The Mexican axolotl (Ambystoma mexicanum) is a urodele amphibian, endemic to the Mexico basin. It is classified as a “Critically Endangered” species in its native habitat by the International Union for Conservation of Nature (IUCN, 2020); this is due to its habitat destruction by pollution and urbanization and also by the introduction of invasive species (SEMARNAT, 2018). This species has been relevant in the Mexican culture and is globally well known as a model to study tissues such as limb regeneration (Bryant et al., 2017).

Even though regeneration and genetic studies have been carried out recently in A. mexicanum, there are other biological topics that have no current information such as embryo development. The first reports on this subject were carried out from 1975 to 1989 (Schreckenberg and Jacobson, 1975; Bordzilovskaya et al., 1989).

To make a complete and accurate description of A. mexicanum embryo development, we have considered the basic stages of embryonic development described in the literature that focused on amphibians. Once amphibian oocytes are fertilized, the entry of the sperm into the oocyte will determine the dorsal–ventral axis of the embryo; that is, the entry site will become the animal pole representing the ventral region of the new individual, while the opposite side will become the vegetal pole giving rise to the dorsal region of the embryo (Alberts et al., 2014). In the first stages, there is a reorganization of the cellular components in which the microtubules and centrioles of the sperm are involved, which generate a rotation of 30° on the equator of the embryo, developing the gray crescent (Gilbert, 2014). Amphibians present uneven holoblastic segmentation because the vegetal pole contains a greater quantity of yolk that decreases the segmentation rate. The first cleavage divides the egg into two equal parts, two blastomeres, along a meridional plane that passes through both animal and vegetal poles, beginning with the formation of a groove or fissure at the animal pole (Puente, 1958). The second division occurs in a meridional plane, perpendicular to the first division, resulting in four equal blastomeres. In the third division, the cleavage groove does not occur exactly at the equator but is displaced toward the animal pole, shaping eight blastomeres (Puente, 1958; Gilbert, 2014). In this stage, a faster segmentation is observed in the animal pole, which causes those cells to look smaller in size (micromeres), while in the vegetal pole, cells are larger in size (macromeres) (Richter and King, 2013); thus, the embryo looks asymmetrical (Nieuwkoop and Faber, 1994) ( Figure 1 ). Cell divisions then continue and the blastomeres increase in number, but not in size due to the absence of cell growth between divisions, until they form a morula of 16 to 64 cells, followed by the transition to the blastula stage, when the embryo has 128 cells (Etkin, 1988).

The blastula has an internal cavity known as blastocoel, and at this stage, maternal genes begin to be replaced by genes from the zygote in a process known as mid-blastula transition (Gilbert, 2014). Regulatory factors, such as transcription factors and epigenetic regulators, play a key role in this transition; the arrangement of blastomeres and the formation of the blastocoel are regulated by specific cellular and molecular signals, such as proteins from the Wnt and BMP families (Etkin, 1988). In addition, numerous cell adhesion molecules hold the blastomeres together, with EP-cadherin being one of the most important (Richter and King, 2013).

In gastrulation, the blastula reorganizes through movements of invagination, involution, and epiboly, forming a gastrula with three well-differentiated germinal layers: ectoderm, mesoderm, and endoderm (Keller, 2005), with the dorsal lip of the blastopore being the main gastrulatory center (Gilbert, 2014). During gastrulation, various morphogens act on cell differentiation regulating the dorsal–ventral axis: Wnt/β-catenin and BMP/Noggin pathways, signals that will give rise to different tissues such as Sonic Hedgehog (Shh), Notch, and Hox genes, which have been described in amphibians, mainly in Xenopus laevis (Stocum, 1992). The gastrula takes on an elongated and slightly convex shape in the dorsal part of the ectoderm due to the formation of the neural plate extended from the dorsal lip of the blastopore toward the anterior part of the embryo. The transverse and lateral neural folds are formed, forming the neural tube that will give rise to the central nervous system; at this point, it is considered a neurula stage (Puente, 1958). Finally, the ectoderm will give rise to the central nervous system, epidermis, and glandular tissue, among others; the mesoderm will give rise to systems such as the muscular, skeletal, urinary, and blood systems, while the endoderm will give rise to more internal organs such as the digestive and respiratory systems (Roberts, 2000; Gilbert, 2014).

