V. General Regulation of Gene Expression

What is a nucleosome? Why is it necessary for dosage compensation to occur in female mammals? What is a Barr body? What is the putative role of the Xist gene?

VI. RNA Processing and Translational Control

What is the mid-blastula transition in Xenopus? What effect does the nucleus/cytoplasm ratio have on the onset of the mid-blastula transition? How can this ratio be altered experimentally? What is heterogeneous nuclear RNA? How can alternative splicing be used to regulate RNA function? How is alternative splicing used to regulate sex determination in Drosophila? What are some ways in which the translation of mRNAs might be regulated? How are the 3' UTRs thought to function in regulation of translation? Can you cite an example involving c-mos? How does the 3'-UTR regulate spatial localization of mRNAs?

VII. Cytoplasmic Determinants

Classic Techniques Used To Study Differentiation

How can cytoplasmic determinants and inductive interactions be used to generate differences in early embryos? You should be able to work with both hypothetical situations and actual examples of each.

Determination in Ascidians

What is myoplasm/yellow crescent? What experiments indicate that early development in ascidians is mosaic? How do these experiments demonstrate a role for myoplasm as a cytoplasmic determinant? How can cytochalasins and compression of embryos be used to study the role of cleavage in muscle formation? How can we use acetylcholinesterase to assess muscle differentiation?


Determination in Molluscs

What classic experiments indicate that early development in molluscs also has mosaic qualities? What is a polar lobe? What experiments show it is important for the formation of larval structures later in development? What happens when lobe formation is suppressed or made symmetric by brief treatments with low levels of cytochalasin?


Germ Cells

What are germ cells? What is "germ plasm"? What is pole plasm in Drosophila? What are pole cells? What experiments show that pole plasm confers special properties on the cells that receive it? How do mutations in the germ cell-less locus affect pole plasm formation and subsequent pole cell formation? What role do oskar and staufen play in localizing pole plasm constituents at the posterior pole of the oocyte? What are the experiments that show that oskar localization determines where pole cells form? What are nurse cells? What is a maternal effect mutation?


Cytoplasmic Determinants in C. elegans

What are P granules? What role do the par genes play in regulating early asymmetries in C. elegans? How are pie-1 and mex-1 thought to regulate the germ cell lineage? What are some components of P granules?

VIII. Anterior-Posterior Pattern in Drosophila

General

How do the early mitoses in the fly embryo allow for the establishment of simple gradients of proteins that might be involved in specifying pattern? What classic experiments suggested that there were posterior and anterior "organizing centers" in the fly egg? What are the three major systems involved in specifying anterior-posterior pattern? What are the effects of mutating important genes of these three systems?

Be able to outline the levels of genetic control of anterior-posterior pattern in the fly (e.g., maternal-effect system, gap genes, pair-rule genes, segment polarity genes, homeotic genes) in an essay or short-answer format. Be able to cite specific examples of each level of control that we talked about in class.

Bicoid and the Anteroir System

How does bicoid function in the anterior system? What are the distributions of bicoid mRNA and protein in normal fly oocytes and zygotes? How is the distribution of bicoid protein altered in eggs from bicoid mutant mothers? What are the effects of more copies of the bicoid gene on protein distributions? On the location and formation of anterior structures? How does the bicoid protein gradient effect the transcription of the hunchback gene? How does the bicoid gradient affect the activation of hunchback transcription? How is nanos thought to repress translation of hunchback protein in the posterior of the early embryo?

Segmentation Genes in Drosophila

What are the basic actions of the gap, pair-rule, and segment-polarity genes? At what approximate stage of development do these various genes act? How do these classes of genes interact with one another in a general way? How do they interact with the maternal effect systems?

Gap Genes

What is the basic expression pattern of a typical gap gene? What happens when one is mutated? What are the basic domains of expression of hunchback, Krüppel, and knirps mRNA? How do the domains of mRNA expression relate to the corresponding domains of protein expression? What sorts of proteins are encoded by the hunchback, Krüppel, and knirps genes? How does this give us clues of their function(s)?

Pair Rule Genes

What is the basic expression pattern of a typical pair-rule gene? What happens when one is mutated? How do the domains of expression of pair-rule genes relate to one another? Using even-skipped as an example, how are the regulatory regions of pair-rule genes constructed in terms of the control of specific stripes? In a very general sense, how could the boundaries of expression of gap proteins regulate the appearance of specific stripes? What sort of proteins are pair-rule genes like even-skipped and fushi tarazu, and what does that suggest about their function?

Segment Polarity Genes

What is the basic expression pattern of a typical segment polarity gene? What happens when one is mutated? In a general way, how are the levels of pair-rule gene products thought to regulate where segment polarity genes are expressed? What sorts of proteins are encoded by segment polarity genes? What does this suggest about their function(s)? How does this differ from earlier stages of development? What role is hedgehog thought to play in communication between wingless expressing cells and engrailed expressing cells? Be sure you know the major components of the wingless signal transduction pathway. What role is beta catenin (aka armadillo) thought to play in the wingless signaling pathway?

