Developmental Dynamics最新文献

Background: Sea urchins have contributed to knowledge of fertilization, embryonic development, and cell physiology for 150 years. Their evolutionary position, as basal deuterostomes, and their long background in developmental biology motivate establishing a genetically enabled sea urchin species. Because of its relatively short generation time of 4-6 months and ease of culture, our lab has focused on the California sea urchin Lytechinus pictus as a multigenerational model and produced knockout and transgenic lines using this species. To ensure that diverse genetic lines can be preserved, methods must be developed to cryopreserve gametes and embryos. We have previously reported methods for cryopreservation of sperm, but robust methods to preserve embryos remain lacking.

Results: Here, we describe a relatively simple method to cryopreserve late gastrulae embryos of L. pictus. Importantly, we show that, after thawing and culturing, the embryos progress through larval development, undergo metamorphosis, and yield juvenile adults, indicating the method is robust.

Conclusion: The cryopreservation of embryos is an important advance that will facilitate the biobanking, sharing, and long-term preservation of diverse genetic lines. This method may also eventually prove useful for cryopreservation of embryos of other marine invertebrates.

Background: 5' Hox genes play crucial roles in limb patterning along the proximal-distal and anterior-posterior axes in mice. However, their functional conservation across tetrapods remains unclear. We previously found that newt Hox13 is essential for digit formation during both development and regeneration. In contrast, the functions of other 5' Hox genes (Hox9-Hox12) in newts remain u[WLYJ-108]nknown. Therefore, we generated 5' Hox knockout newts (Pleurodeles waltl) using CRISPR-Cas9.

Results: Individual knockouts of Hox9, Hox10, and Hox12 disrupted all respective paralogs; however, these newts displayed no apparent abnormalities in limb skeletons. In contrast, Hox11 knockout newts exhibited skeletal defects in the posterior zeugopod and autopod of both the forelimbs and hindlimbs. Moreover, compound knockouts of Hox9 and Hox10 caused substantial loss of stylopod and anterior zeugopod/autopod elements specifically in the hindlimbs.

Conclusion: These findings indicate that Hox9 and Hox10 redundantly regulate stylopod formation in the hindlimbs. Furthermore, Hox9/Hox10 and Hox11 contribute to the development of the anterior and posterior regions of the zeugopod/autopod in the hindlimbs, respectively. These novel roles of 5' Hox genes identified in newts suggest the functional diversification of 5' Hox genes in tetrapod limb development.

Background: The short-lived African turquoise killifish (Nothobranchius furzeri) is an important emerging model organism for gene expression studies, with limited tools for transcript and protein detection, especially methods that are both cost-effective and high-resolution. Brain tissue is particularly challenging to analyze due to its opacity and structural complexity, making whole-organ imaging techniques valuable. However, various tissue-clearing protocols adapted for N. furzeri are long and require specialized equipment.

Results: To address these limitations for gene expression detection techniques, we optimized cryosection-compatible ISH protocols for mRNA detection and adapted the EZ-clear method for whole-brain protein visualization in N. furzeri. Using Gfap and Dat as test markers, we optimized the colorimetric ISH protocol for detecting mRNA in both thick and thin sections, achieved high signal-to-noise ratios, and confirmed expression in expected brain regions. Additionally, we adapted the EZ-clear protocol for brain tissue clearing. We demonstrate the method's compatibility with immunostaining, showing a possible upregulation in Gfap, alongside endogenous fluorescence preservation of transgenic reporter lines.

Conclusions: Our protocols add to the existing cost-effective and accessible methods for gene and protein visualization in N. furzeri. The cryosection-amenable ISH and adapted EZ-clear protocols expand the methodological toolkit for studying gene expression in this emerging model system.

Trichorhinophalangeal syndrome (TRPS) is a rare genetic disease inherited in an autosomal dominant manner. It occurs in 1 in 100,000 people globally and is caused by several types of mutations of the TRPS1 gene. Since the first human patient was reported in 1966, typical and atypical pathologies, disease courses, and treatment case presentations have been reported. TRPS is characterized by sparse slow-growing fine hair, a bulbous nose with tented nares, and brachydactyly with cone-shaped epiphyses on the hands and feet. Growth retardation and hip dysplasia are also frequently observed, suggesting that hair and skeletal phenotypes are the major pathologies of TRPS. Several animal models have been established and studied intensively to address this rare disease. However, comprehensive treatment strategies for TRPS have not been established. In this review, we summarize TRPS pathologies and the characteristics of TRPS1 as an atypical GATA-type transcription factor. We review rodent strains that have contributed to our understanding of the in vivo roles of Trps1 and discuss their validity as animal models of TRPS. We also summarize diseases that demonstrate pathologies similar to TRPS and findings in their animal models.

