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									March 19th 2010

     “Regulation of Organogenesis
     by the Caenorhabditis elegans
          FoxA Protein Pha-4”
      (J. Gaudet and S.E. Mango 2002)

                  Francesco C., William C. & Katryna K.
• Introductory concepts
   – C.elegans, organogenesis, pha-4
• Mango and Gaudet’s paper 2002
   – Results and discussion.
      • Pha-4 a candidate for an organ identity gene
      • Regulation of an organ identity gene
• Mango’s 2009 paper
   – Current/Future work
      • Pha-4 specifies cell fates, morphogenesis, and roles after birth.
• Conclusion
   – Evolutionary implications: multitasking gene products.
 Outline of Introductory Concepts
• C.elegans and pharynx

• Organogenesis

• Forkhead box transcription factor

“Organogenesis’ Model Organism”
• Caenorhabditis elegans (C. elegans)
• Wormatlas.org : C. elegans anatomy resource
              The Pharynx of C. Elegans

Mango, 2009
Pharynx anatomy. (a) Nuclei within the pharynx are shown as muscles (red), neurons
(purple), epithelia (orange), marginal cells (pink), and glands (brown).
Structural Complexity:
Bilobed                           3 Sections

95 Cells                         7 Cell types

Arranged radially                Three fold symmetry (Y-shape)

5 glands                         Twenty neurons
• The process by which the ectoderm, endoderm, and mesoderm
  develop into the internal organs of the organism.

• Text: “…leads to the embryo at last becoming a fully functioning
  organism, capable of independent survival.”

• Text: “ the cellular mechanisms involved in organogenesis are
  essentially similar to those encountered in earlier stages of
  development; they are merely employed in different spatial and
  temporal patterns

• …however; the mechanisms involved are much more
  complex…some general principles (positional information)…no
  unifying principles- it just works that way in that particular organ.”

• Insight from the 2002 paper: Direct transcriptional regulation of
  entire gene networks may be a common feature of organ identity
  genes. Pha-4 is our candidate for the pharynx.
Pha-4: inducing organ identity through
         (a transcription factor for a transcription factor)n?
• The pha-4 locus encodes a gene product that
  most closely resembles a forkhead box A
  transcription factor homolog
• FoxA is a new name for a gene group containing
  HNF3 alpha, beta, gamma-related genes
• FoxA proteins use a conserved, 110-amino acid
  winged helix motif to interact with binding DNA
  and core histones
• FoxA may bind to one side of the nucleosome
  core and occupy a functional linker histone site
“The forkhead box” protein
Building up to Organogenesis in C.

                                THREE PATTERNING SYSTEMS:

                                1) Founder cells (maternal)

                                2) A-P patterning (zygotic: pha-4)

                                3) organ patterning (zygotic: pha-4

              Recall: blastomeres are plastic. Plasticity
              lost after gastrulation.
  Overview: Mango and Gaudet 2002
• Data suggest that pha-4 directly activate most
  or all pharyngeal genes (loss of function shows
  all require pha-4). Therefore, a common
  pharyngeal identity derived from pha-4
• Relative affinity for pha-4 for different
  TRTTKRY elements modulates the onset of
  gene expression (R=A/G, K=T/G, Y=T/C)
  – Affinity modulates the onset of expression of
    targets in combination with additional
    transcription factors.
Identification of Pharyngeal Genes
• To identify genes expressed in the pharynx, Gaudet and
  Mango probed C.elegans microarrays with labelled
  complementary DNA from embryos that produced
  either excess pharyngeal cells (par-1) or no pharyngeal
  cells (skn-1). The ratio of activation in a microarray is
  proportional to the                      .
       • Microarray activity α par-1
                               α 1/skn-1
                               α timing of expression?
                               α binding sites for pha-4?

• 3 independent experiments with microarrays that
  contained 62% of the C.elegans genome and identified
  240 candidate genes.
• CUT OFF= par-1/skn-1 = 2.0
  3 lines of evidence indicate that
microarrays selectively ID pharyngeal
          expressed genes:
1) Microarrays contained 18 genes previously shown to
   be expressed in the pharynx and 15 of these genes
   were identified.

2) mRNA in situ hybridization data demonstrated that
  59/70 positives were actually expressed in pharynx.

3) Made GFP reporters for 10 random microarray positives
   and found that 8/10 were expressed predominantly in
   the pharynx.
• Purpose: to determine whether pharyngeal expressed
  genes were direct pha-4 targets
   – Searched for predicted pha-4 binding sites within microarray
     positive reporters.

