Introducing dorsoventral patterning in adult regenerating lizard tails with gene-edited embryonic neural stem cells.

Lizards regenerate amputated tails but fail to recapitulate the dorsoventral patterning achieved during embryonic development. Regenerated lizard tails form ependymal tubes (ETs) that, like embryonic tail neural tubes (NTs), induce cartilage differentiation in surrounding cells via sonic hedgehog (Shh) signaling. However, adult ETs lack characteristically roof plate-associated structures and express Shh throughout their circumferences, resulting in the formation of unpatterned cartilage tubes. Both NTs and ETs contain neural stem cells (NSCs), but only embryonic NSC populations differentiate into roof plate identities when protected from endogenous Hedgehog signaling. NSCs were isolated from parthenogenetic lizard embryos, rendered unresponsive to Hedgehog signaling via CRISPR/Cas9 gene knockout of smoothened (Smo), and implanted back into clonally-identical adults to regulate tail regeneration. Here we report that Smo knockout embryonic NSCs oppose cartilage formation when engrafted to adult ETs, representing an important milestone in the creation of regenerated lizard tails with dorsoventrally patterned skeletal tissues.

A staging table of embryonic development for a viviparous (live-bearing) lizard Eremias multiocellata (Squamata: Lacertidae).

As the only viviparous reptile in China that has both temperature-dependent sex determination (TSD) and genetic-dependent sex determination (GSD) mechanisms, Eremias multiocellata is considered as an ideal species for studying the sex determination mechanism in viviparous lizards. However, studies on embryonic stage of viviparous lizards and morphological characteristics of each stage are limited. In the present study, the embryonic development process of E. multiocellata is divided into 15 stages (stages 28-42) according to the morphology of embryos. Embryos sizes are measured and continuous dynamic variation of some key features, including limbs, genitals, eyes, pigments, and brain scales are color imaged by a stereoscopic microscope. Furthermore, based on these morphological characteristics, we compare the similarities and differences in the embryonic development of E. multiocellata with other squamate species. Our results not only identified the staging table of E. multiocellata with continuous changes of external morphological characteristics but also developed a staging scheme for an important model species that provides a necessary foundation for study of sex determination in a viviparous lizard.

Thermal sensitivity of lizard embryos indicates a mismatch between oxygen supply and demand at near-lethal temperatures.

Aspects of global change create stressful thermal environments that threaten biodiversity. Oviparous, non-avian reptiles have received considerable attention because eggs are left to develop under prevailing conditions, leaving developing embryos vulnerable to increases in temperature. Though many studies assess embryo responses to long-term (i.e., chronic), constant incubation temperatures, few assess responses to acute exposures which are more relevant for many species. We subjected brown anole (Anolis sagrei) eggs to heat shocks, thermal ramps, and extreme diurnal fluctuations to determine the lethal temperature of embryos, measure the thermal sensitivity of embryo heart rate and metabolism, and quantify the effects of sublethal but stressful temperatures on development and hatchling phenotypes and survival. Most embryos died at heat shocks of 45°C or 46°C, which is ~12°C warmer than the highest constant temperatures suitable for successful development. Heart rate and O2 consumption increased with temperature; however, as embryos approached the lethal temperature, heart rate and CO2 production continued rising while O2 consumption plateaued. These data indicate a mismatch between oxygen supply and demand at high temperatures. Exposure to extreme, diurnal fluctuations depressed embryo developmental rates and heart rates, and resulted in hatchlings with smaller body size, reduced growth rates, and lower survival in the laboratory. Thus, even brief exposure to extreme temperatures can have important effects on embryo development, and our study highlights the role of both immediate and cumulative effects of high temperatures on egg survival. Such effects must be considered to predict how populations will respond to global change.

Phenotypic consequences of maternally selected nests: a cross-fostering experiment in a desert lizard.

Despite the importance of maternally selected nests in shaping offspring phenotypes, our understanding of how the nest environment affects embryonic development and offspring traits of most non-avian reptiles is rather limited largely due to the logistical difficulty in locating their nests. To identify the relative contributions of environmental (temporal [seasonal] and spatial [nest-site]) and intrinsic (clutch) factors on embryonic development and offspring traits, we conducted a cross-fostering experiment by swapping eggs between maternally-selected nests of the toad-headed agama (Phrynocephalus przewalskii) in the field. We found that nest environment explained a large proportion of variation in incubation duration, hatching success, and offspring size and growth. In contrast, clutch only explained a small proportion of variation in these embryonic and offspring traits. More significantly, compared with spatial effects, seasonal effects explained more phenotypic variation in both embryonic development and offspring traits. Eggs laid early in the nesting season had longer incubation durations and produced smaller hatchlings with higher post-hatching growth rates than did later-laid eggs. Consequently, hatchlings from early-laid eggs reached larger body sizes prior to winter. In addition, we found that female toad-headed agama did not select nests specific to reaction norms of their own offspring because hatchlings from original or translocated nests had similar phenotypic traits. Overall, our study demonstrates the importance of seasonal variation in nest environments in determining embryonic development and offspring phenotypes, which has not been widely appreciated at least in non-avian reptiles.

Embryology of the naso-palatal complex in Gekkota based on detailed 3D analysis in Lepidodactylus lugubris and Eublepharis macularius.

The vomeronasal organ (VNO), nasal cavity, lacrimal duct, choanal groove, and associated parts of the superficial (soft tissue) palate are called the naso-palatal complex. Despite the morphological diversity of the squamate noses, little is known about the embryological basis of this variation. Moreover, developmental data might be especially interesting in light of the morpho-molecular discordance of squamate phylogeny, since a 'molecular scenario' implies an occurrence of unexpected scale of homoplasy also in olfactory systems. In this study, we used X-ray microtomography and light microscopy to describe morphogenesis of the naso-palatal complex in two gekkotans: Lepidodactylus lugubris (Gekkonidae) and Eublepharis macularius (Eublepharidae). Our embryological data confirmed recent findings about the nature of some developmental processes in squamates, for example, involvement of the lateral nasal prominence in the formation of the choanal groove. Moreover, our study revealed previously unknown differences between the studied gekkotans and allows us to propose redefinition of the anterior concha of Sphenodon. Interpretation of some described conditions might be problematic in the phylogenetic context, since they represent unknown: squamate, nonophidian squamate, or gekkotan features.

Lizard Embryos Prioritize Posthatching Energy Reserves over Increased Hatchling Body Size during Development.

Embryonic development in oviparous organisms is fueled by maternally allocated yolk, and many organisms hatch before that energy store is used completely; the resultant leftover (residual) yolk is internalized and may support early posthatching life. However, embryos that use most, or all, of their yolk supply before hatching should hatch at a larger size than those that do not exhaust those energy reserves, which could also have benefits for posthatching growth and survival. To examine the trade-off between residual yolk and offspring size, we experimentally reduced yolk quantity at oviposition in lizard eggs (Amphibolurus muricatus) and then quantified offspring size and the amount of internalized residual yolk. This design enabled us to determine whether embryos (1) exhaust yolk supply during development (thereby maximizing neonatal size) or (2) reduce neonatal size by retaining yolk reserves at hatching. Our data support the latter scenario. Eggs from the yolk-reduced treatment produced smaller offspring with a proportion of residual yolk similar to that of offspring from unmanipulated eggs, suggesting that the fitness benefits of posthatching energy stores outweigh those of larger neonatal size.

Embryonic skull development in the gecko, Tarentola annularis (Squamata: Gekkota: Phyllodactylidae).

Tarentola annularis is a climbing gecko with a wide distribution in Africa north of the equator. In the present paper, we describe the development of the osteocranium of this lizard, from the first appearance of the cranial elements up to the point of hatching. This is based on a combination of histology and cleared and stained specimens. This is the first comprehensive account of gekkotan pre-hatching skull development based on a comprehensive series of embryos, rather than a few selected stages. Given that Gekkota is now widely regarded as representing the sister group to other squamates, this account helps to fill a significant gap in the literature. Moreover, as many authors have considered features of the gekkotan skull and skeleton to be indicative of paedomorphosis, it is important to know whether this hypothesis is supported by delays in the onset of cranial ossification. In fact, we found the sequence of cranial bone ossification to be broadly comparable to that of other squamates studied to date, with no significant lags in development.

Ontogeny of the Moorish gecko Tarentola mauritanica with emphasis on morphogenesis of the skin and its derivatives.

