Emilie Bricout-Neveu

Severine Pechberty

Karine Reynaud

Cindy Maenhoudt

Marie José Lecomte

Philippe Ravassard

Paul Czernichow
email: paul.czernichow@wanadoo.fr

Development of the Endocrine Pancreas in the Beagle Dog: From Fetal to Adult Life

Keywords: Fetal development, Pancreas, Dogs, Morphology, Insulin, Glucagon

Our objectives were to describe, in Beagle dogs, the ontogenesis of beta (insulinproducing) and alpha (glucagon-producing) cells from fetal to early postnatal life and adulthood.

In addition, to have some insight into interspecies comparison, Beagle dog pancreases were compared to pancreases from a Labrador and Chow Chow.

At midgestation, the epithelium was dense, beta cells scarce, and alpha cells numerous and concentrated in the center of the pancreatic bud. From 36 to 45 days post conception (pc), beta cell numbers increased and the epithelium expanded and branched out. At 55 days pc, large beta cell aggregates were seen. At weaning, the islets were similar to those in adults, with limited alpha cells intermingled with numerous beta cells. Quantification of the Alpha to Beta cells ratio has shown a gradual increase of beta cells proportion throughout development.

Similar findings were obtained in the 2 other breeds.

In conclusion, in the fetal Beagle dog beta cells emerge from the pancreatic bud at midgestation, but the endocrine structure is mature only in early postnatal life. The ontogenesis of the endocrine pancreas demonstrated in dogs resembles that reported in rats and mice. In contrast, human beta cells appear earlier, at the beginning of the second trimester of gestation.

Our study provides a detailed morphological description of pancreatic development in dogs but supplies no information on alpha-or beta-cell function during fetal life. The morphological data reported here provide a foundation for building physiological studies.

INTRODUCTION

In mammals, fetal growth and maturation depend heavily on maternal nutrition and fetal insulin secretion. Any dysfunction in the process of insulin secretion adversely affects fetal growth and body composition. The important role for insulin in fetal growth is highlighted by the effects on human fetuses of maternal abnormalities in blood glucose control. Poorly controlled diabetes, maternal obesity, and excessive maternal weight gain are associated with fetal and neonatal macrosomia and with intermittent hyperglycemia. In these situations, the high transplacental transfer of glucose causes fetal hyperglycemia and stimulates the fetal release of insulin [START_REF] Obenshain | Human fetal insulin response to sustained maternal hyperglycemia[END_REF] and other growth factors. In turn, the increased insulin secretion stimulates fetal growth and the accumulation of fat and glycogen. The Hyperglycemic and Adverse Pregnancy Outcomes study in 23 316 pregnant women showed that maternal hyperglycemia was associated with birth weight and cord-blood serum C-peptide level values above the 90 th percentile [START_REF] Hapo Study | Hyperglycemia and adverse pregnancy outcomes[END_REF].

Conversely when fetal insulin is low or absent, fetal development is impaired. Intrauterine growth restriction is the rule in newborns with neonatal diabetes, a disease linked to genetic abnormalities in insulin control or production [START_REF] Polak | Neonatal diabetes mellitus: a disease linked to multiple mechanisms[END_REF], and in leprechaunism, a monogenic disease characterized by severe insulin resistance [START_REF] Kosztolanyi | Leprechaunism/Donohue syndrome/insulin receptor gene mutations: a syndrome delineation story from clinicopathological description to molecular understanding[END_REF]. These conditions illustrate the crucial role for insulin in fetal growth, especially during the last trimester of pregnancy.

Elucidating the function of the endocrine pancreas, most notably that of the beta cells that produce and secrete insulin, is therefore important to understand normal fetal development in mammals. In humans, studies of the above-mentioned conditions have provided valuable insights into fetal insulin secretion. Investigations into the physiology of fetal insulin secretion in rodents, sheep, and rhesus monkeys [START_REF] Aldoretta | Maturation of glucose-stimulated insulin secretion in fetal sheep[END_REF][START_REF] Mintz | Fetal insulin and growth hormone metabolism in the subhuman primate[END_REF] consistently demonstrated a pivotal role for fetal insulin. Data from humans, monkeys, and rats indicate that glucose is the main source of energy during fetal 4 life in these species Humans and rats are omnivores and most monkeys are herbivores with a preference for fruit. Among the hormones involved in fetal nutrition in these species, the most important are glucagon and insulin. Little is known about the fetal nutrition of carnivores.

