Skull anatomy and development have been extensively studied due to their significance in evolutionary biology, forensic anthropology, and clinical medicine. Bone collections are an indispensable resource for conducting such anthropological and anatomical studies. However, worldwide there are only few skull collections containing specimens covering the entire fetal and postnatal period. Herein we describe the Zagreb Skull Collection, an identified collection comprising more than 1100 skulls and skull bone sets from the early fetal period to centenarians. The Zagreb Skull Collection consists of two main parts: the unique Collection of Skull Bones containing 386 sets of separated skull bones from the early fetal period to adulthood and the Collection of Skulls containing 742 skulls (age range 4-101 years). The collection was the core source for numerous anatomical studies on the development, postnatal changes, and anatomical variations of the skull. However, the Zagreb Skull Collection is still an underexploited resource for anthropological, forensic, and anatomical studies with translatability to contemporary clinical practice.

Extensive morphological variation found in mammals reflects the wide spectrum of their ecological adaptations. The highest morphological diversity is present in the craniofacial region, where geometry is mainly dictated by the bony skull. Mammalian craniofacial development represents complex multistep processes governed by numerous conserved genes that require precise spatio-temporal control. A central question in contemporary evolutionary biology is how a defined set of conserved genes can orchestrate formation of fundamentally different structures, and therefore how morphological variability arises. In principle, differential gene expression patterns during development are the source of morphological variation. With the emergence of multicellular organisms, precise regulation of gene expression in time and space is attributed to cis-regulatory elements. These elements contribute to higher-order chromatin structure and together with trans-acting factors control transcriptional landscapes that underlie intricate morphogenetic processes. Consequently, divergence in cis-regulation is believed to rewire existing gene regulatory networks and form the core of morphological evolution. This review outlines the fundamental principles of the genetic code and genomic regulation interplay during development. Recent work that deepened our comprehension of cis-regulatory element origin, divergence and function is presented here to illustrate the state-of-the-art research that uncovered the principles of morphological novelty. This article is part of the theme issue \'The mammalian skull: development, structure and function\'.

BACKGROUND: We compared skull shape and variation among genetically modified mice that exhibit different levels of connexin43 (Cx43) channel function, to determine whether Cx43 contributes to craniofacial phenotypic robustness. Specifically, we used two heterozygous mutant mouse models (G60S/+ and I130T/+) that, when compared to their wildtype counterparts, have an ~80% and ~50% reduction in Cx43 function, respectively.
RESULTS: Both mutant strains showed significant differences in skull shape compared to wildtype littermates and while these differences were more severe in the G60S/+ mouse, shape differences were localized to similar regions of the skull in both mutants. However, increased skull shape variation was observed in G60S/+ mutants only. Additionally, covariation of skull structures was disrupted in the G60S/+ mutants only, indicating that while a 50% reduction in Cx43 function is sufficient to cause a shift in mean skull shape, the threshold for Cx43 function for disrupting craniofacial phenotypic robustness is lower.
CONCLUSIONS: Collectively, our results indicate Cx43 can contribute to phenotypic robustness of the skull through a nonlinear relationship between Cx43 gap junctional function and phenotypic outcomes.

Mutations in the gene encoding the gap-junctional protein connexin43 (Cx43) are the cause of the human disease oculodentodigital dysplasia (ODDD). The mandible is often affected in this disease, with clinical reports describing both mandibular overgrowth and conversely, retrognathia. These seemingly opposing observations underscore our relative lack of understanding of how ODDD affects mandibular morphology. Using two mutant mouse models that mimic the ODDD phenotype (I130T/+ and G60S/+), we sought to uncover how altered Cx43 function may affect mandibular development. Specifically, mandibles of newborn mice were imaged using micro-CT, to enable statistical comparisons of shape. Tissue-level comparisons of key regions of the mandible were conducted using histomorphology, and we quantified the mRNA expression of several cartilage and bone cell differentiation markers. Both G60S/+ and I130T/+ mutant mice had altered mandibular morphology compared to their wildtype counterparts, and the morphological effects were similarly localized for both mutants. Specifically, the biggest phenotypic differences in mutant mice were focused in regions exposed to mechanical forces, such as alveolar bone, muscular attachment sites, and articular surfaces. Histological analyses revealed differences in ossification of the intramembranous bone of the mandibles of both mutant mice compared to their wildtype littermates. However, chondrocyte organization within the secondary cartilages of the mandible was unaffected in the mutant mice. Overall, our results suggest that the morphological differences seen in G60S/+ and I130T/+ mouse mandibles are due to delayed ossification and suggest that mechanical forces may exacerbate the effects of ODDD on the skeleton.

