Characterization of a Fetal Liver Cell Population Endowed with Long-Term Multiorgan Endothelial Reconstitution Potential.

Stable reconstitution of vascular endothelial beds upon transplantation of progenitor cells represents an important challenge due to the paucity and generally limited integration/expansion potential of most identified vascular related cell subsets. We previously showed that mouse fetal liver (FL) hemato/vascular cells from day 12 of gestation (E12), expressing the Stem Cell Leukaemia (SCL) gene enhancer transgene (SCL-PLAP+ cells), had robust endothelial engraftment potential when transferred to the blood stream of newborns or adult conditioned recipients, compared to the scarce vascular contribution of adult bone marrow cells. However, the specific SCL-PLAP+ hematopoietic or endothelial cell subset responsible for the long-term reconstituting endothelial cell (LTR-EC) activity and its confinement to FL developmental stages remained unknown. Using a busulfan-treated newborn transplantation model, we show that LTR-EC activity is restricted to the SCL-PLAP+ VE-cadherin+ CD45- cell population, devoid of hematopoietic reconstitution activity and largely composed by Lyve1+ endothelial-committed cells. SCL-PLAP+ Ve-cadherin+ CD45- cells contributed to the liver sinusoidal endothelium and also to the heart, kidney and lung microvasculature. LTR-EC activity was detected at different stages of FL development, yet marginal activity was identified in the adult liver, revealing unknown functional differences between fetal and adult liver endothelial/endothelial progenitors. Importantly, the observations that expanding donor-derived vascular grafts colocalize with proliferating hepatocyte-like cells and participate in the systemic circulation, support their functional integration into young livers. These findings offer new insights into the engraftment, phonotypical, and developmental characterization of a novel endothelial/endothelial progenitor cell subtype with multiorgan LTR-EC activity, potentially instrumental for the treatment/genetic correction of vascular diseases. Stem Cells 2017;35:507-521.Spanish Ministry of Economy and Competitiveness (Grant IDs: BFU2010- 15801, CSD-2007-00008), Junta de Andalucıa Regional Government (Grant ID: CVI-295), European Regional Development Funds, Wellcome Trust, Medical Research CouncilThis is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/stem.249

Neural Crest Cell Survival Is Dependent on Rho Kinase and Is Required for Development of the Mid Face in Mouse Embryos

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia

Neural plate morphogenesis during mouse neurulation is regulated by antagonism of Bmp signalling

© The Company of Biologists Ltd 2007Dorsolateral bending of the neural plate, an undifferentiated pseudostratified epithelium, is essential for neural tube closure in the mouse spinal region. If dorsolateral bending fails, spina bifida results. In the present study, we investigated the molecular signals that regulate the formation of dorsolateral hinge points (DLHPs). We show that Bmp2 expression correlates with upper spinal neurulation (in which DLHPs are absent); that Bmp2-null embryos exhibit premature, exaggerated DLHPs; and that the local release of Bmp2 inhibits neural fold bending. Therefore, Bmp signalling is necessary and sufficient to inhibit DLHPs. By contrast, the Bmp antagonist noggin is expressed dorsally in neural folds containing DLHPs, noggin-null embryos show markedly reduced dorsolateral bending and local release of noggin stimulates bending. Hence, Bmp antagonism is both necessary and sufficient to induce dorsolateral bending. The local release of Shh suppresses dorsal noggin expression, explaining the absence of DLHPs at high spinal levels, where notochordal expression of Shh is strong. DLHPs `break through' at low spinal levels, where Shh expression is weaker. Zic2 mutant embryos fail to express Bmp antagonists dorsally and lack DLHPs, developing severe spina bifida. Our findings reveal a molecular mechanism based on antagonism of Bmp signalling that underlies the regulation of DLHP formation during mouse spinal neural tube closure

Neural plate morphogenesis during mouse neurulation is regulated by antagonism of Bmp signalling