Carnivores, including dogs, must draw most of their energy during fetal life from the breakdown of animal proteins ingested by the mother. The alpha and beta cells in the islets of Langerhans secrete glucagon and insulin. We therefore studied the ontogenesis of alpha and beta cells in dogs. Our goal was to create a foundation for physiological studies of the role for insulin and glucagon during fetal life in this species.

Our primary objective was to determine when hormone-producing cells appeared in the primitive pancreas. To this end, we used immunohistochemistry to detect cells containing insulin or glucagon, A secondary objective was to determine at which stage of fetal or early postnatal development the highly organized structure of the islets of Langerhans was formed. We also studied the immunohistological structure of the endocrine pancreas in adult dogs.

MATERIAL AND METHODS

All procedures involving animals were submitted to and approved by the institutional review board of the Maisons-Alfort Veterinary School, Maisons-Alfort, France. Our methods complied with international regulations for the use of animals in experimental studies. one were from a single strain of Beagle dogs, raised at the Alfort Veterinary School. The exception was the 55-day pc fetus, whose mother resembled a Beagle dog but had no pedigree and was not raised at the Alfort Veterinary School. According to Miller (7), the cutoff between embryonic and fetal life in dogs is 33 days. Thus, our first two specimens were from embryos and the other four from fetuses. All prenatal specimens were obtained by elective caesarean section. The age of the fetuses was determined based on the time of ovulation identified based on the plasma progesterone surge; the exception was the 55-day pc fetus.

Sources of canine pancreatic tissue and sample collection procedure

As shown in

Pancreases were also obtained from two Beagle dogs that died during hospitalization, one on day 1 post delivery and the other at weaning at 8 weeks of age.

Finally, we studied a pancreas from an adult Labrador that was euthanized because of a progressive and severely debilitating neuro-muscular disorder. We also compared the morphology of the Beagle dog pancreases at 46 days pc and 1 day postnatally to those of a Labrador and Chow Chow, respectively, at the same ages (Table 2).

Preparation of canine pancreatic tissue

Immediately after surgery, each pancreas was dissected and fixed in 3.7% formaldehyde then embedded in paraffin. The entire midgut tube including the pancreas and stomach was collected from the 30-day and 33-day pc fetuses, whereas only the pancreas was dissected from the four older fetuses. The pancreases from the 1-day-old and 8-week-old dogs were dissected within 1 hour after death and fixed in phosphate buffer saline/10% formaldehyde prior to paraffin embedding. The same procedure was applied to the pancreatic fragment obtained from the adult Labrador. In that case the pancreatic samples were collected from the right lobe.

Standard hematoxylin-eosin (HE) staining was performed. As shown in Table 1, numerous slices were obtained from each specimen. The slices to be examined were taken at regular intervals and considered representative of the entire gland.

Digital images were taken using an Axio Scan Z1 camera (Zeiss, Oberkochen, Germany).

Quantification of alpha and beta cells

Alpha and beta cells were enumerated by counting the number of nuclei in immunostained cells. Nucleus counts were performed using ImageJ on whole Axio Scan images (Zeiss, Oberkochen, Germany). The percentage of either glucagon-or insulin-positive cells relative to total glucagon and insulin cells was calculated. The number of immunostained sections for each developmental stage varied from 10 to 15 and the sections used for quantification were chosen every 10 to 40 sections, depending on the size of the tissue sample.

RESULTS

Comment citer ce document : Bricout-Neveu, E., Pechberty, S., Reynaud, K., Maenhoudt, C., Lecomte, M. J., Ravassard, P., Czernichow, P. ( 2017 Few insulin-positive beta cells were visible on days 30 and 33 pc. At the earliest stages, only 25% of examined slices contained beta cells, in very small numbers. Thus, insulin-positive cells seemed to emerge around midgestation (30 days pc). Glucagon cells were seen far more frequently. In the pancreatic buds, both cell types were isolated and scattered throughout the bud.

By day 36 pc, counts of both cell types were higher but alpha cells still predominated. The endocrine cells were chiefly seen near the trunks of the branched epithelial system; most were isolated, although small clusters of alpha or beta cells were found. On day 45 pc, the clusters were larger and some of them contained both alpha and beta cells.

