial processes so that the margins between them were finally no longer visible. The interconnection of the right and left medial nasal and maxillary processes gave rise to a continuous upper jaw arch (Fig. 5, ED 17/489 mg). The mandible facial outgrowths were in contact from the beginning of the period of observation. A furrow indicated the J. Exp. Zool. (Mol. Dev. Evol.)

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Figure 3. The development of the forelimbs and hindlimbs in crocodile embryos from embryonic day (ED) 9 to 18. The age in embryonic day (ED) and wet body weight (in mg) are specified at each documented stage. Bar—5 mm.

J. Exp. Zool. (Mol. Dev. Evol.)

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Figure 4. The development of the forelimbs and hindlimbs in crocodile embryos and fetuses from embryonic day (ED) 20 to 53. The age in embryonic days (ED) and wet body weight (in mg) are specified at each documented stage. Bar—5 mm.

contact site on the external surface of the lower jaw arch (Fig. 5, ED 10–17). At ED 20/668 mg, the upper and lower jaws were well formed and started to elongate (Fig. 6). From ED 21, the typical crocodile snout shape started to be apparent due to the increasing snout length (Fig. 6).

The eyes and ears also differentiated progressively. At ED 21/1,335 mg, the eye shape changed from almost round to oval, which was associated with upper and lower eyelid formation (Fig. 6). The Supralabial Scales and Tooth Positions. On the adjacent upper and lower jaw margins, a series of regular protuberances were clearly visible from ED 40/6,000 mg. These protuberances J. Exp. Zool. (Mol. Dev. Evol.)

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Figure 5. Development of the facial outgrowths (lateral nasal (LN), medial nasal (MN), maxillary (MX), and mandibular (MD)) during ED 9–17.

became progressively more pronounced, being separated by furrows. In the developmentally most advanced fetus available for morphological evaluation (ED 53/22329 mg, compare Table 1), the upper jaw showed 5114 (19) and the lower jaw 15 protuberances (Fig. 8). When viewed from the oral aspect, a protuberance had two parts (Fig. 8B and D). The outer (lateral) part corresponded to a supralabial scale, which was easily visible J. Exp. Zool. (Mol. Dev. Evol.)

from the external aspect (Fig. 8A). Histological sections documented that the internal part of a protuberance included a developing tooth at a specific tooth position (Fig. 8E). The Head Size and Body Weight or Day of Incubation In order to search for a simple parameter characterizing the developmental stage, we performed measurement on the heads of

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Figure 6. Head development in crocodile embryos and fetuses from embryonic day (ED) 19 to 37. The age in embryonic days (ED) and wet body weight (in mg) are specified at each documented stage. Bar—5 mm.

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PETERKA ET AL. 62 crocodiles harvested at different stages of prenatal development (Table 3). We measured the total length of the head, length of the maxilla and mandible, the distance between the snout tip and the middle of the eye, the maximum width of the head and the width of the eye (Fig. 9). Among these measures, the maximum antero-posterior length of the head and the distance between the snout tip and the jaw joint exhibited the lowest variability (Fig. 10). All of the measures increased in dependence on both the day of incubation and the body weight of the crocodiles during the prenatal period. The dependence of head measures on chronological age was characterized by a linear curve. The distribution of head measures in dependence on body weight was reminiscent of a part of a growth curve (with the absence of the initial lowest values corresponding to the embryos younger than ED 9). Correlation coefficients were calculated in all measured parameters (A—the maximum head length; B—width of the eye; C—distance between the middle of the eye and the snout tip; D—the upper jaw length; E—the lower jaw length; F—distance between eyes) showed a better correlation with body weight (correlation coefficient 0.99, 0.84, 0.98, 0.99, 0.99, and 0.98 respectively) than with the day of incubation (0.97, 0.78, 0.97, 0.97, 0.97, and 0.92, respectively). Differences between the correlation coefficients were significant (Po0.01) in the distances A, B, D, E, and F. Only the difference in the dimension C was not statistically significant (P40.05).

DISCUSSION

Figure 7. Crocodilian cephalic scales at ED 52 (12,487 mg). The scales according to the terminology of Deraniygala (1939): rostral (1), nasal (2), canthals (3), frontal (4), frontoparietal (5), temporal (6), parietal (7), occipitals (8), postoccipitals (9), infratympanics (10), supratympanic (11), supralabials (12), supraocculars (13), ocular (14), supraciliaries (15), infraciliaries (16).

