Development of Model Systems Xenopus laevis Part II Chapter 7

Xenopus laevis Principles of Development; Lewis Wolpert

There are visible differences between the animal and vegetal hemisphere before fertilization. First two cleavages bisect the poles (meridional) Third divides embryo into animal and vegetal hemispheres.

A number of maternal mRNAs are differentially distributed along the animal-vegetal axis.

Some localized mRNAs code for known signaling molecules. Veg-T

Regional Specification in Xenopus embryos

Xenopus fate mapping Fig 7.9

Xenopus fate map at late blastula stage Animal hemisphere- ectoderm; nervous system on the dorsal side. Vegetal region-- endoderm Marginal region-- mostly mesoderm; some endoderm.

The three germ layers are first specified, and later patterned into different regions. Fate maps don’t always correspond to specification or determination maps.

Mesoderm has been induced but not patterned Specification vs. Fate Map Late Blastula Mesoderm has been induced but not patterned Nervous system has not been induced

Fig 7.16

Formation and Patterning of Germ Layers

Ectoderm Ectoderm is almost like the “default”. Animal hemisphere cells early on will form ectoderm in culture. No specific determinants have been identified, though there is a protein that promotes ectodermal fate and suppresses mesoderm.

Endoderm Specific maternal mRNA localized in the vegetal cortex acts as the endodermal determinant.

Endoderm VegT, a transcription factor, is the endodermal determinant. If injected into a different region, endoderm forms. If VegT is blocked, no endoderm forms.

Endoderm VegT works by turning on (directly or indirectly) genes for a variety of other endodermal transcription factors. These then turn on endodermal genes.

Mesoderm If early animal hemisphere cells are cultured alone, only ectoderm results. If animal cap is combined with endodermal region, mesoderm will be induced. Vegetal hemisphere is able to signal induction of mesoderm following the mid-blastula transition.

Mesoderm Nodal-related proteins are important in mesoderm induction Members of TGFß-superfamily They bind to cell surface receptors to start a signal transduction pathway that leads to changes in gene expression If applied to animal caps, nodal can induce mesoderm If nodal is blocked, mesoderm isn’t induced.

Nodal signaling pathway

Fig 7-17

Cytoplasmic Determinants The unfertilized egg contains three sets of determinants Vegetal (Endodermal) Dorsal Also contains germ plasm with germ cell determinants in the vegetal region

Xenopus development is regulative at the 2-cell stage. If you separate blastomeres at the 2-cell stage, two small, fairly normal embryos result.

Xenopus development is regulative at the 2-cell stage, but… At the 4-cell and 8-cell stages, it depends on how you separate blastomeres.

Fig 7.14

Presence of determinants can be shown by separating blastomeres. Fig 7.14

Vegetal determinants are localized to vegetal cortex during oogenesis in unfertilized eggs. Later they lead to endoderm formation and provide signals for mesoderm induction.

The dorsal determinants (Nieuwkoop center) are in the gray crescent region that forms following fertilization.

Cortical Rotation Fig 7.3

Cytoplasmic Determinants Fig 7.13

Dorsal-Ventral Axis formation The D-V axis is set up by the sperm entry point. The cortex rotates 30º relative to the inner cytoplasm.

Dorsal-Ventral Patterning Dorsal determinants are initially in vegetal cortex. Microtubules grow from the sperm centriole to the vegetal cortex where they anchor. Cortical rotation along these microtubules moves the determinants to the future dorsal side.

Dorsal-Ventral Axis formation The gray crescent forms opposite the SEP. (Not all frogs have a visible gray crescent). Cortical rotation specifies the signaling center-- Nieuwkoop center-- which will have a dorsalizing (and anteriorizing) influence.

Dorsal determinants from the Nieuwkoop center later specify the organizer (Spemann organizer) which is in the future mesoderm near the location where gastrulation will start.

Dorsal-Ventral Axis formation The first cleavage bisects the Nieuwkoop center so it is in both cells of the 2 cell embryo. Second cleavage divides it into dorsal and ventral halves.

