Skull development in the Iberian newt, Pleurodeles waltl (Salamandridae: Caudata: Amphibia): timing, sequence, variations, and thyroid hormone mediation of bone appearance

Abstract

The cranial ossification sequence in Pleurodeles waltl is widely used in phylogenetic analyses of amphibian origin and evolution. However, the patterns published to date are far from completely resolved and contain certain discrepancies. Based on a large sample of P. waltl specimens ranging from early post‐hatching larvae to post‐metamorphic newts, we determined the most common cranial ossification sequence and revealed its intraspecific variations. Since thyroid hormones (THs) are involved in the mediation of skull development in salamanders, we studied the role of THs in the cranial development of P. waltl. The normal sequence and timing of bone appearance were compared with those in larvae reared under conditions of high (in 1 and 2 ng mL‐1 triiodothyronine) and low [in 0.02% thiourea (TU), which inhibits thyroid gland activity] TH levels. Metamorphosis was greatly accelerated in the TH‐treated larvae and was arrested in the TU‐treated larvae, which retained the larval pattern of the palate and rudimentary external gills even after 2 years of the experiment. Early‐appearing bones (the coronoid, vomer, palatine, dentary, squamosal, premaxilla, parasphenoid, pterygoid, prearticular, vomer, frontal, parietal, exoccipital, in this order) arise at the same stages and ages, and follow the same ossification sequence under different TH levels. The timing of the appearance of bones normally arising in the late larval and metamorphic periods (the quadratojugal, orbitosphenoid, prootic, maxilla, nasal, os thyroideum, prefrontal, quadrate, in this order) changes depending on the TH level. The maxilla and nasal display the most pronounced reaction to changes in the TH level: they appear precociously in TH‐treated animals, while their appearance is postponed and they remain rudimentary in TU‐treated animals. Because of different responses to THs, the order in which late‐arising bones appear changes depending on the TH level. Although bones appearing early in larval ontogeny (e.g. the premaxilla, vomer, squamosal, palatine) display no TH‐induced reaction when they start to develop, their further differentiation shows dependence on THs, and these bones become TH‐inducible closer to metamorphosis. These findings indicate that TH involvement in the mediation of cranial development changes from minimal (if at all) in its early stages to maximal during metamorphosis. It is likely that the appearance of bones early in development is mediated by factors other than THs. Their further development is accompanied by changes in the mechanisms mediating their morphological differentiation. That is, likely non‐hormonal mediation becomes replaced or/and complemented by hormonal mediation. The constituent parts of the same bone may exhibit differences in their reactions to changes in TH levels. Although in normal development, the overall cranial ossification sequence is constant, there was variation in the order in which late‐appearing bones was recorded. These observations suggest that this variation results from individual variab...

Increasing Hormonal Control of Skeletal Development: An Evolutionary Trend in Amphibians

“…Interestingly, whereas the appearance of early bones does not depend upon TH-level, their further development and morphological differentiation become TH-inducible. Thus, in P. waltl, changes in the TH-level do not affect the timing of the premaxillar appearance, while later formation of its outgrowth toward the vomer is strictly induced by THs (Smirnov et al, 2020).…”

“…Larvae were reared in aquaria with different experimental media: (i) clear tap water (controls); (ii) alkaline solutions of 3,3 1 ,5-triiodothyronine (T 3 ; Sigma Chemical Co., Germany) at concentrations of 2 × 10 −8 M, 2 × 10 −9 M, and 2 × 10 −10 M; and (iii) 0.02% and 0.04% solutions of goitrogen thiourea (TU; Solins, Russia). Previously, similar concentrations of T 3 and TU were used in experimental studies of the TH-effect on skeletogenesis in urodelan Vassilieva, 2003, 2005;Smirnov et al, 2011Smirnov et al, , 2020 and anuran amphibians (Vassilieva and Smirnov, 2007;Smirnov and Vassilieva, 2014). In Experiment I, treatment of the different groups of larvae with T 3 and TU started at 6 and 7 days posthatching (dph), respectively, soon after the beginning of exogenous feeding.…”

“…In S. keyserlingi, A. mexicanum, L. vulgaris, and P. waltl, the first bones to appear arise at the same time and sequence under different hormonal conditions Vassilieva, 2003, 2005;Smirnov et al, 2011Smirnov et al, , 2020. Midlarval bones display some acceleration and retardation under TH and TU treatments, respectively.…”

“…For this aim, changes in skeletal morphology (timing and sequence of bone appearance) were compared in larvae reared under different TH regimes: normal (in control larvae), low (under treatment with a goitrogen, thiourea, causing TH-deficiency), and high (under treatment with exogenous TH, triidothyronine at different concentrations). This standard protocol of the experiment was applied to both the basal and derived salamanders: the Siberian newt Salamandrella keyserlingii (Hynobiidae), axolotl Ambystoma mexicanum (Ambystomatidae), the Ribbed newt Pleurodeles waltl and Smooth newt Lissotriton vulgaris (Salamandridae) Vassilieva, 2003, 2005;Smirnov et al, 2011Smirnov et al, , 2020. Accumulated evidence suggests that urodelan evolution is accompanied by an increasing TH-impact on cranial metamorphic remodeling (Smirnov, 2006).…”

Amphibian Hormones, Calcium Physiology, Bone Weight, and Lung Use Call for a More Inclusive Approach to Understanding Ossification Sequence Evolution

“…GS 30 is also approximately when frog postembryonic development is believed to become TH mediated, since tissues at earlier stages can be accelerated by exogenous TH, but not delayed by TH inhibitors or thyroid gland inactivation (Allen, 1918;Terry, 1918;Brown, 2005;Kerney et al, 2010;Smirnov and Vassilieva, 2014;Rose and Cahill, 2019). By the same criteria, the onset of TH mediation in salamander ossification occurs between mid and late larval stages (Smirnov et al, 2020). Cells that condense and mineralize around GS 30 might be expected to show accelerated or delayed activation due to the high plasticity in amphibian growth and developmental rates (Rose, 2005(Rose, , 2014a, and the exceptionally low TH involved in their activation.…”