J. Exp. Zool. (Mol. Dev. Evol.)

We documented the external morphology of a series of embryos and fetuses of the Nile crocodile, and described landmarks during the morphogenesis of the head, face, and limbs. We determined the changes in the basic morphometric parameters of the head and the wet body weight during the prenatal development of crocodile embryos and fetuses. Both morphological and morphometric characteristics showed an apparent inter-individual variability among animals of the same age (e.g., Fig. 2). Under natural (open-air) conditions, the incubation temperature of crocodiles alternates during the day and night (the difference can be about 101C), and it also depends on the position of an egg in the nest. Therefore, the hatching time of crocodiles in one nest can differ in 25 days (Hutton, ’87). However, standardized conditions of artificial incubation could also not prevent a high inter-individual variability in developmental progress between embryos/fetuses of the same chronological age. The present data documents a great variability within the group of embryos at each ED in terms of their overall development (body weight, size, and morphodifferentiation). However, the variability in developmental progress between individuals of the same age concerns not only poikilotherm animals. It also exists between mammalian embryos/fetuses of the same age, even among littermates (Peterka et al., 2002). Altogether, these data suggest that chronological age alone (the number of EDs) is not a

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Figure 8. Crocodilian upper and lower jaws at ED 53 (22329 mg). A series of elevations are apparent at the free margin of the upper (A,B) and lower (C,D) jaws corresponding to supralabial scales from the external aspect (A). Histological section of the upper jaw (E) documents that each elevation also corresponds to one tooth position.

sufficiently representative indicator of the developmental stage. For this reason, size measurements are often used as an additional parameter characterizing the developmental stage (e.g., the crown-rump or femur length in humans). In A. mississippiensis, Deeming and Ferguson (’90) have recommended using various morphometric parameters (e.g., head measurements, the length of the body, tail, and forelimb) to characterize the stage of prenatal development. In the present sample of Nile crocodiles, variability in the head size parameters remained relatively high among animals of the same age. Importantly, however, it decreased among the embryos with a similar body weight, irrespective of their age (Fig. 9). Majority of head measures (Table 3) showed significantly better correlation with body weight of animals than with their chronological age (day of incubation). From this aspect, body weight seems to be the most representative and complex parameter reflecting the overall growth of a whole embryo/fetus. We have already gained a considerable experience in employing embryonic body weight to specify the chronological stage (in ED)

of mouse embryos. Large intra- and inter-litter variations in morphological stages exist among mouse embryos of the same strain and similar age (chronological stage). However, we found that the stage of tooth development (tooth morpho-histodifferentiation) correlates much better with age/weight staging than with only age staging (Peterka et al., 2002). An interesting aspect concerns the development of the facial processes in the crocodile. The facial processes (median nasal, lateral nasal, maxillary, and mandibular) appear on the embryonic face separated by narrow spaces. After their fusion, the unit structure of the upper or lower jaw arch, the primary palate, and the nose are formed. The development of these facial processes has been described in detail in A. mississippiensis by Ferguson (’81): Twelve days after egg laying, the characteristic facial processes are evident in the alligator embryo. In the crocodile, the facial processes were clearly distinct on ED 10/143 mg, being separated by narrow spaces (Fig. 5). However, just before, on ED 9/22mg, the facial processes were not yet separated; their distinct primordia were J. Exp. Zool. (Mol. Dev. Evol.)

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Table 3. Head dimensions (mm) in the Nile crocodile during prenatal period. Days 9 9 10 10 10 11 14 15 16 17 17 18 18 19 19 19 20 20 20 20 21 22 22 23 23 24 24 24 25 25 25 25 26 27 27 28 28 29 30 30 31 31 31 32 32 32 33 34

Weight

A

B

C

D

E

F

22 40 132 138 143 168 357 300 429 489 680 463 621 555 575 605 668 669 785 1,222 1,335 1,020 1,467 940 1,662 1,413 1,596 1,993 1,021 1,803 1,994 2,075 1,876 2,202 2,470 1,456 2,588 3,009 2,398 3,354 1,551 3,115 3,683 2,967 4,222 4,571 3,696 5,047

3.4 2.9 4.8 4.5 4.6 4.9 6.2 6.9 7.1 7.3 8.2 7.4 8.3 7.8 8.1 8.3 8.7 8.9 9.3 11.2 12.2 11.2 12.8 9.8 13.4 13 13.4 14.6 10.7 13.8 14.9 14.6 14.6 16.4 16.1 13.3 16.6 19 17.1 18 13.4 17.7 18.7 17 20.2 19.8 18.4 20.8

