![]() Red dashed lines mark the Wnt/β-catenin signaling thresholds and the corresponding positions on the oral-aboral axis where these thresholds are reached. Orange curve: Wnt/β-catenin signaling level in a high concentration of AZK. Lilac curve: Wnt/β-catenin signaling level in a low concentration of AZK. Blue curve: wild type level of Wnt/β-catenin signaling along the oral-aboral axis. ( b) Model of expression of a gene activated at medium Wnt/β-catenin signaling levels (upper row blue highlight represents the range of Wnt/β-catenin signaling levels when the gene can be expressed) versus a gene activated at medium and high Wnt/β-catenin signaling levels (lower row green highlight represents the range of Wnt/β-catenin signaling levels when the gene can be expressed) in the AZK treatments. ( a) Expression of Wnt1, Wnt2, Wnt4, Chd and Dpp expands aborally while clearing from the oral domain and disappears with the increasing AZK concentrations expression of Wnt3, axin1-like, Tcf and Bra expands globally and reaches saturation. Red asterisks on lateral views denote blastopores. The embryo fails to elongate and form aboral structures (e.g., apical tuft - yellow arrowhead on ( l)). The oral surface of the treated embryo carries a re-opened blastopore ( j), the aboral surface shows multiple folds and holes. ( j– l) oral ( j) and aboral ( k) views of the 72 hpf planula after early 5 μM AZK treatment and of the control 72 hpf planula ( l). In treated planula, blastopore starts to re-open and small pits appear throughout the surface. ( h, i) Control 48 hpf planula ( h lateral view) and a planula subjected to the early 2.5 μM AZK treatment ( i, oral view). Gastrulae on ( e) and ( g) were split into halves to make inner structures visible. ( f, g) Small blastopore and pre-endodermal plate on SEM of mid-gastrulae after early treatment with 2.5 μM AZK. Note large blastopore and pre-endodermal plate. ( d, e) SEM of control 24 hpf mid-gastrulae oral ( d) and lateral ( e) views. ( c) Nuclear β-catenin-venus in all cells at 6 and 9 hpf of AZK treated blastulae. ( b) Nuclear translocation of β-catenin-venus fusion protein on one side in 6 and 9 h post fertilization (hpf) embryos injected with β-catenin-venus mRNA. ( a) AZK upregulates Wnt/β-catenin signaling by inhibiting GSK3β. ( f) If EF1a::mOrange2 is co-injected with the Wnt plasmids, mOrange2 fluorescence is observed in the induced secondary head (red arrow). The colour code of the bars is the same as on ( c). Fluorescent aboral ectoderm from Wnt1 and Wnt3 injected embryos also acquires inductive capacity. WntA, Wnt4 and Chd never induce a second axis. ( e) Some embryos injected into single blastomeres at 8–16 cell stage with Wnt1 or Wnt3 or with both these Wnt's develop ectopic axes. ( d) Co-injection of a plasmid with EF1α promoter driving the expression of a gene of interest and Dextran-Alexa488 into single cells in 8–16 cell stage embryos results in formation of a coherent patch of fluorescent cells. Fragment 1 and fragment 2, closest to the bend of the blastopore lip, are inductive. Possible developmental outcomes: no axis duplication (green bars on graph), incomplete axis duplication (an outgrowth with tentacles and, sometimes, pharynx but without mesenteries orange bars on graph), complete axis duplication (two contractile axes with head structures red bars on graph). ( c) Results of transplantation of four sequential blastopore lip fragments from donor gastrulae ( N=31) to four different recipient gastrulae ( N=31 × 4). Black arrowhead points at the bend of the blastopore lip. Gene names are colour-coded as on the scheme showing a lateral view on a mid-gastrula with ectodermal Wnt expression domains depicted as coloured lines. ( b) Five ectodermal Wnt genes are expressed at mid-gastrula, however, Wnt2 is expressed at a distance to the bend of the blastopore lip (red double-headed arrows). Left: wild type polyp right: mosaic mutant polyp. ( a) CRISPR-Cas9 knockout of APC results in the formation of ectopic tentacles and pharynges in F0.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |