Elsevier

Zoology

Volume 121, April 2017, Pages 91-110
Zoology

Three-dimensional reconstruction of the embryonic pancreas in the grass snake Natrix natrix L. (Lepidosauria, Serpentes) based on histological studies

https://doi.org/10.1016/j.zool.2016.11.001Get rights and content

Highlights

  • The pancreas primordium of the grass snake is formed by only two buds: the dorsal and the ventral one.

  • The gall bladder of the grass snake embryos is connected with the liver only by a thin cystic duct.

  • The first endocrine cells appear in the dorsal part of the pancreas, close to the spleen.

  • The differentiating pancreas penetrates the ventral part of the developing spleen and divides it into three parts.

  • The topography of the organ systems in snakes may determine the unique development of their pancreas.

Abstract

The aim of this study was to evaluate two research hypotheses: H0–the embryonic pancreas in grass snakes develops in the same manner as in all previously investigated amniotes (from three buds) and its topographical localization within the adult body has no relation to its development; H1–the pancreas develops in a different manner and is related to the different topography of internal organs in snakes. For the evaluation of these hypotheses we used histological methods and three-dimensional (3D) reconstructions of the position of the pancreatic buds and surrounding organs at particular developmental stages and of the final position and shape of the pancreatic gland. Our results indicate that the pancreas primordium in the grass snake is formed by only two buds – a dorsal and a ventral one – that are not connected until the end of stage II. This differs from the majority of vertebrates investigated so far. The gall bladder of the grass snake embryos is connected with the liver only by a thin cystic duct, which also differs from many other vertebrates. Our histological study also indicates a different distribution of the endocrine cells in the embryonic pancreas of the grass snake because the first endocrine cells appeared in the dorsal part of the pancreas in a region located close to the spleen. During the entire developmental period no evidence of these cells was found in the ventral part of the pancreas. The endocrine cells form elongated, large and irregular-shaped islets. They can also form structures resembling “inverted acini”. Such an arrangement is characteristic of snakes only. The differentiating pancreas penetrates the ventral part of the developing spleen and divides it into three separate parts at developmental stage IX. This is unique among vertebrates. At the end of the embryonic development (stage XI), the pancreas, the spleen and the gall bladder are located in close proximity and form the so-called triad. Our results suggest that the untypical topography of the organ systems in snakes may determine the unique development of the pancreas in these animals.

Introduction

The pancreas is an important glandular organ in all vertebrate species. It comprises two different tissues – exocrine and endocrine – and, consequently, serves as two glands in one.

The branched exocrine part of this gland is composed of ducts and acini that are essential structural and functional units of this part of the gland. The acini are composed of acinar cells filled with zymogen granules including digestive enzymes, while the ducts are formed by ductular cells (Slack, 1995, Gittes, 2009, He et al., 2015). The function of the exocrine pancreas is to supply the gut with the digestive enzymes that are produced and secreted by the acinar cells and are subsequently transported to the intestine via the pancreatic ductal system (Slack, 1995, Edlund, 2002, Pan and Wright, 2011).

The endocrine cells aggregate into islets that are embedded in the exocrine part of the gland (Slack, 1995, He et al., 2015). These hormone-secreting islets have a long evolutionary history and evolved as multiple discrete entities within the exocrine pancreas in early vertebrates (Bonner-Weir and Weir, 1979). The endocrine part of the pancreas produces several important hormones, including insulin, glucagon, somatostatin and pancreatic polypeptides that circulate in the blood (Slack, 1995, Edlund, 2002). The pancreatic islets, which are also called Langerhans hormone-secreting islets, play important roles in regulating blood glucose homeostasis as well as in influencing digestion through the effects of endocrine hormones affecting the exocrine pancreas secretion (Youngs, 1972, Henderson et al., 1981).

The glands of the digestive tract, i.e. the pancreas and the liver, are derived from endoderm of the primitive foregut near the area where the foregut merges into the midgut. The liver and a part of the pancreas differentiate on the ventral wall of the foregut from two distinct fields of the endoderm that are closely apposed to each other. The endodermal field from which the liver bud will be formed is located closer to the stomach primordium. The endodermal field from which the ventral pancreatic buds will be formed is localized more caudally to the endodermal field of the liver (Deutsch et al., 2001). During the embryonic development in vertebrate species, these fields of endoderm evaginate and form the diverticula of the gut – a dorsal one and one or two ventral ones. The dorsal pancreatic bud develops from the dorsal diverticulum of the gut. It forms the dorsal pancreas in adult vertebrates (Choronshitzky, 1900, Tribe, 1918, Slack, 1995). The hepatic bud and the ventral buds of the pancreas develop from the ventral diverticula (Tribe, 1918, Slack, 1995, Deutsch et al., 2001). The origin of the exocrine pancreas in all vertebrates is unambiguous and a literature review indicates that it originates from the dorsal and ventral pancreatic buds (Shih et al., 2013). The origin of the endocrine pancreas has long been controversial (Kelly and Melton, 2000, Assouline et al., 2002, Field et al., 2003), but recent studies suggest that it is similar to the origin of the exocrine pancreas (Merkwitz et al., 2013, Gannon, 2007).

The literature review also indicates that there is a controversy about whether the ventral pancreatic primordia derive directly from the gut wall (first model) or rather from the hepatic diverticulum (second model) which is formed from the gut (Choronshitzky, 1900, Tribe, 1918, Deutsch et al., 2001). According to the first model the ventral pancreatic buds can be formed directly from the ventral wall of the gut as one or two anlagen (Holtfreter, 1925, Deutsch et al., 2001, Tremblay and Zaret, 2005, Murtaugh, 2007, Franklin et al., 2008, Zorn and Wells, 2009). The second model assumes that only one evagination is formed during the early period of embryonic gut differentiation. This evagination is the hepatic diverticulum, which will give rise to the bud of the liver, the bud of the gall bladder and one or two ventral pancreatic buds (Hilton, 1903, Tribe, 1918, Hard, 1944, Severn, 1972, Wells and Melton, 1999, Jørgensen et al., 2007). In general, the part of the hepatic diverticulum that is located nearest the duodenum will form the common bile duct (ductus choledochus). The common bile duct will divide into the cystic duct (this duct will collect the secretions from the gall bladder) and the hepatic duct (this duct will collect the secretions from the liver) (Tribe, 1918, Slack, 1995).

