Does the pancreas of gekkotans differentiate similarly? Developmental structural and 3D studies of the mourning gecko (Lepidodactylus lugubris) and the leopard gecko (Eublepharis macularius)

Abstract

This study investigated the pancreas differentiation of two species of gekkotan families—the mourning gecko Lepidodactylus lugubris (Gekkonidae) and the leopard gecko Eublepharis macularius (Eublepharidae)—based on two‐dimensional (2D) histological samples and three‐dimensional (3D) reconstructions of the position of the pancreatic buds and the surrounding organs. The results showed that at the moment of egg laying, the pancreas of L. lugubris is composed of three distinct primordia: one dorsal and two ventral. The dorsal primordium differentiates earlier than either ventral primordium. The right ventral primordium is more prominent and distinctive, starting to form earlier than the left one. Moreover, at this time, the pancreas of the leopard gecko is composed of the dorsal and right ventral primordium and the duct of the left ventral primordium. It means that the leopard gecko's left primordium is a transitional structure. These results indicate that the early development of the gekkotan pancreas is species specific. The pancreatic buds of the leopard and mourning gecko initially enter the duodenum by separate outlets, similar to the pancreas of other vertebrates. The pancreatic buds (3 of the mourning gecko and 2 of the leopard gecko) fuse quickly and form an embryonic pancreas. After that, the structure of this organ changes. After fusion, the pancreas of both gekkotans comprises four parts: the head of the pancreas (central region) and three lobes: upper, splenic, and lower. This organ develops gradually and is very well distinguished at hatching time. In both gekkotan species, cystic, hepatic, and pancreatic ducts enter the duodenum within the papilla. During gekkotan pancreas differentiation, the connection between the common bile duct and the dorsal pancreatic duct is associated with intestinal rotation, similar to other vertebrates.

Despite the huge evolutionary diversity of non‐ophidian squamates, the embryonic development of its pancreas has been poorly known. The pancreas morphology of adult lizards shows many primitive features, for example, the pancreas containing three branches equivalent to the three pancreatic buds. This type of pancreas is considered plesiomorphic in contrast to the derived morphology of the ophidian pancreas. Studying the development of the pancreas in two different gekkotans will be helpful in determining whether a phylogenetic position affects how the pancreas differentiates.

1. INTRODUCTION

Although squamate reptiles are among the most common amniotes, the gross anatomy and detailed features of the reptilian pancreas are poorly investigated (Buono et al., 2006; Hamny et al., 2016; Miller, 1962; Miller & Lagios, 1970; Moscona, 1990; Penhos & Ramey, 1973; Underwood, 1967). Some findings show that the pancreas of reptiles has the greatest morphological diversity (Miller & Lagios, 1970; Moscona, 1990). It is due to the structural modification of different intestinal parts and differences in the body shape of these animals (Miller & Lagios, 1970). Moreover, within reptilian endocrine pancreas, researcher described two evolutionary trends (Buono et al., 2006; Miller & Lagios, 1970). One of them is a confluence of islets in a large endocrine mass in the splenic lobe simultaneously with a reduction of the number of islets in the duodenal lobe (Epple & Brinn, 1975; Laguesse, 1901; Rhoten, 1987; Rhoten & Hall, 1982; Thomas, 1942). Another one is concerning the presence of PP cells in derivatives of ventral anlagen of embryonic pancreas (Bonner‐Weir & Weir, 1979; El‐Salhy & Grimelius, 1981; El‐Salhy, 1983; Epple & Brinn, 1987; Jackintell & Lance, 1994; Kowalska & Rupik, 2019, 2021; Putti et al., 1992). It is worth highlighting that current knowledge regarding the development of the pancreas in reptiles compared with other vertebrate species is very poor (Chu et al., 2021; Evans, 1934; Miller, 1963; Siwe, 1926). The limited knowledge on the development of this organ in reptiles is associated with the fact that reptiles are difficult to capture, manipulate, and maintain alive under laboratory conditions. In addition, reptilian embryonic development begins while the eggs are still in the female genital tract, so at the time of oviposition, embryos are at an advanced stage of embryogenesis (Rupik, 2011).

A literature review indicated that the pancreas of snakes was the most frequently studied (Moscona, 1990; Thomas, 1942). There are comprehensive pancreas studies that include information on the gross anatomy (Miller & Lagios, 1970; Moscona, 1990; Thomas, 1942) and some aspects of embryonic development (Siwe, 1926). The pancreatic differentiation of grass snake (Natrix natrix) embryos has been studied in more detail. Grass snake embryos are the first specimens of reptiles to have three‐dimensional reconstructions of the differentiating pancreas. These findings indicated that the pancreas in the grass snake embryos differentiates as two buds—the dorsal one and the ventral one—and thus that the initial stages of the development of this gland are different from those described in other vertebrates (see Kowalska et al., 2017, discussion sections). Moreover, the differentiating pancreas penetrates the ventral part of the developing spleen and divides it into three separate parts, which is an exception in vertebrates (see Kowalska et al., 2017, discussion sections). The 3D reconstructions of the developing pancreas of the grass snake first indicated that the main ducts connecting the triad's organs differentiate in such a way that the hepatic duct and the cystic duct merge to form the common bile duct that enters the ventral pancreatic duct (Kowalska et al., 2017). During developmental studies of the pancreas in grass snake embryos, both parts of the pancreas, the exocrine and the endocrine, have been analyzed in detail. Their results have allowed us to describe the origin of islets, mode of islet formation, and cell arrangement within islets (Kowalska & Rupik, 2019), and the process of intrasplenic islet formation, characteristic of the adult grass snake, has also been described (Kowalska & Rupik, 2021). Moreover, the 3D structure of the duct network of the exocrine pancreas in this species has been described in detail (Kowalska & Rupik, 2018).

The interesting results of our studies on the grass snake and a literature review prompted further research into the development of the reptile pancreas. This study investigated the pancreas differentiation of two species of gekkotan families: the mourning gecko Lepidodactylus lugubris (Gekkonidae) and the leopard gecko Eublepharis macularius (Eublepharidae). The results of this study are based on two‐dimensional (2D) histological samples and three‐dimensional (3D) reconstructions of the position of the pancreatic buds and the surrounding organs. These findings can help to compare the differentiation of the pancreas of the two Gekkota species and determine whether the pancreas of the Gekkota differentiates similarly. Moreover, the results are likely to help clarify the morphological differentiation of the reptile pancreas and provide good source material for phylogenetic studies.

2. MATERIALS AND METHODS

2.1. Manipulation of animals and embryos

The pancreas development was studied in two gekkonid lizard species: the leopard gecko E. macularius (BLYTH 1854) and the mourning gecko L. lugubris (Duméril & Bibron, 1836). The research was carried out during the years 2021–2023. The literature review and our other developmental studies indicated that E. macularius is a widely used model organism in laboratory and experimental studies (Agarwal et al., 2022; Hermyt et al., 2020; Vickaryous & Gilbert, 2019). Still, the mourning gecko (L. lugubris) stands out as an ideal model for studying developmental questions (Griffing et al., 2019; Hermyt et al., 2020; Skawiński et al., 2023).

The embryos of both species were obtained from long‐standing non‐commercial breeding at the Institute of Biology, Biotechnology and Environmental Protection. This breeding is registered in the Polish Ministry of Education and Science (number 033). Both gecko species were kept in vivaria in conditions similar to natural ones. Under laboratory conditions, the leopard geckos E. macularius laid eggs from May to August yearly. The mourning gecko L. lugubris lays eggs throughout the year. Both species laid two eggs monthly.