Studying reptilian embryonic development provides answers to many questions related to the development of tetrapods. Reptilian skin has been recently considered in studies at the evo-devo level. The lizard epidermis has to be shed periodically. At the embryonic level, contention exists regarding the first layers to appear, whether the oberhautchen or the clear layer, and whether the shedding complex develops before hatching. Geckos exhibit diverse morphologies independently evolved multiple times within the clade, such as subdigital pad lamellae. Here we investigate the embryonic development of Tarentola mauritanica and establishing its embryonic table. Primarily we follow the development of the integument. This is a closely related species to Tarentola annularis and it is crucial to investigate whether it has the same derived digital condition of claw regression. Eleven embryonic stages are described according to the external morphological characteristics of the embryos. Interestingly, the oviposition stage appears earlier than its close relative T. annularis, and the total incubation time is less. We also describe skin development, adding clear evidence to the debate on the development of the shedding complex, which we found is developed before hatching. We describe one layer of periderm and the clear layer as the first embryonic epidermal layers. Generally, our results show the genus Tarentola to have the advantage of being a unique taxon, easily breed at the laboratory, with multiple clutches per year, and with an earlier stage at oviposition. That could be a model animal for embryonic development and experimental embryology studies.

Effects of glyphosate, cypermethrin, and chlorpyrifos on hematological parameters of the tegu lizard (Salvator merianae) in different embryo stages.

Different studies have demonstrated effects of pesticides during embryo development in vertebrates and stage-dependent effects, but there is no information concerning this for Salvator merianae. We evaluated the effects of the herbicides Glyphosate Roundup (GLY-RU) and Glyphosate Panzer Gold (GLY-PZ); and the insecticides Chlorpyrifos (CPF) and Cypermethrin (CYP), and their complex mixtures, at different concentrations in hematological parameters of S. merianae embryos at two different development stages. The analyzed parameters were Total and Differential White Blood Cells Count, Heterophils/Lymphocytes index (H/L), Lobularity index, and Natural Antibodies (Nabs titres), as well as growth, embryo mortality and birth delay. Heterophils decreased in the intermediate concentrations tested of CYP and GLY-RU, in animals exposed at 33-days development. Lymphocytes increased in the intermediate concentration tested of GLY-RU, and the H/L index decreased in the maximum concentration tested of GLY-RU. NAbs titres increased in those animals exposed to the maximum CYP concentration tested. However, animals exposed at 3/5-days development showed no differences among treatments in most of the analyzed parameters, suggesting a stage-dependent response. Nevertheless, those animals exposed to GLY-PZ showed lower Nabs titres in relation to negative control. These results suggest effects on different hematological parameters related to the immune system of S. merianae, according to the used pesticide (herbicide or insecticide), its concentration and commercial formulation (GLY-RU or GLY-PZ), and the stages of development of the exposed animals. Our results reveal the importance of carrying out studies that evaluate the effects of permanent exposure of living beings and their environments to these toxics.

Expression of specific corneous beta proteins in the developing digits of the Japanese gecko (Gekko japonicus) reveals their role in the growth of adhesive setae.

Geckos possess strong adhesion ability, even can climb on smooth surface. Previous studies have shown that the setae of geckos play a crucial role in their ability to climb on vertical walls. But the biological molecular mechanism of their adhesion ability remains unclear. In the present study, the expression patterns of corneous beta proteins (CBPs) genes related to claws, scales, and feathers development (named as ge-gprp-9, ge-gprp-10, ge-gprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp-15, and ge-gprp-16 respectively) in the developing pad lamellae of different embryonic stages (stage 34, stage 36, stage 39, and stage 42) of the Japanese gecko Gekko japonicus were detected using fluorescence quantitative PCR approach. The results showed that there were significant up-regulated expression of CBPs mRNA at embryonic stage 39 with the embryonic continuous maturation and the highest expression level was detected at embryonic stage 39 or stage 42. Moreover, the expression levels of four CBPs genes ge-gprp-9, ge-gprp-10, ge-gprp-11, and ge-gprp-12 in the embryonic and adult development of gecko were detected by fluorescence in situ hybridization technique. The results from in situ hybridization detection revealed that the positive signals of these CBPs genes expression were the same in the developing pad lamellae of G. japonicus. The positive signals of eight CBPs genes were mainly found in the setae tissue, oberhautchen, and ß layer, which suggests these CBPs genes are involved in the growth of setae.

A comparison of uterine contractile responsiveness to arginine vasopressin in oviparous and viviparous lizards.

