Patterning the Embryo

Transcription

1 Patterning the Embryo Anteroposterior axis Regional Identity in the Vertebrate Neural Tube Fig

2 Brain and Segmental Ganglia in Drosophila Fig. 2.1 Genes that create positional and segment identity Fig

3 A-P Patterning Genes/Proteins Nobel Prize 1995 Christiane Nüsslein-Volhard Eric Weischaus Edward B. Lewis 3

4 Hox Genes and Segment Identity A. Normal Tribolium beetle larva B. Hox gene knockout Fig. 2.4 Hox Genes and Segment Identity Ultrabithorax gene loss transforms T3 to T2 4

5 Hox Genes and Segment Identity Antennipedia gene mutation transforms antenna to leg Hox gene clusters are arranged on the chromosome in their spatial order of expression. In vertebrates there were two duplications of the cluster (4 total). Fig

6 Hox genes are conserved across Bilateria Vertebrate Hindbrain Segmentation: Rhombomeres Hoxa1 -/- Partial transformation of r4/5 to 2/3 Fig

7 Do Hox genes control segment identity in vertebrates? Rhombomere marker red- krox 20, blue- hoxb1a Thus r1 is default Fig. 2.7 A-P Axis Patterning Separate head and tail Organizers? or Activation-Transformation? Regionally specific induction by Spemann's organizer. (A) Otto Mangold's experiment demonstrating regionally specific induction by axial mesendoderm. Transversal segments of the archenteron roof of newt neurulae (left) were implanted into the blastocoel of early gastrulae (middle). At tadpole stage, grafted embryos displayed characteristic regionally specific inductions of balancers, heads, trunks and tails, respectively (right). AE, anterior endoderm; acm, anterior chordamesoderm; mcm, medial chordamesoderm; pcm, posterior chordamesoderm; PME, prechordal mesendoderm. (B) Regionally specific induction by early and late blastopore lips. Dorsal lip tissue was explanted (left) at early or late gastrula stages and grafted into the blastocoel of early newt gastrulae (middle). At tadpole stage, grafted embryos displayed head and trunk duplications, respectively (right). 7

8 Activator-Transformer Hypothesis Activator= gene that turns ectodermal cells into neural tissue Anterior is default state of neural tissue Transformer= gene that turns neural tissue into posterior neural tissue Proposes that posterior-ness requires two signals: Neural positive Posterior positive Activators Activator substances primarily induce anterior neural tissue e.g. Noggin, Chordin, Follistatin- Suggests anterior is a default state for neural tissue Predicts that if these activator signals are removed, rather than posterior-like tissue, should get NO neural tissue (not quite true!) 8

9 Transformers RA Wnts Are higher in posterior region Induce posterior gene expression When inhibited a second head can form Wnt inhibitors: Cerberus FrzB dickkopf Adding Wnt inhibitors anteriorizes activated neural tissue FGFs Induce posterior gene expression The 2 Inhibitor Model (A) BMP antagonism results in trunk duplication, (B) a combination of BMP and Wnt antagonists induces heads. (C) Diagram representing the two inhibitor model. 9

10 Transformers Retinoic acid- regulates hindbrain hox genes posteriorizes embryos at high concentrations Normal RA treatment Source of RA is posterior paraxial mesoderm Fig. 2.8 Wnt/BMP inhibition Block Wnt & BMP Dkk1 expression Inhib BMP & Wnt Inhib BMP and add dkk-1 Antibody control Anti-dkk1 Fig

11 BMP antagonists (e.g. noggin) and the Wnt antagonist dkk1 cooperate to activate neural tissue and promote anterior identity. Co-inhibition of BMP and Wnt antagonists (inhibit the inhibitors) is necessary for formation of anterior structures Wild-type Fig Otx2/Gbx2 cross-repression boundary is necessary for forebrain/hindbrain boundary and MHB specification Control Dkk1 -/- and Noggin -/- Otx2-/- Fig Fig

12 Midbrain-Hindbrain Boundary (MHB) Formation Fig Midbrain-Hindbrain Boundary (MHB) Wnt -/- Fig en1 Fig

13 Forebrain fate mapping Chick-quail chimera technique Fig Fig Prosomeresbased on gene expression patterns Fig

14 Eye Field Induction Genes act in complex networks Otx2 Eye field Eyeless misexpression Fig Fig Patterning the Embryo Dorsoventral axis 14

