Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

AbstractClefts of the lip and/or palate (CL/P) are common anomalies that occur in 1/800 live births. Pathogenic SPECC1L variants identified in patients with rare atypical clefts and syndromic CL/P suggest the gene plays a primary role in face and palate development. We have generated Specc1l gene-trap (Specc1lcGT) and truncation (Specc1lΔC510) alleles that cause embryonic or perinatal lethality, respectively. Specc1lcGT/ΔC510 compound mutants show delayed and abnormal palatal shelf elevation at E14.5. By E15.5, the mutant shelves do elevate and fuse, however, the palatal rugae form abnormally. Palatogenesis requires extensive mesenchymal remodeling, especially during palatal shelf elevation. We posit that this remodeling involves collective movement of neural crest-derived palatal mesenchyme cells. Live time-lapse microscopy was performed to visualize in vitro wound-repair assays with wildtype and SPECC1L-deficient primary mouse embryonic palatal mesenchyme (MEPM) cells. SPECC1L-deficient MEPM cells consistently showed delayed closure in wound-repair assays. To evaluate which features of cellular movement were responsible, we performed automated particle image velocimetry (PIV) and manual cell tracking. The analyses revealed that both cell speed and directionality are disrupted in SPECC1L-deficient cells compared to controls. To determine if primary MEPM cells can move collectively, we assayed stream formation, which is a hallmark of collective movement. Indeed, MEPM cultures displayed correlated movement of neighboring cells. Importantly, correlation length was reduced in SPECC1L-deficient cultures, consistent with a role for SPECC1L in collective migration. Furthermore, we demonstrated that activation of the PI3K-AKT pathway with the 740Y-P small molecule can rescue the wound-closure delay in SPECC1L-deficient MEPM cells. Cell tracking analyses showed that this rescue was due to both increased speed and improved directionality. Altogether, our data showed a novel role for SPECC1L in guided movement through modulation of PI3K-AKT signaling.

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SPECC1L-deficient primary mouse embryonic palatal mesenchyme cells show speed and directionality defects

Scientific Reports ◽

10.1038/s41598-021-81123-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jeremy P. Goering ◽

Dona G. Isai ◽

Everett G. Hall ◽

Nathan R. Wilson ◽

Edina Kosa ◽

...

Keyword(s):

Wound Repair ◽

Cleft Lip ◽

Cell Movement ◽

Live Imaging ◽

Primary Role ◽

Palatal Shelf ◽

Collective Movement ◽

Primary Mouse ◽

Mutant Mouse Embryos ◽

Mesenchyme Cells

AbstractCleft lip and/or palate (CL/P) are common anomalies occurring in 1/800 live-births. Pathogenic SPECC1L variants have been identified in patients with CL/P, which signifies a primary role for SPECC1L in craniofacial development. Specc1l mutant mouse embryos exhibit delayed palatal shelf elevation accompanied by epithelial defects. We now posit that the process of palate elevation is itself abnormal in Specc1l mutants, due to defective remodeling of palatal mesenchyme. To characterize the underlying cellular defect, we studied the movement of primary mouse embryonic palatal mesenchyme (MEPM) cells using live-imaging of wound-repair assays. SPECC1L-deficient MEPM cells exhibited delayed wound-repair, however, reduced cell speed only partially accounted for this delay. Interestingly, mutant MEPM cells were also defective in coordinated cell movement. Therefore, we used open-field 2D cultures of wildtype MEPM cells to show that they indeed formed cell streams at high density, which is an important attribute of collective movement. Furthermore, activation of the PI3K-AKT pathway rescued both cell speed and guidance defects in Specc1l mutant MEPM cells. Thus, we show that live-imaging of primary MEPM cells can be used to assess mesenchymal remodeling defects during palatal shelf elevation, and identify a novel role for SPECC1L in collective movement through modulation of PI3K-AKT signaling.

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Cleavage of Human Embryos: Options and Diversity

Acta Naturae ◽

10.32607/20758251-2016-8-3-88-96 ◽

2016 ◽

Vol 8 (3) ◽

pp. 88-96

Author(s):

Yu. K. Doronin ◽

I. V. Senechkin ◽

L. V. Hilkevich ◽

M. A. Kurcer

Keyword(s):

Time Lapse ◽

Reproductive Technologies ◽

Human Embryos ◽

Imaging Data ◽

Noninvasive Methods ◽

Topographic Features ◽

Time Parameters ◽

Embryo Cleavage ◽

Time Lapse Imaging ◽

Developmental Dynamics

In order to estimate the diversity of embryo cleavage relatives to embryo progress (blastocyst formation), time-lapse imaging data of preimplantation human embryo development were used. This retrospective study is focused on the topographic features and time parameters of the cleavages, with particular emphasis on the lengths of cleavage cycles and the genealogy of blastomeres in 2- to 8-cell human embryos. We have found that all 4-cell human embryos have four developmental variants that are based on the sequence of appearance and orientation of cleavage planes during embryo cleavage from 2 to 4 blastomeres. Each variant of cleavage shows a strong correlation with further developmental dynamics of the embryos (different cleavage cycle characteristics as well as lengths of blastomere cycles). An analysis of the sequence of human blastomere divisions allowed us to postulate that the effects of zygotic determinants are eliminated as a result of cleavage, and that, thereafter, blastomeres acquire the ability of own syntheses, regulation, polarization, formation of functional contacts, and, finally, of specific differentiation. This data on the early development of human embryos obtained using noninvasive methods complements and extend our understanding of the embryogenesis of eutherian mammals and may be applied in the practice of reproductive technologies.

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