Nonapeptides and their receptors regulate a diverse range of physiological processes. We assessed the contractile responsiveness of uteri from the squamate viviparous-oviparous species pair, Pseudemoia entrecasteauxii and Lampropholis guichenoti, as well as the bimodally reproductive species, Saiphos equalis, to arginine vasopressin (AVP). We assessed the resulting uterine contractility as a function of pregnancy status, species and parity mode. We also measured mRNA abundance for the nonapeptide receptor, oxytocin receptor (oxtr), in uteri from P. entrecasteauxii and L. guichenoti and compared expression across pregnancy status and parity mode. We found that pregnant uteri exhibited a significantly greater contractile response to AVP than non-pregnant uteri in all three lizard species studied. Cross-species comparisons revealed that uteri from viviparous P. entrecasteauxii were significantly more responsive to AVP than uteri from oviparous L. guichenoti during both pregnant and non-pregnant states. Conversely, for non-pregnant S. equalis, uteri from viviparous individuals were significantly less responsive to AVP than uteri from oviparous individuals, while during pregnancy, there was no difference in AVP contractile responsiveness. There was no difference in expression of oxtr between L. guichenoti and P. entrecasteauxii, or between pregnant and non-pregnant individuals within each species. We found no significant correlation between oxtr expression and AVP contractile responsiveness. These findings indicate that there are differences in nonapeptide signalling across parity mode and suggest that in these lizards, labour may be triggered either by an increase in plasma nonapeptide concentration, or by an increase in expression of a different nonapeptide receptor from the vasopressin-like receptor family.

Sox2 maintains epithelial cell proliferation in the successional dental lamina.

OBJECTIVES: The successional dental lamina is the distinctive structure on the lingual side of the vertebrate tooth germ. The aim of this study was to investigate the relationship among Sox2, Claudin10 and laminin5 and the role of Sox2 in successional dental lamina proliferation during vertebrate tooth development. MATERIALS AND METHODS: To understand the successional dental lamina, two types of successional tooth formation, that in geckos (with multiple rounds of tooth generation) and that in mice (with only one round of tooth generation), were analysed. RESULTS: Unique coexpression patterns of Sox2 and Claudin10 expression were compared in the successional dental lamina from the cap stage to the late bell stage in the mouse tooth germ and in juvenile gecko teeth to support continuous tooth replacement. Furthermore, Laminin5 expression was shown in the cap stage and decreased after the bell stage. Upon comparing the epithelial cell cycles and cell proliferation in successional dental lamina regions between mouse and gecko molars using BrdU and IdU staining and pulse-chase methods, distinctive patterns of continuous expression were revealed. Moreover, Sox2 overexpression with a lentiviral system resulted in hyperplastic dental epithelium in mouse molars. CONCLUSIONS: Our findings indicate that the regulation of Sox2 in dental lamina proliferation is fundamental to the successional dental lamina in both species.

A developmental synapomorphy of squamate reptiles.

The reptilian clade Squamata is defined primarily by osteological synapomorphies, few of which are entirely unambiguous. Studies of developing squamate eggs have revealed a uniquely specialized feature not known to occur in any other amniotes. This feature-the yolk cleft/isolated yolk mass complex-lines the ventral hemisphere of the egg. During its formation, extraembryonic mesoderm penetrates the yolk and an exocoelom (the yolk cleft [YC]) forms in association with it, cutting off a thin segment of yolk (the "isolated yolk mass" [IYM]) from the main body of the yolk. The YC-IYM complex has been observed and described in more than 65 squamate species in 12 families. In viviparous species, it contributes to the "omphaloplacenta," a type of yolk sac placenta unique to squamates. The only squamates known to lack the IYM are a few highly placentotrophic skinks with minuscule eggs, viviparous species in which it clearly has been lost. Given its absence in mammals, chelonians, crocodylians, and birds, the YC-IYM complex warrants recognition as a developmental synapomorphy of the squamate clade. As in extant viviparous lizards and snakes, the YC-IYM complex presumably contributed to the placenta of extinct viviparous squamates.

Identification of the building blocks of ventricular septation in monitor lizards (Varanidae).

Among lizards, only monitor lizards (Varanidae) have a functionally divided cardiac ventricle. The division results from the combined function of three partial septa, which may be homologous to the ventricular septum of mammals and archosaurs. We show in developing monitors that two septa, the 'muscular ridge' and 'bulbuslamelle', express the evolutionarily conserved transcription factors Tbx5, Irx1 and Irx2, orthologues of which mark the mammalian ventricular septum. Compaction of embryonic trabeculae contributes to the formation of these septa. The septa are positioned, however, to the right of the atrioventricular junction and they do not participate in the separation of incoming atrial blood streams. That separation is accomplished by the 'vertical septum', which expresses Tbx3 and Tbx5 and orchestrates the formation of the electrical conduction axis embedded in the ventricular septum. These expression patterns are more pronounced in monitors than in other lizards, and are associated with a deep electrical activation near the vertical septum, in contrast to the primitive base-to-apex activation of other lizards. We conclude that evolutionarily conserved transcriptional programmes may underlie the formation of the ventricular septa of monitors.

