Formation of Embryonic axis Instructor: Dr. Shahzad A. Mufti Advisor Department of Biosciences

AXIS FORMATION IN AMPHIBIANS Phenomenon of the Organizer Importance of gray crescent to initiate gastrulation, dorsal lip of blastopore. Separation of two halves, bisecting or otherwise of gray crescent. Prospective potency → prospective fate concept. April 11, 2018

AXIS FORMATION IN AMPHIBIANS Phenomenon of the Organizer Fate of cells determined during gastrulation i.e. from “regulative” to “mosaic” development. Spemann and Mangold experiments: concept of primary embryonic induction.Transplantation experiments in 1924 involving Triturus, taeniatus and T. cristatus. Noble prize awarded in 1935. April 11, 2018

Dorsal Mesoderm and Pharyngeal endoderm as Organizer Concept of Neiuwkoop Center. Properties of the dorsal mesoderm (chordamesoderm) were induced by vegetal, presumptive endodermal cells underlying them and these endodermal cells, which induce organizer, constitute Neiuwkoop Center. April 11, 2018

Dorsal Mesoderm and Pharyngeal endoderm as Organizer Functions of Organizer: 4 main functions; (1). Ability to initiate gastrulation movements. (2). Ability to self differentiate (into prechordal plate and chordamesoderm). (3). Induce surrounding mesoderm to form paraxial (somite forming) mesoderm. (4). Induction of overlying ectoderm into neural tube. April 11, 2018

Dorsal Mesoderm and Pharyngeal endoderm as Organizer Search for organizer molecules: The proteins secreted by Nieuwkoop center, activate a set of transcription factors in chordamesoderm (organizer), which in turn activates genes → proteins in organizer. April 11, 2018

Dorsal Mesoderm and Pharyngeal endoderm as Organizer Gooscoid gene activated in organizer, which in turn activate migration and involution proteins overall as well as proteins for self differentiation of overlying chordamesoderm and transformation of above ectodermal cells to form nervous tissue. April 11, 2018

Diffuse able proteins of organizer-Ι The BMP inhibitor First experiments to isolate (& analyze) organizer proteins performed in 1933 by Holtfreter. Formation of “exo-gastrulae” ….i.e. chordamesoderm evaginates in hypersaline solution, so no n.s. Transfilter experiments of Saxen and Toivonen (’60s and ’70s). April 11, 2018

Diffuse able proteins of organizer-Ι The BMP inhibitor Dorsal lip tissue → nervous tissue in competent ectoderm across the filter. A bone morphogenetic protein (BMP 4) is an active inducer of ventral ectoderm (epidermis) and also ventralize of mesoderm (blood cells, connective tissue). April 11, 2018 Fertilization

Diffuse able proteins of organizer-Ι The BMP inhibitor Latest research has shown that NORMAL fate of the potential ectoderm is to become epidermis (through bone morphogenetic proteins (BMP). Organizer tissue secretes certain molecules that BLOCK this induction. April 11, 2018

Diffuse able proteins of organizer-Ι The BMP inhibitor Smith and Harland (1992) isolated one of these molecules….Noggin. Noggin mRNA in dorsal lip of blastopore → into notochord this protein induces nervous system in overlying ecto also dorsalizes the mesoderm. Noggin binds to BMP4 and BMP2. April 11, 2018

Diffuse able proteins of organizer-Ι The BMP inhibitor 2nd organizer protein ….. Chordin (by Sasai et al. 1994). It also binds BMP4 & BMP2. Another protein Nodal-related protein3 (xnr-3) also has similar effects. Fourth organizer secreted protein is Follistatin also prevented BMP from binding to ectoderm and mesoderm near organizer. April 11, 2018

Diffuse able Proteins of Organizer-ΙΙ (The wnt inhibitor) We have seen that the most anterior part of brain (forebrain) is uderlaid by pharyngeal endoderm prechordal (head) mesoderm i.e. “endomesoderm”. It has been now established that the “endomesoderm” does not directly induce the most anterior head structures, but they do it by blocking the Wnt pathway and by blocking BMP 4 (the Wnt family of growth and differentiation, inhibits neural induction i.e. it is anti-neuralization protein). April 11, 2018

Diffuse able Proteins of Organizer-ΙΙ (The wnt inhibitor) Bouwmeester et al (1996) showed that a special protein called Cerberus is formed within pharyngeal endomesoderm, which promotes anterior head structures. It appears that Cerberus binds BMPs as well as Nodal-related proteins and xwnt8 proteins. April 11, 2018

Diffuse able Proteins of Organizer-ΙΙ (The wnt inhibitor) Two other proteins Frzb and Dickkopf are also found to be synthesized in “endomesoderm”, and are found to promote head structures. Both apparently do so through preventing Wnt signaling (i.e. by binding with the Wnt receptos). April 11, 2018

Diffuse able Proteins of Organizer-ΙΙ (The wnt inhibitor) Glinka et al (1997) thus propose a new model for embryonic induction: the induction of trunk structures are induced through blockade of BMP signaling from notochord , but for the induction of head structures, both BMP and Wnt signals must be blocked ( as is done by endomesoderm which thus forms the most anterior part of the organizer). April 11, 2018

Diffuse able Proteins of Organizer-ΙΙ (The wnt inhibitor) Conversion of Ectoderm into Neural plate: Till now we have studied all the factors which prevent the mid dorsal ectoderm becoming epidermis, but other genes then need to be activated to convert the ectoderm into neural tissue. One such factor (protein) is neurogenin. It itself is a transcription factor which then activates a series of genes whose products are responsible for the neural phenoptype. April 11, 2018

Regional Specificity of Induction Fore, mid and hind brain followed by spino-caudal region in an exact anteroposterior direction is highly specific. Can be experimentally verified, through transplantation experiments of successive regions of the organizer (roof of archenteron) by Otto Mangold (1933).