Dorsal-Ventral Axis formation If a 4-cell embryo is divided into dorsal and ventral halves, the dorsal half (with dorsal determinants) will develop into a dorsalized embryo, and the ventral half will be ventralized.

Presence of determinants can be shown by separating blastomeres. Fig 7.14

Dorsal-Ventral Patterning Several experimental treatments interfere with cortical rotation. Drugs that disrupt microtubules UV irradiation of vegetal pole These all disrupt D-V axis and result in radially-symmetrical ventralized embryos.

If cortical rotation is inhibited, the Nieuwkoop center doesn’t form.

UV light disrupts the microtubules involved in cortical rotation.

High UV doses prevent formation of dorsal and anterior structures.

Fig 7.15

Experimental Methods Frog embryos are large and easy to inject. Inject in vitro synthesized specific RNAs to give protein expression in a new place Inject morpholinos to block expression of a gene Morpholinos are short anti-sense RNAs that bind to specific mRNA and prevent it from being translated.

The vegetal region will later be involved in inducing mesoderm. The Nieuwkoop center specifies another signaling center-- the Spemann organizer. Organizer gives signals to pattern dorsal mesoderm and to induce the neural tube.

Nieuwkoop Center Dorsal determinants (Nieuwkoop center) probably work by inhibiting ventral signals. Glycogen synthase kinase 3 (GSK-3) is a ventral signal. If blocked, it results in a dorsalized embryo with overdeveloped anterior structures. A signal from the Nieuwkoop center inactivates GSK-3 to allow dorsal structures to form.

If GSK-3 is expressed on the dorsal side, the If GSK-3 is expressed on the dorsal side, the embryo will be ventralized. Treatment with lithium inactivates GSK-3 giving a dorsalized embryo. Injecting Li into a normal embryo on the ventral side gives a whole new secondary axis, with duplicated dorsal and anterior structures.

Mesoderm Most induced mesoderm is ventral. Small region of dorsal endoderm can induce dorsal mesoderm-- the Nieuwkoop center. Nieuwkoop center induces Spemann Organizer (organizer). Spemann Organizer signals dorsal mesoderm. Cortical rotation  N.C.  S.O.  dorsal mesoderm

Mesoderm Spemann organizer also induces neural tube in the dorsal ectoderm. Cortical rotation  N.C.  S.O.  dorsal mesoderm Induction of dorsal mesoderm and neural tube both block growth factors called BMPs (bone morphogenetic proteins-also in the TGFß superfamily) BMP specifies ventral neural tube

Mesoderm BMP specifies ventral The organizer produces the proteins chordin, noggin and follistatin that interfere with the BMP ventral signal Gives a gradient of BMP signal to pattern ventral to dorsal mesoderm High BMP leads to expression of vent1 and lower BMP leads to expression of vent2, transcription factors (repressors) that interact with other genes to give regional specification, i.e. region with vent 2 gives somites.

BMP inibition BMP specifies ventral. Inhibition of BMP specifies dorsal. Ventral mesoderm treated with BMP inhibitors will be dorsalized Animal cap tissue treated with BMP inhibitors will become neural tissue.

Spemann Organizer Grafting the organizer region into the ventral side of another embryo results in a complete secondary axis The notochord of the second “embryo” comes from the graft, and the neural tube is induced by the notochord. Subsequent inductions give the complete “embryo”

Anterior-Posterior Patterning Spemann Organizer has two regions with different levels of signal to induce neural tissue Anterior organizer induces brain Posterior organizer induces some brain and also spinal cord

Spemann Organizer During gastrulation the organizer becomes part of the chordamesoderm (later notochord) Anterior chordamesoderm induces anterior neural structures and posterior mesoderm induces posterior neural structures.

Spemann’s famous experiment Grafting the dorsal lip of the blastopore to the ventral side of an embryo gives a whole new dorsal axis Grafting the lip at later times during gastrulation results in more posterior dorsal structures being induced.

Anterior chordamesoderm will induce a head with eyes and forebrain. Posterior chordamesoderm will induce a trunk and tail.