The E. macularius eggs were removed from the terrarium after laying, placed in a chick incubator or Zoo Med's ReptiBator, and incubated at 30°C. The eggs were half buried in vermiculite mixed with water at a 1:1 ratio by weight and stored in plastic food storage containers with transparent, translucent sides and tops. Such conditions have been successfully applied in previous studies (Kaczmarek et al., 2017; Kaczmarek & Rupik, 2021; Kowalska et al., 2017; Rupik, 2012, 2013; Rupik et al., 2016; Swadźba & Rupik, 2012). The embryos of E. macularius were isolated at regular intervals starting immediately after the eggs were laid. The developmental stage of embryos was evaluated according to this species' developmental table based on external morphological characteristics (Wise et al., 2009). The embryos of E. macularius represented five stages: 30, 32, 34, 38, and 42.

The eggs of L. lugubris are glued by females to the different vertical surfaces of the terrarium. The eggs were left and incubated in the terrarium to avoid damage to the eggshells at approximately 23–30°C (Hermyt et al., 2020). Embryos of this species were isolated every fortnight. This way of collecting research material allowed embryos at different stages of development to be obtained. Since during the first isolations, all the necessary stages were not obtained during such isolation, during subsequent isolations, only the missing stages were selected. As the developmental table for L. lugubris (Griffing et al., 2019) was not available at the beginning of embryo collection, we employed the developmental table for other gekkonids from Noro et al. (2009). An approximation of the corresponding developmental stages is provided in Table 1. The embryos of L. lugubris represented five stages: 5, 12, 18, 28, and 50.

TABLE 1.

Comparison of corresponding developmental stages in Paroedura picta and Lepidodactylus lugubris based on selected morphological features from Noro et al., 2009 and Griffing et al., 2019, respectively.

Developmental stage		Morphological basis of stage alignment
Paroedura picta (Noro et al., 2009)	Lepidodactylus lugubris (Griffing et al., 2019)
3 dpo	Stage 28	30–32 somites Eye unpigmented Limb buds visible only as small swellings
4–5 dpo	Stage 29	36–41 somites Eye weakly pigmented at margin or pigmentation of the dorsal half of optic cup Buds of forelimb and hind limbs more distinct than before; broader than longer
6–7 dpo	Stage 30	Pigmentation spreading to the front of the eye, or the whole eye is pigmented Paddle‐shaped forelimbs
9–12 dpo	Stage 31	Primordia of forelimbs and hind limbs elongated The proximal part of the forelimbs may be narrowed and the distal part flattened
14–16 dpo	Stage 32	Autopodium, zeugopodium, and stylopodium – easily distinguishable in the forelimbs Mandibular processes extended to the anterior margin of the eye, almost approaching the frontonasal mass
18 dpo	Stage 33	Digit primordia visible as condensations in the autopodia Maxillary prominence and frontonasal mass fused
20–22 dpo	Stage 34	Interdigital webbing starts to regress, freeing the tips of the digits The anterior tip of the mandible extends to the anterior end of the snout
24 dpo	Stage 35	Interdigital webbing reduced to the proximal parts of the forming digits
26 dpo	Stage 36	Interdigital webbing reduced completely or only remnants of the webbing can be seen between the proximal parts of the digits
28 dpo	Stage 37	Digits tapered distally; claws eventually begin to form
30 dpo	Stages 38–39	Distinct claws Pale pigmentation of the trunk; skin remains translucent
35 dpo	Stage 40	Denser pigmentation than before; color pattern begins to appear
40–45 dpo	Stage 41	Distinct color patter
50 dpo	Stage 42	Scales well‐visible Genital organs still everted
55–60 dpo	Stage 43	Adult‐like color pattern Genital organs not visible externally

Embryos of both species were sacrificed by decapitation after sedation. For embryo sedation, the eggs in a plastic Petri dish were cooled in a refrigerator (4–5°C) for about 10 min (L. lugubris) or 15 min (E. macularius). Then, the Petri dish with eggs was placed on ice (still in the fridge) for about 8 min (L. lugubris) or 15 min (E. macularius) (Rollings et al., 2019; Shine et al., 2015). After that, the embryos were immediately isolated, decapitated, and transferred to the cooled fixative.

The stages from both developmental tables (Noro et al., 2009; Wise et al., 2009) do not correspond to each other (the same numbers defining stages in L. lugubris and E. macularius are not “homologous”). Selection of embryos used for the study was based on external morphology. The stage approximation based on external morphology is presented in Table 1 of Wise et al. (2009) (but see Andrews et al., 2013). The stage alignment based on pancreatic features is presented in Table 2 of this paper.

TABLE 2.

Summary of pancreas development of Lepidodactylus lugubris and Eublepharis macularius.

Eublepharis macularius		Lepidodactylus lugubris
Stage	Event		Stage
30 video S1	Three buds (dorsal and two ventral) The left ventral bud is formed by a short duct only The left ventral bud is a transitional structure	Three buds of the pancreas (dorsal and two ventral) Each bud enters the duodenum through a separate duct	5 video S6
	Gallbladder is located above the pancreatic buds, on the ventral side of the liver The cystic duct, the hepatic ducts, and the duct of the ventral bud of the right abdominal primordium all share a common outlet to the duodenum The dorsal bud is more differentiated than the ventral bud The ventral buds are small evaginations of the duodenum
32 video S2	Four parts (head and three lobes: upper, splenic, and lower) can be distinguished in the differentiating pancreas The head of the pancreas differentiates at the level where all three buds are connected Within the head of the pancreas, four ducts (cystic, hepatic, and two pancreatic ducts) enter the duodenum through a common duct: Within the upper lobe two parts can be distinguished: upper and lower The lower part of the upper lobe is short and curved toward the splenic lobe The upper part of the upper lobe is elongated and curved toward the gallbladder The splenic lobe is elongated and located in a place where in stage 32 in E. macularius and 18 in L. lugubris the spleen anlage will appear The lower lobe is located in the loop of the small intestine and is a small evagination found below the head of the pancreas		12 video S7
	The ventral part of the pancreas is located in the liver cavity The gallbladder is located at the level of the pancreas The splenic lobe is elongated and has no characteristic thickening	The pancreas is not embedded in the liver The gallbladder is located just dorsal to the pancreas The splenic lobe is elongated and ends with marked thickening
34 video S3	The spleen anlage is present for the first time in the space between the splenic lobe and the mesonephros		18 video S8
	The lower part of the upper lobe is located close to the splenic lobe The cystic duct is very thin and elongated The gallbladder is present at the level of the lower part of the upper lobe and the splenic lobe	Topographical relations between the lower part of the upper lobe and the splenic lobe are the same as at stage 12 The cystic duct is thick and short The localization of the gallbladder is the same as previously
38 video S4	The shape of the pancreas changes Cystic, hepatic, and pancreatic ducts enter the duodenum within the papilla		28 video S9
	The pancreas is strongly elongated The upper part of the upper lobe extends toward the gallbladder The lower part of the upper lobe expands to form a thickening Within the splenic lobe thickening appears Thickening of the lower part of the upper lobe and thickening of the splenic lobe are in contact with each other The lower lobe is strongly elongated and split in the lower part The gallbladder is arranged at the level of the upper part of the upper lobe	The pancreas elongates but is shorter than the pancreas in the leopard gecko The upper lobe is small, and its upper and lower parts are poorly distinguished Thickening of the splenic lobe enlarges The lower lobe elongates The cystic duct bends toward the upper lobe The gallbladder is arranged at the level of the lower part of the upper lobe of the pancreas
42 video S5	The gallbladder is located just dorsal to the pancreas		50 video S10
	All lobes of the pancreas are strongly elongated The upper part of the upper lobe takes the shape of a pedicle that reaches the lateral surface of the gallbladder The thickening located in the lower part of the upper lobe is offset from the thickening of the splenic lobe for a considerable distance The size and shape of the lower lobe remain unchanged	The fold of the cystic duct disappears The upper part of the upper lobe strongly elongates The splenic lobe is wide and short and moves away from the upper lobe The lower lobe takes the form of thickening and is much shorter than the upper lobe

Note: Similarities for two aligned stages are shown in merged cells.

