Abstract
The NK homeobox gene family comprises an extensive collection of transcription factors which have
been shown to play pivotal roles in cell-fate specification and organogenesis. Murine Bapxl (Nkx3.2)
was isolated as an evolutionarily conserved homologue of Drosophila bagpipe (NK3) which specifies
the visceral mesoderm giving rise to gut musculature. A role for Bapxl in gut development has been
conserved to mammals, where it would appear to be required both to specify the identity and to direct
asymmetric growth of the spleno-pancreatic mesenchyme. Epithelial-mesenchymal signalling is an
essential part of gut organogenesis, however the precise role of the mesenchyme is poorly understood
and critical time points for its presence remain unknown. Initial investigations to probe the
importance of the mesenchyme focussed on attempts to ablate this specific cell population by
directing a toxic phenotype to Bapxl -expressing cells. A recombineering approach using Bapxlcontaining
PACs was employed to generate the targeting construct, however inability to modify the
Bapxl locus necessitated adoption of an alternative approach.
The regulatory elements responsible for directing tissue-specific Bapxl expression have not
yet been determined, therefore to pursue investigation of the role of the mesenchyme in gut
development would require elucidation of a gut-specific Bapxl enhancer. Comparative sequence
analysis between human, mouse and fish identified two candidate control elements, termed ProxB and
DistB, which reside 1 lkb and 18kb respectively downstream of Bapxl. ProxB constitutes a short
region of high sequence conservation between human and mouse, within which 180bp was also found
to be conserved to zebrafish. DistB encompasses a more extensive 8kb genomic region highly
conserved in mammals. Both the ProxB putative regulatory element and a 2kb subfragment of DistB
named DistB 1 were cloned into a LacZ reporter construct and assayed for enhancer activity in
transgenic mice. Both elements exhibited tissue-specific enhancer capacities with ProxB-driven
expression initially confined to the mandibular portion of the first branchial arch and later detected in
Meckel's cartilage and the middle ear. Detailed analysis of the expression pattern and comparison to
evolutionarily conserved expression domains provides good evidence that ProxB constitutes a regionspecific
Bapxl regulatory element. DistB 1 appears to be capable of directing expression to several
different tissues, notably including the digits where it closely resembles endogenous Bapxl
expression. Further parallels between the DistB 1 and Bapxl expression domains suggest that this
element may also contribute to the endogenous regulation of Bapxl.
Phenotypic comparisons between Bapxl null mutant mice and their wildtype counterparts
have contributed significantly to knowledge of the wildtype function of the gene. Within the
developing gut, distinct molecular boundaries are apparent. Bapxl demarcates the domain of the
posterior stomach and asplenic null mutants attest to its being essential for formation of the spleen.
Various gut markers were employed to study the disruption of both gut development and defined
molecular domains in the absence of Bapxl. A marked difference in Sonic hedgehog (Shh) expression
was observed in mutant guts, revealing a fusion of the upper duodenum to the posterior stomach to
contribute to the enlarged stomach phenotype. Furthermore, Shh expression was detected in an
abnormal branch emanating from the duodenum and extending alongside the posterior stomach thus
exposing a novel phenotypic manifestation of the mutation.
A novel anatomical structure, the splanchnic mesothelial plate (SMP) has been implicated in
leftward mesenchymal growth during spleen morphogenesis. Cells within the SMP express Bapxl,
without which leftward growth is disrupted and asplenia results. Prompted by these observations, a
description of spleen development was undertaken to closely examine the precise origin and
subsequent morphogenesis of this organ. Expression of the spleen marker Hoxl 1 was followed in
dissected guts within a defined developmental window. The marked morphological transformation
from a small domain of Hoxl/-expressing cells posterior to the stomach to an extensive layer flanking
the dorsal mesogastrium is described herein. This work prompts questions as to whether a wave of
cell-cell signalling or cell migration is responsible for the observed development of the spleen.