Dorsolateral bending of the neural plate, an undifferentiated pseudostratified epithelium, is essential for neural tube closure in the mouse spinal region. If dorsolateral bending fails, spina bifida results. In the present study, we investigated the molecular signals that regulate the formation of dorsolateral hinge points (DLHPs). We show that Bmp2 expression correlates with upper spinal neurulation (in which DLHPs are absent); that Bmp2-null embryos exhibit premature, exaggerated DLHPs; and that the local release of Bmp2 inhibits neural fold bending. Therefore, Bmp signalling is necessary and sufficient to inhibit DLHPs. By contrast, the Bmp antagonist noggin is expressed dorsally in neural folds containing DLHPs, noggin-null embryos show markedly reduced dorsolateral bending and local release of noggin stimulates bending. Hence, Bmp antagonism is both necessary and sufficient to induce dorsolateral bending. The local release of Shh suppresses dorsal noggin expression, explaining the absence of DLHPs at high spinal levels, where notochordal expression of Shh is strong. DLHPs 'break through' at low spinal levels, where Shh expression is weaker. Zic2 mutant embryos fail to express Bmp antagonists dorsally and lack DLHPs, developing severe spina bifida. Our findings reveal a molecular mechanism based on antagonism of Bmp signalling that underlies the regulation of DLHP formation during mouse spinal neural tube closure

Convergent extension, planar-cell-polarity signalling and initiation of mouse neural tube closure

Planar cell polarity (PCP) signalling is necessary for initiation of neural tube closure in higher vertebrates. In mice with PCP gene mutations, a broad embryonic midline prevents the onset of neurulation through wide spacing of the neural folds. In order to evaluate the role of convergent extension in this defect, we vitally labelled the midline of loop-tail (Lp) embryos mutant for the PCP gene, Vangl2. Injection of DiI into the node, and electroporation of a GFP expression vector into the midline neural plate, reveal defective convergent extension in both axial mesoderm and neuroepithelium, prior to the onset of neurulation. Chimeras containing both wild type and Lp mutant cells exhibit mainly wild type cells in the midline neural plate and notochordal plate, consistent with a cell autonomous disturbance of convergent extension. Inhibitor studies in whole embryo culture demonstrate a requirement for signalling via RhoA/Rho kinase, but not jun N-terminal kinase, in convergent extension and the onset of neural tube closure. These findings identify a cell autonomous defect of convergent extension, requiring PCP signalling via RhoA/Rho kinase, during development of severe mouse neural tube defects

Abnormalities in formation of the craniofacial bones in <i>RockDN;Wnt1-cre</i> embryos.

<p><b>A,B</b>) In severely affected <i>RockDN;Wnt1-cre</i> embryos at E14.5, the frontonasal bones (stained with alcian blue) are absent (arrow in B, compare to A). Meckel's cartilage is also reduced in size (arrowhead in B, compare with A). <b>C,D</b>) Bone (red) and cartilage (blue) staining of a mildly affected <i>RockDN;Wnt1-cre</i> embryo at E18.5 (D), shows that the maxilla (arrowhead) and mandibular (arrow) bones are well formed, although the hyoid bone (red arrow) is reduced in size in mutant embryos. <b>E,F</b>) Inferior views of the base of the skull in mildly affected embryos shows that the basisphenoid and the presphenoid bones are hypoplastic in <i>RockDN;Wnt1-cre</i> embryos, whereas the nasal septum is completely missing. Moreover, the maxillary bones are widely separated in mutant embryos (double arrow in F), compared to control littermates (E). bs = basisphenoid; n = nasal septum; ps = presphenoid. Scale bar = 500 µm.</p

Disruption of the actin cytoskeleton and vinculin-containing focal contacts in E9.5 <i>RockDN;Wnt1-cre</i> embryos.