Primary myogenesis in the sand lizard (Lacerta agilis) limb bud.

Our studies conducted on reptilian limb muscle development revealed, for the first time, early forelimb muscle differentiation at the morphological and molecular level. Sand lizard skeletal muscle differentiation in the early forelimb bud was investigated by light, confocal, and transmission electron microscopy as well as western blot. The early forelimb bud, filled with mesenchymal cells, is surrounded by monolayer epithelium cells. The immunocytochemical analysis revealed the presence of Pax3- and Lbx-positive cells in the vicinity of the ventro-lateral lip (VLL) of the dermomyotome, suggesting that VLL is the source of limb muscle progenitor cells. Furthermore, Pax3- and Lbx-positive cells were observed in the dorsal and ventral myogenic pools of the forelimb bud. Skeletal muscle development in the early limb bud is asynchronous, which is manifested by the presence of myogenic cells in different stages of differentiation: multinucleated myotubes with well-developed contractile apparatus, myoblasts, and mitotically active premyoblasts. The western blot analysis revealed the presence of MyoD and Myf5 proteins in all investigated developmental stages. The MyoD western blot analysis showed two bands corresponding to monomeric (mMyoD) and dimeric (dMyoD) fractions. Two separate bands were also detected in the case of Myf5. The observed bands were related to non-phosphorylated (Myf5) and phosphorylated (pMyf5) fractions of Myf5. Our investigations on sand lizard forelimb myogenesis showed that the pattern of muscle differentiation in the early forelimb bud shares many features with rodents and chicks.

Embryonic development of a parthenogenetic vertebrate, the mourning gecko (Lepidodactylus lugubris).

BACKGROUND: One goal of evolutionary developmental biology is to understand the role of development in the origin of phenotypic novelty and convergent evolution. Geckos are an ideal system to study this topic, as they are species-rich and exhibit a suite of diverse morphologies-many of which have independently evolved multiple times within geckos. RESULTS: We characterized and discretized the embryonic development of Lepidodactylus lugubris-an all-female, parthenogenetic gecko species. We also used soft-tissue µCT to characterize the development of the brain and central nervous system, which is difficult to visualize using traditional microscopy techniques. Additionally, we sequenced and assembled a de novo transcriptome for a late-stage embryo as a resource for generating future developmental tools. Herein, we describe the derived and conserved patterns of L. lugubris development in the context of squamate evolution and development. CONCLUSIONS: This embryonic staging series, µCT data, and transcriptome together serve as critical enabling resources to study morphological evolution and development, the evolution and development of parthenogenesis, and other questions concerning vertebrate evolution and development in an emerging gecko model.

Development and differentiation of the reproductive system of Tropidurus catalanensis (Squamata: Tropiduridae).

Development and differentiation of the reproductive system in lizards begin in the embryonic period, although the stage and time of their occurrence vary according to populations and species. In this study, the events of the development and differentiation of the reproductive system of males and females of Tropidurus catalanensis were characterized during the embryonic, neonatal, and juvenile periods. Embryos at Stages 27, 34, 37, 40, and 41, neonates and juveniles, from Corrientes, Argentina, were analyzed. At Stage 27, the genital ridge was not observed but primordial germ cells were recorded in the yolk sac as well as the mesenteric mesenchyme, indicating the beginning of germ cell migration. Gonadal differentiation commenced at Stage 34. In males from Stage 37, the testes possessed seminiferous cords with Sertoli cells and spermatogonia, while in hatchlings seminiferous tubules and interstitial tissue with mature Leydig cells were present. Spermatogenesis was observed in a specimen of 51.9 mm snout-vent length, corresponding to the minimum reproductive size. In females, from Stage 37 until hatching, the ovaries had a cavernous medulla and a cortex with somatic cells and abundant oogonia. The onset of meiosis and folliculogenesis occurred in the juvenile period.

Novel placental structure in the Mexican gerrhonotine lizard, Mesaspis viridiflava (Lacertilia; Anguidae).