The structure of the pancreas in reptiles – in comparison to that in other vertebrate species – is as yet poorly known. This organ has been described in crocodilian (Rhoten, 1987, Ono et al., 1991), chelonian (Thiruvathukal and Thiruvathukal, 1966, Agulleiro et al., 1985, Lozano et al., 2000, Sarkar and Maiti, 2011) and squamatae species (Rhoten and Hall, 1982, Rhoten, 1984, Putti et al., 1991, Sottovia-Filho and Taga, 1992, Buono et al., 2006). The pancreas of adult snake species was divided into five major morphological types by Moscona in 1990. This classification is based on three major features of the pancreas: the anatomical organization of this gland, its location close to the gall bladder and the spleen, and the distribution of endocrine islets throughout the gland.

The literature survey revealed only fragmentary knowledge about the embryonic development and differentiation of the reptilian pancreas. For this reason, several hypotheses about the differentiation of the pancreas in this group of animals have been put forth on the basis of observations of the evolutionary pancreas development in birds and mammals (Slack, 1995, Pieler and Chen, 2006). There are only a few papers that describe the development of the embryonic pancreas in snakes, such as Coluber natrix (Rathke, 1839), Vipera berus (Saint-Remy, 1893), Vipera aspis (Laguesse, 1901), and in other reptilian species (Siwe, 1926), Xantusia vigilis (Miller, 1963), Anolis carolinensis (Rhoten and Hall, 1982), as well as in Alligator mississippiensis (Jackintell and Lance, 1994). Due to this poor knowledge concerning the developmental processes in reptiles, we considered two hypotheses: H0 – the embryonic pancreas in snakes develops as in all the previously investigated amniotes (from three buds) and its topographical localization within the adult body has no relation to its development; H1 – the snake’s pancreas develops in a different manner because of the different topography of the snake’s internal organs.

The results presented in the majority of publications on the development of internal organs in vertebrates are based on two-dimensional (2D) histological samples that provide only partial and static information about the organogenesis of any organ. This is due to the fact that these samples are usually only snapshots of distinct sections through different parts of a differentiating gland and the surrounding organs at a particular stage of its embryonic development (Zhou et al., 2006, Setty et al., 2008, Radi et al., 2010). In contrast to this method, three-dimensional (3D) reconstruction of the position of the pancreatic buds and the surrounding organs at particular developmental stages as well as of the final position and the shape of the pancreatic gland seems to be a much more accurate method to evaluate our research hypotheses.

3D reconstruction of the internal organs and the topography from 2D images has already been applied to studies on the vertebrate pancreas, but mainly in mammals (Gaubert et al., 2009, Godlewski et al., 2011, Yu et al., 2012, Ciciotte et al., 2014). In our present study of the pancreas development in snakes we applied this method for the first time. In view of the fragmentary data on this issue, our paper can contribute significantly to resolve some questions and doubts put forward by the authors mentioned above.

Section snippets

Manipulation of animals and embryos

Fertilized female grass snakes (Natrix natrix) were caught in Poland in the vicinity of Wroclaw and Lubliniec. The animals were kept in vivaria, in conditions similar to those in the wild, until their eggs were laid, and then released back into their native area. All the specimens used in these experiments were captured according to Polish regulations concerning the protection of wild species (Journal of Laws, 1991, No. 114, item 492; Journal of Laws, 2000, No. 66, item 802; Journal of Laws,

Stage I

The pancreas anlage of the grass snake first appeared at developmental stage I, just after oviposition. It was situated in the loop of the small intestine between the mesonephros and the small elongated ventral end of the liver primordium. The primordium of the pancreas was formed with two buds – the dorsal one and the ventral one. They were located at the same side of the duodenum (Fig. 1A–F). The dorsal end of the ventral bud was located in close proximity to the liver primordium. The dorsal

Pancreatic buds in the grass snake – three, two or one?

Three pancreatic buds have been found in the developing pancreas of Xenopus leavis (Kelly and Melton, 2000), birds (Pictet and Rutter, 1972) and many mammalian species (Tribe, 1918, Slack, 1995, Lammert et al., 2001, Lammert et al., 2003, Edlund, 2002). However, not all of them always participate in the formation of the pancreatic gland. Among the animals that form three embryonic buds of this organ, there are two models of pancreas morphogenesis.

In the first model, one of the two ventral buds

Conclusions

The results of the present study revealed that the pancreas primordium of the grass snake is formed by two buds only, unlike in the majority of the vertebrates investigated so far. At the beginning of their development the localization of the pancreatic buds in the Natrix embryos, between the loop of the duodenum, the stomach and the primordium of the liver, resembles that in other vertebrates. With the appearance and development of neighboring organs (the spleen and gall bladder), this spatial

Acknowledgements

The authors would like to express their most sincere gratitude to Prof. Jerzy Klag and Dr. Andrzej Kędziorski for their critical reading of the manuscript and many helpful suggestions. We are grateful to Dr. Barbara Ruppik for her translations of the French articles referred to in this publication. The authors are deeply indebted to Ms. Michelle L. Simmons, BA, English Language Centre (ELC) (retired), University of Silesia, Katowice, Poland for improving the style. We would like to express our

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