2.2. Light microscopy and 3D reconstructions

The youngest embryos of E. macularius and L. lugubris species as a whole were fixed. Moreover, from the older embryos, pancreatic tissues and adjacent organs were removed and fixed (see Table 3). Embryonic tissues of both species were fixed in Bouin's solution for 48 h at room temperature, dehydrated in graded ethanol, cleared in xylene, and infiltrated in paraffin waxes with a low melting point for 30 min and in paraffin waxes with a high melting point for 24 h (Bagiński, 1969). Finally, they were embedded in paraffin. The paraffin blocks were cut into 6‐μm transverse sections using a rotary microtome (Leica RM2125RT; Leica Biosystems, Nussloch, Germany). All the transverse sections were collected on glass slides, deparaffinized, and stained with Ehrlich's hematoxylin and eosin (Kaczmarek et al., 2017; Skawiński et al., 2023; Swadźba et al., 2009) and with Heidenhain's AZAN (Hermyt et al., 2017; Kiernan, 2008). The sections were analyzed for histological examination and 3D reconstructions, and serial micrographs were taken under the light microscope Olympus BX63 with an Olympus DP28 digital camera (Olympus, Tokyo, Japan). The series of images were loaded using the stacked slice format in the Amira 3D software (version 6.4, FEI, France) and processed as described (Cornillie et al., 2008).

TABLE 3.

Number of gekkotan embryos used for light microscopy (LM) and three‐dimensional reconstruction (3D).

Eublepharis macularius			Lepidodactylus lugubris
Stage	LM	3D a	Stage	LM	3D a
30	3	1	5	3	1
32	3	1	12	3	1
34	3	1	18	3	1
38	3	1	28	3	1
42	3	1	50	3	1
Total	15	5	Total	15	5

^a Specimens used for three‐dimensional reconstructions are the same as for light microscopy.

3. RESULTS

3.1. Morphological and structural description of leopard gecko pancreas differentiation

The report considers only those developmental stages where significant morphological and structural changes of the differentiating organ occur.

3.1.1. Stage 30

At the time of egg laying, the pancreas primordium of the leopard gecko was located dorsally in the body cavity within the loop of the duodenum, below the caudal part of the liver and the gallbladder. The pancreas primordium was formed by three buds: one dorsal and two ventral: left and right. The dorsal bud was the largest and elongated in the cephalic direction. The left and right ventral buds were tiny, connected to the common bile duct; they are located on both sides of it and between it and the duodenum. The left ventral bud was formed only by a short duct (Figure 1a–e).

FIGURE 1.

The embryonic pancreas of the leopard gecko at developmental stage 30. (a–e) 3D reconstructions of the embryonic pancreas and surrounding organs; (a–b) dorsal view, (c) right lateral view, (d) caudal view, (e) cephalic view; (f–i) transverse sections stained with Heidenchan's Azan (f–h), hematoxylin and eosin (i). (f–g) The dorsal bud with large dorsal duct and small agglomerates of endocrine cells; (h) the short left ventral bud comprising by the wide duct; (i) the right ventral bud running along the cystic duct and hepatic duct. Scale bars: e – 100 μm, f – 100 μm, g – 50 μm, h – 100 μm, i – 100 μm. Arrowhead, dorsal pancreatic duct; CBD, common bile duct; CD, cystic duct; D, dorsal; D, duodenum; dashed line, outline of islet; DB, dorsal bud; directions: A, anterior; G, gaster; GB, gall bladder; HD, hepatic duct; I, islets; L, left; L, liver; LVB, left ventral bud; M, mesonephros; N, notochord; P, pancreas; P, posterior; R, right; RVB, right ventral bud; V, ventral.

On the transverse cross sections, the pancreas primordium was visible between the loop of the duodenum and the mesonephros (Figure 1f,h). The pancreas anlage was composed of three buds. All buds had connections to the duodenum at the same level (Figure 1f,h). The dorsal bud was connected to the dorsolateral part of the embryonic duodenum wall. One large duct formed this bud with a stratified epithelium wall and was visible in the median aspect of the embryo body cavity (Figure 1f,g). Small cell agglomerates surrounded by the mesenchyme of the dorsal mesentery were connected to this duct (Figure 1f–i). The cells within agglomerates had round nuclei, and their cytoplasm stained light blue with Azan staining (Figure 1f–h) or pale violet with hematoxylin and eosin staining (Figure 1i). The left and right ventral buds were connected with the common bile duct on the lateral side of the duodenum. The left ventral bud was shorter than the right one and formed only by a small duct (Figure 1h).

3.1.2. Stage 32

At developmental stage 32, the localization of the pancreas toward the liver changed because the caudal part of the liver surrounded the ventral part of the pancreas. Within the structure of the pancreas, the primordium could be distinguished into four parts: three lobes (upper lobe, splenic lobe, and lower lobe) and the head of the pancreas. The splenic lobe was elongated and oriented cephalically. The upper lobe was elongated and oriented similarly to a splenic one. Both lobes were located parallel to each other.

The upper lobe was slightly curved toward the gallbladder. At this time of development, the gallbladder was found at the same level as the pancreatic primordium, differently than in the previous stages. The lower lobe was tiny and located in the loop of the small intestine. The pancreatic, cystic, and hepatic ducts entered the duodenum into the common duct at this developmental stage. It differs from the previous stages (Figure 2a–e).

FIGURE 2.

The embryonic pancreas of the leopard gecko at developmental stage 32. (a–e) 3D reconstructions of the embryonic pancreas and surrounding organs; (a–b) ventral view, (c–d) right lateral view, (e) cephalic view; (f–h) transverse sections stained with hematoxylin and eosin. (f) The pancreatic body comprising ducts and located at the level of the main duct; (g) the lower lobe situated in the loop of the intestine; (h) the splenic lobe with the small islets and the upper lobe located in close proximity to the splenic lobe. Scale bars: e – 200 μm, f–h – 100 μm. CBD, common bile duct; CD, cystic duct; D, dorsal; D, duodenum; directions: A, anterior; dotted line, outline of pancreatic lobes; G, gaster; GB, gall bladder; HD, hepatic duct; I, islets; L, left; L, liver; LL, lower lobe; M, mesonephros; MD, main duct; P, posterior; PB, pancreas body; R, right; S, spleen; SL, splenic lobe; UL, upper lobe; V, ventral.