In general, the embryo of A. mexicanum measures on average 2 mm and is contained in three well-defined jelly layers. In the early stages of development, the difference between the animal pole and the vegetal pole is visible ( Figure 2 ) (Bordzilovskaya et al., 1989).

The incubation and complete embryonic development of A. mexicanum lasts between 9 and 19 days depending on the ambient temperature (Duhon, 1992; Nugas and Byrant, 1996). To carry out a successful incubation, it is necessary to maintain water quality with the physicochemical parameters recommended for this species, including NO₂ (0–40 mg/L), NO₃ (0–1 mg/L), NH₃ + NH₄ (0–0.5 mg/L), and pH (7–8.4) (Mena and Servín, 2014). Additionally, the use of solutions with balanced mineral concentrations for the incubation of embryos has been described, specifically in A. mexicanum (Nugas and Byrant, 1996), using the modified Holtfreter’s solution (NaCl 0.059 M, KCl 0.0067 M, CaCl₂ 0.00076 M, and NaHCO₃ 0.0024 M), showing success in the incubation of embryos in laboratory conditions.

In this work, we studied the embryonic development of the Mexican axolotl (A. mexicanum) as part of our line of research focused on the conservation of the species through gamete and embryo cryopreservation. This information may be useful for different assisted reproduction techniques. For this, we monitored the embryo development of the Mexican axolotl throughout incubation, and then employing high-quality photographs, we examined and analyzed the main and distinctive morphological characteristics of the embryos. The objectives of this work were to expand and update the information reported by previous authors and that findings from this study provide useful information for future research work in cryopreservation processes.

This work was conducted at the Multidisciplinary Research Unit (Reproduction Laboratory and Vivarium) of the Faculty of Superior Studies Cuautitlan (FESC), National Autonomous University of Mexico (UNAM), Mexico.

In this work, we identified a total of 49 stages of axolotl embryo development that were grouped into five phases: 1) fertilized oocyte, 2) cleavage, 3) gastrulation, 4) neurulation, and 5) organogenesis. The time from fertilization to hatching took approximately 350 h (range: 312–388 h) under the management conditions set up in this work.

According to this study, which describes from the fertilization of the oocytes to the hatching of the larvae, we proposed a total of 49 stages, more than those reported by Schreckenberg and Jacobson (1975). Unlike these authors, we have included additional stages (5–12) because our images allowed us to distinguish blastomere segmentation showing a progressive clear reduction in each cell size. In contrast, in the mentioned work, it was not possible to differentiate the limits between individual blastomeres.

Others reported 31 stages according to external anatomical characteristics (Rivera, 2019); in contrast, Holly et al. (2003) reported some stages after hatching, reaching a total of 57, which included the development of the forelimbs and hindlimbs.

It is worth mentioning that this work was based on one individual embryo from three different embryo laying. Embryos were kept all the time in natural conditions within their jelly layers, keeping a constant temperature of 16°C. In contrast, Schreckenberg and Jacobson (1975) removed the embryos from the jelly layers to take photographs; thus, their images showed embryos without any barrier that limited the extension of their body. In our case, embryos within the jelly layers did not have enough space to expand, so they look flexed, which caused the images to be blurred in certain regions. In this work, we decided to reduce the handling of the embryos as much as possible to avoid introducing any artificial change; thus, images showed some blurred areas. In our future work, we will try to improve lighting and optical settings. On the other hand, Holly et al. (2003) fixed embryos in formalin that may cause morphological changes.

The duration of embryogenesis in our work, approximately 350 h at 16°C, was similar to that reported before: from 330 h until the larvae hatched at 18°C (Holly et al., 2003) to 488 h until stage 40, at a constant temperature of 18°C (Schreckenberg and Jacobson, 1975).

The temperature of embryo housing affects embryonic development, and even under the same conditions, each embryo presents a small but different rhythm of development. Despite this difference, it can be considered that the complete development rhythm of the axolotl embryo until hatching takes approximately 2 weeks. It is important to perform more studies for monitoring both external and internal changes of the embryo throughout their development; this may help in understanding and eventually overcome or minimize the effects that cryopreservation protocols produce in the embryos of A. mexicanum.

It is of great interest, that in the future, this study is complemented by histological or molecular techniques, such as PCR and in-situ hybridization, among many others that provide us with more detailed information on the genetic and molecular factors involved in embryo development and differentiation and even in the regeneration of the different tissues of the axolotl; with this, we may understand and correlate the external changes observed with the internal changes that we do not review in this work.