Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Harvard Medical School

Molluscs form their shells out of CaCO(3) and a matrix of biomacromolecules. Understanding the role of matrices may shed some light on the mechanism of biomineralization. Here, a 1401-bp full-length cDNA sequence encoding a novel matrix protein was cloned from the mantle of the bivalve oyster, Pinctada fucata. The deduced protein (Prisilkin-39), which has a molecular mass of 39.3 kDa and an isoelectric point of 8.83, was fully characterized, and its role in biomineralization was demonstrated using both in vivo and in vitro crystal growth assays. Prisilkin-39 is a highly repetitive protein with an unusual composition of Gly, Tyr, and Ser residues. Expression of Prisilkin-39 was localized to columnar epithelial cells of the mantle edge, corresponding to the calcitic prismatic layer formation. Immunostaining in situ and immunodetection in vitro revealed the presence of a characteristic pattern of Prisilkin-39 in the organic sheet and in sheaths around the prisms. Prisilkin-39 binds tightly with chitin, an insoluble polysaccharide that forms the highly structured framework of the shell. Antibody injection in vivo resulted in dramatic morphological deformities in the inner shell surface structure, where large amounts of CaCO(3) were deposited in an uncontrolled manner. Moreover, Prisilkin-39 strictly prohibited the precipitation of aragonite in vitro. Taken together, Prisilkin-39 is the first protein shown to have dual function, involved both in the chitinous framework building and in crystal growth regulation during the prismatic layer mineralization. These observations may extend our view on the rare group of basic matrices and their functions during elaboration of the molluscan shell.

Cloning and characterization of Prisilkin-39, a novel matrix protein serving a dual role in the prismatic layer formation from the oyster Pinctada fucata.

Calcineurin (CN) is a multifunctional protein involved in many important physiological processes in mammalians, but the function of CN in mollusks is still largely unknown. In the present study, through the shell regeneration system, the changes of enzymatic activity of CN were determined in the process of shell regeneration in pearl oyster Pinctada fucata. CN was activated immediately and continuously in the shell regeneration process. The speed of shell regeneration was measured and the ultrastructure of inner shell surface was observed by scanning electron microscopy after inhibiting CN by intramuscular injection of immunosuppresant cyclosporine A (CsA). The results showed that the speed of shell regeneration was delayed and the morphology of calcite and aragonite in the inner shell surface became abnormal when CN was inhibited by CsA. Meanwhile, RT-PCR analysis revealed that the expression of P. fucata BMP-2 in mantle tissue decreased with CsA injection. In vitro secretion level of proteoglycans (PGs) in primary cultures of mantle cells was also decreased when mantle cells were exposed to CsA. Taken together, our results, for the first time, show that CN is involved in the shell formation through regulating the expression of Pf-BMP-2 in mantle tissue, which controls the secretion of PGs/GAGs of the mantle epithelial cells.

Calcineurin plays an important role in the shell formation of pearl oyster (Pinctada fucata).

Regulation of convergence and extension by wnt-frizzled signaling is a common theme in embryogenesis. This study examines the functional requirements of frzb and fzd7a in convergence and extension mechanisms during craniofacial development. Using a morpholino knockdown approach, we found that frzb and fzd7a are dispensable for directed migration of the bilateral trabeculae, but necessary for the convergence and extension of the palatal elements, where the extension process is mediated by chondrocyte proliferation, morphologic change and intercalation. In contrast, frzb and fzd7a are required for convergence of the mandibular prominences, where knockdown of either frzb or fzd7a resulted in complete loss of lower jaw structures. Further, we found that bapx1 was specifically downregulated in the wnt9a/frzb/fzd7a morphants, while general neural crest markers were unaffected. In addition, expression of wnt9a and frzb was also absent in the edn-/- mutant. Notably, over-expression of bapx1 was sufficient to partially rescue mandibular elements in the wnt9a/frzb/fzd7a morphants, demonstrating genetic epistasis of bapx1 acting downstream of edn1 and wnt9a/frzb/fzd7a in lower jaw development. This study underscores the important role of wnt-frizzled signaling in convergence and extension in palate and craniofacial morphogenesis, distinct regulation of upper vs. lower jaw structures, and integration of wnt-frizzled with endothelin signaling to coordinate shaping of the facial form.

Requirement for frzb and fzd7a in cranial neural crest convergence and extension mechanisms during zebrafish palate and jaw morphogenesis.