The pancreas anlage was visible on the cross sections at the left side of the embryo body. It was surrounded by the liver, unlike in the previous stages (Figure 2f). The splenic lobe was formed by a large duct with cylindrical to stratified epithelium. This duct was connected with small cell agglomerates (Figure 2h). The cytoplasm of their cells was stained pink with hematoxylin and eosin staining (Figure 2h). The lower lobe was formed by small ducts and cell agglomerates (Figure 2g). At this developmental stage, the spleen anlage was observed for the first time (Figure 2f,h). It was located near the splenic lobe of the pancreas, between the left mesonephros and the duodenum (Figure 2h). Also, the connection of the pancreas primordium with the duodenum is different from the previous stage. A single sizeable common duct with stratified epithelium entered the duodenum (Figure 2f). Pancreatic, cystic, and hepatic ducts entered this common duct. All these ducts entered the duodenum within the pancreas head (Figure 2f).

3.1.3. Stage 34

At developmental stage 34, the pancreatic lobes were better distinguished than earlier. The splenic lobe was elongated toward the spleen anlage. Also, the upper lobe was more curved into the splenic lobe, and the cystic duct traversed it. In this manner, the upper and splenic lobes were located close to each other. Moreover, the pancreas head was more massive than previously. The gallbladder was located more above the pancreas at this developmental stage than earlier (Figure 3a–e).

FIGURE 3.

The embryonic pancreas of the leopard gecko at developmental stage 34. (a–e) 3D reconstructions of the embryonic pancreas and surrounding organs; (a–b) right lateral view, (c) ventral view, (d–e) dorsal view, (f) cephalic view; (f–h) transverse sections stained with Heidenchan's Azan (g), hematoxylin and eosin (f, h). (f) The lower lobe in the loop of the small intestine, (g) the splenic lobe with the pancreatic islets departing from the pancreatic body, (h) the splenic lobe and the upper lobe. Note the upper lobe is bending toward the splenic lobe. Scale bars: e – 300 μm, f–h – 100 μm. CD, cystic duct; D, dorsal; D, duodenum; directions: A, anterior; dotted line, outline of pancreatic lobes; G, gaster; GB, gall bladder; HD, hepatic duct; L, left; L, liver; LL, lower lobe; MD, main duct; P, posterior; PB, pancreas body; R, right; S, spleen; SL, splenic lobe; UL, upper lobe; V, ventral.

On the transverse cross sections through the lower lobe, more cell agglomerates were observed than at the earliest developmental stages (Figure 3f). Large ducts were visible within the pancreatic head (Figure 3g). At the level of connection of the main duct to the duodenum, the splenic lobe emerged from the pancreas head (Figure 3g). In comparison with the previous stage, the ducts of the splenic lobe were found in the center part of it but the cell agglomerates at the periphery (Figure 3g). The cephalic region of the splenic lobe localized near the spleen anlage included many cell agglomerates (Figure 3h). Moreover, the upper lobe containing small ducts was curved, and its cephalic end was found close to the splenic lobe (Figure 3h).

3.1.4. Stage 38

At developmental stage 38, the pancreas was found in the middle line of the leopard gecko body cavity. It was much more elongated compared with the previous stages. Moreover, it was more significant than the spleen and ended more caudally than the spleen. In addition, at this time of development, the gallbladder moved and settled above the pancreas. The splenic lobe thickened at its cephalic part located near the spleen, but the upper lobe thickened in its middle part, which was curved toward the splenic anlage. Moreover, the cephalic part of the upper lobe was greatly elongated along the cystic duct and ended near the gallbladder. The lower lobe enlarged considerably compared to the previous stages, and its caudal part was divided (Figure 4a–f).

FIGURE 4.

The embryonic pancreas of the leopard gecko at developmental stage 38. (a–f) 3D reconstructions of the embryonic pancreas and surrounding organs; (a–b) ventral view, (c–d) right lateral view, (e) dorsal view, (f) cephalic view; (g–k) transverse sections stained with Heidenchan's Azan; (g–h) the splenic lobe with large pancreatic islets located near the spleen, (i–j) the lower lobe with the small pancreatic islets, (k) the upper lobe with numerous ducts. Note the large accumulation of pancreatic islets within the splenic lobe. Scale bars: f – 300 μm, g – 100 μm, h – 30 μm, i – 100 μm, j – 50 μm, k – 100 μm. CD, cystic duct; D, dorsal; D, duodenum; dashed line, outline of pancreatic islets.; directions: A, anterior; G, gaster; GB, gall bladder; HD, hepatic duct; L, left; L, liver; LL, lower lobe; M, mesonephros; MD, main duct; P, posterior; PB, pancreas body; R, right; S, spleen; SL, splenic lobe; UL, upper lobe; V, ventral.

At this developmental stage, the spleen anlage was significantly enlarged in comparison to previous stages (Figure 4g). In the lower lobe, more cell agglomerates were found than previously (Figure 4i,j), but within the thickening of the splenic lobe, larger cell agglomerates were visible (Figure 4g,h). The upper lobe was built from numerous small ducts (Figure 4g). From this developmental stage, the cystic duct, hepatic duct, and both pancreatic ducts entered the duodenum as separate ducts at the site called the ampulla of Vater. These four ducts' diameters were smaller than in the previous stages (Figure 4k).

3.1.5. Stage 42

At the time of hatching, the pancreas of the leopard gecko was located dorsally to the liver, and the caudal half of the pancreas was located below the liver. It is the final location, similar to that of the adult. The upper lobe was more elongated, and its cephalic part formed a long protrusion which reached the gallbladder. Also, there was greater thickening of the splenic lobe. Both lobes, the upper and the splenic, were farther away from each other than previously (Figure 5a–e).

FIGURE 5.

The embryonic pancreas of the leopard gecko at developmental stage 42. (a–e) 3D reconstructions of the embryonic pancreas and surrounding organs; (a–b) ventral view, (c–d) right lateral view, (e) cephalic view. Note the upper lobe of the pancreas is divided into two parts. Its upper part reaches the gall bladder. Scale bar: e – 400 μm. CD, cystic duct; D, dorsal; D, duodenum; directions: A, anterior; G, gaster; GB, gall bladder; HD, hepatic duct; L, left; L, liver; LL, lower lobe; P, posterior; R, right; S, spleen; SL, splenic lobe; UL, upper lobe; V, ventral.

3.2. Morphological and structural description of L. lugubris pancreas differentiation

The report considers only those developmental stages where significant morphological and structural changes of the differentiating organ occur.

3.2.1. Stage 5

At the time of egg laying, the pancreatic primordium of the mourning gecko was found in the middle right side of the embryo body between the embryonic gut and the caudal part of the liver. The pancreas primordium was located below the gallbladder and was formed by three buds. The dorsal bud was larger than the two other buds and elongated in the cephalic direction. The right and left ventral buds were connected to the common bile duct and formed small evaginations of the embryonic gut wall (Figure 6a–f).

FIGURE 6.

The embryonic pancreas of the mourning gecko at developmental stage 5. (a–f) 3D reconstructions of the embryonic pancreas and surrounding organs; (a–b) caudal view, (c) right lateral view (d–f); (g–j) longitudinal sections stained with hematoxylin and eosin. Note three pancreatic buds: one dorsal and two ventral; (g) the right ventral bud located near the gallbladder, (h) the left ventral bud forming the small evagination of the intestine, (i) the dorsal bud emerged from the intestine as a small duct, (j) agglomerate of the endocrine cells and the cephalic end of the dorsal bud. Ventral buds are located on the opposite side of the duodenum. Scale bars: f – 100 μm, g–j – 50 μm. Arrowhead, dorsal pancreatic duct; CBD, common bile duct; CD, cystic duct; D, dorsal; D, duodenum; dashed line, outline of pancreatic islets; DB, dorsal bud; directions: A, anterior; dotted line, outline of pancreatic buds; G, gaster; GB, gall bladder; HD, hepatic duct; I, islets; L, left; L, liver; LVB, left ventral bud; P, posterior; R, right; RVB, right ventral bud; V, ventral.