Homeotic Genes in Flies

What is a homeotic mutation? What DNA sequence (and therefore domain of the protein) is similar in all of these genes? What sort of domain is this in terms of its function? In flies, what happens when many of these genes are mutated? How are the regions of expression of these genes related to their position on the chromosome? What other classes of genes are thought to regulate the expression of homeotic genes?What role do the torso-like ligand and torso receptors play in terminal system differentiation? What cells produce torso-like? Torso?

IX. Axis Specification in Amphibians

Classic Experiments on Axis Formation in Amphibians

What is the gray crescent, and how can hair loop experiments be used to show the importance of something in this region of the fertilized egg? How does cytoplasmic rotation relate to formation of the dorsal side of the embryo? How can blastomere transplantation be used to show that dorsal, vegetal blastomeres and then dorsal marginal cells have special properties with respect to axis formation (i.e., rescue of UV-treated eggs, production of double axes, etc.)? What is the "Nieuwkoop Center", and what experiments suggest that it exists? What is the organizer experiment? What is an "Einsteck" experiment? What are the signals of the "three-signal model"? What structure functions like an organizer in amniotes?

Molecular Basis of Mesoderm Induction in Amphibians

What roles are beta catenin, Siamois, FGF, and BMP-4 thought to play in mesoderm induction? How can we show this? What experiments indicate that siamois leads vegetal cells to behave like a Nieuwkoop Center? What happens when mRNA for BMP-4 is injected? What is a dominant negative mRNA construct used for as it relates to growth factor receptors? What happens when the function of receptors for FGF are disrupted? BMP receptors?

The Molecular Basis for the Organizer

What role are noggin and chordin thought to play once the organizer is established? How are they thought to interact with BMP-4? How does this compare with what we know about the dorsoventral axis in Drosophila? How conserved are the functions of the Nieuwkoop Center thought to be in other vertebrates?Can you name transcriptional regulators that are expressed in all mesoderm? Organizer mesoderm? What role does Lim-1 appear to play in establishing anterior mesoderm? How can mouse knockouts be used to address this, and what was the result?

Neural Induction

How does mesoderm induce the formation of neural tissue? You should understand the basic "organizer" experiment performed by Hilde Mangold and Spemann. What did it show? How could they tell that real induction of the host tissue by the grafted piece of dorsal lip material was occurring?

"Planar" vs. "Vertical" Signals in Neural Induction

Neural induction could be occurring at two times (or a combination of both!): (1) before the involution of mesoderm during gastrulation (planar signals) or (2) after involution, when mesoderm lies underneath the neural ectoderm (vertical signals). You should understand the following experiments and how the use of molecular markers has shed new light on them: (i) Sandwiches: what happens if mesoderm and ectoderm are placed in a sandwich side-by-side? What happens to transcription factors expressed at specific axial positions in Keller sandwiches? How does the Einsteck experiment of Otto Mangold work, and why is neural induction thought to involve region-specific vertical signals from the mesoderm?

BMP-4 and Neural Induction

What evidence is there that normally, the organizer produces signals that override the epidermalizing influence of BMP-4? What are those signals?

Hox Genes in Vertebrates

What are the similarities between the homeotic complex in flies and the Hox genes in mice? How are they different? How do the positions of rhombomeres relate to the patterns of expression of Hox genes in the central nervous system? What about their expression in branchial arches (pharyngeal arches)? How can the function of homeotic genes in vertebrates be studied? Can you provide some examples in mice? How are rhombomeres and vertebrae affected in selected examples of Hox gene knockouts? How does retinoic acid affect the anterior-posterior axis in vertebrates? Why do single knockouts have little effect? What about the organization of the Hox genes in veterbrates suggests that single knockouts would have relatively small effects? What do single and multiple knockouts in mice tell us about the redundancy of the Hox genes in vertebrate axis specification? How are Hox genes thought to provide positional identity to cells in the developing limbs, especially the arm (or forelimb)? What do muliple knockouts of Hox genes tell us about their role in specifying bones along the proximal-distal axis of the limb? What is synpolydactyly? What Hox gene, when mutated, is responsible for this condition?

X. Limb Development

Review the chapter in your textbook on limb development. It will be helpful.

The Proximal-Distal Axis

What is the role of the apical ectodermal ridge? What happens when the ridge is removed early? What happens if it removed later? What are consequences of ridge removal for the underlying mesoderm? What role does the age of the progress zone play in determining what the next sructures are that form during limb outgrowth?

Molecular Events in the Early Limb Bud

What role do FGF-10, FGF-8, and radical Fringe play in establishing a limb bud and the early AER What role are FGF-2 and FGF-4 thought to play in mediating AER signals?





Limb Identity

What evidence is there that the identity of the mesoderm determines limb type? What is the role of Tbx-5 and Tbx-4 in specifying limb identity?

The Anterior-Posterior Axis

What is the zone of polarizing activity (ZPA)? How are limbs altered when a ZPA is grafted to a new site? How does the identity of the limb mesoderm determine the sort of limb that forms? What effect does retinoic acid have on a limb bud? What role is sonic hedgehog thought to play in ZPA signaling? How does it mimic a ZPA? How can we test this using shh knockout mice? How is shh thought to regulate the Hox genes (e.g., Hoxd13), and how are they, in turn, thought to regulate the final identity of digits?