Late gestation and early postnatal life

Quantification of alpha and beta cells (Figure 4)

The proportion of beta cells increased gradually, from 2.7% on day 30 pc to 42.3% on day 55 pc. After birth, this proportion continued to rise. At weaning (8 weeks of age), the beta cell proportion reached 61.06% , a proportion still significantly lower than the proportion found in adult pancreas (87.02%).

Comparisons across breeds

We compared pancreatic morphology at 45 days pc in a Beagle and a Labrador and on postnatal day 1 in a Beagle and a Chow Chow (Figure 5). At both time points, the two breeds were similar regarding the general morphology of the branched pancreatic epithelium and the number and distribution of alpha and beta cells. On postnatal day 1, clusters or islets of endocrine cells were visible in both breeds. In the mammalian pancreas, endocrine cells cluster into islets of Langerhans, which are embedded in the exocrine tissue and secrete insulin, glucagon, and other polypeptide hormones into the bloodstream. These islets have been extensively studied in adult mammals and are conserved across species [START_REF] Steiner | Pancreatic islet plasticity: interspecies comparison of islet architecture and composition[END_REF][START_REF] Tsuchitani | A comparison of the anatomical structure of the pancreas in experimental animal[END_REF]. The development of the fetal and postnatal endocrine pancreas has been studied in depth in rodents [START_REF] Jørgensen | An illustrated review of early pancreas development in the mouse[END_REF] and humans [START_REF] Béringue | Endocrine pancreas development in growth-retarded human fetuses[END_REF][START_REF] Polak | Early pattern of differentiation in the human pancreas[END_REF] but not in other mammals. The objective of this work was to describe the differentiation and growth of alpha and beta cells from early fetal to postnatal life in the Beagle dog. We found that beta cells emerged from the pancreatic bud at midgestation in dogs. However, in the Beagle dog and two other dog breeds, the fully mature endocrine structure was not present until early postnatal life. During prenatal life, alpha cells outnumbered beta cells. The ratio of beta to alpha cells increased during fetal life and after birth. Importantly even at 8 weeks of age, i.e., at weaning, the ratio remained below the adult value. Since we measured the percentage of either alpha or beta cells relative to the total population of alpha plus beta cells, the increase in the beta/alpha cell ratio does not necessarily indicate expansion of the beta cell mass after weaning. A possible bias is that the size and cellular composition of the canine islets vary according to their location in the adult pancreas and tend to increase along a gradient from the right to the left lobe. [START_REF] Steiner | Pancreatic islet plasticity: interspecies comparison of islet architecture and composition[END_REF][START_REF] Tsuchitani | A comparison of the anatomical structure of the pancreas in experimental animal[END_REF][START_REF] Justice | A comparative immunohistochemical study of pancreatic islets in laboratory animals (rats, dogs, minipigs, nonhuman primates). Exp; toxicol[END_REF]14). Alpha and beta cell counts during antenatal and early postnatal life were determined on several pancreatic slices that were representative of the entire organ. Consequently, comparing them to the values obtained in a single part of the adult pancreas may not be entirely valid. However it should be noted that, Genetic studies in mice and humans [START_REF] Jennings | Human pancreas development[END_REF][START_REF] Cano | Transcriptional control of mammalian pancreas organogenesis[END_REF][START_REF] Habener | Minireview: transcriptional regulation in pancreatic development[END_REF] have led to the identification of a vast number of transcription factors that participate in pancreatic organogenesis Our findings indicate that the key morphological events of pancreatic development described in mice and humans also occur in dogs. Immunohistological studies provide valuable information on morphology, but not on function. Thus, whether the transcriptional regulatory mechanisms governing pancreatic development in dogs are identical to those in rodents cannot be determined from our work.

DISCUSSION

Further studies are needed to elucidate this point.