Background: Thyroid hormones (TH) play critical roles in embryonic vascular development, yet their precise molecular contributions remain inadequately defined. This study investigates how pharmacological blockade of thyroid hormone receptors (TR) by amiodarone disrupts angiogenesis and associated molecular signaling pathways in chick embryos.

Results: Amiodarone-treated embryos exhibited notable morphological defects, including hematomas, anophthalmia, ventral wall defects, and limb anomalies, primarily affecting lateral plate mesoderm-derived tissues. Chorioallantoic membrane analysis revealed significant reductions in vessel density, branching, and total vessel length, along with increased lacunarity, indicating impaired angiogenesis. Molecular profiling showed consistent down-regulation of key angiogenic regulators such as VEGFα, WNT7A, BMP2/6, and phosphatidylinositol 3-kinase/ Ak strain transforming (PI3K/AKT) at both transcript and protein levels. In silico docking confirmed strong TRα and TRβ binding, while deiodinase activity assays and western blotting demonstrated impaired thyroxine-to-triiodothyronine (T3) conversion and reduced T3 levels, confirming systemic hypothyroidism and disrupted thyroid hormone signaling.

Conclusion: Our findings underscore the essential role of thyroid hormone signaling in embryonic angiogenesis. Disruption of TR activation by amiodarone significantly impairs vascular formation through coordinated suppression of Vascular endothelial growth factor alpha (VEGFα), WNT7A, bone morphogenetic proteins, and PI3K/AKT pathways. These insights enhance our understanding of TH-related developmental disorders and may guide therapeutic strategies for managing vascular dysfunctions associated with impaired thyroid signaling.

Background: Mean linear intercept (MLI) is a method of evaluating lung structure and pathology that is widely used in clinical and research settings. Unfortunately, no widely available software for automation of this process is available, and many clinicians and scientists still perform these measurements manually.

Results: To increase the speed and accuracy of obtaining MLI measurements, we have developed a macro for Fiji is just ImageJ (Fiji) to semi-automate the acquisition of these measurements. Twenty to 25 images from each of 43 mouse lungs, a total of 1042 images, were analyzed manually and by macro (automated) to validate the accuracy of the MLI macro. No significant difference was recorded between the manual and automated methods in mouse lung tissue of either different age (P14, P21, 8 weeks) or different condition (healthy vs. emphysema). Optimization of MLI macro parameters showed that additional measurements beyond three lines per image did not further improve accuracy. We also provide an Excel macro that summarizes the airspace data for each image and averages all the image data in a given batch of images.

Conclusion: This Fiji macro can be used to automate MLI measurement in histological sections of lung tissue faster and with lower variance.

Background: Gene transcription is crucial for embryo and postnatal development and is regulated by the Mediator complex. Mediator is comprised of four submodules, including the kinase submodule (CKM). The CKM consists of MED13, MED12, CDK8, and CCNC. In mammals, there are paralogs for CKM components, including MED13L, MED12L, and CDK19. Neurological disorders have been associated with mutations in CKM genes including MED13L syndrome. MED13L syndrome is generally characterized as a haploinsufficiency of MED13L with a broad phenotypic response due in part to a wide range of de novo mutations.

Results: We developed a Med13l heterozygous (HET) mouse model with an exon 11 deletion to evaluate whether Med13l HET mice are a viable research tool to study human phenotypes. We characterized our mouse model using growth, cardiovascular, and skeletal readouts. We observed Med13l HET mice are smaller than wildtype (WT) littermates, and over 60% of them exhibited one of two craniofacial anomalies: a pug snout with midface hypoplasia or a crooked snout. We also observed discontinuous squamosal sutures in a subset of our Med13l HETs.

Conclusions: Med13l HET mice recapitulate MED13L syndrome phenotypes including a developmental growth delay and craniofacial anomalies. Med13l HET mice represent a novel research tool for MED13L syndrome.