– – 1.4 1.6 – 1.9 2.7 – 3.2 3.3 3.8 3.4 3.9 3.5 3.6 3.7 4 4 4.3 4.7 4.9 4.6 4.9 4.4 5.4 5 5.5 5.6 4.1 5.5 5.7 5.4 5.5 5.8 6.2 4.9 5.8 6.1 6.2 5.2 5.7 5.8 5.5 5.6 6.9 5.6 5.6 6.2

– – 2.2 1.9 1.4 1.9 2.3 3.2 2.9 2.8 3.2 2.9 3.2 2.4 2.8 2.8 3.8 3.6 3.2 3.9 4.9 4.1 4.9 3.9 6.1 6.1 6.6 6.5 5.2 – 7.6 – 6.1 7 8.6 5.6 6.9 7.8 8.5 8.3 6.8 8.5 8.9 7.8 9.4 10.4 9.2 9.6

– – – – – – 2.8 3.2 2.4 3 3.6 3.7 3.9 3.3 3.3 3.6 4.8 4.9 4 6.6 7.9 6.8 8.3 5.6 9.7 9.3 9.7 10.7 7.5 10.1 11.2 10.8 10.2 11.6 12 9 12.3 13 12.9 13.1 9.9 13.5 14.1 12.5 15.7 15.4 14.5 16

– – – – – – 1.5 1.6 1.4 2.1 3 2.4 3 2.4 2.6 2.5 3.2 3.6 3.1 5.6 6.9 5.4 7.3 4.9 8.9 8.3 8.5 9.8 6.4 9.2 10.4 10 9.4 10.8 11.9 7.9 11.4 12 11.7 12.3 9.1 12.8 13.3 12.3 14.8 14.4 13.8 15

– – 3.4 3 3.2 3.3 5.9 6.3 6.3 6.5 7.2 – 7.5 6.9 7.3 6.4 6.6 6.7 7.9 8.8 9.7 8 9.6 8 10.1 9.1 10 10 6.6 10 9.8 10 10.4 – 10.9 9.1 11.2 12 11.4 10.6 9.8 11.2 11.6 10.5 11.5 11.5 10.9 12.5

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Table 3. Continued Days

Weight

A

B

C

D

E

F

35 35 35 36 37 37 39 40 40 41 41 45 45 52

2,800 3,659 4,228 5,709 5,158 7,169 7,600 6,620 8,350 7,373 7,580 11,899 12,690 12,487

17.8 19.4 19.6 22.1 20 22.9 23.6 22.7 24.5 23.8 23.8 26.4 29 28.6

4.5 5.4 5.2 6 5.6 6.5 5.7 5.8 5.7 5.2 5 5.7 8.1 8.3

8.4 8.7 8.7 11.5 10.8 12.3 10.7 11.2 13.3 12.6 12.1 14.3 13.1 15

13 14.2 14.2 17.7 16.5 18.9 18.6 17.3 20.5 19.3 19.8 23.3 23.1 24

12.3 13.1 13.5 16.9 15.6 17.7 17.6 16.9 19.8 18.6 18.8 22.6 22.4 23.1

10.3 11.1 11.3 12.5 11.5 13.1 13.3 12.7 13.1 13.1 13.1 14.3 13.8 14

A, maximum head length; B; width of the eye; C, distance between the middle of the eye and snout tip; D, upper jaw length; E, lower jaw length; F, distance between eyes. Po0.01 (dimensions exhibiting a significantly better correlation with body weight than with the day of incubation).

Figure 9. Measured distances on the crocodile head. (A) maximum head length; (B) width of the eye; (C) distance between the middle of the eye and the snout tip; (D) upper jaw length; (E) lower jaw length; and (F) distance between the eyes. interconnected forming a unit structure getting out from surround. The medial and lateral nasal processes looked like a pipe that was connected caudally with the maxillary processes by an isthmus. Thus, the typical separation of the facial processes has to be derived later, as a consequence of their progressive growth. A similar common structure during the initial development of the facial processes has been described in mouse and rat embryos, but not in chick embryos or in man (Peterka and Jelinek, ’78, ’83).

The general plan for the development of the forelimbs and hindlimbs is very similar to that in mammalian embryos. Limb buds represent the initial stage of limb formation. During further development, the buds elongate and autopodia differentiate distally. In the youngest embryo in our collection (ED 9, 22 mg), all limb buds were already present thus we could not determine whether the fore limb buds appear sooner than those of the hindlimbs. However, the collection of C. niloticus by Voeltzkow (’02) comprises earlier stages. It is apparent in the published J. Exp. Zool. (Mol. Dev. Evol.)