On the cross sections, the right and left ventral buds were found on the opposite side of the common bile duct (Figure 6g,h). They formed a small evagination of the embryonic gut wall (Figure 6g,h). At the level of this bud, a small cell agglomeration was found dorsally to the gut surrounded by the mesenchyme of the dorsal mesentery. This agglomeration was connected to the dorsal bud, which was found higher than the previous buds at the dorsal part of the gut (Figure 6i,j). The large duct with columnar epithelium formed the dorsal bud ending with the cell agglomeration (Figure 6j).

3.2.2. Stage 12

At this developmental stage, the localization of the pancreatic primordium differed from the previous stages. The caudal part of the pancreas primordium was found below the caudal part of the embryonic liver (Figure 7a–e).

FIGURE 7.

The embryonic pancreas of the mourning gecko at developmental stage 12. (a–e) 3D reconstructions of the embryonic pancreas and surrounding organs; (a–b) right lateral view, (c–d) ventral view, (e) cephalic view; (f–g) transverse sections stained with hematoxylin and eosin; (f) the main duct departed from the intestine, (g) the dorsal bud forming ducts and small endocrine cell agglomerate and the ventral bud comprising of the one duct. Scale bars: e – 100 μm, f–g – 50 μm. CD, cystic duct; D, dorsal; D, duodenum; DB, dorsal bud; directions: A, anterior; G, gaster; GB, gall bladder; HD, hepatic duct; L, left; L, liver; MD, main duct; P, posterior; R, right; V, ventral; VB, ventral bud.

The large duct entered the embryonic gut on the cross sections (Figure 7f). This duct was divided into two smaller ducts. One of them was connected to the small cell agglomeration (Figure 7g) and extended to the dorsal part of the embryo body (Figure 7g). The second one was divided into four smaller ducts (Figure 7g). One was connected to the gallbladder, while the second was connected with the liver and the two other ducts. The cephalic part of the pancreatic primordium ended at the level of the caudal part of the liver.

3.2.3. Stage 18

At developmental stage 18, the shape of the pancreas changes in comparison to the previous stages. The pancreas was formed from the three lobes. The lower lobe was found between the loops of the intestine. The splenic lobe was located near the spleen and had a bulbous body on the distal end. The upper lobe was connected to the cystic duct. The gallbladder was located at the level of the pancreas (Figure 8a–f).

FIGURE 8.

The embryonic pancreas of the mourning gecko at developmental stage 18. (a–f) 3D reconstructions of the embryonic pancreas and surrounding organs; (a–b) dorsal view, (c) left lateral view, (d–e) ventral view, (f) cephalic view; (g–h) transverse sections stained with Heidenchan's Azan; (g) the splenic lobe departing from the pancreatic body at the level of the main duct, (h) the splenic and upper lobe located near to each other. Note the spleen has appeared for the first time. Scale bars: f – 100 μm, g–h – 50 μm. CD, cystic duct; D, dorsal; D, duodenum; directions: A, anterior; dotted line, outline of pancreatic lobes; G, gaster; GB, gall bladder; HD, hepatic duct; L, left; L, liver; LL, lower lobe; M, mesonephros; MD, main duct; P, posterior; PB, pancreatic body; R, right; S, spleen; SL, splenic lobe; UL, upper lobe; V, ventral.

On the cross sections, this part of the pancreas was formed by the large duct and small cell agglomerations (Figure 8g). During the mourning gecko development, the spleen anlage was observed for the first time (Figure 8g). It was located between the left mesonephros and the pancreas anlage (Figure 8g). The pancreatic ducts and ducts from the gallbladder and the liver joined into the common duct (Figure 8h), which entered the duodenum (Figure 8h). At the cephalic part of the embryonic pancreas, two large cell agglomerations were observed (Figure 8h).

3.2.4. Stage 28

At this developmental stage, the caudal part of the embryonic pancreas was found between the loops of the intestine. The pancreas ended more caudally than the spleen (Figure 9a–f).

FIGURE 9.

The embryonic pancreas of the mourning gecko at developmental stage 28. (a–f) 3D reconstructions of the embryonic pancreas and surrounding organs; (a–b) left lateral view, (c) dorsal view, (d–e) ventral view, (f) cephalic view; (g–h) transverse sections stained with hematoxylin and eosin; (g) the splenic lobe and the pancreatic body connected by the small duct, (h) the thickening of the splenic lobe formed by the cord‐like islets and the upper lobe. Scale bars: f – 200 μm, g–h – 100 μm. CD, cystic duct; D, dorsal; D, duodenum; directions: A, anterior; dotted line, outline of pancreatic lobes; G, gaster; GB, gall bladder; HD, hepatic duct; L, left; L, liver; LL, lower lobe; M, mesonephros; P, posterior; PB, pancreatic body; R, right; S, spleen; SL, splenic lobe; UL, upper lobe; V, ventral.

At the level where the pancreatic lobes were connected, ducts emerging from the pancreas, gallbladder, and liver entered the duodenum as separate small ducts (Figure 9g). At the level of the pancreas and duodenum connection, the splenic lobe emerged from the pancreas. This lobe was formed by small ducts and cell agglomerations (Figure 9g). The end of this lobe was observed near the spleen anlage as a large cell agglomeration connected to the proximal part of the dorsal lobe as a small duct (Figure 9h). Within the aggregation, cells formed a cord‐like structure (Figure 9h). The upper lobe was situated more cephalically and formed small ducts and acini located near the intestine (Figure 9h).

3.2.5. Stage 50

At the time of hatching, the pancreas of the mourning gecko was visible below the liver. The upper lobe was still curved in the spleen direction. Moreover, it elongated cephalically and almost reached the gallbladder, unlike in the earliest stages (Figure 10a–f).

FIGURE 10.

The embryonic pancreas of the mourning gecko at developmental stage 50. (a–f) 3D reconstructions of the embryonic pancreas and surrounding organs; (a–b) ventral view, (c) left ventral view, (d–e) dorsal view, (f) cephalic view; (g–h) transverse sections stained with Heidenchan's Azan (g), hematoxylin and eosin (h); (g) the pancreatic body containing the endocrine cells in the place from which the splenic lobe will be departing, (h) cephalic part of the dorsal lobe containing small ducts. Note the upper lobe is curved into the splenic lobe. Scale bars: f – 300 μm, g–h – 100 μm. CD, cystic duct; D, dorsal; D, duodenum; directions: A, anterior; G, gaster; GB, gall bladder; HD, hepatic duct; L, left; L, liver; LL, lower lobe; P, posterior; PB, pancreas body; R, right; S, spleen; SL, splenic lobe; UL, upper lobe; V, ventral.

Numerous acini were formed on the cross sections within the lower lobe and the head pancreas ducts (Figure 10g). The upper lobe of the pancreas was located deeply within the caudal part of the liver as a thin elongation (Figure 10h). This elongation was formed by ducts and acini near the large blood vessel (Figure 10h).