The neural crest is a unique cell population associated with vertebrate evolution. Neural crest cells (NCCs) are characterized by their multipotent and migratory potentials. While zebrafish is a powerful genetic model organism, the isolation and culture of zebrafish NCCs would provide a useful adjunct to fully interrogate the genetic networks that regulate NCC development. Here we report for the first time the isolation, in vitro culture, and characterization of NCCs from zebrafish embryos. NCCs were isolated from transgenic sox10:egfp embryos using fluorescence activated cell sorting and cultured in complex culture medium without feeder layers. NCC multilineage differentiation was determined by immunocytochemistry and real-time qPCR, cell migration was assessed by wound healing assay, and the proliferation index was calculated by immunostaining against the mitosis marker phospho-histone H3. Cultured NCCs expressed major neural crest lineage markers such as sox10, sox9a, hnk1, p75, dlx2a, and pax3, and the pluripotency markers c-myc and klf4. We showed that the cultured NCCs can be differentiated into multiple neural crest lineages, contributing to neurons, glial cells, smooth muscle cells, melanocytes, and chondrocytes. We applied the NCC in vitro model to study the effect of retinoic acid on NCC development. We showed that retinoic acid had a profound effect on NCC morphology and differentiation, significantly inhibited proliferation and enhanced cell migration. The availability of high numbers of NCCs and reproducible functional assays offers new opportunities for mechanistic studies of neural crest development, in genetic and chemical biology applications.

Characterization of cultured multipotent zebrafish neural crest cells.

Cranial neural crest (CNC) cells are patterned and coalesce to facial prominences that undergo convergence and extension to generate the craniofacial form. We applied a chemical genetics approach to identify pathways that regulate craniofacial development during embryogenesis. Treatment with the nitric oxide synthase inhibitor 1-(2-[trifluoromethyl] phenyl) imidazole (TRIM) abrogated first pharyngeal arch structures and induced ectopic ceratobranchial formation. TRIM promoted a progenitor CNC fate and inhibited chondrogenic differentiation, which were mediated through impaired nitric oxide (NO) production without appreciable effect on global protein S-nitrosylation. Instead, TRIM perturbed hox gene patterning and caused histone hypoacetylation. Rescue of TRIM phenotype was achieved with overexpression of histone acetyltransferase kat6a, inhibition of histone deacetylase, and complementary NO. These studies demonstrate that NO signaling and histone acetylation are coordinated mechanisms that regulate CNC patterning, differentiation, and convergence during craniofacial morphogenesis.

Neural crest development and craniofacial morphogenesis is coordinated by nitric oxide and histone acetylation.

Diabetes is a high risk factor for dementia. Employing a diabetic rat model, the present study was designed to determine whether the content of D-serine (D-Ser) in hippocampus is associated with the impairment of spatial learning and memory ability.

Insulin treatment prevents the increase in D-serine in hippocampal CA1 area of diabetic rats.

The naked carp (Gymnocypris przewalskii), a native teleost, plays an important role in maintenance of the ecological balance in the system of Lake Qinghai (altitude, 3.2 km) on the Qinghai-Tibet Plateau in China. Calcineurin (CN) is the only member of the serine/threonine phosphatase family that can be activated by both Ca2+ and calmodulin (CaM) and involved in many important physiological processes such as salt tolerance/adaption. In this report, cDNAs of CN catalytic subunit paralogue isoforms: GpCAα (GenBank accession no.JQ407043), GpCAγ (GenBank accession no. JQ407043), and CN regulatory subunit (GpCB) (GenBank accession no. JQ410473), were isolated from Gymnocypris przewalskii and their expression patterns in embryos developmentwere characterized. Gene expression profile demonstrated that GpCA and GpCB mRNA was distributed ubiquitously in all embryonic stages and showed decline until final stage of development. Immunohistologicalanalysis revealed CN localization in different tissues including kidney, heart, brain, spermary, and gill. Collectively, these results provide molecular basis and clues to further understand the role of CN during embryos development and its function in tissues for the adaptation mechanism of naked carp.

Cloning and characterization of two subunits of calcineurin cDNA in naked carp (Gymnocypris przewalskii) from Lake Qinghai, China.

Oblique facial clefts, also known as Tessier clefts, are severe orofacial clefts, the genetic basis of which is poorly understood. Human genetics studies revealed that disruption in SPECC1L resulted in oblique facial clefts, demonstrating that oblique facial cleft malformation has a genetic basis. An important step toward innovation in treatment of oblique facial clefts would be improved understanding of its genetic pathogenesis. The authors exploit the zebrafish model to elucidate the function of SPECC1L by studying its homolog, specc1lb.

Yawei Kong

Massachusetts General Hospital,
Center for Regenerative Medicine,
Massachusetts General Hospital, Center for Regenerative Medicine

Harvard Medical School

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage

Yawei Kong

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Harvard Medical School

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage

Massachusetts General Hospital,
Center for Regenerative Medicine,
Massachusetts General Hospital, Center for Regenerative Medicine