<p>A,B,E,F,I,J,M,N (line i in Q) are sections from first pharyngeal arch and C,D,G,H,K,L,O,P (line ii in Q) are from the frontonasal processes. A–H show phalloidin (red) and caspase 3 (green) immunofluorescence, with E–H being magnified regions as shown by the boxes in A–D, respectively. I–L show vinculin (red) and caspase 3 (green) dual immunofluorescence, with M–P being magnified regions as shown by boxes in I–L. The dotted lines in C,D,G,H,K,L,O,P indicate the boundary between the inner ectomesenchyme and the neural ectoderm and the surface ectoderm. <b>A–H</b>) Filamentous actin, labelled with phalloidin (red) outlines the cells in NCC-derived ectomesenchyme and neural ectoderm in the first pharyngeal arch (A,E) and frontonasal processes (C,G) of control embryos. Cortical phalloidin staining is lost in the ectomesenchyme from <i>RockDN;Wnt1-cre</i> mutants (F,H) but is maintained in the neural ectoderm (compare G with H). In addition, intense phalloidin-labelled foci are observed throughout the ectomesenchyme of the <i>RockDN;Wnt1-cre</i> mutants (dense red foci, blue arrow in F,H). Green caspase 3-positive cells are interspersed (white arrow) and overlapping with the phalloidin-intense cells (arrowheads in F,H). <b>I–L</b>) Vinculin and caspase 3 staining. In the pharyngeal arch the vinculin staining is not restricted to the centre of the arch in the mutant (compare J with I). In the frontonasal processes vinculin outlines the boundary between the surface ectoderm and the neural ectoderm with the inner NCC-derived ectomesenchyme (dotted lines in K,L). This discrete vinculin staining is lost in the <i>RockDN;Wnt1-cre</i> mutants (compare P with O). cas3 = activated caspase-3; mes = mesenchyme; ne = neural ectoderm; phall = phalloidin; se = surface ectoderm; vin = vinculin. Scale bar = 50 µm.</p

Disruption of focal adhesions and extracellular matrix in E9.5 <i>RockDN;Wnt1-cre</i> embryos.

<p>A,B,E,F are sections of the first pharyngeal arch (line i in O) and C,D,G,H,I–N are from the frontonasal processes (line ii in O). <b>A–H</b>) E–H are magnified regions shown in the boxes on A–D, respectively. Paxillin has a cortical distribution in the first pharyngeal arch in the control embryos (A,E) and also marks the boundary between the neural ectoderm and NCC-derived ectomesenchyme (arrow in C,G) in the frontonasal processes. This boundary staining is lost in the mutant (arrow in D,H) and there are intense paxillin positive foci in the pharyngeal arch and frontonasal processes (arrowheads in B,F,D,H), confirming loss of cell-substrate adhesion. <b>I–L</b>) Laminin was lost from the ectomesenchyme-neural ectoderm boundary (compare arrowhead in J with L) and was abnormally distributed in the surface ectoderm in the frontonasal processes in the <i>RockDN;Wnt1-cre</i> embryos (compare arrow in I with K). The H&E staining of the same sections are shown in M and N, allowing visualisation of the different cellular layers. fnp = frontonasal process; pa1 = first pharyngeal arch; ne = neural ectoderm; se = surface ectoderm; mes = mesenchyme. Scale bar in A–D,I,K = 50 µm; M,N = 40 µm.</p

Ectopic and excessive cell death in E9.5 <i>RockDN;Wnt1-cre</i> embryos.

<p>A–D are sections through the neural tube (line i in M), E–H through the pharyngeal arch 1 (line i in M) and I–L through the frontonasal processes (line ii in M). A,C,E,G,I,K show caspase 3-expressing cells (red) and Wnt1-cre+ve NCCs (green). B,D,F,H,J,L are the same sections but only showing the caspase 3-expressing cells. <b>A–D</b>) Whereas only very occasional activated caspase 3-expressing, dying, cells (red) are seen in the neural epithelium in control embryos, there are many dying cells observed in the dorsal part of the neural tube, from which the NCC emerge, in <i>RockDN;Wnt1-cre</i> mutant embryos (arrows in C and D). <b>E–L</b>) Very few activated caspase3-expressing cells are observed in the NCC-derived ectomesenchyme of pharyngeal arch 1 (E,F) and the frontonasal region (I,J) in control embryos. In contrast, many dying cells are seen in corresponding regions from <i>RockDN;Wnt1-cre</i> mutants (G,H,K,L). The surface ectoderm in the mutant is more irregular, compared to controls (arrowheads in E,G,I,K) and the inner NCC-derived ectomesenchyme is loosely arranged with gaps between the cells (arrows in G,K). <b>N,O</b>) The mean apoptotic and mitotic indexes were calculated for NCC within E9.5 pharyngeal arches. There is a significant increase in cell death in the mutant samples compared to controls (P = 0.019; * in N). There is no significant difference in cell proliferation between the two samples (P = 0.433; O). Scale bar = 50 µm.</p