4. DISCUSSION

The morphological variation of the reptile pancreas is primarily linked to structural changes in the different parts of the intestine, which are closely related to the variation in body shape of this group of animals (Miller & Lagios, 1970). The structure of the lizard pancreas distinguishes between the upper lobe, splenic lobe, and lower lobe, named branches (Moscona, 1990). These branches of the pancreas in lizard species extend and form their head (Miller & Lagios, 1970; Moscona, 1990). The head of the pancreas containing the ampulla of Vater is located in the loop of the duodenum (Miller & Lagios, 1970; Moscona, 1990). The upper lobe of the pancreas in lizard species runs along the bile duct toward the gallbladder. Still, the lower lobe runs to the small intestine, whereas the splenic lobe runs back to the spleen (Underwood, 1967). The pancreas of the monitor lizard is believed to represent an intermediate morphology between the most common three‐lobed pancreas of the non‐ophidian squamates and the derived compact pancreas of snakes (Moscona, 1990; Stahl, 2003). Although the structure of the lizard pancreas has been described in detail, its development is poorly understood.

Most researchers postulate that the pancreas of lizards develops from three primordial (buds)—one dorsal and two ventral (Bargmann, 1939; Brachet, 1896; Bencosme, 1955; Frye, 1958, 1959; Laguesse, 1901; Miller, 1963; Orrù, 1899; Saint‐Remy, 1893; Siwe, 1926). Although the dorsal primordium always develops in lizards during pancreas differentiation, there is considerable interspecific variation in contributions to the definitive pancreas by ventral primordia (Miller & Lagios, 1970). Hoffmann (1890) denied the presence of ventral primordia in the reptile pancreas's development. Völker (1902) considered that in Lacerta agilis they are present but not involved in pancreatic differentiation. In Podarcis muralis (Brachet, 1896) and the other squamates (Siwe, 1926), only the right ventral pancreatic primordium develops to form the mature ventral pancreas. On the other hand, Miller (1963) found that in the desert night lizard Xantusia vigilis, the right ventral primordium is present only transiently, and the left ventral primordium forms the ventral pancreatic lobe.

A review of the literature indicates that knowledge of the development of the pancreas of lizards, compared to the development of this organ in other vertebrates, is sparse, and the data presented contain many inaccuracies. The inconclusive data on the lizard pancreas development may be due to the use of only 2D histological scrapings in the study, which provide only partial and static information on the organogenesis of this organ. The lack of systematic developmental studies on the lizard pancreas means that only hypotheses are provided regarding the evolutionary differentiation of this organ.

The current study compared the pancreas development in two gekkotan species, the mourning gecko L. lugubris (Gekkonidae) and the leopard gecko E. macularius (Eublepharidae), based on histological slide analyses (2D) and three‐dimensional (3D) reconstruction. The data obtained allowed us to localize the pancreatic primordia and determine their number precisely and made it possible to analyze the connections between the pancreas and the surrounding organs. Due to the diversity of structures described and the processes taking place in them, the discussion has been divided into subsections.

4.1. Early stages of pancreas development

Based on histological studies and 3D reconstructions, at the moment of egg laying, the pancreas of L. lugubris is composed of three distinct primordia: one dorsal and two ventral. It is the same as described in some squamate species such as Podarcis muralis (Brachet, 1896), the ocellated skink (Chalcides ocellatus), Gekko (Siwe, 1926), desert night lizard Xantusia vigilis (Broman, 1937; Miller, 1963), and Chalcides chalcides (Giannelli & Giacomini, 1896). Similarly, three pancreatic buds—one dorsal and two ventral—have also been described in many vertebrate species (Odgers, 1930; Tadokoro et al., 2003).

The dorsal primordium of the pancreas of the mourning gecko differentiates earlier than either ventral primordium. A similar situation has been described in other reptiles as well as many other vertebrate species (Adda et al., 1984; Frye, 1958; Giannelli & Giacomini, 1896; Holtfreter, 1925; Jackintell & Lance, 1994; Jarikji et al., 2009; Pictet et al., 1972; Slack, 1995; Tribe, 1918). At the time of egg laying, it comprises a long duct connecting the gut and a cluster of undifferentiated cells. During embryogenesis, these cells differentiate, forming strands divided into small ducts and acini. Similar histological differentiation of the dorsal part of the pancreas was described in grass snake (Natrix natrix) embryos (Kowalska et al., 2017).

During the early developmental period, the ventral primordia of the mourning gecko pancreas differ in the time of their appearance and vary in size. The right ventral primordium of this species is more prominent, most distinct, and it starts to form earlier than the left one. Both ventral primordia are located symmetrically on both sides of the intestine, similarly as in other vertebrates (Slack, 1995). However, in the mourning gecko, both ventral buds are involved in the development of the pancreas. They merge and participate in the formation of the ventral pancreas. The same has been found in the teleostean fish medaka (Assouline et al., 2002) and in some amphibians (Jarikji et al., 2009; Kelly & Melton, 2000).

Internal organs of the leopard gecko at the time of egg laying were more advanced in development than in the mourning gecko. Based on histological studies and 3D reconstructions, at this time, the pancreas of the leopard gecko is composed of one dorsal and two ventral primordia. Moreover, at this developmental stage, the left ventral primordium was found as a duct. Tracking the development of the pancreas of this species, this duct atrophies at later stages of development. Thus, it can be assumed that in the embryos of this species, the left pancreatic primordium of the differentiating pancreas is a transitional structure. The transient presence of one of the ventral pancreatic attachments has also been described in various reptiles. Complete differentiation of the right ventral pancreatic bud was described in Podarcis muralis (Brachet, 1896), different squamates, and Alligator (Siwe, 1926). Moreover, a study by Miller (1963) also showed regression of one ventral bud in Xantusia. In this species, the right one has disappeared. These findings suggest that in the leopard gecko, the early morphogenesis of the pancreas is similar to that in mammals in terms of the regression of one ventral bud (Edlund, 2002; Kamisawa et al., 2010; Lammert et al., 2003).

Differences are observed in the early stages of pancreas development in mourning and leopard geckos. These differences are related to the fact that in the mourning gecko, all three pancreas buds (one dorsal and two ventral) are involved in the differentiation of the pancreas, whereas in the leopard gecko, only two (dorsal and right ventral) are involved.

4.2. Pancreatic bud differentiation

At the earliest developmental stages, the dorsal pancreas anlage in the studied lizard species is composed of two parts: exocrine and endocrine. A similar structure of the dorsal bud has been described in the embryonic pancreas of other reptilian species (Miller, 1963; Siwe, 1926). However, despite the similar structure, at the time of egg laying, the dorsal buds of these two Gekkota species differ in the degree of their differentiation. In the mourning gecko embryos, the dorsal pancreatic bud is formed by one large duct with cylindrical epithelium ending with the sizeable endocrine cell islet.

In the leopard gecko embryo, the dorsal pancreatic bud is formed by the sizeable branched duct with stratified epithelium and a few smaller pancreatic islets of different sizes. The structure of the dorsal pancreatic bud in the mourning gecko indicates the earlier stage of this bud differentiation, and the single sizeable pancreatic islet can be referred to as the primary islet of Langerhans (Laguesse, 1893; Siwe, 1926), which is a primordium of the pancreatic islets. During the subsequent developmental events within the dorsal bud, the single pancreatic duct starts to branch, which correlates with the disintegration of the primary islet of Langerhans into smaller ones (Siwe, 1926). This dorsal pancreatic bud differentiation step can be found in the embryonic pancreas of leopard geckos just after egg laying.