Several immunocytochemical studies of the distribution of beta and alpha cells in normal adult canine pancreatic islets have been published [START_REF] Béringue | Endocrine pancreas development in growth-retarded human fetuses[END_REF][START_REF] Polak | Early pattern of differentiation in the human pancreas[END_REF]. Our results agree with previous anatomical studies showing that, in addition to fully formed islets, small beta-cell aggregates and a few isolated alpha cells can be seen. In the islets, the alpha (glucagon) cells were usually in a peripheral location [START_REF] Béringue | Endocrine pancreas development in growth-retarded human fetuses[END_REF][START_REF] Polak | Early pattern of differentiation in the human pancreas[END_REF]. In the adult Labrador in our study, the alpha cells were intermingled with the beta cells within fully formed islets instead of being confined to the periphery.

However, only the right lobe of the pancreas was examined, and differences have been reported

between the right and left lobes. Thus, variations in the size and cellular composition of canine islets depending on location in the pancreas may explain the discrepancies between our findings and those reported by others. A detailed morphological comparison between islets from the head (right lobe) and tail (left lobe) is needed.

Although we used Beagle dogs for most of our studies, we also studied two other breeds.

We were thus able to compare the findings in Beagle dogs with those in a Labrador at 45 days pc and in a Chow Chow on postnatal day 1. The endocrine cell clusters were similarly distributed between breeds at both ages. On day 45 pc, alpha cells predominated and beta and alpha cells formed aggregates that did not exhibit the islet structure. On postnatal day 1, in contrast, alpha and beta cells were present in comparable numbers and aggregated into clusters that resembled islets without having the typical architecture of mature islets. These data suggest that pancreatic development may be similar across dog breeds.

In conclusion, our data demonstrate that beta cells are visible by midgestation and that islets form a few days before delivery in dogs. We did not evaluate whether the beta cells secreted insulin, thereby regulating the fetal glucose metabolism. Further studies are needed to elucidate the functional capabilities of the fetal pancreas. The morphological data reported here should prove

Comment citer ce document : Bricout-Neveu, E., Pechberty, S., Reynaud, K., Maenhoudt, C., Lecomte, M. J., Ravassard, P., Czernichow, P. (2017). Development of the endocrine pancreas in the Beagle dog: From fetal to adult life. Anatomical Record, 300 (8), 1429-1438. , DOI : 10.1002/ar.23595

14

We thank the Alfort Veterinary School clinicians for their invaluable help in collecting prenatal and postnatal canine tissues.

Funding: This research project was partly supported by funding from the BPI-France AIMA Programme under grant agreement #DA15005Q00/05

The Anatomical Record

This article is protected by copyright. All rights reserved.

  we obtained antenatal dog pancreas specimens at six developmental stages, namely, days 30, 33, 36, 40, 45, and 55 postconception (pc). All antenatal samples but Comment citer ce document : Bricout-Neveu, E., Pechberty, S., Reynaud, K., Maenhoudt, C., Lecomte, M. J., Ravassard, P., Czernichow, P. (2017). Development of the endocrine pancreas in the Beagle dog: From fetal to adult life. Anatomical Record, 300 (8), 1429-1438. , DOI : 10.1002/ar.23595 5

Figure 1 A

 1 Figure 1 A-B illustrates the development of the dog pancreas from 30 to 45 days pc. As

Figure 2 A 8 Figure 3

 283 Figure 2 A-B shows the pancreases during the perinatal period (55 days pc, postnatal day

  Comment citer ce document : Bricout-Neveu, E., Pechberty, S., Reynaud, K., Maenhoudt, C., Lecomte, M. J., Ravassard, P., Czernichow, P. (2017). Development of the endocrine pancreas in the Beagle dog: From fetal to adult life. Anatomical Record, 300 (8), 1429-1438. , DOI : 10.1002/ar.23595 9