Regional Specificity of Induction The anterior most region induced balancers and part of oral apparatus; the next most anterior part induced head structures, such as nose, eyes, optic vesicles etc, then next section induced Hind brain, followed by dorsal trunk and tail region.

Posterior Transforming Proteins: Wnt signals & Retionic Acid ForToivonen & Saxen (1968), experiment with adult tissues, eg Guinea pig liver → fore brain, G.P Bone marrow → spinal cord; put together → all structures including mid and hind brain

Posterior Transforming Proteins: Wnt signals & Retionic Acid Gradients of “neutralizing” and “mesodormalizing” or “posteriorizing” agents. The posteriorizing agent is most likely a protein of Wnt family, as xwnt8. Suppression of this factor results in more anterior structural development of the nervous system. Thus BMP gradient specifies dorso-ventral axis in frog embryos. However, another factor, retinoic acid has been known to be a “posteriorizing” agent (important in patterning hind brain and further posterior spinal cord etc).

The Anterior Transforming Protiens: Insulin-like growth factors In addition to proteins that block BMP and Wnt signaling in the head, there is also a positive signal that promotes anterior head development such as Isuline-like Growth Factors (IFGs) (Pera et al, 2001).

The Anterior Transforming Protiens: Insulin-like growth factors IGFs accumulate in mid dorsal and anterior most region. Blocking of IGFs result in ack of head formation. Thus cells in the head region are not only protected against Wnt signaling, their conversion to brain and placode etc, is firther facilitated bythe IGFs.

Specifying Left-Right Axis In addition to dorsoventral and anteroposterior axes, the embryos also have a right-left axis, In this region expression of a nodal gene in the lateral plate mesoderm on the left side of the embryo plays an important role. In Xenopus this gene is Xnr1 (Xenopus nodal-related1). How and why this gene is activated only on left side is not very clear.

INDUCTION PROTEINS INHIBITORS BMP Inhibitors: Chordin, Noggin, Xnr3 & Follistatin Wnt Inhabibitors: Cerberus, Frzb, Dickkopf PROMOTORS Anterior : Neurogenin & IGF Poterior: Wnt(Xwnt8) & Retinoic Acid LEFT-RIGHT AXIS Nodal genes (such as xnr1)

Differentiation of donor tissue Conclusion Results of tissue transplantation during early- and late-gastrula stages in the newt Donor region Host region Differentiation of donor tissue Conclusion early gastrula Prospective neurons Prospective epidermis Epidermis Dependent (conditional) development Neurons late gastrula Independent (autonomous) development (determined) April 11, 2018

Determination of ectoderm during newt gastrulation Determination of ectoderm during newt gastrulation. Presumptive neural ectoderm from one newt embryo is transplanted into a region in another embryo that normally becomes epidermis. (A) When the tissues are transferred between early gastrulas, the presumptive neural tissue develops into epidermis, and only one neural plate is seen. (B) When the same experiment is performed using late-gastrula tissues, the presumptive neural cells form neural tissue, thereby causing two neural plates to form on the host. (After Saxén and Toivonen 1962.) April 11, 2018

Spemann's demonstration of nuclear equivalence in newt cleavage Spemann's demonstration of nuclear equivalence in newt cleavage. (A) When the fertilized egg of the newt Triturus taeniatus was constricted by a ligature, the nucleus was restricted to one-half of the embryo. The cleavage on that side of the embryo reached the 8-cell stage, while the other side remained undivided. (B) At the 16-cell stage, a single nucleus entered the as yet undivided half, and the ligature was constricted to complete the separation of the two halves. (C) After 140 days, each side had developed into a normal embryo. (After Spemann 1938.) April 11, 2018

Asymmetry in the amphibian egg Asymmetry in the amphibian egg. (A) When the egg is divided along the plane of first cleavage into two blastomeres, each of which gets one-half of the gray crescent, each experimentally separated cell develops into a normal embryo. (B) When only one of the two blastomeres receives the entire gray crescent, it alone forms a normal embryo. The other half produces a mass of unorganized tissue lacking dorsal structures. (After Spemann 1938.) April 11, 2018

Organization of a secondary axis by dorsal blastopore lip tissue Organization of a secondary axis by dorsal blastopore lip tissue. (A) Dorsal lip tissue from an early gastrula is transplanted into another early gastrula in the region that normally becomes ventral epidermis. (B) The donor tissue invaginates and forms a second archenteron, and then a second embryonic axis. Both donor and host tissues are seen in the new neural tube, notochord, and somites. (C) Eventually, a second embryo forms that is joined to the host. (D) Structure of the dorsal blastopore lip region in an early Xenopus gastrula. (A-C after Hamburger 1988; D after Winklbauer and Schürfeld 1999 and Arendt and Nübler-Jung 1999.) April 11, 2018

Summary of experiments by Nieuwkoop and by Nakamura and Takasaki (1970), showing mesodermal induction by vegetal endoderm. (A) Isolated animal cap cells become a mass of ciliated epidermis, isolated vegetal cells generate gutlike tissue, and isolated equatorial (marginal zone) cells become mesoderm. (B) If animal cap cells are combined with vegetal cap cells, many of the animal cells generate mesodermal tissue. (C) Model for mesoderm induction in Xenopus. A ventral signal (probably FGF2 or BMP4) is released throughout the vegetal region of the embryo. This induces the marginal cells to become mesoderm. On the dorsal side (away from the point of sperm entry), a signal is released by the vegetal cells of the Nieuwkoop center. This dorsal signal induces the formation of the Spemann organizer in the overlying marginal zone cells. The possible identity of this signal will be discussed later in this chapter. (C after De Robertis et al. 1992.) April 11, 2018