At the time when the dorsal pancreatic bud is formed by a small number of ducts and islets, the ventral buds in the studied lizard species form only small ducts, and their differentiation is inhibited. At stage 18 in the mourning gecko, a certain number of ducts and islets form the dorsal pancreatic bud. At stage 32, the leopard gecko ventral bud starts to differentiate, and its differentiation is similar to the mourning gecko at developmental stage 18. Similar differentiation of pancreatic buds was described in Xantusia vigilis (Miller, 1963) and chick embryos (Kim et al., 1997).

4.3. From bud connection to lobe formation

The pancreatic buds (three of mourning gecko and two of leopard gecko) fused quickly and formed an embryonic pancreas. After that, the structure of this organ changed. This change is connected with the differentiation of the final part of the pancreas. After fusion, the pancreas development of both studied gekkotans comprises four parts: the head of the pancreas (central region) and the three lobes: upper, splenic, and lower. The head of the pancreas is the central region of the embryonic gland from which all three lobes emerge. The pancreatic lobes of the studied gekkotans are more or less connected. Moreover, it is a place through which the duct of the gallbladder, hepatic duct, and two pancreatic ducts enter the duodenum. In both species, the head, upper, and lower lobes differentiate from the ventral buds of the pancreas, but the splenic lobe originates from the dorsal pancreatic primordium. These results are similar to other lizard species (Miller & Lagios, 1970; Moscona, 1990).

In both studied Gekkota species, the upper lobe of the pancreas is divided into upper and lower parts. However, it has been noted that the upper lobes in both studied species differ in size and relation to the splenic lobe. The lower part is curved toward the splenic lobe, and the upper part is elongated toward the gallbladder. Furthermore, prior to hatching, the cephalic end of the lower portion exhibits a distinctive thickening. It is different from those described in other lizard species (Moscona, 1990).

The elongated splenic lobe of both studied Gekkota species extends from the pancreas head and is curved into the spleen. The distal end of the splenic lobe, located near the spleen, contains thickening. In the mourning gecko, it is present very early, just after this lobe formation, but in the leopard gecko, it appears much later when this lobe is differentiated. As in other lizards, specifically, the thickening of the splenic lobe in the studied Gekkota species, similarly as in other lizards and boid snakes, can be called the juxtasplenic body. The splenic process of the same lizard species, similar to that in boids, is completed or elongates into a structure called the juxtasplenic body (Moscona, 1990).

The results of this study indicated that within the splenic lobe and especially in that part of it that lies adjacent to the spleen, in both studied Gekkota species, there are pancreatic islands. It is similar to the pancreas of other reptilian species (Miller, 1963; Moscona, 1990). A cluster of pancreatic islands within the juxtasplenic body of the boid and leptotyphlopid pancreas constitutes a distal extension of the dorsal lobe. It is homologous with the islet mass (“principal islet”) located within the compact dorsal lobe of the Colubrid‐type pancreas (Kowalska et al., 2017; Moscona, 1990). Concurrently, this islet cluster is homologous with the juxtasplenic body of the Varanus pancreas and the splenic process of the lizard (Moscona, 1990). In the light of phylogenetic context, it is crucial to consider that an extended lizard pancreas can be regarded as an ancestral condition of the Colubridae pancreas.

Interestingly, pancreatic islets were not found within the splenic lobe in the pancreas of the adult Nile monitor lizard Varanus niloticus (Mohammed et al., 2021) and water monitor lizard Varanus salvator (Hamny et al., 2016). Still, the islets were found in the duodenal lobe (Mohammed et al., 2021).

The upper part of the upper lobe in the studied species is elongated and turned toward the gallbladder. Initially, this part is short but reaches the gallbladder during hatching. This study's findings are similar to those for most lizard species (Moscona, 1990).

The lower lobe in the pancreas of the studied species is located in the loop of the small intestine. Although the location of this lobe in the two species studied is similar, their structure differs markedly. The lower lobe of the leopard gecko is elongated and divided into the caudal part, while in the mourning gecko, it is only a tiny thickening at the time of hatching. These differences in the structure of the lower lobe between the two studied species could be related to the species‐specific differences and anatomy of these lizards (Moscona, 1990). Within the lower lobe, in both studied Gekkota species, small pancreatic islets between the ducts are present. This differs from the observations of Moscona (1990), who reported that in the pancreas of most adult lizards, pancreatic islets were found only in the splenic lobe.

The results of these studies indicated that the pancreas of the mourning gecko and the leopard gecko develops gradually and is very well distinguished at the hatching time. Their structure at hatching is challenging to compare with the adult pancreas studied by Moscona (1990). It can be surmised that this is because there has been no systematic study of the lizard pancreas based on three‐dimensional reconstructions to date.

4.4. Early connection between embryonic pancreas and gallbladder and liver

The literature review indicated that connection between embryonic pancreas and gallbladder and liver in reptilian species has not been systematically studied (Hill, 1926; Kowalska et al., 2017; Siwe, 1926).

Results of our study indicated that the shape of the gallbladder and its topographical relationship to the liver differ among studied lizard species. In the embryonic pancreas of the leopard gecko just after egg laying, the gallbladder is localized at the ventral right side of the embryo. It is oval in shape on the cross sections, and its wall is composed of cylindrical epithelium. The liver surrounds the gallbladder except the ventral side, which is covered only by the connective tissue. Moreover, in the earliest developmental stages of the mourning gecko, the gallbladder is oval or C‐shaped, with cylindrical epithelium. It contacts the liver only from the dorsal side.

From the gallbladder of the leopard gecko emerges the cystic duct, which is elongated toward the duodenum. Slightly below the outlet of the cystic duct from the gallbladder, on the ventral side of the gallbladder, is the termination of the right ventral pancreatic bud duct. At the same level, the hepatic duct exits from the liver. All three ducts elongate in the caudal direction toward the duodenum. First, the cystic duct connects to the hepatic duct, forming the common bile duct. Then, downstream, near the duodenum, at the level of the right ventral pancreatic bud termination, the duct of the right ventral bud connects to the common bile duct. The right ventral duct connects to the common bile duct below, at its dorsal side, near the dorsal pancreatic duct. Moreover, the hepatic duct of the mourning gecko emerges from the liver at the gallbladder level. Below it, the cystic duct emerges from the gallbladder. The cystic duct merges with the hepatic duct to form the common bile duct. The ventral pancreatic buds are connected with the common bile duct. Literature review indicated that similar situation was described in many reptilian species, but numerous variations may be present (Guibé, 1970).

The results of our developmental studies obtained based on 3D reconstructions of the differentiated pancreas of both species indicated that the connection between embryonic pancreas and gallbladder and liver has been very similar. There is only some information about the early connection between embryonic pancreas, gallbladder, and liver in Lacerta, and Gongylus ocellatus. The ducts from the left and right ventral pancreatic buds in the species as mentioned above discharge separately into a common bile duct (Siwe, 1926). The grass snake developmental studies of the pancreas indicated that the common bile duct connects to the ventral pancreatic duct during the early developmental stages. During the subsequent stages, the common bile duct connects to the main pancreatic duct before entering the duodenum (Kowalska et al., 2017). Similar connections were also described in Natrix natrix, Python reticulatus (Hill, 1926), and Varans (Van Dijk, 1935).

4.5. Pancreatic duodenum connection formation

The literature review indicated that the connections of the pancreas with the duodenum are different in reptilian species. In snakes, these connections have two scenarios (Kowalska et al., 2017; Moscona, 1990). For example, in the adder Vipera berus and the Israeli worm snake Typhlops simoni, the dorsal and ventral pancreatic ducts have a distinct outlet into the duodenum. In addition, the cystic duct enters together with the ventral duct (Moscona, 1990). Moreover, in other snakes, such as the grass snake Natrix natrix (Kowalska et al., 2017), small‐banded kukri snake Oligodon melanocephalum, javelin sand boa Eryx jaculus, and long‐nosed worm snake Leptotyphlops macrorhynchus, the pancreatic ducts (dorsal and ventral), cystic and hepatic duct enter a common outlet, the duodenum (Moscona, 1990). In lizards, mainly in Eumeces, the duct of the dorsal lobe, named the duct of Santorini, runs from the juxtasplenic body down the dorsal lobe into the ampulla. Moreover, the duct of the ventral lobe, the duct of Wirsung, has two branches in Eumeces and other lizards with a dual ventral lobe (Gabe, 1970; Moscona, 1990).

The results of these studies indicated that the pancreatic buds of the leopard and mourning gecko initially enter the duodenum by separate outlets, which is similar to the pancreas of other vertebrates (Kowalska et al., 2017; Moscona, 1990; Pictet & Rutter, 1972). The dorsal bud in the studied Gekkota species is connected with the duodenum by the dorsal duct, but the ventral buds and the hepatic duct link to the duodenum by the common bile duct. These connections are similar to those described in all vertebrate species (Moscona, 1990; Pictet & Rutter, 1972; Siwe, 1926).

During the embryonic development of the studied Gekkota species, the cystic duct escapes separately into the ampulla and partially passes through the pancreas. It is quite different from adult lizards. In the lizard species, the cystic duct does not traverse the pancreatic gland as is the case with snakes but enters the ampulla directly (Moscona, 1990).

The findings of these studies indicate that during the development of studied lizards, the common bile duct and the dorsal pancreatic duct join together and enter the duodenum by the common outlet, which could be called the papilla of Vater (Moscona, 1990). The papilla of Vater of the studied Gekkota embryos initially protrudes from the duodenum. Then, during the successive developmental stages, it is probably pulled into the intestinal wall perimeter, connected with the decreasing duct diameters. As a result, it may give the impression that each of the four ducts (cystic, hepatic, and two pancreatic ducts) exits separately into the duodenum, but in fact, they open into a common outlet, the papilla of Vater. These results are different from those in other vertebrate species because in mammalian species, each pancreatic duct enters the duodenum by a separate outlet (Adda et al., 1984).

During gekkotan pancreas differentiation, the connection between the common bile duct and the dorsal pancreatic duct is associated with intestinal rotation. Similar intestinal rotation has been described in all vertebrate species (Gittes, 2009; Kowalska et al., 2017; Slack, 1995).

4.6. Summary

Despite the huge evolutionary diversity of lizards (non‐ophidian squamates), the embryonic development of the pancreas in these reptiles remains poorly known. Unlike snakes, our understanding of the structural differentiation of the pancreas in lizard species remains limited. It may result in an incomplete understanding of evolutionary and developmental aspects of morphological pancreatic variation within squamate reptiles. The adult pancreas of lizards differs from that of adult snakes not only in the minor degree of pancreatic bud fusion but also in its connections to the gallbladder. The pancreas morphology of adult lizard species shows many primitive features, for example, the pancreas containing three branches equivalent to the three pancreatic buds. This type of pancreas is considered plesiomorphic in contrast to the derived morphology of the ophidian pancreas. Two ventral pancreatic buds undergo a smaller or larger fusion in lizards. Complete fusion of the pancreatic buds occurs in snakes, where the pancreas forms a compact organ. It is the highest sign of pancreatic specialization in the squamates. Therefore, studying the development of the pancreas in two different species of geckos will be helpful in determining whether a gecko's phylogenetic position affects how the pancreas differentiates.

The stages of the two studied gekkotans are aligned in Table 2 according to the common developmental events (e.g., the first appearance of the structure or the beginning of specific processes – see 3D movies (Videos [Link], [Link]) in the supplementary online Appendix). The exception is the last stage alignment, in which the adult‐like (pre‐hatch) stages of E. macularius and L. lugubris are compared. The alignment of stages presented in Table 2 does not imply homology of the stages, since developmental sequences are taxon‐specific, and a normal developmental table prepared for one species should not be used for the other species (Andrews et al., 2013). Thus, the stage alignment was used to facilitate between‐species comparison of pancreas development.

AUTHOR CONTRIBUTIONS

WR – funding acquisition, the concept of manuscript, acquisition of data (cutting and staining of histological sections), data analysis, writing‐original draft, writing‐review, and editing. MK – acquisition of data (embryo fixation, embedding, cutting, and staining of histological sections), data analysis, visualization, 3D reconstructions, writing results, and manuscript editing. PK – collecting embryos of both species, external morphological analysis of embryos and stage evaluation, data acquisition (embryo fixation, embedding, cutting, and staining of histological sections), visualization, 3D reconstructions, writing‐review, and editing. All of the authors reviewed and accepted the draft.

CONFLICT OF INTEREST STATEMENT

Authors have no conflicts of interest to declare.

ETHICS STATEMENT

The Institute of Biology, Biotechnology and Environmental Protection obtained approval from the District Veterinary Office in Katowice – Poland (PIW.ZOZ.OZ.5342.5.1/17). The long‐standing non‐commercial breeding of the Institute of Biology, Biotechnology and Environmental Protection is registered in the Polish Ministry of Science and Higher Education (number 033). The gecko species are not included in the Washington Convention of 1973 (Guidelines for appropriate uses of IUCN red list), which was ratified by Poland in 1991 (Journal of Laws, No. 27 item 112 and latest regulations of Journal of Laws, 2000 No. 66 item 802 and Journal of Laws, 2004 No. 112 item 1183). The research was performed following Directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010, on the protection of animals used for scientific purposes and the Act of January 15, 2015, on the protection of animals used for scientific or educational purposes (Journal of Laws 2015 item 266) and later changing (Journal of Laws 2021 item 2338).

Supporting information

Video S1. Eublepharis macularius; stage 30.

Video S2. Eublepharis macularius; stage 32.

Video S3. Eublepharis macularius; stage 34.

Video S4. Eublepharis macularius; stage 38.

Video S5. Eublepharis macularius; stage 42.

Video S6. Lepidodactylus lugubris; stage 5.

Video S7. Lepidodactylus lugubris; stage 12.

Video S8. Lepidodactylus lugubris; stage 18.

Video S9. Lepidodactylus lugubris; stage 28.

Video S10. Lepidodactylus lugubris; stage 50.

Kowalska, M. , Kaczmarek, P. & Rupik, W. (2024) Does the pancreas of gekkotans differentiate similarly? Developmental structural and 3D studies of the mourning gecko (Lepidodactylus lugubris) and the leopard gecko (Eublepharis macularius). Journal of Anatomy, 245, 303–323. Available from: 10.1111/joa.14038

DATA AVAILABILITY STATEMENT

The data supporting this study's findings are available from the corresponding author upon reasonable request.