Sea Urchin Gastrulation Sea urchin gastrulation Skeletogenic primary mesenchyme undergo ingression and form spicules skeletal rods The vegetal plate undergoes invagination to form the archenteron Secondary mesenchyme cells extend long filopodia towards the animal pole The mouth will eventually form where they contact the ectoderm Sea urchin gastrulation vegetal plate archenteron blastopore Gilbert 910e Fig 7 12 Sea urchin gastrulation Gilbert 10e Fig 7 12 blastopore spicules Primary mesenchyme cells detach from the vegetal plate Gilbert 10e Fig 7 13 Primary mesenchyme cells detach from the vegetal plate PMCs detach from neighboring cells and the hyaline layer on the outside PMCs then migrate along the basal lamina on the inside This is an example of an epithelial to mesenchymal transition EMT Gilbert 910e Fig 7 13 PMC ingression involves loss of apical contact PMC Gilbert 7e Fig 8 18 apical outside PMC ingression involves changes in adhesion Conclusion changes in adhesion occur during ingression and other EMTs Gilbert 7e Table 8 2 Invagination of the archenteron occurs in two phases Primary invagination shown here involves simple inpocketing of the vegetal plate including apical constriction Archenteron elongation involves convergent extension of the archenteron once is has formed vegetal plate courtesy Chuck Ettensohn Apical constriction occurs during primary invagination Jeff Hardin Apical constriction occurs in the vegetal plate leading to cells sometimes called bottle cells arrows because their shape looks like old fashioned hand blown glass bottles Apical constriction occurs during primary invagination bottle cells Jeff Hardin Laser killing of bottle blocks invagination locally Laser killing bottle cells leads to loss of invagination blue green compared with other parts of the vegetal plate red orange Conclusion bottle cells contribute to primary invagination Archenteron elongation involves convergent extension Gilbert 10e Fig 7 17 Archenteron elongation involves convergent extension Archenteron The number of cells around the circumference of the archenteron goes down as it elongates The total number of cells is roughly constant at this time Conclusion convergent extension is occurring Gilbert 10e Fig 7 17 Convergent extension occurs during archenteron elongation Jeff Hardin Fluorescent groups of cells introduced into the vegetal plate become elongated and interspersed with unlabeled cells Conclusion This is further evidence for convergent extension Secondary mesenchyme cells become active as the archenteron elongates Secondary mesenchyme cells extend long filopodia towards the animal pole This occurs at about the same time the archenteron beings to elongate across the blastocoel SMCs SMCs can pull on the blastocoel roof where they attach cones of attachment filopodium Secondary mesenchyme cells extend filopodia towards the animal pole Where they attach they pull out regions of the animal pole ectoderm called cones of attachment Laser killing of filopodia does not prevent initial elongation but does prevent complete elongation SMC SMC killed PMC Secondary mesenchyme cells arrow A can be inactivated by a laser beam causing them to withdraw filopodia B arrow When all SMCs are killed the archenteron cannot elongate across the blastocoel completely C Conclusion SMCs are not needed initially but they are needed for full convergent extension likely through their pulling force SMCs are required for full elongation Killing all SMCs stops the archenteron after an initial period of elongation open circles Leaving a few SMCs allows slow elongation compare the solid boxes to a normal embryo shown by the Xs As soon as all are killed late the archenteron stop open boxes Conclusion SMCs are required late probably due to their puling force The archenteron undergoes narrowing in the center evidence of tension Jeff Hardin Archenteron elongation putting it all together Initially the archenteron can elongate on its own via convergent extension Later pulling by secondary mesenchyme is likely required for complete elongation This causes additional cell rearrangement The gastrula has two ECM layers hyaline layer basal lamina The gastrula has two extracellular matrix layers On the outside apical is the hyaline layer On the inside basal is the basal lamina Attachment to the hyaline layer is required for invagination Jeff Hardin Blocking antibodies can be used to block adhesion to the hyaline layer Conclusion Attachment to hyaline is required for invagination The basal lamina is required for morphogenesis Jeff Hardin A drug can be added that prevents crosslinking of collagen weakening the basal lamina This causes defects in archenteron elongation Conclusion The basal lamina is required for normal morphogenesis Skeletogenic mesenchyme form syncytial cables Skeletogenic primary mesenchyme migrate and then fuse to form syncytia with a precise pattern The syncytial cables then secrete skeletal rods spicules Gilbert 10e Fig 7 14 PMCs adopt a precise pattern spicule courtesy C Ettensohn PMCs adopt a precise pattern filopodia Skeletogenic primary mesenchyme adopt a precise pattern Two clusters form on the ventral oral side of the embryo vegetal pole ventral cluster vegetal view Wolpert et al 2e Fig 8 33 vegetal ring NiCl2 disrupts PMC patterning NiCl2 disrupts patterning Instead of two clusters on the ventral oral side of the embryo a radialized pattern of spicules results spicule Jeff Hardin NiCl2 embryos can be used to determine what provide pattern information Ectodermal shells can be made by flushing PMCs out of embryos Then PMCs can be transplanted into such shells Ectoderm from a normal or radialized embryo can be used PMCs from a normal or radialized embryo can be used flush out PMCs normal ectoderm radialized ectoderm from normal add PMCs from radialized NiCl2 embryos can be used to determine what provide pattern information The pattern formed by the transplanted PMCs can then be assessed If it is the ectoderm that provides the information then either type of PMC should form defective spicules when transplanted into a shell from a radialized embryo radialized pattern normal pattern Ectoderm patterns mesenchyme Normal PMCs into NiCl2treated Ectoderm NiCl2 treated PMCs into Normal Ectoderm Jeff Hardin Conclusion ectoderm provides pattern information Fibroblast Growth Factor FGF signaling may underlie patterning of PMCs in situ FGF vegetal view Gilbert 10e Fig 7 15 in situ FGF receptor side view SMCs