To assess skull bone thickness from birth to skeletal maturity at different sites to provide a reference for the correct selection of pin type and pin placement according to age.
270 children and adolescents (age: 0-17 years) with a normal CT scan obtained at Emergency Department for other medical reasons were included. Skull thickness was measured on the axial plane CT scans at eight different sites of the vault: midline anterior (A) and posterior (P), right and left lateral (L), antero-lateral (AL), postero-lateral (PL).
From birth to skeletal maturity, L thickness was increased significantly less (+ 58%) compared with AL (+ 205%), P (+ 233%), PL (+ 247%), and A (+ 269%) thickness (P < 0.01). At the end of growth, the thickest and thinnest points of the vault (absolute value) were found at the P and L measurement sites, respectively (P < 0.01). Children aged < 4 years exhibited the highest variability in AL and PL skull bone thickness, with thickness < 3 mm observed in 85% (64/75 patients) and 92% (69/75 patients) of cases, respectively.
We recommend that the tip of the pin should not exceed 2-3 mm in children aged < 4, and 4 mm in children aged 4-6 years, to decrease the risk of inner table perforation. After the age of 7 years and 13 years, standard-sized pin tips (5 and 6 mm, respectively) may be safely used. Children aged < 4 years show significant variability in skull thickness, and therefore a CT scan may be required for this particular age group.

Homology of turbinals, or scroll bones, of the mammalian ethmoid bone is poorly known and complicated by a varied terminology. Positionally, there are two main types of ossified adult turbinals known as endoturbinals and ectoturbinals, and their cartilaginous precursors are called ethmoturbinals and frontoturbinals, respectively. Endoturbinals are considered to be serially homologous due to similarity in their developmental patterns. Consequently, endoturbinals from mammals with differing numbers of elements cannot be individually homogenized. In this study, the development of the ethmoid of Caluromys philander, the bare-tailed woolly opossum, is described based on serial sections of six pouchlings ranging in age from 20 to 84 days postnatal (PND-84), and computed tomography images of an adult skull. I found that four ethmoturbinals initially develop as seen in PND-20 and PND-30 individuals but by PND-64 an interturbinal (corresponding to endoturbinal III in adults) is present between ethmoturbinals II and III. This developmental pattern is identical to that of Monodelphis domestica, the gray short-tailed opossum, and is probably also present in the marsupials Didelphis marsupialis, and Thylacinus cynocephalus based on work of previous authors. These data suggest that endoturbinal III has a developmental pattern that differs from other endoturbinals, and the name interturbinal should be retained for the adult structure in recognition of this difference. These results may prove useful for homologizing this individual turbinal element across marsupials, the majority of which have five endoturbinals as adults. This might also explain the presumed placental ancestral condition of four endoturbinals if the marsupial interturbinal is lost.

We studied the larval development of compound bones from the otico-occipital and cheek regions in species of the neobatrachian genera Batrachyla, Hylorina, Leptodactylus, Odontophrynus and Pleurodema. Comparisons were made using a set of Ambystoma spp. (Caudata) and Ceratophrys ornata (Anura; Ceratophryidae) larvae. As suggested by previous studies, we verified the compound nature of the exoccipital (two centers, anurans only), frontoparietal (one center, most anurans and Ambystoma; three centers, some anurans), and squamosal (two centers, all anurans and Ambystoma) bones. We discuss old and new homology hypotheses for each of the compound bone centers in the context of the most widely accepted scenario of lissamphibian origins and relationships, i.e., monophyletic Lissamphibia that includes the clade Batrachia (Caudata+Anura) and the most divergent Gymnophiona. Our findings have a direct impact on our understanding of the composition of the skull in Lissamphibia. We recognized the presence of the following bones: (i) opisthotic (fused to the exoccipital) and tabular (fused to the squamosal) in Batrachia (Anura+Caudata) and (ii) supratemporal (fused to the parietal portion of the frontoparietal) in Anura. Separate centers of the parietal were found only in Pleurodema.