  according to most authors cited above, the left right lobe is less rich in beta cells and therefore our decision to compare our data during fetal and post natal life to the right pancreatic adult lobe underestimate the density of beta cells in the adult stage. Comment citer ce document : Bricout-Neveu, E., Pechberty, S., Reynaud, K., Maenhoudt, C., Lecomte, M. J., Ravassard, P., Czernichow, P. (2017). Development of the endocrine pancreas in the Beagle dog: From fetal to adult life. Anatomical Record, 300 (8), 1429-1438. , DOI : 10.1002/ar.23595 10 Since the pioneering work conducted by Pictet et al. in rats and reported in 1972 (15), the morphological development of the pancreas has been studied in great detail, chiefly in mice. Three developmental stages have been identified. The first phase, known as the primary transition period, is the early undifferentiated stage during which morphogenesis occurs. The prepatterned endodermal epithelium of the foregut develops into branching ducts and undifferentiated epithelium. In mice, this occurs between embryonic days (E) 8.5 and E-12.5. The secondary transition extends in mice from E-13 to birth. The buds begin to differentiate into endocrine and exocrine cellular lineages by E-14 and the pancreatic epithelium proliferates and expands extensively. By E-15, the dorsal and ventral pancreatic buds rotate and fuse, and the pancreas is almost fully developed by E-19, i.e., just before birth. At that stage, the pancreas contains endocrine cells organized into isolated clusters that condense into islets of Langerhans (third developmental transition). Finally, the postnatal period of development extends over the first 2-3 postnatal weeks, during which the endocrine cells become mature and acquire full nutrient responsiveness. Recently, Jennings and colleagues (16) have reviewed the development of human pancreas and compared it with mice. In light of such study our data suggests that pancreatic ontogenesis in dogs resembles that in mice. Although the equivalent in dogs to the primary transition in mice cannot be firmly established using endocrine markers only, our results suggest that insulin expression start at midgestation (30 and 33 days pc), i.e., the time of the primary transition in mice. The enlargement of the pancreas and the expansion and branching out of the epithelium with the formation of trunk and tip domains replicate the secondary transition; and the development after 55 days pc matches the postnatal period. In humans, in contrast, beta cells become detectable at the beginning of the second trimester of gestation (16). As illustrated figure Comment citer ce document : Bricout-Neveu, E., Pechberty, S., Reynaud, K., Maenhoudt, C., Lecomte, M. J., Ravassard, P., Czernichow, P. (2017). Development of the endocrine pancreas in the Beagle dog: From fetal to adult life. Anatomical Record, 300 (8), 1429-1438. , DOI : 10.1002/ar.23595 11 6, the time-pattern of beta-cell development in dogs is more closely similar to that in rodents than in humans, although the mature beta-to-alpha cell ratio is reached much later than in mice and other species.

  Comment citer ce document : Bricout-Neveu, E., Pechberty, S., Reynaud, K., Maenhoudt, C., Lecomte, M. J., Ravassard, P., Czernichow, P. (2017). Development of the endocrine pancreas in the Beagle dog: From fetal to adult life. Anatomical Record, 300 (8), 1429-1438. , DOI : 10.1002/ar.23595 12

Figure 1 : 15 Figure 2 :Figure 3 :

 11523 Figure 1: Dog pancreas development and endocrine cell distribution during the third

Figure 4 : 16 t

 416 Figure 4: Comparison of beta and alpha cell proportions throughout the development of

Figure 5 :Figure 6

 56 Figure 5: Comparison of endocrine cell distribution in the developing pancreas of different

Figure 1 :

 1 Figure 1: Dog pancreas development and endocrine cell distribution during the third quarter of dog gestation 252x386mm (300 x 300 DPI)

Figure 2 :

 2 Figure 2: Dog pancreas development and endocrine cell distribution during the perinatal period and before weening 228x307mm (300 x 300 DPI)

Figure 3 :

 3 Figure 3: Endocrine cells in the adult dog pancreas are organized into islets. 139x150mm (300 x 300 DPI)

Figure 4 :

 4 Figure 4: Comparison of beta and alpha cell proportions throughout the development of the dog pancreas and in adulthood 114x103mm (600 x 600 DPI)

Figure 5 :

 5 Figure 5: Comparison of endocrine cell distribution in the developing pancreas of different dog breeds 202x232mm (300 x 300 DPI)

Figure 6

 6 Figure 6 Comparative scheme of endocrine pancreas development in human mouse and dog. 45x26mm (600 x 600 DPI)

Table

CONFLICT OF INTEREST:

Paul Czernichow, and Philippe Ravassard are own shares in Animal Cell Therapy.

ACKNOWLEDGMENTS


Supplementary Figure 1. Transmission of diluted LAN404 sheep scrapie isolate to transgenic mice overexpressing ovine PrP.