14

PETERKA ET AL.

Figure 10. Morphometry of the crocodile head during the prenatal period. (A) Maximum antero-posterior length of the head. (B) The distance between the snout tip and the jaw joint (upper jaw length). Note the head dimensions better correlate with the body weight of the embryos/fetuses than with the day of incubation.

documentation (Table VII in Voeltzkow, ’02) that all the forelimb and hindlimb buds appear at the same time. However, during subsequent development, growth is faster (the size is larger) and also the differentiation of the fingers is more advanced in the J. Exp. Zool. (Mol. Dev. Evol.)

hindlimbs than in the forelimbs. This has been clearly shown by the documentation of Voeltzkow (’02) and also by our present data (Figs. 3 and 4). The difference in the development between the forelimb and hindlimb is interesting from the aspect of

PRENATAL DEVELOPMENT OF CROCODYLUS NILOTICUS comparative embryology. The limb buds appear sooner and limb morphodifferentiation and finger appearance is more advanced in the forelimbs than in the hindlimbs in man (e.g., Larsen, ’93) and mouse (Kaufman, ’92). An interesting situation is found in the chick embryo. Its limb buds appear sooner in the forelimbs than in the hindlimbs. However, during further development, the growth and autopodia differentiation are faster in the hindlimbs than in the forelimbs (Hamburger and Hamilton, ’51). Thus, the limb development in the chick embryo is similar to that of mammals during the initial stage, whereas it is similar to that of crocodiles at later stages. The development of the dentition starts at specific tooth positions in crocodilian embryos being manifested by tooth placodes protruding into the oral cavity. The tooth placodes have been related to the superficial, first-generation teeth that are abortive (Westergaard and Ferguson, ’90). In Nile crocodiles at later stages of prenatal development, there was a series of protuberances on the free jaw margin. The outer part of a protuberance corresponded to a supralabial scale; the inner part comprised a developing tooth (Fig. 8). In the oldest available fetus (Table 1), the upper and lower jaw quadrants showed 19 and 15 protuberances, respectively. These numbers are similar to the adult tooth numbers. There have been documented 18 teeth (5 incisors, 5 canines and 8 molar teeth) in the upper jaw quadrant and 15 teeth (3 incisors, 5 canines, 7 molars) in the lower jaw quadrant of the adult Nile crocodile (Putterill and Soley, 2003). The present data show that tooth positions at later developmental stages of the Nile crocodile are indicated by a regular series of protuberances on the free jaw margin (Fig. 8). The supralabial scales and teeth show a close topographical relationship. We propose that the supralabial scale on the jaw surface could be considered as the external indicator of a tooth position. Future studies should elucidate whether a developmental relationship might exist between the supralabial scales and the adjacent teeth. The number of supralabial scales, which can easily be determined by external inspection, could represent further parameter for crocodile staging.

CONCLUSIONS 1. Both morphological and morphometric characteristics of developing Nile crocodiles showed an apparent inter-individual variability among animals of the same age. This variability decreased among animals of similar body weight, irrespective of their age. 2. In prenatal staging, the body weight can be considered as the most representative and complex parameter reflecting the overall growth of a whole embryo/fetus. 3. The nasal and maxillary facial processes are known to be separated by a narrow space before their fusion. However, before their existence as individual structures, the facial processes are interconnected in a unit structure. Thus, three

15 phases of facial processes development existed in Nile crocodile: union, separation, reunion. 4. In contrast to mammals (humans and mice) the hindlimbs develop faster than the forelimbs in C. niloticus. 5. The head measures showed significantly a better correlation with the body weight of the animals than with their chronological age (day of incubation). 6. The supralabial scales and tooth positions showed a close topographical relationship. The supralabial scales can be used as markers of tooth positions.

ACKNOWLEDGMENTS We thank to Dr. K. Zva´ra for statistical evaluation of data. The technical assistance of Dr. J. Velemı´nska´ and Mrs. B. Rokytova´ is highly acknowledged. The authors thank Dr. F. Sˇpoutil for his scientific comments and Dr. J. Dutt for English revision of the manuscript. This work was supported by the Grant Agency of the Czech Republic (grant 304/07/0223) and by the Ministry of Education, Youth and Sports of the Czech Republic (MSM0021620843).

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