15 Differentiation of the Neural Tube BMP/Wnt Shh Shh Motor neurons Fig Positional Identity in Spinal Cord defined by gene expression patterns Fig

16 Fate Mapping Neural Crest Fig Signals for D-V fate in neural tube- BMP:Shh antagonism Fig Fig

17 D-V nerve cord specification in Drosophila Fig Regional Identity in Cerebral Cortex [but see Molnar 2008] Fig

18 Fgf8 patterning of the cerebral cortex Fig

19 Source for Fig 2.7 Andrew Jan Waskiewicz, Holly A. Rikhof, Cecilia B. Moens (2002) Eliminating Zebrafish Pbx Proteins Reveals a Hindbrain Ground State. Developmental Cell 3(5): Figure 2. MZlzr; pbx2 MO Embryos Lack r2 r6 Identities. RNA in situ hybridizations of wild-type, MZlzr, and MZlzr; pbx2 MO embryos, as shown.(a C) mariposa expression (blue) in rhombomere boundaries is lost in the anterior hindbrain in MZlzr and throughout the hindbrain in MZlzr; pbx2 MO embryos. The arrowhead indicates the r5/r6 boundary.(d F) hoxb1a expression in r4 (blue) is reduced in MZlzr embryos and is eliminated in MZlzr; pbx2 MO embryos.(g I) hoxa2 expression, which in wild-type embryos is expressed from r2 to r5, is limited to r2 in MZlzr embryos and is entirely eliminated in MZlzr; pbx2 MO embryos.(j L) val/mafb expression in r5 and r6 is slightly narrowed in MZlzr embryos, and is also eliminated in MZlzr; pbx2 MO embryos. Embryos in (A) (L) are at hpf. krox20 expression in r3 and r5, and eng3 expression surrounding the mid-hindbrain boundary (MHB) are shown in red in (D) (L).(M and N) Expression of krox20 and val/mafb is never initiated in MZlzr; pbx2 MO embryos (shown here at 11 hpf).(o and P) In contrast, expression of hoxb1b is initiated normally up to the r3/r4 boundary (arrows) in wild-type and MZlzr; pbx2 MO gastrulae, which are shown here at approximately 8 hpf. All embryos are in dorsal views, with anterior to the left in (A) (N) and to the top in (O) and (P). 19

20 Figure 5. Hindbrain Neuron Identity Is Homogenized in Embryos Lacking Pbx Function(A, C, and E) Wild-type embryos; (B, D, and F) MZlzr; pbx2 MO embryos.(a and B) RNA in situ hybridizations with phox2a (blue) and krox20 (red) at 20 hpf showing that the neurons of the locus coeruleus, which differentiate in r0 (arrows), are unaffected in MZlzr; pbx2 MO embryos. Arrowheads indicate hindbrain phox2a-expressing cells in r4 r6 which are absent in MZlzr; pbx2 MO embryos.(c) Cranial motor neurons (isl1-gfp expressing, green) are segmentally organized in 48 hpf wild-type embryos. The motor nuclei of cranial nerves IV (trochlear, in r0), V (trigeminal, in r2 and r3), VII (facial, primarily in r6), and X (vagal, poterior to r7) are indicated. Rhombomeres are also indicated (r).(d) In MZlzr; pbx2 MO embryos, niv are normal and nx neurons are present but reduced; however, very few motor neurons are detected in the intervening region (asterisks).(e) Overlay of cranial motor neurons (isl1-gfp, green) and Engrailed staining (red 4D9 antibody) at 28 hpf. Motor neurons of cranial nerves are indicated as in (A) and (B). At this earlier stage, niv neurons are not yet detectable, but a set of unidentified motor neurons are present in r1 (white asterisks; Higashijima et al., 2000). Eng-expressing neurons differentiate in r1 r3 (red staining; white arrowheads; Hatta et al., 1991). In this dorsal view, the r1 neurons are ventral to the broad Eng domain that extends through r0, and so in this confocal projection, they are not distinguishable.(f) In MZlzr; pbx2 MO embryos, isl1- GFP and Eng-expressing neurons are found homogeneously throughout the transformed region of the hindbrain (white arrowheads). All embryos are in dorsal view with anterior to the left. 20