(a) Primary transmission of 1:1000 diluted LAN404 sheep scrapie isolate and subpassage to tg338 mice (6 mice per brain inoculated). On primary transmission, all the mice showed a 21K PrP res signature in their brain. On secondary transmission, made with undiluted brain homogenate from mouse no. 4 and 7, divergent phenotypes were observed: either a short ID

typical of LA21K fast prions (red), or a longer ID together with 21K (grey) and mixed 21K/19K (grey + blue) PrP res profiles. (b) PrP res electrophoretic patterns at primary passage (P1) and secondary passage (P2). The 21K PrP res signatures (fragment size, glycoform ratio) are similar between P1 and P2 for mouse no.7, whereas for mouse no.4 the brains exhibit a 21K fast-like signature at P2. Electrophoretic patterns of LA21K fast and LA19K prions serially passaged in tg338 mice are shown as controls. (c) Representative histoblots (2 nd passage) of antero-posterior coronal brain sections at the level of the septum (i), hippocampus (ii), midbrain (iii) and brainstem (iv). The pattern observed at P2 for mouse no.4 is specific of LA21K fast, with prominent PrP res deposition in the corpus callosum, lateral hypothalamic nuclei and raphe nuclei of the brain stem. The pattern observed at P2 for mouse no.7 is different, with plaque-like PrP res deposits scattered in the corpus callosum, the dorsal thalamus, the optic tract, the periaqueductal area, the tegmental nuclei and the cerebellum.

  Scale bar = 1 mm. Supplementary Figure 3. Transmission of LA19K and LA21K fast prions to mice expressing ovine PrP at middle or low levels. LA19K (blue) and LA21K fast (red) prions at the second passage on tg301 +/--(see Fig. 1a) were further transmitted to lines expressing PrP C at lower levels, as indicated. Segmented circles are used to indicate the proportion of mice with LA19K (blue) or LA21K fast (red) PrP res profiles in their brains. Both LA19K and LA21K fast prions can be faithfully propagated in low or middle expresser mice.

Supplementary Table 1: Duration of disease in mice expressing PrP C at variable levels, inoculated with LAN404 sheep brain or tg338-derived LAN404 prions (2 nd passage, 19K PrP Sc signature).

	Transgenic mouse line PrP C level *	Days to death	Difference
			tg338-LA19K	LAN404
	tg338 +/-	4	175	316	-141
	tg328 +/-	3.5	242	314	-72
	tg211 +/-	2.8	258	341	-83
	tg143 +/-	1.5	327	351	-24
	tg335 +/-	1.2	444	424	+20

*Expressed as x-fold that in sheep brain

The brain stem of the infected mice was enriched in PrP res species with a 21K signature, and prominent proportion of diglycosylated species as compared to LA21K fast (shown as control). A mixed 21K/19K profile was observed in the cerebellum.

Supplementary Note 1. Kinetic simulation of substrain selection as a function of PrP C expression level.

In order to simulate the outcome of LAN404 isolate transmission to transgenic mice expressing PrP C at different, levels, we assumed the following:

1-Two substrains A and B exist in the same isolate.

2-The kinetic order of the templating process for each substrain is different. The replication of substrain A occurs by the integration of three PrP C molecules (n=3) when for substrain B, four molecules are required (n=4). This replication mode contrasts with the generic model proposed by Weissman and colleagues for Sup35, for which the templating occurs through the integration/conversion of one monomer 1 . Recent developments in our group strongly suggest that mammalian prion assemblies are constituted of oligomeric, elementary bricks. As an example, for 263K hamster prions, this elementary brick would correspond to a trimer (Igel-Egalon et al., to be published).

3-The conversion rate remains constant during the evolution of the pathology 4-The fragmentation rate is not considered in order to have a constant conversion process.

5-PrP C expression level does not vary (overexpressing mice) or to a limited extent during the early phase of replication 2 .

Then we defined C i and respectively as the concentration of substrain A and B. i defines the size of assemblies as was defined in the simulation as adimensional parameter.

Kinetics constants used for simulations are : k1 = 100 M -3 s -1 k2 = 0.16 s -1 n=3 k3 = 1.05 M -4 s -1 k4= 0.01 s -1 p=4

Simulations were run using ODE 15s from MATLAB routine for a concentration range of 10 - 8 to 10 -6 as µM. Results were reported as adimentional variable. Phase space were analysed using Jacobin and Rout-Hurwitze criteria using a homemade MATHLAB code.