• The consensus binding site: TRTTKRY.
   – R=A or G (purine), K= T or G and Y= T or C (pyramidine). (IUPAC
     IUB Nomenclature)
   – Pha-4 binds a TRTTKRY promoter of myo-2.
       • Only known direct target at the time

• All eight of the pharyngeally expressed reporters
  identified carried three or more TRTTKRY elements
• interesting to note: avr-15, eat-20 and peb-1 expression
  dependent on intron containing TRTTKRY element.
                          Figure 1
• Figure 1 A is a diagram of the promoter fragments used to drive
  expression of a GFP:H1S2B reporter.

• Triangles indicate whether element is on top or bottom strand.

• X indicated eliminated element.

• # indicates 5’ end relative to the predicted start codon of each

• “pharynx expression” (+/++/+++) is the relative strength of
  pharyngeal GFP at its peak in embryogenesis. This is assessed
                        Figure 1
• pharynx-specific expression of eight out of eight reporter
  constructs depended on TRTTKRY elements.

• for every promoter analyzed elimination of one or more binding
  elements resulted in either a complete loss of pharyngeal GFP (i
  through vi) or a substantial decrease in expression and a delay in
  the observed onset of expression (vii and viii).

• Findings suggest that expression in the pharynx is critically
  dependent on TRTTKRY elements for genes expressed early (
  M05B5.2) or late (ZK816.4) in development.

• “because our reporter constructs relied on randomly selected
  genes, our findings suggest that many, perhaps all, genes
  selectively expressed in the pharynx depend on TRTTKRY.”
   Pha-4 works during early and late
        pharynx development
• Identified pharyngeal genes & TRTTKRY
• Pha-4 works during early and late pharynx
• temperature-sensitive pha-4 (pha-4(ts))
   Pha-4 works during early and late
        pharynx development
• Pha-4 was required for viability
• Early embryo to late embryo
   – After pharyngeal precursors before pharynx was fully
• Late embryo to L1
   – Arrest at L1 (larvae stage with a “stuffed pharynx”
     phenotype which looks it’s defective in pharyngeal
• L1
   – Small, unhealthy, with feeding defect
Pha-4 works during early and late
     pharynx development
     Pha-4 works during early and late
          pharynx development
•   D2096.6
•   C44H4.1
•   Coelomocytes
•   myo-2
    – CEH-22
    – PEB-1
Pha-4 works during early and late
     pharynx development
Pha-4 works during early and late
     pharynx development
Pha-4 binds TRTTKRY elements in vitro
•   Pha-4 & TRTTKRY
•   TGTTTGC labelled probe from myo2-WT
•   TGTTGAC from M05B5.2 (myo2-M)
•   TGTTTGT from D2096.6 (myo2-D)
•   TATTTGT from ceh-22 (myo2-ceh)
•   TGAACAG from myo2-non
•   Electrophoretic mobility shift assays
Pha-4 binds TRTTKRY elements in vitro
Pha-4 binds TRTTKRY elements in vitro
• Pha-4 binds to different TRTTKRY sequences
  with different affinities
• TATTTGT from myo2-ceh had 4x less affinity
  than TGTTTGC from myo2-WT
• The rest of them had similar affinities
Pha-4 binds TRTTKRY elements in vitro
• EMSA with labelled myo2-WT and myo2-D
  (wt) and myo2-D (mut) cold competitors
• D2096.6-mut had 2x higher affinity
Figure 4: The onset expression of pharyngeal genes due to affinity of TRTTKRY
elements for pha-4
                     Figure 5
• If the affinity of pha-4 for TRTTKRY elements
  regulates onset of expression, then a decrease in
  pha-4 levels should mimic a down mutation of a
  pha-4 binding site.

• This prediction was tested with the early
  expressed M05B5.2 reporter in a pha-4
  temperature sensitive mutant strain.
                    Figure 5 (2)
• (figure 5 a-d) In animals grown at non-permissive
  temperature (20 ), the pharynx was formed but often

• This shows that pha-4 was present at insufficient levels
  to promote proper development.

• Under these conditions the M05B5.2 reporter was
  delayed as compared to the same strain grown at a
  permissive temperature.

• Eventually, expression of this reporter reached a level
  equivalent to that of unshifted controls.
• -(figure 5e) Effect of affinity on onset of expression
  may be generally true. An analysis of promoter for
  microarray positives revealed a correlation between
  the rank of positive genes and the probability of a
  gene having a high-affinity pha-4 binding site in its

• -x-axis shows gene rank (1 is the highest par-1/skn-1 =
  25)and y-axis shows frequency of genes with TRTTKRY
  elements in upstream region of microarray positives.

• -Black bars represent the frequency of genes with
  high- and moderate-affinity sites. Shaded bars
  represent the frequency of genes containing only a
  low affinity pha-4 site.
• Genes that were highly ranked were more likely to
  contain a moderate- to high-affinity site than
  were lower ranked genes ( Chi-square goodness of
  fit, P=0.07).