The evolution of viviparity alters the physical relationship between mothers and offspring and the prevalence of viviparity among squamate reptiles presents an opportunity to uncover patterns in the evolution of placental structure. Understanding the breadth of this diversity is limited because studies of placental structure and function have emphasized a limited number of lineages. We studied placental ontogeny using light microscopy for an embryological series of the Mexican gerrhonotine lizard, Mesaspis viridiflava. This species develops an elaborate yolk sac placenta, an omphaloplacenta, which receives vascular support arising in a structure known only from other gerrhonotine lizards. A prominent feature of the omphaloplacenta is a zone of uterine and embryonic epithelial cell hyperplasia located at the upper shoulder of the yolk mass, often extending above the yolk mass. The omphaloplacenta covers more than one-half of the surface area of maternal-embryonic contact. The chorioallantoic placenta has a more restricted distribution because the allantois remains in the embryonic hemisphere of the egg throughout development and lies internal to the vascular support for the omphaloplacenta in areas where they overlap. The structural profile of the chorioallantoic placenta indicates a potential for respiratory exchange and/or hemotrophic nutritive transport, while that of the omphaloplacenta suggests that nutritive transfer is primarily via histotrophy. An eggshell is present in the earliest embryonic stages examined but regresses relatively early in development. Placental specializations of this species are consistent with a pattern of matrotrophic embryonic nutrition and have evolved in a unique lineage specific developmental pattern.

Effects of regional hypoxia and incubation temperature on growth, differentiation, heart mass, and oxygen consumption in embryos of the leopard gecko (Eublepharis macularius).

Oviparous reptile embryos must tolerate fluctuations in oxygen availability and incubation temperature during development. In this study, regional hypoxia was simulated by painting eggs of Eublepharis macularius with melted paraffin wax to decrease the available surface area for gas exchange by approximately 80%. Experimental and control eggs were incubated at either 28 or 34 °C and embryo mass, stage, heart mass, relative heart mass, and oxygen consumption (V̇O2) were measured at 15 and 30 days of incubation. Embryo mass from the regional hypoxia treatment was reduced by about 50% at day 15 and by about 30% at day 30 of incubation, independent of incubation temperature compared to controls. Embryo stage from the regional hypoxia treatment was reduced by about 2 stages at day 15 independent of incubation temperature but there was no effect of hypoxia treatment at day 30. Absolute heart mass was reduced by about 60% in regional hypoxia embryos sampled at day 15 while relative heart mass was increased by about 30% in regional hypoxic embryos at day 30 compared to controls, suggesting that heart mass is conserved at the expense of somatic growth. Embryo V̇O2 was affected by incubation temperature at both 15 and 30 days of incubation but not by regional hypoxia treatment. These results indicate that embryos of E. macularius possess plasticity in their capacity to respond to reduction in oxygen availability during incubation, and are able to survive and continue developing when gas exchange surface area is severely limited.

Review: The Regenerating Tail Blastema of Lizards as a Model to Study Organ Regeneration and Tumor Growth Regulation in Amniotes.

Tail regeneration in lizards is a unique case of organ regeneration among amniotes. The Review summarizes past and recent studies indicating that tail regeneration utilizes numerous signaling pathways typical for tumor growth. The regenerative blastema-cone contains sparse proliferating cells that utilize coding and noncoding RNAs in an environment rich in water and hyaluronate, as typical for tumor outgrowth. Differently from tumors, the blastema appears as a polarized outgrowth where the distal region contains proliferating cells mainly driven by the up-regulation of Wnt, snoRNAs, and associated onco-genes. The down-regulation of immune-genes coupled with the high production of hyaluronate coating blastema cells likely protect them from attach by immune cells. Immunoevasion of blastema cells allows the proliferation and migration necessary for the morphogenesis of a new tail. Transcriptome and immunolabeling data suggest that gradients for wnts, shh, msx, and signaling receptors are present in the tail blastema. It is hypothesized that cells along these gradients activate different genes, including tumor suppressors that are expressed in more proximal regions where cells stop proliferating and differentiate into tissues of the new tail. The continuous proliferation at the apex of the blastema is turned into a regulated growth in more proximal regions near the original tail. In contrast, it is hypothesized that no or nonresponding gradients of signaling proteins are present in tumor outgrowths so that cell proliferation but no differentiation occurs in expanding tumors. Considering signaling gradients, the lizard model of regeneration can help in understanding the lack of regulation of tumor growth. Anat Rec, 302:1469-1490, 2019. © 2018 American Association for Anatomy.