• Conversely, lower ranked genes were slightly
  more likely to contain only a low-affinity pha-4

• The paper suggests that this correlation was
  revealed because RNA was isolated from embryos
  at mid-stages of development, when the
  abundance of a given mRNA reflected onset of
     Affinity model for pharyngeal
• Proposed two ideas
  – Pha-4 directly regulates a wide array of genes
    expressed at multiple stages of pharyngeal
    expression (temporal regulation)
  – The affinity of TRTTKRY elements for pha-4
    regulates the relative onset of the expression of
    target genes (TRTTKRY affinity)
            Temporal Regulation
• Suggest that sites with high-affinity to pha-4 bind
  in early embryo development, whereas low-
  affinity binding sites may not bind until later
  development (when pha-4 levels are high)
   – Similar to gradient of bicoid along the anteroposterior
     axis in Drosophila embryos
• Threshold responses that allow genes required
  for early development to be able to be expressed
  prior to those genes necessary to later
  components of development  temporal
             TRTTKRY Affinity
• Found that the affinity of a pha-4 binding site
  may or may not relative to the absolute onset
  of pharyngeal gene expression in all cases
• Based on example, high-affinity pha-4 site
  may be necessary but not enough for early
  activation for transcription
• Depending, the availability of other activators
  may determine the onset of expression
 Implications for other developmental
• FoxA proteins occur in multiple other species and
  may directly regulate most or all genes with an
• Mammals and Drosophila  required for early
  developmental stages of digestive tract formation
  and activates late development genes
• Suggest that organ identity factors may use a
  strategy, which resembles the pha-4 strategy, in
  order to establish gene expression during
     Evolution of organogenesis
• Based on findings, suggest that a gene
  network regulated by pha-4 has evolved by
  serial recruitment of target genes
      The Molecular Basis of Organ
    Formation: Insights From the C.
    elegans ForegutMango (2009)
• Transition to Zygotic Control: pha-4 and
  Tissue/Organ Patterning
  – Pha-4 is the key regulator of pharynx development
  – Important because it is the only gene that if deleted
    the entire pharynx is mutated
  – Suggests that it plays a role in transcribing many, or
    even all, the genes involved in the pharynx
  – Organ identity gene within the pharynx
  – Suggest that the role that FoxA factors play in foregut
    specification, differentiation, and function is
    evolutionarily ancient.
• Developmental Plasticity and Commitment to
  Pharyngeal Fate
  – Three strategies that ensure embryonic
    blastomeres commit to pharyngeal fate
     1. Positive-feedback loops between Pha-4 and
        regulators of pharyngeal cell types
     2. Positive autoregulation
     3. Transcriptional repression, which is important to
        inhibit cell fates
• Downstream of Pha-4
  – Multiple inputs ensure that the pharyngeal muscles
    are made and myo-2 is expressed
  – Combinatorial control is important, but it is not the
    only input for pharyngeal patterningthe affinity of
    pha-4 for its binding sites within promoters
• Pha-4 and aging
  – It is required after birth for the development of both
    the pharynx and gonad
  – Pharynx ages over time, which could contribute to the
    health and longevity of animals and the rate of
    pharyngeal pumping declines with age
• Recap of findings
  – PHA-4 is required for pharynx development
  – PHA-4 is involved in or directly activates all pharyngeal
  – Relative affinity for TRTTKRY elements modulates the
    onset of gene expression
  – Direct transcriptional regulation of entire gene
    networks may be a common feature of organ identity
  – Pha-4 is involved in regulating the increase in
    longevity that is induced by diet restriction
•   Gaudet, J. & Mango, S.E. 2002. Regulation of Organogenesis by the Caenorhabditis
    elegans FoxA Protein PHA-4. Science 295 821-825.
•   Hannenhalli, S. & Kaestner, K.H. 2009. The Evolution of Fox Genes and Their Role in
    Development and Disease. Nature Review: Genetics 10 233-240.
•   Mango, S.E. 2009. The Molecular Basis of Organ Formation: Insights From the C.
    elegans Foregut. Annual Review of Cell and Developmental Biology 25: 597-628.
•   Detailed protocols and a list of genes with a par-1/skn-1 ratio 2 are available on
    Science Online at www.sciencemag.org/cgi/content/full/295/5556/821/DC1.
•   http://www.kiwicrossing.com/drrussell/images/worm.jpg
•   http://www.imsc.res.in/~sitabhra/research/neural/celegans/celegans.gif
•   http://upload.wikimedia.org/wikipedia/commons/d/d4/Cell_differentiation.gif
•   http://www.cisreg.ca/tfe/structures/Foxa2-15376-3bpyA.pdb.png_thumb150.png

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